Function of Root Border Cells and their Exudates on Plant Defense in Hydroponic Systems

Item Type text; Electronic Dissertation

Authors Curlango-Rivera, Gilberto

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.

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Link to Item http://hdl.handle.net/10150/202535 FUNCTION OF ROOT BORDER CELLS AND THEIR EXUDATES ON PLANT DEFENSE IN HYDROPONIC SYSTEMS

by

Gilberto Curlango-Rivera

______

A Dissertation Submitted to the Faculty of the

SCHOOL OF PLANT SCIENCES

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY WITH A MAJOR IN PLANT PATHOLOGY In the Graduate College

THE UNIVERSITY OF ARIZONA

2011

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THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Gilberto Curlango-Rivera entitled Function of Root Border Cells and their Exudates on Plant Defense in Hydroponic Systems and recommend that it be accepted as fulfilling the dissertation requirement for the

Degree of Doctor of Philosophy

th ______Date: July 20 , 2011 Martha C. Hawes

th ______Date: July 20 , 2011 Hans D. VanEtten

th ______Date: July 20 , 2011 Chieri Kubota

th ______Date: July 20 , 2011 Mary Olsen

Final approval and acceptance of this dissertation is contingent upon the candidate‟s submission of the final copies of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

______Date: July 20th, 2011 Dissertation Director: Martha C. Hawes

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STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED: Gilberto Curlango-Rivera

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ACKNOWLEDGEMENTS

I want to honestly express my gratitude to Dr. Martha Hawes for giving me the opportunity of doing my doctoral studies in her research laboratory. Her advice and guidance helped me to accomplish this important goal of my academic career and life.

In the same manner, I want to thanks to Dr. Hans VanEtten for his advice and disposition. His example as a scientist has been a very important part of my formation.

Thanks to Dr. Chieri Kubota, Dr. Sandy Pierson, and Dr. Mary Olsen for being part of my committee and for their advice and help on my research.

I want to say thanks to all my friends that helped me during my studies in different ways. Special thanks to Dr. Fushi Wen, Dr. Gerard White, Dr. Yolanda Flores-Lara, and

MS Rhodesia Celoy for their help, advice on research, and friendship.

Thanks to all members of my family for the strength they have given me throughout my studies. Thanks to my father, to my mother, to my sisters, and to my grandparents for their support.

Thanks to my wife for her help and the good moments.

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DEDICATION

To all members of my family,

To my parents,

To my sisters,

To my grandparents,

To my wife,

and

Specially to my mother and grand mother

Julieta and Julieta

6

TABLE OF CONTENTS

LIST OF FIGURES……………………………………………………………….... 8

ABSTRACT……………………………………………………………………….... 9

I INTRODUCTION………………………………………………………………… 11

I.1 Context of Research…………………………………………………………... 11

I.2 Literature Review…………………………………………………………….. 12

I.2.1 Controlled Environment Agriculture…………………………………….. 12 I.2.2 Diseases in Controlled Environment Agriculture…………………...... 14 I.2.3 Root Border Cells……………………………………………………...... 16 I.2.4 Defense Mechanisms by Border Cells…………………………………… 19 I.2.5 Border Cell Exudates: Lectins…………………………………………… 21 I.2.6 Border Cell in Hydroponics…………………………………………...... 28

I.3 Objectives…………………………………………………………………….. 28

I.4 Format of this Dissertation…………………………………………………… 29

II PRESENT STUDY………………………………………………………………. 30

II.1 Summary of appendix A: Dynamics of root border cells in hydroponic systems………………………………………………………………………… 30

II.2 Summary of appendix B: Proteins among the polysaccharides. A new perspective on root cap slime………………………………………………….. 31

II.3 Summary of appendix C: Contribution of the root cap to soil fertility: Extracellular plant lectins……………………………………………………… 33

II.4 Summary of appendix D: Transient exposure of root tips to primary and secondary metabolites: Impact on root growth and production of border cells.. 34

II.5 Summary of appendix E: Root tips moving through soil. An intrinsic vulnerability...... 36

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TABLE OF CONTENTS – Continued

II.6 Summary of appendix F: Extracellular DNA: The tip of root defenses?...... 37

II.7 Summary of appendix G: Altered root tip morphology in pea hairy roots with altered expression of a border cell specific gene………………………… 38

REFERENCES……………………………………………………………...………. 40

APPENDIX A DYNAMICS OF BORDER CELLS IN HYDROPONIC SYSTEMS: PRODUCTION AND FUNCTION……………….………………... 48

APPENDIX B PROTEINS AMONG THE POLYSACCHARIDES: A NEW PERSPECTIVE ON ROOT CAP „SLIME‟……………………….……………... 70

APPENDIX C CONTRIBUTION OF THE ROOT CAP TO SOIL FERTILITY: EXTRACELLULAR PLANT LECTINS ……………………………………….. 75

APPENDIX D TRANSIENT EXPOSURE OF ROOT TIPS TO PRIMARY AND SECONDARY METABOLITES: IMPACT ON ROOT GROWTH AND PRODUCTION OF BORDER CELLS……...…………………………………... 92

APPENDIX E ROOT TIPS MOVING THROUGH SOIL: AN INTRINSIC VULNERABILITY ………………………………………...…………………… 103

APPENDIX F EXTRACELLULAR DNA: THE TIP OF ROOT DEFENSES?…... 107

APPENDIX G ALTERED ROOT TIP MORPHOLOGY IN PEA HAIRY ROOTS WITH ALTERED EXPRESSION OF A BORDER CELL SPECIFIC GENE….. 114

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LIST OF FIGURES

Figure 1. Root tips and border cells………………………………………………… 17

Figure 2. Root cap structure organized in structured tiers………………………….. 18

Figure 3. Fungal infection zone…………………………………………………….. 21

Figure 4. Lectins in the root cap……………………………………………………. 25

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ABSTRACT

Controlled environment agriculture offers a solution to challenges including less available land, water deficits, and consumer demand for pesticide free produce. However, control of soil-borne diseases is a major limiting factor. The goal of this dissertation was to examine predictions of the hypothesis that border cells function to protect plant health by controlling microorganisms associated with plants grown in hydroponic culture.

Border cells separate from root tips upon immersion in water, and appear to have important roles in the defense mechanisms of plant roots.

The general objectives were (1) to study the delivery of border cells in hydroponics; (2) to evaluate interactions between border cells and microorganisms in hydroponics; and (3) to explore approaches to alter border cell production for improved root disease control.

In this study it was confirmed that border cells can be released continuously into the solution of hydroponic culture suggesting that plants grown in this system may use extra energy in the production of new border cells. Free border cells interacted with microorganisms present in the hydroponic solution by secreting an extracellular capsule.

Previous studies showed that proteins are a key component of this capsule, including lectins. The interaction of pea lectin and Nectria haematococca spores therefore was explored. Results demonstrated that pea lectin agglutinates fungal spores in a hapten- specific manner, and inhibits their germination. Lectin had no negative effect on root development suggesting that it could be used as a potential control for soil-borne diseases in hydroponics. 10

To control the production of border cells, subsequent studies measured the impact of a transient exposure of root tips to different metabolites secreted by root caps and border cells. Exposure to specific metabolites altered the production of border cells without measurable effects on root growth and development. This is in contrast to results obtained with altered gene expression. For example, gene silencing of a border cell specific gene resulted in altered root growth.

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I. INTRODUCTION

I.1 Context of Research

Traditional agriculture faces emerging challenges to be both more productive and environmentally friendly (Schnotzler et al., 2003). The development of controlled environment agriculture techniques offers a viable alternative to obtain a high efficiency in water use, and higher productivity of safe produce. As a result, the development of controlled environment agriculture has been increasing worldwide (Enoch and Enoch,

1998). The North American region plays an important role especially in the use of high- technology approaches such as closed-hydroponics systems, achieving average yields of

378 metric tons of tomato per hectare and obtaining better efficiencies in the use of water

(Cook and Calvin, 2005).

However, although in these systems the crop is protected and the diversity of plant pathogens is reduced considerably, disease remains a significant problem that may limit application of the economic and cultural potential of controlled environment systems (Dasberg, 1998). In most cases, the problem is soil-borne pathogens

(Stanghellini and Rasmussen, 1994). In soil ecosystems, plants have different defense mechanisms to overcome soil-borne infections, including those carried out by root border cells and their exudates (reviewed in Hawes et al., 1998, 2000, 2003). Border cells are plant cells present on root tips with the particular characteristic that they separate from the root cap upon immersion in water. An important function of root caps is that they are programmed to replace those cells that have separated. Border cells and their exudates 12

appear to have important roles in the defense mechanisms of plant roots, such as protecting the growing root tip from pathogenic organisms and harsh conditions in the rhizosphere (Miyasaka and Hawes, 2000; Wen et al., 2007a).

The goal of this dissertation work is to examine predictions of the hypothesis that border cells function to protect plant health by controlling microbial populations associated with plants grown using hydroponic cultures. If correct, then control of border cells and their products may prove to be a key target for improved efficiency of safe, ecologically sound crop production using controlled environmental systems.

I.2 Literature Review

I.2.1 Controlled Environment Agriculture. In the course of the development of conventional agriculture, there have been modifications in the cultural practices used to increase productivity. Higher yields have been reached by the management of higher plant densities, monocultures, and high amounts of synthetic fertilizers (Chrispeels and

Sadava, 1994). However, these practices have caused serious outbreaks of diseases, which have been partially controlled with toxic chemicals causing serious environmental and human health problems (Lenteren, 2000). Other limiting factors are the reduced availability of water (Yeston et al., 2006) and arable land acreage (Liang et al., 2005).

