Sucrose Synthase Localization during Initiation of Seed Development and Trichome Differentiation in Cotton Ovules.

Sucrose synthase in cotton (Gossypium hirsutum L.) ovules was immunolocalized to clarify the relationship between this enzyme and (a) sucrose import/utilization during initiation of seed development, (b) trichome differentiation, and (c) cell-wall biosynthesis in these rapidly elongating "fibers." Analyses focused on the period immediately before and after trichome initiation (at pollination). Internal tissues most heavily immunolabeled were the developing nucellus, adjacent integument (inner surface), and the vascular region. Little sucrose synthase was associated with the outermost epidermis on the day preceding pollination. However, 1 d later, immunolabel appeared specifically in those epidermal cells at the earliest visible phase of trichome differentiation. The day following pollination, these cells had elongated 3- to 5-fold and showed a further enhancement of sucrose synthase immunolabel. Levels of sucrose synthase mRNA also increased during this period, regardless of whether pollination per se had occurred. Timing of onset for the cell-specific localization of sucrose synthase in young seeds and trichome initials indicates a close association between this enzyme and sucrose import at a cellular level, as well as a potentially integral role in cell-wall biosynthesis.

Genetic variability for stomatal conductance in Pima cotton and its relation to improvements of heat adaptation.

Responses of stomata to environment have been intensively studied, but little is known of genetic effects on stomatal conductance or their consequences. In Pima cotton (Gossypium barbadense L.), a crop that is bred for irrigated production in very hot environments, stomatal conductance varies genetically over a wide range and has increased with each release of new higher-yielding cultivars. A cross between heat-adapted (high-yielding) and unadapted genotypes produced F2 progeny cosegregating for stomatal conductance and leaf temperature. Within segregating populations in the field, conductance was negatively correlated with foliar temperature because of evaporative cooling. Plants were selected from the F2 generation specifically and solely for differing stomatal conductance. Among F3 and F4 populations derived from these selections, conductance and leaf cooling were significantly correlated with fruiting prolificacy during the hottest period of the year and with yield. Conductance was not associated with other factors that might have affected yield potential (single-leaf photosynthetic rate, leaf water potential). As breeders have increased the yield of this crop, genetic variability for conductance has allowed inadvertent selection for "heat avoidance" (evaporative cooling) in a hot environment.

Temperature-Dependent Water and Ion Transport Properties of Barley and Sorghum Roots : II. Effects of Abscisic Acid.

Water flux through excised roots (J(v)) is determined by root hydraulic conductance (L(p)) and the ion flux to the xylem (J(i)) that generates an osmotic gradient to drive water movement. These properties of roots are strongly temperature dependent. Abscisic acid (ABA) can influence J(v) by altering L(p), J(i), or both. The effects of root temperature on responses to ABA were determined in two species differing in their temperature tolerances. In excised barley (Hordeum vulgare L.) roots, J(v) was maximum at 25 degrees C; 10 micromolar ABA enhanced J(v), primarily by increasing L(p), at all temperatures tested (15-40 degrees C). In sorghum (Sorghum bicolor L.) roots, J(v) peaked at 35 degrees C; ABA reduced this optimum temperature for J(v) to 25 degrees C by increasing L(p) at low temperatures and severely inhibiting J(i) (dominated by fluxes of K(+) and NO(3) (-)) at warm temperatures. The inhibition of K(+) flux by ABA at high temperature was mostly independent of external K(+) availability, implying an effect of ABA on ion release into the xylem. In sorghum, ABA enhanced water flux through roots at nonchilling low temperatures but at the expense of tolerance of warm temperatures. These effects imply that ABA may shift the thermal tolerance range of roots of this heat-tolerant species toward cooler temperatures.

Enhanced Photosynthesis and Stomatal Conductance of Pima Cotton (Gossypium barbadense L.) Bred for Increased Yield.

