AN ABSTRACT OF THE THESIS OF

Carlos L. Ballare for the degree of Doctor of Philosophy in Crop Science presented on July 10, 1992. Title: Photomorphogenic Processes in the Agricultural EnvAr,nment

Abstract approved: Redacted for Privacy Steven R. Radosevich

I review recent advances in photomorphogenic research and use information derived from physiological experiments to explain patterns of space occupation and competition among neighboring plants. I found compelling evidence that the presence of surrounding vegetation influences plant growth and development not only because it modifies the availability of light energy and materials, but also because it affects aspects of the environment that are specifically used by seeds and plants to obtain information about the proximity of neighbors (e.g. red:far-red ratio, thermal regime, photon-fluence-rate gradients). The significance of recent findings on signal perception by plants is discussed in connection with the development of mechanistic models of plant competition.

I used a photomorphogenic mutant of cucumber (Cucumis sativus L.) to explore the roles of the photoreceptor phytochrome B in morphological responses to UV-B radiation and in processes that influence morphological development in patchy light environments. The studies on UV-B responses provided strong circumstantial evidence that some morphological effects of UV-B on green plants can be separated from unspecific damage and involve the action of a UV-B photoreceptor. Experiments in natural and artificial light/shade mosaics showed that, when compared over periods of several weeks, wild-type (WT) plants are far more efficient than phytochrome-B-deficient mutants (lh) at invading and maintaining their shoots in light gaps. The reasons for this difference were multiple and appeared to result from several actions of phytochrome B on WT plants. These actions include:(i) establishment of a phototropic system that responds to lateral red:far-red gradients,(ii) modulation of gravitropic responses of stems,(iii) delay of tendril production and reduction of stem-bending responses to mechanical stresses exerted by tendrils, and (iv) switching plants grown under full light from the "shade- avoiding" (default) developmental program to the one that results in a phenotype with short internodes and reduced apical dominance.

In the final section of this thesis I discuss the agricultural significance of photomorphogenic research. Three areas are considered: photocontrol of seed germination, morphological and pigmentation responses to UV- B radiation, and photomodulation of plant morphology in canopies. Photomorphogenic Processes in the Agricultural Environment by Carlos L. Ballare

A THESIS submitted to Oregon State University

in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Completed July 10, 1992 Commencement June 1993 APPROVED: Redacted for Privacy Pro essor of Crop and Soil Science in charge of major Redacted for Privacy Head of department of Crop and Soil Science

Redacted for Privacy

Dean of Grad Schoo

Date thesis is presented July 10, 1992

Typed by Carlos L. Ballare ACKNOWLEDGEMENTS

I wish to express here my sincere gratitude to the individuals and institutions that contributed to thesuccess of this endeavor. I am deeply indebted to Dr. Steven R. Radosevich for his gracious advise in all the steps that led to the completion of my degree, and for his hospitality and support during my stay at Oregon State University. He and Dr. Mary Lynn Roush significantly contributed to broaden my scientific background and forced me to review most ofmy ideas on ecological research and the relationships between science and society. I am also grateful to Dr. Joe B. Zaerr for his advise, for discussions on aspects of science and education, and for helping me with the translation of German papers. He and Dr. Radosevich gave me opportunities to lecture in their Tree Physiology and Weed Ecology classes. My appreciation is also extended to Dr. Terri Lomax, for serving as a committee member and for organizinga class based on seminars during the fall of 1990 where I learneda great deal on molecular aspects of plant development. I had also the good fortune of having Dr. Leslie Fuchigamias the Graduate Council Representative in my committee. Leswas collaborative in every respect and I am most grateful for his cordiality and encouragement. The cooperation ofDr. Paul W. Barnes during the first months after my arrival to Corvallis was very stimulating. Paul allowedme to work in the facilities of the U. S. Environmental Protection Agency and spent many hours discussing with me aspects of photobiology and physiological ecology.

I thank the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) of Argentina for granting me an External Fellowship that made this visit to the United States possible and to the Dept of Forest Science, O. S. U., for additional financial support. I am deeply indebted to Claudio M. Ghersa and M. Alejandra Ghersa, two Argentinean colleagues and friends who helped me and my family in many aspects of our daily life in Corvallis. The Ghersa's place was also the locus of many stimulating discussions on science and ecology. Tim Brewer, Abul Hashem, Andre Laroze, Doris Prehn, the Olivas, Thomanns, Barbara and Gil Vera, and Barbara Yoder were among the friends and mates that contributed to make our stay more enjoyable.

I also wish to thank several O.S.U. staff members for their competence and cordiality, specially Lu Berger, Gretchen Bracher, Fred Friedow, Bud Graham, Randy Hopson, and Mayvin Sinclair.

My greatest appreciation goes to my wife, Ani, and to our son Nicolas, for their love and strength. This work is dedicated to them and to my parents, Pedro and Dora, andmy sister Cecilia. CONTRIBUTION OF AUTHORS

Co-authors of individual papers are identified on the first page of each chapter. The following is brief list of their contributions to each manuscript.

Paul W. Barnes advised me on techniques used for UV-B irradiation and dosimetry, collaborated in the design and execution of the experiments presented in chapter 3, in the discussion of hypotheses and results, and edited successive drafts of the manuscript.

Richard E. Kendrick corresponded with me on some of the ideas presented in chapters 3 and 4 and reviewed the manuscripts.

Steven R. Radosevich advised me on many steps along the completion of this thesis. He contributed to the ideas discussed in chapters 2, 4, 5, and 6 and edited early versions of the manuscripts presented in chapters 4, 5, and 6.

Rodolfo A. Sanchez discussed with me several of the thoughts and hypotheses presented in chapter 6 and commented on an early draft of that paper.

Ana L. Scopel was involved in the discussion of the hypotheses of chapters 4, 5, and 6, collaborated in the design and execution of experiments, and reviewed and improved successive drafts of the manuscripts. TABLE OF CONTENTS

page CHAPTER 1. GENERAL INTRODUCTION 1 CHAPTER 2. LIGHT GAPS: SENSING THE LIGHT OPPORTUNITIES IN HIGHLY DYNAMIC CANOPY ENVIRONMENTS 6 Introduction 7 Sensing and responding to light opportunities 8 Competition for light during early succession 31 Signals, decisions, and models of plant competition 34 Summary 38 References 49 CHAPTER 3. PHOTOMORPHOGENIC EFFECTS OF UV-B RADIATION ON HYPOCOTYL ELONGATION IN WILD-TYPE AND STABLE- PHYTOCHROME-DEFICIENT MUTANT SEEDLINGS OF CUCUMBER 64 Abstract 65 Introduction 66 Materials and Methods 68 Results 71 Discussion 73 References 83 CHAPTER 4. PHYTOCHROME-MEDIATED PHOTOTROPISM IN DE-ETIOLATED SEEDLINGS. OCCURRENCE AND ECOLOGICAL SIGNIFICANCE 87 Abstract 88 Introduction 89 Materials and methods 91 Results 94 Discussion 97 References 111 CHAPTER 5. FORAGING FOR LIGHT IN HORIZONTALLY- PATCHY ENVIRONMENTS BY WILD-TYPE AND PHYTOCHROME-B- DEFICIENT CUCUMBER PLANTS 115 Abstract 116 Introduction 118 Materials and Methods 120 Results 123 Discussion 130 Conclusions 135 References 146 CHAPTER 6. PHOTOMORPHOGENIC PROCESSES IN THE AGRICULTURAL ENVIRONMENT 150 Abstract 151 Introduction 153 Photocontrol of weed seed germination in arable land 153 Plant responses to solar UV-B 157 Photomorphogenic signals in process of plant competition 162 R:FR-signals and phytochrome 162 Fluence-rate signals 164 Implications for crop improvement 166 Summary and outlook 173 References 176 LIST OF FIGURES

Figure page 2.1 Spectral distribution of daylight and wavelengths absorbed by known plant photoreceptors. 41 2.2 Effects of sunlight pulses on the germination of buried seeds of Datura ferox under field conditions. 42 2.3 Elongation rates of the first internode of Datura ferox seedlings growing in even canopies of different densities under natural radiation and effect of density on PAR interception by individual seedlings. 43 2.4 Effect of the proximity of a green grass canopy on the elongation of the first internode of Sinapis alba seedlings growing under full sunlight. 44 2.5 Effects of neighboring seedlings on the spectral distribution of the radiation scattered within the stems of Datura ferox seedlings in an even canopy of LAI = 0.72. 45 2.6 Effects of increasing density in even canopies of dicotyledonous seedlings on light interception by leaves and the light environment of the stems. 46 2.7 Elongation responses of Datura ferox first internodes when seedlings were placed in the center of an even canopy of LAI -0.9 with the internodes surrounded by annular cuvettes containing distilled water (clear filter) or a CuSO4 solution that absorbed FR radiation and maintained the R:FR ratio at ca. 1.1 (FR-absorbing filter). 47 2.8 The effect of green filters that reduced the R:FR ratio of side-light on the azimuthal orientation of main shoots of Portulaca oleracea seedlingsgrown in the field. 48 3.1 Spectral photon distributions measured at plant level under control (polyester-filtered) and UV-B (cellulose-acetate filtered) lamp systems. 78 3.2 Effect of 6-h exposures to UV-B on hypocotyl elongation, cotyledon area expansion and dry matter accumulation in de-etiolated lh and WT cucumber seedlings. 79 3.3 Recovery from UV-B inhibition of hypocotyl elongation in de-etiolated lh and WT cucumber seedlings. 80 3.4 Effects of GA3 on hypocotyl elongation growth in de-etiolated lh and WT cucumber seedlings exposed to UV-B. 81 3.5 Effects of covering different parts of the seedling with a UV-B-excluding polyester filter on hypocotyl elongation in de-etiolated cucumbers exposed to UV-B for 6 h. 82 4.1 Spectral photon distributions of the light sources used in the controlled-environment experiments on phototropism. 103 4.2 Effect of the proximity of a green canopy on the orientation of the hypocotyl of WT seedlings at solar noon. 104 4.3 Time courses of hypocotyl bending in WT and lh seedlings growing under white light with or without exposure to unilateral FR light. 105 4.4 Effects of increasing the fluence rate of unilateral FR light on hypocotyl curvature in WT and lh seedlings. 106 4.5 Effects of increasing the fluence rate of bi- lateral FR light on hypocotyl elongation in WT and lh seedlings (the latter with or without application of GA3). 107 4.6 Effect of shading the hypocotyl during irradiation with unilateral FR light (121.7 gmol m2 s4)on hypocotyl curvature in WT seedlings. 108 4.7 Effects of the proximity of a green maize canopy and B-absorbing acetate filters on the spectral photon distribution of the light flux measured parallel to the ground. 109 4.8 Effects of the proximity of a green maize canopy and B-absorbing acetate filters on the orientation of the hypocotyl of WT and lh seedlings at solar noon. 110 5.1Morphological development of WT and lh plants under conditions of uniform illumination in the controlled environment. 138 5.2 Graviotropic responses of mature WT and lh plants under conditions of full light in the controlled environment. 139 5.3 Spatial distribution of nodes produced by WT and lh plants after four weeks of growth at the border of a maize canopy in the field. 140 5.4 Internode production, spatial distribution of main stems and tendril dynamics in WT and lh plants grown for 6 weeks at the border of simulated canopies in a controlled environment. 141 5.5 Angular orientations of leaves and main-stem internodes of WT and lh plants grown for 5 weeks at the border of artificial canopies in the controlled environment. 142 5.6 Time courses of artificial gap invasion by six WT and six lh shoots in the controlled environment. 143 5.7 Effects of tendril attachment to supports on tendril morphology and internode orientation. 144 5.8 Changes of internode curvature after severing the attached tendril in lh plants. 145 LIST OF TABLES

Table page 4.1 Effects of the proximity of green plants on the light flux measured parallel to the ground. 102 5.1 Stem length and angle of inclination toward the artificial light gaps in WT and lh-mutant seedlings. 137 PHOTOMORPHOGENIC PROCESSES IN THE AGRICULTURAL ENVIRONMENT

CHAPTER 1

GENERAL INTRODUCTION

Life on Earth is dependent on the process of photosynthesis performed by plants. Apart from providing the energy for photosynthesis, light has a dramatic influence on the development of living organisms. Light is a vehicle of information. Many life forms, but particularly photoautotrophic plants, have evolved fascinating systems to obtain information about their environment by "measuring" certain aspects of the impinging radiation. The acquisition of information by higher plants is the principal focus of subsequent chapters, but I will give some general examples here. Seeds of weedy species do not germinate at random through time. Light is one of the environmental factors that signals seeds to germinate. A specific photoreceptor in the seeds (phytochrome) can detect short light pulses when the surrounding soil is disturbed by tillage,or changes in the spectral distribution of radiation when a tree-fallopens a gap in the canopy. When seedlings or sprouts penetrate through the soil toward the surface their morphology is completely different from the one we see after emergence. The stems are elongated, the leaves rudimentary, and tissues contain little or no chlorophyll. It is light, perceived by specific photoreceptors while the seedlingsare breaking through the soil surface, that triggers all the changes in morphology and physiology that make the seedling competent to photosynthesize. The enzymes that are responsible forCO2 fixation in the chloroplasts are not active all the time. 2 The reducing pathway is shut off every night. It is the activity of the photosystems, fueled by sunlight, that changes the chloroplast environment and activates the photosynthetic catalysts. The morphology of plants is tremendously plastic. Plants grown with plenty space are often profusely branched, short, and form extended canopies (i.e. umbrella-type trees). Plants of the same genotype grown in a crowded population are commonly less branched and form vertically-extended canopies or crowns (i.e. lolly-pop type trees). It is light, detected by specialized photoreceptors, that informs plants about the proximity of neighboring individuals and elicits changes in morphological development. Finally, many plants go through seasonal cycles of vegetative and reproductive activity. The duration of the photoperiod, detected by internal mechanisms re-phased by light signals, is one of the factors that informs plants about seasonal changes.

Photomorphogenesis is a term used to refer to all regulatory actions of light on plant growth and development that are independent of photosynthesis. A milestone in the study of photomorphogenesis was the discovery, forty years ago, of the plant photoreceptor phytochrome, by a scientific group led by H. A. Borthwick and S. B. Hendricks at the U.S.D.A. Plant Industry Station, Beltsville. We know today that phytochrome is not a single pigment. In Arabidopsis, for example, three different phytochromes and five different phytochrome genes have been discovered over the last few years. In addition, we have learned that other photoreceptors, such as the blue/ultraviolet-A-, and the ultraviolet-B-absorbing photoreceptors, are involved in the control of photomorphogenesis.

How do these photoreceptors act? The first step is light absorption by the photoreceptor (e.g. phytochrome) 3 accompanied by some change in the properties of the pigment molecule. This alteration initiates a transduction process, that is a series of molecular events that culminate in altered growth or development. The precise sequence for any response is not yet known, but the gap in understanding of how the "black boxes" work between light absorption and observable response has been dramatically narrowed during the last few years. Phytochrome-mediated processes are being studied intensively. One of the things that we have learned is that there is more than just one primary mechanism of phytochrome action. In some cases it is clear that plant responses mediated by phytochrome do not involve changes in gene expression (e.g. chloroplast orientation in the alga Mougeotia). Yet, in others (probably the majority of phytochrome responses), light absorption by phytochrome leads to altered expression of selected genes. There is little evidence for a direct interaction between phytochrome and the genome and, possibly, there is more than one mechanism whereby phytochromes affect transcriptional activity.

Two areas of photomorphogenic research have made rapid progress during the past two decades. The explosion of molecular biology gave photobiologists a whole spectrum of new techniques to study the molecular properties of photoreceptors and to unravel signal transduction chains. A significant example is the recent characterization of several phytochrome genes and molecular species, which opened the possibility that different phytochromes have different mechanisms of action and physiological roles. On the other hand, many plant physiologists and ecologists became interested in the significance of photomorphogenesis in the natural environment.

This thesis is focused on the ecological significance 4 of photomorphogenesis in the agricultural environment. In Chapter 2, I review some recent developments on how seeds and growing plants perceive the light environment and discuss the role of information acquired via photomorphogenic pigments on mechanisms of competition in plant communities. The style of chapter 2 is deliberately provocative. Its last section contains a great deal of speculation, and I invite the reader to takesome of the ideas expressed there as an evolving conceptual model, from which constructive discussion and experimentation could be derived.

In the next three chapters I explore the involvement of photomorphogenic mechanisms and phytochrome on selected responses of plants to the light environment. Each chapter was written as an individual manuscript and can be read independently of the others. The experiments reported in Chapter 3 constitute and attempt to understand morphological (more specifically stem-elongation) responses of plants to ultraviolet-B radiation. This is an issue of importance for agriculture given the increased concern about stratospheric ozone depletion and its potential effects on ultraviolet-B levels received by plant communities. Although there is considerable information on effects of ultraviolet-Bon plant growth, the underlying mechanisms are still poorly understood. Chapter 4 is concerned with the ecological significance of plant phototropic responses and, by usinga phytochrome-B-deficient mutant of cucumber, I investigated the role of phytochrome in phototropism. The major conclusions of that series of experimentsare that (i) phytochrome functions as a phototropic sensor ingreen plants and (ii) phototropic responses to red:far-red gradients created by neighboring plants allowyoung seedlings to actively project leaves into lightgaps when colonizing patchy canopies. This study is expandedupon in 5 Chapter 4, where I compared wild type and phytochrome-B- deficient plants of cucumber in regard to their efficiency to invade light gaps in heterogeneous canopies. Several critical actions of phytochrome B on plant morphological development that are of key importance in the processes of "foraging" for light in patchy environmentsare identified and discussed. The evidence derived from these physio- ecological studies with a photomorphogenic mutant has significant implications for our perceptions about mechanisms of competition among neighboring plants.

In the last Chapter, I review recent advances in the understanding of the ecology of photomorphogenesis from the perspective of an agronomist. Three majorareas are covered: photocontrol of seed germination, responses to solar ultraviolet-B, and morphological and developmentalresponses in plant canopies. The chapter closes witha discussion on implications of recent discoveries for the improvement of cropping systems. One of the points emphasized in that review is the importance to pursue photo-physiological research under field conditions. Such an approach is urgently needed if the goal is to discover how the expression and agronomic significance of photomorphogenic responses is affected by other factors of the crop's environment. 6

CHAPTER 2

LIGHT GAPS: SENSING THE LIGHT OPPORTUNITIES IN HIGHLY DYNAMIC CANOPY ENVIRONMENTS

Carlos L. Ballar6

Depts of Forest Science and Crop and Soil Science, Oregon State Univ., Corvallis, OR 97331-5705, USA, and Dept de Ecologia, Facultad de Agronomia, Univ. de Buenos Aires, (1417) Buenos Aires, Argentina.

Exploitation of Environmental Heterogeneity by Plants: Ecophysiological Processes Above and Below Ground. Edited by M. M. Caldwell and R. W. Pearcy, Academic Press, In review. 7 I. INTRODUCTION In natural environments the amount of photosynthetic light energy received at a given point in space is extremely variable over time. Part of this variability is associated with seasonal changes, part is due to the activity of nearby vegetation and, at a finer time scale, to weather factors and changes in solar angle during the course of the day. Because plant fecundity and carbon gain are likely to be positively correlated in most situations, the ability to exploit light opportunities in temporally and spatially patchy environments should be regarded as a major determinant of evolutionary success. The first (obvious?) condition for a plant to exploit a pulse of light availability is that it must be growing at the right time. That is germination or sprouting must be synchronized with the onset of periods of high light availability. The second condition is that the plant has to manage to position and orient its light absorbing surfaces so as to maintain high rates of light capture during the growing period. Finally, the photosynthetic apparatus must be adapted to operate under the prevailing light environment.

Plants exhibit a fascinating variety of mechanisms for acquiring information about the light environment. These mechanisms operate at several stages controlling, inter allia, the timing of seed germination, the transition to the photoautotrophic stage, the spatial orientation of branches and leaves, the elongation of stems and hence the height of leaf insertion, the production of new branches or tillers, and the performance of the photosynthetic apparatus.

The consequences of these mechanisms, that is the association between germination flushes and canopy openings or soil disturbances, or between plant morphology and canopy density and structure, have been the subject of previous reviews or are covered by other authors. This chapter deals 8 with the processes involved in the perception of light opportunities by plants in systems characterized by rapid changes of plant cover over time, such as those dominated by herbaceous species in early secondary succession. In the first and major part of the paper I concentrate on what is known about the nature of the environmental signals and sensory systems that regulate physiological and morphological responses to changes in light environment. The focus is on germination responses of seeds and morphological reactions of plants of early successional species. Then I present an overview of the likely roles of information- acquiring systems for plants that exploit patchy and highly dynamic light environments. Finally, I discuss the necessity of considering these systems in mechanistic models of plant competition. Most of the examples I use in the discussion are derived from research on herbaceous species from agricultural plant communities.

II. SENSING AND RESPONDING TO LIGHT OPPORTUNITIES A. Environmental signals and plant receptors Seeds and growing plants use many environmental cues to obtain information about the prevailing light climate. These cues are factors of the environment that, under natural conditions, are directly or indirectly associated with important aspects of the flux of photosynthetically active radiation (PAR), such as its intensity, direction, and likelihood of future change. Examples of these cues are the amplitude of the daily fluctuation of soil temperature sensed by a seed, the differential intensity of blue light received at opposite flanks of a stem, or the ratio between the fluxes of red and far-red radiation. The way in which seeds and plants perceive these signals is considered in this section. As it will be seen light signals, perceived by specific photoreceptors play in many cases a fundamental role in the detection of light opportunities. Therefore, a brief description of plant photoreceptors is necessary here.

As far as we know today, higher plants contain at least three families of photoreceptors (e.g. Mohr 1986)(Fig. 2.1): 1. Phytochromes, which absorb radiation over a wide range from the ultraviolet (UV) to the far-red (FR) with maxima 9 around 660 nm for the Pr form, and 730 nm for the Pfr form 2. A series of blue (B)/ UV-A absorbing pigments 3. A little characterized receptor for UV-B radiation.

For the purposes of this paper we can assume that these receptors are devices that transform light into biological signals (see Smith 1982, for a discussion of partial processes involved in light perception by plants). It should be kept in mind that, in addition to the specific photoreceptors listed above, the two photosystems in chloroplasts (PSI and PSII) may act as sensors of variations in light environment and play a role in processes such as the long-term photosynthetic acclimation to total irradiance (e.g. Chow et al. 1990). More indirectly, plants may accommodate to variations in light conditions by sensing and reacting to changes in levels of photosynthetic products, which may act as internal developmental signals (see an example in Section II.B.5.).

Phytochrome is the best characterized of all plant photoreceptors and, because it is involved in many of the responses discussed in this chapter, a brief overview of its major photochemical properties is included in this section. Detailed coverage can be found in several recent books (e.g. Kronenberg and Kendrick Eds. 1986, Furuya Ed. 1987, Attridge 1990). Phytochromes are now known to be a family of proteins associated with a very similar chromophore (Sharrock and Quail 1989, Furuya 1989). Phytochrome molecules exist in two relatively stable forms, Pr and Pfr, with absorption maxima in the R and FR regions of the electromagnetic spectrum, respectively Fig. 2. 1). Each form is converted into the other upon absorption of light. Because the absorption spectra of Pr and Pfr overlap to a large extent, the pigment cannot be pushed to pure Pr or Pfr. Thus, under conditions of continuous illumination, a photoequilibrium (0)between the two forms is reached that may be characterized by the proportion of phytochrome molecules that are in the Pfr form, i.e. the Pfr/P ratio. Values of 0 may vary from 0.02 under monochromatic FR to 0.86 under monochromatic R (e.g. Mancinelli 1988). If the pigment is exposed to polychromatic light, as is the case under natural conditions, the Pfr/P ratio at 0 is highly dependent on the ratio of R to FR 10 photon fluxes in the incident radiation (R:FR ratio) (Smith and Holmes 1977). Thus, 0 values obtained by exposing purified phytochrome preparations to natural light fields typically vary from ca. 0.6 (direct sunlight; R:FR = 1.15) to 0.15 (light filtered through dense leaf canopy; R:FR = 0.2). The effects of light treatments on a number of physiological functions are well correlated with their effects on 0. Values of 0 can be estimated analytically (Hartmann and Cohnen-Unser 1972) using spectral irradiance data and published values of phytochrome photoconversion- coefficients (e.g. Smith 1986, Mancinelli 1988). The same model can be used to calculate the rate of cycling between Pr and Pfr, which might modulate certain responses to total irradiance (Section II.B.3.), and to estimate how much Pfr is formed after exposures to light that are too short to drive the pigment to photoequilibrium (e.g. light pulses perceived by seeds during soil cultivations, Section II.B.1.)(see Cone and Kendrick [1985], Scopel et al. [1991]). It should be noted here that physiological responses to different aspects of the light environment (e.g. total irradiance, R:FR ratio) might be mediated by different molecular species of phytochrome.

B. Plant responses to light opportunities 1. Seed Germination. Successional environments are characterized by transient increases in light availability at the soil surface, which are caused by disturbances that eliminate or reduce the existing vegetation. Canopy gaps may be created by natural agents (i.e. fire, storms, or herbivores) or by man in managed ecosystems. Gaps may be of very different sizes, from minute ones created by small herbivores foraging on herbaceous vegetation to those involving hundreds of hectares caused by tillages in cultivated areas. Frequently, disturbance is followed by pulses of germination of seeds of colonizing species.

The environmental changes brought about by disturbances that are responsible for triggering seed germination are, as can be anticipated, many and diverse. To a large extent the subject is reviewed by Vdsquez-Yanez (1993); therefore, coverage here will be selective and restricted to the 11 perception by seeds of light opportunities in cultivated lands.

In arable lands, light at the soil surface level varies in a more or less cyclical fashion due to the managing practices associated with the production of a particular crop. The major disturbances, and hence the major causes of light opportunities in these systems are grazing, mowing, and soil cultivation. Several factors have been proposed to work as environmental signals in the mechanisms whereby seeds detect these events, including soil temperature changes, light, changes in the soil atmosphere, and modification of chemical composition of the soil solution. These are briefly considered in the remainder of this section. a. Light opportunities created by canopy removal One of the major consequences of canopy removal is to modify the thermal regime of the topsoil. In particular, the daily thermal amplitude (i.e., temperature - temperaturemin) in the upper strata is much larger in bare soil than under a dense vegetation canopy. Seeds of weed species usually germinate better at alternating than at constant temperatures (e.g. Aldrich 1984). This observation led Thompson et al. (1977) to propose that, under natural conditions, increased temperature fluctuations may signal to seeds when canopy gaps occur. The best evidence for the operation of a gap sensing-mechanism dependent on soil temperature fluctuations was provided by Benech Arnold et al.(1988) working with Johnsongrass (Sorghum halepense). They found that germination of this weed was greater in experimental canopy openings than under undisturbed vegetation, and were able to demonstrate that the promotive effect of clipping could be reproduced under the canopy by manipulating the soil temperature regime to mimic the temperature fluctuations that occurred in the adjacent bare plots.

In other cases, the environmental factor that informs seeds about the occurrence of openings in the leaf canopy appears to be light itself. From an optical point of view, green leaves are made of a strongly scattering material with 12 well defined absorption bands (Knipling 1970). Flavonoids and photosynthetic pigments absorb most of the radiation in the UV and visible wavelengths (i.e. 290-700 nm), and water absorbs strongly in the infrared (> 1200 nm). There is very little absorption between 700 and 1200 nm so that most of the FR emerges from either side of the leaf in the form of scattered radiation. This accounts for the low R:FR ratios that are observed in transmittance and reflectance spectra of green plant organs and in light measurements taken beneath vegetation canopies.

