Water Deficit and Spatial Pattern of Leaf Development. Variability in Responses Can Be Simulated Using a Simple Model of Leaf Development1

We analyzed the effect of short-term water deficits at different periods of sunflower (Helianthus annuus L.) leaf development on the spatial and temporal patterns of tissue expansion and epidermal cell division. Six water-deficit periods were imposed with similar and constant values of soil water content, predawn leaf water potential and [ABA] in the xylem sap, and with negligible reduction of the rate of photosynthesis. Water deficit did not affect the duration of expansion and division. Regardless of their timing, deficits reduced relative expansion rate by 36% and relative cell division rate by 39% (cells blocked at the G0-G1 phase) in all positions within the leaf. However, reductions in final leaf area and cell number in a given zone of the leaf largely differed with the timing of deficit, with a maximum effect for earliest deficits. Individual cell area was only affected during the periods when division slowed down. These behaviors could be simulated in all leaf zones and for all timings by assuming that water deficit affects relative cell division rate and relative expansion rate independently, and that leaf development in each zone follows a stable three-phase pattern in which duration of each phase is stable if expressed in thermal time (C. Granier and F. Tardieu [1998b] Plant Cell Environ 21: 695–703).

Protein Changes in Response to Progressive Water Deficit in Maize1 : Quantitative Variation and Polypeptide Identification

Three-week-old plants of two unrelated lines of maize (Zea mays L.) and their hybrid were submitted to progressive water stress for 10 d. Changes induced in leaf proteins were studied by two-dimensional electrophoresis and quantitatively analyzed using image analysis. Seventy-eight proteins out of a total of 413 showed a significant quantitative variation (increase or decrease), with 38 of them exhibiting a different expression in the two genotypes. Eleven proteins that increased by a factor of 1.3 to 5 in stressed plants and 8 proteins detected only in stressed plants were selected for internal amino acid microsequencing, and by similarity search 16 were found to be closely related to previously reported proteins. In addition to proteins already known to be involved in the response to water stress (e.g. RAB17 [Responsive to ABA]), several enzymes involved in basic metabolic cellular pathways such as glycolysis and the Krebs cycle (e.g. enolase and triose phosphate isomerase) were identified, as well as several others, including caffeate O-methyltransferase, the induction of which could be related to lignification.

Oxidative Damage in Pea Plants Exposed to Water Deficit or Paraquat1

The application of a moderate water deficit (water potential of −1.3 MPa) to pea (Pisum sativum L. cv Lincoln) leaves led to a 75% inhibition of photosynthesis and to increases in zeaxanthin, malondialdehyde, oxidized proteins, and mitochondrial, cytosolic, and chloroplastic superoxide dismutase activities. Severe water deficit (−1.9 MPa) almost completely inhibited photosynthesis, decreased chlorophylls, β-carotene, neoxanthin, and lutein, and caused further conversion of violaxanthin to zeaxanthin, suggesting damage to the photosynthetic apparatus. There were consistent decreases in antioxidants and pyridine nucleotides, and accumulation of catalytic Fe, malondialdehyde, and oxidized proteins. Paraquat (PQ) treatment led to similar major decreases in photosynthesis, water content, proteins, and most antioxidants, and induced the accumulation of zeaxanthin and damaged proteins. PQ decreased markedly ascorbate, NADPH, ascorbate peroxidase, and chloroplastic Fe-superoxide dismutase activity, and caused major increases in oxidized glutathione, NAD+, NADH, and catalytic Fe. It is concluded that, in cv Lincoln, the increase in catalytic Fe and the lowering of antioxidant protection may be involved in the oxidative damage caused by severe water deficit and PQ, but not necessarily in the incipient stress induced by moderate water deficit. Results also indicate that the tolerance to water deficit in terms of oxidative damage largely depends on the legume cultivar.

Molecular and Physiological Responses to Water Deficit in Drought-Tolerant and Drought-Sensitive Lines of Sunflower1 : Accumulation of Dehydrin Transcripts Correlates with Tolerance

To investigate correlations between phenotypic adaptation to water limitation and drought-induced gene expression, we have studied a model system consisting of a drought-tolerant line (R1) and a drought-sensitive line (S1) of sunflowers (Helianthus annuus L.) subjected to progressive drought. R1 tolerance is characterized by the maintenance of shoot cellular turgor. Drought-induced genes (HaElip1, HaDhn1, and HaDhn2) were previously identified in the tolerant line. The accumulation of the corresponding transcripts was compared as a function of soil and leaf water status in R1 and S1 plants during progressive drought. In leaves of R1 plants the accumulation of HaDhn1 and HaDhn2 transcripts, but not HaElip1 transcripts, was correlated with the drought-adaptive response. Drought-induced abscisic acid (ABA) concentration was not associated with the varietal difference in drought tolerance. Stomata of both lines displayed similar sensitivity to ABA. ABA-induced accumulation of HaDhn2 transcripts was higher in the tolerant than in the sensitive genotype. HaDhn1 transcripts were similarly accumulated in the tolerant and in the sensitive plants in response to ABA, suggesting that additional factors involved in drought regulation of HaDhn1 expression might exist in tolerant plants.

