Growth-induced water potentials and the growth of maize leaves

Profiles of water potential (Psi(w)) were measured from the soil through the plant to the tip of growing leaves of fully established maize (Zea mays L.). The profiles revealed gradients in transpiration-induced Psi(w) extending upward along the transpiration path, and growth-induced Psi(w) extending radially between the veins in the elongating region of the leaf base. Water moving upward required a small gradient while that moving radially required a much larger gradient primarily because the protoxylem vessels were encased in many small, undifferentiated cells that were likely to act as a barrier to radial flow. Upon maturation, these small cells enlarged and some began to conduct water, probably decreasing the barrier. In the mature leaf, the growth-induced Psi(w) were absent but the transpiration-induced Psi(w) remained. When leaves were growing, the growth-induced Psi(w) moved water into the elongating cells during the day and night, and it shifted with changes in transpiration-induced Psi(w). The shift involved solutes accumulating in the growing region. When water was withheld, the growth-induced Psi(w) disappeared and leaf elongation ceased even though turgor pressure was at its highest. Turgor was maintained by osmotic adjustment that doubled the osmotic potential of the elongating cells. If elongation resumed at night or with rewatering, the growth-induced Psi(w) reappeared. If pressure was applied to the soil/root system to cause guttation and re-establish the growth-induced Psi(w), elongation resumed immediately. These findings support the hypothesis that the primary control of growth is the disappearance and reappearance of the growth-induced Psi(w) because the potential changed in the xylem and nearby cells, blocking or permitting radial water movement and thus blocking or permitting growth.     

Xylem tension affects growth-induced water potential and daily elongation of maize leaves

Diurnal rates of leaf elongation vary in maize (Zea mays L.) and are characterized by a decline each afternoon. The cause of the afternoon decline was investigated. When the atmospheric environment was held constant in a controlled environment, and water and nutrients were adequately supplied to the soil or the roots in solution, the decline persisted and indicated that the cause was internal. Inside the plants, xylem fluxes of water and solutes were essentially constant during the day. However, the forces moving these components changed. Tensions rose in the xylem, and gradients of growth-induced water potentials decreased in the surrounding growing tissues of the leaf. These potentials, measured with isopiestic thermocouple psychrometry, changed because the roots became less conductive to water as the day progressed. The increased tensions were reversed by applying pressure to the soil/root system, which rehydrated the leaf. Afternoon elongation immediately recovered to rapid morning rates. The rapid morning rates did not respond to soil/root pressurization. It was concluded that increased xylem tension in the afternoon diminished the gradients in growth-induced water potential and thus inhibited elongation. Because increased tensions cause a similar but larger inhibition of elongation if maize dehydrates, these hydraulics are crucial for shaping the growth-induced water potential and thus the rates of leaf elongation in maize over the entire spectrum of water availability.     

Leaf Age as a Determinant in Stomatal Control of Water Loss from Cotton during Water Stress

The stomatal resistance of individual leaves of young cotton plants (Gossypium hirsutum L. var. Stoneville 213) was measured during a period of soil moisture stress under conditions of constant evaporative demand. When plants were subjected to increasing soil water stress, increases in stomatal resistance occurred first on the lower leaves and the stomata on the upper surfaces were the most sensitive to decreasing leaf-water potential. Stomatal closure proceeded from the oldest leaves to the youngest as the stress became more severe. This apparent effect of leaf age was not due to radiation differences during the stress period. Radiation adjustments on individual leaves during their development altered the stomatal closure potential for all leaves, but did not change the within-plant pattern. Our data indicate that no single value of leaf water potential will adequately represent a threshold for stomatal closure in cotton. Rather, the stomatal resistance of each leaf is uniquely related to its own water potential as modified by age and radiation regime during development. The effect of age on stress-induced stomatal closure was not associated with a loss of potassium from older leaves. Increases in both the free and bound forms of abscisic acid were observed in water-stressed plants, but the largest accumulations occurred in the youngest leaves. Thus, the pattern of abscisic acid accumulation in response to water stress did not parallel the pattern of stomatal closure induced by water stress.     

