The inhibition of mitochondrial metabolic activity in etiolated pea seedlings under water stress

In the present work, we studied the influence of water (osmotic) stress on mitochondrial metabolic activity in etiolated pea (Pisum sativum L.) seedlings. Three-day-old pea seedlings were subjected to stress by placing their roots in 0.6 M mannitol for 48 h. Epicotyl growth was severely suppressed, and tissue water content was decreased. We revealed the negative influence of the water stress on mitochondrial metabolic activity of seedlings, which effect was retained also after organelle isolation. In particular, in the mitochondria of stressed seedlings, the rate of oxidation of malate and other respiratory substrates (in state 3) was severely decreased, as well as respiratory control ratio. The rate of proline oxidation was reduced most seriously (by 70%). The efficiency of oxidative phosphorylation, according to the ADP/O ratio was not changed or was increased as compared to mitochondria in control plants. Activation of CN-resistant oxidase and other alternative pathways of electron transport in the mitochondrial electron-transport chain in stressed plants were not observed. In the epicotyl tissues under water stress, no MDA was accumulated and proline accumulation was insignificant. The role of mitochondria in adaptation responses of young seedlings is discussed.     

Polyamine Metabolism and Osmotic Stress : I. Relation to Protoplast Viability

Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine, and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, andVigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.     

Changes in Mulberry Leaf Metabolism in Response to Water Stress

A series of experiments were conducted to characterize the water stress-induced changes in the activities of RuBP carboxylase (RuBPCO) and sucrose phosphate synthase (SPS), photosystem 2 activity, and contents of chlorophylls, carotenoids, starch, sucrose, amino acids, free proline, proteins and nucleic acids in mulberry (Morus alba L. cv. K-2) leaves. Water stress progressively reduced the activities of RuBPCO and SPS in the leaf extracts, the chlorophyll content, and PS2 activity in isolated chloroplasts. Plants exposed to drought showed lower content of starch and sucrose but higher total sugar content than control plants. While the soluble protein content decreased under water stress, the amino acid content increased. Proline accumulation (2.5-fold) was noticed in stressed leaves. A reduction in the contents of DNA and RNA was observed. Reduced nitrogen content was associated with the reduction in nitrate reductase activity. SDS-PAGE protein profile showed few additional proteins (78 and 92 kDa) in the water stressed plants compared to control plants.     

Distinct responses to copper stress in the halophyte Mesembryanthemum crystallinum

Selective gene expression allows the halophyte Mesembryanthemum crystallinum to survive a salt stress. To broaden our understanding of the environmental cues initiating diverse stress responses in this higher plant, unstressed and 0.4 M NaCl‐stressed plants were compared to plants treated with several concentrations of copper (CuSO₄), an increasingly relevant environmental heavy metal pollutant. Comparisons of control and copper‐stressed plants included germination, chlorophyll content, accumulation of proline, heat shock protein (HSP) 60 and a Crassulacean acid metabolism (CAM)‐specific marker enzyme, phospho enol pyruvate carboxylase (PEPCase). In germination and whole plant tests, M. crystallinum was significantly more tolerant to copper than Arabidopsis thaliana. Mature M. crystallinum plants stressed with 50 ppm CuSO₄ for 48 h became dehydrated. These plants produced a 4‐fold increase in proline concentration and accumulated both the CAM‐specific PEPCase and HSP 60 compared to controls. Higher levels of copper stress resulted in a 10‐fold increase in leaf proline content, 10‐fold HSP 60 accumulation but no detectable PEPCase protein compared to unstressed controls. HSP 60 did not accumulate under NaCl stress. Concurrent with copper‐induced genetic responses to stress, copper was accumulated and concentrated in leaves (3 500 ppm). Together, these results suggest that this halophyte copes with copper metal exposure through distinct genetic mechanisms.     