Yield of Pima cotton (Gossypium barbadense L.) has tripled over the last 40 years with the development of new cultivars. Six genetic lines representing successive stages in the breeding process (one primitive noncultivated accession, four cultivars with release dates from 1949 to 1983, and one unreleased breeding line) were grown in a greenhouse, and their gas exchange properties were compared. Among the cultivated types, genetic advances were closely associated with increasing single-leaf photosynthetic rate (A) and stomatal conductance (g(s)), especially in the morning. The A and g(s) of the primitive line approached those of the cultivated types early in the morning, but were much lower for the rest of the day. In both morning and afternoon, A was correlated with g(s) across genotypes but was not correlated with leaf thickness, concentrations of chlorophyll or starch, or intercellular CO(2) concentration (c(i)). In the oldest cultivar, the relationship of A to c(i) did not change between morning and afternoon. In the two most recent lines, the slopes of the A:c(i) curves at limiting c(i) exceeded that of the oldest cultivar by 25 to 50% in the morning, but the differences were much smaller in the afternoon. The maximum A of the newer lines at high c(i) exceeded that of the oldest cultivar only in the morning. Breeding for increasing yield has enhanced the photosynthetic capacity and stomatal conductance of Pima cotton and altered the diurnal regulation of photosynthesis.

Temperature-dependent water and ion transport properties of barley and sorghum roots : I. Relationship to leaf growth.

Root temperature strongly affects shoot growth, possibly via "nonhydraulic messengers" from root to shoot. In short-term studies with barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) seedlings, the optimum root temperatures for leaf expansion were 25 degrees and 35 degrees C, respectively. Hydraulic conductance (L(p)) of both intact plants and detached exuding roots of barley increased with increasing root temperature to a high value at 25 degrees C, remaining high with further warming. In sorghum, the L(p) of intact plants and of detached roots peaked at 35 degrees C. In both species, root temperature did not affect water potentials of the expanded leaf blade or the growing region despite marked changes in L(p). Extreme temperatures greatly decreased ion flux, particularly K(+) and NO(3) (-), to the xylem of detached roots of both species. Removing external K(+) did not alter short-term K(+) flux to the xylem in sorghum but strongly inhibited flux at high temperature in barley, indicating differences in the sites of temperature effects. Leaf growth responses to root temperature, although apparently "uncoupled" from water transport properties, were correlated with ion fluxes. Studies of putative root messengers must take into account the possible role of ions.

Responses of transpiration and hydraulic conductance to root temperature in nitrogen- and phosphorus-deficient cotton seedlings.

Suboptimal N or P availability and cool temperatures all decrease apparent hydraulic conductance (L) of cotton (Gossypium hirsutum L.) roots. The interaction between nutrient status and root temperature was tested in seedlings grown in nutrient solutions. The depression of L (calculated as the ratio of transpiration rate to absolute value of leaf water potential [Psi(w)]) by nutrient stress depended strongly on root temperature, and was minimized at high temperatures. In fully nourished plants, L was high at all temperatures >/=20 degrees C, but it decreased greatly as root temperature approached the chilling threshold of 15 degrees C. Decreasing temperature lowered Psi(w) first, followed by transpiration rate. In N- or P-deficient plants, L approached the value for fully nourished plants at root temperatures >/=30 degrees C, but it decreased almost linearly with temperature as roots were cooled. Nutrient effects on L were mediated only by differences in transpiration, and Psi(w) was unaffected. The responses of Psi(w) and transpiration to root cooling and nutrient stress imply that if a messenger is transmitted from cooled roots to stomata, the messenger is effective only in nutrient-stressed plants.

Water transport properties of cortical cells in roots of nitrogen- and phosphorus-deficient cotton seedlings.

