Effect of water stress on protein content in two maize cultivars differing in drought resistance

The changing pattern, of WSD and concomitant protein changes were scanned in twoZea mays cultivars, with differential sensitivity to water stress. Increasing protein values were recorded in resistant cv. Ageti-76 with decreasing osmotic potentials of substrate (on 3rd day of stress), although the values in susceptible cv. Vijay remained almost on a par with the controls. In another experiment both the cultivars revealed initial increase of protein on 2nd day at osmotic potentials of - 2 and -4 × 10⁶ Pa, however, values declined up to day 7 of stress. WSD showed an increasing trend in both the experiments, although slightly higher values of WSD were registered in cv. Ageti-76 in comparison with cv. Vijay. The significance of protein changes in reference to drought is discussed.     

Water balance in leaf tissue

Samples of the leaf tissue (14cm²) were placed in a plexiglass chamber which consisted of three parts. Water absorbed by the leaf tissue on one side of the sample was transported through the middle part of the sample to the opposite side and was transpirated there. The intensity of transpiration the intensity of water absorption and water saturation deficit (w.s.d.) were determined simultaneously in this tissue by gravimetry. Water balance was studied either in saturated samples of leaf tissue or in tissue where w.s.d. (10%, 20%, 30%, 40%) was established in advance. Although conditions for water absorption in leaf segments were optimal, w.s.d. originated in the saturated leaf tissue under all given external conditions (evaporation from 41.7 to 17.8 mg cm^?2 h^?1). W.s.d. which was established in advance for the most part increased during the experiment and reached even high values (more than 60%). the equilibration was reached only under conditions of low evaporation and initial w.s.d. higher than 20% in young leaves and higher than 30% in adult leaves. A positive correlation between the ratio of the intensity of water absorption to the intensity of transpiration and w.s.d. was found only under conditions of lower evaporation (17.8 and 23.2mg cm^?2h^?1). The maximal values of w.s.d. were limited in this way. Water balance was studied: 1. in leaf tissue of upper, middle and lower leaves of fodder cabbage, 2. in leaf tissue of middle leaves of young and adult plants of fodder cabbage, 3. in leaf tissue of dicots (fodder cabbage) with different vessel orientation in respect to water transport, 4. in leaf tissue of monocots (banana-tree) with water transport upright to the vessel orientation. Considerable change of water balance was observed when the water transport was prolonged by two incisions in the middle part of the sample. Results of all these experiments revealed the possibility of water stress origin even in leaf tissue sufficiently supplied with water.     

Leaf cell water and enzyme activity

This work supports further the thesis that under conditions of water stress, cell water content may supersede hormonal regulation in effecting enzyme activity, thus becoming a regulatory factor in cellular metabolism. Addition of NaCl to the root medium of barley plants (Hordeum vulgare L.) markedly increased leaf RNase activity parallel to an increase of leaf water saturation deficit (WSD). Kinetin and abscisic acid, applied to the salinated plants, also modified RNase activity, as well as leaf-WSD. The familiar pattern of effects of these hormones on leaf RNase as well as leaf chlorophyll content was inverted, kinetin effected a relative increase in RNase activity and a decrease in leaf chlorophyll, whereas abscisic acid effected a relative decrease in RNase activity and maintained chlorophyll content. A close relationship between enzyme activity and leaf WSD became evident when leaf RNase and protease activities in the salinated plants were plotted against leaf WSD. This close relationship was maintained irrespective of the hormonal treatments, which in themselves markedly modified leaf WSD. As predicted, high relative humidity which relived the leaves from salt-induced water stress prevented the salt-induced rise in RNase activity.     

Hydroreactivity of stomata in kale leaves of different insertion level as determined by analysis of transpiration curves

Values of the water saturation deficit (WSD) for hydroactive stomatal movements of kale leaves were estimated using the method of transpiration curve analysis. Stomata of young leaves started closing at WSD values of 5 to 6 per cent and were completely closed at 18 to 20 per cent WSD. During maturation and ageing of leaves these WSD values increased to 12.5 and 18 to 23 per cent respectively. Thus the stomatal reaction is more sensitive to changes in WSD in adult leaves than in young ones. After maturation is attained both values decrease. In apparently withering leaves the individual phases of transpiration curves can barely be distinguished, probably for the reason that even under optimal conditions their stomata remain half-closed and at high WSD values an incomplete closing of the aperture occurs. The injured cuticle of withering leaves affects the shape of the transpiration curve as well.     

Visual estimation of leaf water stress inMercurialis perennis L.