Photosynthetic, Physiological and Biochemical Responses of Tomato Plants to Polyethylene Glycol-Induced Water Deficit

Polyethylene glycol （PEG 6000）-induced water deficit causes physiological as well as biochemical changes in plants. The present study reports on the results of such changes in hydroponically grown tomato plants （Lycopersicon esculentum Mill. cv. Nikita）. Plants were subjected to moderate and severe levels of water stress （i.e. water potentials in the nutrient solution of- 0.51 and -1.22 MPa, respectively）. Water stress markedly affected the parameters of gas exchange. Net photosynthetic rate （Pn） decreased with the induction of water stress. Accordingly, a decrease in the transpiration rate （E） was observed. The ratio of both （Pn/E） resulted in a decrease in water use efficiency. One of the possible reasons for the reduction in Pn is structural damage to the thylakoids, which affects the photosynthetic transport of electrons. This was indicated by an increase in non-photochemical quenching and a reduction in the quantum yield of photosystem Ⅱ. Furthermore, a decrease in both leaf water potential and leaf osmotic potential was observed, which resulted in a significant osmotic adjustment during stress conditions. Analysis of the physiological responses was complemented with a study on changes in proline content. In stressed plants, a 10-fold increase in proline content was detected compared with control plants. It is clear that water stress tolerance is the result of a cumulative action of various physiological and biochemical processes, all of which were affected by PEG 6000-induced water stress.     

Water stress induced alterations in ornithine aminotransferase of ragi (Eleusine coracana): Protection by proline against heat inactivation and denaturation by urea and guanidinium chloride

Water stress resulted in a specific response leading to a large and significant increase (80-fold) in free proline content of ragi (Eleusine coracana leaves and seedlings. L-Proline protected ornithine aminotransferase, an enzyme in the pathway for proline biosynthesis, isolated from normal and stressed ragi leaves against heat inactivation and denaturation by urea and guanidinium chloride. The protection of the stressed enzyme by L-proline was much more complete than that of the enzyme isolated from normal leaves. While L-ornithine, one of the substrates, protected the stressed enzyme against inactivation, it enhanced the rate of inactivation of the normal enzyme. α-Ketoglutarate protected both the normal and stressed enzyme against inactivation and denaturation. These results support the suggestion that ornithine aminotransferase has undergone a structural alteration during water stress. In view of the causal relationship between elevated temperature and water stress of plants under natural conditions, the protection afforded by proline against inactivation and denaturation of the enzyme from stressed leaves assumes significance. These results provide an explanation for a possible functional importance of proline accumulation during water stress.     

Diurnal Fluctuations in Relative Water Content, Nitrate Reductase and Proline Content in Water-stressed and Non-stressed Wheat

Diurnal variations in relative water content (RWC), nitrate reductase (NR) and proline content (PC) were studied at 3 h intervals during a 24 h cycle in the flag leaf of wheat (Triticum vulgare, v. Kalyansona) grown under stressed and non-stressed conditions. RWC was lower in stressed plants than in non-stressed ones throughout the 24 h period. Although it was lowest at 12 h, it recovered by 15 h. Non-stressed plants maintained higher NR activity compared to those under stress. The enzyme activity during night was about the same as during day time in both types of plants. Compared to non-stressed plants, stressed ones had lower NO₃^? content. Proline accumulation occurred under stress conditions and had a maximum at 12–15 h. Non-stressed plants exhibited higher PC during night than day time. Changes in temperature and relative humidity were noted during the period and their influence on RWC, NR and proline was discussed.     

Stress responses in two genotypes of mulberry (Morus alba L.) under NaCl salinity

Changes in biomass yield rates, cell membrane stability (CMS), malondialdehyde (MDA) content and in the levels of physiological stress markers such as proline and glycine betaine in two high yielding genotypes (S1 and ATP, salt tolerant and salt sensitive, respectively) of mulberry under NaCl salinity were studied. Biomass yield rates and CMS were significantly decreased in both the genotypes under stress conditions. Per cent of decrease in biomass yield rate and CMS was relatively less in S1 than in ATP. Salt stress results a significant increase in the accumulation of proline, by 6-fold in S1 and 4-fold in ATP. Glycine betaine content was also increased significantly in stressed plants. However, the per cent increase was more in S1 than in ATP. The level of lipid peroxidation as indicated by MDA formation was greater in ATP than in S1. These results clearly support the better salt tolerant nature of S1 compared to ATP genotype.     

