Proline transport increases growth efficiency in salt-stressed Streptomyces griseus

Streptomyces griseus synthesizes proline for osmoregulation under salt stress. Uptake of exogenous [14C]proline and internal synthesis of proline were quantified in cells growing at salt concentrations from 0 to 1 M NaCl. Externally supplied proline accounted for an increased proportion of the intracellular pool of free proline as salt concentration was increased, but neither the concentration nor the composition of the internal amino acid pool was substantially altered by supply of exogenous proline. Uptake of exogenous proline significantly increased the specific growth yield of S. griseus growing under salt stress; the increased yield was proportional to reductions in proline synthesis.     

Proline accumulation and glutamine synthetase activity are increased by salt-induced proteolysis in cashew leaves

In this study cashew (Anacardium occidentale) plants were exposed to a short- and long-term exposure to NaCl in order to establish the importance of the salt-induced proteolysis and the glutamine synthetase activity on the proline accumulation. The cashew leaf showed a prominent proline accumulation in response to salt stress. In contrast, the root tissue had no significant changes in proline content even after the drastic injury caused by salinity on the whole plant. The leaf proline accumulation was correlated to protease activity, accumulation of free amino acid and ammonia, and decrease of both total protein and chlorophyll contents. The leaf GS activity was increased by the salt stress whereas in the roots it was slightly lowered. Although the several amino acids in the soluble pool of leaf tissue have showed an intense increment in its concentrations in the salt-treated plants, proline was the unique to show a proportional increment from 50 to 100 mol m-3 NaCl exposure (16.37 to 34.35 mmol kg-1 DM, respectively). Although the leaf glutamate concentration increased in the leaves of the salt-stressed cashew plants, as compared to control, its relative contribution to the total amino acid decreased significantly in stressed leaves when compared to other amino acids. In addition, when the leaf discs were incubated with NaCl in the presence of exogenous precursors (Glu, Gln, Orn or Arg) involved in the proline synthesis pathways, the glutamate was unique in inducing a significant enhancement of the proline accumulation compared to those discs with precursor in the absence of NaCl. These results, together with the salt-induced increase in the GS activity, suggest an increase in the de novo synthesis of proline probably associated with the increase of the concentration of glutamate. Moreover, the prominent salt-induced proline accumulation in the leaves was associated with the higher salt-sensitivity in terms of proteolysis and salt-induced senescence as compared to the roots. In conclusion, the leaf-proline accumulation was due, at least in part, to the increase in the salt-induced proteolysis associated with the increments in the GS activity and hence the increase in the concentration of glutamate precursor in the soluble amino acid pool.     

Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities

Proline and betaine accumulate in plant cells under environmental stresses including salt stress. Here, we investigated effects of proline and betaine on the growth and activities of antioxidant enzymes in tobacco Bright Yellow-2 (BY-2) culture cells in suspension under salt stress. Both proline and betaine mitigated the inhibition of growth of BY-2 cells under salt stress and the mitigating effect of proline was more than that of betaine. Salt stress significantly decreased the activities of superoxide dismutase (SOD), catalase and peroxidase in BY-2 cells. Exogenous application of proline or betaine alleviated the reduction in catalase and peroxidase activities but not SOD activity under salt stress. In addition, proline was found to be effective in alleviating the inhibition of salt stress-induced catalase and peroxidase activities in BY-2 cells. Neither proline nor betaine directly scavenged superoxide (O(2)(-)) or hydrogen peroxide (H(2)O(2)). It is concluded that exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine because of its superior ability to increase the activities of antioxidant enzymes.     

Salt stress enhances proline utilization in the apical region of barley roots

Accumulation of an osmoprotectant, proline, is enhanced in response to salinity in plants. Here, by immunohistochemical analysis, we demonstrated that proline transporter (HvProT) was highly expressed in the apical region of barley roots under salt stress. Free proline was accumulated more in the basal region than in the apical region of barley roots under salt stress, although expression level of HvProT was higher in the apical region. On the other hand, salt stress increased proline and hydroxyproline contents in the cell wall fraction of the root apical region, suggesting increment of proline utilization. Expression of the genes encoding cell wall proteins (proline rich protein and extensin) and cellulose synthase was induced in barley roots by salt stress. These findings indicated that free proline transported by HvProT presumably behaved as a component of cell wall synthesis in the apical region of barley roots under salt stress.     

