Leaf Expansion – an Integrating Plant Behaviour

Plant, Cell and Environment (1999) 22, 1463–1473

COMMISSIONED REVIEW Leaf expansion – an integrating plant behaviour

E. VAN VOLKENBURGH

Department of Botany, Box 355325, University of Washington, Seattle, WA 98195, USA

ABSTRACT the phase of leaf development contributing most to surface area and shape of the lamina. Leaves expand to intercept light for photosynthesis, to take Leaves can be considered, functionally, as iterated green up carbon dioxide, and to transpire water for cooling and antennae specialized for trapping light energy, absorbing circulation. The extent to which they expand is determined carbon dioxide, transpiring water, and monitoring the envi- partly by genetic constraints, and partly by environmental ronment. The leaf canopy may be made up of many or few, conditions signalling the plant to expand more or less leaf small or large leaves. They may be simple in shape, like the surface area. Leaves have evolved sophisticated sensory monocotyledonous leaves of grasses or dicotyledonous mechanisms for detecting these cues and responding with leaves of sunflower and elm. Or leaves may be more their own growth and function as well as influencing a complex, with intricate morphologies as different as the variety of whole-plant behaviours. Leaf expansion itself is delicate, sensitive structure of the Mimosa leaf is from the an integrating behaviour that ultimately determines canopy magnificent blade of Monstera. Some species, such as development and function, allocation of materials deter- cactus, do not develop leaves at all, but carry out leaf func- mining relative shoot : root volume, and the onset of repro- tions in the stem. Other plants display small leaves in order duction. To understand leaf development, and in particular, to conserve water, but with the consequence of limiting how leaf expansion is regulated, we must know at the mol- photosynthetic productivity. ecular level which biochemical processes accomplish cell Plant species can be lumped into groups of slow-growers growth. Physiological experimentation focusing on ion and rapid-growers, with contrasting ecological strategies fluxes across the plasmamembrane is providing new mol- paralleled by differences in leaf area (Lambers, Poorter & ecular information on how light stimulates cell expansion Van Vuuren 1998). Slowly growing species live in harsher in some dicotyledonous species. Genetic analyses in Ara- environments, and their low relative growth rate can be bidopsis, corn, and other species are rapidly generating attributed to production of small, or slowly expanding leaf a list of mutations and enzyme activities associated with area. More rapidly growing species may have evolved from leaf development and expansion. Combination of these slowly growing ones, or vice versa, and have a faster rela- approaches, using informed physiological interpretations of tive growth rate associated with more rapid development phenotypic variation will allow us in the future to identify of leaf area (Poorter & van der Werf 1998). Size and shape genes encoding both the processes causing cell expansion, of leaves is to a large extent genetically controlled, imply- and the regulators of these events. ing that these are adaptive features lending advantage to plants in specific habitats. Yet, developmental flexibility Key-words: cell expansion; ion transport; osmoregulation; exists even within an individual plant, with leaf size and phytochrome; proton pump; wall extensibility. shape depending on environmental circumstances prevailing during leaf formation. Leaves will remain small if cir- INTRODUCTION cumstances are unfavourable, or they will expand a large surface area when the necessary nutrients are available The diversity of leaf shapes and sizes is a compelling and (Chapin 1991). Plants have developed sophisticated sensing curious feature of our natural surroundings. Leaves attract mechanisms for determining the availability of resources in our attention, and their many distinct characteristics have the soil, atmosphere and light. They respond to these cues aided the catagorization of plants into taxonomic groups. by regulating biochemical processes that control leaf The functions of all these shapes and sizes remain an eco- expansion, developing their leaf canopies accordingly. logical and evolutionary puzzle, one which will be more Given the prominence of leaves in our environment, approachable once we understand the cellular mechanisms their importance for plant function, and our fascination at work in creating the leaf blade. These mechanisms will with their appearance, it is astounding that so little is known be considered here under the description of leaf expansion, about the physiological processes giving rise to these organs. Genetic studies have identified a small but growing Correspondence: Fax: 1 206 6851728; number of genes involved in controlling leaf development, e-mail: [email protected] and physiological studies have described a few biochemical