Classic studies on the photocontrol of seed germination, carried out mainly on freshly dispersed seeds or with seeds kept in dry storage for some time, have shown that, in order to be effective, light must establish a relatively high level of the Pfr form of phytochrome (see references in Bewley and Black 1982). Thus, the potential effect of light can be nullified if the Pfr content is immediately reduced to about 2 % with a saturating pulse of FR radiation. Seeds exposed at the soil surface to light filtered through a leaf canopy (low R:FR ratio) would be prevented from germinating. Opening the canopy will increase the R:FR ratio and this would form enough Pfr in the seeds to promote germination (see Vasquez-Yanez 1993). The evidence for a gap-sensing mechanism based on perception of light quality changes in herbaceous plant communities under natural conditions is scant. Perhaps one of the drawbacks of a R:FR-driven dormancy mechanism is that seeds would be required to stay at or very near the soil surface in order to perceive changes in light environment. Rates of seed predation have been shown to be overwhelming in abandoned (Mitelbach and Gross 1984, van Esso and Ghersa 1989) and intensively cultivated fields (Scopel et al. 1988), which may prevent accumulation of significant seed banks on the ground surface.

Besides temperature and light, there are other environmental factors associated in a predictable way with variations in above-ground biomass that, on theoretical grounds at least, might act as effective signals in the control of seed germination under natural conditions. Pons (1989) proposed that by responding to NO3 concentration in 13 the soil, seeds might detect gaps in the canopy in herbaceous communities. Organic acids produced by decaying vegetation (e.g. Simpson 1990) or reduction in levels of allelopatic substances (e.g. Angevine and Chabot 1979) might also act as germination signals, but the evidence for these mechanisms remains largely circumstantial.

b. Light opportunities preceded by soil disturbances In agricultural land the event that most frequently heralds a period of high light availability is disturbance of the topsoil by tillage operations. That seeds have the sensory machinery capable of detecting these events is fairly obvious when one looks at the patterns of seed germination in arable soils. Tillage during the season(s) of germination usually results in large cohorts of seedlings of weedy species (e.g. Chancellor 1964, Roberts and Potter 1980, Ballard et al. 1988b). In some species, seeds must spend some period of time in the soil before they can be induced to germinate by exposure to light or temperature fluctuations. Thus, in these cases, germination seems to depend on the occurrence of at least two tillage episodes: one to bury the seeds and other to provide the necessary environmental signal to the (in some way sensitized) seeds. This suggests a remarkable degree of adjustment to the intensively-disturbed agricultural environment (Soriano et al. 1970). What are the mechanisms that allow such a degree of synchronization?

Seeds of many species enter the soil bank with a high degree of primary dormancy that may be imposed by a variety of factors. During burial, seed populations undergo dramatic changes in the dormancy status, as gauged from results of germination tests on exhumed seed samples. In some cases these changes are cyclical, with dormancy reaching a minimum by the time of the year when germination usually occurs, and then increasing again in seeds that remained in the soil bank after that period (secondary dormancy) (reviews by Karssen 1982, Baskin and Baskin 1985).

Germination tests carried out on unearthed seed samples in the laboratory are useful to show variations in the germination behavior over time, particularly the broadening 14 and narrowing of environmental conditions in which germination can occur. Nevertheless, these tests may provide little insight into the nature of the environmental conditions that actually trigger germination under real field conditions (see Bouwmeester and Karssen 1989). At the scale of the individual seed, tillages produce a great variety of environmental changes, and different species or ecotypes appear to have evolved to rely on different signals to detect soil disturbances.

The mechanism that depends on temperature fluctuations (Section II.B.1.a.) is likely to be important for many species. Daily thermal amplitude diminishes rapidly with depth and, particularly at the beginning of the growing season, seeds buried more than a few cm are subjected to very small temperature fluctuations in most soils. Tillages, particularly plowing, can carry seeds from deep to shallow soil strata, where higher thermal amplitudes may elicit germination (see Koller 1972, Ghersa et al. 1992).

In many cases light appears to play a decisive role in the mechanisms whereby buried seeds detect cultivation episodes. Classic simulation experiments by Sauer and Struik (1964) and Wesson and Wareing (1969) have shown that, if steps are taken to exclude the light, fewer seedlings emerge from disturbed soil samples. More recently, Scopel et al. (1991) induced germination of buried seeds of Datura ferox, an annual weed of summer crops, by piping sunlight from the soil surface without disturbing other microenvironmental factors during illumination. This demonstrated that, for a fraction of the seed population, germination in the field was limited only by the lack of light. The experiment was carried out during the spring, with large daily temperature- fluctuations even at -10 cm. When seeds were unearthed using light-tight equipment, exposed to sunlight, and placed again at the initial depth (simulating the effects of soil cultivation), germination exceeded 90%. The most significant aspect of these results is the observation that a short period of burial produced a dramatic (ca. 10,000-fold) increase in the sensitivity of Datura ferox seeds to light. In fact, after a period in the soil, the majority of the seed population could be induced to germinate in the field 15 by irradiations equivalent to 0.1 to 10 ms of full sunlight Fig. 2. 2). The amount of Pfr established by these short irradiations would be on the order of 0.0001 to 0.01 %, that is 2 to 4 orders of magnitude lower than the level established by a saturating exposure to pure FR (Scopel et al. 1991).

It is now well established that many plant responses to light, particularly in dark-grown material, can be elicited by irradiations that form only minute amounts of Pfr and, therefore, can not be reversed by FR. This type of photoresponse has been termed very-low-fluence (VLF) response, to distinguish it from the classic, FR-reversible, low-fluence (LF) response (Mandoli and Briggs 1981, Blaauw- Jensen 1983, Kronenberg and Kendrick 1986). Seeds of several species displaying VLF responses have been obtained in the laboratory by means of artificial pretreatments. Based on the fluence response curves obtained in laboratory and field conditions, Scopel et al. (1991) interpreted the sensitization observed in buried seeds as a natural transition from the LF to the VLF mode of phytochrome action. Previous laboratory experiments by Taylorson (1972) and Baskin and Baskin (1979) also indicate a very high light sensitivity in seeds retrieved from soil. Theoretical calculations of the amount of light that individual seeds would get during soil tillage suggest that VLF responses are of great importance for the perception of light signals of soil cultivation (Scopel et al. 1991).

2. Stem phototropism and leaf movements After germination, the transition to the photoautotrophic stage, that involves leaf and plastid development and reduced axis elongation is also controlled by light acting through photomorphogenic pigments. Once seedlings have emerged from soil, they have to adjust to a complex light environment that is characterized by extreme variations in intensity and direction of the incident rays. These variations are caused by changes of solar angle during the course of the day, degree of cloudiness, and light absorption and scattering by neighboring objects and plants. In this section, I briefly discuss how plants or individual organs detect the direction of illumination and how they 16 react with morphological changes that improve light interception. Typical morphological responses to diurnal variations of light direction are phototropic movements of young stems and modifications of leaf inclination and azimuth.

Phototropism is the bending of elongating shoots towards the brighter side in an uneven light field. This bending results from redistribution of growth, i.e. inhibition on the illuminated flank and parallel stimulation on the shaded side, with little change in net growth (Rich et al. 1987 and references therein; see also Briggs and Baskin 1988, lino 1990). The subject has been reviewed fairly recently (Firn 1986, lino 1990) and only some general aspects will be considered here.

Most of our understanding of phototropic mechanisms in higher plants comes from experiments with etiolated (dark- grown) seedlings. These studies have shown that detection of the direction of illumination is based on fluence rate gradients that are created between the illuminated and dark flanks of the phototropic organ (i.e. coleoptile tip, stem). These gradients are the consequence of internal light scattering (mainly at the air / cell-wall interphases) and absorption (Seyfried and Fukshansky 1983, Vogelmann and Haupt 1985, Vogelmann 1986). The B / UV-A region of the spectrum, sensed by one or more as yet unidentified specific receptors, is used by plant phototropic-systems to obtain information about changes in light direction (e.g. lino 1990). Most studies indicate that phytochrome does not function as a detector of the direction of illumination in etiolated seedlings (see references in lino 1990), although this idea has been challenged by recent studies with dark- adapted corn (Zea maize) (Iino et al. 1984) and etiolated pea (Pisum sativum) (Parker et al. 1989) seedlings. What is clear is that phytochrome is involved in accessory processes (e.g. sensitivity and responsivity adjustment) that may be essential for the phototropic machinery to operate under natural light conditions (see Woitzik and Mohr 1989, lino 1990, and references therein).

In light-grown, green seedlings phytochrome appears to 17 participate in the detection of internallight gradients. Early action spectra obtained usingbroad-band light sources show a peak in the R region inaddition to the B / UV-A peak (Atkins 1936). The hypothesis put forward bylino (1990) to explain how phytochrome may act as aphototropic sensor in plant receiving polychromatic lightis based on the differential penetration of light of differentwavelengths into green organs. Due to absorption bychlorophyll, the fluence rate of R light declines abruptlywithin the first mm of tissue, while thefluence rate gradient in the FR region is comparatively much less steep(Seyfried and Fukshansky 1983, Vogelmann and Bjorn 1984,Vogelmann 1986). Therefore, in shoots exposed to unilaterallight, there would be a gradient of R:FR ratio and0 between the illuminated and shaded flanks of the organ.Since low Os stimulate elongation (Section II.B.3.), theshaded side would elongate faster than theilluminated one, and this will cause the shoot to bend towards thelateral light source. Although this proposedmechanism is consistent with the available data we can not yet concludethat it actually plays a role in determining phototropic responsesin nature. Plants are seldom exposed to trulyunilateral illumination. The "shaded" flank of the stem mayactually receive substantial amounts of diffuse radiationwhich, in open conditions, will be unaltered skylight,with even higher R:FR ratios than direct sunlight(Holmes and Smith 1977a).

Recent results with potted cucumber(Cucumis sativus) seedlings provide some indication about theinvolvement of different photoreceptors in natural phototropism(Ballare et al. 1991b). Seedlings grown in a glasshousewith no supplemental lighting naturally bent along thehypocotyl towards the Northern side (the experiments werecarried out in the Southern hemisphere). The degree ofcurvature in seedlings of the lh-mutant, which isdeficient in a stable phytochrome (see Section II.B.3.a.), was the same asin wild-type individuals. Furthermore, in bothgenotypes, bending was totally prevented by removing the Bcomponent from direct sunlight with a celluloseacetate filter. This suggests that the B waveband, actingthrough a sensor other than phytochrome, is essential forphototropic responses, at least in plants grown in sparsesettings. The situation 18 might be different for plants growing in the vicinity of other vegetation. In fact, light scattering by nearby plants can substantially increase the amount of FR that impinges laterally on vertically oriented shoots (Figs 2.5 and 2.6, Section II.B.3.b.). This will create R:FR gradientsacross stems that would be related to the distribution of neighbors around the plant. For instance, if a plant is growing at the edge of a gap, the R:FR ratio received at the stem surface will tend to be lower on the side that faces thecanopy than on the side that faces open space. Under these conditions, the phytochrome-dependent phototrophic mechanism considered by lino (1990) might, in addition to the B-driven system, signal the elongating organ to bend towards thegap.

Stem bending responses to directional light signalsare probably an important component of the mechanisms that help the plant to forage for light and improve PAR capture in heterogeneous light environments. The above, admittedly superficial consideration of phototropism suggests thatmore work is needed in order to clarify the contribution of different photoreceptor systems to the responses observed under natural conditions.

Apart from being able to rapidly change the direction of growth of young stems, many plants can adjust the spatial orientation of their leaves in response to directional light signals (reviews by Ehleringer and Forseth 1989, Koller 1990). Diaphototropic leaf movements reorient the lamina throughout the day so as to maintain it nearly perpendicular to the direct sunlight vector (solar-tracking). Solar tracking by young leaves is analogous to phototropic stem movements because reorientation depends on redistribution of growth between opposite flanks of the petiole. In mature leaves, reorientation is based on reversible structural deformations of a specialized tissue, the pulvinus,located between the lamina and the petiole (or at the base of each leaflet in species with compound leaves). Information about changes in the direction of illumination is acquired by leaves through a specific receptor for B light. The available information about sites of light perception and the fascinating complexity of the mechanisms ofresponse were reviewed recently by Koller (1990). 19 Solar tracking contributes to maintain a high and relatively constant PAR interception during the day and may also have some positive influence on photosynthesis via increased leaf temperature at the extremes of the photoperiod (Mooney and Ehleringer 1978, Shackel and Hall 1979). The adaptive value of solar tracking has been frequently considered in relation to the ecology of species that occur in arid habitats with short growing seasons (e.g. Mooney and Ehleringer 1978, Ehleringer and Forseth 1980, 1989). Solar tracking is also common in seedlings of pioneer species of secondary succession, and most likely contributes to increase plant growth rate during the early stages of canopy development.

3.Stem elongation Rapid responses to directional light signals are important to adjust the orientation of light harvesting organs to increase PAR interception in temporally variable environments. In many situations, particularly in rapidly growing canopies formed by plants of similar stature, there may be little advantages to change orientation without rapid growth in height. Not surprisingly, the key feature of elongation growth in species of early succession is plasticity and responsivity to local canopy conditions.

The rate of stem elongation is influenced by many factors, including all those that affect total plant growth (i.e. PAR, water and nutrient supply), and a number of others with specific morphogenic activity. Some of these factors have more than one effect: light, for instance, may stimulate stem elongation via promoting total growth but, under certain conditions, increased light intensity leads to inhibitory morphogenic effects. In the short term at least, shade-intolerant species with orthotropic stems typically respond to leaf shading or increased population density by accelerating internode elongation. Plants with diagraviotropic stem systems may show the opposite reaction to shading (i.e. decreased elongation), and elevation of the foliage depends on changes in shoot inclination (Section II.B.5.) or increased petiole length (Solangaarachchi and Harper 1988, Thompson and Harper 1988, Methy et al. 1990). 20 a. Responses to shading in multi-story canopies Stimulation of stem elongation in the lowest strata of multi-story canopies may be caused by several microenvironmental changes brought about by the presence of a leaf cover. Under controlled conditions, stem elongation rate is highly sensitive to changes in air humidity, mechanical disturbances, total irradiance, and spectral light quality. Although the effects of air humidity (Mc Intyre and Boyer 1983) and mechanical perturbations (e.g. movements caused by wind, Neel and Harris 1971, Telewsky and Jaffe 1986) are well documented, little is known about the extent to which responses to these variables contribute to the putative effect of shading on elongation under field conditions.

Differences in light environment between open and leaf- covered sites do certainly influence elongation growth. The optical characteristics of leaves (Section II.B.1.b.) determine that, compared to open situations, the radiation environment beneath canopies is characterized by low levels of UV and visible wavelengths and reduced R:FR ratios (e.g. Vezina and Boulter 1966, Holmes and Smith 1977b). All these factors are known to influence elongation, at least under controlled conditions.

The UV-B waveband inhibits shoot elongation in many species. Evidence is accumulating to suggest that this is not merely a by-product of unspecific growth inhibition, but a true photomorphogenic response to UV-B (e.g. Steinmetz and Wellmann 1986, Barnes et al. 1990, Ballard et al. 1991a), which most likely involves the action of a specific UV-B receptor. Attenuation of UV-B by a leaf canopy would thus lead to increased elongation in sensitive species growing in areas that naturally receive high UV-B levels.

Another aspect of the light environment beneath leaf canopies that may stimulate elongation is the reduced fluence rate in the 320-800 nm waveband (which comprises the UV-A, PAR and FR regions, and that I will call "white light" for brevity). Short-term experiments with young seedlings (grown in the light for a few hours) have demonstrated marked inhibitory effects of increased white light 21 irradiance on hypocotyl elongation (e.g. Meijer 1959). These effects are mediated by a specific photoreceptor(s) operating in the B / UV-A region (Gaba and Black 1979, Thomas and Dickinson 1979, Ritter et al. 1981) and phytochrome(s) (e.g. Ritter et al. 1981, Wall and Johnson 1981, Holmes et al. 1982, Gaba and Black 1985; see also Cosgrove 1986). The effects of white light irradiance on more mature, fully de-etiolated plants are not as clear (see references in Ballare et al. 1991c), and paradoxically, there is some controversy about the actual significance of this variable in the control of stem elongation under leaf canopies (see Frankland 1986, Child and Smith 1987). Britz (1990) has recently shown that stem elongation in fully de- etiolated soybean plants can be substantially promoted by reducing white-light fluence rate, and suggested that this response was, at least in part, mediated by a specific B light receptor.

The effect of reduced R:FR ratio in promoting stem elongation beneath vegetational canopies has received considerable attention in recent years and the subject has been reviewed by Smith (1982, 1986) and Smith and Morgan (1983). The work of Smith and associates has substantiated the following key points. (i) The reduction of R:FR ratio beneath canopies is a function of the amount of leaf area and is closely correlated with the reduction of PAR (Holmes and Smith 1977b). (ii) Changes in R:FR beneath canopies are within the range of high phytochrome sensitivity, that is where variations of R:FR lead to approximately proportional changes in 0(Smith and Holmes 1977). (iii) In controlled environments, under light levels comparable to those that can be found beneath dense canopies (ca. 10 % of full sunlight), plants of shade-intolerant species respond to reduced R:FR with an increase of elongation rate that is quantitatively related to the estimated effect of the R:FR treatment on 0(Morgan and Smith 1978, 1979).

The importance of light signals (relative to other microenvironmental factors) in determining elongation responses to leaf shading has been tested by using photomorphogenic mutants. The lh mutant of cucumber (Adamse et al., 1987) has a longer hypocotyl compared to its near 22 isogenic wild-type (WT), andis deficient in a stable type of phytochrome (Adamseet al. 1988, Peters et al.1991, Lopez et al. 1991), thatis in the form that appears to mediate stem elongation responsesto R:FR in light-grown plants. Seedlings of the lhmutant do not respond to R:FR treatments (Adamse et al. 1987,Lopez-Juez et al. 1990, Ballare et al. 1991b).Furthermore, the lack of stable phytochrome does, in some way,eliminate the elongation responses to B lightmediated by other photoreceptor(s) (Ballare et al. 1991b; see Mohr1986 and Gaba and Black 1987 for discussions aboutinteractions among photoreceptor systems). The lh-mutant istherefore very useful as an experimental tool because, exceptfor the responses to UV-B radiation, it appears to be(naturally) blind to photomorphogenic signals of leafshading. When WT seedlings under-story of a were transferredfrom full sunlight to the dense canopy in a glasshouseexperiment (Ballare et al. 1991b), the average rate ofhypocotyl elongation was promoted by a factor of ca. 3.5.This reaction was totally absent in the lh mutant.Applications of gibberellin A3 promoted elongation of lhseedlings in the under-story, suggesting that the lack of responseto shading was not a consequence of assimilatelimitation, but a reflection of the inability of the mutantto sense variations in the Band R-FR environment.Qualitatively similar results were obtained in parallel fieldexperiments during the summer at Buenos Aires. From thesestudies one is forced to conclude that mechanisms controlledby light signals play amajor role in determining short-termelongation responses to shading (Ballare et al. 1991b).

One of the issues thatreceived little attention in the above discussion is the effectof the illumination history on the sensitivityof plants to light stimuli.The responses to shading in the cucumberexperiments were measured two days after the beginning oftreatments. In the long term (i.e. 1 or 2 weeks) thedifferences in elongation rate between "sun" and "shade"plants tended to disappear (Ballare et al. 1991b). Thismight reflect either that shaded plants could notelongate more rapidly because of assimilate limitations or that theyhad changed their pattern of response toshading. The latter does not appear 23 to be an unreasonable possibility, since in cases of strongly asymmetric competition (i.e. seedlings v. established plants) the "escapist" response to shading is likely to be of very limited value to the subordinated plants. Photosensory adaptation refers to the processes whereby the sensitivity of a system to lighttreatments is compensated for fluctuations in the illumination background (Galland 1989). Adaptation has been studied indetail in various systems, but we know relatively little about how sensitivity of higher plants to changes in irradiance and R:FR ratio is affected by prolongedshading.

b. Early reactions in even canopies The assertion that stem elongation responses tomassive shading are unlikely to contribute to the success ofpioneer species in the natural environment is indirectly supported by many studies on the demography of weedsin secondary succession. It is a common observation that plants that emerge late in the season, whenthe aerial and below-ground spaces have already been occupiedby earlier individuals, tend to make little growth and usually experience veryheavy mortality (e.g. Black and Wilkinson 1963, Weaver and Cavers 1979, Gross 1980, Peters 1984, Ballare et al.1987b, Panetta et al. 1988, Scopel et al. 1988). It would seem,therefore, that when plants of early successional species become severely shaded and experience low levels of other resources, the time for initiatingshade-avoidance reactions is already over. Sophisticated, environmentallycontrolled dormancy mechanisms (Section II.B.1.), tend to assurethat germination of pioneer species is restricted to the beginning of periods of high light availability. Therefore, competition for light tends to occur among individuals of relatively similar size, in canopies with little vertical organization and low species diversity (see Bazzaz 1990). In this scenario, precise modulation of elongation growthis a major strategic priority, for slight differencesin height may result in disproportionatelylarge differences in PAR capture between neighboring plants (Grime and Jeffrey 1965, Ballare et al. 1988a, see also Section III). Thereis now solid evidence that the control of stem elongation in even canopies entails a great deal of mechanistic sophistication, and that responses to the proximity of other plants can 24 begin well before the leaves becomeseverely shaded by neighboring individuals.

Fig. 2.3 shows the elongationresponses of seedlings of the annual weed Datura feroxwhen they were placed in the center of even canopies of differentdensities. The average leaf area index (LAI) of the canopies variedfrom 0 (isolated plants) to a maximum of1.5, so that shadingamong neighboring seedlings was very small inthese experiments. However, in that range of LAIs, increasingneighborhood density led to a marked, rapid increasein stem elongation rate. In a related study (Ballare etal. 1987a), seedlings of white mustard (Sinapis alba)were grown on the Northern (directly illuminated in the Southernhemisphere) side of grass hedges, half of which had been bleached byspraying with a herbicide. Seedlingsgrown in front of green hedges developed longer internodes than thosenear bleached neighbors even though, in bothcases, the seedlings were continuously exposed to unfiltered sunlight(Fig. 2.4). What follows is a discussion of the roleof light signals in these early elongationresponses to the proximity of other plants.

As pointed out in section II.B.1.a.,transmittance and reflectance spectra of green leavesare very similar and show a steep rise in the FR region.Indeed, the distinction between transmitted and reflected lightis mainly operational (based onlyon the relative positions of the light source, the leaf, and the lightreceiver), and should not obscure the important fact thatthe two components have the same origin and, therefore,very similar spectral properties. Ballare et al. (1987a)hypothesized that the differential reflection of R andFR radiation, acting through phytochrome, providesan environmental cue for the detection of non-shading neighbors inthe early stages of canopy development. Measurements obtained withconventional light sensors (e.g. Kasperbaueret al. 1984) or cuvettes containing phytochrome preparations(Smith et al. 1990) pointed at illuminated plant partstypically show low R:FR or Pfr/P ratios. However, to predict theeffects of neighboring plants on the state ofphytochrome in a particular organ of an intact,green plant is usually 25 cumbersome. To a large extent this is because plants are much more complex structures than a flat light receiver. Individual organs are usually exposed, at the same time, to light scattered by nearby objects and foliage and to direct sunlight. Ballare et al. (1989) used a fiber optic probe implanted in different plant organs and attached to a spectroradiometer in order to investigate how the quality of radiation scattered within plant tissues was affected by the proximity of other plants. One limitation of the fiber optic approach is that we do not know the internal distribution of the phytochrome molecules involved in the control of straight growth. However, it is the only way to visualize the integrated product of light beams entering the plant from all possible directions. Fiber optic studies showed that, in even canopies, the proximity of neighboring plants is unlikely to bring about a significant spectral change inside leaves that are almost perpendicular to the direct light vector if shading among neighbors is small. This is due to the overwhelming contribution of direct sunlight to the internal light regime.

The situation is very different in the case of vertical stems or leaves. In these cases, light reflected by neighboring plants may cause a substantial increase in the amount of FR light scattered inside the tissues, even if the LAI of the canopy is very low (Fig. 2.5). Calculations showed that spectral changes of the magnitude shown in Fig. 5 may cause a 30 % decrease in the projected value of 0 inside the stem (Ballare et al. 1989, see also Mancinelli 1991).

Stem internodes have some degree of autonomy to respond to local light-quality conditions (Garrison and Briggs 1975, Morgan et al. 1980, Casal and Smith 1988a,b). Figure 2.6 shows a detailed picture of the effects of increasing canopy density on the light environment of the stems in even-aged populations of Sinapis alba and Datura ferox. Measurements were taken with the fiber optic probe (Fig. 2.6c), or with an integrating cylinder (Ballare et al. 1987a) that accepts side-light received from all compass points (Fig. 2.6b). It can be seen that the only detectable change when LAI increases in the very-low range (i.e. LAI between 0 and 1) 26 received by is a substantial increasein the amount of FR the stems. The canopyworks as aneffective FR trap, and the R:FR ratio at the stemsurface typically dropsfrom ca. 0.9 to 0.4 (Ballare etal. 1987a). Otherwavelengths are of the virtually unaffected inthis LAI range. If the LAI rapid reduction in the canopy increasesfurther, there is a fluence rates of B, R,and FR radiationreceived by the all these drastic stems. The mostsignificant aspect is that changes in the stem'slight environment takeplace well before shading at leaflevel becomes a factor.In fact, measurements of lightinterception by leaves (Fig.2.6a) there is only a show that even incanopies with a LAI of 2, small effect ofneighbors on light availablefor photosynthesis.

How do all thesechanges in the stems'light projection environment influenceelongation rate and, hence, here some of new leaves intouncolonized space? I describe to address of the experimentswhich have been carried out this question. In oneseries of field experiments(Ballare species from open et al. 1987a),seedlings of three annual habitats, Sinapis alba,Datura ferox, andChenopodium album mirrors that were grown infront of selective-reflecting provided additional FR so asto mimic the presenceof other seedlings (cf. Figs. 2.5,2.6), without affectingPAR receipt at leaf level. Themirrors reduced the R:FRratio received by the stems(measured with theintegrating cylinder) from ca. 0.8 to 0.5and the estimated 0inside the with the fiber stem (calculated fromspectral scans obtained optic probe) from ca. 0.34to 0.25 (see Ballareet al. 1989). Plants exposedto additional FR producedlonger internodes than the controls growneither without or in front of mirrors thatreflected R and FR lightwith have approximately similarefficiencies. Comparable results FR been obtained inexperiments were the additional treatment was appliedduring the central hours ofthe photoperiod only (Ballard etal. 1991c).

In a series ofglasshouse studies, 2-weekold seedlings short of Datura ferox andSinapis alba were placed for a period of time in canopiesof different densitiesformed by plants of similarstature. Transplanting ledto a rapid 27 increase in the rate of stem elongation that was related to the LAI of the surrounding canopy. This reaction to the proximity of neighbors was much reduced when the internodes of test plants were covered by a FR-absorbing filter collar that maintained a high R:FR ratio at the stem level (Fig. 2.7). These experiments demonstrated that in seedling canopies of very low LAI (i.e. LAI < 1), where interference among neighbors for PAR capture is almost nil, the R:FR ratio perceived at the internode level plays a major role driving adjustments of stem elongation rate to local density conditions (Ballare et al. 1990).

In canopies with LAI > 1, the efficiency of the FR- absorbing filters was generally reduced, even when they still maintained a relatively high R:FR (ca. 0.9). It appeared that some factor, other than the reduced R:FR, promoted elongation growth in those populations. Light measurements presented in Fig. 2.6 showed that, when LAI reaches about 1, PAR interception by leaves continues to be high, but total irradiance at the stem level begins to decline rapidly. In most of the experiments that addressed the influence of total irradiance on elongation growth, the light treatments were applied to the whole shoot (Section II.B.3.a.), which is clearly not equivalent to the effects of neighboring plants in even canopies. In experiments under natural radiation, annular filters that reduced the fluence rate of side-light by less than 50 % without influencing PAR interception by leaves promoted internode elongation (Ballare et al. 1991c). This response to fluence rate was not obviously affected by R:FR in the range of ratios typical of canopies of low LAI (i.e. 0.3 < R:FR < 0.9). This suggests that, when the canopy begins to close, both R:FR- and fluence-rate-dependent mechanisms modulate elongation responses to neighbors proximity (Ballare et al. 1991c).

