Shoot-tip derived callus cultures of Sorghum bicolor were transformed by Agrobacterium tumefaciens as well as by bombardment methods with the mutated pyrroline-5-carboxylate synthetase (P5CSF129A) gene encoding the key enzyme for proline biosynthesis from glutamate. The transgenics were selfed for three generations and T4 plants were examined for 100 mM NaCl stress tolerance in pot conditions. The effect of salt stress on chlorophyll and carotenoid contents, photosynthetic rate, stomatal conductance, internal carbon dioxide concentration, transpiration rates, intrinsic transpiration and water use efficiencies, proline content, MDA levels, and antioxidant enzyme activities were evaluated in 40-day-old transgenic lines and the results were compared with untransformed control plants. The results show that chlorophyll content declines by 65% in untransformed controls compared to 30-38% loss (significant at P < 0.05) in transgenics but not carotenoid levels. Photosynthetic rate (PSII activity) was reduced in untransformed controls almost completely, while it declined by 62-88% in different transgenic lines. Salinity induced ca 100% stomatal closure in untransformed plants, while stomatal conductance was decreased only by 64-81% in transgenics after 4 days. The intercellular CO2 decreased by ca 30% in individual transgenic lines. Malondialdehyde (MDA) content was lower in transgenics compared to untransformed controls. The activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6) and glutathione reductase (GR; EC1.8.1.7) were quantified in leaves exposed to 100 mM NaCl stress and found higher in transgenics. The results suggest that transgenic lines were able to cope better with salt stress than untransformed controls by protecting photosynthetic and antioxidant enzyme activities.

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Five popularly grown mulberry cultivars (K-2, MR-2, TR-10, BC2-59 and S-13) were subjected to drought stress by withholding irrigation, to obtain leaf water potentials (W w ) ranging from -0.75, -1.50 and -2.25 MPa. Accumulation of proline, glycine betaine and abscisic acid (ABA) were quantified in control and water stressed mulberry leaves. The activities of enzymes involved in proline accumulation including glutamate dehydrogenase (EC1.4.1.2-4), pyrroline-5-carboxylate synthetase (EC 1.2.1.41), pyrroline-5-carboxylate reductase (EC1.5.1.2), ornithine transaminase (EC 2.6.1.13) were significantly enhanced in the leaves of all the cultivars with decreasing leaf water potentials, while the activities of proline dehydrogenase (EC 1.5.1.2) were reduced with progressive increase in water stress. Accumulation of proline, glycine betaine and abscisic acid was relatively higher in S-13 and BC2-59 compared to K-2, MR-2 and TR-10 under water deficit conditions. Our results demonstrate that S-13 and BC2-59 have superior osmoprotectant mechanisms under water-limited growth regimes.

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The unicellular photosynthetic alga Chlamydomonas reinhardtii was propagated in iron deficiency medium and patterns of growth, photosynthetic efficiency, lipid accumulation, as well as the expression of lipid biosynthetic and photosynthesis-related proteins were analysed and compared with iron-sufficient growth conditions. As expected, the photosynthetic rate was reduced (maximally after 4 days of growth) as a result of increased non-photochemical quenching (NPQ). Surprisingly, the stress-response protein LHCSR3 was expressed in conditions of iron deficiency that cause NPQ induction. In addition, the protein contents of both the PSI and PSII reaction centres were gradually reduced during growth in iron deficiency medium. Interestingly, the two generations of Fe deficiency cells could be able to recover the photosynthesis but the second generation cells recovered much slower as these cells were severely in shock. Analysis by flow cytometry with fluorescence-activated cell sorting and thin layer chromatography showed that iron deficiency also induced the accumulation of triacylglycerides (TAG), which resulted in the formation of lipid droplets. This was most significant between 48 and 72 h of growth. Dramatic increases in DGAT2A and PDAT1 levels were caused by iron starvation, which indicated that the biosynthesis of TAG had been increased. Analysis using gas chromatography mass spectrometry showed that levels of 16:0, 18:0, 18:2 and 18:3 fatty acids were significantly elevated. The results of this study highlight the genes/enzymes of Chlamydomonas that affect lipid synthesis through their influence on photosynthesis, and these represent potential targets of metabolic engineering to develop strains for biofuel production.