Growth-limiting deficiencies of N or P substantially decrease the hydraulic conductance of cotton (Gossypium hirsutum L.) roots. This shift could result from decreased hydraulic conductivity of cells in the radial flow pathway. A pressure microprobe was used to study water relations of cortical cells in roots of cotton seedlings stressed for N or P. During 10 days of seedling growth on a complete nutrient solution, root cell turgor was stable at 0.4 to 0.5 megapascal, the volumetric elastic modulus increased slowly from 6 to 10 megapascals, and the half-time for water exchange increased from 10 to 15 seconds. In seedlings transferred to N-free solution for 10 days, final values for each of those parameters were approximately doubled. Root cell hydraulic conductivity (cell Lp) was 1.4 x 10(-7) meters per second per megapascal at the time of transfer. In the well-nourished controls, cell Lp decreased over 10 days to 38% of the initial value, but in the N-stressed plants it decreased much more sharply, reaching 6% of the initial value after 10 days. Transfer to solutions without P or with an intermediate level of N also decreased cell Lp. The changes in root cell Lp were consistent with nutrient effects on intact-root water relations demonstrated earlier. However, cell Lp was about half that of the intact root, implying that substantial water flow may follow an apoplastic pathway, bypassing the cortical cells from which these values were derived.

Correlation of Stomatal Conductance with Photosynthetic Capacity of Cotton Only in a CO(2)-Enriched Atmosphere: Mediation by Abscisic Acid?

Some evidence indicates that photosynthetic rate (A) and stomatal conductance (g) of leaves are correlated across diverse environments. The correlation between A and g has led to the postulation of a "messenger" from the mesophyll that directs stomatal behavior. Because A is a function of intercellular CO(2) concentration (c(i)), which is in turn a function of g, such a correlation may be partially mediated by c(i) if g is to some degree an independent variable. Among individual sunlit leaves in a cotton (Gossypium hirsutum L.) canopy in the field, A was significantly correlated with g (r(2) = 0.41, n = 63). The relative photosynthetic capacity of each leaf was calculated as a measure of mesophyll properties independent of c(i). This approach revealed that, in the absence of c(i) effects, mesophyll photosynthetic capacity was unrelated to g (r(2) = 0.06). When plants were grown in an atmosphere enriched to about 650 microliters per liter of CO(2), however, photosynthetic capacity remained strongly correlated with g even though the procedure discounted any effect of variable c(i). This "residual" correlation implies the existence of a messenger in CO(2)-enriched plants. Enriched CO(2) also greatly increased stomatal response to abscisic acid (ABA) injected into intact leaves. The data provide no evidence for a messenger to coordinate g with A at ambient levels of CO(2). In a CO(2)-enriched atmosphere, though, ABA may function as such a messenger because the sensitivity of the system to ABA is enhanced.

Abscisic Acid Movement into the Apoplastic solution of Water-Stressed Cotton Leaves: Role of Apoplastic pH.

Leaves of cotton (Gossypium hirsutum L.) were subjected to overpressures in a pressure chamber, and the exuded sap was collected and analyzed. The exudate contained low concentrations of solutes that were abundant in total leaf extracts, and photosynthetic rates and stomatal conductance were completely unaffected by a cycle of pressurization and rehydration. These criteria and others indicate that the experimental techniques inflicted no damage upon the leaf cells. The pH and abscisic acid (ABA) content of the apoplastic fluid both increased greatly with pressure-induced dehydration. Although ABA concentrations did not reach a steady state, the peak levels were above 1 micromolar, an order of magnitude greater than bulk ABA concentrations of the leaf blades. Treatment of leaves with fusicoccin decreased the K(+) concentration, greatly reduced the pH rise, and completely eliminated the increase in ABA in the apoplast upon dehydration. When water-stressed leaves were pressurized, the pH of the exuded sap was increased by 0.2 units per 1 megapascal decrease in initial leaf water potential. Buffer capacity of the sap was least in the pH range of interest (6.5-7.5), allowing extremely small changes in H(+) fluxes to create large changes in apoplastic pH. The data indicate that dehydration causes large changes in apoplastic pH, perhaps by effects on ATPases; the altered pH then enhances the release of ABA from mesophyll cells into the apoplastic fluid.

The apoplastic pool of abscisic acid in cotton leaves in relation to stomatal closure.