What are the photoreceptors responsible for the perception of changes in fluence rate? Photosynthetic pigments are unlikely candidates. In fact, shading the stems with a pink collar that reduced PAR by filtering the green waveband (with minimal effects on spectral regions absorbed by phytochromes and B / UV-A receptors), did not result in increased elongation in Sinapis alba (Ballare et al. 1991c). 28 Phytochrome(s) and B / UV-A sensors have been implicated to explain the fluence-rate-dependent photoinhibition of hypocotyl elongation in seedlings de-etiolated for a few hours (see Section II.B.3.a.) and the same photoreceptors would seem to control elongation responses to stem shading in the case of more mature, fully de-etiolated plants (Ballare et al. 1991c).

4. Branching and production of new ramets In the long term, most species rely on the production of new branches for colonization of open space in canopies, and it is well established that local conditions of crowding have a dramatic influence on the amount of branches that a plant produces throughout its life span (e.g. Langer 1966, Harper 1977). Multiple factors, including responses to resource levels and morphogenic signals, are most likely involved in the control of branching and ramet production under field conditions. The possible involvement of external morphogenic signals was first suggested by Kirby and Faris (1972) after observing that, in barley (Hordeum vulgare) crops, the effects of planting density on the fate of individual tiller buds appeared to be decided quite early during the bud --> tiller transition. In this section I briefly cover the effects of light (sse Casal et al. 1986, and Hutchings and Slade 1988 for more complete treatment).

Reduced total irradiance has been shown to result in increased apical dominance and reduced production of new branches in growth cabinet (e.g. Mitchell 1953), greenhouse (Slade and Hutchings 1987, Methy et al. 1990) and field experiments (Bubard and Morison 1984, Thompson and Harper 1988). It is not clear whether this detrimental effect of shading is a consequence of assimilate limitations (which might act as an internal signal) or a more direct response to light fluence-rate mediated by specific photoreceptors. Direct responses to B and R fluence rates might contribute to determine that the inhibitory effects of leaf shading on branching are usually more pronounced than those caused by roughly similar reductions in PAR obtained with neutral filters (e.g. Solangaarachchi and Harper 1987). Direct responses to UV-B fluence rates should also be considered when interpreting results of field studies, because moderate 29 levels of these wavelengths areknown to promote tillering in some species (Barneset al. 1990).

Alterations of the ratiosbetween wavelengths (i.e. Mansfield R:FR ratio) influenceapical dominance (Tucker and 1972) and are certainlyinvolved in the branching responses to the proximity ofother plants. Deregibus etal. (1983) demonstrated that tilleringin grasses is depressed by FR light acting through phytochromein a R:FR reversible manner (see also Casal et al.1985, Kasperbauer andKarlen 1986, Casal 1988). Deregibus etal. (1985) also showedthat photomorphogenic mechanisms doplay a role in the controlof tillering under fieldconditions. Working in a natural grassland, they were able toincrease tillering ratesduring the growing season bysupplementing the radiationreceived at the base of Paspalumdilatatum and Sporobolusindicus plants with small amountsof R light from lightemitting diodes that increased theR:FR ratio.

The interactions betweenlight signals and resource have levels in the control ofbranching and ramet production not been thoroughlyinvestigated in any system. The experiments of Casal et al.(1986) with Paspalumdilatatum show that in grasscanopies of low LAI, wheremutual shading other is small, tillering responsesto the proximity of plants are most likelymediated by the altered R:FRbalance. Grasses were shown to be verysensitive to small reductions of R:FR (Casal et al.1987), particularly if thetreatments are appliedduring daytime (as opposed toend-of-day pulses) (Casal et al. 1990). Thus,changes in light environment produced, for example, by thedifferential reflection of R and FR light by nearbyleaves (e.g. Figs 2.5 and2.6) may signal grass plants to reducetillering rate early in the development of the canopy (seeCasal et al. 1987). Thisis probably advantageous forthe internal economy ofthe plant in environments where latebranches have little chanceof contributing to reproduction.It should be noted thatthe factors that influencebranch or tiller mortalityin canopies are still unclear.The popular belief thatbranch senescence istriggered by resource (light)starvation has been questioned recentlyby Lauer and Simmons (1989). 30

5. Spatialorientation of branches The phototropic responsesof leaves and stems considered in Section II.B.2. areexamples of rapid, reversible movements thatallow plants to re-arrangetheir architecture in continuouslychanging light conditions. Plants can also changethe orientation (zenithand azimuth angles) of whole branchesin response to more permanent features of their lightenvironments. Casal et al.(1990) found that the tillers ofannual ryegrass (Lolium multiflorum) tended to adopt a moreerect position in the canopy as plantdensity was increased, andthere is some evidence that R:FR signalsperceived by phytochromeactivate mechanisms that control shootinclination (e.g. Casal et al. 1990, Aphalo et al.1991). Effects of totalirradiance may also be involved in the responsesof shoot inclination to that neutral- canopy density.Grime et al. (1986) reported shading treatments led tothe production of morevertical shoots in a number ofbryophytes that, under natural conditions, tend to exploitthe upper strata of pasture canopies. Montaldi andco-workers suggested that,in grasses of with diagraviotropic stems,the angle of inclination shoots is modulated by sucrosecontent (Montaldi 1963, Willemods et al. 1988), andpostulated that the effectsof irradiance and R:FR ratio oninclination are consequences of the effects of thesevariables on carbohydrateproduction and partitioning (e.g.Beltrano et al. 1991).

An interesting exampleof how plants usedirectional light signals to adjustthe orientation ofbranches in patchy canopies wasprovided by Novoplansky etal. (1990). oleracea, a weed of They found thatseedlings of Portulaca intensively disturbed areaswith a diagraviotropicshoot neighbors when system, tended toavoid growing towards their colonizing bare soil. Thisappeared to be a consequenceof effects of neighbors on(i) the azimuthalorientation adopted by the main stemat the time it becamehorizontal and (ii), the patternof lateral shootinitiation. The after authors implicatedlight signals in these responses observing that seedlings alsotended to grow away from green plastic objects that absorbedPAR and reduced the R:FR with gray ratio. Furthermore, whenseedlings were confronted transmission and green plasticsthat provided similar PAR 31 with different effects on the R:FR ratio, only a small proportion of the plants became recumbent toward thegreen (low R:FR) sector (Fig. 2.8). Since the green plastic reflected more FR than the neutral gray, the response observed in Portulaca might have been a consequence of the phytochrome-mediated phototropism discussed earlier (Section II.B.2).

III. COMPETITION FOR LIGHT DURING EARLY SUCCESSION In Section II, I have condensed the available information about the mechanisms whereby plants of early succession perceive and react to changes in light conditions with emphasis on germination responses of arable weeds and morphological plasticity of vegetative shoots. In this Section, I present an overview of how species of early secondary succession apply the acquired environmental information to actively locate and exploit light opportunities in highly dynamic canopy systems.

A. The acquisition of information and the race for light By definition, secondary successions are generated by some type of disturbance that brings about an increase in light availability at the ground surface. Disturbance is followed by recolonization of the site by individuals of pioneer species (and cultivated plants in agricultural environments) that rapidly begin to compete with each other for access to light and other resources. We can visualize the process as a race, where the success of an individual relative to its neighbors depends essentiallyon (1) its relative ability to capture light energy at the beginning of the period of interaction (which is mainly a function of its relative initial size), and (2) its relative ability to maintain high rates of light capture as thecanopy develops.

Differences in initial size are largely due to differences in propagule (seed) size and emergence time (or emergence order). The production of large seeds carrying large embryos may have a number of associated costs (Harper et al. 1970, Thompson 1987) and small seeds appear to prevail among pioneer species in many different systems (Fenner 1987). As mentioned earlier (Section II.B.1.), germination does not occur at random through time in 32 habitats subjected to periodic disturbance. Environmental signals such as light and daily temperature fluctuations synchronize germination events with variations in light receipt at the ground surface. Field experiments are beginning to uncover the complexity of the perceptual processes involved and are contributing to determine which are the relevant cues for different species and environments. Given the importance of relative time of emergence in determining growth and fecundity (see references in Section II.B.3.b. and Weiner 1988), one can hardly dispute the notion that the ability of seeds to input environmental information into the systems that control the degree of dormancy is a key element of success in frequently disturbed ecosystems.

Maintaining high rates of light interception as the canopy develops is obviously necessary to sustain growth. The amount of leaf area generated per unit of dry matter produced (i.e. the leaf area ratio), a quotient that is strongly influenced by light conditions (Blackman and Wilson 1951, Evans and Hughes 1961), is certainly a major determinant of the dynamics of light capture. Light interception per unit leaf area depends on how foliage pieces are spatially arranged in relation to the pattern of light availability. Since this pattern is characterized by major fluctuations imposed by solar movement, atmospheric factors and rapid growth of neighboring plants, a high degree of morphological plasticity, based on continuous acquisition and interpretation of environmental information, is most likely an essential requirement for species of early secondary succession. In Section II, I discussed how information acquired by B / UV-A receptors (and probably by phytochrome as well) is used to orient leaves and young growing stems to maximize PAR capture in variable light environments. Changes in branch inclination (zenith angle) in response to local conditions of crowding are also known to occur, and although the picture is still incomplete, there is clear evidence that photomorphogenic signals are involved in the controlling mechanisms (Section II.B.5.). Perception of patchiness in the light environment may lead to non-random spatial patterns of vegetative spreading in clonal plants. Phytochrome-perceived light cues are known to 33 mediate tillering responses to canopy density under certain conditions (Section II.B.4.) and they also appear to influence the processes whereby plants with diagraviotropic shoot systems choose a direction for spreading (Section II.B.5.). Considerable evidence has been accumulated to suggest that these morphogenic events may take place very early in the process of space colonization. Thus, the observed patterns of branching and site occupation reflect both the limitations imposed by the resources available at each particular spot of the environmental mosaic, and the "decisions" that the invading shoots took even before experiencing discontinuities in resource supply.

Stem elongation is important for maintaining young leaves within the reach of direct sunlight in rapidly growing even canopies. Although stem internodes in herbaceous plants appear to be relatively cheap in terms of the opportunity value of the carbon invested in their construction (Ballare et al. 1991d), different types of indirect costs are most likely associated with the production of longer stems (e.g. reduced mechanical strength, increased exposure to herbivores). Therefore, for each particular plant in a population, there should be an optimum rate of stem elongation dictated by the rate of growth in height of the surrounding vegetation (see discussions by Givinish 1982, and Waller 1988). I have presented evidence in Section II.B.3.b. that light signals of different nature, perceived by phytochromes and B / UV-A sensors, modulate the rate of stem elongation in even canopies of dicotyledonous seedlings. Furthermore, it was shown that elongation responses to changes in canopy density and vertical distribution begin before the amount of light available to leaves for photosynthesis is affected by neighboring individuals. These early reactions are, to a large extent, a consequence of effects of adjacent plants on the stems' light conditions and the ability of stem sections to respond to local light signals. Thus, in a sense, when responding to these local light changes, plants are "guessing" about future variations in PAR receipt at leaf level. Morgan and Smith (1979) and Corre (1983) found that elongation responses to experimental changes in R:FR ratio are usually more dramatic in pioneer species than in species 34 of shaded habitats, which is consistent with the notion that these responses are valuable only for plants that compete for access to light against individuals of similar morphology in relatively open canopies. Ballare et al. (1988a) grew seedlings of the arable weed Datura ferox at three densities in the field and monitored the time course of stem elongation, leaf area expansion and PAR interception per plant. Two weeks after the beginning of the study, the LAI of the densest population was - 2 and shading among neighboring seedlings was still small. However, a significant effect of canopy density on seedling height was already evident, and seedlings transferred from low- to high-density canopies intercepted one third of the PAR captured by their local neighbors. These results suggest that a short lag in stem elongation would be enough to seriously compromise seedling survival in rapidly growing communities.

The examples discussed above suggest that the systems that permit acquisition of information about present and future changes in PAR availability imposed by abiotic and biotic factors play a fundamental role as determinants of competitive success in early successional communities. Curiously, however, and with few exceptions (e.g. Grime et al. 1986), the value of information-acquiring systems has been largely overlooked in experimental and theoretical approaches to the study of plant interactions. The likely reasons and potential consequences of this apparent neglect are discussed in the following section.

IV. SIGNALS, "DECISIONS", AND MODELS OF PLANT COMPETITION Two major avenues of research have contributed the most to shape our current perception of the mechanisms of plant competition. Phenomenological studies, on the one hand, provided a wealth of information on the consequences of competition in relatively simple systems like monocultures and mixtures of cultivated species and arable weeds (reviews by Willey and Heat 1969, White and Harper 1970, Harper 1977, Weiner 1988, Firbank and Watkinson 1990, Radosevich and Roush 1990). These studies examined the effects of crowding on plant growth, morphology and mortality. Classic physiological experiments, on the other hand, generated 35 information on plant responses to light and other environmental resources that are presumably or certainly affected by changes in canopy density. These two disciplines grew up rather independently from each other and, traditionally, there has been very little exchange of ideas between groups working in descriptive population or community ecology and plant physiology. Physiological ecologists historically appeared to be more inclined to study plant responses to abiotic factors in extreme habitats (e.g. alpine, desert; see Mooney 1991) than to unravel physiological mechanisms of plant-plant interactions in more productive environments.

During the last few years, a number of mechanistic models of plant competition have been developed by ecologists and agronomists. These models vary greatly in complexity and purpose; the common denominator is that they are designed to predict the outcome of plant competition from assumptions about the way plants affect and respond to environmental resources (Goldberg 1990). Models of this kind are proving to be useful to study competition for single resources over short periods of time in cultivated plant associations (e.g. Rimmington 1984), or to predict results of competitive interactions among plants with contrasting life forms or allocation patterns along gradients of resource supply or disturbance intensities (Tilman 1988). However, their value as analytic tools may be limited when the goal is to pin-point specific physiological traits that determine variations in competitive ability.

Most competition models assume that plants are "blind" organisms. They are assumed to grow over a fixed period at a rate that is essentially proportional to the instantaneous rate of resource supply. Developmental and biomass allocation patterns are most commonly fixed (i.e. genotype- or age-dependent) or, in a few cases, some degree of phenotypic plasticity is incorporated into the model by assuming that allocation among organs or parts depends on resource levels or ratios (e.g. Tilman 1988). Most commonly plants are forced to co-occur, and the only possible way by which plants may influence each other's activity is by decreasing (or increasing) resource availability. These 36 assumptions are false.Previous sections (II.B.1)have presented an overview ofthe systems usedby seeds to (see also acquire information aboutthe aerial environment specific Vasquez Yanez 1993). Thesesystems, based on rather environmental signals, wereshown to be responsiblefor synchronizing growth activitywith periods of increased with light availability. Inhighly disturbed habitats, differences in pulsed supplies oflight and other resources, formation of ability to coordinateseed germination with the natural or artificial gapsare likely tobe far more ability important determinants ofdifferences in competitive (e.g. than variations inresource-assimilation physiology photosynthetic capacity) orallocation patterns. Information-acquiring systems mayhelp reduce the costs associated with theproduction of competitiveoffspring. mechanisms to Thus, weed speciesthat evolved efficient afford to detect indicators oflight opportunities may produce small seeds,which are cheap andsuitable for with chances of dispersal but unlikely togenerate seedlings success in alreadyoccupied systems. Models orexperimental species or designs that assumeuniform germination across part of the genotypes certainlymiss a very important competition story in earlysuccessional environments. regulation of green Our presentunderstanding of the also plant development byspecific photoreceptor systems highlight the significanceof informationcarried by light in plant signals as determinantsof morphological responses canopies (Sections II.B.2.to II.B.5.). Thesignals that to match the plants use foraccommodating their architecture prevailing conditions ofillumination are, bydefinition, of light directly or indirectlyassociated with the amount ask available for photosynthesis.Therefore, one can rightly why it is necessaryto recognize theinformation-acquiring systems as separatecomponents in models ofplant competition. From a conceptualpoint of view it isimportant in the canopy is to keep in mind thateach individual plant they alter the influenced by itsneighbors not only because availability of resourcesbut also because theymodify aspects of theenvironment that are usedby the plant to The use of decide amongalternative developmental programs. specific cues to keeptrack of variations inlight supply 37

the plant over may offerseveral strategic advantages to direct monitoring of the resource,the most significant of which is probably theability to appropriatelytime the responses. InSection II.B.3.b., Idiscussed how perception rapid of R:FR changes byindividual internodes may allow variations in light stem elongationreactions that precede interception at leaf level. Thus,particularly where light supply is mainly afunction of the size andactivity of likely to neighboring individuals,natural selection is favor specific mechanismsof phenotypic responseto environmental factors that arereliable and sensitive indicators of plant proximity.These mechanisms should, therefore, be considered as anintegral part of the traits that determinevariations in competitiveability.

From a practicalpoint of view, thedistinction between plant responses to resourcesand signals may be of importance in applied fieldslike agronomy or forestry. In a part of every crop, neighboringindividuals are an important plant's environment. The wayin which each plantresponds to the proximity cues affects itsperformance and the yield of whole population.Elongation growth (SectionII.B.3), tillering (Section II.B.4),developmental timing (e.g. Mondal et al. 1986), andcarbon allocation toreproductive structures (Heindi and Brun1983) are all affectedby canopy density in field crops, anddifferent types of evidencehave effects are been presented showingthat at least part of the light indeed photomorphogenic responsesto alterations of climate caused by nearbyplants. Some of the response mechanisms could have beenselected because they conferred competitive advantages underconditions that were different from the ones normallyfaced by crop plants inmodern agricultural systems. Moreover,selection did most likely the favor physiological responsesthat were beneficial to individual plant, rather thanto the whole population.If we accept these possibilities, weshould accept that under certain combinations ofplanting density andenvironment, crop yields arelikely to be limited by the presence(or lack of) particularphysiological responses tospecific proximity cues.

The design of cropideotypes is based on identification 38 of factors that limit commercial yield in a given cultural/environmental scenario and consists of deciding what morphological or physiological traits should be added or dropped from the crop plant in order to make it more efficient at converting resources into commercial yield. Until recently, the only way available for translating ideotypes into real plants was by repeated hybridization and selection. But with the arrival of molecular biology techniques, the opportunities for engineering plants with very specific physiological traits is increasing enormously. Therefore, knowledge on the mechanistic details of plant responses to their neighborhood environment is likely to be now more important than in the past. Based on the limited information we have today, it would seem that manipulation of photomorphogenic behavior to induce or suppress developmental responses to signals of neighbors' proximity may be rewarding in commercial crops.

V. SUMMARY Under natural conditions, abiotic factors and the activity of vegetation determine dramatic variations in the flux of photosynthetically active radiation received at the ground surface. Light opportunities in this heterogeneous environment are perceived by terrestrial plants through a great variety of specialized mechanisms. At the seed level, aspects of the thermal regime of the soil and light signals perceived by phytochrome are used to detect the onset of periods of high light availability. In the particular case of seeds of arable weeds, it seems that daily fluctuations of soil temperature and pulses of light operating through the very-low-fluence mode of phytochrome action, are very important to inform seeds about the occurrence of soil disturbances that usually prelude periods of abundant light. At the green plant level, photoreceptors play a fundamental role driving architectural plasticity and morphological responses to variations in the light environment. One or more specific photoreceptors for B light are essential for phototropic movements of leaves and stems, which, in many species, allow fine adjustment of leaf display to rapid changes in the direction of incoming light. The role of phytochrome in the detection of light gradients during phototropism under natural conditions is not completely 39 clear at present. Precise control of stem elongation rate may be essential for plants that forage forlight in plant communities formed by individuals of similar height. A number recent of experiments have indicated that phytochrome can detect FR-rich radiation scattered byneighboring plants and trigger changes in stem elongation rate before the plant is subjected to a reduction in light availability. In addition to this R:FR-mediated response, fluence rate signals, perceived by phytochrome(s) and B / UV-A sensors may influence stem elongation in even canopies. In the case of multi-story canopies, glasshouse and field experiments with photomorphogenic mutants indicated that, at least in the short term, elongation responses to leaf shading depend to a large extent on processes in which a stable pool of phytochrome is directly or indirectly involved. Evidence has also been obtained over the last few years for the involvement of phytochrome in the control branching and lateral spreading in clonal plants.

The acquisition of information on present and future light conditions appears to be a major priority for plants of early successional communities, where dramatic changes of plant cover (and hence light availability) occur over relatively short periods of time due to disturbances and plant growth. Mechanistic models of plant competition usually fail to acknowledge that each plant in the community is affected by neighboring individuals not only because they modify the availability of energy and materials necessary for growth, but also because they affect aspects of the environment that are specifically used by the plant to obtain information about neighbors' activities and to make developmental "decisions". Responses to proximity signals may, in turn, have a dramatic impact on the resource- harvesting capacity of the plant, and influence competitive ability. A better understanding of the mechanisms whereby plants acquire and "interpret" environmental information should provide new insights into the factors that determine differences between genotypes in the ability to compete for light. From a practical standpoint, this knowledge would be an useful aid to the design of more productive crop genotypes. 40 Acknowledgements. Many of the ideas expressed in this chapter are the product of discussions with Pedro Aphalo, Jorge Casal, Claudio Ghersa, Steve Radosevich, Rodolfo Sanchez, and Ana Scopel. I thank the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET, Argentina) and the University of Buenos Aires for supporting the research presented here. I am also indebted to CONICET and the Dept. of Forest Science for financingmy stay at Oregon State University. Les Fuchigami, Mary Lynn Roush, andJoe Zaerr gave useful suggestions on an earlier draft of the text and Bracher Gretchen Bracher prepared the figures. 41

UV B A PAR FR

0L1.1 Z < E) ..--.. < ccE eC c 4 z in HC0 0E o_I Z. E E E --I c c c < c) CC c o I- m co NCO -cl- co 0 CC w m m LL o_ (/)

I 200 300 400 500 600 700 800 900 WAVELENGTH(nm)

PHYTOCHROME

A MAX Pr = 660 (R) A MAX Ph = 730 (FR)

B/UV-A P

UV -B P

FIG. 2.1. Spectral distribution of daylight and wavelengths absorbed by known plant photoreceptors. The shaded region is the visible portion of the spectrum. Abbreviations: B, blue; B/UV-A P, blue/ultraviolet-A absorbing photoreceptor(s); FR, far-red; PAR, photosynthetically active radiation; Pfr, far- red absorbing form of phytochrome; Pr, red absorbing form of phytochrome; R, red; UV-B P, ultraviolet-B absorbing photoreceptor. The spectrum was scanned near noon on a clear summer day at Corvallis, OR, USA. 42

100

T

80 SL FR T

0 4 1 0z 60 z 40- cc w

20-

0-

0 10-1 100 101 102 103 104 106 106 SUNLIGHT (400-800 nm) FLUENCE (.1 mol m-2)

0 0.0001 0.0010.01 0.1 1 10 100 FULL SUNLIGHT EQUIVALENT (seconds)

FIG. 2.2. Effects of sunlight pulses on the germination of buried seeds of Datura ferox under field conditions. Seeds were unearthed near midday using light-tight equipment, exposed to sunlight and buried again at -7 cm. The open symbol indicates germination of seeds irradiated with FR after a saturating exposure to sunlight. After Scopel et al. 1991. 43

7-

1-- 5

-215E5 >< I I -0< 0.3 0.5 1.0 1.5 2.0 licz LEAF AREA INDEX a_

FIG. 2.3. Elongation rates of the first internodeof Datura ferox seedlings growing in even canopies of different densities under natural radiation. Also shown isthe effect of density on PAR interception by individual seedlings. After Ballare et al. 1990. 44

9 8 E 7 1I 6 z0 Jw 5 12; 4- 0 z 3_ cc iw2 Z GREEN FENCE

1 0 BLEACHEDFENCE

0 II I III I 10 12 14 1618 20 22 24 DAYS OF TREATMENT

FIG. 2.4. Effect of the proximity ofa green grass canopy on the elongation of the first internode of Sinapisalba seedlings growing under full sunlight. Controlseedlings were grown in front of canopies bleached with an application of paraquat. After Ballare et al. 1987b. 45

1.0 SECOND INTERNODE MIDDAY

0.8 LAI _0.00 it4 fA. ,14 .1 I ---.0/2 1 1 I I 1 I 0.6 I e I I I I o ig 0.4

0.2 R FR

a_ 0 600 700 800 WAVELENGTH (nm)

FIG. 2.5. Effects of neighboring seedlingson the spectral distribution of the radiation scattered withinthe stems of Datura ferox seedlings in an even canopy of LAI= 0.72. AbbrevitifiTiiis: FR, far-red; R, red. AfterBallare et al. 1989. 46 0z 17- uj Z -E 0

ZI CCId -a.E 0 3 INTEGRATING CYLINDER FR R B

1-i_f_21 wea.rliZ es,'" 111" are.

FIBER OPTIC PROBE 2_

A AAttA A .4.4.

:1 as.

S.

0 .

0.05 0.1 0.5 1 2 5 LEAF AREA INDEX

FIG. 2.6. Effects of increasing density in even canopies of dicotyledonous seedlings on light interception byleaves (top) and the light environment of the stems. The integrating cylinder collects side light received by the stem surface; the fiber optic probe collects light scattered within the stem tissue. Abbreviations: B, blue;FR, far-red; R, red. After Ballare et al. 1991c. 47

6

E w4

cc z 0 z 0 w_1 2

0 ISOLATED WITH NEIGHBORS

FIG. 2.7. Elongation responses of Daturaferox first internodes when seedlings were placed inthe center of an even canopy of LAI -0.9 under natural radiation.During the experiment the internodeswere surrounded by annular cuvettes containing distilled water (clearfilter) or a CuSO4 solution that absorbed FR radiationand maintained the R:FR ratio at ca. 1.1 (FR-absorbing filter). Ballare et al. 1990. Adapted from 48 2 R:FR the main field. of only of different inthe the reduced frequency of extended transmission. grown PAR that orientation each relative in filters similar filters seedlings the gray azimuthal and had 1990. green the developing and of on oleracea Green al. represents stems rim et effect light bars their pot's The side Portulaca of the with directions. 2.8. of of Novoplansky length above FIG. ratio shoots The plants compass cm After 49 REFERENCES Adamse, P., P. A. M. P. Jaspers, J. A. Bakker, R. E. Kendrick, and M. Koornneef. 1988. 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CHAPTER 3

PHOTOMORPHOGENIC EFFECTS OF UV-B RADIATION ON HYPOCOTYL ELONGATION IN WILD-TYPE AND STABLE-PHYTOCHROME-DEFICIENT MUTANT SEEDLINGS OF CUCUMBER

Carlos L. Ballare*, Paul W. Barnes and Richard E. Kendrick

Depts of Forest Science and Crop and Soil Science, Oregon State Univ., Peavy Hall 154, Corvallis, OR 97331-5705, U.S.A.(C. L. B.); Dept of Biology, Southwest Texas State Univ., San Marcos, TX 78666-4616, USA (P. W. B.); Dept of Plant Physiological Research, Wageningen Agricultural Univ., Generaal Foulkesweg 72, NL-6703 BW Wageningen, The Netherlands (R. E. K.).