The development of controlled environment agriculture techniques has been shown to be a viable alternative. The productivity in these protected systems is very high leading to much higher yields per unit of land, and increased efficiencies in the use of water (Schnotzler et al., 2003). For instance, water usage in field conditions is at least 13

two fold higher than in greenhouses (Rouphael et al., 2005). Also, since the crop is protected against harsh environments and disease agents, the production of free-pesticide produces is possible resulting in an environment-friendly agriculture system. Other advantages of controlled environment agriculture is that it needs a higher number of trained workers (8-10 persons per hectare versus one in open fields) which may help to reactivate the agricultural economy by the generation of more employees, mainly in developing countries (Tiwari, 2003).

With these advantages, the development of controlled environment agriculture has been increasing worldwide. According to Enoch and Enoch (1998), the total area of greenhouses around the world is about 800,000 hectares. This area is distributed mainly in Asiatic countries, the European Union and recently North America.

China is the world‟s leader in protected agriculture area with seventy-five percent of the world‟s total, and specifically this area is designated to vegetable production

(Enoch and Enoch, 1998). Japan and South Korea are important regions of Asia with

42,000 and 3,807 hectares respectively (Enoch and Enoch, 1998).

The European community has a representative area of greenhouse designated to the production of vegetables (Enoch and Enoch, 1998). Spain represents the largest area of greenhouses with 18,500 hectares, followed by Italy, France, and the Netherlands with

9,000, 6,450, and 4,500 hectares respectively. Countries including Belgium, United

Kingdom, and Germany have an area that ranges between 2,000 and 2,500 hectares, while Portugal, Denmark, and Ireland have 1250, 140, and 55 hectares respectively. This

European region represents about twenty-five percent of the global greenhouse 14

distribution excluding China (Enoch and Enoch, 1998). However, recently this number has been increasing because of the increasing development of controlled environment agriculture in other regions in North America. The use of techniques as closed- hydroponics systems, where plants are grown in a nutrient solution that is recovered and recycled through the root system of plants, allows achievement of higher yields and higher efficiencies in the use of water (Cook and Calvin, 2005).

In 2003, the tomato industry of Canada, Mexico and United States produced

528,000 metric tons of greenhouse tomatoes, which represent around thirty-seven percent of all fresh tomatoes sold in United States. This production was accomplished in an area of 1,726 hectares, with average yields of 378 metric tons/hectare compared with 25 metric tons/hectare from conventional agriculture. The North American fresh tomato industry represents thirteen percent of the total worldwide greenhouse production (Cook and Calvin, 2005).

I.2.2 Diseases in Controlled Environment Agriculture. In controlled environment agriculture the crop is protected from external environmental conditions such as excessive heat, cold, wind, and rains, and from pests and pathogens organisms such as bacteria, viruses and fungi. However, organisms can enter the protected system by a number of ways. Examples include the ventilation system, water source, dust, humans, insects and contaminated plant material. Also, in spite of the reduction of the diversity and amount of disease causing agents within these systems, serious outbreaks of diseases remain a problem (Dasberg, 1998). These are mainly caused by soil-borne pathogens since the root system is difficult to monitor in order to carry out a disease 15

preventive program. Causal agents are mostly fungal species such as Fusarium,

Verticillium, Rhizoctonia, and also oomycetes, such as Phytophthora, Olpidium,

Plasmopara and several species of Pythium (Stanghellini and Rasmussen, 1994).

Oomycetes are of considerable importance in hydroponics because they are favored by aquatic environments that allow rapid spread through the nutrient solution, infecting roots through the whole system causing serious economical losses or even losing the whole crop (Stanghellini and Rasmussen, 1994). Fungi such as Fusarium spp. also cause serious diseases in many plant species worldwide (Fujinaga et al., 2003; Katan et al., 1997; Punja and Parker, 2000; Stanghellini and Rasmussen, 1994).

An approach to prevent soil-borne diseases in controlled environment systems is the use of grafted transplants, which also enhance plant productivity (Lee, 1994). Another approach to solve this problem is the disinfection of the nutrient solution by ultraviolet radiation, which increases the costs of an already expensive technology. This radiation penetrates the cell wall of microorganisms causing damage to their DNA, so that they cannot multiply and eventually are eradicated. Ultraviolet radiation has been effective in control of Pythium aphanidermatum (Stanghellini, 1984), but unfortunately iron chelates are broken down by this radiation causing iron chlorosis in plants (Daughtrey, 1980;

Nederhoff, 2001).

Plants have different defense mechanisms in order to prevent infections from soil- borne pathogens. One of these defense mechanisms is carried out by border cells and their exudates (Hawes et al, 2000). 16

I.2.3 Root Border Cells. Border cells (Fig. 1) are living plant cells generated at root caps (Fig. 2) forming a sheath of cells that surrounds and gives protection to the root tip, having the characteristic that they separate from the root cap once in contact with water (reviewed in Hawes et al., 1998, 2000, 2003). This phenomenon of border cell separation is visualized with the aid of a stereoscope (Fig. 1). For instance, when root tips from pea (Pisum sativum L.) seedlings are submerged in water, border cells disperse immediately. This triggers cell cycle activation within the root cap meristem, with immediate production of border cell production. After 24 h a new set of border cells is completed, the root cap turns off the border cell cycle and no more border cells are produced, unless border cells are again removed by immersion in water or other signals

(Brigham et al., 1998). It is hypothesized that once the whole set of border cells is formed, border cells release an unknown signal which is sensed by the root cap, which consequently blocks cell cycle (Brigham et al., 1998). Therefore, in order to stop the border cell cycle, the root cap must sense the unknown signal; otherwise the production of border cells by the root cap will be continuous requiring a constant use of energy. In addition, it has been shown that the interruption of the border cell cycle increases plant susceptibility against pathogenic organisms (Woo et al., 2004).

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C A B

Figure 1. Root tips and border cells. Root tip in dry conditions visualized with a stereoscope (A). Visualization of border cells attached to root tip in dry conditions with scanning electron microscopy (From Hawes and Brigham, 1992; photo by Perkins S, Calvert HE and Bauer WD) (B). Detached border cells from root tip in the presence of free water, visualized with a stereoscope (C). Magnification A,C 10X; B 20X. (From Hawes et al., 2003).

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Figure 2. Root cap structure organized in structured tiers (from Barlow, 1975).

Root caps release a variable number of border cells depending on plant species. A single radicle of tomato, pepper, corn, pea, and cucumber will produce a set of 20, 90,

2700, 3300, and 3800 border cells, respectively (Hawes and Pueppke, 1986). The energy required to produce such numbers of cells is regulated within roots and the photosynthetic mechanism of plants (Farrar et al., 2003). For example, it has been shown that tomato plants translocate forty-three percent of photosynthates to the root system of tomato plants grown in soil, and from this percentage, seventy percent is released as rhizodeposition (Lynch and Whipps, 1991), including border cells. Nevertheless, in soil ecosystems the energy necessary for the extra production of border cells may be limited 19

by the normal control of border cell production. Thus, at ninety-nine percent humidity which is typical for soil conditions most of the time, border cell production ceases

(Brigham et al., 1998; Guinel and McCully, 1986). Only upon exposure to continuous film of water do border cells disperse rapidly, inducing renewed production.

I.2.4 Defense Mechanisms by Border Cells. To date, results obtained under controlled laboratory conditions, have been consistent with the hypothesis that border cells function to protect plant health by protecting the root tip, which houses the root meristem from which all new cells are derived (reviewed in Hawes et al., 1998, 2000,

2003, 2005).

Root tips and border cells are of considerable importance for plants because of their interaction with the microbial population in the rhizosphere (Jaroszuk-Scisel et al.,

2009; Liljeroth et al., 1991; McCully and Canny, 1988). For example, root border cell production is positively correlated with root colonization by beneficial organisms such as arbuscular micorrhizae (Niemira, et al., 1996). Also, Tsai and Phillips (1991) found that flavonoids from alfalfa (Medicago sativa) root exudates promote spore germination, hyphal elongation and branching in the vesicular-arbusclar mycorrhizae Glomus etunicatum. Moreover, Arriola (1997) found that the mycorrhizal fungus Glomus intraradices was positively correlated with maximum border cell production by four species from the Amaranthaceae family (Gomphrena globosa, Amaranthus tricolor,

Amaranthus caudatus, and Celosia cristata).

In addition, root border cells may attract pathogenic microorganisms as a 'decoy' defense mechanism. For instance, nematodes are attracted toward border cells, but after 20

30 min of interaction, nematodes are immobilized in response to exudates release by border cells. After a few hours or few days, nematodes regain motility. By that time the growing root tip has escaped from the danger of infection (Zhao et al., 2000b; Hubbard et al., 2005). A similar defense mechanism against pathogenic fungi is carried out by root border cells. Gunawardena et al. (2001, 2002, 2005) observed that fungal spores of N. haematococca (mating population VI) are localized among pea (P. sativum L.) border cells. As a result, a “mantle” is formed among border cells, their secretions and fungal hyphae (Fig. 3) (Gunawardena et al., 2005). The mantle detaches spontaneously allowing the uninfected root tip to keep growing, thus escaping infection. New border cells start to regenerate right away forming a new set in 24 h giving renewed protection to the growing root tip. Similar responses are shown when root tips are in adverse abiotic conditions such as the presence of aluminum or high carbon dioxide levels (Chen et al., 2008; Liu et al., 2007; Miyasaka and Hawes, 2001; Zhao et al., 2000a).

Recent discoveries have revealed the mechanism by which border cells may function in prevention of infection by fungal spores (Wen et al., 2007b). When a set of ca. 120 proteins secreted by pea root tips (the root cap „secretome‟) were treated with protein degrading agents, infection by the pea pathogen N. haematococca was increased from lees than five percent to one hundred percent. Multidimensional protein identification technology analysis revealed that border cells secrete a number of different proteins including those involved in plant defense mechanisms (Wen et al., 2007b). Of particular interest was the discovery that lectins are among the secretome. The possibility that this protein plays a key role in border cell function, and therefore is a target for 21

increased crop protection in hydroponics, was explored. Their rationale for this is summarized in the following paragraphs.

