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.

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.

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.

Amino Acid interactions in the regulation of nitrate reductase induction in cotton root tips.

Glycine, asparagine, and glutamine inhibited the induction by nitrate of nitrate reductase activity in root tips of cotton (Gossypium hirsutum L.). This inhibition was partially or entirely prevented when the inhibitor was applied in combination with any of several other amino acids. Studies of (14)C-labeled amino acid uptake showed that, in most cases, the apparent antagonism resulted simply from competition for uptake. However, certain antagonists did not curtail uptake. The most effective of these were leucine (against all three inhibitors), and isoleucine and valine (against asparagine or glutamine, but not glycine). These results show that interactions among amino acids in the regulation of nitrate reductase induction result from at least two mechanisms, one acting on uptake of inhibitory amino acids, and the other involving true antagonism.

Control of Enzyme Activities in Cotton Cotyledons during Maturation and Germination: I. Nitrate Reductase and Isocitrate Lyase.

Actinomycin D at 10 mug/ml strongly inhibited the increase in isocitrate lyase activity during germination of seeds and 40-day-old embryos of cotton (Gossypium hirsutum L.) when the germination period was preceded by 3 hours of soaking in the inhibitor solution. No inhibition was observed without the presoaking. Induction of nitrate reductase activity by nitrate was never inhibited by actinomycin D under the same conditions, and was frequently stimulated about 50%. Thus, the method of applying actinomycin D to the seeds and ovules could affect interpretation of its action. Abscisic acid at 5 mug/ml blocked production of isocitrate lyase activity in both pregermination treatments, but did not inhibit induction of nitrate reductase activity. Induction of nitrate reductase activity became insensitive to the two inhibitors during ovule maturation, at about 32 days after anthesis. The results indicate that isocitrate lyase, a germination enzyme, is not synthesized on performed mRNA. In this respect, the appearance of activity in cotton resembles that in other species of fatty seeds. In contrast, induction of nitrate reductase activity, which is unnecessary for germination, apparently is not regulated at the level of transcription except in young ovules.

Differential regulation of nitrate reductase induction in roots and shoots of cotton plants.

The induction of nitrate reductase activity in root tips of cotton (Gossypium hirsutum L.) was regulated by several amino acids and by ammonium. Glycine, glutamine, and asparagine strongly inhibited induction of activity by nitrate and also decreased growth of sterile-cultured roots on a nitrate medium. Methionine, serine, and alanine weakly inhibited induction, and 11 other amino acids had little or no effect. Ammonium also decreased induction in root tips, but was most effective only at pH 7 or higher. The optimum conditions for ammonium regulation of induction were identical to those for growth of sterile-cultured roots on ammonium as the sole nitrogen source. Aspartate and glutamate strongly stimulated induction, but several lines of evidence indicated that the mechanism of this response was different from that elicited by the other amino acids. The effects of amino acids on induction appeared to be independent of nitrate uptake.In green shoot tissues, all attempts to demonstrate regulation of induction by amino acids failed. The great difference in observed responses of root and shoot to amino acids suggests that their nitrate reductase activities are regulated differently. Differential regulation of this enzyme is consistent with the responses of root and shoot nitrate reductase activity to nitrate.

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.

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.

Distribution and development of nitrate reductase activity in germinating cotton seedlings.

Activity of nitrate reductase in roots and cotyledons of cotton seedings (Gossypium hirsutum L. cv. Deltapine 16) increased rapidly on germination, reaching a maximum after 1 day of imbibition. Thereafter, activity declined until emergence and greening of the cotyledons, when it again began to increase steadily. Germinating soybean (Glycine max (L.) Merrill cv. Merit) and sunflower (Helianthus annuus L. cv. Peredovic) seedlings did not show the early peak of activity. The early peak depended on nitrate and was sensitive to cycloheximide, but not to actinomycin D or other inhibitors of RNA synthesis. The second, light-dependent increase was sensitive to actinomycin D. In roots, the early peak of activity occurred before any growth. After emergence of the root tip from the seed coat, activity was localized in the terminal 2 millimeters, whether expressed on a fresh weight, protein, or root basis. The difference in activity between the apical (0-2 millimeter) and subapical (2-4 millimeter) segments did not result from differences in nitrate availability, energy supply, or turnover rates of nitrate reductase. Root activity was similar to that of the cotyledons after emergence, in that both were sensitive to actinomycin D.