Physiologia Plantarum 83:652-658 (1991). 65 ABSTRACT Hypocotyl elongation responses to ultrviolet-B (UV-B) radiation were investigated in glasshouse studies using de- etiolated seedlings of a long-hypocotyl mutant (lh) of cucumber (Cucumis sativus L.) deficient in stable phytochrome, its near isogenic wild type (WT), and a commercial cucumber hybrid (cv. Burpless). A single 6-or 8- h exposure to UV-B applied against a background of white light inhibited hypocotyl elongation rate by ca 50 % in lh and WT seedlings. This effect was not accompanied by a reduction in cotyledon area expansion or dry matter accumulation. Plants recovered rapidly from inhibition and it was possible to stimulate hypocotyl elongation in plants exposed to UV-B by application of gibberellic acid. In all genotypes inhibition of elongation was mainly a consequence of UV-B perceived by the cotyledons; covering the apex and hypocotyl with a filter that excluded UV-B failed to prevent inhibition. These results indicate that reduced elongation does not result from assimilate limitation or direct damage to the apical meristem or elongating cells, and strongly suggest that it is a true photomorphogenic response to UV-B. The fact that UV-B fluences used were very low in relation to total visible light, and the similarity in the responses of lh and wild-type plants, are consistent with the hypothesis that UV-B acts through a specific photoreceptor. It is argued that, given the weak correlation between UV-B and visible-light levels in most natural conditions, the UV- B receptor may play an important sensory function providing information to the plant that cannot be derived from light signals perceived by phytochrome or blue/UV-A sensors.

Key words - Cucumber, Cucumis sativus, photomorphogenesis, phytochrome mutants, hypocotyl elongation, UV-B radiation. 66 INTRODUCTION Photomorphogenesis in higher plants involves the action of at least three photoreceptor systems: phytochromes, a variety of receptors for blue / ultraviolet-A (UV-A) radiation, and an as yet little- studied UV-B receptor (e.g. Mohr 1986). The existence of the latter photoreceptor has been inferred from studies on light-mediated synthesis of anthocyanins and flavonoids (Wellmann 1971, 1976, 1983, Yatsuhashi et al. 1982, Mohr and Drumm-Herrel 1983, Beggs et al. 1986). Besides influencing pigment synthesis, UV-B radiation is known to induce a variety of changes in plant morphology, including inhibition of elongation growth (Steinmetz and Wellmann 1986, Tevini and Teramura 1989, Barnes et al. 1990, and references therein). Barnes et al. (1988) and Ryel et al. (1990) suggested that differential morphological responses to UV-B may be important in determining competitive relations between species in communities subjected to increased levels of UV-B.

The mechanisms underlying the effects of UV-B on plant morphology are poorly understood and, for a number of reasons, the involvement of a specific receptor in the responses is far from established. In many studies whereUV- B was applied against a background of white light, altered plant morphology was accompanied by a reduction in photosynthesis and total biomass production. Thus, it is difficult to determine whether reduced elongation of stem and leaves is due to specific photomorphogenic effects of UV-B orissimply a consequence of a general reduction in growth capacity resulting from photosynthetic inhibition (Tevini and Teramura 1989). UV-B inhibition of elongation may also result from direct cellular damage at the level of meristems and elongating organs. In fact, the action spectrum obtained by Steinmetz and Wellmann (1986) for the inhibition of hypocotyl elongation in etiolated seedlingsof 67 Lepidium sativum resembles those related to DNA damage in other systems (see Caldwell 1971). Even when damaging processes are ruled out as a cause of inhibition, the possible involvement of other photoreceptors must be considered. Thus, the interpretation of experiments with monochromatic UV-B sources is complicated by the fact that the protein moiety of phytochrome also absorbs in the UV-B region, and part of the energy can be transferred to the chromophore and can be effective in photoconversion (Pratt and Butler 1970). There have been suggestions that some morphological responses to UV-B might be a consequence of direct absorption of UV-B by growth regulators (e.g. auxins, see Curry et al. 1956, Tevini and Teramura 1989).

Recent field and glasshouse experiments with grasses have indicated subtle effects of UV-B on shoot (leaf) elongation (10 to 15 % reduction in total height) that were not accompanied by similar changes in total dry matter (Barnes et al. 1988, 1990) or photosynthesis (Beyschlag et al. 1988). It is very unlikely that this effect of UV-B was mediated by phytochrome, because the fluence rates of UV-B used were very small in relation to the total phytochrome- absorbable radiation that plants received under natural conditions. However, phytochrome might still have played a role, either by influencing the synthesis of UV-B screening pigments, or as a result of interactions with the putative UV-B receptor system. Several types of interactions between UV-B and light treatments that affect phytochrome are well documented in the case of anthocyanin and flavonoid synthesis in various plant systems (e.g. Wellmann 1971, Mohr and Drumm-Herrel 1983, Beggs and Wellmann 1985, Arakawa 1988) .

We have investigated the roles played by UV-B damage and phytochrome in the inhibition by UV-B of hypocotyl 68 elongation by means of physiological experiments with de- etiolated seedlings of the lh mutant of cucumber and its near isogenic wild type (WT). The lh mutant has been proposed to be deficient in the stable type phytochrome on the basis of spectrophotometric (Adamse et al. 1988, Peters et al. 1991), immunochemical (Lopez et al. 1991, E. Lopez- Juez and A. Nagatani, personal communication), and physiological (Adamse et al. 1987, 1988, Lopez-Juez et al. 1990, Ballare et al. 1991) evidence.

Abbreviations - lh, long-hypocotyl mutant; UV-A, Ultraviolet-A (320-400 nm); UV-B, Ultraviolet-B (280-320 nm); WT, wild-type.

MATERIALS AND METHODS Seed sources and growth conditions Seeds of the long-hypocotyl (lh) mutant and near isogenic wild type (WT) of Cucumis sativus L. (Koornneef and van der Knaap 1983, Adamse et al. 1987) were multiplied in the departments of Genetics (Wageningen Agricultural Univ., The Netherlands) and Ecology (Facultad de Agronomia, Univ. de Buenos Aires, Argentina). Seeds of the commercial hybrid cv. Burpless used in some experiments were obtained from Fredonia Seeds Co., Fredonia, NY, USA.

All the experiments were carried out during the autumn and winter months under glasshouse conditions at Corvallis, OR, USA. Seeds were germinated on moist filter paper under continuous white light provided by fluorescent tubes at 25°C. Two days after germination (3.5 days after sowing) seedlings were transferred to the glasshouse, planted in 10- cm pots filled with standard 1:1 (v/v) perlite (Supreme Perlite Co., Portland, OR, USA) / promix (Promix Premier Brands Inc., New Rochelle, NY, USA) mixture, and grown for 69 an additional 4 days under natural radiation supplemented with light from sodium vapor lamps (see below). Air temperature fluctuated daily between 18 and 26°C (experiments in Figs 3.2 to 3.4), or between 17 and 33°C. (experiments in Fig. 3.5); photoperiod was 14 h.

Solutions of gibberellic acid (GA3)(Sigma) were applied to seedlings by gently painting the apex, both sides of the cotyledons and the hypocotyl with a brush. The concentration of GA3 used (1 mM) was at least 10 times greater than the concentration required to saturate elongation responses under our conditions.

Light sources and filters During de-etiolation in the glasshouse and during the experimental period plants were grown under natural radiation supplemented with light from high pressure sodium vapor lamps (Lucalox LU1000, General Electric Co., Cleveland, OH, USA). Maximum instantaneous photosynthetically active radiation (PAR) at plant level, measured with a cosine-corrected sensor (Quantum type, Q4888, Li-Cor, Lincoln, NE, USA), typically varied between 150 and 480 umol m2 s4 depending on sky conditions. PAR levels > 1,200 umol m2 s4 were recorded only occasionally. UV-B was supplied by panels of 8 fluorescent tubes (UV-B 313, Q-Panel, Cleveland, OH, USA) enclosed by either 0.13 mm -thick clear cellulose acetate film (UV-B treatment) or 0.13-mm-thick clear polyester (optically equivalent to Mylar, Hillcor Plastics, Santa Fe Springs, CA, USA; control treatment) as described by Barnes et al. (1990). The UV-B lamps were turned on and off 3 h (4 h in some experiments) before and after solar noon, respectively. Filters were pre- aged for 6 h before the experiments and replaced after 18-24 h of use. 70 Spectral irradiance measurements were taken with a double-monochromator spectroradiometer (Optotronic Model 742, Orlando, FL, USA). Details about instrument calibration and wavelength-accuracy tests are given by Barnes et al. (1990). Representative scans of the light sources are shown in Fig. 3.1. The estimated effective UV-B fluence at plant level in the UV-B treatment was 9.6 kJ m2 d4 (6 h) or 12.7 kJ m2 d4 (8-h exposures). These figures were calculated by using the generalized plant action spectrum of Caldwell (1971) normalized to unity at 300 nm, and are given to facilitate comparison with other studies. Effective UV-B fluences calculated in this way are comparable to those that naturally exist in tropical locations.

In studies on the site of perception, different plant organs were covered with UV-B-excluding clear polyester during irradiations. Two plastic 'leaves' were placed over the adaxial surface of the cotyledons and were held in place by means of small wire arms. The filters did not project shadows beyond the edge of the cotyledons and left the apex of the seedling uncovered. The apex and hypocotyl were covered by means of an inverted folder made of clear polyester in which two small windows were open to exclude the cotyledons.

Growth measurements and statistics Hypocotyl length (defined as the distance between the connection with the seed and the base of the apex) was measured to the nearest 0.5 mm with a ruler. Leaf area was measured with a LI-3100 area meter (Li-Cor, Lincoln, NE, USA). Dry weights (shoot + root) were obtained after tissue was dried at 70°C for 48 h. Nondestructive estimations of initial cotyledon areas and dry weights were obtained by relating these attributes to measurements of cotyledon 71 length and width. The regressions models were fitted using WT and lh seedlings of the same age and grown under the same conditions as the experimental plants. Values of the coefficients of determination ranged from 0.85 to 0.99. Individual plants were assigned at random to each of the light treatments and the type of filter (clear polyester or cellulose acetate) used in each of the light banks was re- randomized at every filter change. Each experiment was repeated at least twice with similar results; data from different experimental days were pooled for analysis. Mean comparisons were made with Student's t-tests using SAS algorithms (SAS Institute Inc. 1987); in factorial experiments (e.g. Fig. 3.5) only probabilities associated with pre-planned comparisons are reported. Appropriate transformations were used to obtain variance homogeneity whenever necessary.

RESULTS Six day-old, fully de-etiolated WT and lh seedlings were exposed to UV-B during the central 6 h of the photoperiod. The increment in hypocotyl length was evaluated 24 h after the beginning of the UV-B treatment. Hypocotyl elongation was much more rapid in the stable-phytochrome- deficient lh mutant than in WT seedlings. UV-B strongly inhibited hypocotyl elongation and the relative effect was similar in both genotypes (inhibition was 53 and 47 % in lh and WT seedlings, respectively, Fig. 3.2A).

Photosynthetic activity is frequently lowered by UV-B treatments, particularly in plants receiving low PAR levels (Warner and Caldwell 1983, Mirecki and Teramura 1984), and cucumber has been reported to be very sensitive in this regard (e.g. Takeuchi et al. 1989, Tevini and Teramura 1989). Therefore, the observed inhibition of elongation might be simply another consequence of a drastic drop in 72 photosynthetic assimilation. Plants in our experiments were growing very rapidly (relative growth rates 25-30 % dry weight per day). However, under our conditions, we did not find any effect of 6-h exposures to UV-B on cotyledon area expansion or dry matter accumulation (Fig. 3.2B,C). It is important to note here that differences between means on the order of 50 % (that is an effect comparable to that found in the case of hypocotyl elongation, Fig. 3.2A) would have been rated as highly significant in these experiments, in which standard errors of means were ca 12 % in the case of dry weight increments.

In order to study the effects of UV-B over time we conducted another series of experiments. Plants were given an 8-h exposure to UV-B on the sixth day after germination (7.5 days after sowing) and elongation rates during and after the UV-B treatment were estimated from length measurements. These experiments showed that: 1) the immediate effect of UV-B on elongation (i.e. inhibition during the UV-B treatment) was very similar in WT and lh seedlings, and 2) the recovery from UV-B inhibition was rapid. This was particularly true in the case of WT seedlings, where the difference between control and UV-B treated plants disappeared overnight. The lh mutant recovered the day after UV-B exposure (Fig. 3.3).

In another group of experiments with a similar irradiation schedule, plants were coated with saturatingGA3 (1 mM) at the beginning of the UV-B treatment. Control plants received distilled water only. Applications ofGA3 increased hypocotyl elongation by two-fold in both control and UV-B treated plants (Fig. 3.4). Thus, the relative inhibition was not affected by GA3 treatments and, in the case of the WT seedlings, recovery after UV-B was delayed by 73 GA3 (data not shown).

In order to determine the sites of UV-B actionwe selectively covered the adaxial surfaceof the cotyledons or the apex and the hypocotyl with clearpolyester film. The filter excluded UV-B with minimal effectson UV-A and PAR levels received by the tissue. These experimentswere carried out with WT and lh seedlingsand with the commercial hybrid cv. Burpless. In allcases the inhibitory effect of UV-B on hypocotyl elongation was abolishedby covering the cotyledons (Fig. 3.5). Inhibitionwas not prevented by covering the apex and hypocotyl in WTand cv. Burpless seedlings, but was reduced tosome extent in seedlings of the lh mutant. When plantswere not exposed to UV-B (i.e. lamps enclosed by clear polyester film),elongation rates of covered and uncovered plantswere not significantly different (0.18 < P < 0.84).

DISCUSSION UV-B radiation strongly inhibited hypocotylelongation in the three genotypes of cucumberinvestigated (Figs 2-5). Several lines of evidence suggest that thiseffect of UV-B is not a consequence of nonspecificmetabolic damage.

It is clear that the strong inhibitoryeffect of UV-B on hypocotyl elongation is not accompanied by similar effects on dry matter accumulationor cotyledon area development (Fig. 3.2), even when PAR levelswere relatively low. Although we have observedsome growth inhibition by UV- B in longer term experiments (i.e.,1 week or more of treatment), that effect seems to lag wellbehind the rapid elongation response. Our results withcucumber hypocotyls parallel previous observationson leaf elongation responses to UV-B. In Rumex patientia leaves,UV-B radiation appears 74

and Caldwell (1978) to reduce celldivision rate (Dickson that is not and causes a rapidinhibition of elongation (Sisson and correlated withphotosynthetic responses (1988, 1990) Caldwell 1977).Similarly, Barnes et al. elongation in reported that UV-B reducessheath and blade affecting total field- and glasshouse-growngrasses without shoot biomass.

elongation Our experimentsalso showed that hypocotyl increased rates of UV-B treatedplants can be substantially weight to by GA3 applications(Fig. 3.4). This adds extra is not merely a the conclusion thatinhibition of elongation to UV-B consequence ofassimilate limitation. Some responses with in other systems havebeen found to be correlated changes in GA3 content(Rau et al. 1988,cited in Tevini and Teramura 1989). It isinteresting to note that evenat saturating doses, GA3 failedto negate theinhibitory effect inhibition is not of UV-B (Fig. 3.4),suggesting that the mediated by effects on GA3synthesis or activity.

Time course experiments(Fig. 3.3) showed that recovery that, if any from UV-B inhibitionis rapid, suggesting without having cellular damage exists,it is easily repaired long-lasting consequences onelongation growth. The differences between WT andlh seedlings in thekinetics of recovery mightexplain observationsshowing a slightly higher inhibition in thelh mutant in long-termexperiments (e.g. field where plants were notexposed to continuous UV-B experiments by Ballare et al.1991).

the entire In most previousstudies UV-B was applied to experiments shoot; a significantobservation in the present extension is exerted was that UV-Binhibition of hypocotyl mainly through thecotyledons (Fig. 3.5).Therefore, direct 75 action on the dividing or elongating cells cannot explain the UV-B effect.

Our experiments have eliminated the most common explanations for the inhibition of elongation by UV-B that include some component of damage (i.e. reduced photosynthetic assimilation and damage to meristems and elongating regions). Although it would be difficult to reject all of them, it should be noted that our data are totally consistent with a model in which hypocotyl elongation is controlled by UV-B acting through a receptor located in the cotyledons. This is analogous to the inhibition of hypocotyl elongation by red light in cucumber (Black and Shuttleworth 1974), a response that appears to be mediated by the stable type of phytochrome (Adamse et al. 1987). However, it is clear from the relatively low fluences of UV-B used in our experiments (Fig. 3.1), and the similarity in the responses of WT and lh seedlings, that UV- B is not acting through phytochrome. A specific UV-B receptor is therefore implicated.

Phytochrome and the UV-B receptor(s) are known to interact in several plant systems. These interactionsare well documented in the case of the photoregulation of flavonoid and anthocyanin synthesis. Thus, in etiolatedcorn seedlings, red-light pretreatments, probably acting through phytochrome, enhance the response to UV-B (Beggs and Wellmann 1985). In apple fruits (Arakawa 1988) and in etiolated wheat seedlings (Mohr and Drumm-Herrel 1983) the effect of UV-B on anthocyanin production is greatly enhanced by subsequent red-light treatment. In most cases it would seem that Pfr is necessary for the expression of the response to UV-B, although the precise mode of coaction between the two photoreceptor systems is still unclear (Beggs et al. 1986). In our experiments the responses to UV- 76 B of the phytochrome-deficient lh mutant werevery similar to those of the near isogenic WT. We therefore conclude that the effect of UV-B on hypocotyl elongation in de-etiolated cucumbers is independent of the presence of stable phytochrome. Steinmetz and Wellmann (1986) reported that the inhibitory effect of monochromatic-UV-B plus red light pulses on hypocotyl elongation in etiolated Lepidium sativum was not affected by subsequent modifications in phytochrome status.

Morphological effects of UV-B have most commonly been interpreted as by-products of nonspecific damage caused by UV-B radiation. As pointed out by Wellmann (1983)some of these effects may represent active responses that confer some adaptive advantage or protection to the plant (e.g. reduced exposure to UV-B). As a result ofvery effective Rayleigh (molecular) scattering through the atmosphere,a large proportion (usually >50 %) of global UV-B is received at the Earth's surface as diffuse radiation (Caldwell1971, Grant 1991). Strong scattering, and absorption in theozone layer, contribute to determine that the incoming irradiance and spectral photon distribution are muchmore dramatically affected by solar elevation in the UV-B region than in the visible part of the electromagnetic spectrum(Caldwell 1971). An important implication of this, which is often overlooked, is that it might be impossible for the plantto retrieve information about how much UV-B is being received by a particular organ at a given point in time from light signals sensed by other photoreceptors (i.e. phytochromeand cryptochrome). Thus, if morphological responses to UV-Bare adaptive to plants, they may requirea specific receptor to sense the UV-B environment. 77 Acknowledgements. We are most grateful toDr. Maarten Koornneef and members of the Genetics Dept (Wageningen Agricultural Univ.) for provision of originalseed batches and to Marjorie Storm and Dave Olszyk for theirhelp in setting up the experiments in the facilitiesof the U. S. Environmental Protection Agency (Corvallis).C.L.B. was supported by the Consejo Nacional de Investigaciones Cientificas y Tecnicas (Republica Argentina)and thanks Dr Steve Radosevich for his hospitality atOSU. 78

-2 mW m n '1 80 Control UV-B

40

296 306 316

11111111111111111111111111111111111111111 300 350 400 450 500 550 600 650 700 750 800 Wavelength (nm)

FIG. 3.1. Spectral photon distributions measured at plant level under control (polyester-filtered) and UV-B (cellulose-acetate filtered) lamp systems. Measurements were taken under completely overcast sky. Spectral irradiances for the UV region (inset) are given in energy units to facilitate comparison with previous works. 79

A PoNM:01 Control 1.6 ED uv -a

0.6 1.1,7110007

0

B - control C 1ZO in, -a Pr4".0.2406

PoT-0.9437

0

C 10 - contra Pr,T.O.WM CMuv-e

P1,T.0.6352 6

4

2

0 /h-mutant Wild-Type

FIG. 3.2. Effect of 6-h exposures to UV-Bon hypocotyl elongation, cotyledon area expansion and dry matter accumulation in de-etiolated lh and WT cucumber seedlings. Initial values of leaf area and dry weightwere estimated from measurements of leaf width and length. Final measurements were taken 24 h after the beginning of theUV-B treatment. Significance levels are indicated for each difference; n=16. 80

A: Wad-Type Coatiel ENS uv-s en Day I

0.2

PoT-0.7995 PoT43.0027 Pr)T-0.4996

day 1 night 1 day 2

Time Period

FIG. 3.3. Recovery from UV-B inhibition of hypocotyl elongation in de-etiolated lh and WT cucumberseedlings. Day 1 refers to the 8-h period during which plantswere exposed to UV-B in the middle of a 14-h photoperiod,day 2 is the equivalent period on the next day, night1 is the intervening 16-h (10 h night) period. Significancelevels are indicated for each difference; n=12. Note difference between panels in y-axis scale. 81

A: 0.4 Wild -Type I= controlCM .uv-e

Por-00001 0.3

0.2

0.1

0

Water GA3

FIG. 3.4. Effects of GA3 on hypocotyl elongation growth in de-etiolated lh and WT cucumber seedlings exposed to UV-B. Eight-hour exposures were used; length increments were measured at the end of the UV-B treatment. Significance levels are for the effect of GA3; n=14. Note difference between panels in y-axis scale. 82

0.5

0.2

0.1

0

2

1

0

0.3

0.2

0.1

0 none cotyledons hypocotylapex

Organ Covered

FIG. 3.5. Effects of covering different parts of the seedling with a UV-B-excluding polyester filteron hypocotyl elongation in de-etiolated cucumbers exposed toUV-B for 6 h. Length increments were measured 24 h afterthe beginning of the UV-B treatment. Significance levelsare indicated for each difference; n=14-20. Note differencesamong panels in y-axis scale. 83 REFERENCES Adamse, P., P. A. M. P. Jaspers, J. A. Bakker, R. E. Kendrick, and M. Koornneef M. 1988. Photophysiologyand phytochrome content of long-hypocotyl mutant and wild- type cucumber seedlings. Plant Physiology 87:264-268. Adamse, P., P. A. P. M. Jaspers, R. E. Kendrick, andM. Koornneef. 1987. Photomorphogenicresponses of a long hypocotyl mutant of Cucumis sativus L. Journal ofPlant Physiology 127:481-491. Arakawa, 0. 1988. Photoregulation of anthocyanin synthesis in apple fruit under UV-B and red light. Plantand Cell Physiology 29:1385-1389. Ballare, C.L., J. J. Casal, and R. E. Kendrick.1991. Responses of light-grown wild-type and long-hypocotyl mutant seedlings of cucumber to natural and simulated shadelight. Photochemistry and Photobiology54:819-826. Barnes, P.W., P. W. Jordan, W. G. Gold, S. D. Flint, andM. M. Caldwell. 1988. Competition, morphology andcanopy structure in wheat (Triticum aestivum L.) and wild oat (Avena fatua L.) exposed to enhanced ultraviolet-B radiation. Functional Ecology 2:319-330. Barnes, P. W., S. D. Flint, and M. M. Caldwell.1990. Morphological responses of crop and weed speciesof different growth forms to ultraviolet-B radiation. American Journal of Botany 77:1354-1360. Beggs, C.J., and E. Wellmann. 1985. Analysis of light- controlled anthocyanin formation in coleoptiles ofZea mays L.: The role of UV-B, blue and far-red light. Photochemistry and Photobiology 41:481-486. Beggs, C. J., E. Wellmann, and H. Grisebach. 1986. Photocontrol of flavonoid biosynthesis. Pages467-499 in R. E. Kendrick and G. H. M. Kronenberg, editors. Photomorphogenesis in plants. Martinus Nijhoff, Dordrecht. The Netherlands. Beyschlag, W., P. W. Barnes, S. D. Flint, andM. M. 84

Caldwell. 1988. Enhanced UV-B irradiation hasno effect on photosynthetic characteristics of wheat (Triticum aestivum L.) and wild oat (Avena fatua L.) under greenhouse and field conditions. Photosynthetica 22:516-525. Black, M., and J. E. Shuttleworth. 1974. The role of the cotyledons in the photocontrol of hypocotyl extension in Cucumis sativus L. Planta 117:57-66. Caldwell, M. M. 1971. Solar UV radiation and the growth and development of higher plants. Pages 131-177 in A. C. Giese, editor. Photophysiology, Vol. 6. AcademicPress, New York, NY, USA. Curry, G. M., K. V. Thimann, and P. M. Ray. 1956. The base curvature response of Avena seedlings to the ultraviolet. Physiologia Plantarum 9:429-440. Dickson, J. G., and M. M. Caldwell. 1978. Leafdevelopment of Rumex Datientia L. (Polygonaceae) exposed to UV irradiation (280-320 nm). American Journal of Botany 65:857-863. Grant, R. H. 1991. Ultraviolet and photosynthetically active bands: plane surface irradiances at corncanopy base. Agronomy Journal 83:391-396. Koornneef, M., and B. J. van der Knaap. 1983. Anotherlong hypocotyl mutant at the lh locus. Cucurbita Genetics Cooperative Reports 6:13-18. Lopez, E., A. Nagatani, R. E. Kendrick, M.Koornneef, J. Wesselius, and M. Furuya. 1991. The lh mutantof cucumber lacks a type II phytochrome protein andshows an extreme shade-avoidance reaction. Page 7 in Book of Abstracts, Proceedings of the Annual Meeting and 31d Symposium of the Japanese Society of PlantPhysiology. Okayama. Japan. LOpez-Juez, E., W. F. Buurmeijer, G. H. Heeringa,R. E. Kendrick, and J. C. Wesselius. 1990.Response of light- 85 grown wild-type and long-hypocotyl mutant cucumber plants to end-of-day far-red light. Photochemistry and Photobiology 52:143-150. Mirecki, R. M., and A. H. Teramura. 1984. Effects of Ultraviolet-B irradiance on soybean V. The dependence of plant sensitivity on the photosynthetic photon flux density during and after leaf expansion. Plant Physiology 74:475-480. Mohr, H. 1986. Coaction between pigment systems. Pages 547- 564 inR. E. Kendrick and G. H. M. Kronenberg, editors. Photomorphogenesis in plants. Martinus Nijhoff, Dordrecht, The Netherlands. Mohr, H., and H. Drumm-Herrel. 1983. Coaction between phytochrome and blue/UV light in anthocyanin synthesis in seedlings. Physiologia Plantarum 58:408-414. Peters, J. L., R. E. Kendrick, and H. Mohr. 1991. Phytochrome content and hypocotyl growth of long- hypocotyl mutant and wild type cucumber seedlings during de-etiolation. Journal of Plant Physiology 137:291-296. Pratt, L., and W. Butler. 1970. Phytochrome conversion by ultraviolet light. Photochemistry and Photobiology 11:503-509. Ryel, R. J., P. W. Barnes, W. Beyschlag, M. M. Caldwell, and S. D. Flint. 1990. Plant competition for light analyzed with a multispecies canopy model I. Model development and influence of enhanced UV-B conditionson photosynthesis in mixed wheat and wild oat canopies. Oecologia (Berlin) 82:304-310. SAS Institute Inc. 1987. SAS/STAT Guide for Personal Computers. SAS Institute Inc., Cary, NC, USA. Sisson, W. B., and M. M. Caldwell. 1976. Photosynthesis, dark respiration, and growth of Rumex patientia L. exposed to ultraviolet irradiance (288 to 315 nanometers) simulating a reduced atmosphericozone 86 column. Plant Physiology 58:563-568. Steinmetz, V., and E. Wellmann. 1986. The role of solarUV-B in growth regulation of cress (Lepidium sativum L.) seedlings. Photochemistry and Photobiology 43:189-193. Takeuchi, Y., M. Akizuki, H. Shimizu, N. Kondo, and K. Shugahara. 1989. Effect of UV-B (290-320 nm) irradiation on growth and metabolism of cucumber cotyledons. Physiologia Plantarum 76:425-430. Tevini, M., and A. H. Teramura. 1989. UV-B effectson terrestrial plants. Photochemistry and Photobiology 50:479-487. Warner, C.W., and M. M. Caldwell. 1983. Influence of photon flux density in the 400-700 nm waveband on inhibition of photosynthesis by UV-B (280-320 nm) irradiation in soybean leaves: separation of indirect and immediate effects. Photochemistry and Photobiology 38:341-346. Wellmann, E. 1971. Phytochrome-mediated flavone glycoside synthesis in cell suspension cultures of Petroselinum hortense after preirradiation with ultraviolet light. Planta 101:283-286. Wellmann, E. 1976. Specific ultraviolet effects in plant photomorphogenesis. Photochemistry and Photobiology 24:659-660. Wellmann, E. 1983. UV radiation in photomorphogenesis. Pages 745-756 in W. Shropshire Jr. and H. Mohr, editors. Encyclopedia of Plant Physiology, New Series, Vol. 16B. Springer Verlag, New York, NY, USA. Yatsuhashi, H., T. Hashimoto, and S. Shimizu. 1982. Ultraviolet action spectrum for anthocyanin formation in broom sorghum first internodes. Plant Physiology 70:735-741. 87

CHAPTER 4

PHYTOCHROME-MEDIATED PHOTOTROPISM IN DE-ETIOLATED SEEDLINGS: OCCURRENCE AND ECOLOGICAL SIGNIFICANCE

Carlos L. Ballare., Ana L. Scopel, Steven R. Radosevich, and Richard E. Kendrick

Depts of Forest Science and Crop and Soil Science, Oregon State Univ., Peavy Hall 154, Corvallis, OR 97331-5705, USA (C. L. B., A. L. S., S. R. R), Dept de Ecologia, Facultad de Agronomia, Univ. of Buenos Aires, (1417) Buenos Aires, Argentina (C. L. B., A. L. S.) and Dept of Plant Physiological Research, Wageningen Agricultural Univ., Generaal Foulkesweg 72, NL-6703, The Netherlands (R. E. K.)