Dehydration-Stress-Regulated Transgene Expression in Stably Transformed Rice Plants1

To confer abscisic acid (ABA) and/or stress-inducible gene expression, an ABA-response complex (ABRC1) from the barley (Hordeum vulgare L.) HVA22 gene was fused to four different lengths of the 5′ region from the rice (Oryza sativa L.) Act1 gene. Transient assay of β-glucuronidase (GUS) activity in barley aleurone cells shows that, coupled with ABRC1, the shortest minimal promoter (Act1–100P) gives both the greatest induction and the highest level of absolute activity following ABA treatment. Two plasmids with one or four copies of ABRC1 combined with the same Act1–100P and HVA22(I) of barley HVA22 were constructed and used for stable expression of uidA in transgenic rice plants. Three Southern blot-positive lines with the correct hybridization pattern for each construct were obtained. Northern analysis indicated that uidA expression is induced by ABA, water-deficit, and NaCl treatments. GUS activity assays in the transgenic plants confirmed that the induction of GUS activity varies from 3- to 8-fold with different treatments or in different rice tissues, and that transgenic rice plants harboring four copies of ABRC1 show 50% to 200% higher absolute GUS activity both before and after treatments than those with one copy of ABRC1.

Drought-Induced Effects on Nitrate Reductase Activity and mRNA and on the Coordination of Nitrogen and Carbon Metabolism in Maize Leaves1

Maize (Zea mays L.) plants were grown to the nine-leaf stage. Despite a saturating N supply, the youngest mature leaves (seventh position on the stem) contained little NO3− reserve. Droughted plants (deprived of nutrient solution) showed changes in foliar enzyme activities, mRNA accumulation, photosynthesis, and carbohydrate and amino acid contents. Total leaf water potential and CO2 assimilation rates, measured 3 h into the photoperiod, decreased 3 d after the onset of drought. Starch, glucose, fructose, and amino acids, but not sucrose (Suc), accumulated in the leaves of droughted plants. Maximal extractable phosphoenolpyruvate carboxylase activities increased slightly during water deficit, whereas the sensitivity of this enzyme to the inhibitor malate decreased. Maximal extractable Suc phosphate synthase activities decreased as a result of water stress, and there was an increase in the sensitivity to the inhibitor orthophosphate. A correlation between maximal extractable foliar nitrate reductase (NR) activity and the rate of CO2 assimilation was observed. The NR activation state and maximal extractable NR activity declined rapidly in response to drought. Photosynthesis and NR activity recovered rapidly when nutrient solution was restored at this point. The decrease in maximal extractable NR activity was accompanied by a decrease in NR transcripts, whereas Suc phosphate synthase and phosphoenolpyruvate carboxylase mRNAs were much less affected. The coordination of N and C metabolism is retained during drought conditions via modulation of the activities of Suc phosphate synthase and NR commensurate with the prevailing rate of photosynthesis.

Overexpression of Nitrate Reductase in Tobacco Delays Drought-Induced Decreases in Nitrate Reductase Activity and mRNA1

Transformed (cauliflower mosaic virus 35S promoter [35S]) tobacco (Nicotiana plumbaginifolia L.) plants constitutively expressing nitrate reductase (NR) and untransformed controls were subjected to drought for 5 d. Drought-induced changes in biomass accumulation and photosynthesis were comparable in both lines of plants. After 4 d of water deprivation, a large increase in the ratio of shoot dry weight to fresh weight was observed, together with a decrease in the rate of photosynthetic CO2 assimilation. Foliar sucrose increased in both lines during water stress, but hexoses increased only in leaves from untransformed controls. Foliar NO3− decreased rapidly in both lines and was halved within 2 d of the onset of water deprivation. Total foliar amino acids decreased in leaves of both lines following water deprivation. After 4 d of water deprivation no NR activity could be detected in leaves of untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much more stable than NR activity. NR mRNA abundance increased in the leaves of the 35S-NR plants and remained constant in controls for the first 3 d of drought. On the 4th d, however, NR mRNA suddenly decreased in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls. The phosphorylation state of the protein was unchanged by long-term drought. There was a strong correlation between maximal extractable NR activity and ambient photosynthesis in both lines. We conclude that drought first causes increased NR protein turnover and then accelerates NR mRNA turnover. Constitutive NR expression temporarily delayed drought-induced losses in NR activity. 35S-NR expression may therefore allow more rapid recovery of N assimilation following short-term water deficit.

Energetics of the interaction between water and the helical peptide group and its role in determining helix propensities

The alanine helix provides a model system for studying the energetics of interaction between water and the helical peptide group, a possible major factor in the energetics of protein folding. Helix formation is enthalpy-driven (−1.0 kcal/mol per residue). Experimental transfer data (vapor phase to aqueous) for amides give the enthalpy of interaction with water of the amide group as ≈−11.5 kcal/mol. The enthalpy of the helical peptide hydrogen bond, computed for the gas phase by quantum mechanics, is −4.9 kcal/mol. These numbers give an enthalpy deficit for helix formation of −7.6 kcal/mol. To study this problem, we calculate the electrostatic solvation free energy (ESF) of the peptide groups in the helical and β-strand conformations, by using the delphi program and parse parameter set. Experimental data show that the ESF values of amides are almost entirely enthalpic. Two key results are: in the β-strand conformation, the ESF value of an interior alanine peptide group is −7.9 kcal/mol, substantially less than that of N-methylacetamide (−12.2 kcal/mol), and the helical peptide group is solvated with an ESF of −2.5 kcal/mol. These results reduce the enthalpy deficit to −1.5 kcal/mol, and desolvation of peptide groups through partial burial in the random coil may account for the remainder. Mutant peptides in the helical conformation show ESF differences among nonpolar amino acids that are comparable to observed helix propensity differences, but the ESF differences in the random coil conformation still must be subtracted.