© 1999 Blackwell Science Ltd 1463 1464 E. Van Volkenburgh mechanisms involved in cell division and expansion in for nutrients, thus attracting sufficient carbohydrate and stems and roots. But most studies of leaf function have nitrogen to signal continued division cycles? What if the focused on photosynthetic capacity, light interception, and expansion of meristematic cells were stalled for a time responses of leaves to environmental stresses (sometimes during mid-cycle, perhaps by a transient water deficit – including inhibition of growth), without including detailed would that be enough to reduce carbon/nitrogen import study of the mechanisms regulating leaf expansion. It is and prevent completion of the cell cycle? What do we know, the intent of this brief, and selected, review to promote actually, about the minute-to-minute regulation of cell thoughtful investigation into the mechanisms controlling expansion, and the dependence of the cell cycle on cell leaf expansion. With emerging molecular genetic methods, expansion? it should soon be possible to determine the biochemical Cellular mechanisms for controlling growth are likely to basis for both the processes that drive leaf expansion, and be genetically redundant. This means that identifying phe- the signalling pathways that control it. notypes for genetic variants may be difficult without precise physiological information. The following focuses on mechanisms that may explain short-term regulation of cell Regulation of leaf expansion at the growth, with the recognition that meristematic cells must cellular level enlarge in volume prior to mitosis and cell division. An Once a leaf primordium has been initiated within an apical emphasis on the molecular basis for the physiological meristem, the newly formed organ embarks on a predic- processes controlling cell expansion will help us, in the long table developmental programme leading to the formation run, to identify functions of genes known to affect leaf of a small but recognizable leaf. This early phase of leaf growth and morphology. development is largely accomplished by production of new cells that become anatomically committed to form orga- Biophysical considerations nized tissues creating a dorsiventral structure. At this stage, in dicotyledonous leaves, the blade and petiole become dis- The question of how leaf cells, and for that matter, plant tinct, and vascular patterning is evident. In monocotyledo- cells in general, enlarge is a complex one without many nous leaves, the blade grows to a considerable length before answers as yet. For the last several decades, the regulation the sheath is formed, but even at early stages the vascular of cell expansion has been described and investigated from pattern is clear. As is the case for growing roots and stems, a biophysical point of view starting with the theoretical it is possible in leaves to identify zones of cell division and treatment of Lockhart (1965). Since then, the theory has zones of cell expansion, although these zones are much been amplified and revised (e.g. Passioura & Fry 1992) but more distinct in monocot leaves. In dicot leaves, the in general, these revisions share a similar basis. Cell growth processes of cell division and expansion may overlap spa- theory is based on the observation that the relative growth tially as well as temporally to a considerable extent (Dale rate of cells is a function of the internal hydrostatic or 1988). No matter how much cell division occurs, it is the turgor pressure in excess of the yield threshold of the cell process of cell expansion that creates the surface area of wall, and the extensibility of the cell wall. Turgor pressure the mature organ. Even in the zone of cell division, cells itself is a dependent variable, determined by the osmotic must increase in volume prior to mitosis and cytokinesis gradient attracting water into the cell, the reflection coeffi- (Ray 1987). After cell replication ceases, leaf cells continue cient of the plasmamembrane, the hydraulic conductance of to expand and may obtain a final volume 20 to 50 times that the membranes to water, and the biomechanical properties of their meristematic progenitors (Maksymowych 1973; of the cell wall material (Cosgrove 1981). In theory, growth Becraft 1999). rate could be controlled by any one of these variables, or Variation in leaf size has been attributed to differences by complex changes involving several. in cell number, or cell size, or combinations of the two Over the past 20 years, emphasis has shifted from the (e.g. Granier & Tardieu 1998; see also references cited in view that changes in leaf growth rate are mostly due to fluc- Humphries & Wheeler 1963). In general, it is known that tuations in turgor (e.g. Boyer 1968;Wenkert, Lemon, & Sin- cell division in plants is correlated with carbohydrate clair 1978). It is now also recognized that leaf cells can supply, and that leaves starved for photosynthate or other regulate cell wall extensibility and thus the rate of expan- nutrients will develop fewer cells than those growing amid sion (Van Volkenburgh & Cleland 1980; Taylor et al. 1995; plenty (Dale 1988; Chapin 1991).The mechanisms for main- Bogoslavsky & Neumann 1998). The elegant data of Boyer taining cell divisions, giving rise to a larger leaf, or to cur- showing the effect of water deficit on various physiological tailing division and inhibiting leaf growth, are not clear. processes (Boyer 1970) focused attention on the vulnera- Considerable information is being reported on regulation bility of leaf expansion to reductions in turgor (Hsiao 1973). of cell cycle in plant tissues and in growing leaves (Francis This way of thinking was challenged by the advent of more 1998), yet the connection between these data, and regula- precise methods for measuring turgor, in particular the tion of leaf expansion rate are unclear. Perhaps this is development of the micropressure probe (Husken, Steudle because we do not yet understand the interdependence of & Zimmermann 1978), and the recognition that growth- cell expansion and cell division. Is it possible, for instance, related turgor must be measured directly in the cells that that rapidly expanding meristematic cells act as strong sinks are growing (Michelena & Boyer 1982). Gradually it was