Influence of exogenous proline on embryogenic and organogenic maize callus subjected to salt stress

The effect of exogenous proline (6 mM) and increasing NaCl doses (from 0.4 to 1.2% w/v) on the maintenance of organogenic and embryogenic callus lines derived from the salt-sensitive maize inbred W64Ao2 were studied. To this end, total protein, free amino acid and polyamine content were analyzed. The demand of exogenous nitrogen and especially of proline, even in the presence of salt, differed in the two types of morphogenic calluses. The total protein content of embryogenic calluses was higher in the presence of proline than in its absence, in all the cases studied. An opposite effect of proline was observed in organogenic calluses: the presence of proline and salt decreased significantly their protein content. With respect to amino acid and polyamine contents, the organogenic calluses showed physiological characteristics of salt-adaptation, whereas the embryogenic calluses were more sensitive to NaCl. Although endogenous proline increased in the organogenic calluses cultured in the presence of salt, in embryogenic calluses it only rose at the lowest salt concentration. Furthermore, the endogenous arginine content under saline conditions was higher in organogenic calluses. A compensatory effect between proline and polyamine metabolism related to the endogenous arginine content in response to salt stress was also observed. This effect differed in the two types of calluses.     

Influence of exogenous proline on embryogenic and organogenic maize callus subjected to salt stress

The effect of exogenous proline (6 mM) and increasing NaCl doses (from 0.4 to 1.2% w/v) on the maintenance of organogenic and embryogenic callus lines derived from the salt-sensitive maize inbred W64Ao2 were studied. To this end, total protein, free amino acid and polyamine content were analyzed. The demand of exogenous nitrogen and especially of proline, even in the presence of salt, differed in the two types of morphogenic calluses. The total protein content of embryogenic calluses was higher in the presence of proline than in its absence, in all the cases studied. An opposite effect of proline was observed in organogenic calluses: the presence of proline and salt decreased significantly their protein content. With respect to amino acid and polyamine contents, the organogenic calluses showed physiological characteristics of salt-adaptation, whereas the embryogenic calluses were more sensitive to NaCl. Although endogenous proline increased in the organogenic calluses cultured in the presence of salt, in embryogenic calluses it only rose at the lowest salt concentration. Furthermore, the endogenous arginine content under saline conditions was higher in organogenic calluses. A compensatory effect between proline and polyamine metabolism related to the endogenous arginine content in response to salt stress was also observed. This effect differed in the two types of calluses.     

一氧化氮参与调节盐胁迫下脱落酸诱导的小麦幼苗叶片脯氨酸的累积

不同浓度(0.01～5.00mmol/L)的外源一氧化氮(NO)供体硝普钠(SNP)以浓度依赖性的性式诱导150mmol/LNaCl胁迫下小麦(Triticum aestivum L.cv.Yangmai 158)幼苗叶片脯氨酸的累积.其中0.1 mmol/L的SNP效果最明显,而结合采用NO清除剂c-PTIO和血红蛋白的处理均分别逆转了该效应.研究结果还发现:0.1 mmol/L SNP诱导的脯氨酸累积还可能有利于盐胁迫下小麦幼苗的保水性;0.1 mmol/L的SNP显著激活了内源ABA的合成,而结合血红蛋白的处理则证实,在外源ABA诱导脯氨酸累积的过程中NO可能作用于ABA信号分子的下游,但NO和ABA信号分子在此诱导反应中不存在累积效应.进一步研究脯氨酸合成和降解的酶促反应途径,发现外源NO处理前4天内可能主要是通过提高△'-吡咯啉-5-羧酸合成酶(P5CS)的活性来促进脯氨酸的合成,以后直至第8天主要是通过抑制脯氨酸脱氢酶(ProDH)的活性来抑制脯氨酸的降解;ABA对于P5CS和ProDH活性的调节能力弱于NO.此外,Ca2 在NO诱导的盐胁迫下小麦叶片脯氨酸累积的信号分子途径中起重要的介导作用.     

