Pramod A. Shirke

Desiccation-induced physiological and biochemical changes in resurrection plant, Selaginella bryopteris

Selaginella bryopteris is a lycophyte resurrection plant, which incurves during desiccation and recovers on availability of moisture. The aim of the study was to test and understand the various physiological and biochemical changes the fronds undergo during desiccation and rehydration, to get an insight as to how this plant adapts and survives through the dry phase. Upon desiccation, S. bryopteris fronds showed drastic inhibition in net photosynthesis (A) and maximal photochemical efficiency of PSII (F(v)/F(m)) however, chlorophyll content did not show much variation. Dark respiration (R(d)) continued even at 10% relative water content (RWC), and showed a burst after rehydration, which is proposed to be crucial to establish protection mechanisms. Desiccation caused an enhanced production of reactive oxygen species (ROS) and increased lipid peroxidation. Proline accumulation increased substantially by 11-fold. Sucrose and starch contents decreased upon desiccation as compared to control. The antioxidative enzymes viz. superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) along with soluble acid invertase increased during desiccation. S. bryopteris shows mechanical as well as physiological mechanisms for tolerance to extreme levels of desiccation stress. The rapid and almost complete recovery of F(v)/F(m) after rehydration clearly indicates the absence of marked photoinhibitory or thermal injury to PSII during desiccation. This along with the homoiochlorophyllous characteristics enables S. bryopteris to recover its A. The antioxidant metabolism further plays an important role in the desiccation tolerance of S. bryopteris.

Midday depression in photosynthesis: Effect on sucrose-phosphate synthase and ribulose-1,5-bisphosphate carboxylase in leaves of Prosopis juliflora (Swartz) DC

The midday depression in net photosynthetic rate (PN) and stomatal conductance (gs) in Prosopis juliflora was studied in relation to two key enzymes of carbon metabolism. Diurnal gas exchange measurements carried out in autumn on P. juliflora showed a pronounced depression in PN and gs along with a decrease in apparent carboxylation efficiency (CE*) during midday. The activities of sucrose-phosphate synthase (SPS) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) also showed large diurnal fluctuations. Initial RuBPCO activity (that present in vivo) and total activity (fully carbamylated activity) increased gradually with increase in irradiance, in the morning reaching a maximum by 08:00 h. The RuBPCO activity declined sharply during midday due to decrease in initial activity. The drop in the % activation of RuBPCO indicated that the deactivation of RuBPCO was achieved via a decarbamylation mechanism. There was a marked similarity in the diurnal patterns of SPS activity and the PN. During a diurnal rhythm, SPS activity increased after irradiation, reaching a maximum at 08:00 h and then declined during midday. Diurnal fluctuations in SPS activity could be due to the changes in the amount of protein (changes in Vmax) as well as to the changes in kinetic properties (changes in Vlim). Hence the midday decline in photosynthesis is closely associated with the regulation of RuBPCO and SPS activities.

Leaf Photosynthesis, Dark Respiration and Fluorescence as Influenced by Leaf Age in an Evergreen Tree, Prosopis Juliflora

P. juliflora trees produce leaves during two growth periods. The first cohort of leaves is produced during spring in cool conditions, while the second cohort is produced during monsoon under warm conditions. I studied photosynthetic characteristics of young, mature, and old leaves of the previous season (monsoon) in the spring season. Maximum net photosynthetic rate of a young leaf was lower than that of the mature and old leaves. The total CO2 fixed per day by the young leaves was just 36 % of that in the mature leaves while the old leaves fixed 76 % of that of the mature leaf. The total transpiration rate and water use efficiency (WUE) were similar in the mature and old leaves, while they were much lower in the young leaves. Dark respiration rate was maximal in the young leaves as compared to the mature and old leaves. About 92 % of the total CO2 fixed per day were respired by the young leaves. The diurnal fluorescence characteristics (ΔF/Fm′, qp, and qN) of the young, mature, and old leaves showed that photochemical efficiency of photosystem 2 during midday decreased more in the young and old leaves than in the mature ones. However, the fluorescence characteristics showed that in all the three leaf types there was complete recovery of the photochemical efficiency at sunset from the midday depression. Fv/Fm in the young and mature leaves also confirmed this. Hence the young and old leaves were photosynthetically less efficient than mature leaves, but they were well adapted to withstand the harsh environmental conditions.