Figure 3. Fungal infection zone (From Gunawardena, 2005).

I.2.5 Border Cell Exudates: Lectins. Lectins are plant, animal and microbial proteins that bind to certain specific carbohydrate groups on cell surfaces and agglutinate them (Lehninger, 1982). Lectins are abundant in plants, particularly those of the legume family. Plant extracts with the ability to agglutinate human cells such as erythrocytes were first described as ricin from seeds of the castor bean (Ricinus commuinis) and abrin 22

from Abrus precatorius (reviewed in Lis and Sharon, 1972, 1977, 1981, and 2004;

Reviewed in Sharon, 2007). The first plant lectin was isolated by Sumner and Howell

(1936) in a purified form from seeds of the jack bean (Canavalia ensiformis) and named concanavalin A. The same authors demonstrated that this lectin was hapten-specific, this is, it precipitated glycogen and starch from solution and this agglutination activity was inhibited by sucrose from sugar cane. Cell specificity of lectins was demonstrated by lima bean (Phaseolus lunatus syn. limensis) lectin agglutination on human erythrocytes type

A, but not those of type B or O; based on these findings, Boyd and Shapleigh (1954) proposed the term lectin (Latin lego, to choose or to pick out) for these sugar-binding proteins.

Lectin composition. Most lectins are glycoproteins with carbohydrate content that can be as high as fifty percent. The sugar components are the same as those found in other plant glycoproteins, with the exception of L-arabinose. However, concanavalin A, wheat germ, and peanut lectins are exceptions that do not contain covalently bound carbohydrates. The amino acid pattern of lectins is characteristic of plant proteins, and there are no structural features common to all of them. Many are rich in aspartic acid, serine, and threonine, which comprise about thirty percent of their amino acid content.

Plant lectins are low or devoid of sulfur-containing amino acids. A few exceptions include potato, pokeweed and wheat germ lectins which are rich in cysteine with 11.5,

18, and 20 percent of the total amino acid residues, respectively. Most lectins are soluble cell components and also they are membrane-bound (reviewed in Lis and Sharon, 1981). 23

Properties of lectins. Lectins are important tools in cell differentiation and cell surface characterization, as well as detection, isolation, and characterization of carbohydrate-containing materials such as polysaccharides, glycoproteins, and glycolipids (reviewed in Lis and Sharon, 1981). For example, lectins were used in the

1950s to show that the determinants of the specificity of the blood types in humans were sugars: α-N-acetylgalactosamine of type A, α-galactose of type B, and α-L-fucose of type

O specificity (reviewed in Lis and Sharon, 1981). In addition, cell differentiation by lectins was shown in experiments in the 1960s, when the wheat germ agglutinin from

Triticum vulgare was found to agglutinate tumor cells at concentrations much lower than those required for the agglutination of normal cells. In the same manner, concanavalin A, soybean (Glycine max) lectin, and many others agglutinated malignant cells preferentially, suggesting that tumor cells must have a different surface structure from normal cells since the specific carbohydrate residues to which lectins bind are apparently more exposed on tumor cell surfaces. These findings led to the use of lectins for studying changes on cell surfaces during growth, differentiation, and malignant transformation

(Lehninger, 1982; reviewed in Lis and Sharon, 1981). Moreover, lectins from the red kidney bean (P. vulgaris) cause lymphocyte cells to enlarge. This cell enlargement facilitated the study of chromosomes increasing the understanding of relationships between chromosomes abnormalities and diseases, and the expansion of cytogenetics

(Nowell, 1960). Others functions of lectins include enzymatic properties such as the mung bean lectin which has a strong enzymatic activity comparable to that of α- galactosidase (Hankins and Sharon, 1978). 24

Localization of plant lectins. In plants, lectins occur mostly in seeds representing three to four percent of the dry mass (Summer and Howell, 1936). Mishkind et al. (1983) showed the presence of wheat germ agglutinin in different tissues of wheat, barley, rye, and rice plants. These included the germ portion of the grain, such as the surface layers of various organs including the coleoptile, scutellum, coleorhiza, and the first adventitious roots. The functions remain unknown. Of special interest to this dissertation was the discovery that lectins were restricted to the root cap and root border cells (Fig. 4).

A small population of cells in the embryo contains most of the lectin present in the grain, which represent one percent or less. In addition, Gatehouse and Boulter (1980) have also shown the presence of lectins in seeds, and besides that, they reported the detection of lectin in pea roots. Pea lectins purified from seeds appear to be different from pea lectins purified from roots, since they have different sugar specificity and agglutination activity, suggesting that they may have different function and structures. Specifically, root lectins were detected in the root cortical cells and on the surface of root hairs, showing their presence at sites of rhizobial infection. At higher magnifications, lectins were detected in root cap cells and border cells (Mishkind et al. 1983).

25

A B C D

Figure 4. Lectins in the root cap. Mishkind et al. (1984) used immunolocalization to demonstrate that in the embryonic root caps of wheat (A) lectin is localized at the cap periphery (B). As the root emerges (C), lectin is secreted into the extracellular environment of border cells (D).

The presence of lectins in phloem exudates from cucurbits species such as pumpkin (Cucurbita maxima Duch.), cucumber (Cucumis sativus), and melon (Cucumis melo) has been reported by Sabnis and Hart (1978). Lectins were found in five day old seedlings, and no detection was possible in seeds. The agglutinating activity was shown at concentrations as low as 0.1 μg/mL, and interestingly, sugars such as raffinose and stachyose (which are transported in the phloem of these species), sucrose, galactose, glucose, fucose, mannose, xylose, and arabinose, do not inhibit agglutination. From the total phloem protein content, fifteen to twenty-five percent was found as lectin, which is a higher content compared to that of seeds from other plant species. According to these authors, one possible function of phloem lectin may be the protection of the sugar-rich 26

phloem from invasions of microorganisms, since inhibition of pathogenic microorganisms has been reported as described below.

Lectin-microbe interaction. For the majority of plants in the leguminosae family, emergent root hairs are the sites of infection by Rhizobium spp. Diaz et al (1986), found a positive correlation on the colonization of the bacterial symbiont Rhizobium leguminosarum on sites where lectins from pea (P. sativum L.) roots were accumulated.

Lectins were observed on the tips of newly formed, growing root hairs and on epidermal cells located just below the young hairs. Most nodulation (seventy-three to ninety percent) occurred on sites where lectin was localized. Contrary, few plants showed nodulation at sites where the lectin was absent.

Plant lectins also interact with pathogenic organisms, showing antifungal, antiviral, antibacterial, and anti-insect properties, as well as toxicity toward herbivorous organisms (reviewed in Peumans and Van Damme, 1995a; 1995b). For example, wheat germ lectin agglutinates and inhibits growth of Trichoderma viride and Fusarium solani

(Mirelman et al., 1975; Cristinzio et al., 1988). Wheat (Triticum aestivum L.) germ agglutinin levels in seedling roots are induced two-fold by fungal species such as

Rhizoctonia solani, Fusarium culmorum, Pythium ultimum and Neurospora crassa, as well as by fungal elicitors. These findings suggest that lectins could be involved in the defense of wheat against fungal attack. Moreover, lectin from stinging nettle (Urtica dioica L.) rhizomes bind chitin, inhibiting the growth of phytopathogenic and saprophytic chitin-containing fungi such as Botrytis cinerea, Collectotrichum lindemuthianum,

Phoma betae, Phycomyces blakesleeanus, Septoria nodorum, Trichoderma hamatum and 27

T. viride (Broekaert et al., 1974) This lectin inhibition is different from the one caused by chitinase, but both the nettle lectin and chitinase acts synergistically against fungal growth.

In the case of pathogenic bacteria, Pueppke et al. (1982), showed that the lectin concanavalin A binds to Agrobacterium tumefaciens strains, reducing the number of tumors produced on potato. In contrast, the potato lectin did not bind to A. tumefaciens and neither reduced the number of tumors produced. Isolated lectins from maize (Zea mays) were tested for agglutination of virulent and avirulent Erwinia stewartii strains.

Results showed that lectins bind better to the avirulent strains rather than to the virulent ones, which produce higher amounts of extracellular polysaccharide. When the extracellular polysaccharide was washed from virulent E. stewartii strains, lectin agglutination was higher than in unwashed virulent-bacterial cells. According to these results, the extracellular polysaccharide may be involved in pathogenicity, since it may prevents bacterial agglutination in the host and consequently increasing its multiplication

(Bradshaw-Rouse et al., 1981).

Lectins found in cell walls of organisms are implicated in host-microbe interactions (Sharon, 1987). For example, the human pathogen Escherichia coli produces mannose specific surface lectins, functioning primarily in the initiation of infection by mediating bacterial adherence to epithelial cells. This mannose specific lectin also acts as recognition molecules in lectinophagocytosis by mouse, rat, and human peritoneal macrophages and human polymorphonuclear leukocytes (Sharon, 1987). 28

I.2.6 Root Border Cells in Hydroponics. Root border cells have been implicated in protection of the root from injury and infection. Also, it has been shown that, the phenomenon of border cell separation is influenced by aquatic conditions (Yu et al., 2006; Endo et al., 2011). Yet their impact in hydroponics remains unexamined.

Border cells were first reported by Knudson (1919), who found free border cells released into the nutrient solution of pea and maize plants grown in hydroponics. Also, he made the important observation that these cells remained metabolically active for at least forty- five days after they were released by root tips. This observation was later supported by

Hawes and Pueppke (1986) who additionally found that border cells are able to divide in response to the proper supply of nutrients. Griffin et al. (1976) estimated that root exudates including border cells, released by peanut plants in hydroponics may contribute ninety-five to ninety-eight percent of the rhizodeposition present in the nutrient solution.

