Evaluation of Abiotic Stress Resistance in Mutated Populations of Cauliflower (Brassica oleracea var. botrytis)

An efficient in vitro screening method has been developed for cauliflower to create NEU and NMU induced mutant lines selected on hydroxyproline containing medium. Mutant lines and control plants were sub-cultured many times on maintenance medium and stored at 5°C for 2 years and then tested for salt and hydroxyproline resistance as in vitro and in vivo plants. In vitro shoot tips were also sub-cultured to media containing hydroxyproline and NaCl for 28 days and then assessed for their leaf proline content. Non-acclimated and acclimated in vivo plants were also assessed for resistance to freezing. Populations of control and selected lines were created by mass pollination and subsequently tested for their NaCl and frost resistance. Control plants had little or no NaCl or hydroxyproline resistance whilst selected plants showed varying degrees of resistance. In vitro and in vivo responses of selected lines were correlated. Leaf proline content was increased markedly in the mutant lines and the greatest proline contents occurred following NaCl stress with the most respondent line having 100-fold levels compared to the controls. Both non-acclimated and acclimated selected lines showed improved frost resistance over controls. Improvements in frost resistance were heritable but improvements in NaCl resistance were not. The results clearly demonstrated that NaCl, frost and hydroxyproline resistance were stable traits over repeated in vitro sub-cultures and prolonged low temperature storage. A complete range of mutants with single, double or triple resistance traits were produced. The level of resistance however was not necessarily correlated with the level of proline and some lines showed resistance without elevated proline. It is concluded that elevated proline is not essential for improved resistance to abiotic stress in cauliflower, but where it does occur it does improve resistance.     

Tobacco transformants expressing antisense sequence of proline dehydrogenase gene possess tolerance to heavy metals

Analysis of resistance of genetically modified tobacco plants bearing antisense suppressor of proline dehydrogenase gene and characterized with higher content of proline to elevated concentrations of heavy metals was performed. It was demonstrated that progeny of transgenic plants have high resistance to lead, nickel and cadmium ions.     

Evaluation of salt tolerance in Nicotiana tabacum plants bearing an antisense suppressor of the proline dehydrogenase gene

We studied the stress resistance of genetically modified (GM) tobacco plants bearing an antisense suppressor of the gene for proline dehydrogenase. Such plants are characterized by elevated proline content. The progeny of the transgenic plants were shown to have elevated salt tolerance.     

Dormancy development during cold hardening of in vitro cultured Malus domestica Borkh. plants in relation to their frost resistance and cryotolerance

The development of dormancy, frost resistance and cryotolerance of in vitro apple plants (Malus domestica Borkh.), cv. Greensleeves during their exposure to cold hardening was studied. In vitro cultures were cold hardened at 4°C under a short photoperiod up to 25?weeks. The dormancy status, non-structural saccharides, proline, water content and frost resistance were evaluated for optimization of cryopreservation. According to regrowth tests, in vitro cultures exhibited endogenous dormancy after the maximal frost resistance was reached. The highest regeneration ability of shoot tips after cryopreservation by encapsulation–dehydration method coincided with the period of the plant’s dormant state and maximum of frost resistance. All studied saccharides and proline exhibited the maximal values at the beginning of cold hardening and/or the dormancy phase. Contrary to the accumulation of saccharides and proline, water content showed the inverse time behaviour. According to these results, the cold hardening-induced endodormancy, high frost resistance and accumulation of saccharides and proline are the important prerequisites for the successful cryopreservation of shoot tips of in vitro grown apple plants.     

Evaluation of salt tolerance in Nicotiana tabacum plants bearing an antisense suppressor of the proline dehydrogenase gene

We studied the stress resistance of genetically modified (GM) tobacco plants bearing an antisense suppressor of the gene for proline dehydrogenase. Such plants are characterized by elevated proline content. The progeny of the transgenic plants were shown to have elevated salt tolerance.     

Hardening, abscisic acid, proline and freezing resistance in two winter wheat varieties

Two varieties of winter wheat ( Triticum aestivum L.) differing in freezing resistance ("Holme" from Sweden, freezing resistant, and "Amandus" from Germany, less freezing resistant) were hardened for five weeks by gradually reducing the day/night temperature from 20°C/15°C during the first week to 2° C/0° C during the fifth week and the photoperiod from 15 to 9 h. This treatment increased the freezing resistance of both varieties in comparison to unhardened control plants. Hardening caused an increase in osmolarity of cell sap and in the levels of proline and abscisic acid (ABA). Increase in osmolarity preceded the increase in ABA level, and proline levels increased later than ABA levels. Holme had higher values of osmolarity as well as higher levels of ABA and proline. but the differences between the two varieties were significant only for proline. Since the pressure potential remained constant or increased slightly during the hardening period, it is suggested that the accumulation of ABA is due to the hardening process and not to simple water stress caused by cold-induced inhibition of water uptake by the root.
Spraying hardened plants with 10⁻⁴ M ABA 24 h before a freezing test increased freezing resistance in both varieties, but did not obliterate the differences in freezing resistance between the two varieties. Spraying hardened plants with an aqueous proline solution (10%, w/v) was without effect on freezing resistance. It is concluded that the hardening procedure causes an accumulation of ABA in winter wheat leaves and that ABA is involved in the chain of events leading to freezing resistance.     