Suboptimal nitrogen nutrition, leaf aging, and prior exposure to water stress all increased stomatal closure in excised cotton (Gossypium hirsutum L.) leaves supplied abscisic acid (ABA) through the transpiration stream. The effects of water stress and N stress were partially reversed by simultaneous application of kinetin (N(6)-furfurylaminopurine) with the ABA, but the effect of leaf aging was not. These enhanced responses to ABA could have resulted either from altered rates of ABA release from symplast to apoplast, or from some "post-release" effect involving ABA transport to, or detection by, the guard cells. Excised leaves were preloaded with [(14)C]ABA and subjected to overpressures in a pressure chamber to isolate apoplastic solutes in the exudate. Small quantities of (14)C were released into the exudate, with the amount increasing greatly with increasing pressure. Over the range of pressures from 1 to 2.5 MPa, ABA in the exudate contained about 70% of the total (14)C, and a compound co-chromatographing with phaseic acid contained over half of the remainder. At a low balancing pressure (1 MPa), release of (14)C into the exudate was increased by N stress, prior water stress, and leaf aging. Kinetin did not affect (14)C release in leaves of any age, N status, or water status. Distribution of ABA between pools can account in part for the effects of water stress, N stress, and leaf age on stomatal behavior, but in the cases of water stress and N stress there are additional kinetinreversible effects, presumably at the guard cells.

Intracellular pH of Cotton Embryos and Seed Coats during Fruit Development Determined by P Nuclear Magnetic Resonance Spectroscopy.

The pH of the phosphate-containing compartments of developing cotton seed coat and embryo tissues was determined by means of (31)P nuclear magnetic resonance spectroscopy. The pH values of these tissues varied as a function of developmental age. From 27 to approximately 38 days postanthesis, a strong pH differential existed between the two tissues; the seed coat was up to 1.4 pH units more acid than developing cotton embryos. The pattern of pH values found with this technique agrees with pH values of tissue homogenates in distilled water. The results confirm an earlier suggestion that seed coat cells are more acidic than embryo cells during key developmental stages of the seed. The pH differential between these two tissues causes abscisic acid to diffuse from seed coats to embryos against its apparent concentration gradient to prevent viviparous germination, despite a higher abscisic acid concentration in the embryo.

Photosynthesis of cotton plants exposed to elevated levels of carbon dioxide in the field.

The cotton (Gossypium hirsutum L.) plant responds to a doubling of atmospheric CO2 with almost doubled yield. Gas exchange of leaves was monitored to discover the photosynthetic basis of this large response. Plants were grown in the field in open-top chambers with ambient (nominally 350 µl/l) or enriched (nominally either 500 or 650 µl/l) concentrations of atmospheric CO2. During most of the season, in fully-irrigated plants the relationship between assimilation (A) and intercellular CO2 concentration (ci) was almost linear over an extremely wide range of ci. CO2 enrichment did not alter this relationship or diminish photosynthetic capacity (despite accumulation of starch to very high levels) until very late in the season, when temperature was somewhat lower than at midseason. Stomatal conductance at midseason was very high and insensitive to CO2, leading to estimates of ci above 85% of atmospheric CO2 concentration in both ambient and enriched chambers. Water stress caused A to show a saturation response with respect to ci, and it increased stomatal closure in response to CO2 enrichment. In fully-irrigated plants CO2 enrichment to 650 µl/l increased A more than 70%, but in water-stressed plants enrichment increased A only about 52%. The non-saturating response of A to ci, the failure of CO2 enrichment to decrease photosynthetic capacity for most of the season, and the ability of the leaves to maintain very high ci, form in part the basis for the very large response to CO2 enrichment.

Carbon Accumulation during Photosynthesis in Leaves of Nitrogen- and Phosphorus-Stressed Cotton.

Leaves of cotton (Gossypium hirsutum L.) accumulate considerable dry mass per unit area during photosynthesis. The percentage of C in that accumulated dry mass was estimated as the regression coefficient (slope) of a linear regression relating C per unit area to total dry mass per unit area. Plants were grown on full nutrients or on N- or P-deficient nutrient solutions. In the fully nourished controls, the mass that accumulated over a 9-hour interval beginning at dawn contained 38.6% C. N and P stress increased the C concentration of accumulated mass to 49.7% and 45.1%, respectively. Nutrient stress also increased the starch concentration of accumulated mass, but starch alone could not account for the differences in C concentration. P stress decreased the estimated rate of C export from source leaves, calculated as the difference between C assimilation and C accumulation. The effect of P stress on apparent export was very sensitive to the C concentration used in the calculation, and would not have been revealed with an assumption of unchanged C concentration in the accumulated mass.