recognized that leaf growth rate could change dramatically rate in elevated CO2 (Ranasinghe & Taylor 1996). Given with no change in leaf turgor (e.g. Shackel, Matthews & the complexity of the plant primary cell wall, and the fact Morrison 1987; Van Volkenburgh & Boyer 1985). This that the growing cell must continually be making new wall shifted attention to the regulation of wall extensibility (e.g. to enclose its ever-increasing volume, it is likely that the Neumann 1995) as a mechanism for regulating the rate of composition of the extruded wall material is regulated such leaf expansion, and in recent years several mechanisms that wall extensibility (and growth rate) is consistent with have been described for cellular regulation of wall yielding prevailing environmental conditions and capacity for the properties (Cosgrove 1997). But, the relevant stress on the leaf tissue to feed itself. walls is not turgor per se, but turgor (P) in excess of the yield threshold (Y) (P – Y). Changes in either wall prop- Osmotic regulation erty, extensibility or yield threshold, could alter growth rate without any change in turgor.Alternatively, if (P-Y) is small Cells expand by accumulating solutes, absorbing water, (in the range of 0·1 MPa, Van Volkenburgh & Cleland generating turgor pressure, and extending the cell wall. 1986), growth rate could be dramatically affected by small Although the evidence strongly suggests that changes in increases or decreases in apoplastic solute concentration cell wall biochemistry play a significant role in determining causing undetectable changes the absolute value of turgor. the rate of cell expansion, it is likely that cells will utilize more rapidly responsive pathways to regulate growth rate over the short-term. The most rapid response of growing Wall extensibility cells to light, or hormone application, is an electrical depo- When light stimulates expansion of bean or pea leaves, it larization of the plasmamembrane (Elzenga, Prins & Van does so by increasing the rate of proton efflux from epi- Volkenburgh 1995; Keller & Van Volkenburgh 1996a). This dermal and mesophyll cells (Van Volkenburgh & Cleland response could either be part of the signal transduction 1980; Staal et al. 1994). It is thought that a proton pump in pathway leading to growth rate regulation (Ward, Pei & the plasmamembrane, most likely a proton ATPase, carries Schroeder 1995), or more likely, reflect a part of the growth out this activity (Linnemeyer, Van Volkenburgh & Cleland mechanism itself. 1990; Stahlberg & Van Volkenburgh 1999). Acidification of Membrane potential of growing leaf cells is strongly the apoplast loosens the cell wall, making it more respon- polarized (– 120 to – 220 mV; Elzenga et al. 1995; Stahlberg sive to stress imposed by turgor (Rayle & Cleland 1992). It & Van Volkenburgh 1999) as a result of the activity of the is not clear how lower pH loosens cell walls, but one possi- proton pump. This strong membrane polarization is essen- bility is that protein molecules called expansins (Cosgrove tial for passive uptake of cations, and is a consequence of 1998) are generally located in growing cell walls.At low pH, the proton motive force used in cotransport of sugars and these proteins release hydrogen-bonding between cellulose amino acids. Active efflux of protons hyperpolarizes the microfibrils and hemicelluloses and allow them to slip past membrane beyond (more negative than) the Nernst poten- one another (Keller & Cosgrove 1995) relaxing stress on tial for potassium (Ek) as long as the passive conductance the wall. Another mechanism for causing wall loosening for ions is minimal. An increase in conductance for potas- may involve xyloglucan endotransglycosylases, XETs (Fry sium, by opening K channels for example, will depolarize + et al. 1992), which accomplish a breakage and reformation the membrane potential to Ek because K will be attracted of glycosidic linkages among polymers in the wall. XETs into the cell. Similarly, an increase in conductance for are most active at pH 6, and are not thought to participate anions will depolarize the cell because these ions will be in acid-induced wall loosening (Purugganan, Braam & Fry repelled from the cytoplasmic side of the plasmamembrane 1997). and flow into the apoplast. Cellular control of ionic con- The pH of the wall is only one of the variables that can ductances, as well as concentrations of ions on either side influence acid-induced wall loosening; the other is the of the membrane, will determine the membrane potential. capacity for the wall to loosen when acidified (Cleland, Cos- Leaf cells are ‘irritable’ in an electrical sense, and in par- grove & Tepfer 1983;Van Volkenburgh, Schmidt & Cleland ticular they are highly sensitive to light which elicits a 1985). In principle, the wall could be loosened by increas- complex electrical response. In growing leaves, light stimu- ing proton efflux or in some other way lowering wall pH, lates a transient calcium influx, chloride efflux and or, with the pH remaining constant the composition of wall membrane depolarization (Elzenga et al. 1995; Shabala & polymers could be modified making it more responsive to Newman 1999). This also occurs in elongating stems which pH-sensitive loosening mechanisms (e.g. increasing the require anion channel activity for growth regulation (Cho expansin level). Although it has not yet been shown that & Spalding 1996). Light also stimulates proton efflux cells can increase growth rate by synthesizing and export- which hyperpolarizes the membrane in growing pea leaves ing more expansin to the wall, this possibility is suggested (Stahlberg & Van Volkenburgh 1999), and a transient by the demonstration that expansin placed upon the apical calcium influx depolarizing the membrane in Chara (Wayne meristem can cause formation of a leaf buttress (Fleming 1994) and in growing moss protonema (Ermolayeva, et al. 1997). Similarly, it has not been shown that synthesis Sanders & Johannes 1997). The depolarization of growing or export of XET to the wall regulates leaf growth rate, but leaf cells by light is transient in photosynthetically compe- the activity of XET has been correlated with leaf growth tent cells (Elzenga et al. 1995; but see Stahlberg & Van

