Proline Metabolism and Transport in Maize Seedlings at Low Water Potential

The growing zone of maize seedling primary roots accumulatesproline at low water potential. Endosperm removal and excisionof root tips rapidly decreased the proline pool and greatlyreduced proline accumulation in root tips at low water potential.Proline accumulation was not restored by exogenous amino acids.Labelling root tips with [¹⁴C]glutamate and [¹⁴C]proline showedthat the rate of proline utilization (oxidation and proteinsynthesis) exceeded the rate of biosynthesis by five-fold athigh and low water potentials. This explains the reduction inthe proline pool following root and endosperm excision and theinability to accumulate proline at low water potential. Theendosperm is therefore the source of the proline that accumulatesin the root tips of intact seedlings. Proline constituted 10% of the amino acids released from the endosperm. [¹⁴C]Prolinewas transported from the scutellum to other parts of the seedlingand reached the highest concentration in the root tip. Less[¹⁴C]proline was transported at low water potential but becauseof the lower rate of protein synthesis and oxidation, more accumulatedas proline in the root tip. Despite the low biosynthesis capacityof the roots, the extent of proline accumulation in relationto water potential is precisely controlled by transport andutilization rate.     

Accumulation of Free Proline at Low Temperatures

The accumulation of free proline in the first leaves of barley, Hordeum distichum L., and wheat, Triticum aestivum L., in response to a range of low temperatures was examined with 10-day-old plants. In barley (cv. Prior) no proline accumulated at 8°C or above, but in wheat (cv. Gabo) proline accumulated at 12°C and lower temperatures. In barley, the first leaf survived for 29 days following transfer to 5°C and continued to accumulate proline throughout this period. In contrast, the first leaves of plants maintained at 20°C survived for 13 days only and accumulated no proline. Proline accumulation at low temperature was shown to be light-dependent, both in intact plants and excised leaf sections, and the light requirement could not be replaced by supplying leaf segments with precursors of proline. Proline accumulation in response to water stress was not light-dependent at 20°C but was at 5°C. Inter-specific and intra-specific variation in the extent of accumulation in response to low temperature was also examined. Considerable variation was encountered but there was no clear relationship with geographical distribution or chilling sensitivity for the species and no correlation with accumulation in response to water stress in the cultivars of barley examined.     

Mycorrhizal-Mediated Lower Proline Accumulation in Poncirus trifoliata under Water Deficit Derives from the Integration of Inhibition of Proline Synthesis with Increase of Proline Degradation

Proline accumulation was often correlated with drought tolerance of plants infected by arbuscular mycorrhizal fungi (AMF), whereas lower proline in some AM plants including citrus was also found under drought stress and the relevant mechanisms have not been fully elaborated. In this study proline accumulation and activity of key enzymes relative to proline biosynthesis (▵¹-pyrroline-5-carboxylate synthetase, P5CS; ornithine-δ-aminotransferase, OAT) and degradation (proline dehydrogenase, ProDH) were determined in trifoliate orange (Poncirus trifoliata, a widely used citrus rootstock) inoculated with or without Funneliformis mosseae and under well-watered (WW) or water deficit (WD). AMF colonization significantly increased plant height, stem diameter, leaf number, root volume, biomass production of both leaves and roots and leaf relative water content, irrespectively of water status. Water deficit induced more tissue proline accumulation, in company with an increase of P5CS activity, but a decrease of OAT and ProDH activity, no matter whether under AM or no-AM. Compared with no-AM treatment, AM treatment resulted in lower proline concentration and content in leaf, root, and total plant under both WW and WD. The AMF colonization significantly decreased the activity of both P5CS and OAT in leaf, root, and total plant under WW and WD, except for an insignificant difference of root OAT under WD. The AMF inoculation also generally increased tissue ProDH activity under WW and WD. Plant proline content significantly positively correlated with plant P5CS activity, negatively with plant ProDH activity, but not with plant OAT activity. These results suggest that AM plants may suffer less from WD, thereby inducing lower proline accumulation, which derives from the integration of an inhibition of proline synthesis with an enhancement of proline degradation.     