Leaf inclination of three upper leaf pairs of a plant was used as a visual parameter for estimation of leaf (plant) water status inM. perennis. Negative correlation was found between leaf angle and leaf water saturation deficit (WSD). Large angles (between 130 and 80–90°) indicated WSD below approx. 12%, narrow angles expressed negative water balance of the plant and indicated usually strong or moderate water stress. The correlation may be expressed by two regression lines differing in slope. Some differences were observed between three leaf pairs investigated: At the same water deficits, leaves of the first pair (from apex) were inclined to a less degree than leaves of the other two leaf pairs. Leaves of the third pair wilted the most rapidly, therefore their WSD were highest at moderate and strong water stress of the plant. The approximation method tested is suitable for judgment of the plant water status inM. ptrennis and may be used in ecological investigations in forest ecosystems.     

Effects of Age and Water Stress on Endogenous Levels of Plant Growth Regulators in Euphorbia lathyrus L.

The effect of droughting and waterlogging on changes in endogenouslevels of hormones have been studied in Euphorbia lathyrus L.with particular reference to plant age and leaf age. Younger tissues (i.e. immature leaves and young plants) synthesizedsignificantly greater amounts of ABA per unit fresh weight thandid corresponding older tissue. The increased ABA levels correlatedwell with corresponding increases in water saturation deficit(WSD) and stomatal resistance in a given treatment but the relationshipbetween the absolute values of these three parameters variedgreatly between seedlings of different ages. Endogenous ethylenelevels in leaves and stems were unaffected by droughting irrespectiveof age. Imposition of waterlogging led to marked increases in ABA levelsin young plants though this was not strictly related to changesin WSD or stomatal resistance. Endogenous levels of ethyleneincreased in response to waterlogging, this being marked inthe older seedlings which also had a higher incidence of senescenceand abscission. The more complex effects of waterlogging, the significance ofage of tissue on hormonal responses to stress, and the adaptivevalue of such responses to younger tissues are discussed.     

Partial Restoration of the High Rate of Plastid Pigment Development and the Ultrastructure of Plastids in Detached Water-stressed Wheat Leaves

Detached etiolated wheat (Triticum aestivum L. cv. Chris) leaves accumulated plastid pigments at a high rate, developed chloroplasts with stacked thylakoids, and stored plastid starch when wetted on filter paper in light. A moderate water deficit of — 10 bars markedly reduced the accumulation of chlorophyll and carotenoids in the 8-day-old detached leaves during greening. δ-Aminolevulinic acid treatment of stressed leaf segments resulted in slightly increased pigment accumulations but benzyladenine application restored plastid pigment formation in stressed tissue to within 15% of the pigment content of the nonstressed detached leaves. The addition of δ-aminolevulinic acid to benzyladenine-treated stressed leaf segments improved both chlorophyll and carotenoid formation to nearly the amounts found in nonstressed leaf tissue. Stressed leaf sections developed plastids that were small, lacked starch, contained few thylakoids per granum, and possessed dilated thylakoids. Benzyladenine application to the stressed leaf segments did not restore normal plastid stacking but benzyladenine induced the formation of extended intergranal lamellae and stimulated pigment accumulations in both stressed and nonstressed detached leaves. Starch was absent in plastids of benzyladeninetreated leaf sections.     

Microbial Decomposition of Elm and Oak Leaves in a Karst Aquifer

Dry Chinquapin oak (Quercus macrocarpa) and American elm (Ulmus americana) leaves were placed in four microcosms fed by groundwater springs to monitor changes in dry mass, ash-free dry mass, and microbial activity over a 35-day period. Oxygen microelectrodes were used to measure microbial activity and to estimate millimeter-scale heterogeneity in that activity. Oak leaves lost mass more slowly than elm leaves. Generally, there was a decrease in total dry weight over the first 14 days, after which total dry weight began to increase. However, there were consistent decreases in ash-free dry mass over the entire incubation period, suggesting that the material remaining after initial leaf decomposition trapped inorganic particles. Microbial activity was higher on elm leaves than on oak leaves, with peak activity occurring at 6 and 27 days, respectively. The level of oxygen saturation on the bottom surface of an elm leaf ranged between 0 and 75% within a 30-mm² area. This spatial heterogeneity in O₂ saturation disappeared when the water velocity increased from 0 to 6 cm s^-1. Our results suggest that as leaves enter the groundwater, they decompose and provide substrate for microorganisms. The rate of decomposition depends on leaf type, small-scale variations in microbial activity, water velocity, and the length of submersion time. During the initial stages of decomposition, anoxic microzones are formed that could potentially be important to the biogeochemistry of the otherwise oxic aquifer.     