Plant Physiology, in press (1992). 88

ABSTRACT Phototropic responses to broadband far-red (FR) radiation were investigated in fully de-etiolated seedlings of a long-hypocotyl mutant (lh) of cucumber (Cucumis sativus L.), which is deficient in phytochrome-B, and its near- isogenic wild type (WT). Continuous unilateral FR light provided against a background of white light induced negative curvatures (i.e. bending away from the FR light source) in hypocotyls of WT seedlings. This response was fluence-rate dependent and was absent in the lh mutant, even at very high fluence rates of FR. The phototropic effect of FR light on WT seedlings was triggered in the hypocotyls and occurred over a range of fluence rates in which FR was very effective in promoting hypocotyl elongation. FR light had no effect on elongation of lh-mutant hypocotyls. Seedlings grown in the field showed negative phototropic responses to the proximity of neighboring plants that absorbed blue (B) and red light and back-reflected FR radiation. The bending response was significantly larger in WT than in lh seedlings. Responses of WT and lh seedlings to lateral B light were very similar; however, elimination of the lateral B light gradients created by the proximity of plant neighbors abolished the negative curvature only in the case of lh seedlings. More than 40% of the total hypocotyl curvature induced in WT seedlings by the presence of neighboring plants was present after equilibrating the fluence rates of B light received by opposite sides of the hypocotyl. These results suggest that:(i) phytochrome functions as a phototropic sensor in de-etiolated plants, and (ii) in patchy canopy environments, young seedlings actively project new leaves into light gaps via stem bending responses elicited by the B-absorbing photoreceptor(s) and phytochrome. 89 INTRODUCTION Phototropism occurs when a lateral light stimulus induces a difference in extension rate between the two sides of an elongating organ. The difference in extension rate is a response to the internal light gradient created by light scattering and absorption between the "illuminated" and "shaded" flanks of the phototropic organ (Reviews by Firn [1986], Briggs and Baskin [1988], and lino [1990]). Action spectra for phototropism in a variety of plant systems show peaks of quantum effectiveness between 320 and 500 nm (References in Dennison [1979] and lino [1990]), indicating that a B/UV-A photoreceptor plays a key role in the detection of internal light gradients. The roles of phytochrome in phototropism are not completely clear. Studies with etiolated oats (Briggs 1963, Dennison 1979) and etiolated dicotyledonous seedlings (Shropshire and Mohr 1970) suggested that phytochrome does not function as a detector of radial light gradients, and it has been proposed that its main function in phototropism is to modulate the response to B light mediated by another receptor (Woitzik and Mohr 1988). However, recent work with dark-adapted maize (lino et al. 1984, Kunzelmann and Schafer 1985) and totally etiolated pea seedlings (Parker et al. 1989) indicted that the radial Pfr gradient formed upon exposing the shoots to short pulses of lateral light can explain observed curvature responses. The picture is more complex in the case of de- etiolated seedlings, because detailed information on spectral sensitivity is scant. lino (1990) suggested that the R:FR gradient that exists across green organs exposed to unilateral polychromatic light (Seyfried and Fukshansky 1983, Vogelmann 1986) should generate a radial gradient of Pfr/P. This radial Pfr/P gradient might, in turn, lead to a gradient of growth inhibition and eventually to phototropic bending. Rough action spectra obtained by Atkins (1936) using broadband light sources for de-etiolated seedlings of 90 two dicotyledonous species do, in fact, show a small peak in the R region. Shuttleworth and Black (1977) obtained positive curvatures in green cucumber seedlings using broadband R light; the response was not observed in sunflower seedlings, where only one fluence rate was tested. Winning (1938) reported a phototropic effect of R light in sunflower seedlings de-etiolated under reduced sunlight; the same treatment was ineffective in the case of Sinapis alba. Recent studies with cucumber indicated that the bending of hypocotyls toward direct sunlight under glasshouse conditions is mainly a response to the B component; experimental reversal of the natural B light gradient between opposite sides of the seedlings led to reversal of the direction of curvature, and the effect of this treatment was the same in seedlings of wild type (WT) and the phytochrome-B-deficient (lh) mutant (Ballare et al. 1991b).

In parallel with the uncertainty on the role of phytochrome in the detection of radial light gradients, there is little information on the influence of the spectral distribution of natural radiation on phototropic movements of light-grown plants (Morgan and Smith 1981). Remarkably few studies on phototropism have been conducted in natural light environments, and the gaps in our understanding of the ecological aspects of phototropic responses have been discussed recently by Firn (1988) and Iino (1990). In this study we set out to test the role of phytochrome in the detection of radial light gradients in de-etiolated seedlings exposed to continuous unilateral illumination. We also tested phototropic responses in plant canopies and evaluated the possibility that changes in the spectral composition of scattered radiation, brought about by nearby plants, may elicit phototropic responses in young seedlings. Our approach is based on physiological experiments with seedlings of the lh mutant of cucumber and itsnear isogenic 91 WT. The lh mutant lacks a phytochrome-B polypeptide (LOpez- Juez et al. 1992), shows reduced levels of spectrophotometrically-detectable phytochrome after de- etiolation (Adamse et al. 1988, Peters et al. 1991), and lacks (or exhibits severely reduced) elongation responses to end-of-day (Adamse et al. 1988, Lopez-Juez et al. 1990) and day-time (Ballare et al 1991b) R:FR treatments.

Abbreviations: B, blue; UV-A, ultraviolet-A (320-400 nm); FR, far-red; R, red; P, total phytochrome; WT, wild type; lh, long-hypocotyl mutant; WL, white light.

MATERIALS AND METHODS Plant material and growth conditions Seeds of the long-hypocotyl (lh) mutant and the near isogenic wild-type (WT) of Cucumis sativus L. (Adamse et al. 1987) were obtained from the Department of Genetics (Wageningen Agricultural Univ., The Netherlands). For the field experiments, cucumber seeds were germinated under white light (WL) as described previously (Banat6 et al. 1991a). Two-day-old seedlings were transferred to a glasshouse, planted in 10-cm pots containing a standard soil-less mixture and grown for an additional 4 d under natural radiation supplemented with light from high-pressure sodium vapor lamps (Banat-6 et al. 1991a). The pots were then transferred to the field and distributed among light treatments. Maize seeds (Zea mays L. cv Early Jubilee) were sown directly in the field in recently cultivated soil on 15 May, 1991. Six plots (1.0 x 0.5 m; east-west orientated) were planted at a rate of ca. 4000 seeds m2 in order to produce dense, homogeneous canopies. The experiments were carried out between early July and early September, 1991; during this period maize plants were periodically clipped to maintain canopy height at 50 + 5 cm. All field experiments 92 were carried out at the Vegetable Research Farm, Department of Horticulture, Oregon State University, near Corvallis, OR. For the controlled-environment experiments, cucumber seeds were sown in 2.5-cm pots at a depth of 0.5 cm. Pots were incubated in the same growth room where the phototropic experiments were carried out. The cotyledons began to emerge from the soil 7 d after sowing; after two more days of growth, seedlings were selected for uniformity and allocated to the different light treatments. The WL (Fig. 4.1) in the growth room was provided by a mixture of 110-W F96T12/CW/HO fluorescent tubes (Sylvania) and 300-W incandescent lamps (Photoperiod = 12 h d4; PPFD [plant level] = 160 gmol s4; day/night temperature = 22/16°C).

Light treatments and experimental procedures In the field experiments, potted cucumber seedlings were placed ca. 8 cm to the south of the directly sunlit edge of dense maize canopies or at the center of a 10 x 10 m plot where all weedy vegetation was removed periodically by soil cultivation. In the initial experiments only WT seedlings were used. The pots were fitted inside 10 cm (i.d.) x 30-cm high clear-polyester open cylinders in order to prevent movement of the seedlings by wind. In experiments designed to test the role of phytochrome in natural phototropism, pots were fitted inside 10 cm (i.d.) x 30-cm (WT seedlings) or 40-cm high (lh seedlings) acetate cylinders that were prepared as follows. The hemi-cylinder that faced south (i.e. the one that received direct sunlight) was made of a sheet of Roscolux #21 (Golden Amber) acetate (Rosco Labs., Port Chester, NY), that absorbed most of the B/UV-A radiation with minimal effects on R-FR wavelengths, or a sheet of Roscolux #34 (Flesh Pink) acetate, that reduced PPFD to a similar extent due to strong absorption in the green region (Transmission of the pink 93 film in the B and R-FR regions was 50 % and >80 %, respectively). This southern wall of the cylinder filtered all direct sunlight received by the seedlings. The hemi- cylinder that faced north (i.e. the one that received diffuse skylight or light scattered by the nearby canopy) was made of Roscolux #34. Seedlings were placed in front of the maize canopies or at the center of a clear plot as described above.

In the controlled-environment experiments, continuous unilateral FR light (Fig. 4.1) was provided from the side against a WL background (from above) by a bank of incandescent lamps. The light beam was filtered trough 3 cm of water and one sheet of each of the following Roscolux filters: #83 (Medium Blue), #12 (Straw), and #42 (Deep Salmon). The resulting beam was a rectangle 60 cm wide and ca. 13 cm high which struck the entire shoot of the seedlings. Different fluence rates were obtained by inserting neutral density filters constructed of 1.1-mm mesh plastic netting between the light sources and the seedlings. A mock source covered with black plastic was used as a control. In preliminary experiments we found that seedlings tended to bend away from the simulated and FR light sources even if the lights were off. This was remedied by placing, on the opposite side of the seedlings, a 30-cm high B- absorbing curtain (Roscolux #12) at a distance of 23 cm. In the experiments where FR light was applied bi-laterally, the seedlings were positioned midway between the light source and a mirror; FR fluence rates were calculated by doubling the measured output of the lateral light source. In the experiments to determine the site of perception, carried out with WT seedlings, a small, "U"-shaped aluminum piece (1 x 0.3 [lateral wings) x 2.5 [height] cm), was used to prevent lateral FR from reaching the hypocotyl. The shield was positioned at a distance of 5 mm from the hypocotyl. 94

Applications of GA3 (100 gM) were carried out as described previously (Ballare et al. 1991a).

Phototropic responses were determined by measuring the angle between the vertical vector and the upper 1.5-cm section of the hypocotyl on the plane parallel to the lateral light beam (controlled-environment experiments) or on the north-south plane (field experiments). The angle of curvature was measured 48 h after the beginning of treatments in the field experiments and at different intervals (see figures and figure legends) in the controlled environment. Hypocotyl length (defined as the distance between the rim of the pot and the base of the apex) was measured to the nearest 0.5 mm with a ruler. All light measurements were obtained by using a cosine-corrected receiver and a calibrated LI-COR 1800 spectroradiometer (Li- Cor, Lincoln, NE).

Statistics Individual plants (=pots) were assigned at random to each of the light treatments (i.e. light source or canopy x filter combination). Each experiment was repeated at least three times with similar results; data from different experimental days were pooled for analysis. Results are presented as means ± SE, except where indicated otherwise.

RESULTS Cucumber seedlings placed on the southern (sunlit) side of a green, dense maize crop bent toward the south (i.e. away from the neighboring crop) within 2 d of the beginning of the treatment. The hypocotyls of their isolated counterparts were almost vertical (Fig. 4.2). This neighbor- induced negative tropism could have been mediated by a B- absorbing photoreceptor or by phytochrome, since the 95 proximity of green plants can change the horizontal component of radiation absorbed by these two photoreceptors due to absorption and reflection of sunlight (Ballare et al. 1987, 1991c, Kasperbauer et al. 1984, Smith et al. 1990).

Induction of hypocotyl curvature by lateral B light gradients is a well documented phenomenon in de-etiolated dicotyledonous seedlings. A series of controlled-environment experiments were carried out to test whether a phytochrome- mediated phototropism can be demonstrated in de-etiolated cucumbers. Seedlings de-etiolated for 2 d were given additional FR light from one side against a background of WL provided from above. In WT seedlings, continuous unilateral FR induced a negative phototropic bending that was statistically detectable after 2 h of treatment; curvature was about 90 % of the maximal after 4 h. No curvature was detected in the lh mutant (Fig. 4.3). In WT seedlings, increasing the fluence rate of unilateral FR light between 17 and 122 gmol m2 s4 had a log-linear effect on hypocotyl curvature (Fig. 4.4). When WT seedlings were irradiated bi- laterally, variation of FR fluence rate over such a range led to a ca. 2 fold increase of hypocotyl elongation rate (Fig. 4.5). In seedlings of the lh mutant, additional FR light failed to promote elongation; however, applications of GA3 were clearly effective in FR-treated and control seedlings (Fig. 4.5). This effect of GA3 corroborates results obtained under a variety of other lighting conditions (Adamse 1988, Ballare et al 1991a,b) showing that the potential for longitudinal growth promotion exists in lh seedlings.

In our experiments the seedlings were positioned in front of the FR light source with the long axis of their cotyledons perpendicular to the lateral light beam. This 96 positioning was done in order to minimize differences in light environment between cotyledons, which might have indirectly induced hypocotyl curvature (the so called "simulated phototropism" [Shuttleworth and Black 1977]). Under these conditions, interposing an opaque barrier between the FR source and the hypocotyl, without affecting the light environment of the cotyledons, drastically reduced the negative phototropic response (Fig. 4.6).

Spectral scans of the light received by cucumber seedlings in the field showed that plants grown close to the sunlit edge of a maize canopy received less B and R light from the northern side and much more FR than isolated seedlings (Table 4.1). In order to assess the relative contribution of each of these changes to the bending response showed in Fig. 4.2, we:(i) compared the curvature responses to green neighbors of WT and lh seedlings, and (ii) tested the effect of eliminating the steep south-to- north B-light gradient created by the canopy by placinga B- absorbing filter on the southern side of the seedlings. To compensate for the reduction in PPFD caused by this treatment, control seedlings were shaded by a green- absorbing acetate (see Materials and Methods). The effects of the acetate filters and plant neighbors on the light received by the plant from the northern and southern sides are shown in Fig. 4.7. Seedlings grown without neighbors (i.e. isolated seedlings) experienced a natural south-to- north gradient of B light (Fig. 4.7A), and their hypocotyls were slightly curved toward the south (Fig. 4.8, A and B). The B-absorbing filter placed on the southern side reversed the direction of the natural B-light gradient (Fig. 4.7B) and caused isolated seedlings to bend toward the north (Fig. 4.8, A and B). The response to the B barrier was of thesame magnitude in isolated WT and lh seedlings. Thepresence of a green canopy on the northern side exaggerated the south-to- 97 north B-light gradient and, in addition, createda steep R:FR gradient due to absorption of R light and back- reflection of FR radiation (Fig. 4.7C; cf. Fig.4.7A and Table 4.1). Plants grown in front of a greencanopy were markedly curved toward the south (i.e. away from the canopy) (Fig. 4.8, C and D; see also Fig. 4.2). The bendingresponse to the proximity of the canopy was significantly larger in WT than in lh seedlings (Fig. 4.8, C and D). Eliminating the B-light gradient induced by the canopy bymeans of a B- absorbing filter (Fig. 4.7D) abolished the negative tropic response in lh seedlings (Fig. 4.8C). In contrast, a residual curvature of ca. 11 degrees (i.e. about 46 %of the maximum response) remained in WT plants (Fig. 4.8D)

DISCUSSION Our results strongly suggest that phytochrome functions as a detector of light gradients established across the hypocotyls of green cucumber seedlings and mediates phototropic responses to long-wavelength (> 600 nm) radiation. Unilateral FR radiation administrated againsta background of WL provided from above induceda negative phototropic response in WT seedlings (Fig. 4.3). This response was:(i) fluence rate dependent (Fig. 4.4), (ii) not an indirect consequence of differential illuminationof the cotyledons (Fig. 4.6; cf. Shuttleworth and Black1977), and (iii) not observed in the phytochrome-B-deficientlh mutant (Fig. 4.3), even at very high fluence rates ofFR light (Fig. 4.4). Comparison of these results with previous work is difficult because other authors used different species or experimental protocols. Atkins (1936) obtained rough action spectra for the induction of phototropic responses to continuous irradiation using broadband light sources. The spectra for de-etiolated dicotyledonous seedlings (Lepidium sativum and Celosia cristata)showed a small (positive) peak in the R region in addition to the 98 peak in the B/UV-A. No data pointswere reported between ca. 710 and 850 nm (L. sativum) or 650 and 850 nm (C. cristata). No such R-peak was observed for etiolated oat coleoptiles, where the ineffectiveness of radiation above 500 nm has been documented extensively (e.g. Briggs 1963, Dennison 1979). Shropshire and Mohr (1970) stated that continuous Ror FR light did not induce hypocotyl curvature in etiolated seedlings of two dicotyledonous species: Fagopyrum esculentum and Sinapis alba. Continuous R light was also ineffective in de-etiolated S. alba, but elicited curvature in de-etiolated sunflowers (BUnning 1938). Shuttleworthand Black (1977) found that continuous unilateral R light induced positive curvature in hypocotyls of de-etiolated cucumbers. However, the authors were suspicious of the spectral purity of the R light source (i.e. B light contamination) and did not discuss that result further.More recently, Parker et al. (1989) interpreted the phototropic response of totally etiolated pea epicotyls to short B light pulses on the basis of phytochrome action. Based on previous results with dark-adapted maize mesocotyls (lino et al. 1984, Kunzelmann and Schafer 1985), they proposed that epicotyl curvature in their experimental conditionswas induced by a Pfr gradient established across the epicotyls after illumination with unilateral B light. A related mechanism may account for the negative phototropism induced by continuous FR light in our experiments. Although fluence rate gradients for 660 and 730 nm have been measured in green hypocotyls of other species (Vogelmann 1986), extrapolation to the present experiments is difficult because our seedlings received omnidirectional WL simultaneously with the unilateral FR treatment. The important point is that, in our experiments, curvaturewas induced over a range of FR fluence rates (Fig. 4.4)where a 2-fold change of fluence rate (i.e. equivalent to the fluence-rate differential that could be expected between 99 opposite flanks of a 2-mm diameter, green hypocotyl [Vogelmann 1986]) should produce a sizable (ca. 0.1 mm 114) difference in elongation rate (Fig. 4.5). Simple geometrical relations indicate that such a difference in elongation between the "illuminated" and "shaded" flanks would yielda bending rate of about 2.9 degrees h4, which is roughly similar to the bending rates measured during the initial2 h in our experiments (Fig. 4.3).

The question at this point is whether a phytochrome- mediated phototropic mechanism plays a role in the curvature responses of young seedlings to the proximity of green neighbors (Fig. 4.2). The observation that hypocotyl-bending responses were larger in WT than in lh seedlings (Fig. 4.8, C and D, controls) is consistent with such a possibility, but certainly can not be taken as definite evidence. In fact, it is well known from a number of other studies that an important action of phytochrome in phototropism is to alter the pattern of response to unilateral B light mediated by a specific photoreceptor (e.g. Woitzik and Mohr 1988). Therefore, the observed differences between WT and lh seedlings might simply reflect an effect of phytochrome-Bon the curvature response to the steep south-to-north B-light gradient (Table 4.1 and Fig. 4.7C) created by thepresence of a green canopy. However, when that B-light gradientwas experimentally eliminated by placing a B-absorbing filteron the southern side of the plants (Fig. 4.7D),a significant southward curvature was still observed in the hypocotyls of WT plants (Fig. 4.8D). Moreover, when the B-absorbing barrier was placed on the southern side of isolatedplants, the northward curvature responses of WT and lh seedlings were similar (Fig. 4.8, A and B). These and previous data (Ballare et al. 1991b) suggest that the lack of phytochrome- B does not affect the (steady state) curvature induced by 100 lateral B light in de-etiolated cucumbers under natural light fluences. If we therefore assume that the actions of phytochrome and the B-absorbing photoreceptor are roughly additive in this system, we can calculate that about 46% of the total bending induced by the proximity of a green canopy is due to radial R:FR gradients perceived by phytochrome.

The observation that the hypocotyl is the main site of perception of lateral FR (Fig. 4.6) is consistent with results obtained in other dicotyledonous seedlings irradiated with B light or WL (Franssen and Bruinsma 1981, Hart and MacDonald 1981, Schwartz and Koller 1980, Shuttleworth and Black 1977) and with the hypothesis that it is the R:FR gradient established across the hypocotyl that elicits the bending response. This observation is very important with respect to the ecological significance of stem phototropism. In open plant stands, the presence of neighboring individuals can alter the light environment of vertically-oriented stems well before there is any mutual shading at leaf level (Ballare et al. 1987, 1990, 1991c, and Fig. 4.7, this paper). These alterations influence the rate of internode elongation and may convey critical information to plants that are competing with neighbors for colonizing the aerial environment (Ballare et al. 1990). According to our data (Fig. 4.7), the radial light gradients perceived by the stems are related to the spatial distribution of neighbors around the plant. Tropic responses to these gradients would improve the efficiency with which a seedling collects light in a patchy light environment, as the probability of new leaves being actively projected into light gaps would increase. It has been reported recently that seedlings of Portulaca oleracea, a plant with a plagiotropic shoot system, tend to avoid growing toward plant neighbors or plastic objects that back-scatter radiation with a low R:FR ratio. Part of the response in 101 this case appeared to be because main stemswere unlikely to become recumbent toward natural or simulated neighbors (Novoplansky et al. 1990). Our results indicate that, in light-grown cucumbers, phototropic mechanisms controlled by the B-absorbing photoreceptor(s) and by phytochromeare responsible for the negative bendingresponses elicited by the proximity of neighboring plants.

Acknowledgements. We are most grateful to Dr. Maarten Koornneef and members of the Department of Genetics (Wageningen Agricultural Univ.) for provision of the cucumber seeds. We also wish to thank Randy Hopson (Veg. Res. Farm, OSU) and Lloyd Graham (Forest Res. Lab., OSU)for practical assistance, and Gretchen Bracher for preparingthe figures. C.L.B. and A.L.S. were supported by the Consejo Nacional de Investigaciones Cientificasy Tecnicas (Argentina) and the Dept. of Forest Science (OSU). 102

TABLE 4.1. Effects of the proximity of green plants on the light flux measured parallel to the ground.