These findings suggest that in hydroponics, there is a frequent release of border cells which are metabolically active after separation of their root caps. The significance of these cells in plant development and root disease, and potentially as a target for improved crop protection, is the focus of this dissertation.

I.3 Objectives

I.3.1 To define the dynamics of border cell production in model hydroponic systems, using agronomically important species such as cereals and legumes, and with special emphasis on common greenhouse crops including tomato, cucumber, pepper and lettuce. 29

I.3.2 To compare results of the dynamics of border cell production in model hydroponics with the dynamics of border cell production in semi-commercial hydroponics.

I.3.3 To explore specifically the ways border cells may interact with microbial populations to influence plant health.

I.3.4 To examine the functional impact of border cells on root function, with special emphasis on the role of secreted compounds in root growth and resistance to infection.

I.4 Format of this Dissertation.

Results of this dissertation will be presented as appendices containing a copy of either a published article or a draft manuscript in preparation for publication. A summary of each appendix containing the contribution of the author of this dissertation is presented in the following section.

30

II. PRESENT STUDY

The methods, results, and conclusions of this study are presented in the papers appended to this dissertation. The following is a summary of the most important findings in this document.

II.1 Summary of appendix A: Dynamics of root border cells in hydroponic systems.

Appendix A is a draft manuscript in progress to be submitted to Journal of

American Society for Horticultural Science. Root border cells have been implicated in protection of the root from injury and infection (reviewed in Hawes et al., 1998). It has been shown that border cell separation occurs in the presence of free water (reviewed in

Wen et al., 2007a), but little is known about their impact on the rhizosphere of hydroponic systems. Border cells of roots grown in hydroponic systems were first described by Knudson (1919), who observed that border cells from pea (P. sativum L.) and maize (Zea mays L.) remained alive for at least 45 days after they separated from root tips. Griffin et al. (1976) estimated that border cells and their associated exudates from the root cap (ca. 1 mm of the apex) are up to ninety-eight percent of the rhizodeposition released by peanuts roots grown in hydroponic culture. The observation that these cells continue metabolically active after separation of their root caps was later supported by Hawes and Pueppke (1986). Considering these findings it is hypothesized that in hydroponics there is a constant release of border cells that are metabolically active after separation of their root caps. In this study, the number of border cells released into 31

the nutrient solution from different important crops grown in model hydroponic systems was determined and compared to experimental semi-commercial hydroponic systems.

Results of the current study confirmed results reported by Knudson (1919) regarding the presence of viable border cells released into the nutrient solution. During the first days of culture root tips remain free of border cells, as border cells detach continuously into liquid. Pea and cucumber (Cucumis sativus L.) root tips were shown to accumulate a sheath of border cells surrounding the root tip. In contrast, root tips remain free of border cells border cells in semi-commercial hydroponic settings. Samples analyzed were taken during the day when the crop was being irrigated. In samples analyzed before the irrigation system started, root tips showed accumulation of border cells, suggesting that the presence of free water due to frequent irrigation caused border cell separation. However, border cells were not found in the nutrient solution at any time.

These results suggest that in hydroponic culture the dynamics of border cell separation is different from soil systems and it may influence the rhizosphere in different ways. For example, the constant release of border cells may cause extra production of border cells by plants and a change in the establishment of a microbial community on the rhizosphere.

My contribution to this study was the collection of samples and detailed microscopic analysis of the number and viability of border cells from plants of different crops grown in model hydroponic cultures and in a semi-commercial hydroponic system.

II.2 Summary of appendix B: Proteins among the polysaccharides: A new perspective on root cap ‘slime.’ 32

Appendix B is an article published in Plant Signaling and Behavior (2007), 2(5):

410-412. Root border cells, whose long-term viability in hydroponic culture was described by Knudson (1919), have been implicated in protection of the root from injury and infection (Hawes et al., 1998). In the previous appendix Knudson‟s (1919) observation that metabolically active border cells are released into the nutrient solution of model hydroponic systems was confirmed. However, the impact of these cells in this type of culture is unknown. Thus, in this study the ways border cells may interact with microbial populations present in hydroponic cultures were measured. In previous studies it has been demonstrated that root caps are covered by border cells contained in a slime- mucilage material that is mainly composed of polysaccharides and a small portion of proteins (Bacic, 1986; Chaboud, 1983; Wright 1975). This small part includes ca. 120 proteins that are secreted into the extracellular environment with different functions such as signaling, structure, and defense (Brigham et al., 1995; Wen et al., 2007b).

In the current study India ink was used to measure dynamics of the slime- mucilage material in which border cells are contained. Also, a slime-mucilage layer or capsule surrounding single border cells was visualized. When border cells were treated with proteinase, this capsule was eliminated and could not be visualized with the India ink assay, suggesting that proteins are a key component of this slime-mucilage material.

In addition, capsule surrounding single border cells responded differently to the presence of different microorganisms, which was determined by measuring the increase of the border cell capsule. For example, the presence of a seed-born epiphyte, Bacillus sp., caused a dramatic 50 X increase in size of the capsule of corn border cells grown for 7-10 33

days in model hydroponic culture. The impact in crop production is unknown. In conclusion, this study establishes that further research is necessary to examine the cost- benefit of border cell release into hydroponic crop systems. Metabolically active border cells released in hydroponic culture may have a positive impact since they interact with the microbial populations present in these systems. If so, the extra energy used by plants in hydroponics for the continuous production of border cells might foster a beneficial microbial population in the hydroponic rhizosphere. An alternative hypothesis is that uncontrolled border cell production wastes energy and stimulates growth and microbial populations that are not beneficial.

My contribution to this article was setting up and running a model hydroponic system in which pea and corn plants were grown, detailed microscopic analysis of border cells from hydroponic solution samples using India ink assays, and training of graduate student Mariana Del Olmo-Ruiz during her rotation program.

II.3 Summary of appendix C: Contribution of the root cap to soil fertility:

Extracellular plant lectins.

Appendix C is an invited research article published in Soil Fertility (2009),

Lucero DP and Boggs JE (eds.), Nova Science Publishers, Inc. pp. 65-79. ISBN: 978-1-

60741-466-7. In this study it was determined that pea lectins are involved in recognition and agglutination of fungal spores. Previous studies have demonstrated that border cells respond to specific signals released by microorganisms, which triggers defense responses

(reviewed in Hawes et al., 1998, 2000, 2003). These signals are cell wall components, 34

such as sugars, and it has been shown that these components recognize and bind to lectins

(Halverson and Stacey, 1986). Lectins are part of a set of ca. 120 proteins secreted by border cells (Wen et al., 2007b), and have been reported to be involved in plant defense mechanisms (Peumans and Van Damme, 1995a, 1995b). Based on that, it was hypothesized that lectins function as signals involved in border cell defense mechanisms such as recognition and agglutination of pathogens. During this study I tested the prediction that recognition of the pathogen is carried out by the carbohydrate component of lectins from border cells, implementing agglutination assays and using the pea – N. haematoccoca model system.

Results revealed that N. haematococca spores are recognized and agglutinated by pea lectin. This response is correlated with inhibition of spore germination and growth.

The results suggest that aspects of recognition involve lectins from border cells.

My contribution to the content of this article is the implementation of agglutination assays and detailed microscopic examination of border cell-fungal spore binding. Training of undergraduate Gabriela Albala (University of Arizona) during her independent study, winter term students David Olsen (Luther College), Jimp Kemp

(Luther College), and Emily Hildebrand (Centre College), was also a component of this study.

II.4 Summary of appendix D: Transient exposure of root tips to primary and secondary metabolites: Impact on root growth and production of border cells. 35

Appendix D is an article published in Plant and Soil (2010), 332: 267-275. In this study the impact of pea lectin, and other root secreted metabolites on root development was evaluated. Specifically, the effect of pea lectin, sugars, amino acids and secondary metabolites on root cap cell cycle was measured based on border numbers. Border cell viability, root growth, and morphology of pea seedlings also were evaluated. If lectins are involved in pathogen recognition and agglutination during plant defense mechanisms

(appendix C), then the accumulation of root border cell-secreted lectins in the nutrient solution of a hydroponic system may be predicted to have a positive effect in plant health.

If so, then lectins are a potential tool in the control of soil borne pathogens in hydroponics. However, glucose and mannose as sugar components of lectins may be a limiting factor in this control strategy, mainly in closed agricultural systems where the accumulation of different compounds including sugars and different metabolites may reach a high concentration in the nutrient solution becoming toxic to plants (Jung, 2003;

Knudson, 1917). These hypotheses were explored by developing a transient-exposure assay to measure effects of metabolites released from roots.

Results demonstrated that pea lectin (1 µg/mL) and the amino acids valine, alanine, threonine, and asparagine do not have a negative effect on pea at a concentration of 10 mM. The sugars galactose, glucose, mannose, fucose, arabinose, xylose, rhamnose, acetylglucosamine and acetylgalactosamine did not have a negative effect as well, at concentrations up to 50 mM. In contrast, rhamnose reduced the root growth and border cell production. Metabolites such as ferulic acid and naringenin decreased the root length and border cell production at 1 mM concentrations, and salicylic acid at 50 mM. Our 36

results show that pea lectin (1 µg/mL) and its carbohydrate contents glucose and mannose are not toxic to pea seedlings, which supports the use of lectins as potential tool to control soil-borne disease.

My contribution to this research was to setting up the transient treatments and analysis of growth, detailed microscopic analysis of border cell number and viability.

Also, training of undergraduate Gabriela Albala (University of Arizona) during her independent studies, and helping Dr. Denise Duclos to design and set up experiments during her post-doctoral research.

II.5 Summary of appendix E: Root tips moving through soil: An intrinsic vulnerability.

Appendix E is an article published in Plant Signaling and Behavior (2011), 6(5):

726-727. In this article, results show the attraction of Pythium dissotocum to cotton root tips where border cells are present, and that this attraction occurs in a transient manner, within seconds. This suggests that a transient presence of metabolites secreted by border cells into the rhizosphere can play a critical role in plant defense.