Cyclic Variations in Plant Properties under Constant Environmental Conditions

Cyclic fluctuations in stomatal aperture, transpiration rate and leaf water potential under constant environmental conditions have been investigated in intact plants of cotton, pepper, and sunflower. Stomatal aperture and transpiration rate were least when leaf water potential was high and were greatest when leaf water potential was low. Lowest leaf water potential values lagged behind the occurrence of highest transpiration rates, and high overall resistance to water flow occurred in cycling plants. Both of these are considered essential for the occurrence of persistent cyclic behaviour. Hydropassive opening of stomates as the leaves wilted facilitated cycling in cotton and pepper, but not in sunflower, where hydropassive opening did not occur. The roots were identified as the site of the major resistance to water flow in the plant and further experiments directly showed the importance of this root resistance in initiating cycling by causing water stress in the leaves as the stomates opened. Root resistance varied diurnally, becoming increasingly important at night. Root resistance naturally rose to high levels in cotton. High levels were induced in pepper or sunflower by having the roots in deionized water for several days or by anoxia. Quantitative measurements of overall plant resistance were made from leaf water potential and transpiration rate data. The results are discussed and it suggested that plant resistance may indirectly be of importance in the movement of water from the plant to the air.     

Effects of Water Stress and Hardening on the Internal Water Relations and Osmotic Constituents of Cotton Leaves

Experiments were designed to test the hypothesis that the internal water relations of leaves are altered when cotton plants (Gossypium hirsutum L.‘Acala SJ-2′) are conditioned by several cycles of water stress. Preliminary experiments suggested that plants so conditioned are less sensitive to water deficits and that the change might be partly explained by an accumulation of solutes or by structural alterations attendant on development under conditions of water stress. Leaves of preconditioned plants maintained turgor to lower values of water potential than did leaves of well-watered plants. Accompanying this change was a lower osmotic potential at any given leaf water content in preconditioned plants. Tissue analysis of several osmotically active solutes indicated that soluble sugars and malate accumulate to about the same levels (dry-weight basis) in both conditioned and unconditioned plants exposed to stress. These accumulations could not account for the turgor change. Analysis of the data on relative water content indicated that the leaves of conditioned plants had less water per unit dry weight than did leaves of controls. This change accounts for a substantial fraction of the difference between the osmotic potential of conditioned and control plants. The results of a simple model suggest that structural changes may play a significant role in explaining differences in the responses of conditioned and control plants to water stress.     

Do the antiherbivore traits of expanding leaves in the Neotropical tree Inga paraensis (Fabaceae) vary with light availability?

Treefall gaps in tropical forests have a profound effect on plants growing in the understory, primarily due to increased light availability. In higher light, mature leaves typically have increased anti-herbivore defenses. However, since the majority of herbivory occurs while leaves are expanding, it is important to determine whether defense expression during the short period of leaf expansion is canalized (invariant) or plastic in response to variation in light. Therefore, we examined young leaves of Inga paraensis (Fabaceae) saplings growing along a light gradient in a terra-firme forest in Central Amazonia. We quantified leaf production and expansion time, dry mass of phenolics, saponins, and nitrogen, ants attracted to extrafloral nectaries, and leaf consumption. Over the entire light gradient, the number of leaves produced per flush increased by 50?% and the mass of phenolic compounds by 20?%, but no other traits changed. On average, 39?% of leaf area was consumed with no difference across the light gradient. Alone, none of the leaf traits was a significant predictor of leaf consumption, except for phenolics, which showed a positive relationship. Multiple regressions showed that leaf consumption was positively related to more leaves per flush and a higher concentration of phenolics in leaves. Unlike studies of mature leaves, young leaves of I. paraensis show low plasticity in defense traits across a light gradient, suggesting that leaf development is canalized.     

Expansion of pea leaves subjected to short water deficit: cell number and cell size are sensitive to stress at different periods of leaf development