Bioremediation of adverse impact of cadmium toxicity on Cassia italica Mill by arbuscular mycorrhizal fungi

Cassia italica Mill is an important medicinal plant within the family Fabaceae. Pot experiment was conducted to evaluate cadmium stress induced changes in physiological and biochemical attributes in C. italica with and without arbuscular mycorrhizal fungi (AMF). Cadmium stressed plant showed reduced chlorophyll pigment and protein content while AMF inoculation enhanced the chlorophyll and protein content considerably. AMF also ameliorated the cadmium stress induced reduction in total chlorophyll and protein contents by 19.30% and 38.29%, respectively. Cadmium stress enhanced lipid peroxidation while AMF inoculation reduced lipid peroxidation considerably. Increase in proline and phenol content was observed due to cadmium stress and AMF inoculation caused a further increase in proline and phenol content ensuring better growth under stressed conditions. AMF alone also enhanced proline and phenol content. Activity of antioxidant enzymes enhanced under cadmium treatment and AMF inoculation further enhanced their activity thereby strengthening the antioxidant system. Enhanced activities of antioxidants and increased accumulation of osmolytes help plants to avoid damaging impact of oxidative damage. The research has shown that AMF inoculation mitigated the negative impact of stress by reducing the lipid peroxidation and enhancing the antioxidant activity. The present study strongly supports employing AMF as the biological mean for enhancing the cadmium stress tolerance of C. italica.     

Proline, Ornithine, Arginine and Glutamic Acid Contents in Detached Rice Leaves

The effects of water stress on the contents of proline, ornithine, arginine and glutamic acid in detached rice leaves were examined. In water stressed leaves, the content of proline was elevated to a content approximately 8-, 14- and 17-fold higher than in control leaves after 4, 8 and 12 h, respectively. We also observed that omithine and arginine contents were much higher under water stress than in control leaves. However, the content of glutamic acid in water stressed leaves was higher after 4 and 8 h and lower after 12 h than that in control leaves.     

Alterations in photosynthetic pigments, protein and osmotic components in cotton genotypes subjected to short-term drought stress followed by recovery

In order to assess drought tolerance mechanism in cotton, short-term drought-induced biochemical responses were monitored in two cotton (Gossypium hirsutum L.) genotypes contrasting their tolerance to water deficit. The seeds of two genotypes, namely GM 090304 (moderately drought tolerant) and Ca/H 631 (drought sensitive), were sown in pots containing soil, sand and peat in the ratio of 1:1:1, and irrigated every alternate day up to 45 days after sowing when each genotype was subjected to a cycle of water stress by withholding irrigation for 7 days. The stress cycle was terminated by re-watering the stressed plants for 7 days. The leaf of the drought tolerant genotype (GM 090304) maintained higher relative water content under water stress than that of the drought sensitive genotype (Ca/H 631). The levels of biochemical components, such as chlorophylls, carotenoids, total protein, free proline, total free amino acids, sugars, starch and polyphenols, were measured during the stress as well as the recovery periods. The chlorophylls, carotenoids, protein and starch contents decreased in drought stressed plants as compared to control and tended to increase when the plants were recovered from stress. The degree of decrease in chlorophylls, carotenoids and protein contents under drought was higher in the sensitive genotype (Ca/H 631) as compared to the moderately tolerant genotype (GM 090304). However, proline, total free amino acids, total sugars, reducing sugars and polyphenol contents were increased in drought stressed plants and tended to decrease during the period of recovery. Drought-induced increases in total free amino acids, proline, sugars and polyphenols were significantly higher in the moderately tolerant genotype (GM 090304) than in the sensitive genotype (Ca/H 631). These results suggest that proline, sugars and polyphenols act as main compatible solutes in cotton in order to maintain osmotic balance, to protect cellular macromolecules, to detoxify the cells, and to scavenge free radicals under water stress condition.     