Free proline accumulation in drought-stressed plants under laboratory conditions

Summary Free proline accumulation was measured in leaves of intact sorghum (Sorghum bicolor L. cv. Pioneer 846) and soybean (Glycine max. L. cv. Calland) grown in growth chambers and subjected to ‘normal’ drought stress. Stomatal diffusive resistance and leaf water potential were used to determine the degree of stress at the time of proline analysis. Free proline did not accumulate markedly in either species until each was severely stressed, indicating that proline is not a sensitive indicator of drought stress. Free proline accumulated under less stress in soybean than in sorghum. Since soybean is less drought resistant than sorghum, proline accumulation may be an indicator of drought resistance or susceptibility. re]19731003     

Proline accumulation and nitrate reductase activity in contrasting sorghum lines during mid-season drought stress

Six lines of sorghum ( Sorghum bicolor L. Moench) with differing drought resistance (IS 22380, ICSV 213, IS 13441 and SPH 263, resistant and IS 12739 and IS 12744, susceptible) were grown under field conditions in the semi-arid tropics and analysed for proline and nitrate reductase activity (NRA; EC 1.6.6.1) during a mid-season drought. The resistant lines accumulated high levels of proline, while the susceptible lines showed no significant proline accumulation. Most of the proline was accumulated after growth of the plants had ceased. In a separate greenhouse experiment, most of the proline was found in the green rather than the fired portions of leaves. The levels returned to that of irrigated controls within 5 days of rewatering. Proline levels increased as leaf water potential and relative water content fell, and there was no apparent difference among the different sorghum lines with change in plant water status. Susceptible lines accumulated less proline than resistant lines as leaf death occurred at higher water potentials. Proline accumulation may, however, contribute to the immediate recovery of plants from drought. Leaf NRA reached high levels at about 35 days after sowing in both the stressed and irrigated plants, after which it declined. The decline in NRA was more pronounced in the stressed than in the irrigated plants and closely followed changes in the growth rate. Upon rewatering, NRA increased several-fold in all the lines and, in contrast to proline accumulation, genotypic differences in NRA were small, both during stress and upon rewatering. The high sensitivity of NRA to mild drought stress was reflected in the rapid decline of activity with small changes in leaf water potential and relative water content. The results are discussed in the light of a possible role for proline during recovery from drought, and the maintenance of NRA during stress and its recovery upon rewatering.     

Physiological responses to water stress following a conditioning period in berseem clover

Berseem clover (Trifolium alexandrinum L.) is an important crop in semi-arid regions; its herbage and seed yields are often reduced by water stress. Our objectives were (i) to determine the effect of water stress, applied after a conditioning period, on water relations, proline accumulation and plant dry weight, and (ii) to investigate if some physiological responses differed in varieties of berseem. Five cultivars (Axi, Bigbee, Lilibeo, Sacromonte and Saniros) were grown in a controlled environment, and subjected to four irrigation treatments (T1, T2, T3 and T4 referring to plants irrigated to field capacity every 1, 2, 3 or 4 d, respectively) during a conditioning period (12 d). T1 treatment indicated the well-watered control, whereas T2, T3 and T4 treatments represented the conditioned plants. Leaf water potential (Ψ), osmotic potential (Ψπ), relative water content (RWC), gravimetric soil water content (GSWC) and leaf proline concentration were recorded during the conditioning period and a subsequent water deficit period (3 d) applied at early flowering growth stage. The conditioned plants subjected to subsequent water deficit maintained higher values of Ψ, Ψπ, RWC and GSWC, and lower values of leaf proline concentration. Reductions in parameter values were inversely related to the water stress severity that plants had previously experienced. At the end of the experiment, T1 showed 42%, 58% and 31% lower values for Ψ, Ψπ and RWC, respectively, than those of T4. Conditioned plants were also shorter and accumulated less leaf, stem and total dry weight. The conditioning treatments did not affect the relation between Ψ and Ψπ since conditioned plants show similar values of Ψπ as the control at the same Ψ value. Thus, drought acclimation in berseem clover contributed to water stress tolerance by the maintenance of tissue hydration. The berseem cultivars examined showed differences in plant growth parameters, but they were very similar for physiological responses to water deficit. The main genetic difference was recorded for turgor maintenance capacity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Field Estimates of Root and Xylem Resistances in Pinus contorta using Root Excision