甘薯愈伤组织对干旱胁迫和盐胁迫的生理反应对比

研究干旱胁迫和盐胁迫对“芦选一号”。日‘薯愈伤组织可溶性蛋白、可溶性糖、脯氨酸含量、SOD活性等的影响,从而在细胞水平上探讨甘薯抵御渗透胁迫的生理机制。并分析甘薯细胞对干旱处理（PEG-6000）和盐处理（NaCl）的反应差异。结果表明,可溶性蛋白质含量在干旱胁迫下缓慢升高,在轻度和中度盐胁迫的生长前期和中期有较大幅度的上升。但后期下降,表明短时间盐胁迫下,Na^＋可能促进可溶性蛋白的合成;MDA在重度干旱胁迫下的含量显著低于重度盐胁迫,而SOD活性显著高于盐胁迫。表明在盐胁迫下细胞膜透性增加的主要原网是膜脂过氧化作用。干旱处理则是PEG-6000脱水的直接结果;重度干旱胁迫下,可溶性糖含量在短期内迅速升高,然后下降,而脯氨酸含量则在胁迫中后期迅速上升。脯氨酸可能有补偿可溶性糖含量降低的作用。     

外源脯氨酸对盐胁迫下甜瓜幼苗硝酸还原的影响

采用营养液水培方法,以＂雪美＂品种甜瓜（Cucumis melo L.）为材料,研究了外源脯氨酸（Proline）对盐胁迫下甜瓜幼苗叶片和根系硝酸还原的影响。结果表明：（1）盐胁迫提高了甜瓜幼苗叶片和根系内铵态氮（NH4＋-N）和可溶性蛋白含量;降低了硝态氮（NO-3-N）含量和硝酸还原酶（nitrate reductase,NR）活性。（2）外源施用脯氨酸明显地提高了盐胁迫下甜瓜幼苗叶片和根系内NO-3-N和可溶性蛋白含量;降低了盐胁迫下甜瓜幼苗叶片和根系内NH＋4-N含量;增强了盐胁迫下甜瓜幼苗体内NR活性。研究结果表明,外源脯氨酸可以通过调节甜瓜幼苗体内硝酸还原酶活性和氮化合物含量来缓解盐胁迫对甜瓜幼苗植株的伤害。     

外源脯氨酸对盐胁迫下甜瓜幼苗根系抗坏血酸-谷胱甘肽循环的影响

以2个不同耐盐强度的甜瓜品种‘玉皇’（耐盐性强）和‘雪美’（耐盐性弱）为材料,采用营养液栽培方法,研究外源脯氨酸对盐胁迫下甜瓜幼苗根系抗坏血酸-谷胱甘肽循环的影响。结果显示：（1）盐胁迫下,2个甜瓜品种根系内的还原型抗坏血酸（ASA）、还原型谷胱甘肽（GSH）含量降低,氧化型谷胱甘肽（GSSG）含量升高,且‘雪美’变化幅度大于‘玉皇’;（2）盐胁迫下,施用外源脯氨酸提高了2个甜瓜品种根系中ASA和GSH的含量,降低了GSSG含量,同时也提高了GSH/GSSG的比值,且对‘雪美’的作用大于‘玉皇’;（3）盐胁迫处理3 d时,2个甜瓜品种根系的抗坏血酸过氧化物酶（APX）、脱氢抗坏血酸还原酶（DHAR）、谷胱甘肽还原酶（GR）活性均下降,且‘雪美’下降的幅度较大;随着胁迫时间的延长（5 d时）,‘玉皇’幼苗根系内APX、DHAR、GR活性有所上升,‘雪美’根系中这3种酶活性则进一步降低;（4）盐胁迫下,施用外源脯氨酸提高了2个甜瓜品种根系内的APX、DHAR和GR的酶活性,且对‘雪美’的作用大于‘玉皇’。本研究结果表明,外源脯氨酸可以通过增加非酶促抗氧化物质ASA、GSH的含量和抗氧化酶活性,提高抗坏血酸-谷胱甘肽循环清除活性氧的能力,从而缓解盐胁迫对甜瓜植株的伤害。     

碱性盐胁迫下星星草幼苗中几种渗透调节物质的变化

模拟松嫩盐碱草地碱化土壤的离子组成配制碱性盐溶液处理星星草（Ｐｕｃｃｉｎｅｌｌｉａｔｅｎｕｉｆｌｏｒａ）幼苗,测定了碱性盐胁迫下星星草幼苗地上部分几种渗透调节物质含量的变化,随着碱性协浓度及胁迫天数的增加,Ｎａ＾＋可溶性盐,游离脯氨酸和可淀粉糖的含量逐渐增加,其中游离脯氨酸的变化倍数最大,Ｋ＾＋含量在浓度小于２０ｇＬ＾－１碱性胁迫下随胁迫天数增加而略为减少,在４０ｇｒＬ＾－１和６０ｇＬ＾－１碱性胁     