We have followed the expansion of individual pea leaves frominitiation to maximum area, over two markedly different periods.During the first one (2/3 of total leaf development time), cellproduction occurred while cell and leaf expansions were slow.Rapid expansion (95% of total) occurred for a second periodlasting 1/3 of total development time, whereas cell divisionwas virtually completed. Water deficits of 15 d were appliedduring either slow or rapid expansion, and characterized bymeasurements of soil water potential, stomatal conductance,leaf water potential and xylem [ABA]. Plants which experiencedwater deficit during the slow expansion period had markedlyreduced expansion during the second period (i.e. 1 or 2 weeksafter cessation of deficit), while all variables characterizingwater status were returned to the level of the control. This‘after effect’ was accounted for by a reduced cellnumber per leaf, while individual cell area was not affected.In contrast, water deficit occurring during rapid leaf expansionimmediately reduced leaf expansion via cell area, without affectingcell number per leaf. These experiments indicate a role, inthe response to water deficits, for events occurring very earlyin the development of pea leaves, while leaf expansion is tooslow to be measured with macroscopic methods. This role wouldbe accounted for by cell production during the first 2/3 ofleaf development while cell expansion would account for changesin the area of leaves experiencing a later stress. These resultssuggest that long-term temporal analysis may be essential inthe study of dicot leaf expansion compared to monocot leaveswhere temporal analysis can be inferred from spatial analysis. Key words: Leaf growth, dicot leaves, water stress, ell division, cell expansion, Pisum sativum L.     

Function of leaf hamamelitol as a compatible solute during water stress treatment of Hedera helix L.

Hamamelitol is an unusual branched-chain sugar alcohol previously suggested to function as a leaf compatible solute. In this study, we have examined the leaf metabolism and intracelluiar compartmentalization of hamamelitol and other soluble sugars during long-term water stress treatment of Hedera helix (English ivy). Total leaf hamamelitol content was relatively low in greenhouse control plants, but increased 2-fold during water stress treatment to levels approaching those observed in field-grown plants (6–7 μmol g^?1 fresh weight). Using density gradient fractionation with non-aqueous solvents, we showed that hamamelitol occurs primarily in the cytoplasm and vacuoles of leaf mesophyll cells. During water stress treatment most of the increase in leaf hamamelitol occurred in the mesophyll cytoplasm, compensating osmotically for a decrease in cytoplasmic sucrose concentration. The maximum concentration of cytoplasmic hamamelitol was 155 mol m^?3 and occurred in field-grown plants. Labelling experiments showed that hamamelitol is slowly synthesized from ¹⁴CO₂ in leaves of H. helix, but is very long-lived (estimated t_1/2 of 4 years). Together, these data indicate that hamamelitol probably functions during long-term stress conditions as an osmotically active, compatible solute in plant leaves. We suggest that the signal for enhanced accumulation of hamamelitol during the water stress treatment was initiated by decreased plant growth and increased leaf sucrose hydrolysis.     

Stomatal response of cotton to water stress and abscisic Acid as affected by water stress history

The threshold leaf water potential required to initiate stomatal closure in cotton (Stoneville 213) became progressively more negative when plants were subjected to a series of water stress cycles. The shift in the threshold water potential required for induction of stomatal closure was dependent on the number of previous stress cycles and leaf age. The basal level of endogenous abscisic acid (ABA) in fully turgid leaves increased in response to the stress treatments, whereas the amount accumulated in response to a subsequent stress did not differ greatly among plants that had experienced different degrees of stress conditioning.     

Diurnal osmotic cycling in cotton leaves as an indicator of source–sink balance

Abstract Diurnal cycling of osmotic potential was studied in leaves of cotton plants (Gossypium hirsutum L.) grown in the field. Osmotic potential was determined by a pressure-volume procedure as the value coinciding with zero turgor. In plants grown under favourable conditions (no water stress or N stress), osmotic potential at zero-turgor measured at midday was initially about 0.3 MPa lower than before dawn, but this cycling disappeared during the season as the number of fruits per plant increased. In water-stressed or N-deficient plants, osmotic cycling was decreased or even eliminated. Across treatments, cycling of osmotic potential occurred only when plants carried at least 560 cm² of leaf area per fruit. The results are interpreted to mean that diurnal cycling of osmotic potential reveals a ‘sink-limited’ condition within the plant.     

Soil Water Stress and Photosynthesis in Cotton

An experiment was conducted in SPAR systems at Florence, S.C., to obtain a data set for use in the simulation of the effect of drying soil on photosynthetic rates in cotton. The plant water status was monitored using leaf water potential and stem diameter meaurements. Reductions were noted in apparent photosynthesis rates after only 5 days of soil drying, and as anticipated, there was uniform displacement of the diurnal cycle of leaf water potential, and corresponding decreases in transpiration and CO₂ uptake. The photosynthesis-light response curves indicated that an average two-fold reduction in photosynthesis rates occurred for solar radiation greater than 250 W/m². Stem diameter change (from a nonstress pre-sunrise value) and integrated stem stress were found to be good indicators of maximum daily plant water stress. The integrated stem stress gave a measure of the duration of the stress along with its magnitude. A simulation method for predicting leaf water potential from stem diameter measurements was used to show that the magnitude and duration of plant water stress increased uniformly during the experiment. This increase was representative of the decreased rates of photosynthesis measured. These data will be used in the simulation of cotton growth and yield.     