Different responses of two ecotypes of C<Subscript>3</Subscript>–C<Subscript>4</Subscript> xero-halophyte <Emphasis Type="Italic">Bassia sedoides</Emphasis> to osmotic and ionic factors of salt stress

The effects of low and moderate salinity (100 and 200 mM NaCl, respectively) and iso-osmotic stress generated by polyethylene glycol PEG (1) (–0.3 MPa) and PEG (2) (–0.6 MPa) on maximum quantum yield of photosystem II (PSII), growth, photosynthesis, transpiration, dark respiration, water use efficiency (WUE), water content, chlorophyll, proline, Na⁺ and K⁺ concentrations were investigated in shoots of two ecotypes С₃–С₄ xero-halophyte Bassia sedoides (Pall.) Aschers. Plants were grown from seeds of two Southern Urals populations (Makan and Podolsk) differing in their bioproductivity. Aboveground biomass of the Makan plants was approximately 10-fold higher than that of the Podolsk ecotype. The plants of both ecotypes were sensitive to water deficit. They showed similar decrease in biomass, water content, net photosynthesis and transpiration intensity under both low and moderate osmotic stress (PEG). However, the content of сhlorophyll and free proline in shoots of the Podolsk plants increased under moderate osmotic stress (PEG(2)). Under salinity the differences between transpiration, F_v/F_m, WUE, water content, chlorophyll and proline concentrations in shoots of two ecotypes were no found. But, the Podolsk plants showed decrease in the growth parameters (1.5-fold), increase in the dark respiration intensity (2-fold) and the Na⁺/K⁺ ratio (1.2-fold) under moderate salinity (200 mM NaCl). Thus, the reduction of bioproductivity of the Podolsk ecotype under salinity was the result of ionic rather than osmotic factor of salinity. In the Podolsk plants the additional transpiration costs and consumption of assimilates (correspondingly) increased with the toxic sodium ion accumulation under salinity. This led to decrease in the growth parameters. Thus, two B. sedoides ecotypes have different adaptive strategies of tolerance to the ionic factor of salt stress at the level of the physiological processes associated with the dark CO₂ gas exchange. Moreover, in less tolerant and productive Podolsk ecotype the increase in proline content in shoots characterized comparatively low adaptation to osmotic factor, and the increase in dark respiration and the Na⁺/K⁺ ratio pointed to relatively low resistance to ion factor of salinity as compared with the Makan ecotype.     

Alterations in the activities of the enzymes of proline metabolism in Ragi (Eleusine coracana) leaves during water stress

Free proline content in Ragi (Eleusine coracana) leaves increased markedly (6 to 85 fold) as the degree of water stress, created by polyethylene gylcol treatment, was prolonged There was also a marginal increase in soluble proteins in the stressed leaves as compared to that in the controls. Water stress stimulated the activities of ornithine aminotransferase and pyrroline-5-carboxylate reductase, the enzymes of proline biosynthesis and markedly inhibited the enzymes involved in proline degradation viz., proline oxidase and pyrroline-5-carboxylate dehydrogenase. These results suggest that increase in free proline content of Ragi leaves could be due to enhanced activities of the enzymes synthesizing proline but more importantly due to severe inhibition of the enzymes degrading proline. These observations establish for the first time, the pathway of proline metabolism in plants by way of detection of the activities of all the enzymes involved and also highlight the role of these enzymes in proline accumulation during water stress.     