外源NO对NaCl胁迫下辣椒幼苗氧化损伤的保护效应

以辣椒品种陇椒2号为试验材料,研究了外源NO供体硝普钠(SNP)对辣椒幼苗氧化损伤的影响.结果显示,在100 mmol/L NaCl胁迫下,辣椒叶片的MDA含量、质膜相对透性和脯氨酸含量均增加,保护酶SOD、CAT活性降低,而POD活性只在胁迫18 d时降低.0.1 mmol/L SNP处理可减缓NaCl胁迫下辣椒幼苗叶片MDA含量的上升,降低叶片质膜相对透性,并诱导SOD、POD和CAT活性增加,提高脯氨酸含量,表明外源NO可以通过提高盐胁迫下辣椒幼苗叶片组织的抗氧化能力来缓解氧化损伤.而SNP相似物NaNO2和K3Fe(CN)6处理对盐胁迫引起的氧化损伤并没有起到明显的缓解作用,进一步证实了NO对辣椒幼苗耐盐性具有专一性的调节作用.     

Effect of salt stress on cucumber: Na+–K+ ratio, osmolyte concentration, phenols and chlorophyll content

A pot experiment with 17 diverse genotypes of cucumber with four levels of salt stress viz., 0, 2, 4 and 6 dS m^?1 was carried out during 2006. ANOVA revealed significant differences amongst genotypes and genotype × salt stress interaction indicating the genetic variability and differential response of the genotypes to different salt stress levels. The salt stress adversely affected the biochemical parameters; effects were severe under 4 dS m^?1. No genotype could survive at 6 dS m^?1. Sodium content, Na⁺–K⁺ ratio, proline, reducing sugars, phenol and yield reduction (%) increased significantly as the salt stress increased. Potassium, chlorophyll, membrane stability index and fruit yield decreased significantly under salt stress in all genotypes. However, the genotypes CRC-8, CHC-2 and G-338 showed lower accumulation of sodium, lesser depletion of potassium, lower Na⁺–K⁺ ratio and higher accumulation of proline, reducing sugars, phenols, better membrane stability and lower yield reduction (%) under salt stress, while CH-20 and DC-1 were sensitive to salt stress. Thus, a combination of traits such as higher membrane stability, lower Na⁺–K⁺ ratio, higher osmotic concentration and selective uptake of useful ions and prevention of over accumulation of toxic ions contribute to salt stress tolerance in cucumber. These traits would be useful selection criteria during salt stress breeding in cucumber.     

Abscisic acid, indole-3-acetic acid and mineral–nutrient changes induced by drought and salinity in longan (Dimocarpus longan Lour.) plants

Longan species (Dimocarpus longan Lour.) exhibit a high agronomic potential in many subtropical regions worldwide; however, little is known about its responses to abiotic stress conditions. Drought and salinity are the most environmental factors inducing negative effects on plant growth and development. In order to elucidate the responses of longan to drought and salinity, seedlings were grown under conditions of drought and salt stresses. Drought was imposed by suspending water supply leading to progressive soil dehydration, and salinity was induced using two concentrations of NaCl, 100 and 150 mM in water solution, for 64 days. Data showed that salt concentrations increased foliar abscisic acid (ABA) and only 150 mM NaCl reduced indole-3-acetic acid (IAA) and increased proline levels. NaCl treatments also increased Na⁺ and Cl^? content in plant organs proportionally to salt concentration. Drought increased leaf ABA but did not change IAA concentrations, and also increased proline synthesis. In addition, drought and salt stresses reduced the photosynthesis performance; however, only drought decreased leaf growth and relative leaf water content. Overall, data indicate that under severe salt stress, high ABA accumulation was accompanied by a reduction of IAA levels; however, drought strongly increased ABA but did not change IAA concentrations. Moreover, drought and high salinity similarly increased (or maintained) ion levels and proline synthesis. Data also suggest that ABA accumulation may mitigate the impact of salt stress through inducing stomatal closure and delaying water loss, but did not mediate the effects of long-term drought conditions probably because leaves reached a strong dehydration and the role of ABA at this stage was not effective to detain leaf injuries.     