Leaf expansion of four sunflower (Helianthus annuus L) cultivars in relation to water deficits. I. Patterns during plant development

Abstract. The influence of a slow stress and recovery cycle on the pattern of leaf expansion in four diverse sunflower cultivars ( Helianthus annuus L. cvs. Hysun 31, Havasupai, Hopi and Seneca) was studied in a glasshouse. Stress had no significant effect on the time of flower bud emergence and anthesis, or on final leaf number, but delayed the appearance of leaves at high insertions in all cultivars except Hysun 31.
Leaf expansion was markedly reduced as the predawn leaf water potential decreased from −0.35 to −0.60 MPa, and the predawn turgor pressure decreased from 0.3 to 0.2 MPa, and expansion ceased at a predawn leaf water potential of about −1.0 MPa, i.e. when the predawn turgor pressure reached zero.
The leaves most reduced in final size when water was withheld were those at the insertions which grew the most rapidly in unstressed plants. The maximum reduction in final leaf size of 25–35% was similar in all cultivars and was due to retardation of the rate of leaf expansion: the duration of leaf expansion was actually increased by stress. However, leaves that were initiated during stress, but emerged after rewatering, had final leaf areas at least equal to those in the unstressed plants: in the cultivar Seneca, the final size of leaves of high insertion was significantly greater in stressed than unstressed plants, whereas in the three other cultivars the final leaf sizes were similar in both treatments. All four cultivars examined adjusted osmotically to the same degree, but leaf water potentials in one, Seneca, increased more rapidly after rewatering than in the other three, and this may have contributed to the greater relative leaf size in the leaves of high insertion in this cultivar.     

Measurement of a growth-induced water potential gradient in tall fescue leaves

Spatial distribution of cell turgor pressure, cell osmotic pressure and relative elemental growth rate were measured in growing tall fescue leaves ( Festuca arundinacea ). Cell turgor pressure (measured with a pressure probe) was c . 0.55 MPa in expanding cells but increased steeply (+0.3 MPa) in cells where elongation had stopped. However, cell osmotic pressure (measured with a picolitre osmometer) was almost constant at 0.85 MPa throughout the leaf. The water potential difference between the growth zone and the mature zone (0.3 MPa) was interpreted as a growth-induced water potential gradient. This and further implications for the mechanism of growth control are discussed.     

An abscisic acid-related reduced transpiration promotes gradual embolism repair when grapevines are rehydrated after drought

* Proposed mechanisms of embolism recovery are controversial for plants that are transpiring while undergoing cycles of dehydration and rehydration. * Here, water stress was imposed on grapevines (Vitis vinifera), and the course of embolism recovery, leaf water potential (Psi(leaf)), transpiration (E) and abscisic acid (ABA) concentration followed during the rehydration process. * As expected, Psi(leaf) and E decreased upon water stress, whereas xylem embolism and leaf ABA concentration increased. Upon rehydration, Psi(leaf) recovered in 5 h, whereas E fully recovered only after an additional 48 h. The ABA content of recovering leaves was higher than in droughted controls, both on the day of rewatering and the day after, suggesting that ABA accumulated in roots during drought was delivered to the rehydrated leaves. In recovering plants, xylem embolism in petioles, shoots, and roots decreased during the 24 h following rehydration. * A model is proposed to describe plant recovery after rehydration based on three main points: embolism repair occurs progressively in shoots and further in roots and in petioles, following an almost full recovery of Psi(leaf); hydraulic conductance recovers during diurnal transpiring hours, when formation and repair of embolisms occurs in all plant organs; an ABA residual signal in rehydrated leaves hinders stomatal opening even when water relations have recovered, suggesting that an ABA-induced transpiration control promotes gradual embolism repair in rehydrated grapevines.     

Water-deficit stress effects on pistil biochemistry and leaf physiology in cotton (Gossypium hirsutum,L.)