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Microalgae accumulate lipids during stress such as that of nutrient deprivation, concomitant with cessation of growth and depletion of chloroplasts. By contrast, certain small chemical compounds selected by high-throughput screening in Chlamydomonas reinhardtii can induce lipid accumulation during growth, maintaining biomass. Comprehensive pathway analyses using proteomics, transcriptomics, and metabolomics data were acquired from Chlamydomonas cells grown in the presence of one of two structurally distinct lipid activators. WD10784 stimulates both starch and lipid accumulation, whereas WD30030-treated cells accumulate only lipids. The differences in starch accumulation are largely due to differential effects of the two compounds on substrate levels that feed into starch synthesis and on genes encoding starch metabolic enzymes. The compounds had differential effects on photosynthesis, respiration, and oxidative stress pathways. Cells treated with WD10784 showed slowed growth over time and reduced abundance of photosynthetic proteins, decreased respiration, and increased oxidative stress proteins, glutathione, and reactive oxygen species specific to this compound. Both compounds maintained central carbon and nitrogen metabolism, including the tricarboxylic acid cycle, glycolysis, respiration, and the Calvin-Benson-Bassham cycle. There were few changes in proteins and transcripts related to fatty acid biosynthesis, whereas proteins and transcripts for triglyceride production were elevated, suggesting that lipid synthesis is largely driven by substrate availability. This study reports that the compound WD30030 and, to a lesser extent WD10784, increases lipid and lipid droplet synthesis and storage without restricting growth or biomass accumulation by mechanisms that are substantially different from nutrient deprivation.

SUMMARYThe present study documents critical analysis of drought-induced physiological responses in mulberry (Morus spp.) with insights into growth dynamics and leaf productivity. The study was performed for two years in a two-phase experimental design combining both field (experiment no. 1) and glasshouse (experiment no. 2) observations. In field assays, we surveyed 15 mulberry genotypes under two irrigation regimes: well-watered (20 to 24 irrigations in each growing season) and water-limited (irrigated once in a fortnight in each growing season). The genotypes were assessed for variation in key leaf gas exchange characteristics: net photosynthetic rates (Pn), stomatal conductance of CO2 (gs), transpiration rates (E) and instantaneous water use efficiency (WUEi). Leaf yield/plant was considered to determine the tolerance index (TI). Drought stress severely down-regulated leaf-level physiological variables in the susceptible genotypes resulting in poor leaf yield. However, genotypes S-13 and V-1 performed better in terms of leaf gas exchange and proved their superiority over other genotypes in drought tolerance. Conversely, genotypes DD and Bogurai were highly susceptible to drought. Under glasshouse conditions, the combined leaf gas exchange/chlorophyll a fluorescence measurements further dissected out stomatal and non-stomatal restrictions to Pn. As internal/ambient CO2 ratio (Ci/Ca) decreased concurrently with gs in non-irrigated stands, it appeared that greater stomatal limitation to Pn was associated with decreased photo-assimilation and leaf yield production. Further, higher leaf temperature (TL) (>35 °C) and down-regulation of maximum quantum yield of photosystem II (Fv/Fm) were apparent in the susceptible compared to the tolerant genotypes, which indicated chronic photoinhibition due to photo-inactivation of photosystem II centres in the susceptible genotypes. Drought-induced trade-offs in biomass allocation were also highlighted. Overall, our results suggest that greater rooting vigour and leaf hydration status, minimal stomatal inhibition and stabilized photochemistry might play major roles in maintaining higher Pn and associated gas exchange functions in drought-tolerant mulberry genotypes under water stress conditions. The higher leaf yield production in tolerant than susceptible genotypes can be attributed to minimal plasticity in foliar gas exchange traits and better quantitative growth characteristics under low water regimes.