Tobacco transformants expressing the <Emphasis Type="Italic">Medicago truncatula</Emphasis> ornithine aminotransferase cDNA

The Medicago truncatula ornithine aminotransferase cDNA was cloned under the potent constitutive 35S RNA promoter of the cauliflower mosaic virus and transferred into the genome of tobacco Nicotiana tabacum SR1 plants. Transformed tobacco plants grew better in salinity stress, but did not differ in proline content under normal or stress conditions from control plants. It was assumed that the role of ornithine aminotransferase in the molecular mechanisms of stress resistance is not associated with additional proline synthesis.     

Importance of N source on heat stress tolerance due to the accumulation of proline and quaternary ammonium compounds in tomato plants

Proline and quaternary ammonium compounds (QAC), in addition to being N-rich, are known to accumulate in plants under different environmental stress conditions. The accumulation of N-rich compounds in plants has been shown to confer stress resistance. The aim of our work is two-fold: first, to study the influence of temperature on proline, QAC, and choline metabolism in tomato leaves; and second, to investigate the relationship between N source applied (NO3- or NH4+) and thermal stress resistance in these plants. To do this, experiments were conducted at three different temperatures (10 degrees C, 25 degrees C, 35 degrees C); at each temperature half of the plants received NO3-, and the other half received NH4+. At 35 degrees C the plants had the lowest biomass production with respect to 25 degrees C (optimal temperature) and 10 degrees C (cold stress), suggesting that tomato plants were most affected by heat stress. At 35 degrees C, there were also high levels of choline and proline due to the activation of Delta1-pyrroline-5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT), and simultaneous inhibition of proline dehydrogenase (PDH) and proline oxidase (PO). However, plants with NH4+ as the N source exhibited reduced growth with respect to the plants fed with NO3-. This is interesting because, under heat stress (35 degrees C), biomass production, as well as proline and choline accumulation, in NH4+ fed plants was higher than in NO3- fed plants. From this, we concluded that tomato plants fed with NH4+ as the N source show higher tolerance to heat stress (35 degrees C) than plants fed with NO3-.     

Moderately increased constitutive proline does not alter osmotic stress tolerance

Proline-overproducing carrot cell lines were isolated by selection in medium containing hydroxyproline, a toxic analogue of proline. During growth of the cells in culture, length of lag phase, doubling time, and maximum fresh weight were the same for the hydroxyproline-resistant cell line (HP) and the wild-type cell line (JW). Proline content and resistance to hydroxyproline in the HP and JW lines were not strictly correlated indicating that another reason besides the constitutive level of proline is involved in hydroxyproline resistance. Tolerance to polyethylene glycol-induced desiccation stress was not different between the two lines except perhaps at the early stages of culture growth when the proline levels of the two cell lines were nearly the same. The complexity of the relationship between proline accumulation and osmotolerance is discussed and strategies to achieve constitutive high levels of proline accumulation in plants are proposed.     

利用P5CS基因转化白三叶的研究

为了培育抗旱白三叶,构建了植物表达载体pBPC-P5CS,利用基因枪法转化白三叶的愈伤组织,PCR检测和Southern blot鉴定证实白三叶中已导入P5CS基因。对转P5CS基因白三叶植株的不同抗旱指标进行了分析。结果表明,与对照相比,转P5CS基因株系的抗旱能力得到了较大的提高。干旱胁迫下,与对照相比,转P5CS基因植株的脯氨酸含量和相对含水量分别比对照高20.0%-21.2%和5.6%-8.5%。     

Effect of flooding on tomato cultivars: The relationship between proline accumulation and other morphological and physiological changes

Flooding of the root system of tomato plants ( Lycopersicon esculentum ) caused cessation of leaf elongation, leaf epinasty, formation of adventitious roots, and increase in diffusive resistance associated with the wilting of leaves at the first stage of the stress. Upon development of adventitious roots, the wilted leaves regained their turgor and the diffusive resistance slowly decreased at a rate slower than that at which water potential increased. In the course of flooding, proline accumulated but after 11 days dropped back to the control level. The extent of proline accumulation in various tomato cultivars was positively correlated with the extent to which their leaf water potential dropped, but was not correlated with the changes in their diffusive resistance. Cultivars which accumulated the highest proline levels were those which showed the most severe injury, with only one cultivar as an exception. However, only in the cultivars producing high levels of proline was the return of leaf turgor followed by resumption of leaf elongation. In cv. 'Hosen', which was severely injured by the stress, but accumulated a low level of proline, leaf elongation was not resumed. The results suggest that proline accumulation is an indicator of the cultivar's sensitivity to dehydration associated with the flooding stress, and confirm the notion that proline may play a role in the post-stress recovery process.     