My contribution to this study supports the general significance of this phenomenon by demonstrating the attraction of microorganisms to root tips using different model systems, specifically, the attraction of Erwinia carotovora carotovora and Pseudomonas fluoresncens to pea root tips. Studies to determine host specificity of the attraction of different microorganisms to border cells is in progress.

37

II.6 Summary of appendix F: Extracellular DNA: The tip of root defenses?

Appendix F is an invited review article published in Plant Science (2011), 180:

741-745. In human pathogenesis, the role of extracellular DNA (exDNA) is not limited to specific types of pathogens but appears to play a role in bacterial, fungal, and protozoan infection. Similarly the role of exDNA in plant defense has been documented (Wen et al.,

2009). To evaluate a broader role for exDNA in plant pathogenesis, we selected the causal agent of bacterial wilt, whose invasion of legume roots has recently been characterized in detail, using Medicago truncatula as a model (Turner et al. 2009).

Results revealed that susceptibility to infection of pea roots by Ralstonia solanacearum GMI1000, occurred when the bacteria were co-inoculated with DNase 1.

This increased infection was ameliorated when actin, a specific inhibitor of DNase 1 activity, was added. Control plants treated with DNase 1, alone, showed no change in growth or development. The results obtained by treating with DNase 1 at the time of inoculation of plants with N. haematococca or R. solanacearum suggest that, as in mammalian systems, the presence of exDNA influences host susceptibility to bacterial as well as fungal plant pathogens. If correct, the capacity to degrade exDNA by the production of extracellular DNase will be predicted to be a virulence factor in plant pathogens as it is now known to be in human pathogens. If so, then inhibition of pathogen exDNase is a promising target for disease prevention in hydroponic systems. Gene silencing of a N. haematococca exDNase to test its role in disease is in progress.

My contribution to this article is the survey carried out to identify the production of exDNase activity by plant pathogens using in vitro assays, to measure the effect of 38

exDNases in bacteria root rot using a model hydroponic system, and to use gene silencing to study exDNase role in virulence root pathogens.

II.7 Summary of appendix G: Altered root tip morphology in pea hairy roots with altered expression of a border cell specific gene.

Appendix G is a draft manuscript in progress to be submitted to Annuals of

Botany. The preceding appendixes show that the controlled production and release of border cells and their exudates may provide a tool to modify root-microbe associations in hydroponic cropping systems. Altered production of the secreted products by border cells could be a feasible approach to deliver specific compounds into the rhizosphere in order to control diseases in a more efficient manner (Hawes et al., 1998, 2000, 2003; Lilley et al., 2010). In this study, a specific gene expressed only in root border cells was identified.

The coding region of this gene, showed a putative motif that includes a flavin binding protein site. Flavins have been implicated in plant defense and root growth (Bonner,

1942; Liu et al., 2010, Mishina and Zeier, 2006; Verdrengh and Tarkowski, 2005).

The impact of flavins in the transient exposure assay was measured. Pea and corn seedlings grown in hydroponic culture and treated with different flavins showed a neutral impact on root growth, border cell viability and production, and mucilage-slime capsule response. No protection by riboflavin was observed on pea seedlings inoculated with N. haematoccoca, a pea pathogen. In addition, transformed hairy roots with altered expression of this gene caused negative effects on root development such as altered root growth and abnormal border cell formation. Similar results were obtained in efforts to 39

modify other root cap genes (Table 1 in Curlango-Rivera et al., 2010). This suggests that genetic modification as an approach to deliver specific compounds into the rhizosphere may not be a viable alternative for the control of soil-borne pathogens.

My contribution to this article was to carry out transient assays for root tip responses to flavins, infection assays in model hydroponics systems to evaluate the influence of flavins on disease protection, and detailed microscopic examination of border cell mucilage-slime capsules in response to different flavins.

40

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48

APPENDIX A

DYNAMICS OF BORDER CELLS IN HYDROPONIC SYSTEMS:

PRODUCTION AND FUNCTION

Gilberto Curlango-Rivera

Martha C. Hawes

Draft manuscript in progress to be submitted to Journal of American Society for

Horticultural Science.

49

Dynamics of root border cells in hydroponic systems: Production and function.

Gilberto Curlango-Rivera,1 Martha C. Hawes*

Draft manuscript in progress to be submitted to Journal of American Society for

Horticultural Science.

Author affiliation:

1Division of Plant Pathology and Microbiology, Controlled Environment Agriculture

Center. School of Plant Sciences, University of Arizona, Tucson, AZ, USA.

*Corresponding author; Department of Soil, Water, and Environmental Science,

University of Arizona, Tucson, AZ, USA; E-mail [email protected].

50

ABSTRACT

Root border cells have been implicated in protection of the root from injury and infection. It has been shown that border cell separation occurs in the presence of water, and that the cells can remain alive after they separate from root tips. Root tip exudates, including border cells, can contribute with up to ninety-eight percent of the rhizodeposition from peanuts and other legumes grown in hydroponics. However, little is known about their impact on growth, development, and susceptibility to disease. We hypothesized that in hydroponics there is a constant release of border cells that are metabolically active after separation of their root caps. In this study, the number of border cells released into the nutrient solution from different important crops grown in model hydroponic systems was determined and compared to experimental semi-commercial hydroponic systems. Results confirmed the presence of active border cells released into the nutrient solution. During the first days of culture root tips remained free of border cells, and overtime, pea and cucumber root tips accumulated a sheath of border cells surrounding the root tip. In contrast, root tips remain free of border cells border cells in semi-commercial hydroponic settings. However, border cells were not found in the nutrient solution at any time. These results suggest that in hydroponic culture the dynamics of border cell separation is different from soil systems and it may influence the rhizosphere in different ways. For example, the constant release of border cells may stimulate changes in associated microbial populations on the rhizosphere.

51

INTRODUCTION

The development of controlled environment agriculture techniques has been shown to be a viable alternative to conventional agriculture (Cook and Calvin, 2005;

Tiwari 2003). The use of techniques such as closed-hydroponics systems, where plants are grown in a nutrient solution that is recovered and recycled through the root system of plants, allows achievement of higher yields and higher efficiencies in the use of water

(Cook and Calvin, 2005; Schnotzler et al., 2003). According to Enoch and Enoch (1998), the total area of greenhouses around the world is about 800,000 hectares. In spite of the protection these systems offer, diseases remain a problem (Dasberg, 1998), mainly caused by soil-borne pathogens (Stanghellini and Rasmussen, 1994). Prevention of soil- borne diseases in controlled environment systems increases the costs of operation, and relies on the use of pesticides or are not totally effective (Daughtrey, 1980; Lee, 1994;

Nederhoff, 2001; Stanghellini, 1984).

Plants have different defense mechanisms in order to prevent infections from soil- borne pathogens. One of these defense mechanisms is carried out by border cells and their exudates (Hawes et al, 2000). Border cells are living plant cells generated at root caps forming a sheath of cells that surrounds and protects the root tip (reviewed in Hawes et al., 1998, 2000, 2003). Border cells separate from the root tip upon immersion in water (Hawes et al., 1998). After border cell separation the root cap starts to produce new border cells within minutes, completing a whole set in 24 h. After the new set of border cells is complete, the root cap cell cycle is blocked and no more border cells are produced, unless border cells are again separated by immersion in water. It is 52

hypothesized that once the whole set of border cells is formed, border cells release an unknown signal which is sensed by the root cap, which consequently blocks the root cap cell cycle (Brigham et al., 1998). Therefore, in order to stop the cell cycle, the root cap must sense the unknown signal; otherwise the production of border cells by the root cap will be continuous requiring a constant use of energy. Conversely, it has been shown that the interruption of the root cap cell cycle to inhibit border cell production increases plant susceptibility against pathogenic organisms (Woo et al., 2004).

Root caps release a variable number of border cells depending on plant species. A single radicle of tomato, pepper, corn, pea, and cucumber will produce a set of 20, 90,

2700, 3300, and 3800 border cells, respectively (Hawes and Pueppke, 1986). The energy required to produce such numbers of cells is regulated within roots and the photosynthetic mechanism of plants (Farrar et al., 2003). For example, it has been shown that tomato plants translocate forty-three percent of photosynthates to the root system of tomato plants grown in soil, and from this percentage, seventy percent is released as rhizodeposition (Lynch and Whipps, 1991), including border cells. Nevertheless, in soil ecosystems the energy necessary for the extra production of border cells may be limited by the normal control of border cell production. Thus, at ninety-nine percent humidity which is typical for soil conditions most of the time, border cell production ceases

(Brigham et al. 1998). Only upon exposure to free water do border cells disperse rapidly, inducing renewed production.

Metabolically active border cells were first reported by Knudson (1919), who found free border cells released into the nutrient solution of pea and maize plants grown 53

in hydroponics. Also, he made the important observation that these cells remained one hundred percent viable for at least forty-five days after they were released by root tips.

This observation was later supported by Hawes and Pueppke (1986) who additionally found that border cells are able to divide in response to the proper supply of nutrients.

Griffin et al. (1976) estimated that root exudates including border cells, released by peanut plants in hydroponics may contribute ninety-five to ninety-eight percent of the rhizodeposition present in the nutrient solution. These findings suggest that in hydroponics, there is a frequent release of border cells which are metabolically active after separation of their root caps.

Since border cells are the main product released into the rhizosphere, and evidence suggests that they function in defense, it is reasonable to propose that their production in hydroponic culture may influence growth, development, and susceptibility to disease. The objective of this work will be to study the release of border cells of important agronomic plant species and greenhouse crops using model hydroponic cultures and experimental semi-commercial hydroponic systems.

METHODOLOGY

Border cell production in model hydroponic systems.