Measurement Photon Irradiance (Amol m4 s4) R: FRt B R FR

Receiver pointed toward the south 137.5 223.7 238.3 1.00

Receiver pointed toward the north: (a) no neighbors 36.3 42.8 72.6 0.63 (b) in front of a green canopy 8.0 21.6 128.1 0.16

*Measurements were taken near midday under clear sky on 28 July 1991. The surface of the light receiver was normal to the ground and received direct solar radiation when pointed toward the south. The receiver was positioned 8 cm to the south of the maize canopy to obtain the last row of data. B=400-500 nm, R=600-700 nm, FR=700-800 nm. t(654- 664/726 -736) quantum flux ratio. 103

1.0 WL 11 FR ee c_ / m III ....-2 I 0 I 0 I C I CIS I "1:3 I 20.5- I c I 0 I I t I 0 I I ELI I t0 I 0. I u) r el I I 1 400 500 600 700 800 Wavelength (nm)

FIG. 4.1. Spectral photon distributions of the light sources used in the controlled-environment experiments. The quantum integral between 300 and 400 nm in the FR beam was < 0.01 % of the output between 700 and 800 nm. 104

Isolated Canopy

FIG. 4.2. Effect of the proximity of a green canopy on the orientation of the hypocotyl of WT seedlings at solarnoon. Seedlings were grown for 2 d at the center ofa clear plot (isolated) or 8 cm to the southern (sunlit) side ofa dense maize crop (canopy). Data are means of 4 independent experiments (n=12-16). 105

4 2-

0

-4,3' a)-2- e WT O Ih a)-4- -0 WT + FR -6- lh + FR =92 48 8_ m 0 -10- -12- i'--...... -14- -16 0 45 67 II Time (h)

FIG. 4.3. Time courses of hypocotyl bending in WT and lh seedlings growing under WL with or withoutexposure to unilateral FR light (86.5 gmol m2 s-1). Time= 0 indicates the onset of FR irradiation; negative valueson the y-axis indicate bending away from the FRsource; II indicates the end of the second day of treatment (i.e. two 8-hexposures to unilateral FR light). Data are means of 4 independent experiments (n=15-28); for clarity of presentation,SE (all <1 degree) were omitted in the upper curves. 106

4 2 - - 0 4-, 1;-2- -8 -4- =-6- o wr 0 lh -10- WT + FR Ih+ FR -12- -14- -16 0 2 3 4 5 In FR fluence rate (Imo! m-2 s-1)

FIG. 4.4. Effects of increasing the fluence rateof unilateral FR light on hypocotyl curvature inWT and lh seedlings. Curvatures were measured after5 h of continuous FR light; negative values on the y-axis indicatebending away from the FR source. Data are means of 4 independent experiments (n=68-80, controls,or 8-29, intermediate fluences, or 29-37, highest fluence). 107

1.4 + GA3 1.2- 'T

7e--'1.0- Control E E 1 0.8a) - Iris WI' oc 0.6- O lh WT + FR lh + FR ga)0.4' El 0.2-, 0..-1,,---r--.T--r---6 2 3 4 5 In FR fluence rate (gmolm-2 s-1)

FIG. 4.5. Effects of increasing the fluencerate of bi- lateral FR light on hypocotyl elongation inWT and lh seedlings (the latter with or without applicationof GA3). Elongation rates were estimated from lengthmeasurements taken at the beginning of the experimentand after 5 h of continuous FR irradiation. The solid lineparallel to the abscissa denotes the range of FR fluencerates used in the curvature experiments. Data aremeans of 3 independent experiments (n=13-28). 108

Control FR Treatment

FIG. 4.6. Effect of shading the hypocotyl during irradiation with unilateral FR light (121.7 Amol m-2 sl)on hypocotyl curvature in WT seedlings. Curvatures were measured after 5 h of continuous FR irradiation; negative valueson the y- axis indicate bending away from the FRsource. Data are means of 9 independent experiments (n=28-37). 109

..../ .... I 1 1 ....f %., E i i i I 1 II I %.1 II -esE. --, -.---...... -.--. .r. -?.-- 0 0 500 c400 600 700 800400 500 600 700 800 as Cue3 3 , Ccanopy (control) D canopy (B barrier) 0 15 .cca. 2- e t 0.a) (1) 1 ,_-...... 0 I , I 0'--:----si" a i I I 400 500 600 700 800 400 500 600 700 800

Wavelength (nm)

FIG. 4.7. Effects of the proximity of a green maizecanopy and B-absorbing acetate filters on the spectral photon distribution of the light flux measured parallel to the ground. Measurements were taken near midday under clearsky on 28 July 1991. The surface of the light sensor was normal to the ground and pointed toward the southern (solid line) or northern (dashed line) hemisphere. When pointed toward the south, the sensor received either direct sunlight filtered by a Roscolux #34 acetate (control)or direct sunlight filtered by a B-absorbing acetate #23 (B-barrier). When pointed toward the north, the sensorwas always shielded by the acetate #34 and received either diffuse skylight (isolated) or light back-scattered byan 8-cm distant maize canopy (canopy). 110

12 A /h/isolated B WT/isolated _c 8 0 4

0

4 =0 8

12

T/canopy

Control B barrier Control B barrier

FIG. 4.8. Effects of the proximity of agreen maize canopy and B-absorbing acetate filters on the orientation ofthe hypocotyl of WT and lh seedlings at solarnoon. Seedlings were grown for 2 d at the center of a clear plot (isolated) or 8 cm away from the southern edge of a dense maizecrop (canopy). Data are means of 11 independent experiments (n=19-26, isolated, or 42-56, canopy). 111 REFERENCES Adamse, P. 1988. Mutants as an aid to the study of higher plant photomorphogenesis. PhD thesis. Wageningen Agricultural University, Wageningen, The Netherlands. Adamse, P., P. A. M. P. Jaspers, J. A. Bakker, R. E. Kendrick, and M. Koornneef. 1988. Photophysiology and phytochrome content of long-hypocotyl mutant and wild- type cucumber seedlings. Plant Physiology 87:264-268. Adamse, P., P. A. M. P. Jaspers, R. E. Kendrick, and M. Koornneef. 1987. Photomorphogenic responses of a long hypocotyl mutant of Cucumis sativus L. Journal of Plant Physiology 127:481-491. Atkins, G. A. 1936. 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CHAPTER 5

FORAGING FOR LIGHT IN HORIZONTALLY-PATCHY ENVIRONMENTS BY WILD-TYPE AND PHYTOCHROME-B-DEFICIENT CUCUMBER PLANTS 116 ABSTRACT

In this paper we address the following questions:(i) Do plants that colonize horizontally-patchy environments actively project leaf area into light gaps and avoid poorly illuminated sites? (ii) What are the roles of the photoreceptor phytochrome in gap colonization? Our approach was based on dynamic studies of gap colonization by cucumber (Cucumis sativus L.) plants and comparisons of morphological development between wild-type (WT) and lh-mutants. The lh mutant of cucumber is deficient in phytochrome B and, when de-etiolated, seedlings show reduced or noresponse to alterations of red:far-red (R:FR) ratio, one of the environmental factors that plants use to monitor the proximity of other plants. WT plants grown at the edges ofa maize canopy in the field projected outside thecanopy nearly all the nodes produced over a period of four weeks. In contrast, about 50 % of the nodes and more than 80 % of the apical buds of lh plants were positioned within the maize crop. Controlled-environment experiments showed that seedlings of both genotypes are able to detect horizontal light / shade mosaics and actively project leaves toward light gaps via phototropic (stem-bending)responses. These responses are presumably mediated by a specific blue-light- absorbing photoreceptor. A mechanism independent of phytochrome B also appeared to control the angle of display of individual leaves with respect to the direction of illumination. Our controlled-environment experiments showed that, when compared over periods of several weeks, WT plants are far more efficient than lh mutants at invading and maintaining their shoots in light gaps. Thereasons for this difference were multiple and appeared to result fromseveral actions of phytochrome B on WT plants that received full light with high R:FR ratio. These actions included: (i) Influencing the gravitropic response of stems,so that WT shoots grown under full light were diagravitropic, whereas 117 lh shoots were orthogravitropic. The diagravitropic habit, coupled with an efficient phototropic system,appears to help WT shoots to escape from the shaded sectors and invade light gaps.(ii) Delaying tendril production and reducing stem-bending responses to mechanical stresses exertedby tendrils. These two actions combine with theones listed above to determine that the direction of shoot growth in WT plants is mainly dictated by the spatial distribution of light gaps, and not by the distribution of potential points of support for tendril attachment. (iii) Switching plants from the "shade-avoiding" (default) developmentalprogram to the one that results in a phenotype with short internodes and reduced apical dominance. These two actions would favor concentration of nodes and leaf area in lightgaps. 118 INTRODUCTION The light environment in plant canopies is heterogeneous in space and time. Plants are considered to "forage" for light if they tend to adjust the spatial distribution of their light-absorbing surfaces to trackthe spatial distribution of the light resource (Grime 1979, Slade and Hutchings 1987, Hutchings and Mogie 1990). Although the ultimate manifestation of foraging for light in space is concentration of plant modules or biomass in high- light patches, such a pattern of asymmetric growthmay originate for various reasons, i.e.:(i) simple differences in plant growth rate between low- and high-light patches, (ii) differences in growth rate preceded by specific morphological responses to the local light climate, and (iii) active projection of modulesor leaf area into light gaps and concomitant avoidance of poorly illuminated sites. The first case is probably trivial. If the plant has equal access to patches of different quality, one would expect to find most of the biomass concentrated under high-light conditions after a sufficiently long period of growth.The only necessary condition for such asymmetry tooccur is that individual modules of the plant havesome degree of growth independence. The second case can be exemplified with the observations of Pefialosa (1983) on the morphological development of the vine (Ipomoea phillomega) in tropical rain-forests of Mexico. This liana "travels" through the forest floor by means of creeping stolons with long internodes and rudimentary leaves. The transformationof stolons into twinning shoots with short internodes and large leaves appears to be favored by the changes of light environment that the stolons experience when theyreach sunny patches. These morphological changes can be interpreted as a form of foraging for light, becausethey increase the light-harvesting capacity of theshoot when it reaches a gap in the forest canopy (fora more complete 119 discussion see Penalosa 1983, Hutchings and Slade1988, and Hutchings and Mogie 1990). The third case (selective projection of leaf area toward light gaps) isa clear example of true foraging for light, and probably the most efficient mode of space occupation in patchy light environments. These considerations suggest that static descriptions of plant architecture can be interpreted in several ways. Therefore, they are not sufficient to decide whether or not a selective pattern of space invasion is the principal mechanism that causes a non-random distributionof plant biomass between high- and low-light patches.

For selective invasion to occur, plants needsensors capable of monitoring environmental changes associated with variations in light conditions. A considerable bodyof evidence derived from experiments in controlled-environments (reviewed by Smith 1986) and conditions of naturalradiation (reviewed by Ballare et al. 1992b) indicates that projection of leaves into the upper strata of thecanopy is a process modulated by at least two specific lightsensors: phytochrome and a blue- (B-) absorbing photoreceptor.In plant canopies formed by plants of similar height (e.g.crop stands), the light signals that trigger changes in stem elongation rate via these photoreceptorsmay be perceived by an individual plant well before its leaves become shaded by neighbors (Ballare et al. 1987; Ballare et al. 1991c). Therefore, the acceleration of stem elongation elicitedby high population densities in even-aged monocultures(Ballare et al. 1988; Ballare et al. 1990) can be regardedas a true process of foraging for light in the vertical dimension.

Light signals perceived by phytochrome alsohave been implicated in mechanisms that influence the directionof growth on the horizontal plane in patchycanopy environments. Novoplansky et al.(1990) observed that the 120 shoots of Portulaca oleracea, a rapidly-growing recumbent herb, showed a consistent tendency to avoid developing toward neighboring individuals when colonizing patchy canopies. They also found that main stems of E., oleracea seedlings did not normally become recumbent toward plastic objects which light absorption spectrumwas similar to that of green leaves.

Recently, it has been demonstrated that the proximity of green neighbors elicits a rapid phototropicresponse (stem-bending) in young seedlings of cucumbergrown in the field. This response is mediated bya B-absorbing photoreceptor and phytochrome (Ballare et al., 1992a). The phototropic response might be the initial step ofa mechanism leading to neighbor avoidance and selective invasion of high-light patches in heterogeneous canopies. The experiments reported in this paperwere designed to gain mechanistic insight into the process of foraging for light in horizontally-patchy canopies and to test the involvement of the photoreceptor phytochrome B (phyB)(See Smith and Whitelam 1990, and Quail 1991, fora description of the phytochrome family). To this end, we compared the morphological development of wild-type and phyB-deficient mutant seedlings of cucumber in experimental mosaicsof light and shade and studied the influence ofphytochrome on morphological processes that defined the dynamicsof gap colonization.

MATERIALS AND METHODS Plant material Seeds of the long-hypocotyl (lh) mutant and thenear isogenic wild-type (WT) of Cucumis sativusL. (Adamse et al. 1987, Kendrick and Nagatani 1991) were obtained from the Department of Genetics (Wageningen Agricultural University, The Netherlands). The lh mutant lacksa PHYB polypeptide 121 (Lopez Juez et al. 1992) and shows reduced levels of spectrophotometrically-detectable phytochrome after de- etiolation (Adamse et al. 1988; Peters et al. 1991). phyB- deficient lh plants grown under white light of high red (R)

: far-red (FR) ratio have a phenotype that resembles that of WT plants grown under deep vegetational shade, where the low R:FR ratios maintain low levels of the active form of phytochrome (Pfr) at photoequilibrium. Thus, compared with the WT, lh plants have much longer hypocotyls and internodes, increased apical dominance and tendril formation (e.g. Lopez Juez et al. 1990), and higher shoot:root ratios (Casal J. J. and Ballare C. L., unpublished data). Mutant lh seedlings lack (or exhibit severely reduced) elongation responses to end-of-day (Adamse et al., 1988, Lopez Juez et al. 1990) and day-time (Ballare et al. 1991b, 1992a) R:FR treatments. The phyB deficiency severely reduces the elongation responses to natural shadelight andappears to eliminate elongation responses to B light mediated bya B- absorbing photoreceptor (Ballare et al. 1991b). Light-grown mutant seedlings retain phototropic (hypocotyl-bending) responses to B light, but lack phytochrome-mediated tropic responses to FR radiation (Ballare et al. 1992a).

Experimental procedures (1) Field experiment. The experiment was carried out in the fields of the Vegetable Research Farm, Department of Horticulture, Oregon State University, near Corvallis, OR. Maize seeds (Zea mays L. cv Early Jubilee)were sown in rows at a density of 100 plants m2 in a 3 x 9 m plot laid out in recently cultivated soil on 15 May, 1991. A 2-m widezone was delimited around the crop and kept free of weeds. Maize leaves that extended beyond the perimeter of the plotwere clipped periodically to produce a distinctcanopy / open- ground border. Weeds that emerged within the limits of the 122 maize crop were not controlled. The mostcommon species were Amaranthus retroflexus and Solanum nigrus.

Cucumber seeds were germinated in a growth chamber under white light provided by fluorescent tubes. Two-day old seedlings were transferred to a glasshouse, planted in10-cm pots containing a standard potting mixture andgrown for an additional 4 days under natural radiation supplemented with light from sodium vapor lamps (see Ballare et al.1991a). The pots were then transferred to the field, where the seedlings were grown for another 3 days before being transplanted into holes dug at the edges of the maizecrop. Differences in initial height between WT and lh seedlings were minimized by adjusting planting depth. Seedlings were well watered and fertilized with a standard 12:12:12NPK mixture. By the time of transplanting (July 1991),the maize crop was 50-cm tall, and reduced midday photosynthetically- active radiation (PAR) and R:FR ratio at groundlevel (measured with a LiCor 1800 spectroradiometer, Lincoln,NE) by 85 % and 59 %, respectively. One month after transplanting, the number of new nodes produced byWT and lh seedlings, their location in relation to the maizecanopy, and the height of the main-stem apex were recorded.

(2) Controlled-environment experiments. These experiments were carried out in a 2.5 x 2.5 x 2.5-m walk-in growthroom with controlled environment. Lightwas provided by a mixture of 110-W F96T12/CW/HO fluorescent tubes (Sylvania)and 300-W incandescent lamps (Photoperiod= 14 h day''; photosynthetic photon flux density at plant level= 160 gmol m'a s'; R:FR ratio = 2.24; day/night temperature= 22/16°C). Cucumber seeds were sown in 1-L pots at a depth ofca. 0.5 cm. The cotyledons began to emerge from soil 7 days after sowing; after seven more days, four metal rods (1-m long)were 123 driven into each pot in order to provide potential points of attachment for the cucumber tendrils. The morphological development of seedlings was followed over periods of different duration in conditions of uniform illuminationor in artificial light / shade-support mosaics. In this latter case, areas of reduced irradiance were created in the growth room by means of opaque screens that were suspended 80 cm above the pot rim level. Stray light in the shaded areaswas reduced by covering the walls of the chamber with black plastic sheets. A large number of rods were placed under the artificial canopies (shaded sectors) in order to simulate the presence of stems or culms that might serve assources of support for climbing plants like cucumber. The pots containing the seedlings were placed at the light / shade- support border. Variations of this general setup were used in individual experiments as explained below. In eachcase, cucumber plants (=pots) were assigned at random to the different treatments and were considered as replicates for statistical analysis. Results are givenas means ±SE except when otherwise is indicated.

RESULTS Morphology of WT and lh seedlings under uniform illumination Wild type and lh-mutant seedlings of cucumberwere grown in a controlled environment and their morphological development was followed over a period ofone month focusing on attributes that, a priori, were considered to be of consequence in processes of space colonization. The generation of main-stem nodes occurred ata similar rate in both genotypes (Fig. 5.1A) but, as observed in other studies (e.g. L6pez-Juez et al. 1990), internode elongationwas much faster in lh plants. This was reflected in large differences between genotypes in total plant height (Fig. 5.1B).In WT seedlings the first axillary brancheswere produced at the second main-stem node, about three weeks afteremergence 124 (Fig. 5.1C). The onset of branching was comparatively retarded in lh seedlings and, more remarkably, all the laterals produced remained undeveloped during the period of observations (Fig. 5.1D). Undeveloped lateral stems consisted of a short internode (length < 0.5 mm), a leaf primordium, and a very small apex. Lateral branches produced by WT seedlings elongated rapidly (Fig. 5.1D) and were almost horizontal (inclination with respect to vertical= 79.2 ± 3.5 degrees; n = 12; 39 days).

Mutant lh seedlings produced the first tendrils on the third main-stem node (Fig. 5.1E) and, by the third week of observations each plant had, on the average, 2 tendrils coiled around the vertical supports established at the perimeter of the pots (Fig. 5.1F). In comparison, WT plants started to produce tendrils later and at higher node numbers (Fig. 5.1E), which resulted in delayed attachment to the supporting rods (Fig. 5.1F).

None of the main-stems became recumbent during the period comprised by this set of observations, but we noticed that WT shoots tended to become horizontal as plants entered the reproductive phase (data not shown). Differences between WT and lh-mutant in the gravitropic response of their stems were therefore investigated using mature plants. The 12-th main-stem internode was fixed onto a horizontal plane and the inclination of successive internodes measured 48 h later. The substrate was covered with aluminum foil to create a nearly-isotropic light environment in the vicinity of the shoot tips. No potential sources of support for tendrils were provided in order to avoid possible effects of tendril attachment on stem orientation (see below). Shoots of mature WT plants maintained low deflection angles; in contrast, lh-mutant internodes rapidly tended toresume a vertical stance (Fig. 5.2). A similar diagravitropicre- 125 orientation of WT shoots was observed 96 h after placing mature internodes in a vertical, upside-down position (not shown). In summary, these observations showed that the lack of phyB results in increased stem elongation, reduced branch development, stimulated tendril production, and altered stem gravitropism in mature plants.

Morphological development of WT and lh seedlings ina patchy canopy environment Wild type and lh seedlings were grown in the field for 3 days and transplanted to the edges of a maize crop. After four weeks of growth, the number and spatial location of all new nodes generated by the seedlings were recorded. WT plants produced 8.9 (± 0.8) nodes compared with 6.9 (± 0.4) generated by lh plants. Eighty percent of the total population of nodes produced by WT plants were placed outside the maize canopy; in contrast, aboutone half of the nodes produced by lh plants were found within the crop (Fig. 5.3A). All WT plants had approximately horizontal stems after one month, and their apical buds were placed close to the ground surface (Fig. 5.3B). The direction of horizontal spreading was clearly non-random, since more than 80 % of WT plants had their apical bud outside the canopy (Fig. 5.3C). In contrast, only 5 % of the lh seedlings developed a diagravitropic stance. The main-stem apices of lh plants were at an average height of 24 cm and, most significantly, nearly all of them were positioned within the maizecrop (Fig. 5.3C). This experiment revealed that the lack ofa single photoreceptor (phyB) has a striking impacton the way in which cucumber seedlings position their growth points relative to the distribution of the lightresource in a patchy canopy. 126 Morphological development of WT and lh seedlings in artificial canopies Artificial canopies were created in a growthroom using neutral screens supported at a height of 80 cm bymeans of numerous vertical rods. This experimental setup mimicked the situation in dense natural canopies, where shading by overhanging leaves is normally associated with thepresence of large numbers of (vertically-oriented) stems, culmsor petioles that provide potential points of attachment for tendrils of climbing plants. WT and lh seedlingsgrown in individual pots were placed at the edges of these artificial canopies and the changes in plant morphology and shoot orientation were followed over time.

As they emerged from the soil, seedlings of both genotypes bent along the hypocotyls and projected their cotyledons toward the full-light sector (Table 5.1). Seedlings of the lh mutant initially invaded the artificial gaps more rapidly than the WTs (Fig. 5.4B). In fact, although the angles of hypocotyl curvaturewere very similar in both genotypes under our lighting conditions,hypocotyl elongation was much faster in lh than in WT seedlings(Table 5.1).

Wild type and lh seedlings produced main-stemnodes at a similar rate (Fig. 5.4A). By the third week, most lh seedlings had produced a tendrilon the second node (Fig. 5.4C). When tendrils were first visible, they appearedas tightly-packed coils at the axil of the youngest unfolding leaf. As tendrils uncoiled, their tipswere projected within a few hours ca. 7 cm ahead of the apical bud. Tendrils at that stage elongated very rapidly (2.4 + 0.3mm h4; n = 10) and nutated actively (not shown). The maximal lengthof functional tendrils recorded underour conditions was about 127

25 cm. At that point tendrils usually developedseveral free coils or buckles at the tip and ceasedto elongate. By the fourth week, two thirds of the lh plantshad at least one tendril coiled around the supporting rodsor around stems of neighboring plants (Fig. 5.4C). No tendrilshad been produced by WT seedlings. After the attachmentof the first tendrils of lh plants to the outerrow of vertical rods, there was a very rapid, backward movementof the terminal buds from the full-light to the shade-supportsectors (Fig. 5.4B). In turn, as the apical meristems movedcloser to the full-light / shade-support border,new tendrils contacted and coiled around more internal supports.At that point the direction of growth appeared to be primarilydictated by the spatial arrangement of potential supportingpoints. More than one half of the apical budswere in the shaded areas after 6 weeks of growth. WT plants projectedall new growth into the full-light areas throughout theexperiment (Fig. 5.4B). Their internodes were short (< 2cm)and fewer than 10 % of the plants produced tendrils after6 weeks (Fig. 5.4C).

Measurements of leaf orientation, takenon the fifth week, showed that, in both genotypes, the laminaswere inclined toward the full-lightarea (Fig. 5.5). The angle of leaf display showed remarkably little variabilityamong replicate leaves, and was completely independentof the spatial orientation of the main stem. Thatconstancy appeared to be a consequence of adjustment inthe angle between the stem and the petiole, not betweenthe petiole and laminae (not shown). Main stems ofWT plants were consistently inclined toward the artificialgaps. There was much more variation in stem angleamong lh plants, where the average angle was negative, indicating preferential inclination toward the shade-support sectorafter five weeks (Fig. 5.5). Thus, the lack of phyB had markedconsequences 128 on the distribution of nodes through the light / shade- support mosaic, but did not affect orientation of individual leaves.

The effects of tendrils on gap colonization We noticed that, after several weeks of growth, WT plants of the above experiment started to produce tendrils at the axil of every new leaf and some of the tendrils eventually became attached to the supports (not shown).Two additional experiments were carried out to investigatehow tendril production influences stem motility and foragingfor light in a patchy environment.

In the first experiment we tested whetheror not the ability to project stems into lightgaps is limited by the activity of thigmotropic tendrils. WT and lh plantswere grown to the 11-th node stage in the controlled environment. The ninth internode of six WT and six lh plantswas affixed with its long axis oriented vertically inan artificial light gap, 2 cm from the full-light / shade border.A row of vertical rods was installed along that border,but no sources of support were provided in the shaded sectors. The angular orientation of the internodes produced above the clamped stem section was followedover time (Fig. 5.6). WT plants projected all new nodes to the full-lightsector and the shoots tended to become horizontal with time (Fig.5.6). This was so in spite of the fact that WT plantsat that stage produced a tendril in every new node, andsome of the older tendrils reached and coiled around the supporting rods. Shoot of the lh mutant tended togrow upwardly and, by the end of the experiment, the total populationof nodes produced by the six plants was almost equally distributed between the full-light and shade sectors (Fig. 5.6).In some cases failure of early tendrils to attach to the supporting rods resulted in partial "lodging" of the shoot towardthe 129 shade or full-light areas (see second, fifth, and sixth plant in Fig. 5.6). However, the stems rapidly returned toa nearly vertical stance. Elongation rates of tendrils produced by WT and lh plants were similar (See Fig. 5.7A), and the maximum length reached by the tendrils before becoming non-functional was about 25 cm in both genotypes.

In a second experiment we tested whether the internodes modify their direction of elongation when their distal-node tendril becomes attached to supports. Plantswere grown in the controlled environment up to the 12-th node stage. The tenth internode was marked, laid horizontally ona flat surface and fixed with masking tape. The tendril produced at the distal node of internode number 12 was placed in contact with a small plastic rod and allowed to coil thigmotropically (See Fig. 5.7, upper diagram). Changes in tendril morphology and angular orientation of the internodes were followed over a period of 12 hours. The tendril section between the point of contact with the support and the node elongated very little during the experimental period (Fig. 5.7A). In addition, free coils or buckles were formed along the tendrils (Fig. 5.7B). Internodes of lh mutant plants bent along their axis toward the side of tendril attachment (Fig. 5.7C). This bending could not be detected inWT internodes. The basis of this difference betweenWT and lh internodes is not clear at present. Bending of lh internodes was most likely initiated by the pulling exerted by the coiling tendril. In fact, when tendrilswere attached to mobile supports no bending was observed (data notshown). However, it is clear that during the process of bending in lh there is a concomitant realignment of stem growth. This is demonstrated by the observation that the orientationof "curved" lh internodes changes very littleupon severing the attached tendril (Fig. 5.8). Irrespective of the mechanism, this experiment demonstrated that tendrilattachment can 130 actively alter the direction of growth of lh stems.

DISCUSSION This study showed that cucumber plants tend to selectively invade light gaps when growing ina patchy light environment, and revealed that the photoreceptor phyB hasa dramatic and multiple influence on the efficiency ofgap colonization. Our results have important implications for the understanding of mechanisms of foraging for light and the role of information acquired by photoreceptors in shaping plant architecture.

Mechanisms of foraging for light Wild type and phyB-deficient lh-mutant seedlingsof cucumber actively projected their main-stem apical buds toward artificial light gaps set up in the growthroom (Fig. 5.4B). The hypocotyls of both genotypes bent toward the light gaps soon after seedlingemergence (Table 5.1). This phototropic response has been characterized in severalplant systems (reviewed by lino 1990). In dicots, phototropism is triggered in the hypocotyls themselves, although differential illumination of the cotyledonsmay cause hypocotyl curvatures under some circumstances (Shuttleworth and Black 1977). Two photoreceptor systemsare involved in the detection of lateral light gradients establishedacross elongating organs: a B-light photoreceptor and phytochrome (lino 1990; Ballar6 et al. 1992a, and referencestherein). In the artificial full-light / shade gradient,we used neutral screens to modify total fluence rate without changing the R:FR ratio. Therefore, stem bendingresponses observed in this experiment (Table 5.1)can be attributed solely to the fluence rate gradient established betweenthe illuminated and shaded flanks of the hypocotyl, whichwas most likely perceived by the B-absorbing photoreceptor. Previous studies (Ballar6 et al. 1991b, 1992a)showed that 131 WT and lh seedlings have similar phototropic responses to B light under conditions of continuous illumination. In natural canopies, the changes in R and FR radiation imposed by neighboring individuals can trigger similar phototropic responses via phytochrome (Ballare et al. 1992a). We have previously demonstrated that hypocotyls of cucumber seedlings grown in the field tend to bend away from neighboring plants that absorb R light and back-reflect FR radiation, even if the lateral B-light gradient created by the presence of neighbors is experimentally eliminated. This bending response elicited by lateral R:FR gradients is absent in the phyB-deficient lh mutant (Ballare et al. 1992a). The lack of phytochrome-mediated phototropism might be therefore one of the reasons why the lh mutant was less efficient than the WT at positioning nodes outside the maize canopy in the field experiment (Fig. 5.3).

The observation that shoots of WT and lh mature plants have different responses to gravity (orthogravitropic in lh, diagravitropic in WT; Figs. 5.2 and 5.6) is broadly consistent with previous findings in other species showing that treatments that lower the Pfr level in plant tissue (e.g. high plantation densities or supplemental FR light) tend to produce more vertically-oriented shootsor leaves (Casal et al. 1990; Aphalo et al. 1991). We suggest that this regulation of gravitropism by Pfr levels playsan important role in the adjustment of plant morphology to patchy situations. Thus, orthogravitropic stems would confer an advantage to plants that compete for light in a crowded patch, but diagravitropic stems are likely to be farmore effective at invading the gaps that the shoots eventually find when moving through the canopy.

Phototropic and gravitropic responses would establish the initial direction of spreading in relation to the light 132 / shade mosaic. However, an important determinant of the ability to colonize light gaps is the extent to which the modules that eventually reach a high-light environmentare able to stay within the gap and take advantage of the increased PAR (Slade and Hutchings 1987, Hutchings and Mogie, 1990). Simple models of optimal plant architecture in heterogeneous media (Sutherland and Stillman 1988) predict that the production of short internodes and profuse branching in "good" sites would improve the overall efficiency of resource acquisition by the plant. This isthe morphological pattern that we observed in WT cucumbersgrown under full light in the controlled environment (Fig. 5.1,B, C, and D). In contrast, lh plants grown under unaltered white light produced extremely long hypocotyls and internodes and their axillary branches remained undeveloped (Fig. 5.1 B, C, and D). Acceleration of stem elongationand reduction of branching are typicalresponses of WT cucumbers to natural (Banat6 et al. 1991b) and simulated (Lopez-Juez et al. 1990) shadelight.

This and previous studies (Lopez-Juez et al. 1990) with the lh mutant have revealed that tendril production in cucumbers is under phytochrome control. WT plants respond with increased tendril production when exposed to light conditions that simulate aspects of vegetational shade(e.g. end-of-day FR pulses, Lopez-Juez et al. 1990). WT plants in our controlled-environment experiments (high R:FR ratio) produced the first tendril at node number 6, betweenone and one-and-a-half months after emergence (Figs. 5.1 and5.4). In contrast, lh-mutant seedlings started to tendril at node number 2 (Figs. 5.1 and 5.4), and plants became attachedto the rods installed in the shaded sectorssoon after emergence (Fig. 5.4C). By that time, stems of lh plantswere inclined toward the gaps (Table 5.1, and Fig. 5.4B),but attachment of successive tendrils to the rodswas 133 accompanied by a re-direction of main-stem growth. Since lh internodes elongated exceptionally fast (Fig. 5.1) and curved in response to the "pulling" exerted by attached tendrils (Fig. 5.7 and 5.8), the apical buds were rapidly projected back into the shaded areas (Fig. 5.4B).