Photosynthesis in Two Wheat Cultivars Differing in Salt Susceptibility

Salinised (150 mM NaCl for 15 d) roots excised from salt sensitive wheat cultivar Giza 163 showed about 15-fold increase in the ratio of Na/K while salt tolerant Sakha 92 exhibited only 7.5-fold increase compared to their control ratios. Root ratio of saturated/unsaturated fatty acids was stimulated twice in the sensitive cultivar versus 1.7-fold increase in the tolerant ones. Salinity enhanced greatly the accumulation of spermine (Spm) and spermidine (Spd) contents associated with a decrease in putrescine (Put) content in both wheat cultivars. Higher ratios of Spm+Spd/Put associated with lower content of proline and low ethylene evolution were detected in shoots and roots of salt tolerant cultivar. Chlorophyll a/b ratio showed an increase from 1.3 in control of both cultivars to 1.6 and 1.4 in stressed Giza 163 and Sakha 92, respectively. A reduced Hill reaction activity (19 %) was observed in stressed chloroplasts isolated from leaves of the tolerant cultivar versus 40 % inhibition in the sensitive ones. Moreover, chloroplasts isolated from stressed leaves of the sensitive cultivar showed about 25 % reduction in fluorescence emission at 685 nm as well as shifts in the peaks in the visible region.     

Proline Accumulation in Water-stressed Barley Leaves in Relation to Translocation and the Nitrogen Budget

Mobilization of N from leaves of barley (Hordeum vulgare L.) during water stress, and the role of proline as a mobilized species, were examined in plants at the three-leaf stage. The plants responded to water stress by withdrawing about 25% of the total reduced N from the leaf blades via phloem translocation. Most of this N loss was during the first 2 days while translocation of ¹⁴C-photosynthate out of the stressed blade still remained active. Free proline accumulation in the blade was initially slow, and became more rapid during the 2nd day of stress. Although a major free amino acid, proline accounted for only about 5% of the total N (soluble + insoluble) retained in severely stressed blades. When the translocation pathway in water-stressed leaves was interrupted just below the blade by a heat girdle, a cold jacket, or by blade excision, N loss from the blade was prevented and proline began to accumulate rapidly on 1st day of stress. Little free proline accumulated in the blades until after the ability to translocate was lost. Proline was, however, probably not a major species of N translocated during stress, because proline N accumulation in heat-girdled stressed leaves was five times slower than the rate of total N export from intact blades.     

Proline Accumulation, Nitrogenase (C2H2 reducing) Activity and Activities of Enzymes related to Proline Metabolism in Drought-Stressed Soybean Nodules

We examined the effect of drought stress on proline accumulation,nitrogenase activity and activities of enzymes related to prolinemetabolism in soybean (Glycine max [L.] Merr.) nodules. Nitrogenase(C₂H₂ reducing) activity was inhibited 90% or more as a resultof drought stress. This inhibition was substantially reversedafter a 4 h recovery period. Pyrroline-5-carboxylate reductaseactivity in extracts of drought-stressed nodules from 25-d-oldplants was 55% higher than in unstressed nodules, but the sameactivity in preparations from 55-d-old plants was similar tothat of control plants. Extracts of recovering nodules on plantsof both ages had activities near those of controls. Droughtstress increased the activity of the pentose phosphate pathwayby about 65% in extracts of nodules from 55-d-old plants, butthere was no effect in extracts of nodules from younger plants(25-d-old). Proline dehydrogenase activity was 3.7 and 1.6 timeshigher in bacteroids isolated from nodules taken from 25- and55-d-old stressed plants, respectively, than in comparable controlbacteroids. This activity remained high in bacteroids from bothsets of recovering nodules. The amount of proline in extractsfrom stressed nodules was 3- to 4-fold higher than in unstressednodules, despite increased proline dehydrogenase activity andremained high in nodules collected 4 h after rewatering. Thisincrease was observed in both cytoplasmic and bacteroid fractions.The possible physiological significance of these results isdiscussed. Key words: Proline metabolism, pentose phosphate pathway, drought stress, soybean nodules     

Stress-induced Δ1-pyrroline-5-carboxylate synthetase (P5CS) gene confers tolerance to salt stress in transgenic sugarcane