A root excision technique was used to estimate the proportionof total resistance to water flux residing in the soil, theroot, and the xylem of lodgepole pine (Pinus contorta Douglex. Loud.) trees in the field. Root excision at mid-day alwaysresulted in rapid recovery of leaf water potential when waterwas supplied to the cut stem, suggesting a high soil-root resistance.Transpiration was unaffected if leaf water potential beforecutting was not limiting leaf conductance. By mid-June wateruptake by the excised stem always exceeded calculated crowntranspiration indicating recharge of internal sapwood storage.Predawn leaf water potential before root excision was highlycorrelated with total soil-plant resistance (r² = 0·89)and calculated root water uptake (r² = 0·92).     

Proline accumulation in canola leaf discs subjected to osmotic stress is related to the loss of chlorophylls and to the decrease of mitochondrial activity

Many stress studies use simplified experimental models like leaf discs, but the validity of such approaches remains a matter of debate, especially when attempts are made to obtain suitable biotests. We report here the analysis of the resistance of canola leaf discs to osmotic stress, as related to the accumulation of proline. For that purpose, the explanted tissues were incubated for 24 h under continuous light and at 28°C on polyethylene glycol solutions of external potentials (Π_ext) ranging from −0.1 to −8.0 MPa. Proline content, water deficit and chlorophyll content were quantified. The cell membrane stability, which is a structural component of viability, was assessed according to electrolyte leakage. Biomembrane oxidative damage was measured through the production of malondialdehyde and the mitochondrial activity was quantified by assessing the ability of the tissues to reduce 2,3,5-triphenyltetrazolium chloride. Although the water deficit of the tissues reached high values (i.e. up to 70%), the cell membrane stability remained high. Furthermore, the oxidative damage to biomembranes was quite low, even in highly dehydrated tissues. In contrast, osmotic stress induced a significant decrease in the chlorophyll content and a strong accumulation of proline. These two processes each reached a maximum at a Π_ext of −3.0 MPa, although the tissues appeared to be viable at even lower Π_ext, suggesting they are not responsible for primary resistance. The mitochondrial activity was strongly decreased in the stressed leaf discs, although this activity was at least partially restored in tissues incubated for 24 h on a non-stressing medium. It appears that the cell components of the osmotic stress resistance assessed observed in this study do not reflect the modes of resistance of an intact plant.     

Proline Metablism During Water Stress in Mulberry

The influence of water stress on proline metabolism was studiedin 3-month-old mulberry plants at four levels of water stress.Leaf water potential was drastically decreased in all treatments.Though leaf area and relative water content were decreased,drastic decrease was observed only in very severe stress treatments.Proline accumulation was observed both in roots and leaves instress treatments; but accumulation was greater in roots thanin leaves. The enzymes, proline dehydrogenase and proline oxidase,were inhibited under stress conditions. Proline oxidase wasmore inhibited in roots than in leaves. The significance ofthe relative activities of these two enzymes is discussed. Key words: Water stress, proline dehydrogenase, proline oxidase     

Does proline accumulated in leaves of water deficit stressed barley plants confine cell membrane injury? I. Free proline accumulation and membrane injury index in drought and osmotically stressed plants

The aim of this work was to examine the relationship between proline accumulation and membrane injury in barley leaves suffering from the effects of water deficit. Water deficit stress was induced by water withholding or by immersing the roots in polyethylene glycol (PEG 6000) solution of osmotic potential −1.5 MPa. The effect of water stress on proline accumulation and on membrane injury was evaluated in leaf blades of several barley genotypes. Substantial differences in proline accumulation and membrane injury indices among most of the genotypes investigated were observed. It was found that in drought stressed plants a higher ability to accumulate proline positively correlates with lower membrane injury. Whereas, in osmotically stressed plants the highest proline accumulation in the leaves was noticed in genotype with the largest membrane injury. The possible role of proline in membrane protection under conditions of slow-acting drought or shock-acting osmotic stress is discussed.     