脯氨酸对不结球白菜（Brassica chinensis）镉积累的影响

采用溶液培养的方法,研究了过量Cd对不结球白菜（Brassica chinensis）幼苗内源脯氨酸含量的影响及外源脯氨酸在不结球白菜Cd积累中的作用。100μmol？L-1 Cd处理明显增加不结球白菜叶片脯氨酸（Pro）的含量,并且随着Cd胁迫时间的延长,其含量也显著上升。100μmol？L-1 Cd处理明显降低脯氨酸脱氢酶（PDH）活性,而增加δ1-吡咯啉-5-羧酸合成酶（P5CS）的活性。1~5 mmol？L-1外源Pro处理显著降低不结球白菜幼苗地上部Cd含量,对根系Cd含量无显著影响,但外源Pro处理并不能缓解过量Cd对不结球白菜幼苗生物量的抑制作用。外源Pro处理下,不结球白菜叶片中Pro含量与其地上部干重呈显著的负相关关系。     

Salt stress increases ferredoxin-dependent glutamate synthase activity and protein level in the leaves of tomato

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) catalyzes an essential step in the pathway of glutamate biosynthesis. Exposing detached tomato ( Lycopersicon esculentum ) leaves for 6 h to 12 g l⁻¹ NaCl resulted in a significant two-fold increase in the activity of ferredoxin-dependent glutamate synthase extracted from the leaves. Western blot studies demonstrated that salt treatment also increased the ferredoxin-dependent glutamate synthase content of the leaves. A similar effect of salt on the concentration of this enzyme was found in the leaves of hydroponically-grown tomato plants. The induction of ferredoxin-dependent glutamate synthase under salt stress may provide the glutamate required for the proline synthesis which is a common response to salt stress.     

盐胁迫下唐古特白刺对外源Ca2＋的生理响应

为探讨Ca2＋与盐生植物耐盐性的关系,以唐古特白刺（Nitrariatangutorum）为试验材料,研究不同浓度外源C矛’对盐胁迫下唐古特白刺的生理响应,结果表明,盐浓度不高于300mmol·L-1时,施加一定浓度Ca2＋（≤15mmol·L-1）可增加唐古特白刺叶片相对含水量,提高叶片水势与根系活力,降低电解质外渗率,减少MDA含量,增加脯氨酸的积累,同时提高SOD和POD活性,且这种趋势随Ca2＋浓度的增加而增加。而高浓度Ca2＋（〉15mmol·L-1）对唐古特白刺各生理指标均表现出不同程度的抑制作用,影响唐古特白刺的正常代谢活动。说明一定浓度的Ca2＋（≤15mmol·L-1）能有效缓解盐胁迫（NaCI≤300mmol·L-1）对唐古特白刺造成的伤害,高盐胁迫下外施Ca2＋缓解作用不明显,甚至表现为抑制作用。     

Hydrogen peroxide-induced salt tolerance in the <Emphasis Type="Italic">Arabidopsis</Emphasis> salicylate-deficient transformants <Emphasis Type="Italic">NahG</Emphasis>

The effect of hydrogen peroxide treatment on the salt tolerance of wild-type Arabidopsis thaliana L. plants (Col-0) and plants transformed with the bacterial salicylate hydroxylase gene (NahG) was studied. The base tolerance to salt stress caused by 200 mM of NaCl in solution culture was higher in plants with the NahG genotype in comparison with the wild-type plants. Growth inhibition was observed for wild-type plants under the action of exogenous hydrogen peroxide, which was not observed for the NahG transformants; salt tolerance increased in the both types of plants after treatment, which was assessed based on the growth indicators and the ability to preserve the chlorophyll pool following NaCl treatment. The content of endogenous Н2О2 in the leaves of wild-type plants increased significantly following exogenous hydrogen peroxide treatment and salt stress, while it practically did not change in the leaves of the NahG genotype. The SOD activity increased in both genotypes after treatment with exogenous hydrogen peroxide, and remained at an elevated level after salt stress in comparison with the nontreated plants. Furthermore, the catalase activity increased in leaves of the salicylate-deficient genotype but not in the Col-0 genotype. The guaiacol peroxidase activity increased in plants of both genotypes under the action of hydrogen peroxide and salt stress, with the NahG plants demonstrating a higher degree of increase. The Н₂О₂ treatment facilitated the increase of the proline content in leaves of the plants of both genotypes under conditions of salt stress. It was concluded that there were hydrogen peroxide signal transduction pathways in Arabidopsis plants that were salicylic acid independent and that the antioxidant system functioned more effectively in salicylate-deficient Arabidopsis plants.     