Physiological performance and differential expression profiling of genes associated with drought tolerance in contrasting varieties of two Gossypium species

Cotton is mostly cultivated under rain-fed conditions in India, thus faces frequent drought conditions during its life cycle. Drought being a major stress factor responsible for yield penalty, there has always been a high priority to generate knowledge on adaptation and tolerance of cotton. In the present study, four cotton varieties, JKC-770 and KC-2 (Gossypium hirsutum), and JKC-717 and RAHS-187(Gossypium herbaceum), were imposed to drought. Under drought condition, differential changes in physiological characters like net photosynthesis, transpiration, stomatal conductance, chlorophyll fluorescence, relative water content (RWC), and predawn water potential (ψ 0) showed a change. While proline, malondialdehyde (MDA), and glutathione-S-transferase (GST) content increased along with a concomitant change in the expression of their associated genes. Under moderate stress, tolerant varieties maintain lower ψ 0 probably due to higher proline content as compared to sensitive varieties. Cyclic electron flow (CEF) also plays an important role in tolerance under mild water stress in G. hirsutum varieties. CEF not only activates at high light but also initiates at a very low light intensity. Expression analysis of genes reveals that drought-tolerant varieties showed enhanced detoxifying mechanism by up-regulation of asparagine synthase (AS), glutathione-S-transferase (GST), and methyl glyoxalase (GlyI) genes under drought stress. Up-regulation of Δ1-pyrroline-5-carboxylase synthase (Δ1P5CS) enhanced accumulation of proline, an osmolyte, under drought in tolerant varieties. While the drought-sensitive varieties showed up-regulation of ethylene responsive factor (ERF) and down-regulation of WRKY70 responsible for senescence of the leaf which correlated well with the high rate of leaf fall in sensitive varieties under water stress.

Diurnal and seasonal changes in photosynthesis and photosystem 2 photochemical efficiency in Prosopis juliflora leaves subjected to natural environmental stress

The plants of Prosopis juliflora growing in northern India are exposed to large variations of temperature, vapour pressure deficits (VPD), and photosynthetic photon flux density (PPFD) throughout the year. Under these conditions P. juliflora had two short periods of leaf production, one after the winter season and second after summer, which resulted in two distinct even aged cohorts of leaves. In winter with cold nights (2–8 °C) and moderate temperatures during the day, the plants showed high rates of photosynthesis. In summer the midday temperatures often reached <45 °C and plants showed severe inhibition of photosynthesis. The leaves of second cohort appeared in July and showed typical midday depression of photosynthesis. An analysis of diurnal partitioning of the absorbed excitation energy into photochemistry showed that a smaller fraction of the energy was utilised for photochemistry and a greater fraction was dissipated thermally, further the photon utilisation for photochemistry and thermal dissipation is largely affected by the interaction of irradiance and temperature. The plants showed high photochemical efficiency of photosystem 2 (PS2) at predawn and very little photoinhibition in all seasons except in summer. The photoinhibition in summer was pronounced with very poor recovery during night. Since P. juliflora exhibited distinct pattern of senescence and production of new leaves after winter and summer stress period, it appeared that the ontogenic characteristic together with its ability for safe dissipation of excess radiant energy in P. juliflora contributes to its growth and survival.

Physiological performance, secondary metabolite and expression profiling of genes associated with drought tolerance in Withania somnifera.

Physiological, biochemical, and gene expression responses under drought stress were studied in Withania somnifera. Photosynthesis rate, stomatal conductance, transpiration rate, relative water content, chlorophyll content, and quantum yield of photosystems I and II (PSI and PSII) decreased in response to drought stress. Comparative expression of genes involved in osmoregulation, detoxification, signal transduction, metabolism, and transcription factor was analyzed through quantitative RT–PCR. The genes encoding 1-pyrroline-5-carboxylate synthetase (P5CS), glutathione S-transferase (GST), superoxide dismutase (SOD), serine threonine-protein kinase (STK), serine threonine protein phosphatase (PSP), aldehyde dehydrogenase (AD), leucoanthocyanidin dioxygenase/anthocyanin synthase (LD/AS), HSP, MYB, and WRKY have shown upregulation in response to drought stress condition in leaf tissues. Enhanced detoxification and osmoregulation along with increased withanolides production were also observed under drought stress. The results of this study will be helpful in developing stress-tolerant and high secondary metabolite yielding genotypes.

Reflectance and cyclic electron flow as an indicator of drought stress in cotton (Gossypium hirsutum).