Relationship of water potential to growth of leaves

A thermocouple psychrometer that measures water potentials of intact leaves was used to study the water potentials at which leaves grow. Water potentials and water uptake during recovery from water deficits were measured simultaneously with leaves of sunflower (Helianthus annuus L.), tomato (Lycopersicon esculentum Mill.), papaya (Carica papaya L.), and Abutilon striatum Dickson. Recovery occurred in 2 phases. The first was associated with elimination of water deficits; the second with cell enlargement. The second phase was characterized by a steady rate of water uptake and a relatively constant leaf water potential. Enlargement was 70% irreversible and could be inhibited by puromycin and actinomycin D. During this time, leaves growing with their petioles in contact with pure water remained at a water potential of —1.5 to —2.5 bars regardless of the length of the experiment. It was not possible to obtain growing leaf tissue with a water potential of zero. It was concluded that leaves are not in equilibrium with the potential of the water which is absorbed during growth. The nonequilibrium is brought about by a resistance to water flow which requires a potential difference of 1.5 to 2.5 bars in order to supply water at the rate necessary for maximum growth.

Leaf growth occurred in sunflower only when leaf water potentials were above —3.5 bars. Sunflower leaves therefore require a minimum turgor for enlargement, in this instance equivalent to a turgor of about 6.5 bars. The high water potentials required for growth favored rapid leaf growth at night and reduced growth during the day.

     

Transport of the Auxin 2,4-Dichlorophenoxyacetic Acid Through Absiccion Zones, Pulvini, and Petioles of Phaseolus vulgaris

Measurements were made of the transport of 2,4-dichlorophenoxyacetic acid-¹⁴C (2,4-D) through segments cut from the region of the distal abscission zone in young and old primary leaves of Phaseolus vulgaris L. When old leaves were used basipetal transport of 2,4-D in segments including pulvinar tissue, abscission zone, and petiolar tissue was much less than in wholly petiolar segments. In both young and old plants, segments consisting entirely of pulvinar tissue transported 2,4-D basipetally at a velocity about half that in petiolar tissue. At both ages the flux of 2,4-D through pulvinar tissue was less than that through petiolar tissue. In segments from old leaves the flux through pulvinar tissue was much less than in young plants; the flux through petiolar tissue changed little with age. There was no change with age in the velocity of basipetal transport. The distribution of ¹⁴C along segments including the abscission zone showed no marked discontinuity. It was concluded that the pulvinus limited the basipetal movement of 2,4-D through segments from old leaves which included both pulvinar and petiolar tissue, but there was no evidence that the abscission zone itself was a barrier to auxin transport.     

Toxicity of Leaf and Stem Extracts to Tylenchorhynchus dubius

Plant extracts, made by grinding 2 g of fresh tissue in 5 ml of water, were toxic to Tylenchorhynchus dubius and Hoplolaimus spp. Such extracts from leaves and stems of bean (Phaseolus vulgaris L.) and leaves of tobacco (Nicotiana tabacum L.) were most toxic; those from leaves of corn (Zea mays L.), tomato (Lycopersicon esculentum Mill.) and rhododendron (Rhododendron catawbiense L.) were less toxic; and extracts of bean roots were nontoxic. Nematode movement slowed markedly within 1 hr in tobacco leaf extract, and within 4 hr in bean leaf extract; both extracts completely inactivated or killed 95% of the nematodes in 24 hr. Heating leaf extract 10 min at 80 C eliminated toxicity. Absorption of fusicoccin, a phytotoxin produced by Fusicoccum amygdali Del., increased the toxicity of tomato leaf extracts, whereas water extracts of acetone-extracted powder preparations of leaves were about 15-fold more toxic than water extracts of fresh tissue. Addition of homogenized leaves of bean, tobacco and tomato to soil significantly reduced nematode populations within 3 days.     

Transpiration, water absorption, and internal water balance of cotton plants as affected by light and changes in saturation deficit

In controlled environment studies of cotton plants (Gossypium barbadense L.) a light-induced acceleration of transpiration upset the water balance established in the dark because of a lag in water absorption. A plant-water deficit could be generated either by sudden illumination at a given saturation deficit (sd) of the air, or by raising the sd in conjunction with illumination, without different effects.

Direct water balance measurements were confirmed in every experiment by beta ray gauge detection of changes in leaf-water content resulting from unequal gain and loss of water by the whole plant.

Recovery from the initial loss of turgidity always was faster and more complete at the higher than at the lower values of sd. Recovery occurred even in the light at the higher values of sd, but was enhanced by return to darkness and a lower sd, which at times resulted in superhydration.