Volkenburgh 1999), and is similar in duration to the lag (Assmann & Shimazaki 1999). In both cell types, re- phase preceding acceleration of growth. The lag phase sponses to light are complex and include both photo- could be explained by transient opening of anion channels synthetic responses mediated by chlorophyll, and non- which allow efflux of chloride (Elzenga & Van Volkenburgh photosynthetic responses. One of the initial problems in 1997a,b) followed by cation efflux and a temporary reduc- determining how light stimulates leaf expansion was to tion in turgor that slows or stops growth. At the end of the describe the photobiology of this response and identify the lag period, closure of anion channels, stimulation of the photoreceptors involved. Early work showed that for leaf proton pump, membrane hyperpolarization, acidification of strips containing both mesophyll and epidermal layers, both the apoplast, solute uptake and cell expansion occur simul- blue light- and red light-mediated pathways could support taneously. Inhibition of the pump, but not of the depolar- cell expansion apart from chlorophyll-mediated photosys- ization, prevents light-stimulated growth (Stahlberg & Van tem II (Van Volkenburgh et al. 1990). In Arabidopsis cotyle- Volkenburgh 1999). dons, cryptochrome1 mediates blue light-stimulated cell Ion fluxes across the plasmamembrane will affect the expansion via a whole plant response involving inhibition osmotic concentration of the apoplast, with its limited of hypocotyl elongation (Blum, Neff & Van Volkenburgh aqueous volume.Addition or removal of relatively few ions 1994). Phytochrome B is required in a cell-autonomous will change the apoplastic solute concentration to a much fashion for red light-stimulated cell expansion (Neff & Van larger extent than in the cytoplasm. Thus, ‘dumping’ of Volkenburgh 1994), although in some cases phytochromeB- solutes into the apoplast, or extraction of solutes from the deficient leaves expand better than wild-type leaves (Chory wall space represents an efficient way for cells to control 1992; Robson, Whitelam & Smith 1993). It has been diffi- the osmotic gradient across the plasmamembrane, and con- cult however, to determine whether phytochrome B acts sequently turgor and growth rate (Cosgrove & Cleland directly on a mechanism related to cell expansion (e.g. 1983). changing ion channel conductance). Just as likely would be Although very little is known about the regulation of the phytochrome-mediated enhancement of photosynthesis proton pump (but see, for example, recent information on and consequent fueling of growth. Recently we have the role of 14-3-3 proteins in activating this pump; Bauns- demonstrated that the stimulation of mesophyll cell growth gaard et al. 1998), it is clear that its activity is of primary by light is dependent on light-stimulated proton efflux importance for growth. The proton pump plays a dual role (Stahlberg & Van Volkenburgh 1999) and does not require during cell expansion. As discussed above, one role is to photosynthetic activity.This result is consistent with a direct lower wall pH and contribute to wall loosening. However, interaction between phytochrome, or a blue light photore- the proton pump is critical for maintaining a hyperpolar- ceptor with the proton pump. ized plasmamembrane to drive solute uptake for nutrition, The precise roles of light-stimulated ion fluxes, apart metabolism, electrical and osmotic regulation. Inhibition of from proton efflux, are obscure. To determine whether they the pump will depolarize the membrane, allowing efflux of influence wall osmotic potential, the concentrations of ions cations. It could be that pump activity is primarily regulated in the walls and their effect on cell turgor needs to be mea- for these latter reasons having to do with ion distributions sured in growing tissue. If they are primarily fluxes to across the plasmamembrane. A consequence of membrane control membrane potential, or to control cytoplasmic ion transport of an actively growing cell would naturally be content, then it might be possible to identify these roles by increased proton efflux and lowered apoplastic pH. Perhaps investigating the irreversible swelling of protoplasts. rapidly growing species evolved metabolic reactions in the Uniform sets of protoplasts are not difficult to isolate from cell walls (expression of expansins) that optimize expansion guard cells, and swell when exposed to blue light (Zeiger & of the acidic cell wall. Hepler 1977). Protoplasts isolated from etiolated wheat leaves swell upon exposure to red light which acts via phytochrome (Bossen et al. 1988), and auxin stimulates swelling Photobiological control of ion fluxes of protoplasts from oat coleoptiles (Keller & Van Volken- In growing pea epidermal cells, both red and blue light stim- burgh 1996b) dependent on availability of external potas- ulate growth by increasing the rate of proton efflux, appar- sium. Protoplast volume can increase only when membrane ently by separate mechanisms (Van Volkenburgh, Cleland vesicles fuse with the plasmamembrane increasing the & Watanabe 1990; Staal et al. 1994). It has been proposed surface area; the membrane cannot stretch appreciably that blue light stimulates the proton pump by direct inter- (Wolfe, Dowgert & Steponkus 1986). It seems likely that