In vivo assay of nitrate reductase in cotton leaf discs: effect of oxygen and ammonium.

Factors affecting nitrate reduction by leaf discs of cotton (Gossypium hirsutum L.) were investigated. When incubated in 30 mm nitrate, discs reduced nitrate much more slowly under air or O(2) than under N(2). Inhibition by O(2) did not occur at nitrate levels of 100 mm or greater. Treatment with arsenate had little effect under N(2) but stimulated nitrate reduction under air. Similarly, ammonium inhibited nitrate reduction, with the inhibition being partially relieved by arsenate. Uptake of nitrate was unaffected by ammonium. The NAD/NADH ratio increased in response to both oxygen and ammonium. The effects of these treatments on nitrate reduction can be explained by competition with nitrate for NADH generated by glycolysis.

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.

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.

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.

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.

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: I. Dependence upon Leaf Structure.

Cotton plants (Gossypium hirsutum L.) grown on deficient levels of N exhibited many of the characteristics associated with drought resistance. In N-deficient plants, leaf areas and leaf epidermal cells were smaller than at the same nodes in high-N plants. N-deficient leaves lost only about half as much water per unit change in water potential as did high-N leaves. In addition, they maintained a greater relative water content than high-N leaves at any given potential. Osmotic potentials (determined from pressure-volume curves) were slightly lower in N-deficient leaves. This difference in solute concentration was not from organic acids, which were almost unchanged. Sugar concentrations could account for only about 25% of the difference.Leaves of N-deficient plants contained considerably more dry matter per unit moisture. Most of this difference in dry weight was in the crude cell wall fraction. The pressure-volume curves and other indirect evidence strongly suggested that cell walls of N-deficient leaves were substantially more rigid than cell walls of high-N leaves. The effects of N deficiency on cell wall properties mimic the changes which occur during drought adaptation.

Water Relations of Cotton Plants under Nitrogen Deficiency: II. Environmental Interactions on Stomata.

Nitrogen deficiency in cotton plants (Gossypium hirsutum L.) considerably increased the sensitivity of stomata to water stress. At air temperatures of 27, 35, and >/=40 C, threshold potentials for complete stomatal closure were -10, -15, and -26 bars in N-deficient plants and -20, -20, and -30 bars in high-N plants, respectively. This three-way interaction among N supply, water potential, and air temperature was similar to that exerted on leaf expansion. The effects of N supply on stomatal behavior could not be explained on the basis of either osmotic or structural considerations. Rather, effects of N deficiency on mesophyll and stomata were independent and divergent. Stomatal behavior may impart a stress avoidance type of drought resistance to N-deficient plants.

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.

Polar transport of kinetin in tissues of radish.

Polar transport of kinetin-8-(14)C occurred in segments of petioles, hypocotyls, and roots of radish (Raphanus sativus L.). The polarity was basipetal in petioles and hypocotyls and acropetal in roots. In segments excised from seedlings with fully expanded cotyledons, indole-3-acetic acid was required for polarity to develop. In hypocotyl segments isolated at this stage, basipetal and acropetal movements were equal during the first 12 hours of auxin treatment after which time acropetal movement declined. Pretreatment with auxin eliminated this delay in the appearance of polarity. In hypocotyl segments excised from seedlings with expanding cotyledons, exogenous auxin was unnecessary for polarity. Potassium cyanide abolished polarity at both stages of growth by allowing increased acropetal movement. The rate of accumulation of kinetin in receiver blocks was greater than the in vivo increase in cytokinin content of developing radish roots.

Ethylene and carbon dioxide in the growth and development of cultured radish roots.

Ethylene is produced by cultured radish roots in amounts large enough to be physiologically important. When roots were grown in controlled atmospheres, applied ethylene was generally inhibitory to elongation, lateral root initiation, and cambial activity. 1% CO(2) similarly affected roots not given ethylene. In contrast, elongation and lateral root production of ethylene-treated roots were stimulated by 1% CO(2). The results suggest that the often-observed stimulation of root growth by CO(2) is due to an interaction with endogenous ethylene.