Plant species used for border cells quantification in laboratory conditions are described in Table 1. Z. mays and C. sativus were surface sterilized in 95% (v/v) ethanol for ten minutes and 50% (v/v) commercial bleach (6 % sodium hypochlorite) for ten minutes. Legume seeds were surface sterilized in 95% ethanol for ten minutes (v/v), 54

followed by full strenghth commercial bleach (6 % sodium hypochlorite) for thirty minutes, eliminating floating seeds. Seeds from the other species were surface sterilized in 95% (v/v) ethanol and 1% (v/v) commercial bleach (6 % sodium hypochlorite) for five minutes each. All seeds were rinsed five times and imbibed in distilled water for 1 hours, with the exception of P. sativum which was imbibed for 6 hours. After imbibition, seeds were plated onto 1 % water-agar plates which were overlaid with Whatman # 1 filter paper in order to prevent loss of border cells by root penetration of the agar. Seeds were incubated at 25 °C (Hawes and Pueppke, 1986).

Quantification of border cells.

After seedlings reached a length of 25 mm, root tips were washed from border cells, as described (Hawes and Pueppke, 1986). Then, one set of three seedlings was put back onto Petri plates, and another was kept separated in 1.5 ml microfuge tubes with 1 ml of distilled water simulating hydroponic conditions. After 24 hours, border cells from each seedling on agar plates were harvested in 100 μl of distilled water, and then three samples of 10 μl each were counted using a light microscope. For seedlings in water, a sample of 100 μl and a subsample of 10 μl were used, repeating three times for each microfuge. This experiment was repeated three times for each plant species.

After quantifying border cells in water-agar plates, we proceeded to quantify border cell number in hydroponics just by setting seedlings in 1.5 ml centrifuge tubes containing 1 ml of distilled water. Previously, border cells were removed and incubated.

After 24 hours, the new set of border cells formed was counted. 55

Border cells in semi-commercial hydroponic systems.

In order to determine the presence of border cells in the root system of hydroponic systems, we proceeded to take root samples of different greenhouse crops grown under aggregate-hydroponic conditions and determine the presence of border cells. In these systems, rock wool is used as an artificial substrate which gives plant support. This substrate is drip-irrigated during 10 hours per day with irrigation frequencies of 3-4 times per hour.

Most of the root system was distributed on the bottom and out of the substrate.

Thus, root tips samples were taken from the bottom of the substrate, and the number of root tips with and without border cells was determined. After that, root tips were plated overnight on water-agar plates, and next day were analyzed for border cell number and viability.

To measure the effect of irrigation effect on border cell production, root tip samples were taken after the last schedule of the irrigation system (6:00 p.m.). To see if root tips were viable and able to produce more border cells during the overnight period when the irrigation system was off, samples were taken before the initiation of the irrigation system (7:00 a.m.). In addition, samples were taken from plants irrigated with different electrical conductivity.

RESULTS

Border cell production in model hydroponic culture. 56

Table 2 presents the number of border cells produced by root tips from seedlings grown for 24 hours on water agar and in hydroponics for 24 h. These results show differences in border cell production depending in the plant species. Cucumber, tomato and pepper species produced a high number of border cells in water culture. Alfalfa produced the same number of border cells either growing on water-agar or in water culture conditions. Corn, pea and lettuce released fewer border cells in water culture than on water agar. Fluorescein diacetate used as a vital stain to measure cell viability (Hawes and Pueppke, 1986) showed that detached border cells were metabolically active. Also, root tips in water culture conditions were free of border cells or had only a few attached to the root cap, with the exception of cucumber and pea seedlings. During the first days of culture root tips from all species remained free of border cells, and overtime, pea and cucumber root tips accumulated a sheath of border cells surrounding the root tip (Fig. 1).

A similar phenomenon was observed on roots of water hyacinth grown in ponds in

Gainesville, Florida (Hawes, unpublished results).

Border cells in semi-commercial hydroponic systems.

Plants grown in hydroponics had a higher number of root tips without border cells, and when these roots tips were placed on water-agar conditions root caps restored the ability to accumulate border cells (Table 3). It is important to mention that root tips registered as “roots with border cells” were root tips with a few cells attached showing or not showing border cell separation after immersion in water. Root tips with a normal set of border cells were not observed. All the root tips analyzed for high and low electric 57

conductivity conditions were free of border cells after a 10 hour period of irrigation, and after overnight some root tips showed accumulation of a few border cells. Fluorescein diacetate assays showed that accumulated border cells were metabolically active.

DISCUSSIONS

Our results support previous studies by Knudson (1919) and Griffin et al. (1976).

This is, in model hydroponics under laboratory conditions, the roots of important agronomic crops (Table 2) can constantly release border cells. Interestingly, some plant species release even more than one set of border cells daily. In addition, root caps remained free of border cells suggesting that plant species tested release border cells continuously, as they are produced. Moreover, the use of the vital stain fluorescein diacetate, confirmed Knudson's (1919) observations that metabolically active border cells are delivered into the nutrient solution of hydroponic systems.

Results from semi-commercial hydroponic systems (Tables 3 and 4), support the observations seen in model hydroponic cultures. In commercial hydroponic systems, plants are drip-irrigated frequently for a 10 h period during the day, with irrigation frequencies of 3-4 times per hour. Root tips analyzed confirmed that this constant irrigation left root tips largely free of border cells. Border cells remains on root tips sampled after overnight without irrigation.

These results suggest that fundamental differences may exist in border cell function in hydroponics, compared with soil ecosystems. One prediction of this hypothesis is that due to the constant production and release of border cells, defense 58

mechanisms of border cells are no longer functional in hydroponic systems because of the absence of border cells surrounding the root tip leave it susceptible to pathogen infection. Another prediction of this hypothesis is that a larger proportion of fixed carbon would be translocated to roots, consequently increasing the rhizodeposition rate (Lynch and Whipps, 1991). If these predictions are true, then production of border cells in hydroponics may be deleterious to crop production.

CONCLUSION

Results confirmed that border cells are released into nutrient solution in model hydroponic culture. A cost-benefit analysis to determine the impact of this process on plant growth, development, and disease response throughout the growing season is needed. Methods to control border cell production may facilitate crop improvement.

59

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Table 1. Plant species tested in model hydroponic systems.

Plant species Pisum sativum L. “Little Marvel” 1 Medicago truncatula Zea mays L. “Golden Bantam” 2 Lycopersicon esculentum L. “Mariachi RZ” 3 F1 hybrid L. esculentum L. “DRO 83” 4 F1 hybrid rootstock Cucumis sativus L. “Langley Hybrid” 5 Capsicum annuum L. “Crusader Hybrid” 5 Lactuca sativa L. “Black Seeded Simpson” 6 L. sativa L. “Paris White Romain” 7 1 Meyer Quality Seeds, 2 Vegetable Seed Warehouse, 3 Rijk Zwaan, 4 De Ruiter, 5 Rogers, 6 Lawn and Garden, 7 Burpee.

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Table 2. Border cell production from plant species in model hydroponic cultures.

Plant species # of border cells per root tip # of border cells per root tip in Agar in water Corn, Z. mays 3,500 900 Alfalfa, M. truncatula 2,090 1,970 Pea, P. sativum (25 °C) 4,700 2,560 Pea, P. sativum (15 °C) 5,870 2,100 Cucumber, C. sativus 1,440 4,870 Tomato, L. esculentum 360 1,010 Tomato rootstock, L. 135 250 esculentum Lettuce, L. sativa 170 40 Pepper, C. annuum 6 165

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Table 3. Presence of border cells attached to the root tips of different greenhouse crops grown in aggregate hydroponics (CEAC‟s greenhouse).

Plant Roots Roots with Roots without Roots with Roots species sampled Bcells Bcells Bcells (ON without (hydroponics) (hydroponics) on agar Bcells plates) (ON on agar plates) Cucumber 32 11 21 21 6 Pepper 32 10 22 16 11 Tomato 70 22 48 No tested No tested

ON = overnight; Bcells= border cells

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Table 4. Presence of border cells attached to root tips of tomato plants irrigated with high and low electric conductivity (EC).

Plant Roots Roots Roots Roots Roots Roots species sampled with without sampled with without Bcells Bcells Bcells Bcells (7 am) (7 am) (6 pm) (6 pm) Tomato 7 2 5 12 0 12 (high EC) Tomato 11 4 7 6 0 6 (low EC)

Bcells = border cells

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Figure Captions:

Figure 1. Normal separation of border cells. Normal pea root tip (A). Pea root tip in free water showing normal separation of border cells (B).

Figure 2. Cucumber root tip showing a sheath of border cells formed after days of culture in model hydroponics. Normal microscopic visualization (A). (B) Visualization using

India ink stain (B).

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Figure 1.

A B

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Figure 2.

A B

70

APPENDIX B

PROTEINS AMONG THE POLYSACCHARIDES: A NEW PERSPECTIVE ON

ROOT CAP ‘SLIME’

Fushi Wen

Gilberto Curlango-Rivera

Martha C. Hawes

Plant Signaling and Behavior (2007), 2(5): 410-412.

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APPENDIX C

CONTRIBUTION OF THE ROOT CAP TO SOIL FERTILITY:

EXTRACELLULAR PLANT LECTINS

Gilberto Curlango-Rivera

Gabriela Albala

Jim P. Kemp

Denise V. Duclos

Martha C. Hawes

Soil Fertility (2009), Lucero DP and Boggs JE (eds.), Nova Science Publishers, Inc. pp.

65-79. ISBN: 978-1-60741-466-7. 76

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APPENDIX D

TRANSIENT EXPOSURE OF ROOT TIPS TO PRIMARY AND SECONDARY

METABOLITES: IMPACT ON ROOT GROWTH AND PRODUCTION OF

BORDER CELLS

Gilberto Curlango-Rivera

Denise V. Duclos

Jean J. Ebolo

Martha C. Hawes

Plant and Soil (2010), 332: 267-275.