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.     

Protein Turnover in Isolated Barley Leaf Segments and the Effects of Stress

Leaf segments prepared from the first leaves of barley (Hordeumvulgare) exhibit a rapid loss of protein when given a matricstress with polyethylene glycol. Protein synthesis was reducedby the stress but a greater effect of stress was seen on proteindegradation. Growing leaves were exposed to ³H₂O for 4 d ormore to label total protein, and the half-life of protein 2-³H,in the isolated segments prepared from such leaves, was shownto be c. 140 h in the absence of stress. Stress reduced thisto c. 62 h. A short pulse with ³H₂O preferentially labels rapidlyturning-over protein and a 24 h pulse given to isolated leafsegments labelled proteins with a half-life of c. 64 h in thepresence or absence of stress. Degradation of the 24 h pulse-labelledproteins was inhibited by cycloheximide. Proline accumulationoccurred in the stressed segments and was inhibited by cycloheximide.The results are discussed in the light of current views concerningprotein degradation and possible relationships between proteolysisand proline accumulation.     

Influence of Proline and Glycinebetaine on Salt Tolerance of Cultured Barley Embryos

Lone, M. I., Kueh, J. S. H., Wyn Jones, R. G. and Bright, S.W. J. 1987. Influence of proline and glycinebetaine on salttolerance of cultured barley embryos.—J. exp. Bot. 38:479–490. The addition of exogenous proline and glycinebetaine to culturedbarley (Hordeum vulgare L. cv. Maris Mink) embryos increasedshoot elongation under saline conditions. Inhibition of shootelongation by NaCl was relieved by proline when plantlets weregrown in deep crystallizing dishes but not in Petri dishes whereshoots come into direct contact with the medium. The effectof proline could be related to a decrease in shoot Cl^–and Na⁺ accumulation which was only observed in plantlets grownin crystallizing dishes. Proline but not betaine uptake intocultured plantlets was stimulated by NaCl while each organicsolute inhibited the endogenous synthesis of the other soluteunder salt stress. Comparison of the effects of exogenously supplied proline withenhanced endogenous proline accumulation in the mutant lineR5201 suggested that the increased proline accumulation in themutant is an order of magnitude too low to have a significantphysiological effect. The implications of the effect of prolineon ion transport, discrimination and accumulation are discussed. Key words: Salt tolerance, proline, ion transport, barley embryo culture     

Novel Light-Dark Change of Proline Levels in Halophyte (Mesembryanthemum crystallinum L.) and Glycophytes (Hordeum vulgare L. and Triticum aestivum L.) Leaves and Roots under Salt Stress

Proline accumulation was determined in a facultative halophyte,Mesembryanthemum crystallinum and glycophytes, barley (Hordeumvulgare L.) and wheat (Triticum aestivum L.) Proline accumulationpreceded the shift of CAM in M. crystallinum and did not occurin the continuous darkness. The novel light-dark change of prolinelevel (high in the light and low in the dark) was observed inleaves of all three plants. Proline levels of shoots in barleyand wheat also showed the same light-dark change, suggestingthat proline accumulated in the leaves in the light was nottranslocated to other tissues in the dark period. These resultssuggest that proline has a bifunctional role in the acclimationto high salt stress; an osmoregulant role in the light, anda substrate for dark respiration to supply energy to compartmentationof ions into vacuole in the dark. ¹Present address: Kyoto Biological Res. Lab., Bio-Chiba Inc.Watsuka,Soraku, Kyoto, 619-12 Japan ²Present address: Kobayashi Pharmaceutical Co., Ltd. Doshomachi,Chuo-ku, Osaka, 541 Japan     