action between a blue-light photoreceptor and the pump regulation of vesicle fusion contributes to the rate of pro- (Elzenga 1997). Red light may influence pump activity indi- toplast swelling. Vesicle fusion, or exocytosis, is influenced rectly by modulating passive ion conductances, such as by cytosolic Ca2+ (Thiel & Battey 1998), suggesting a role calcium and potassium channels (Staal et al. 1994; Elzenga, for phytochrome-mediated calcium influx across the plas- Staal & Prins 1997). A calcium-dependent potassium mamembrane, enhancement of vesicle fusion, wall synthe- channel has been described in growing epidermal cells of sis and growth in leaf cells. pea (Elzenga & Van Volkenburgh 1994). At the cellular level, we know that for leaves of several In growing mesophyll cells, the situation is a bit more species, the rates of proton efflux, solute uptake and wall complex, and is perhaps similar to that of guard cells loosening determine the rate of cell expansion. We have

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1463–1473 Leaf expansion 1467 started to identify the molecular components, in particular shrinking are reversible (Satter 1990), but the difference the proton pump, ion channels, and expansins, that partici- among these examples may rest simply in the ability of the pate in processes leading to cell expansion. How these mol- cell wall to extend irreversibly (Wetherell 1990).When sen- ecules, which can be considered parts making up the sitive Mimosa leaflets fold downward in response to touch, motor(s) driving expansion, are connected to the switches the cells on the lower surface of the pulvini shrink rapidly – the photoreceptors and hormones regulating leaf cell because they lose solutes (potassium) and water, whereas expansion, are important questions to address. It is hoped the cells on the upper surface actively take up solutes that genetic analysis of plants displaying altered leaf growth (potassium), water, and swell. The ion movements are characteristics will lead us to these connections. However, driven by regulation of the proton pump, and resultant a genetic approach requires precise analysis of phenotype, acidification of the apoplast would cause wall loosening in which is confounded at present both by lack of detailed irreversibly swelling tissues.The circadian regulation of leaf information along the lines of what has been described movement is directed in part by red and blue light, and a above, as well as failure to identify the tissue(s) within phytochrome-dependent potassium channel activity has which the mechanisms are operating. been described in protoplasts isolated from pulvini (Kim, Cote & Crain 1993). In both flytraps and pulvini, expansion of cells is differ- Roles of tissues in leaf expansion entially expressed across the organ to accomplish an ap- A major difficulty in understanding leaf expansion is to propriate, organ-based behaviour. Development of leaf imagine how growth of the various tissues within the leaf is morphology must be similarly determined by coordinated, coordinated so that a flat dorsiventral structure is created. differential expansion of cells. By analogy with pulvini and Of course, this does not always occur, as in curly leafed flytraps, a good place to look for the coordinating mecha- spinach, or leaves on plants infected with rol genes from nism in leaf expansion is in regulation of ion distribution, Agrobacterium rhizogenes (Schmulling, Schell & Spena and in particular, potassium flux across plasmamembranes. 1988). In these cases, it appears that extension of the vas- The action potentials generating flytrap movement, and the cular tissue does not keep up with expansion of the inter- osmotic flux causing swelling of cells in both organs, are veinal tissues. Other variations in relative tissue expansion based on movement of potassium. In addition, information are less dramatic but still well-known. Consider the differ- from guard cells, in particular with respect to the control of ence in anatomical structure between sun and shade leaves, potassium fluxes, may be significant for regulation of leaf with the thicker sun leaves developing many mesophyll cell expansion. layers and elaborated periveinal tissues, while thinner The role of the epidermis in growth of organs has been shade leaves produce relatively undeveloped mesophyll, discussed (Green 1986), in particular for leaves (Becraft smaller veins and extended epidermis. Or, consider the 1999), and is considered to restrict expansion of underlying nastic behaviours of leaves exposed to pathogens or other tissues. Mature leaves of argenteum peas readily display the stresses. concept of a restrictive epidermis. In this variety of Pisum Leaf epinasty (curling under) and hyponasty (curling up) sativum, mesophyll cells become detached from the epi- are growth responses resulting in curvatures of the leaf dermis (Hoch, Pratt & Marx 1980). As the leaf matures, the blade from uneven growth of upper or lower sides of the epidermal layer stops expanding first and the expanding leaf, respectively. A stunning example of hyponasty with a mesophyll layer buckles within the two epidermal layers. If function is the rapid closure of Venus Flytraps when trig- epidermis normally restricts expansion of leaves, either the gered by a visiting insect. Closure of the trap is caused by mesophyll or vascular tissue must be responsible for driving the two halves of the leaf blade folding up along the axis of growth. In mature dicot leaves, mesophyll cells are often the main vein. Cells on the lower surface of the leaf, but not pulled apart from one another leaving considerable air the upper surface, undergo a rapid growth response to an spaces. It has been suggested that the metabolic events electrical signal generated by sensory hairs on the upper leading to mesophyll cell separation may contribute to less- surface.This growth response has been attributed to proton ening the resistance to expansion and perhaps be one of the efflux, and acid-growth of the cells on the lower surface mechanisms controlling the rate of leaf expansion (Jeffree, (Williams & Bennett 1982; but see discussion in Simons Dale & Fry 1986). 