Stomatal responses to water stress and to abscisic Acid in phosphorus-deficient cotton plants.

Cotton (Gossypium hirsutum L.) plants were grown in sand culture on nutrient solution containing adequate or growth-limiting levels of P. When water was withheld from the pots, stomata of the most recently expanded leaf closed at leaf water potentials of approximately -16 and -12 bars in the normal and P-deficient plants, respectively. Pressure-volume curves showed that the stomata of P-deficient plants closed when there was still significant turgor in the leaf mesophyll. Leaves of P-deficient plants accumulated more abscisic acid (ABA) in response to water stress, but the difference was evident only at low water potentials, after initiation of stomatal closure. In leaves excised from unstressed plants, P deficiency greatly increased stomatal response to ABA applied through the transpiration stream. Kinetin blocked most of this increase in apparent sensitivity to ABA. The effect of P nutrition on stomatal behavior may be related to alterations of the balance between ABA and cytokinins.

Hydraulic conductance as a factor limiting leaf expansion of phosphorus-deficient cotton plants.

Suboptimal levels of phosphorus (P) strongly inhibited leaf expansion in young cotton (Gossypium hirsutum L.) plants during the daytime, but had little effect at night. The effect of P was primarily on cell expansion. Compared to plants grown on high P, plants grown on low P had lower leaf water potentials and transpiration rates, and greater diurnal fluctuations in leaf water potential. Hydraulic conductances of excised root systems and of intact transpiring plants were determined from curves relating water flow rate per unit root length to the pressure differential across the roots. Both techniques showed that low P significantly decreased root hydraulic conductance. The effects of P nutrition on hydraulic conductance preceded effects on leaf area. Differences in total root length, shoot dry weight, and root dry weight all occurred well after the onset of differences in leaf expansion. The data strongly indicate that low P limits leaf expansion by decreasing the hydraulic conductance of the root system.

Seed development in cotton: feasibility of a hormonal role for abscisic Acid in controlling vivipary.

In maturing cotton (Gossypium hirsutum L.) fruits, embryos acquire the capacity to germinate in vitro about 16 days before fruit maturity and dehiscence. Vivipary is believed to be prevented by abscisic acid (ABA) originating in the seed coat and diffusing to the embryo (the Ihle-Dure hypothesis). Although endogenous ABA levels are much greater in embryos than in seed coats during the period of germinability, in «donor-receiver¼ experiments movement of (14)C-ABA is strongly polar in favor of the embryo. Compartmental efflux analysis showed that embryos contain 90% of their ABA in a vacuole-like compartment and an insignificant amount in a cytoplasm-like compartment. In contrast, seed coats have only 60% of their ABA in the «vacuole¼ and a much greater fraction than embryos in the «cytoplasm¼. As a result, efflux across the plasma membranes of seed coat cells is much faster than from embryo cells. Increasing external pH strongly inhibits ABA uptake by isolated seed coats and embryos, indicating a role of pH gradients in its partitioning (i.e. ABA tends to be transferred from acidic to alkaline compartments). Aqueous extracts of seed coats are much more acidic than those of embryos. This difference, presumably originating in the «vacuoles¼, can account for the different intracellular distributions of ABA in the two tissues and therefore can account for the polarity of ABA diffusion between tissues. The results implicate intracellular pH gradients in the control of ABA movement between seed coat and embryo. Demonstration of the feasibility of inward ABA movement, despite apparently unfavorable diffusion gradients, provides direct support for the Ihle-Dure hypothesis.

Abscisic Acid accumulation in cotton leaves in response to dehydration at high pressure.