The response and the functioning of the photosynthetic machinery of cotton, Gossypium hirsutum during water stress was studied by leaf optical properties, linear (ETRII) and cyclic electron flow (CEF) and chlorophyll a fluorescence. We observed that in G. hirsutum, during water limitation, Chlorophyll b showed the best correlation with reflectance at 731 nm and is a better indicator of drought. Fv/Fm was observed to be very insensitive to mild water stress. However, during severe water stress the leaves exhibit considerable inhibition in Fv/Fm and an increase in anthocyanin levels by about 20‐fold. CEF was very responsive to mild water stress. The mild drought stress caused large decrease in the ability of the leaves to utilize the light energy. Photosystem I and photosystem II is protected from photoinhibition by high CEF and nonphotochemical quenching under mild water stress. While during severe drought stress, linear electron flow showed a sharp decrease in comparison to CEF. CEF play a major role in G. hirsutum leaves during mild as well as under severe water stress condition and is thus a good indicator of water stress.

Sucrose-phosphate synthase in tree species: light/dark regulation involves a component of protein turnover in Prosopis juliflora (SW DC).

Light dependent modulation of sucrose‐phosphate synthase activity (SPS; EC 2.4.1.14) was studied in a tree species, namely Prosopis juliflora. In this paper we demonstrate that cycloheximide, an inhibitor of cytoplasmic protein synthesis, when fed to detached leaves of P. juliflora through transpiration stream in the dark or in light completely prevents in vivo light activation of Vlim and Vmax activities of SPS. In case of spinach, however, cycloheximide feeding affects only Vlim activity while Vmax activity remained unchanged. In contrast, chloramphenicol, an inhibitor of protein synthesis in chloroplast has no effect on the light activation of SPS in Prosopis. The treatment with cycloheximide showed slight reduction in the rate of O2 evolution indicating that cycloheximide had very little effect on overall photosynthesis. These results indicate that short term protein turnover of the SPS protein and some other essential component(s) (e.g., a putative protein that modifies SPS activity) is one of the primary steps in a complex and unique regulatory cascade effecting the reversible light activation of SPS.

Physiological performance and differential expression profiling of genes associated with drought tolerance in root tissue of four contrasting varieties of two Gossypium species

Root growth in drying soil is generally limited by a combination of mechanical impedance and water stress. As the major function of root tissue is water and nutrient uptake, so it imparts an important role in plant growth and stress management. Previously, we have studied physiological performance and expression profiling of gene associated with drought tolerance in leaf tissue of four cotton varieties. Here, we have further continued our studies with the root tissue of these varieties. The Gossypium hirsutum species JKC-770 is drought-tolerant and KC-2 is drought-sensitive, while Gossypium herbaceum species JKC-717 is drought-tolerant and RAHS-187 is drought-sensitive. JKC-770 and JKC-717 the drought-tolerant varieties showed a comparatively high glutathione-S-transferase, superoxide dismutase, proline along with their gene expression, and low malondialdehyde content indicating low membrane damage and better antioxidative defense under drought condition. The expression levels of cellulose synthase, xyloglucan:xyloglucosyl transferase, and glycosyl hydrolases suggest modulation in cell wall structure and partitioning of sugars towards osmoprotectants instead of cell wall biosynthesis in tolerant varieties. Heat shock proteins and serine/threonine protein phosphotases show upregulation under drought condition, which are responsible for temperature tolerance and protein phosphorylation, respectively. These effects many metabolic processes and may be playing a key role in drought tolerance and adaptability of JKC-770 towards drought tolerance. The long-term water use efficiency (WUE) estimated in terms of carbon isotope discrimination (∆13C) in the root tissues showed maximum depletion in the ∆13C values in JKC-770 variety, while minimum in RAHS-187 under drought stress with reference to their respective control, suggesting a high WUE in JKC-770 variety.

Diurnal and Seasonal Modulation of Sucrose Phosphate Synthase Activity in Leaves of Prosopis juliflora

Diurnal changes of sucrose-phosphate synthase (SPS) activity in different seasons were measured in Prosopis juliflora (Swartz) DC. leaves. SPS activity showed large variations with two distinct peaks, one around 09:00 and another at 21:00. The diurnal pattern was apparently not due to circadian rhythm since the activities were directly related to the changes in environmental parameters (irradiance, temperature, and leaf to air vapour pressure deficit) in different seasons. During the day, the enzyme showed changes in kinetic properties, differential sensitivity to allosteric modulators, differential response to ATP concentration, to concentration of endogenous sucrose, and to protein phosphatase inhibitors. These results taken together indicate the modulation of SPS in synchrony with photosynthesis and suggest the existence of multiple levels of modulation, presumably as an adaptive response to changing environmental extremes.