1992). Significant potassium and water flux occurs out of For the leaf to develop without tears or wrinkles, the upper, and into lower, cells. Cells on the upper surface expansion of the several tissues must be co-ordinated. One shrink, whereas cells on the lower surface increase turgor, way to accomplish this would be for the expansion of each and grow – rapidly. tissue to depend on the other, with each assigned a unique The mechanism described for closure of the traps is but interdependent role for driving expansion. For instance, similar to that of circadian and sensory leaf movements in the epidermis may restrict expansion, the veins drive grasses (Brock & Kaufman 1990) and in leaves that do not expansion, and the mesophyll may provide substrate or form pulvini (Wetherell 1990). In these cases, movement of signal to regulate both. If this were so, growth of epidermal the leaf is accomplished by irreversible swelling of tissue on cells would be limited by wall loosening mechanisms, one side, and shrinking of tissue on the other side of the whereas growth of vascular tissues would be turgor- base of the leaf. In leaves with pulvini, the swelling and regulated. This possibility is supported by several observa-

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1463–1473 1468 E. Van Volkenburgh tions: epidermal cells have lower turgor than mesophyll and Volkenburgh 1994), and as was discussed above, involves vascular cells (Fricke, Leigh & Tomos 1994), epidermal light-stimulated proton efflux from both mesophyll and layers peel off the lower surface of closing fly traps (Simon epidermal cells. The role of photosynthesis, and thus 1992), growth of leaf discs is enhanced by removal of epi- chlorophyll as the photoreceptor, has been somewhat con- dermal layers in argenteum pea (Van Volkenburgh, unpub- troversial in discussions of light-stimulated leaf growth lished observation), and photosynthesis is not required for, (Stahlberg & Van Volkenburgh 1999). Both growth and but enhances proton efflux and growth (Stahlberg & Van proton efflux are considerably enhanced when photosyn- Volkenburgh 1999). Compact mesophyll tissue would result thetic reactions are occurring. It is important to note that from failure of epidermis or vascular tissue to expand. Con- this is the result of increased photosynthetic activity within versely, air spaces among the mesophyll cells will develop the growing leaf cells themselves, not a result of increased when veins and epidermis continue to expand after the sugar supply from remote sources. mesophyll stops. Control over the osmotic force generated The growth rate of leaf cells is influenced by other factors by the driver cells (vascular parenchyma?) and the mechan- besides light, most notably by all of the known growth reg- ical resistance offered by the restraining cells (the epider- ulators. Endogenous gibberellin levels are correlated with mis?) would coordinately influence the rate of leaf leaf size and shape in pea (Ross, Murfet & Reid 1993) and expansion. It is likely that both of these growth regulatory tomato (Nagel 1998). Exogenous cytokinin and gibberellin mechanisms, osmotic swelling and biochemical wall loos- both stimulate cell expansion (Engelke, Hamzi & Skoog ening, simultaneously operate in meristematic cells. It is 1973), but neither acts via an ‘acid-growth’ mechanism in suggested here as a possibility worth considering that as bean leaves (Brock & Cleland 1989). Abscisic acid inhibits epidermal cells develop, pathways leading to control of wall leaf expansion, perhaps by interacting with potassium con- extensibility are elaborated, whereas developing vascular ductance and interfering with proton efflux (Van Volken- parenchyma will express proteins contributing to solute burgh & Davies 1983; Bacon, Wilkinson & Davies 1998). accumulation and water flux. One way to determine how Ethylene is associated with leaf epinasty, but mainly affects tissue growth is coordinated in the organ is to characterize the growth of petioles in that response (Abeles, Morgan & these regulatory pathways at the cellular level within a Saltveit 1992). The mechanisms for these growth responses developmental context. are not known. Auxin has been thought to have very little effect on the expansion of dicot leaves, but is known to stimulate growth of monocot leaves, and especially coleoptiles, Regulation of leaf cell expansion by by an acid-growth mechanism (Cleland 1964; Van Volken- exogenous factors burgh 1994). Auxin also acts via an ethylene-independent Leaf development is highly dependent on environmental pathway to stimulate growth of the upper palisade and epi- conditions, in particular the quantity and quality of light, dermis, causing epinasty in tobacco leaves (Hayes 1977; and the availability of water. These, as well as other condi- Keller & Van Volkenburgh 1997). This growth response is tions act via various sensory systems to regulate the meta- not associated with enhanced proton efflux (Keller & Van bolic reactions controlling growth at the cellular level. Volkenburgh 1998). Fusicoccin, on the other hand, stimu- Within the plant as a whole, phytohormones and other sig- lates short-term leaf expansion by stimulating proton efflux nalling mechanisms integrate growth responses among and