Pressure-volume techniques were utilized to examine the control of abscisic acid (ABA) accumulation in dehydrated cotton (Gossypium hirsutum L. cv Tamcot SP 37) leaves. Leaves were rapidly dehydrated in a pressure chamber to a balance pressure coincident with the loss of cellular turgor, and then the pressure was either maintained at that level or released. Rapid accumulation of ABA began within two hours after the balance pressure was achieved, whether or not the high pressure potential of the cells was maintained by the externally imposed pressure. The results show that loss of pressure per se does not trigger ABA accumulation in dehydrated leaves. Rather, the stimulus may be related to cellular shrinkage and relaxation of the elastic wall.

Water Relations of Cotton Plants under Nitrogen Deficiency: V. Environmental Control of Abscisic Acid Accumulation and Stomatal Sensitivity to Abscisic Acid.

Suboptimal N nutrition increased the water potential for stomatal closure in water stressed cotton (Gossypium hirsutum L.) leaves. This increased sensitivity to water stress had two components, increased accumulation of abscisic acid (ABA) and increased apparent stomatal sensitivity to ABA. Low N increased the threshold water potentials for stomatal closure and ABA accumulation by about 4 bars and 2 bars, respectively. Low N also greatly increased stomatal response to low concentrations of exogenous ABA applied to excised leaves through the transpiration stream. In low N leaves, kinetin decreased stomatal response to ABA to the level observed with high N leaves. Kinetin by itself had little effect on stomata, nor did it alter stomatal response to ABA in high N leaves. The results suggest a cytokinin-ABA balance which is altered by suboptimal N nutrition to favor stomatal closure during stress.Ambient temperature and N nutrition interacted to alter stomatal response to water stress. Stress-induced ABA accumulation and apparent stomatal sensitivity to ABA were independently affected. The effects of each treatment, and their interaction, could be explained as the net result of changes in both accumulation and apparent sensitivity. Although the results document environmental control of stomatal response to ABA, either altered partitioning of ABA between active and inactive pools, or altered sensitivity of the guard cells, could account for the data.

Control of Leaf Expansion by Nitrogen Nutrition in Sunflower Plants : ROLE OF HYDRAULIC CONDUCTIVITY AND TURGOR.

Nitrogen nutrition strongly affected the growth rate of young sunflower (Helianthus annuus L.) leaves. When plants were grown from seed on either of two levels of N availability, a 33% decrease in tissue N of expanding leaves was associated with a 75% overall inhibition of leaf growth. Almost all of the growth inhibition resulted from a depression of the daytime growth rate. Measurements of pressure-induced water flux through roots showed that N deficiency decreased root hydraulic conductivity by about half. Thus, N deficiency lowered the steady-state water potential of expanding leaves during the daytime when transpiration was occurring. As a result, N-deficient leaves were unable to maintain adequate turgor for growth in the daytime. N deficiency also decreased the hydraulic conductivity for water movement into expanding leaf cells in the absence of transpiration, but growth inhibition at night was much less than in the daytime. N nutrition had no detectable effects on plastic extensibility or the threshold turgor for growth.

Water Relations of Cotton Plants under Nitrogen Deficiency: III. STOMATAL CONDUCTANCE, PHOTOSYNTHESIS, AND ABSCISIC ACID ACCUMULATION DURING DROUGHT.

Nitrogen nutrition exerted a strong effect on stomatal sensitivity to water stress in cotton. In well-watered plants grown with 0.31 millimolar N in the nutrient solution, stomata closed at a water potential of -9 bars even though the wilting point was below -15 bars. For each doubling of nutrient N level, the water potential for stomatal closure decreased by about 2 bars. Elevated intercellular CO(2) concentrations caused only slight stomatal closure regardless of N nutrition. Exogenous abscisic acid (ABA) greatly increased stomatal sensitivity to elevated CO(2) concentrations.PLANTS SUBJECTED TO WATER STRESS GAVE THE FOLLOWING RESPONSES: (a) decreased stomatal conductance at ambient external CO(2) concentration; (b) increased stomatal sensitivity to elevated CO(2) concentrations; (c) decreased mesophyll conductance to CO(2); and (d) increased endogenous ABA content. All of these responses to stress occurred at a higher water potential in N-deficient plants than in normal plants. The results show that N nutrition and water stress interact to control ABA accumulation and the events regulated by that accumulation.