Proline Accumulation in Transgenic Tobacco as a Result of Expression of Arabidopsis Δ1-Pyrroline-5-carboxylate synthetase (P5CS) During Osmotic Stress

The entire coding sequence of the bi-functional enzyme, Δ¹-Pyrroline-5-carboxylate synthetase (P5CS) from Arabidopsis thaliana was reverse-transcribed, amplified and expressed under the control of CaMV 35S promoter in transgenic tobacco plants. Several lines were established and tested for the expression of P5CS. Drought and salinity were applied as osmotic stresses and proline content of the transformed plants was compared with that of non-transformed controls. Results indicate that transgenic lines express higher levels of proline and show enhanced resistance to the applied osmotic stress as compared to the non-transgenic plants.     

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.     

渗透调节参与循环干旱锻炼提高烟草植株抗旱性的形成

对漂浮育苗的烟草幼苗进行控水一半萎焉．复水一恢复的循环干旱锻炼。结果表明,这种干旱锻炼能显著提高烟草幼苗根、茎、叶中的渗透调节物质可溶性糖和脯氨酸的含量,降低细胞渗透势。当干旱锻炼过的烟草植株遭受后续的干旱胁迫时,与未锻炼的对照相比,其根、茎、叶能积累更多的可溶性糖和脯氨酸,从而降低了细胞渗透势,使叶片能维持较高的膨压。这些结果表明渗透调节参与了循环干旱锻炼提高的烟草植株抗旱性的形成过程。此外,干旱锻炼提高了烟草幼苗的根／冠比。循环干旱锻炼过程中烟草植株一方面使其各部位通过渗透调节来对干旱环境进行生理适应,另一方面通过调节光合产物在地上部和地下部的分配以影响根／冠比来对干旱环境进行形态适应,以最终提高其抗旱性。     

Changes in abscisic acid and proline levels in maize varieties of different drought resistance

Four varieties of maize differing in drought resistance and geographical origin (Swabi White and Shaheen from Pakistan, Garbo and Goldprinz from Germany) were analyzed for their proline and abscisic acid (ABA) accumulation during a prolonged water stress period. Proline levels increased continuously during the stress period in all the four varieties, but to different amounts. The drought-susceptible varieties Shaheen and Goldprinz produced higher levels of proline than the drought-resistant varieties Swabi White and Garbo. A negative correlation was also found between maximal ABA contents and degree of drought resistance during prolonged stress of younger plants. ABA levels did not increase steadily, but reached a maximum long before the end of the stress phase, and then declined. The results are discussed in relation to the possibility of using proline and ABA levels as biochemical indicators of resistance against drought.     

Effect of 24-epibrassinolide on drought stress-induced changes in Chorispora bungeana

Brassinosteroids (BRs) have been proposed to increase the resistance of plants to drought stress. The effect of foliar application of 0.1 μM 24-epibrassinolide (EBR) on chlorophyll (Chl) content, photosystem 2 (PS 2) photochemistry, membrane permeability, lipid peroxidation, relative water content (RWC), proline content, and the antioxidant system in drought-stressed Chorispora bungeana plants was investigated. The results showed that polyethylene glycol (PEG) induced water stress decreased RWC, Chl content and variable to maximum Chl fluorescence ratio (F_v/F_m) less in plants pretreated with EBR than in non-pretreated plants. In addition, lipid peroxidation, measured in terms of malondialdehyde content, membrane permeability and proline content in drought-stressed plants were less increased in EBR pretreated plants, while antioxidative enzyme activities and reduced ascorbate and glutathione contents were more increased in EBR pretreated than in non-pretreated plants. These results suggested that EBR could improve plant growth under drought stress     

Is proline involved in stomata regulation of Commelina communis plants recovering from salinity stress?

Plants of Commelina communis L. were grown in culture solution to which NaCl was added for 48 h. The solutions were then replaced with normal medium, so that the plants could recover from the stress. The water potential increased almost to that of the controls during 4 h of recovery, but stomatal resistance stayed high. Cytokinin treatment of leaf discs failed to enhance recovery of stomatal aperture, although it enhanced stomatal recovery of identically treated epidermal tissue. Proline levels in leaves correlated well with stomatal resistance. Incubation of epidermal tissue in D-proline inhibited stomatal opening. NaCl and benzyladenine interacted with the effect of proline, and the effect of abscisic acid and was additive to that of proline.     

Effect of Didymella applanata (Niessl/Sacc.) on proline and hydroxyproline containing cell wall proteins of red raspberry

Structural cell wall proteins and their immobilisation through formation of covalent cross-links belong to important defense response mechanisms. In this work, the D. applanata — induced increase of wall-bound proline and hydroxyproline contents were associated with red raspberry resistance. The effect did not depend on the developmental stage and lignification degree of infected primocanes. Pathogen-induced accumulation of hydroxyproline noted in susceptible plants suggested the post-translational hydroxylation of proline-rich proteins and/or may be of other wall proteins. Moreover, the developmentally related increase of hydroxyproline content was associated with the resistance acquirement noted at the end of the summer period. The increase of cell wall structural protein levels was accompanied by free proline accumulation in the cytosol fraction.