wall loosening (Van Volkenburgh & Cleland 1980). roots, stems and leaves. It is important to distinguish direct Over-expression of the auxin-binding protein ABP1 in effects on growing leaf cells from indirect effects caused by developing tobacco leaves causes cells to grow larger than other responses such as stomatal closure, or a change in normally, but interestingly this has little effect on leaf mor- stem growth, when determining mechanisms of leaf growth phology (Jones et al. 1998). regulation. More curious is the potential role of brassinosteroids in There is no doubt that leaf expansion is regulated by light promoting leaf expansion (Chory & Li 1997). These com- acting via phytochrome and other photoreceptor signalling pounds were found to be important in development of pathways (Dale 1988). During de-etiolation, red light, plants from the discovery of the genetic basis of the det2 reversible by far red light, stimulates cell division and phenotype in Arabidopsis. Cotyledon and leaf expansion in expansion in young leaves (Downs 1955; Van Volkenburgh the dark was promoted by the det2 mutation which caused et al. 1990).The growth response of de-etiolating cotyledons a defect in the synthesis pathway of brassinosteroids. to red light is considerably reduced in plants lacking phy- Depletion of these compounds causes leaves to expand as tochrome B (Neff & Van Volkenburgh 1994). Plants lacking if exposed to light, at least up to a point. It is not clear, cryptochrome1 or cryptochrome 2 fail to expand cotyle- however, whether the role of the brassinosteroids is in reg- dons in blue light (Blum et al. 1994; Lin et al. 1998). Even ulating the phytochrome control pathway, or in controlling in fully greened leaves, cell expansion continues to be the processes at the end of the pathway that leads to cell regulated by non-photosynthetic photosystems including expansion. phytochromes (Smith 1995; Stahlberg & Van Volkenburgh Leaf expansion is most certainly also influenced by the 1999). growth and condition of other organs on the plant. Gener- The cellular mechanism for light-stimulated leaf expan- ally, expanding leaves will reach a mature size and stop sion has been characterized in bean and pea leaves (Van growing at about the same time as the next younger leaf

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1463–1473 Leaf expansion 1469 begins its rapid growth phase. Removal of young expand- Volkenburgh 1994) as well as leaves (Stahlberg, R. & Van ing leaves will provoke mature bean leaves to begin Volkenburgh, E., unpublished results) expand very little in growing again (Van Staden & Carmi 1982). It would be response to red light. The cry1 mutation causes cotyledons useful to know whether this response is based on renewed on intact plants not to grow in blue light, although, as dis- substrate availability to the older leaf, or disruption in a sig- cussed above, this could be primarily a mutational effect on nalling pathway that had inhibited growth in the older leaf. hypocotyl elongation. The det and cop mutations cause Similar questions have been raised with respect to the stim- seedlings to de-etiolate in darkness, and affect leaf devel- ulation of leaf growth, and inhibition of stem growth, upon opment in light-grown plants (Staub & Deng 1996), but de-etiolation. Normally, light will stimulate leaf expansion again, these mutations are acting on plant development in of both intact and excised pieces of leaves. In the case of general, and are not specific to leaf expansion. the blue-light photoreceptor mutants of Arabidopsis, cry1, A few mutations have been described that are leaf- blue light fails to stimulate cotyledon expansion in intact specific, and these may give us more information about how plants, but is fully stimulatory when the cotyledons are leaf cell expansion is controlled. The CURLY LEAF gene excised (Blum et al. 1994).This means that CRY1 is not nec- is thought to control both leaf cell division and expansion; essary for blue light-stimulated cotyledon expansion. clf plants have normally sized cotyledons and smaller leaves However, the result also indicates that failure of blue light (Kim, Tsukaya & Uchimiya 1998a). The as1 mutation to inhibit elongation of hypocotyls in cry1 seedlings indi- causes leaves to develop asymmetrically due to failure to rectly inhibits expansion of cotyledons in these seedlings. produce cells at the leaf margins (Tsukaya & Uchimiya Implied is a role for substrate allocation in controlling the 1997). REVOLUTA is necessary for apical meristem and rate of leaf expansion. Is the role of CRY1 to regulate thus whole plant development, and for limiting cell divi- hypocotyl elongation by a cell-autonomous mechanism, sions in the leaves (Talbert et al. 1995). Cell expansion in such that blue light-inhibition of CRY1 hypocotyls releases cotyledons is affected by the ANGUSTIFOLIA and substrate for cotyledons to use? If this is true, it is puzzling, ROTUNDIFOLIA3 genes. Cotyledons of an plants are because the substrates in the Arabidopsis seedling must be narrow due to failure of cells to expand the blade in width, entirely supplied by the cotyledons.What makes the cotyle- while rot3 cotyledons are short and fat due to failure of cells dons fail to utilize stored substrates in the dark, and succeed to elongate the blade (Tsukaya, Tsuge & Uchimiya 1994; in utilizing them in the light? Alternatively, CRY1 may be Kim, Tsukaya & Uchimiya 1998b). ROT3 encodes a located within tissues connecting the hypocotyl and cotyle- member of the cytochrome P-450 family, but its relation- don, either in the vascular tissues that extend from one to ship to processes controlling cell elongation is unknown. the other, or the hook region situated