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APPENDIX E

ROOT TIPS MOVING THROUGH SOIL: AN INTRINSIC VULNERABILITY

Gilberto Curlango-Rivera

Martha C. Hawes

Plant Signaling and Behavior (2011), 6(5): 726-727.

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APPENDIX F

EXTRACELLULAR DNA: THE TIP OF ROOT DEFENSES?

Martha C. Hawes

Gilberto Curlango-Rivera

Fushi Wen

Gerard J. White

Hans D. VanEtten

Zhongguo Xiong

Plant Science (2011), 180: 741-745.

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APPENDIX G

ALTERED ROOT TIP MORPHOLOGY IN PEA HAIRY ROOTS WITH

ALTERED EXPRESSION OF A BORDER CELL SPECIFIC GENE

Fushi Wen

Gilberto Curlango-Rivera

Lindy A. Brigham

Martha C. Hawes

Draft manuscript in progress to be submitted to Annuals of Botany.

115

Altered root tip morphology in pea hairy roots with altered expression of a border cell specific gene.

Fushi Wen, Gilberto Curlango-Rivera1, Lindy A. Brigham, Martha C. Hawes*

Draft manuscript in progress to be submitted to Annuals of Botany.

Author affiliation:

1Division of Plant Pathology and Microbiology, Controlled Environment Agriculture

Center. School of Plant Sciences, University of Arizona, Tucson, AZ, USA.

*Corresponding author; Department of Soil, Water, and Environmental Science,

University of Arizona, Tucson, AZ, USA; E-mail [email protected]

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Background and Aims. Root tips of higher plants are dynamic sensory organs. Three distinct tissues within the apex--the root cap, root apical meristem, and elongation zone-- control root development. Surrounding the apex in most species are populations of root border cells programmed to detach from the root cap periphery into the external environment. Gene expression in border cells is markedly distinct from that of the root.

Methods. mRNA differential display was used to identify border cell specific sequences.

Expression was confirmed using RNase protection assay, Southern and mRNA northern blot analysis. Reporter gene and antisense mRNA were expressed in transgenic hairy roots.

Key results. BRD13 was identified as a low abundance message expressed constitutively in border cells but not in leaves, stems, or roots without border cells. The predicted protein shares sequence similarity with flavin binding proteins. Transgenic hairy roots expressing antisense mRNA exhibited abnormal growth and morphology. Root tip exposure to riboflavin and other flavins had no effect on border cell production or viability, or on direction or rate of root growth.

Conclusions. Flavin binding proteins play key roles in development, defense, and local auxin biosynthesis. Altered expression of a border cell specific gene with a putative flavin binding motif resulted in altered root morphology and direction of growth.

117

INTRODUCTION

Root growth occurs by the generation of cells within the apical meristem followed by rapid growth of the new cells within the region of elongation. A second, independently regulated meristem generates cells of the root cap. The root cap, at the apex, controls direction of growth in response to gravity and environmental stimuli. Root border cell populations are programmed to separate from the root cap periphery into the external environment. Root border cells, previously termed 'sloughed root cap cells' are programmed to detach as a population of single cells from the root periphery into the external environment. These populations constitute a differentiated tissue which influences microbial colonization in a manner which appears to be parallel to that of mammalian neutrophils (Hawes and Brigham, 1992; Hawes et al. 2011; Wen et al. 2009).

As such, genes expressed in border cells potentially provide a tool for delivery of chemicals into the root tip region where water and nutrient uptake as well as infection by pathogens and symbionts is initiated (Lilley et al. 2010).

The separation of border cells from the root cap is the endpoint of a coordinated set of processes within the root cap. As cell division occurs in the root cap meristem, older cells are displaced towards the periphery of the cap. Tiers of cells have distinct morphological characteristics that reflect distinct functions including cell division, gravity sensing and mucilage production. Once a cell reaches the periphery of the cap, pectolytic enzymes degrade the middle lamella such that the cell, while still lodged in its original position, is physically separated from the root cap and from adjacent border cells. 118

The entire population of separated border cells is encased within a high molecular weight mucilage consisting of DNA, proteins, polysaccharides, and diverse soluble metabolites.

The cells disperse immediately into suspension if the cap is placed in water as the surrounding mucilage absorbs water.

Border cell populations synthesize a set of proteins whose profile is markedly distinct from that of proteins from progenitor cells in the root cap (Brigham et al., 1995).

The change in function and morphology that occurs when border cells separate from the cap is controlled at least in part at the level of transcription (Brigham et al., 1995). This switch was exploited to identify a gene whose expression is specific to root border cells and shares sequence homology with flavin binding proteins. Altered expression resulted in morphological changes including altered direction of growth.

MATERIALS AND METHODS

Plant material and handling

Seeds of Pisum sativum L. cv. 'Little Marvel' (Royal Seed Company, Kansas City,

MO, USA) and Zea mays cv. Golden Bantam were surface sterilized and germinated as described (Brigham et al., 1995). Border cells were isolated from the root tips of seedlings when the radicle was 2.5 cm long. Each root tip was immersed for 30-60 seconds in 2 mL of sterile distilled water which was agitated to release the cells into suspension (Hawes and Pueppke, 1989). Border cell preparations were assayed for 119

microbial contamination by plating samples onto plates with solidified Luria broth (10% tryptone (w/v), 5% yeast extract (w/v), 5% NaCl (w/v)); any samples that developed bacterial or fungal colonies were discarded.

mRNA differential display

mRNA differential display was used to identify border cell-specific messages, by comparing mRNA patterns of root border cells with those of cells in the root tip (Liang and Pardee 2007). First strand cDNA was synthesized from either 100 ng of poly A+- mRNA or 200 ng of total RNA by Superscript reverse transcriptase (Gibco-BRL Co.,

Bethesda, MD, USA). Total RNA was treated with RNase-free DNase I (Ambion Inc.,

Austin, TX, USA) to remove chromosomal DNA contamination. Poly A+-mRNA was isolated using the PolyATtract mRNA isolation system (Promega Co., Madison, WI,

USA). First strand cDNA synthesis was primed by one of the T12MN primers (M stands for the degenerate primer except T and N is one of the four dNTPs). A portion of this first strand reaction was used for polymerase chain reaction (PCR) amplification using sets of the arbitrary 10-mer primer for the 5'-end and same T12MN primer for the 3'-end. PCR was performed with Taq DNA polymerase (exonuclease-) (Boehringer Mannheim Corp.,

Indianapolis, IN, USA) and [-35S]dATP for 40 cycles at 940C for 30 s, 400C for 2 min, and 720C for 30 s, and 5 min extension at 720C. The amplified PCR products were size- fractionated on a 6% denaturing PAGE gel for 4 h. After drying, the gel was exposed to

XAR-5 film. Each set of experiments was repeated three times with independent batches of RNA samples, as described (Brigham et al. 1995). One mRNA found to be specific to 120

border cells, designated BRD13, was subjected to detailed sequence and functional analysis.

mRNase protection assay and Southern blot analysis

Radioactive riboprobe of BRD13 was generated by in vitro transcription of cDNA with 32P-CTP. For RNase protection, the radioactive riboprobe was hybridized with 20 ug of total RNA from different tissues. After RNase A/T1 treatment, the resulting radioactive riboprobe was analyzed by a denaturing polyacrylamide gel. After drying of the polyacrylamide gel, X-ray film was exposed for 1-24 hour. As a control to document equal loading of total RNA in each lane, ribonuclease protection of PsUBC4 mRNA (pea ubiquitin conjugating enzyme) (Brigham et al. 1995; Woo et al., 1994) was also performed.

Cloning and sequencing of cDNA

Using BRD13 as a probe, a -ZAP (Strategene) cDNA library of whole root tips

(root cap and border cells) was screened. A full length cDNA was designated BRD13 and sequenced using the T7 and T3 promoter sequences of the pBlueScript vector

(Strategene). Internal primers were designed to complete the sequencing. Sequencing was performed 3 times in each direction to verify accuracy. Sequence analysis was performed using the Wisconsin GCG software. Southern blot analysis was performed using the full length cDNA, as described.

121

Construction of transformation vectors and transgenes

The promoterA 1244 bp fragment of BRD13 was PCR-amplified with primer 1,

5‟-ATCAGGAGCTCAGACTATGGTGACGCCATTG-3‟ containing a created Sst1 site and primer 2, 5‟-AGCTCCCGGGTCTATGAGAGAGAATTAATTT-3” containing a created SmaI site (position 10 and 1254 in the BRD13 sequence, respectively). This

PCR-amplified fragment was digested by SmaI and SstI simultaneously, then inserted in antisense orientation under the control of the BRD13 promoter in vector pBI121 whose

GUS gene was removed by digestion with SmaI and SstI. The resulting constructs, or reporter genes using GFP, were mobilized into A. rhizogenes strain R1000 through triparental mating using pRK2013 as helper strain and kanamycin as selectable markers

(Ditta et al., 1980; Tieman et al., 1992).

Antisense mRNA expression in pea hairy roots

Constructs were transformed into pea stems using A. rhizogenes R1000 containing a kanamycin resistance gene as a selectable marker. Pea seeds were sterilized as described at the beginning of methods. Sterilized seeds were germinated on 1% water agar in magenta vessels at 24oC in the dark until hypocotyls reached approximately 1 cm in length. Subsequently, seedlings were incubated at 24oC with a 16 hr light period.

Sterile stem segments (1.5-2 cm long) were transferred aseptically in an inverted position to TM-1 solid medium (Shahin, 1985) containing 500 mg/L carbenicillin. A drop of bacterial suspension (A600 is about 1.0) was then placed on the upper surface of the stem section with a 10 l Pippetman. The plates were incubated at 24oC, with a 16 hr 122

photoperiod, and 2 E m-2sec-1 light intensity. Ten to 15 days after inoculation, hairy roots emerge from the upper surface of the inoculated stem (Nicoll et al., 1995).