These dynamic studies suggest that differences between WT and phyB-deficient lh plants in regard to the spatial placement of nodes in natural (Fig. 5.3) and artificial (Fig. 5.4) full-light / shade-support mosaicsare a function of differences in (i) internode elongation rate, (ii) rate of tendril production, (iii) gravitropic responses of stems and, probably, (iv) stem bending responses to mechanical stimuli. In the lh mutant, the orthogravitropic habit would limit exploration of the surroundings, tendrils would limit motility and cause stems to bend toward points of attachment, and, under these conditions, fast internode elongation would entail the risk of rapid penetration into shaded environments, where further tendrils would find abundant sources of support. We therefore conclude that phyB is responsible for detecting the occurrence ofgaps in the canopy and switching the plant from the "shade-avoiding" (default!) developmental program to the one that results in the short-internoded, more branched, and diagravitropic phenotype. In addition to these functions, phyB is the sensor of a phototropic system that would help WT plants to actively avoid crowded patches in natural canopies (Ballare et al. 1992a).

The above-mentioned response factors would determine the three-dimensional distribution of stems and nodes in the canopy space as a function of the spatial distribution of light gaps and potential points of attachment. However, in cucumbers, each individual leaf retains the capacity to orientate itself in relation to the local light climate, 134 independently of the orientation of the supporting internodes (Fig. 5.5). The similarities observed betweenWT and lh in regard to leaf display are consistent with the information derived from physiological experiments showing that the B-light region of the spectrum, perceived bya photoreceptor different from phytochrome, playsa central role determining the direction of leaf orientation (e.g. Koller 1990).

Role of information acquired by photoreceptors in shaping plant architecture The idea that plants "select" the direction of radial expansion on the basis of mechanisms that detect environmental heterogeneity has been suggested and discussed (e.g. Schellner et al. 1982, Billow-Olsen et al. 1984, Eriksson 1986, Schmid 1986, Novoplansky et al.1990, Bazzaz 1991). Yet, observed canopy asymmetries induced by the proximity of neighbors are commonly interpretedas products of different levels of resource availability experienced by different parts of an uniformly-expanding genet.Moreover, models of foraging plants (Sutherland and Stillman1988, 1990, de Kroon and Schieving 1991) do not consider the possibility that plants acquire informationon the spatial distribution of resources and "choose" to project their stems toward the most prospective sectors, although they often incorporate morphological responses of plant modules to the local light environment. The data usedas the basis for discussion of foraging processes commonly consistof maps showing that the spatial distribution of leaves, buds, or some other plant parts is non-random, and that modules tend to concentrate in "good" sites. With few exceptions (Billow -Olsen et al. 1984), observationson morphology and biomass distribution are mademany weeks or months after the beginning of the experiments, which provides little information on mechanisms and makes it difficultto 135 distinguish among simple growth responses to local stresses, local morphological adjustment, or foraging involving selection of the direction of spreading (see Eriksson 1986).

This study incorporated two key elements: timecourses of morphological development and comparison between genotypes that differ only in a morphogenic photoreceptor. Cucumber plants grown in a heterogeneous light environment were shown to selectively project their main stems toward the gaps (Table 5.1, and Fig. 5.4B). The ability to persist within the invaded high-light patches was found to be severely impaired in the phyB-deficient lh-mutants (Fig. 5.4B). Apart from the mechanisms that determine the overall placement of stem nodes in the light / shade mosaic,a precise regulation of leaf display in relation to the direction of illumination was found to be operative inboth genotypes (Fig. 5.5). According to these data,we suggest that the main effect of local differences of PAR availability on the spatial distribution of plant growth would be to exaggerate asymmetries thatare triggered by photomorphogenic systems that acquire information aboutthe radiation environment.

CONCLUSIONS Our experiments showed that the photoreceptor phyB substantially enhances the capacity of cucumber plantsto forage for light in horizontally-patchycanopy environments. From these and previous experiments it is suggested that this enhancement results froma combination of several regulatory actions of phyB on plant growth anddevelopment. The critical actions identified by the studies withcucumber are: 1) Establishment of a phototropic system thatresponds to lateral R:FR gradients created by asymmetric distributionof 136 neighbors around the plant (Ballare et al. 1992a). 2) Delay of tendril production under high light / high R:FR conditions, and reduction of stem bending responses to lateral forces created by attaching tendrils. These elements would combine to determine that the direction of growth is primarily dictated by the spatial distribution of lightgaps and not by the availability of support (which is likely to be more abundant in the vicinity of other plants) 3) Inhibition of stem elongation and promotion of axillary branching under high-light / high-R:FR conditions, which would favor occupation of high-light gaps. 4) Modulation of mature stem gravitropic response by the level of phyB Pfr, so that light conditions that establish relatively low Pfr levels (vegetational shadelight) result in orthogravitropic shoots and those that establish high Pfr concentrations result in diagravitropic stems. The diagravitropic habit, coupled with an efficient phototropic system (driven in part by phyB), would help plants to rapidly colonize light gaps and escape from shadedareas.

Acknowledgements. I am most grateful to Dr. Maarten Koornneef and members of the Department of Genetics (Wageningen Agricultural Univ.) for provision of the cucumber seeds. I also wish to thank Randy Hopson (Veg. Res. Farm, OSU) and Lloyd Graham (Forest Res. Lab., OSU) for practical assistance, and Gretchen Bracher for preparingthe figures. Financial support from the Consejo Nacional de Investigaciones Cientificas y Tecnicas (Argentina) and the Dept. of Forest Science (OSU) is gratefully acknowledged. 137

TABLE 5.1. Stem length andangle of inclinationtoward the artificial light gapsin WT and lh-mutantseedlings.*

Time after emergence genotype length angle (days) (cm) (degrees)t

4 WT 2.6+0.1 35.6+1.9 lh 7.4+0.6 38.9+1.8

17 WT 4.3+0.1 30.7+1.9 lh 22.5+0.1 27.0+3.4

*Data are means ±SE (n=14, WT or n=12, lh). "'Vertical = 0 degrees. 138 18 were or branch. (whenever one. E appearance plants controlled (WT) nodes and lh the 14 +SE oldest the and in of number C WT Main-stem node second panels before (days) of mean indicate as the in respectively. 6- _ 4- 2- 0' 8 6- 4- 2- the bars -E0, 0 is node on Emergence illumination symbols). produced 40 after brackets (3) development point the tendrils, (2) 30 Time vertical nodes or 20 uniform of determined of datum cotyledonary between 10 of plants; size were - C - Each number 12 10_ 8. 6. 6- the the given branches E.° E za-C 2 0 8 E4 co Morphological the S', -0 z 2ci5 C Tin x < than from lengths 2 5.1. conditions replicate figures axillary FIG. under environment. (1h) larger counted Branch The indicate of 139

WT 48 h

lh

FIG. 5.2. Gravitropicresponses of mature WT and lh plants under conditions of full light inthe controlled environment. The 12-th internodewas attached to a horizontal substrate (indicatedwith an X), and the angular orientation of three successiveinternodes was measured 48 h later. The segments delimitedby circles indicate the average angles of the internodes; therange is indicated by the arcs (n=7). 140

4

2

0

2

4

6

8

30 B

0- 80C w c;;- 60- C c7) Tac 0cl-40- -E. a"c20-

0 WT lh

FIG. 5.3. Spatial distribution of nodesproduced by WT and lh plants after four weeks of growthat the border of a maize canopy in the field. Theupper panel indicates the average number of nodes positioned insideor outside the canopy by WT and lh plants and includes nodesof the main stem (except the cotyledonary node) andaxillary branches if present. Thin bars represent +SE (n=20). 141 8-A o, p WT lh

12

10

8

6

4

2

0 -2 -4 -6 -8

10

Time after Emergence (days)

FIG. 5.4. Internode production, spatial distribution of main stems and tendril dynamics in WT and lh plantsgrown for 6 weeks at the border of simulated canopies ina controlled environment. Main-stem nodes were numbered fromthe cotyledonary node as node number one. Main-stem projection is the distance between the apical bud andthe base of the plant measured on a horizontal plane and perpendicularlyto the shade-support / full-light border (see diagram inthe upper-right corner). Positive values indicateprojection of apical buds toward the artificialgaps. Each datum point is the average of 12(WT) or 16 (lh) plants +SE. Plantswere considered to be "attached" when they had at leastone tendril coiled around the rods placed in theshade-support sector. 142

Shade-support Full-light

FIG. 5.5. Angular orientations of leaves and main-stem internodes of WT and lh plants grown for 5 weeks atthe border of artificial canopies in the controlled environment. Angles were measured on a vertical plane perpendicular to the shade-support / full-light border and correspond tothe third youngest leaf of each plant and the internodebelow its point of insertion; arcs indicate +SE (n=13).The angles and lengths of petioles Were not recorded; the dashedlines were drawn to clarify presentation assuming that the angle between lamina and petiole was 110 degrees. 143

is

N

ca 0-

U) ca 0-

co is 0-

0 1 2 3 4 5 6 7

Time (days)

FIG. 5.6. Time courses of artificial gap invasion by six WT (0) and six lh () shoots in the controlled environment. The gray-tone and white sectors in the figure indicate the shaded and full-light areas in the growth room, respectively, and the dashed line denotes the row of vertical rods established at the border of the gaps. The ninth internode of each shoot was oriented vertically and clamped 2 cm toward the gaps on day -1. The segments delimited by circles indicate the angular orientation of the successive internodes (from 10 to 14) produced by the plants. Angles were measured on a vertical plane perpendicular to the shade / full-light border. 144

Free coils = t Bending angle = a A distance = (rfo - n o)

A B lh Zr) 1.0- ,c) WT t E 2 . 0) 0" 0.5- a) 22 u.

0

c 12C 1 T m 0 ^ v u 0 F 6 < m c 3 15c 0 m 0 -3

WT Ih WT Ih

FIG. 5.7. Effects of tendril attachment to the supports on tendril morphology and internode orientation. The upper diagram shows a top view of the experimental setup and the meaning of the different variables measured. The duration of the experiments, from the beginning of tendril coiling (dashed lines), to the final set of measurements (solid lines) was 12 h. Tendril elongation rates given in panel A were calculated by measuring the length of the tendril segment between the node and the point of contact with the support. The lines parallel to the abscissa indicate elongation rates of tendrils before attachment to the supports. Positive bending angles (panel B) and negative Adistances (panel C) indicate that the internodes curved toward the support after tendril attachment. Each value is the average of 22 (bending angles) or 15 observations +SE. 145

100

FIG. 5.8. Changes of internode curvature after severing the attached tendril in lh plants. The setup sketched in Fig. 5.7 was used. Twelve hours after tendril attachment, the internode bending angle was measured (a=+12.3 ±2.4; n=21) and the curvature remaining after cutting the tendrils was measured again at the indicated times. 146 REFERENCES Adamse, P., P. A. M. P. Jaspers, J. A. 1988. R. E. Kendrick, and M. Koornneef. 1988. Photophysiology and phytochrome content of long-hypocotyl mutant and wild-type cucumber seedlings. Plant Physiology 87:264-268. Adamse, P., P. A. M. P. Jaspers, R. E. Kendrick, and M. Koornneef. 1987. Photomorphogenic responses of a long hypocotyl mutant of Cucumis sativus L. Journal of Plant Physiology 127:481-491. Aphalo, P. J., D. J. Gibson, and A. H. DiBenedetto. 1991. Responses of growth, photosynthesis, and leaf conductance to white light irradiance and end-of-day red and far-red pulses in Fuschsia magellanica Lam. New Phytologist 117:461-471. Ballare, C. L., P. W. Barnes, and R. E. Kendrick. 1991a. Photomorphogenic effects of UV-B radiation on hypocotyl elongation in wild-type and stable-phytochrome- deficient mutant seedlings of cucumber. Physiologia Plantarum 83:652-658. Ballare, C. L., J. J. Casal, and R. E. Kendrick. 1991b. Responses of wild-type and lh-mutant seedlings of cucumber to natural and simulated shadelight. Photochemistry and Photobiology, 54:819-826. Ballare, C. L., R. A. Sanchez, A. L. Scopel, J. J. Casal, and C. M. Ghersa. 1987. Early detection of neighbour plants by phytochrome perception of spectral changes in reflected sunlight. Plant Cell and Environment 10:551- 557. Banat-6, C. L., R. A. Sanchez, A. L. Scopel, and C. M. Ghersa. 1988. Morphological responses of Datura ferox L. seedlings to the presence of neighbors. Their relationships with canopy microclimate. Oecologia (Berlin) 76:288-293. Ballare, C. L., A. L. Scopel, S. R. Radosevich, and R. E. Kendrick. 1992a. Phytochrome mediated phototropism in 147 de-etiolated seedlings. Occurrence and ecological significance. Plant Physiology, in press. Ballard, C. L., A. L. Scopel, and R. A. Sanchez. 1990. Far- red radiation reflected from adjacent leaves: an early signal of competition in plant canopies. Science 247:329-332. Ballard, C. L., A. L. Scopel, and R. A. Sanchez. 1991c. Photocontrol of stem elongation in plant neighbourhoods: effects of photon fluence rate under natural conditions of radiation. Plant Cell and Environment 14:57-65. Ballard, C. L., A. L. Scopel, R. A. Sanchez, and S. R. Radosevich. 1992b. Photomorphogenic processes in the agricultural environment. Photochemistry and Photobiology, in press. Bazzaz, F. A. 1991. Habitat selection in plants. American Naturalist 131: S116-S126. Bulow-Olsen, A., N. R. Sackville Hamilton, and M. J. Hutchings. 1984. A study of the growth of Trifolium repens L. as affected by intra- and interplant contacts. Oecologia (Berlin) 61:383-387. Casal, J. J., Sanchez, R. A., and D. Gibson. 1990. The significance of changes in the red/far-red ratio, associated with either neighbour plants or twilight, for tillering in Lolium multiflorum Lam. New Phytologist 116:565-572. de Kroon, H. and F. Schieving (1991) Resource allocation patterns as a function of clonal morphology: A general model applied to a foraging clonal plant. Journal of Ecology 79:519-530. Eriksson, 0. 1986. Mobility and space capture in the stoloniferous plant Potentilla anserina. Oikos 46:82- 87. Grime, J. P. 1979. Plant strategies and vegetation processes. John Wiley and Sons, New York, New York, 148

USA. Hutchings, M. J., and M. Mogie. 1990. The spatial structure of clonal plants: Control and consequences. Pages 57-76 in I. van Groenendael and H. de Kroon, editors. Clonal growth in plants: regulation and function. SPB Academic Publishing, The Hague, The Netherlands. Hutchings, M. J., and A. J. Slade. 1988. Morphological plasticity, foraging and integration in clonal perennial herbs. Pages 83-109 in A. J. Davy, M. J. Hutchings, and A. R. Watkinson, editors. Plant population ecology. Blackwell Scientific Publications, Oxford, Great Britain. lino, M. 1990. Phototropism: mechanisms and ecological implications. Plant Cell and Environment 13:633-650. Kendrick, R. E. and A. Nagatani 1991 Phytochrome mutants. Plant Journal 1:133-139. Koller, D. 1990. Light-driven leaf movements. Plant Cell and Environment 13:615-632. Lopez-Juez, E., W. F. Buurmeijer, G. H. Heeringa, R. E. Kendrick, and J. C. Wesselius. 1990. Response of light- grown wild-type and long-hypocotyl mutant cucumber plants to end-of-day far-red light. Photochemistry and Photobiology 58:143-150. Lopez-Juez, E., A. Nagatani, K.-I. Tomizawa, M. Deak, R. Kern, R. E. Kendrick, and M. Furuya. 1992. The cucumber long-hypocotyl mutant lacks a light-stable PHYB-like phytochrome. Plant Cell, 4:241-251. Novoplansky, A., D. Cohen, and T. Sachs. 1990. How portulaca seedlings avoid their neighbors. Oecologia (Berlin) 82:490-493. Peflalosa, J. 1983. Shoot dynamics and adaptive morphology of Ipomoea phillomega (Nell.) House (Convolvulaceae), a tropical rainforest liana. Annals of Botany 52:737-754. Peters, J. L., R. E. Kendrick, and H. Mohr. 1991. Phytochrome content and hypocotyl growth of long- 149 hypocotyl and wild type cucumber seedlings during de- etiolation. Journal of Plant Physiology 137:291-296. Quail, P. H. 1991. Phytochrome: A light-activated molecular switch that regulates plant gene expression. Annual Review of Genetics 25:389-249. Schmid, B. 1986. Spatial dynamics and integration within clones of grassland perennials with different growth forms. Proceedings of the Royal Society of London B228: 173-186. Schellner, R. A., S. J. Newell, and 0. T. Solbrig. 1982. Studies on the population biology of the genus Viola. IV. Spatial pattern of ramets and seedlings in three stoloniferous species. Journal of Ecology 70:273-290. Shuttleworth, J. E., and M. Black. 1977. The role of cotyledons in phototropism of de-etiolated seedlings. Planta 135:51-55. Slade, A. J., and M. J. Hutchings. 1987. The effect of light intensity on foraging in the clonal herb Glenchoma hederacea. Journal of Ecology 75:639-650. Smith, H. 1986. The perception of light quality. Pages 187- 217 in R. E. Kendrick and G. H. M. Kronenberg, editors. Photomorphogenesis in plants. Martinus Nijhoff, Dordrecht, The Netherlands. Smith, H. and G. C. Whitelam. 1990. Phytochrome,a family of photoreceptors with multiple physiological roles. Plant, Cell and Environment 13:695-706. Sutherland, W. J., and R. A. Stillman. 1988. The foraging tactics of plants. Oikos 52:239-244. Sutherland, W. J., and R. A. Stillman. 1990. Clonal growth: Insights from models. Pages 95-111 in I. van Groenendael and H. de Kroon, editors. Clonal growth in plants: regulation and function. SPB Academic Publishing, The Hague, The Netherlands. 150

CHAPTER 6

PHOTOMORPHOGENIC PROCESSES IN THEAGRICULTURAL ENVIRONMENT

Carlos L. Bellaire*, Ana L.Scopel, Rodolfo R. Sanchezand Steven R. Radosevich

Depts of Forest Science andCrop and Soil Science,Oregon State Univ., Peavy Hall 154,Corvallis, OR 97331-5705, USA (C. L. B., A. L. S., S. R.R), Dept de Ecologia, Facultadde Agronomla, Univ. of BuenosAires, (1417) Buenos Aires, Argentina (C. L. B., A. L. S., R.A. S.)

Photochemistry and Photobiolgy,in press (1992). 151

ABSTRACT'S Studies under natural lightconditions are revealing how photomorphogenic mechanismsinfluence processes of significance for agriculture. In this paper wereview recent advances in three areas: photocontrolof weed seed germination, acclimation to solar UV-Bradiation, and plant interactions in canopies.

In the area of the photocontrolof germination there is increasing interest for understandingthe role of phytochrome in the mechanisms wherebysoil tillages trigger weed seed germination. It hasbeen demonstrated recently that seeds of some weed speciesacquire the ability to respond to very low photon fluencesduring burial. Field studies showed that germination ofsensitized seeds can be triggered by millisecond-exposures tosunlight, and there are indications thatshort light pulses of this nature may be important light signals ofsoil disturbance.

In the area of UV-Bacclimation, there is increasing evidence from action spectroscopyindicating that a specific UV-B photoreceptor participatesin the control of flavonoid accumulation in the leaf epidermis. Recentstudies provided experimental support for the notion thatUV-B-induced flavonoids play an important roleprotecting the photosynthetic system against damage by UV-Bradiation. Some effects of UV-B on plant morphologyhave also been interpreted as photomorphogenic responsesmediated by an UV- B receptor.

In the area of plantinteractions in canopies, progress has been made studying the rolesof phytochrome and blue / UV-A sensors under naturalconditions. It has been shown that, by perceiving lightsignals through these two photoreceptors, plants obtaininformation about the 152 characteristics of the surroundingvegetation well before they experience reductionsin the availability of resources as a consequence ofneighbors' activities.

There appears to be a largepotential for improving existing cropping systems bymanipulating light environment or by deliberatelychanging the photomorphogenicbehavior of crop plants. However,important gaps still exist in our understanding of how the expressionand agronomic significance of photomorphogenic responses areinfluenced by other factors of the plant'senvironment.

tThis Abstract has beenrewritten after the paper was accepted by Photochemistry andPhotobiology and is more detailed than the published version. 153 INTRODUCTION In this paper we review some recent advances in our understanding of how photomorphogenic mechanisms operate under natural conditions and influence processes that are important in the agricultural environment. Many significant developments have occurred over the last few years in such disparate areas as grassland ecology and transgenic plant technology. Our strategy is to focus on areas where a substantial proportion of the information has been derived from experiments carried out under conditions of natural radiation, and where recent developments (i) imply a qualitative change in the way an important agronomic issue is addressed, and (ii), are likely to influence applied agricultural research in the near future. The three areas we discuss are: 1) photocontrol of weed seed germination in arable land, 2) plant responses to solar UV-B radiation, and 3) photomorphogenic signals in processes of plant competition in crop canopies.

Abbreviations: B, blue; FR, far-red; LAI, leaf area index; LD, long day; LF, low fluence; P, total phytochrome; PAR, photosynthetically active radiation (400-700 nm); Pfr, FR- absorbing form of phytochrome; PhyA, phytochrome-A gene; PHYA andPHYB, phytochrome-A and -B polypeptides; R, red; VLF, very low fluence.

PHOTOCONTROL OF WEED SEED GERMINATION IN ARABLE LAND Soil cultivation in arable fields is typically followed by pulses of weed seedling emergence (e.g. Chancellor 1964; Banat-6 et al. 1988a). Moreover, seeds of some weed species or ecotypes appear to require periodic disturbance of the soil to lose primary dormancy and germinate (e.g. Soriano et al. 1970). Sauer and Struik (1964) and Wesson and Wareing 154

(1969) first established a connection between light signals and germination flushes in disturbed soil. Since then, work in different laboratories has focused on such aspects as the penetration of light through soil (reviewed by Tester and Morris 1987), variations in light requirements for germination during burial (see references in Karssen 1982), and interactions between light and other environmental factors (e.g. Egley 1989). Most experimentation in the last two areas has been carried out on unearthed seed samples that were subjected to standard germination tests under controlled conditions. Only a small number of light treatments were usually compared in the experiments (e.g. darkness vs. one or two broad-band sources at one fluence). Yet, an important generalization that can be derived from such studies is that the light responses of weed seeds may change dramatically over relatively short periods of time (i.e. weeks). In a number of cases (Taylorson 1972; Baskin and Baskin 1980, 1985), and for reasons that are not clear even at the simplest physiological level, the light requirement for germination appears to fluctuate in a seasonal fashion during burial.

Most laboratory studies on phytochrome control of germination have been carried out with seeds that displayed red (R)+-far-red (FR) reversible photoresponses (i.e. low fluence [LF] responses). In recent years, effects of light on germination that are not reversible by FR have received attention. Using suitable pretreatments (e.g. chilling, giberellic acid, ethanol), the sensitivity of seeds of some species to Pfr can be increased several orders of magnitude (VanDerWoude and Toole 1980; VanDerWoude 1985; Cone et al. 1985; Rethy et al. 1987; Taylorson and Dinola 1989). Germination of sensitized seeds can be triggered by light treatments that establish < 0.01 % Pfr. Because a small 155 number of light quanta are required to obtain such low levels of Pfr, this photoresponse has been termed very-low- fluence (VLF) response. Although VLF responses can be readily demonstrated in the laboratory, the ecological significance of the very high light sensitivity may be not immediately obvious (Smith and Whitelam 1990).

Bouwmeester and Karssen (1989) showed that the expression of the dormancy status of seeds retrieved from soil can be substantially influenced by pretreatments (e.g. drying of the seed sample) and incubation conditions during the germination tests. Therefore, little of the information derived from standard tests under laboratory conditions can be directly used to establish the nature of the light signals that trigger weed seed germination in the field. Indeed, we still know relatively little about how important light signals are in relation to other environmental variables that are also affected by tillage (see also Woolley and Stoller 1978). The frequently held view that light is the ultimate signal for germination induction in light-requiring seeds should not be accepted without scrutiny. In Datura ferox, where a light requirement for germination has been well established, formation of Pfr must be followed by a period of alternating temperatures in order to obtain full expression of the germination response. Pfr can be stored for several hours or days until the seeds are exposed to temperature fluctuations (Casal et al. 1991). In turn, tillages can profoundly affect the thermal environment sensed by seeds by affecting the amount of residues left on the ground surface and the vertical distribution of the seed population in the soil profile.

Some recent studies have specifically focused on the interphase between photomorphogenesis and seed ecology in arable land. In a series of laboratory experiments, Scopel 156 et al. (1991) studied the germination responses of seeds of D. ferox to R and FR light pulses that established different calculated percentages of Pfr. Seeds that had been kept in dry storage in the laboratory only germinated when the Pfr level established by the light treatments was above 2 % and the germination-%Pfr response function resembled those obtained for typical LF responses. Similar responses are usually found in freshly collected seeds of this species (Casal et al. 1991). In contrast, most of the seed population retrieved from soil could be induced to germinate by pulses that provided < 0.01 % Pfr, and the response curve was clearly biphasic. This increase in lightsensitivity was interpreted as a natural transition from the LF response to the VLF response. A period of cold temperature did not appear to be required for the increase in light sensitivity in D. ferox. In a related field experiment, sunlight pulses were given to seed samples that were incubated in thesoil at 7 cm depth after exposures to sunlight. The goal was to mimic the effect of soil cultivation. The major findings were: (1) exposure to light was essential totrigger germination in the soil,(2) most of the seeds could be induced to germinate by light pulses equivalent to 10 ms of full sunlight, which would establish < 0.01 %Pfr (temperature fluctuations were not limiting in this experiment), and (3) less than 10 % of the effect of saturating exposures to sunlight could be reversed by subsequent FR. These results show that VLF responses can be demonstrated in the field. Other observations of high light sensitivity in exhumed seeds have been reported (Taylorson 1972; Baskin and Baskin 1979), which suggests that germination responses to VLFs are probably very frequent among seeds that have spent some time in the soil.

Scopel et al.(1991) have suggested that the natural switch to the VLF state may be essential for detection by 157 seeds of very short exposures to sunlight while the soil is being disturbed through tillage. How important these short pulses are in relation to other light signals is not clear at present. Scopel et al. (1991) hypothesized that such factors as desiccation, predation, and photoinhibition may reduce the contribution of seeds left at the soil surface to the flush of germination. It should be noted, however, that seeds buried a few mm might be protected from some of these hazards and could integrate a weak light signal (Tester and Morris 1987; Mandoli et al. 1990) over several photoperiods after tillage, which might yield enough Pfr to trigger germination in a population of sensitized seeds. In principle, this possibility is not unreasonable since, in Datura ferox at least, promotion of seed germination by light is mediated by a stable type of phytochrome (Casal et al. 1991; see also Lercari and Lipucci di Paola 1991). The interesting observations of Hartmann and Nezadal (1990) indirectly support the suggestion that light pulses received during the actual episode of disturbance are key signals for germination. They found that weed populations declined dramatically in experimental plots where all soil disturbing operations were conducted during the night. The effect of artificial lighting during the night tillage was not tested. Given the increased concern about massive herbicide inputs and the need to find alternative methods of weed control (e.g. Frisbie and Smith 1990), the issue has substantial practical interest and warrants further attention.