Flowering in cotton (Gossypium hirsutum L.) is a sensitive stage to water-deficit stress, but the effects on metabolism are not well understood. The objective of this study was to monitor gas exchange responses of cotton plants under conditions of limited water supply and evaluate the effects on the carbohydrate concentrations and glutathione reductase levels in the cotton flower. Growth chamber experiments were conducted in 2008 and 2009, with normal day/night conditions of 32/24 °C and optimum quantities of Hoagland's nutrient solution applied until flowering. Treatments were imposed at flowering and consisted of control (Control), where optimum quantities of water were applied, and water stress (WS) where 50% of optimum quantity of water was supplied. Water-deficit stress resulted in a significant decrease in leaf stomatal conductance. Leaf photosynthetic and respiration rates were similarly decreased compared to the control. Ovary and style water potential of water-stressed leaves were significantly higher compared to the water potential of water stressed leaves, indicating that cotton flowers are fairly resistant to changes in the water status of the plant. However, carbohydrate concentrations of water-stressed pistils (ovary and style) were significantly increased compared to the control and a similar pattern was observed in the levels of glutathione reductase of water-stressed pistils. In conclusion, water-deficit stress during flowering resulted in significant decreases in leaf gas exchange functions as well as leaf water potential. Cotton pistils appeared to be less sensitive since they were able to maintain water potential similar to the control under limited water supply and increase glutathione reductase levels. However, pistil carbohydrate metabolism was significantly affected resulting in accumulation of both hexose and sucrose indicating a perturbation in sucrose cleaving and hexose utilizing enzymes that could potentially have as a consequence a decrease in fertilization and seed set efficiency.     

Stomatal behaviour and leaf water potential of chilled and water-stressed Solanum melongena, as influenced by growth history

Abstract Leaf diffusion resistance and leaf water potential of intact Solanum melongena plants were measured during a period of chilling at 6 °C. Two pretreatments, consisting of a period of water stress or a foliar spraying of abscisic acid (ABA), were imposed upon the plants prior to chilling. The control plants did not receive a pretreatment. In addition to intact plant studies, stomatal responses to water loss and exogenous abscisic acid were investigated using excised leaves, and the influence of the pretreatment observed. Chilled, control plants wilted slowly and maintained open stomata despite a decline in leaf water potential to –2.2 MPa after 2 d of chilling. In contrast plants that had been water stressed or had been sprayed with abscisic acid, prior to chilling, did not wilt and maintained a higher leaf water potential and a greater leaf diffusion resistance. In plants that had not received a pretreatment, abscisic acid caused stomatal closure at 35 °C, but at 6°C it did not influence stomatal aperture. The two pretreatments greatly increased stomatal sensitivity to both exogenous ABA and water stress, at both temperatures. Stomatal response to water loss from excised leaves was greatly reduced at 6°C. These results are discussed in relation to low temperature effects on stomata and the influence of preconditioning upon plant water relations.     

Modulation of Competition between Fruits and Leaves by Flower Pruning and Water Fogging, and Consequences on Tomato Leaf and Fruit Growth

The effects of water fogging and reducing plant fruit load werestudied in a tomato crop grown in a glasshouse under Mediterraneansummer conditions. The objective of these treatments was toreduce competition between leaves and fruits for carbohydratesand water. Flower pruning increased plant leaf area and increasedfruit, stem, lamina and petiole dry mass (DM). This indicatesthat leaf area growth was limited during the summer due to competitionbetween fruits and leaves for assimilates. In contrast, reducingthe air vapour pressure deficit (VPD) by water fogging had noeffect on plant leaf area or aerial plant DM. Interestingly,there was a significant interaction between plant fruit loadand VPD: the higher the leaf[ratio]fruit ratio the greater theresponses to a reduction in VPD (increase in fruit DM, fruitdiameter, fruit and leaf expansion rate). The data suggest thatunder high fruit loads, water and carbohydrates limit growthunder Mediterranean summer conditions. However, reducing VPDwas not always sufficient to enhance fruit and leaf growth.This might be due to the lower leaf area under high fruit load.In contrast, reducing VPD under low fruit load triggered higherrates of leaf and fruit expansion; this is probably linked toa greater availability of water and carbohydrates. Copyright2001 Annals of Botany Company Assimilate competition, assimilate supply, flower pruning, fruit load, fruit growth, generative/vegetative growth, leaf growth, Lycopersicon esculentum, specific leaf weight, tomato, vapour pressure deficit, water stress     