Rehydration in the light could be attributed to at least 2 processes: A) a diminished transpiration rate if earlier water loss was sufficient to induce stomatal closure, and B) an increased rate of water absorption. The data suggest that a water deficit, temporary or persisting, does not cause a significantly lowered transpiration rate; thus, recovery must depend on increased absorption. The communicative link between the 2 processes appears weak, transmitting strong signals only.

     

Changes in spectral reflectance of a foliar lichen <Emphasis Type="Italic">Umbilicaria hirsuta</Emphasis> during desiccation

Water potential (ψ_w) and water saturation deficit (WSD), and several reflectance (R) indexes were assessed in an aerophytic lichen Umbilicaria hirsuta (Sw. ex Westr.) Hoffm. The water index (WI, R₉₀₀/_R970) and normalized difference vegetation index, NDVI [(R₉₀₀-R₆₈₀)/(R₉₀₀+R₆₈₀)] were strongly correlated both with the ψ_w and the WSD of lichen thalli. No significant changes during desiccation were found in structural independent pigment index, SIPI [(R₈₀₀-R₄₄₅)/(R₈₀₀-R₆₈₀)]. Sensitivity of the spectral detection of water status was rather small at high hydration level (WSD < 25 %, or ψ_w > −1 MPa), but this is not much limiting its value and potential use, because physiological processes in lichens are usually inhibited at much lower values of ψ_w than in leaves of vascular plants.     

The relationship between the resistance to water transport and the water saturation deficit in leaf tissue of kale and tobacco

Positive linear correlation between the resistance to water transport in liquid phase and water saturation deficit (w.s.d.) in the tissue ofBrassica oleracea andNicotiana tabacum leaves was observed. At the same values of w.s.d. corresponding values of the resistance to water transport were higher when dehydration of the leaf tissue occurred during the experiment and lower when water balance was in equilibrium or resaturation of the leaf tissue occurred.     

Response of the Succulent Leaves of Peperomia magnoliaefolia to Dehydration: Water Relations and Solute Movement in Chlorenchyma and Hydrenchyma

Relative water content, solute concentrations, and osmolality were determined in the water storage tissue (hydrenchyma) and the assimilatory tissue (chlorenchyma) of the succulent leaves of Peperomia magnoliaefolia (Jac) (Piperaceae) during slow desiccation. Relative water loss was significantly greater for the hydrenchyma than for the chlorenchyma. When whole leaves had lost 50% of their initial water content, the concomitant decrease of the relative water content of the hydrenchyma was 75 to 85%, but of the chlorenchyma only 15 to 25%. In spite of this differential water loss, the osmolality in both tissues increased to the same extent, indicating solute flow from the hydrenchyma to the chlorenchyma during desiccation. Solute translocation appeared to be unspecific, probably reflecting symplastic mass flow from one tissue to the other. The observed volume preservation of the chlorenchyma stabilized photosynthesis of Peperomia magnoliaefolia (Jac) leaves, which was less inhibited by a given decrease of the relative water content of the whole leaves than in nonsucculent leaves.     

Rapid estimates of relative water content

Relative water content may be accurately estimated using the ratio of tissue fresh weight to tissue turgid weight, termed here relative tissue weight. That relative water content and relative tissue weight are linearly related is demonstrated algebraically. The mean value of r² for grapevine (Vitis vinifera L. cv. Shiraz) leaf tissue over eight separate sampling occasions was 0.993. Similarly high values were obtained for maize (Zea mays cv. Cornell M-3) (0.998) and apple (Malus sylvestris cv. Northern Spy) (0.997) using a range of leaf ages. The proposal by Downey and Miller (1971. Rapid measurements of relative turgidity in maize (Zea mays L.). New Phytol. 70: 555-560) that relative water content in maize may be estimated from water uptake was also investigated for grapevine leaves; this was found to be a less reliable estimate than that obtained with relative tissue weight. With either method, there is a need for calibration, although this could be achieved for relative tissue weight at least with only a few subsamples.     

Solutes in the free space of growing stem tissues

The concentration of osmotically active solutes in the cell wall free space of young stem tissues was studied using a variety of extraction methods. When the intercellular air spaces of etiolated pea (Pisum sativum L.) internodes were perfused with distilled H₂O, the resulting solution contained a solute concentration of about 70 milliosmoles per kilogram. A second procedure involving vacuum infiltration of segments followed by centrifugation to collect the free space solution gave similar results. Apical stem segments yielded free space extracts about twice as concentrated as those from basal portions of the stem. After correcting for dilution of the free space solution by the infiltrated water, the osmotic pressure of the undiluted free space in pea stem tissue was estimated to be 2.9 bars for apical segments, 1.8 bars for basal regions. These values may be somewhat overestimated due to solute efflux from intracellular pools during the extraction procedure. Similar results were obtained for stem regions of etiolated soybean (Glycine max [L.] Merr.) and cucumber (Cucumis sativus L.) seedlings.