inbetween. There it An interesting mutation noted originally for its effect on could be acting as a control gate, directing substrates to chloroplast division, arc6 causes mesophyll cells to develop hypocotyls in the dark, and to cotyledons in the light. If this with only two, rather large chloroplasts. The effect of this is true, CRY1 is not functioning in a cell-autonomous mutation on leaf development is to slow the rate of expan- fashion either to inhibit hypocotyl, or to stimulate cotyle- sion particularly during the later phase of leaf expansion don growth.This possibility is supported by the observation (Pyke et al. 1994). This can be interpreted, from the discus- that illumination of the hypocotyl causes growth of non- sion of the mechanisms contributing to cell growth, as an illuminated leaves (De Greef et al. 1978), and that systemic effect of the mutation on photosynthetic output. The rate acclimation can be induced by exposure of only a portion of cell expansion is enhanced by photosynthesis (Blum et al. of a seedling (Karpinski et al. 1999). The mode of commu- 1992; Stahlberg & Van Volkenburgh 1999). The large nication among organs following exposure to light is not at chloroplasts in arc6 mutants almost certainly have reduced all clear. diffusional capacity due to lower surface-to-volume ratio compared with small chloroplasts. This would reduce photosynthetic rate and the dependent processes (proton Information gleaned from genetic approaches efflux) required for driving cell expansion. Mutational analysis is a promising approach for discover- Analysis of mutations in other species, in particular ing processes critical for regulating leaf expansion monocotyledons, will contribute to our overall picture of (Tsukaya 1995). A rapidly increasing number of mutations leaf development and expansion (see discussion in Becraft have been described recently in Arabidopsis giving rise to 1999). For example, the tangled mutation affects cell divi- altered leaf morphologies. The identified genes are, for the sion and expansion in the blade of corn leaves but surpris- most part, involved with regulation of developmental stages ingly, the leaf shape and size remains largely unaffected by prior to cell expansion and leaf blade formation. For these defects (Smith, Hake & Sylvester 1996). For both pea example, the ago mutations underlying the argonaut phe- and tomato, much genetic information has been accumu- notype give rise to rosette leaves that lack leaf blades, but lated relating to leaf shape, and the formation of compound plant architecture in general is affected (Bohmert et al. leaves. Among other genes, a knotted-like homeobox gene 1998). More pertinent sets of mutants describe pathways (KNOX) seem to be involved in compound leaf develop- for regulation of development by light, including effects on ment (Goliber et al. 1999). Extension of molecular studies leaf expansion. In particular, leaves of phyB mutants are carried out in model species such as Arabidopsis and maize elongated and narrow, and phyB cotyledons (Neff & Van to include the wealth of traditional genetic information

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1463–1473 1470 E. Van Volkenburgh from other species will ultimately provide much informa- (Schmitt 1997). A genetic approach with an evolutionary tion about the genetic basis for differences in leaf shape. perspective (Dudley & Schmitt 1995; Schmitt, McCormac Connection of this genetic information to biochemical func- & Smith 1995) could lead to a wealth of information on the tion will challenge our current understanding of mecha- variety of mechanisms species have developed to expand nisms driving leaf expansion. their leaves. Quantitative genetic methods are promising for analysis Expansion of leaf cells is absolutely required for cell divi- of complex traits such as leaf shape or size. Provided large sion, it is a prerequisite for meristematic tissues to produce enough progeny populations of parental species with dis- new organs, and differential expansion of cells (usually tinct phenotypes, and depending on the quality of the phe- accompanied by divisions) determines leaf shape. It is notypic information, it is possible to find quantitative trait necessary to understand the fundamental physiological loci (QTLs) specific for chosen developmental processes. processes governing cell expansion in order to interpret For example, QTLs associated with bud break and forma- phenotypes resulting from developmental genetic studies. tion in Populus have been indentified (Howe et al. 1998). Discovery of individual genes responsible for altered leaf Using a candidate gene approach, the PHYB and ABI-1 shape will lead us to pathways regulating leaf growth. Com- genes were colocalized to several of these QTLs, suggest- plementary understanding of physiological mechanisms ing that both phytochrome B and abscisic acid play a role will make it possible to develop sophisticated phenotypic in bud break and formation. A similar project could be screens leading to identification of genes encoding ele- carried out to find QTLs associated with leaf cell division, ments of the biochemical processes driving leaf expansion. or expansion. Although cloning QTLs remains extremely difficult, once this technological difficulty is overcome, we will be presented with a multitude of genes causing REFERENCES subtle differences in developmental processes including Abeles F.B., Morgan P.W. & Saltveit M.E. Jr (1992) Ethylene in leaf expansion. Plant Biology, 2nd edn. Academic Press, San Diego, CA. Assmann S.M. & Shimazaki K. (1999) The multisensory guard cell. Stomatal responses to blue light and abscisic acid. 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