Role of proline and leaf expansion rate in the recovery of stressed white clover leaves with increased phosphorus concentration

Osmotic adjustment (OA) and increased cell-wall extensibility required for expansive leaf growth are well defined components of adaptation to water stress in dry soil, which might interact with soil phosphorus (P) concentration and defoliation frequency for intensively grazed white clover in legume-based pastures. Experiments were conducted with frequently and infrequently defoliated mini-swards of white clover growing in dry soil with low and high P concentrations. The higher yielding high-P plants were able to dry the soil to greater soil water suctions; their leaves had lower water potential values, yet they showed fewer water stress symptoms and underwent a more complete recovery from the water stress symptoms on rewatering, than the low-P plants. High- P plants had greater OA, proline concentration and leaf expansion rate. On the other hand, low-P plants showed an increased osmotic concentration when there was no change in the total solute content per unit of leaf d. wt, indicating more loss of water from the leaf tissue. The key measures that appeared to be directly associated with plant recovery over a short period following water stress were increased proline concentration and leaf expansion rate, probably resulting from increased cell-wall extensibility rather than increased production of cells for the high-P plants.     

Heterogenous stomatal closure in response to leaf water deficits is not a universal phenomenon

The extent and occurrence of water stress-induced “patchy” CO₂ uptake across the surface of leaves was evaluated in a number of plant species. Leaves, while still attached to a plant, were illuminated and exposed to air containing [¹⁴C]CO₂ before autoradiographs were developed. Plant water deficits that caused leaf water potential depression to −1.1 megapascals during a 4-day period did result in heterogenous CO₂ assimilation patterns in bean (Phaseolus vulgaris). However, when the same level of stress was imposed more gradually (during 17 days), no patchy stomatal closure was evident. The patchy CO₂ assimilation pattern that occurs when bean plants are subjected to a rapidly imposed stress could induce artifacts in gas exchange studies such that an effect of stress on chloroplast metabolism is incorrectly deduced. This problem was characterized by examining the relationship between photosynthesis and internal [CO₂] in stressed bean leaves. When extent of heterogenous CO₂ uptake was estimated and accounted for, there appeared to be little difference in this relationship between control and stressed leaves. Subjecting spinach (Spinacea oleracea) plants to stress (leaf water potential depression to −1.5 megapascals) did not appear to cause patchy stomatal closure. Wheat (Triticum aestivum) plants also showed homogenous CO₂ assimilation patterns when stressed to a leaf water potential of −2.6 megapascals. It was concluded that water stress-induced patchy stomatal closure can occur to an extent that could influence the analysis of gas exchange studies. However, this phenomenon was not found to be a general response. Not all stress regimens will induce patchiness; nor will all plant species demonstrate this response to water deficits.     

Developmental Changes in Direct and Indirect Defenses in the Young Leaves of the Neotropical Tree Genus Inga (Fabaceae)

Plant fitness is affected by herbivory, and in moist tropical forests, 70 percent of herbivore damage occurs on young leaves. Thus, to understand the effects of herbivory on tropical plant fitness, it is necessary to understand how tropical young leaves survive the brief, but critical, period of susceptibility. In this study, we surveyed three species of Inga during young leaf expansion. Three classes of toxic secondary metabolites (phenolics, saponins, and tyrosine), extrafloral nectar production, leaf area, and extrafloral nectary area were measured at randomly assigned young leaf sizes. In addition, all defenses were compared for potential trade‐offs during leaf expansion. No trade‐offs among defenses were found, and the concentration of all defenses, except tyrosine, decreased during leaf expansion. We suggest that plants continued to increase phenolic and saponin content, but at a rate that resulted in decreasing concentrations. In contrast, tyrosine content per leaf steadily increased such that a constant concentration was maintained regardless of young leaf size. Nectar production remained constant during leaf expansion, but, because young leaf area increased by tenfold, the investment in extrafloral nectar per leaf area significantly decreased. In addition, nectary area did not change during leaf expansion and therefore the relative size of the nectary significantly decreased during young leaf expansion. These results support the predictions of the optimal defense hypothesis and demonstrate that the youngest leaves have the highest investment in multiple defenses, most likely because they have the highest nitrogen content and are most susceptible to a diversity of herbivores.