An inland and a coastal population of the Mediterranean xero-halophyte species Atriplex halimus L. differ in their ability to accumulate proline and glycinebetaine in response to salinity and water stress

Soil salinity and drought compromise water uptake and lead toosmotic adjustment in xero-halophyte plant species. These importantenvironmental constraints may also have specific effects onplant physiology. Stress-induced accumulation of osmocompatiblesolutes was analysed in two Tunisian populations of the Mediteraneanshrub Atriplex halimus L.—plants originating from a salt-affectedcoastal site (Monastir) or from a non-saline semi-arid area(Sbikha)—were exposed to nutrient solution containingeither low (40 mM) or high (160 mM) doses of NaCl or 15% polyethyleneglycol. The low NaCl dose stimulated plant growth in both populations.Plants from Monastir were more resistant to high salinity andexhibited a greater ability to produce glycinebetaine in responseto salt stress. Conversely, plants from Sbikha were more resistantto water stress and displayed a higher rate of proline accumulation.Proline accumulated as early as 24 h after stress impositionand such accumulation was reversible. By contrast, glycinebetaineconcentration culminated after 10 d of stress and did not decreaseafter the stress relief. The highest salt resistance of Monastirplants was not due to a lower rate of Na⁺ absorption; plantsfrom this population exhibited a higher stomatal conductanceand a prodigal water-use strategy leading to lower water-useefficiency than plants from Sbikha. Exogenous application ofproline (1 mM) improved the level of drought resistance in Monastirplants through a decrease in oxidative stress quantified bythe malondialdehyde concentration, while the exogenous applicationof glycinebetaine improved the salinity resistance of Sbikhaplants through a positive effect on photosystem II efficiency. Key words: Atriplex halimus, glycinebetaine, halophyte, NaCl, osmotic adjustment, proline, salinity, water stress     

The effect of jasmonic acid on the accumulation of ABA,proline and spermidine and its influence on membrane injury under water deficit in two barley genotypes

Seedlings of two barley genotypes (‘Maresi’ and wild form of Hordeum spontaneum) were treated with jasmonic acid (JA 5 μM and 15 μM) for 24 h, and then subjected to water stress (PEG 6000 solution of − 1.5 MPa). JA caused an increase in the content of ABA but not in that of proline and spermidine in the two studied genotypes. The effect of the treatment did not depend on the applied JA concentration. The pre-stress treatment with JA changed plant response to water deficit with regard to membrane injury. Treatment with a lower JA concentration (5 μM) caused a substantial reduction of the stress-induced membrane damage in the both genotypes. A higher JA concentration (15 μM) caused the reduction of membrane injury only in H. spontaneum and was ineffective in ‘Maresi’. JA had no influence on the leaf water status in water-stressed plants. A possible role of JA in leaf ABA accumulation and alleviation of cell membrane injury under water deficit is discussed. The work was partly supported by the Polish Committee For Scientific Research, grant No 5 PO6A 036 18     

Antioxidant enzyme activity,proline accumulation,leaf area and cell membrane stability in water stressed Amaranthus leaves

Traditional crops are extremely important for food production in low income, food-deficit countries (LIFDCs) where they continue to be maintained by socio-cultural preferences and traditional uses. Significant potential exists to improve these crops, one of which is to select for improved productivity during moisture stress conditions. Germplasm of Amaranthus tricolor, Amaranthus hypochondriacus and Amaranthus hybridus were subjected to various screening methods to measure metabolic and physiological changes during water stress. The activities of enzymes involved in the oxygen-scavenging system during abiotic stress conditions (superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR)), free proline production, leaf area (LA), cell membrane stability (CMS), leaf water potential (LWP) and relative water content (RWC) were measured in these three amaranth species during induced water stress. This study showed significant differences in metabolic responses during water deficit of the three species tested. Moisture stress and a decrease in RWC and LWP were first experienced in A. hybridus and A. hypochondriacus, followed by A. tricolor. There was an indirect correlation between leaf water status (RWC and LWP), enzyme activity, proline production and leaf area. The combined effect of GR, APX and SOD could ensure higher levels of regulation of the toxic effect of H₂O₂ which could be associated with drought tolerance in Amaranthus. Distinct differences in onset of proline accumulation and the amount of accumulated in leaves upon induced water stress was noticed for the three amaranth species tested. Proline accumulation during water stress conditions in amaranth seems to be indirect and could possibly have a protective role apart from osmoregulation during stress conditions. This contention is supported by the decrease in leaf area and high cell membrane stability for two of the species tested. This study forms part of a project aimed at the development of improved traditional crops to contribute to food production and quality for subsistence farmers in areas with low precipitation or variable rainfall patterns.     