One to 2 weeks after emergence, primary hairy roots were excised and cultured on hormone free Gamborg‟s B5 medium (Sigma), pH 5.8, with 1% Difco agar, 100 mg kanamycin, 500 mg carbenicillin, and 20 g of sucrose per L. Putative positive hairy root clones (selected on kanamycin) were subcultured once a month on the same medium without kanamycin. Two to four weeks after subculture, sufficient material was available for RNA gel blot analysis. For confirmation of transformation, genomic DNA from independent transformants was digested with BamHI and analyzed by DNA gel blotting using a 32P-labeled CaMV 35S promoter fragment as a probe. The frequency of transformed stems that gave rise to hairy roots was approximately 85%. Results reported here represent ten independent transformations conducted over an 18-month period, with dozens of replicate plate cultures and hundreds of roots.

Transient assay for root tip responses to flavins

Impact of riboflavin, lumichrome, flavin adenine dinucleotide (FAD) and flavin adenine mononucleotide (FMN) on root growth, development, border cell production, viability, and slime production, and susceptibility to infection were assayed as described

(Curlango-Rivera et al. 2010).

RESULTS AND DISCUSSION

123

Border cell expressed sequences were compared to root tip expressed sequences using mRNA differential display (Brigham et al. 1995). BRD13, consisting of 250 base pairs of the 3‟ end of a target cDNA, was one of several sequences identified in border cells but not the root tip. This sequence was further analyzed to verify specificity. Using

Brd13 as a probe to identify the cDNA, full length BRD13 was cloned and sequenced

(Genbank Accession Number AF139187). The cDNA of BRD13 consists of 1723 nucleotides. Southern blot analysis of pea genomic DNA revealed the presence of a single major band (Figure 1).

The largest open reading frame (frame 3) consists of 975 nucleotides. There are two

ATG start sequences at the beginning of the sequence consistent with other pea genes

(Zhu, 2000). An unknown protein from Arabidopsis Chromosome III (Accession #

AAF00629.1) shows 70% identity within a 200 amino acid region in the middle of the longest reading frame for the deduced protein sequence – nucleotides 144 to 743. Motifs identified within the coding region include 3 casein kinase II (CK-2) phosphorylation sites, 2 N-myristoylation sites, 5 protein kinase C phosphorylation sites, and a flavin binding protein site. RNase protection was used to analyze the expression patterns.

BRD13 expression was detected only in border cells and root tips which included border cells (Figure 2). Northern blot analysis yielded the same outcome (Figure 3). The

BRD13 promoter linked to green fluorescent protein (GFP) confirmed the pattern of border cell specific expression (Figure 4).

Root tips of some species, including legumes, produce riboflavin which is found in the extracellular environment (Bonner 1942, Higa et al 2010, Rovira and Campbell 1961, 124

Susin et al. 1994, Vorweiger et al. 2007, Welkie et al. 1988). Riboflavin and its derivatives have been implicated in root infection by Rhizobium, and in activation of defense responses (Liu et al. 2010, Mishina and Zeier 2006, Ramamani et al. 2008, Streit et al. 1996). Experiments were carried out to examine the possibility that a border cell specific flavin binding protein might condition altered defense response in response to dynamic changes in flavins (Table 1, 2). No signficant changes in response of corn or pea roots to fungal infection were seen.

Soluble chemicals in the mucilage surrounding the root tip and border cells have been implicated as modulators of root growth (Baluska et al. 1996). Altered riboflavin in cultured roots has been reported to result in changes in root growth (Drew et al. 1993,

Gorst et al 1983). Moreover, flavin binding proteins increasingly are recognized as key regulators of local auxin biosynthesis and associated growth responses (Chandler 2009,

Forneris et al. 2009, Roje 2007, White et al. 1988). Transient changes in riboflavin and derivatives resulted in no significant alterations in corn and pea root growth, border cell production and viability, or response to gravity (Table 3, 4). However, transformed hairy roots expressing antisense mRNA exhibited marked phenotypic changes commonly observed in response to auxin (Figure 5). The root tip appears enlarged with altered morphology including direction of growth. It will be of interest in future studies to determine if BRD13 plays a role in local auxin synthesis fostering dynamic changes in tip growth.

125

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Table 1. Transient efffect of riboflavin on pea infection by Nectria haematococca. Pea seedlings inoculated at 24 h post riboflavin treatment.

Treatment Seedling

1 2 3 4 5

H2O 0 0 0 0 0

0.1 mM 0 0 0 0 0

1 mM 0 0 0 0 0

2mM 0 0 0 0 0

H2O + N 3 3 3 3 EZ 2

H2O + 0.1 2 3 3 EZ 2 EZ 2

mM

H2O + 1 3 T 3 EZ 1 EZ 2 3 3

mM

H2O + 2 3 EZ 2 3 3 EZ 2

mM

T = root tip, EZ = elongation zone, 0 = no symptoms, 1 = brownish, 2 = either T or EZ brownish/blackish, 3 = both T and EZ brownish/blackish.

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Table 2. Transient efffect of riboflavin on pea infection by Nectria haematococca. Pea seedlings inoculated at 2 h post riboflavin treatment.

Treatment Seedling

1 2 3 4 5

H2O 0 0 0 0 0

0.1 mM 0 0 0 0 0

1 mM 0 0 0 0 0

2mM 0 0 0 0 0

H2O + N 3 T 2 EZ 1 3 3

H2O + 0.1 mM EZ 2 T 1 EZ 1 3 EZ 2

H2O + 1 mM EZ 2 3 3 3

H2O + 2 mM T 2 T 2 T 2

T = root tip, EZ = elongation zone, 0 = no symptoms, 1 = brownish, 2 = either T or EZ brownish/blackish, 3 = both T and EZ brownish/blackish.

134

Table 3. Effect of flavins on root length (RL), viability (V) and border cell (BC) number of pea at 24 h.

Trial Treatment n RL (mm) A V % BC # Gravity ± SD test 1 H2O 3 9.3 ± 0.6 85 5633 0.1 mM 3 11.7 ± 4.0 86 7333 Riboflavin 1 mM 3 12.3 ± 2.9 84 6100 Riboflavin

2 H2O (8 h) 1 1940 0.1 mM 2 2450 Riboflavin (8 h) 1 mM 3 1870 Riboflavin (8 h) H2O (24 h) 6 4690 ± 1117 0.1 mM 7 3890 ± 495 Riboflavin (24h) 1 mM 8 3530 ± 976 Riboflavin (24 h)

3 H2O 10 8.1 ± 4.9 90 ± 1.4 5870 ± 1853 0.1 mM 10 9.9 ± 3.4 88 ± 4.2 5440 ± 792 Riboflavin 1 mM 10 13.1 ± 4.2 89 ± 2.1 4640 ± 1075 Riboflavin

4 H2O 10 11.5 ± 2.8 88 ± 1.4 5220 ± 198 5 nM 10 14.1 ± 4.0 88 ± 4.2 4980 ± 85 Lumichrome 1 μM 10 13.5 ± 4.2 82.5 ± 2.1 5800 ± 226 Lumichrome H2O (48 h) 10 37.6 ± 5.2 5 nM 10 44.6 ± 5.1 Lumichrome (48 h)

135

Table 3. Continued.

Trial Treatment n RL (mm) A V % BC # Gravity ± SD test 5 H2O 4 11.5 ± 3.5 64 4325 1 μM FAD 4 14.3 ± 1.5 57 3200 100 μM FAD 4 9.3 ± 1.5 76 4175

6 H2O 4 13.5 ± 1.7 39 5025 + 1 mM FAD 4 13.5 ± 7.4 51 5800 + 10 mM FAD 4 11.0 ± 3.6 64 6050 +

7 H2O 4 11.5 ± 3.5 64 4325 1 μM FMN 4 10.0 ± 2.2 64 3900 100 μM FMN 4 12.0 ± 3.7 67 4750

8 H2O 4 13.5 ± 1.7 39 5025 + 1 mM FMN 4 14.0 ± 3.9 49 5000 + 10 mM FMN 4 13.8 ± 6.8 75 5000 +

Data show the average ± standard deviation (Avg ± SD) from at least two replicates; other data not showing standar deviation are from a single observation.

136

Table 4. Effect of flavins on root length (RL), viability (V) and border cell (BC) number of corn at 24 h.

Trial Treatment n RL (mm) V % BC # Gravity test

1 H2O 4 13.3 ± 19 97 1325 +

1 μM FAD 4 21.0 ± 6.8 100 1150 +

100 μM FAD 4 16.5 ± 2.4 93 1300 +

2 H2O 4 20.3 ± 4.2 100 850 +

1 μM FMN 4 21.0 ± 4.1 100 1800 +

100 μM FMN 4 19.0 ± 1.6 100 1850 +

3 H2O 4 13.5 ± 1.3 100 1150 +

100 μM Riboflavin 4 15.3 ± 3.2 100 975 +

1 mM Riboflavin 4 12.8 ± 4.0 100 925 +

Data show the average ± standard deviation from four replicates; other data not showing standard deviation are from a single observation.

137

Figure captions:

Figure 1. Southern blot analysis.

Figure 2. RNase protection assay.

Figure 3. mRNA northern.

Figure 4. Border cell specific expression of GFP under the control of the BRD13 promoter.

Figure 5. Morphological changes in transgenic hairy roots expressing BRD13 antisense mRNA under the control of the BRD13 promoter.

138

Figure 1.

139

Figure 2. RNase Protection of BRD13

1 2 3 4 5 6 7 1 = Leaves 2 = Stems 3 = Roots 4 = Roots without root tips 5 = Border cells 6 = Internal control 7 = Undigested Probe ( ) and Internal control , Indicates the protected probes

, Internal control

140

Figure 3.

141

Figure 4.

142

Figure 5.

A B

C D