外源ABA对楸树幼苗NaCl胁迫的缓解效应及其生长生理响应特征

以2年生楸树(苏楸1号和008-1)扦插苗为材料,采用盆栽试验法,分析盐胁迫(0.5%NaCl)处理下楸树幼苗生长、生理的变化,并分析不同浓度外源ABA(15、25、35 mg/L)对盐胁迫(30 d)楸树幼苗的缓解效应及其生理生化特性,以探索重度盐胁迫下适合楸树幼苗生长的适宜外源ABA浓度,为增强盐碱地楸树的耐盐性、提高盐碱地的利用提供理论依据。结果显示:(1)0.5%NaCl胁迫下,两品种楸树幼苗叶片表现出不同程度的盐害症状,且‘苏楸1号’叶片盐害症状较‘008-1’严重;随胁迫时间延长,两品种楸树幼苗的相对电导率(REC)均呈先上升后下降的变化趋势,叶绿素(Chl)、相对含水量(RWC)均呈降低趋势,可溶性糖(SS)、可溶性蛋白(SP)、脯氨酸(Pro)以及超氧化物歧化酶(SOD)活性均呈先上升后下降趋势,但‘008-1’的REC显著低于‘苏楸1号’,Chl、RWC、SS、SP、Pro、SOD均显著高于苏楸1号,表明‘008-1’的耐盐性较‘苏楸1号’更强。(2)喷施外源ABA使得盐胁迫下‘008-1’楸树的苗高显著增加、新叶提前萌发,表明外源ABA在一定程度上能够缓解盐胁迫对楸树生长的影响;喷施外源ABA降低了盐胁迫下‘008-1’楸树幼苗叶片的REC,提高了Chl、RWC、SS、SP、Pro、SOD、过氧化物酶(POD)以及过氧化氢酶(CAT)活性,促进了内源激素生长素(IAA)、脱落酸(ABA)、赤霉素(GA3)以及玉米素核苷(ZR)的积累。研究表明,楸树品种‘008-1’的耐盐性更强;外源喷施适宜浓度ABA能够缓解盐胁迫对楸树幼苗生长的影响,降低幼苗叶片细胞膜透性,促进幼苗渗透调节物质的积累,增强渗透调节能力,并提高盐胁迫下幼苗的抗氧化酶活性,促进植物对内源激素含量的调节,从而提高楸树的耐盐性,且以25 mg/L ABA处理的效果最好。     

Proline accumulation inside symbiosomes of faba bean nodules under salt stress

A two‐week salt treatment (NaCl, 100 m M ) induced a 50% inhibition of acetylene reduction activity (ARA) of faba bean ( Vicia faba L. var. minor cv. Soravi) nodules, associated with a large increase in the nodule pool of amino acids. The concentration of proline in the different nodule compartments was determined after calculating their respective volumes from their areas on electron micrographs. The proline concentration exhibited a large increase, especially in the cytosol where its amount was 8‐fold enhanced under salt stress, whereas the low proline content of bacteroids was less affected. Increase of proline concentration in faba bean nodules subjected to salt stress was correlated with an enhancement of the cytosolic Δ¹‐pyrroline‐5‐carboxylate synthetase (EC 2.7.2.11 + EC 1.2.1.41; P5CS) activity. Experiments with purified symbiosome preparations showed that the greatest proline content occurred in the peribacteroid space (PBS), where proline was the most abundant amino acid, with a concentration reaching 15.3 m M under salt stress. Proline accumulation in the PBS resulted both from a diffusive transport from the host cell to the symbiosomes through the peribacteroid membrane (PBM) and from the very low rate of uptake by faba bean bacteroids. This accumulation could be partly responsible for the 1.7‐fold enlargement of the symbiosome volume observed in salt‐stressed nodules. In incubations of bacteroids, isolated from salt‐stressed or unstressed plants and supplied with O₂ by purified oxyleghemoglobin, addition of proline stimulated neither O₂ consumption nor ARA. These results were consistent with proline playing a role as osmoticum, rather than energy source for bacteroid N₂ fixation in amide‐exporting legumes such as faba bean.