Implication of ABA and proline on cell membrane injury of water deficit stressed barley seedlings

The aim of this work was to examine the ability of ABA and proline to counteract the deleterious effect of water deficit stress on cell membrane injuries. Six-day-old seedlings of two barley genotypes (cv. Aramir, line R567) were treated with ABA (2·10⁻⁴ M) or proline (0.1 M) for 24 h, and then subjected to osmotic stress for 24h, by immersing their roots in polyethylene glycol (PEG 6000) solution of osmotic potential of −1.0 MPa and −1.5 MPa or by submerging the leaf pieces in PEG solution of osmotic potential of −1.6 MPa. Pretreatment of plants with ABA and proline caused an increase of free proline level in the leaves. Plants treated with ABA exhibited a lower membrane injury index under water stress conditions than those untreated even when no effect of this hormone on RWC in the leaves of stressed plants was observed. Pretreatment of plants with proline prevented to some extent membrane damage in leaves of the stressed seedlings, but only in the case when stress was imposed to roots. Improvement in water status of leaves was also observed in seedlings pretreatment with proline. The protective effect of both ABA and proline was more pronounced in line R567 that exhibited higher membrane injury under water deficit stress conditions.     

Assessing the drought tolerance variability in Mediterranean alfalfa (Medicago sativa L.) genotypes under arid conditions

This work investigated the variability in drought tolerance under arid conditions of Mediterranean alfalfa genotypes with the overall aim to assess the main criteria that are associated with the relative tolerance and to discover the most tolerant ecotypes. For this, 16 alfalfa genotypes originating from seven countries of the Mediterranean basin were tested in an experimental station in south of Tunisia. The trial was conducted under two irrigation treatments. The first was normally irrigated by providing an amount of water corresponding to the potential evapotranspiration of the crop, and in the second with water deficit which was applied by stopping the irrigation during 8 weeks in summer. A significant decrease was observed under water deficit for biomass production, leaf stem ratio and water use efficiency. The sensitive index, in stress treatment, varies between 13.8% and 46.2% for dry yield. Results showed that some genotypes exhibited high-forage yield potential even in the presence of stress, mainly Amerist, Sardi10 and Siriver. Proline accumulation in leaves was greater in water-stressed plants, while the K⁺ osmo-regulatory role was not definite. High biomass production, accumulation of proline and constancy of K⁺ in leaves are the most important criteria for tolerant alfalfa.     

Drought-induced proline synthesis depends on root-to-shoot communication mediated by light perception

Proline accumulation in roots and shoots is one of the most evident responses to environmental stresses such as drought, which is currently one of the main threats for agriculture. Based on this response, in this work, we hypothesize that proline accumulation is dependent on root-to-shoot communication through light perception. Thus, we used exaggerated light response (hp1) and phytochrome-deficient (au) mutants of tomato, which were combined through self-grafting and reciprocal grafting and subjected to drought stress, for posterior determination of shoot and root growth and proline content. Light-affected proline metabolism, as hp1, had the highest accumulation, while au presented the lowest proline values. Reciprocal grafting showed that hp1 and MT as scion or rootstock improved MT and au proline content, respectively, indicating shoot-to-root and root-to-shoot communication modulate the metabolism of this compatible osmolyte. Dry weight, leaf area, and root area presented similar patterns to proline content, indicating the importance of this compound for plant growth under stress conditions. These results provide a new perspective on light mediation of long-distance proline translocation in stressed plants.