PLANT RESPONSES TO SOLAR UV-B Concern about the impact of high levels of solar UV-B radiation on natural and cultivated plant communities has increased after recent indications of a global reduction of the stratospheric ozone layer (Kerr 1988, 1991; Solomon 1990) and a trend of increasing surface UV-B irradiances in some regions (Lubin and Frederick 1989; Blumthaler and 158

Ambach 1990). Tevini and Teramura (1989) andCaldwell et al. (1989) have reviewed most of the currentliterature on ecophysiological effects of UV-B on higher plants. Wewill briefly discuss recent work on pigmentation and morphological responses to UV-B and its agricultural significance. Light induction of photoreactivating enzymes will not be covered here (See Langer and Wellmann1990; Pang and Hays 1991).

Light promotes the accumulation of anthocyanins and flavonoids in many plant systems. Visible light issometimes effective (acting through phytochrome and blue / UV-A receptors) but, in many cases, exposure to UVradiation is required for flavonoid induction (See Beggs et al. 1986a,b). Action spectra for UV-induced synthesis of anthocyaninsand flavonoids are characterized by maximum quantum effectiveness in the UV-B region (i.e. 290 - 300 nm; reviewed by Wellmann 1983; Beggs et al. 1986b; Hashimoto 1990). In contrast, action spectra for damagingeffects of UV radiation in plant systems, such asinhibition of photosynthesis in intact leaves (Caldwell et al. 1986), inhibition of photosystem II activity in thylakoid suspensions (Bornman et al. 1984) and inhibition of R- or R+UV-B-induced anthocyanin synthesis (Wellmann et al. 1984; Hashimoto et al. 1991) show greater photon effectiveness at shorter wavelengths. This observation has lent tothe suggestion that the effect of UV-B on flavonoid synthesisis mediated by a specific photoreceptor (Beggs et al. 1986b; Hashimoto 1990). Apparently complex interactions between UV- B and light absorbed by other photoreceptorshave been reported and Beggs et al. (1986b) concluded thatin most systems it would seem that the presence of Pfris at least necessary for full expression to the responseto UV-B.

Several enzymes of the flavonoid synthetic pathway are 159 induced by light and in some cases it has been possible to relate the increases in enzyme activity with increases in the rates of transcription of the relevant genes (Refs. in Beggs et al. 1986b and Hahlbrock and Sheel 1989). Rapid effects of light on the activity of phenylalanine ammonia- lyase (PAL), an enzyme of the general phenylpropanoid pathway, mediated by isomerization of the PAL inhibitor trans-cinnamic acid has also been reported (Tevini and Teramura 1989). Work with mustard (Sinapis alba) cotyledons has indicated that mechanisms regulated by phytochrome beyond enzyme induction also influence the rate of accumulation of some products of the flavonoid pathway (Brodenfeldt and Mohr 1988). Considerable progress has been made over the last few years in elucidating the molecular mechanisms that mediate the effects of light and other environmental factors on the expression of genes encoding enzymes of the flavonoid pathway (See Hahlbrock and Sheel 1989; Fritze et al. 1991 and references therein).

Flavonoids absorb effectively in the UV, show little absorption in the visible region and accumulate in the vacuoles of epidermal cells. In parsley (Petroselinum crispum) leaves the whole sequence of light-triggered biosynthetic events leading to flavonoid production have been localized in the epidermis (Schmelzer et al. 1988). These observations are consistent with the view that flavonoids play an important role as natural UV filters, protecting plant tissues from deleterious effects of UV-B radiation (Caldwell et al. 1983). Under natural conditions the fluxes of UV-B and visible radiation are poorly correlated (Caldwell 1971). Therefore, the involvement of a specific UV-B photoreceptor in the control of flavonoid accumulation might be adaptive, as this sensor would provide information on the UV-B environment that plants can not derive from light signals sensed by other photomorphogenic 160 pigments. Indirect evidence supporting an important role of flavonoids in acclimation to high UV-B levels has been obtained by examining the natural variation in leaf epidermal transmittance along geographic gradients that are associated with large changes in solar UV-B irradiance. Robberecht et al. (1980) found that in plants from low- latitude alpine environments (high UV-B), UV epidermal transmittance was always very low (<2 %). In contrast, at high and medium latitudes (lower UV-B) there was a larger variability in epidermal transmittance among species. Plants exposed to experimentally enhanced UV-B levels under field conditions often respond with increased flavonoid accumulation and decreased UV-epidermal transmittance (Flint et al. 1985). More direct tests of the protective role of flavonoids have recently been reported. Tevini et al. (1991) used variable chlorophyll fluorescence to measure the effects of UV-B on photosynthetic activity in rye (Secale cereale) primary leaves that had accumulated different amounts of UV-B absorbing compounds in the epidermis. They found that short white-light + UV-B pretreatments, which stimulated flavonoid accumulation and decreased UV transmittance of the epidermis, reduced the negative impact of short-wave UV-B treatments on the estimated quantum yield and photosynthetic capacity. A similar approach had been previously used by Beggs et al. (1986a) to evaluate the role of flavonoid pigments in protecting parsley roots against UV-B damage. The UV-B fluences required to produce a significant accumulation of flavonoids in the rye system were roughly similar to those received at the Earth's surface during a clear summer day at mid latitudes. However, as the authors acknowledge, a full assessment of the ecological significance of their findings requires more study. In part this is because the spectral distributions of artificial and solar UV-B are not identical, which makes it necessary to use some type of "weighing" function for 161 comparisons (see Caldwell et al. 1986). In addition, other factors may influence the degree of protection achieved by leaves under field conditions. For instance, increases in leaf thickness in response to high levels of photosynthetically active radiation (PAR) (Warner and Caldwell 1983) and UV-B (Cen and Bornman 1990; Bornman and Vogelmann 1991) may afford some additional protection against UV-B.

Besides influencing pigment synthesis, UV-B may induce a number of changes in plant morphology. Attempts to unravel the underlying mechanisms are typically complicated by the fact that, particularly in controlled-environment experiments with low PAR, morphological responses to UV-B are usually accompanied by reductions in photosynthesis and total growth (Tevini and Teramura 1989). However, a number of effects of UV-B on morphology are difficult to interpret as mere by-products of photosynthetic inhibition. Sisson and Caldwell (1976) found that UV-B inhibits leaf elongation in Rumex patientia, and the effect is not correlated with changes in photosynthesis. Recent field and glasshouse experiments have shown that UV-B reduces shoot (leaf) lengthening in grasses without affecting total growth (Barnes et al. 1988, 1990) or photosynthesis (Beyschlag et al. 1988). UV-B also inhibits axis elongation in etiolated seedlings (Steinmetz and Wellmann 1986). In de-etiolated cucumbers (Cucumis sativus) growing in a glasshouse, 8-h exposures to supplemental UV-B inhibit hypocotyl elongation up to 50 % without affecting growth rate or cotyledon area expansion (Ballare et al. 1991a). Morphological effects of UV-B may be the consequence of disturbing effects of UV-B on cellular functions other than photosynthesis (e.g. damage to meristems or elongating cells). In fact, the action spectrum for the inhibition of hypocotyl elongation in Lepidium sativus resembles those obtained for DNA damage (Steinmetz 162 and Wellmann 1986). In cucumbers the inhibitory effect of UV-B on elongation is triggered mainly in the cotyledons; therefore, direct damage to hypocotyl or apical cells is obviously not involved in the response (Ballare et al. 1991a). Barnes et al. (1990) have shown that moderate levels of UV-B applied to glasshouse-growing plants stimulate tillering in some grass species. These lines of evidence suggest that some responses to UV-B radiation should be regarded as true photomorphogenic effects. Recent field studies (Barnes et al. 1988) and simulations using a multi- species canopy model (Ryel et al. 1990) suggest that differential morphological responses to UV-B are important in determining the balance of competition between species in cultivated plant associations and should be taken into account when trying to predict the consequences of stratospheric ozone depletion at the plant community level.

PHOTOMORPHOGENIC SIGNALS IN PROCESSES OF PLANT COMPETITION The idea that neighboring plants in a cultivated plant association compete with each other for access to environmental resources is familiar. Yet, the factors that determine variations in competitive success among plant individuals are often hotly discussed among ecologists and agronomists. Recent studies highlight the potential significance of information acquired by plant photoreceptors in the processes of competition for light. We will review some of the major findings and discuss them in relation to their potential significance for crop improvement.

R:FR signals and phytochrome Spectroradiometric studies of the natural light environment (e.g. Kasperbauer 1971; Holmes and Smith 1977) and physiological experiments with light-grown plants (e.g. Morgan and Smith 1978) have demonstrated that a number of typical morphological reactions to shadelight (increased 163 stem elongation, reduced leaf to stem dry-weight ratio) can be triggered by phytochrome in response to the low R:FR ratios that prevail under leaf canopies (See reviews by Smith 1982, 1986). Results of field- and glasshouse- experiments with light-grown seedlings of the lh mutant of cucumber, which is deficient in a PHYB polypeptide (Lopez- Juez et al. 1992), are fully consistent with the notion that this phytochrome species plays a fundamental role in elongation responses to shadelight (Ballare et al. 1991b).

It has been shown recently that, in even canopies (i.e. canopies formed by plants of similar height), several morphological responses to the proximity of other plant individuals, such as reduced tillering rate in grasses (Casal et al. 1986) and increased stem elongation rate in dicotyledonous seedlings (Ballare et al. 1987, 1988b), can be detected well before the onset of severe mutual shading among neighbors. This observation led Ballare et al. (1987) to propose that the FR radiation reflected by nearby leaves may work as an early signal of oncoming competition during canopy development.

Plant tissues absorb very little in the FR region, and most of the photons in this waveband exit plant organs as scattered radiation. Kasperbauer et al. (1984) found that the R:FR ratio measured with cosine-corrected light receivers at the outer strata of soybean canopies varied between the east and west side of north-south oriented rows. The differences were attributed to variations in the contribution of FR reflected by adjacent plants. Using cuvettes containing purified phytochrome, Smith et al. (1990) found that the Pfr/P ratio at photoequilibrium decreases progressively as the cuvettes are moved closer to sunlit plant fences. Measurements obtained in even canopies of dicotyledonous seedlings of very low density (leaf area 164 index [LAI] < 1), where mutual shading amongneighbors is negligible, indicated that the proximity of plant individuals causes a marked increase in the amountof FR received at the surface of (vertically oriented)stems (Ballare et al. 1987, 1991c). Fiber opticstudies showed that this increase in FR receipt is paralleledby a detectable increase in the amount of FR scatteredinside the stems, a concomitant drop of the R:FRratio, and a reduction in the estimated phytochrome photoequilibriumwhich are quantitatively related with the LAI of the population (Ballare et al. 1989, 1991c).

Several experiments in which mirrors and plant hedges of different colors were used to provide additional FR to plants growing in the field demonstrated that stem elongation can be promoted by spectral changes thatmimic the proximity of other plants, even if theseedlings are growing under full sunlight (e.g. Ballare et al. 1987, 1989). Comparable results have been obtainedin studies of other morphological responses (e.g. Casal et al.1987; Novoplansky et al. 1990; Davies and Simmons 1991).Ballare et al. (1990) provided direct evidence for theinvolvement of R:FR ratio by showing that, in seedlingcanopies of low LAI, the typical stem elongation responses toincreased population density can be reduced or abolished bycovering individual stem sections with FR-absorbing filters. Phototropic responses elicited by non-shading neighbors and mediated by phytochrome have also been demonstrated (Ballare et al. 1992).

Fluence-rate signals Recent work under natural conditions ofradiation has shown that, in even plant canopies, there are at leasttwo more photomorphogenic factors thatinfluence stem elongation apart from the change in R:FR ratio. First, when the canopy 165 begins to close, there is a rapid drop in blue (B) light irradiance at the stem level. Simulation experiments have shown that local depressions of B light trigger an acceleration of stem elongation rate in plants growing under sunlight. This effect is not mediated by phytochrome or photosynthetic pigments and, most likely, involves the action of a specific B light photoreceptor (Ballare et al. 1991c). Second, the drop in R and FR irradiances that occurs under similar conditions may stimulate elongation independently of changes in R:FR ratio. An inverse relationship between R+FR irradiance received by the stem and elongation rate has been documented in sunlight-grown mustard and Datura ferox seedlings (Ballare et al. 1991c). Responses to R:FR ratio and irradiance might be mediated by different phytochrome species. Transgenic tobacco plants that overexpress an oat phyA gene, and maintain abnormally high levels of oat PHYA polypeptide when grown in the light, are dwarf and respond to additional FR with further reduction in stem elongation (McCormac et al. 1991).

An important conclusion derived from spectroradiometer studies in even canopies of dicotyledonous seedlings is that, at low LAIs, increases in plant density can bring about significant spectral and fluence-rate changes at the level of the stem with minimal effects on the light climate of the leaves (Ballare et al. 1989, 1991c; see Mancinelli (1991] for a discussion of the influence of plant geometry on light perception). In these conditions, therefore, there is a clear separation between the flux of photomorphogenic information received by the plant and the effects of neighbors on the flux of photosynthetic light energy. Collectively, these results provide a mechanistic explanation for the early morphological responses to canopy density that are observed in even-aged populations of seedlings. 166

Implications for crop improvement Going back to the starting point of this discussion, the role of photoreceptors in plant competition, these studies provide clear evidence for the notion that plant growth and morphology in crop canopies is affected by neighboring individuals not only because they directly affect the availability of resources that are necessary for growth but also because they modify aspects of the light environment that are used by plants to make developmental "decisions". Thus, in even plant stands, the R:FR ratio and fluence-rate perceived by the stems convey information on the proximity of neighbors that each individual plant uses to make decisions about morphology and assimilate allocation as the canopy develops. Sincemorphological changes elicited by these signals (e.g. increased stem elongation rate) may have a major impact on the resource-harvesting capacity of the plant (e.g. Ballard et al. 1988b), it may be anticipated that variations in the ability to gather and interpret photomorphogenic information would dramatically affect competitive ability in fast-growing plant stands.

The concept is important in attempts to understand weed-crop competition or to explain yield-density responses of field crops. Regarding possible strategies for crop improvement, it is valid to ask: Can we engineer better crop plants by altering specific aspects of their photomorphogenic behavior? (See also Smith 1991). We will discuss some potentially rewarding research avenues in the remaining of this section.

Stem elongation responses. Factors that confer competitive ability to the individual crop plant are not necessarily "desirable" agronomic traits (See discussions by Donald [1968] and Marshall [1991]). As discussed earlier in this paper, the initial response of seedlings of shade- 167 intolerant species to an increase in canopy LAI is a rapid acceleration of stem growth that is triggered by a variety of light signals. It is tempting to speculate, therefore, that genotypes showing reduced stem elongation responses to photomorphogenic signals of plant proximity may save assimilates that could be used to grow more leaves, roots, or reproductive structures. However, two important points should be considered. First, the increase in stem length may be proportionally larger than the increase in stem biomass, and second, the increase of stem biomass may imply more than a simple redistribution of a fixed pool of photosynthate. In a series of glasshouse experiments with amaranths (Amaranthus quitensis), Ballare et al. (1991d) showed that canopies accumulate much more dry matter in the internodes when they receive the full sunlight spectrum than when they are grown under FR-absorbing filters. However, screening the FR did not promote the growth of leaves or roots in these experimental crops. In other words, the extra-carbon used to sustain higher rates of stem growth under full-spectrum conditions resulted from an increase in canopy net photosynthesis. In turn, the increase in canopy productivity appeared to be related to the stem elongation responses, because filtering the FR had no effect on biomass accumulation when plants were grown as isolated individuals. There are at least two possible explanations for these results. First, an increase in internode elongation (triggered by low R:FR ratios) might alter the pattern of light penetration in the canopy, resulting in increased light interception and/or more efficient utilization of the intercepted light. Second, an increase in stem growth may stimulate leaf-source photosynthesis through a feedback mechanism. This latter possibility has been indirectly supported by experiments with isolated plants showing that, if the R light irradiance received by the stem is reduced, elongation and biomass accumulation in the internodes can be 168 substantially increased without affecting the growth of other plant parts (i.e. total plant biomass increases as a result of a localized reduction of R:FR ratio) (Ballare et al. 1991d). Responses of this type are not likely to be observed in experiments carried out under low irradiances. In wheat (Triticum aestivum) crops, the Rhtl and Rht2 alleles are known to reduce plant height and increase grain yield, but the causal links between these two effects are still not completely clear (e.g. Borrell et al. 1991). Moreover, in some species the assimilate invested in stems contributes a significant proportion of the carbohydrate used to fill harvestable organs towards the end of the crop cycle (Incoll and Neales 1970). Testing different varieties of one such crop, Helianthus tuberosus, Soja and Haunold (1991) found that tuber yield was significantly and positively correlated with mid-season stem dry weight; mid- season leaf area and maximum leaf photosynthesis werenot good predictors of crop yield. Low assimilate-storage capacity, whether in stems or in tubers, appeared to be an important limitation to crop yield. It is not clear to what extent the storage capacity of the stems is influenced by the amount of carbon invested in structural stem material, which, in turn, is likely to be modulated by photomorphogenic factors. To conclude, the evidence reviewed in this section indicates that the estimation of the opportunity cost of the assimilate invested in stem elongation would require careful experimentation under field conditions.

It should be noted that photomorphogenic elongation responses may affect crop productivity independently of their effects on carbon allocation. Thus, a high sensitivity to light signals of plant proximity will have detrimental effects whenever lodging is a factor affecting yield, but might be desirable in the case of timber trees to favor 169 rapid growth in height and reduced branching.

Branching. It is well established that apical dominance is influenced by the R:FR ratio of the incident radiation acting through phytochrome in dicotyledonous plants (e.g. Tucker and Mansfield 1972) and grasses (Deregibus et al. 1983, Kasperbauer and Karlen 1986). The axillary buds are one of the sites of light perception: local reductions of R:FR obtained by increasing FR (Casal et al. 1987) or by reducing R (Begonia et al. 1988, their "FR" treatment) lead to reduced branching. Experiments under natural radiation have demonstrated that in open grass canopies the drop in R:FR ratio caused by neighboring plants can reduce branching (tillering) rate even if the production of new branches is not limited by the availability of photosynthetic light energy (Casal et al. 1986). The evidence is based on the following observations: (1) tillering in a number of temperate grasses was found to be very sensitive to small depressions in R:FR below sunlight values (Casal et al. 1985, 1987), and (2), in sparse grass populations, the detrimental effect of neighbors on tillering rate can be countered by delivering small amounts of R light to the base of the plants (Deregibus et al. 1985, Casal et al. 1986). Little is known about the role played by photomorphogenic signals in the control of branching in dense canopies, where resource limitations are likely to be severe, and where many aspects of the light environment sensed by buds and other plant parts are simultaneously altered. Frequently, the inhibitory effects of heavy natural- or simulated-leaf shading (i.e.: low B and R, high FR) on branching and ramet production are more severe than those produced by comparable reductions of PAR obtained by means of neutral filters (Solangaarachchi and Harper 1987, Methy et al. 1990). This is most likely due to differences between shading treatments in the R:FR ratio, but the role of direct responses to B and 170 R fluence rate has not been investigated. A better understanding of the photomorphogenic regulation of branching would provide the bases for incorporating specific patterns of response to plant population density. In grasses and cereals, a high sensitivity to small reductions in R:FR would be desirable in species or agronomic scenarios where tillering does not contribute to yield stability and reduces the harvest index (i.e. high-input agricultural systems; Donald 1968, 1979, Bugbee and Salisbury 1989), but may be deleterious in less predictable environments (e.g. Kirby and Faris 1972).

Developmental timing. One potential mechanism for crop improvement is to alter the relative lengths of successive developmental phases (e.g. Rawson 1988). Thus, crop genotypes may be designed so that their phenology is better adjusted to match the temporal pattern of resource availability in a given environment. Mondal et al. (1986a) reported detailed studies on onion (Allium cepa) development in different cultural environments. They found that a number of key developmental events, such as the transition to bulb scale production and bulb maturity, are substantially advanced when planting density is increased. Bulbification in onions is stimulated by long days (LD) and it is well established that addition of FR during the photoperiod can accelerate development in this (Lercari 1982, Mondal et al. 1986b) and other (e.g. Deitzer et al. 1979) LD plants. The effect of FR has the characteristics of a classical high- irradiance response and, therefore, it is most likely mediated by Type I phytochrome (Carr-Smith et al. 1989). Further field experiments by Mondal et al. (1986c) showed that in onion plants shaded by green leaves (low PAR, high FR), leaf production ceased before and maturity occurred earlier than in plants shaded by neutral filters (low PAR, low FR). The effects of varying PAR alone on bulb 171 development were small. These studies suggest that changes in the canopy light environment perceived by phytochrome may be involved in the modulation of the observed developmental responses of onion plants to population density. It is interesting to note that in other species where flowering is promoted by LD, such as barley (Hordeum vulgare) (Kirby 1967), radish (Raphanus sativus) (Weston 1982) and wheat (Yu et al. 1988), increasing population density typically has the effect of accelerating reproductive development. In many LD plants, floral formation is closely associated with rapid stem elongation ("bolting") (e.g. Metzger 1987). Therefore, one of the advantages that an individual plant may gain from accelerating reproductive development in a crowded population would be to anticipate its neighbors in the race for colonizing the aerial space. A better understanding of the action of phytochrome(s) in the photoperiodic induction of LD plants, and more detailed physiological experiments in the field, would help to interpret the observed relationships between plant density and developmental timing and may provide clues to crop improvement programs.

Flower and fruit abscission and reproductive allocation. As evidenced by some of the work discussed earlier in this review, the study of the photoregulation of assimilate partitioning among plant organs and its relation to crop productivity has considerable agronomic interest (see also Kasperbauer et al. 1984, Britz 1990 and references therein). In seed crops, the allocation of photosynthate to reproductive structures is obviously a major determinant of yield. Heindl and Brun (1983) found that the abscission of reproductive structures in soybean (Glycine max) crops can be, in certain cases, reduced under field conditions by lighting the lower canopy strata during flowering and early pod growth with R or white light. Responses to R and white light were similar in their experiments (in spite of the 172 fact that the R source provided -3 times less PAR quanta) and were not observed when the reproductive primordia were artificially shaded. Subsequent studies (Myers et al. 1987) showed that R light (compared with FR or darkness) increases sucrose accumulation by soybean racemes cultured in vitro. Similar results had been obtained previously by Mor et al. (1980) working with dark-adapted rose (Rosa hvbrida) shoot tips. In growth chamber experiments with whole soybean plants, R light applied to the racemes via fiber optic guides was found to increase fruit set compared with FR and control (no lighting) treatments (Myers et al. 1987). These observations led to the conclusion that reproductive abscission in the lower soybean canopy and assimilate accumulation by young fruits are modulated by photomorphogenic signals perceived by the primordia (Heindl and Brun 1983, Myers et al. 1987). In other crops it has been shown that abscission of reproductive structures induced by shading can be prevented by applications of inhibitors of ethylene action (Wien and Zhang 1991). This suggests that the actual level of abscission may be set by factors other than assimilate supply. Although it is not easy to visualize an ecological scenario in which such a response mechanism could have been selected, the results obtained with soybeans suggest that there is scope for reducing abscission by manipulating photomorphogenic responses of reproductive primordia. The impact of such manipulations on crop yield will depend on (i) the relative importance of reproductive abscission as a determinant of seed numbers per unit area, and (ii) whether or not increases in seed numbers are accompanied by compensatory reductions in individual seed weight. A number of observations suggest that soybean yields are limited by canopy photosynthesis (Egli and Yu 1991 and references therein), which would imply that the chances of improving crop yields by reducing reproductive abscission are small. 173 On the other hand, other field studies with soybeans suggest that leaf photosynthesis can be enhanced by treatments that increase the number of reproductive sinks (reviewed by Shibles et al. 1987). These considerations indicate that carefully designed field experiments will be required in order to asses the agronomic consequences of interfering with the photocontrol of flower and fruit abscission.

SUMMARY AND OUTLOOK Plant photomorphogenesis has evolved in dark rooms and growth cabinets as a vigorous research area. Studies in controlled environments provided the template, and indeed much of the inspiration for more field-based projects. In turn, recent research in natural light environments has raised issues and questions that had not received attention before. Field experiments on the photocontrol of seed germination drew attention to the dynamics of light sensitivity and demonstrated that seeds of some weed species naturally switch to the VLF state after a short period of burial. This transition might be a crucial step in the sequence of events that enable light-requiring weed seeds to detect soil disturbances in arable fields. This hypothesis is supported by circumstantial evidence, butmore direct experimentation is needed to test the ecological and management consequences of changes in light sensitivity. In the area of UV-B photophysiology, it has been clearly shown that UV-B irradiation may cause alterations in plant morphology without affecting total growth or photosynthesis. These morphological responses, that could be mediated by a specific UV-B photoreceptor, may have importantconsequences on competitive interactions among plant species in mixed canopies. Field experiments also revealed the limitations of studies of UV-B effects on plant productivity carried out under low levels of PAR. The hypothesis that a specific UV-B photoreceptor is involved in the induction of flavonoid 174 accumulation in the epidermal cells by UV radiation is supported by action spectroscopy, and the protective role played by these compounds as natural UV-filters has been clearly demonstrated in controlled-environment experiments. In the area of photomorphogenesis in plant canopies, recent studies have shown that, by perceiving R:FR and fluence-rate signals through phytochrome(s) and a specific B light receptor, plants obtain information about the proximity of other plant individuals well before the PAR received by leaves is reduced by the activities of neighbors. This demonstration suggest that competitive interactions among plants in crop canopies may be much more complex than previously thought. The ability of individual plants to obtain photomorphogenic information may indeed be crucial for success in the competition for light in dense crops. Studies under natural light conditions are also contributing to clarify the role of photomorphogenic signals in processes such as organ abscission and assimilate partitioning. The observation that stem-perceived light signals may stimulate stem growth without affecting biomass accumulation by other plant parts suggests that the assumptions that underlie calculations of the costs associated with photomorphogenic responses should be tested experimentally under field conditions. In addition to the well known changes in morphology, some species respond to increased plant density with accelerated development. The photomorphogenic bases of this phenomenon are still not well understood.

These developments are very likely to influence more applied agricultural research in the near future. The general conclusion that we draw from this review is that the acquisition of information through morphogenic photoreceptors plays a substantial role controlling plant behavior in agricultural environments. This is usually overlooked in conventional texts on physiological plant 175 ecology and crop physiology. The increasesin crop yield that occurred over the last decades havebeen largely based on increasing the amount of resourcesavailable to "desirable" plants via fertilization, irrigation,and weed control. The evidence reviewed here suggeststhat further improvement can be achieved by manipulating the wayin which plants gather and interpret photomorphogenicinformation. One way to this end is theproduction of genetically engineered crop plants with specific alterationsin photomorphogenic behavior. Such endeavors would require more knowledge on the photobiology of plants under naturalfield conditions. In fact, most photomorphogenic responses may have a large impact on fluxes of energy andmaterials between the plant and the environment and amongplant parts. Therefore, their occurrence and agronomicimpact will be influenced by other environmental variables that affectthe "economy" of the plant, particularly theavailability of resources (i.e. light andnutrient levels). A particularly important question posed by some of the examplesdiscussed in this review is to what extent carbon fixation bythe canopy and assimilateallocation patterns in the field are influenced by photomorphogenic processes taking placein the assimilate sinks (e.g. stem internodes, reproductive primordia). Experiments under natural conditions of radiation are necessary to solve this question and to evaluate the potential benefits of modifying the photophysiology of sink organs.

Acknowledgements. This review was written while C.L.B.and A.L.S. were on a leave at Oregon StateUniversity financed by the Consejo Nacional de InvestigacionesCientificas y Tecnicas (Argentina) and the Dept. of ForestScience (Oregon State University). We thank Barbara Yoder forcritical comments on an early draft of the manuscript. 176

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