From measurements of the electrical conductivity and refractive index of free space extracts before and after ashing, it appears that 25% of the solutes are inorganic electrolytes and 75% are organic nonelectrolytes with an average size similar to that of glucose.

A significant osmotic pressure in the wall space offers an explanation for the frequent observation that nontranspiring plants have negative water potentials. Calculations of hydraulic resistance from water potential data must take into account solutes in the free space, else `apparent,' but unreal, changes in resistance may be calculated.

     

Surface wax esters contribute to drought tolerance in Arabidopsis

Waxes are components of the cuticle covering the aerial organs of plants. Accumulation of waxes has previously been associated with protection against water loss, therefore contributing to drought tolerance. However, not much information is known about the function of individual wax components during water deficit. We studied the role of wax ester synthesis during drought. The wax ester load on Arabidopsis leaves and stems was increased during water deficiency. Expression of three genes, WSD1, WSD6 and WSD7 of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT or WSD) family was induced during drought, salt stress and abscisic acid treatment. WSD1 has previously been identified as the major wax ester synthase of stems. wsd1 mutants have shown reduced wax ester coverage on leaves and stems during normal or drought condition, while wax ester loads of wsd6, wsd7 and of the wsd6wsd7 double mutant were unchanged. The growth and relative water content of wsd1 plants were compromised during drought, while leaf water loss of wsd1 was increased. Enzyme assays with recombinant proteins expressed in insect cells revealed that WSD6 and WSD7 contain wax ester synthase activity, albeit with different substrate specificity compared with WSD1. WSD6 and WSD7 localize to the endoplasmic reticulum (ER)/Golgi. These results demonstrated that WSD1 is involved in the accumulation of wax esters during drought, while WSD6 and WSD7 might play other specific roles in wax ester metabolism during stress.     

Correction of flow resistances of plants measured from covered and exposed leaves

The difference in water potential between an enclosed nontranspiring leaf and an adjacent exposed transpiring leaf, and the transpiration rate of a similarly exposed leaf, were used to calculate the change in hydraulic resistance of sorghum (Sorghum bicolor [L.] Moench) and sunflower (Helianthus annuus L.) leaves throughout the day and at various rates of transpiration. Since cotton (Gossypium hirsutum L.) leaves enclosed in aluminum foil alone had enclosed leaf water potentials about 0.06 megapascals lower than similar leaves enclosed in a polyethylene bag shielded with aluminum foil, the sorghum and sunflower leaves were enclosed in polyethylene bags shielded with aluminum foil. Enclosing the exposed leaf in a plastic sheath just prior to excision led to the water potential measured by the pressure chamber technique being 0.3 to 0.4 megapascals higher at rapid transpiration rates than in exposed leaves not sheathed just prior to excision. This error, previously shown to arise from rapid water loss after excision, led to an overestimation of the leaf hydraulic resistance in both species. Correction of the error reduced the resistance by 40 to 90% in irrigated sorghum and by about 40% in irrigated and unirrigated sunflower. After correction, the hydraulic resistances were still flow-dependent, but the dependency was markedly reduced in sorghum.     

Drought tolerance depends on the age of the spring wheat seedlings and differentiates patterns of proteinases

The majority of plant species lose their ability to tolerate severe water deficit after germination at the beginning of seedling growth, in the time of emergence of the radical from the seed. The experiment was designed to compare the differences in proteolytic response between 4-and 6-days old spring wheat (Triticum aestivum L.) seedlings of Eta cultivar, respectively tolerant and sensitive to severe drought inducing up to 90% water saturation deficit (WSD). In coleoptiles the changes of proteolytic activity had the same trend regardless on the seedlings age and increased about fourfold upon 85% WSD as compared to the control, from about 4 to 19 (U/mg protein h). The dehydration of roots of 4 day old seedlings resulted in sharp, fivefold activity increase at 85% WSD (from 11 to >50 U/mg protein h). In roots of 6 days old seedlings dehydrated to 55% WSD the proteolytic activity raised twofold and was about 2.5 times higher than in roots of 4 days old seedlings dehydrated to the same WSD. In coleoptiles of both the 4- and 6-days old seedlings subjected to drought appearance of new bands of serine proteinases was observed. Presented results indicate that roots are more sensitive to drought than coleoptiles, which brings an argument for breeders showing that programs involving roots phenotyping have a full biochemical rationale.