High salinity interferes in sugarcane growth and development, affecting not only crop yield but also reducing sucrose concentration in culms. Sugarcane plants submitted to salt stress can accumulate compatible solutes, such as proline, which may counteract the effects of salt accumulation in the vacuole and scavenge reactive oxygen species. The objective of this study was to evaluate the response to salt stress of sugarcane plants transformed with the Vigna aconitifolia P5CS gene, which encodes ?1-pyrroline-5-carboxylate synthetase, under the control of a stress-induced promoter AIPC (ABA-inducible promoter complex). For this, 4-month-old clonally multiplied sugarcane plants from two transformation events were irrigated every 2 days with 1/10 Hoagland’s solution supplemented with 100, 150 and 200 NaCl, progressively, during 28 days. Transgenic lines showed increased transgene expression in 3.75-fold when compared with the control plants after 9 days of irrigation with saline water, which can explain the higher proline concentration found in these plants. At the end of the experiment (day 28), the transgenic lines accumulated up to 25 % higher amounts of proline when compared with non-transformed control plants. Stress response in transgenic plants was also accompanied by a reduction of malondialdehyde (MDA) derived from cellular lipid peroxidation in leaves, lower Na⁺ accumulation in leaves and maintenance of photochemical efficiency of PSII. Thus, proline contributed to the protection of the photosynthetic apparatus and the prevention of oxidative damage in transgenic sugarcane under salt stress.     

Solute accumulation, solute potential, germinability and seedling vigour of seeds of finger millet (Eleusine coracana Gaertn.) raised under rain-fed conditions and under irrigation

Abstract The nature of the solutes accumulated in the grains of ten finger millet genotypes grown under rain-fed conditions with moisture stress during panicle and grain development were determined. Although grain yield decreased under stress, the solute potential (SP) of seeds from stressed plants was significantly lower (more negative), probably due to the increase in sucrose content. There were no significant changes in the content of total amino acids, free proline, reducing sugars or potassium. The contribution of sucrose to total solute potential was approximately 32% in seeds obtained from stressed plants as compared with only 15% in seeds from irrigated treatment. The germinability and seedling vigour of the seeds from stressed plants was significantly higher under simulated stress with polyethylene glycol (PEG), but was similar to that of seeds of non-stressed plants under normal conditions.     

Water stress-induced alterations in the proline metabolism of drought-susceptible and -tolerant cassava (Manihot esculenta) cultivars

The free proline levels and activities of ornithine aminotransferase (EC 2.6.1.13) and proline oxidase (EC 1.5.2.2), two of the enzymes involved in proline metabolism were studied during the induction of water stress in a drought susceptible (M-4) and a drought tolerant (S-1315) cultivar of cassava ( Manihot esculenta Crantz). Water stress induced by polyethylene glycol (MW 6000, osmotic potential — 1.65 MPa) caused a ca 25-fold increase in proline in young excised leaves of the susceptible cultivar (M-4) while the increase was about 9-fold in the tolerant cultivar (S-1315). The activity of ornithine aminotransferase (OAT), a key enzyme involved in the biosynthesis of proline, was found to increase 3-fold in water stressed leaves of M-4 and about 2-fold in those of S-1315. The activity of proline oxidase, which is involved in the degradation of proline to pyrroline-5-carboxylate, was reduced by 50% in M-4 and nearly 25% in S-1315 on water stress. Comparison of the kinetic properties of OAT showed that the enzyme from water-stressed leaves is more stable to heat inactivation compared to that of control. These results indicate that during water stress there are alterations in the metabolism of proline in cassava, and the extent of alteration varies between drought-susceptible and -tolerant cultivars.     

Photosynthetic responses and proline content of mature and young leaves of sunflower plants under water deficit

In mature and young leaves of sunflower (Helianthus annuus L. cv. Catissol-01) plants grown in the greenhouse, photosynthetic rate, stomatal conductance, and transpiration rate declined during water stress independently of leaf age and recovered after 24-h rehydration. The intercellular CO₂ concentration, chlorophyll (Chl) content, and photochemical activity were not affected by water stress. However, non-photochemical quenching increased in mature stressed leaves. Rehydration recovered the levels of non-photochemical quenching and increased the F_v/F_m in young leaves. Drought did not alter the total Chl content. However, the accumulation of proline under drought was dependent on leaf age: higher content of proline was found in young leaves. After 24 h of rehydration the content of proline returned to the same contents as in control plants.