Metabolic regulation of polyamines and γ‐aminobutyric acid in relation to spermidine‐induced heat tolerance in white clover

• Heat stress decreases crop growth and yield worldwide. Spermidine (Spd) is a small aliphatic amine and acts as a ubiquitous regulator for plant growth, development and stress tolerance.
• Objectives of this study were to determine effects of exogenous Spd on changes in endogenous polyamine (PA) and γ‐aminobutyric acid (GABA) metabolism, oxidative damage, senescence and heat shock protein (HSP) expression in white clover subjected to heat stress. Physiological and molecular methods, including colorimetric assay, high performance liquid chromatography and qRT‐PCR, were applied.
• Results showed that exogenous Spd significantly alleviated heat‐induced stress damage. Application of Spd not only increased endogenous putrescine, Spd, spermine and total PA accumulation, but also accelerated PA oxidation and improved glutamic acid decarboxylase activity, leading to GABA accumulation in leaves under heat stress. The Spd-pretreated white clover maintained a significantly higher chlorophyll (Chl) content than untreated plants under heat stress, which could be related to the roles of Spd in up‐regulating genes encoding Chl synthesis (PBGD and Mg‐CHT) and maintaining reduced Chl degradation (PaO and CHLASE) during heat stress. In addition, Spd up‐regulated HSP70, HSP70B and HSP70‐5 expression, which might function in stabilizing denatured proteins and helping proteins to folding correctly in white clover under high temperature stress.
• In summary, exogenous Spd treatment improves the heat tolerance of white clover by altering endogenous PA and GABA content and metabolism, enhancing the antioxidant system and HSP expression and slowing leaf senescence related to an increase in Chl biosynthesis and a decrease in Chl degradation during heat stress.

     

Silicon‐mediated changes in polyamines participate in silicon‐induced salt tolerance in Sorghum bicolor L.

Silicon (Si) is generally considered a beneficial element for the growth of higher plants, especially under stress conditions, but the mechanisms remain unclear. Here, we tested the hypothesis that Si improves salt tolerance through mediating important metabolism processes rather than acting as a mere mechanical barrier. Seedlings of sorghum (Sorghum bicolor L.) growing in hydroponic culture were treated with NaCl (100 mm ) combined with or without Si (0.83 mm ). The result showed that supplemental Si enhanced sorghum salt tolerance by decreasing Na⁺ accumulation. Simultaneously, polyamine (PA) levels were increased and ethylene precursor (1‐aminocyclopropane‐1‐carboxylic acid: ACC) concentrations were decreased. Several key PA synthesis genes were up‐regulated by Si under salt stress. To further confirm the role of PA in Si‐mediated salt tolerance, seedlings were exposed to spermidine (Spd) or a PA synthesis inhibitor (dicyclohexylammonium sulphate, DCHA) combined with salt and Si. Exogenous Spd showed similar effects as Si under salt stress whereas exogenous DCHA eliminated Si‐enhanced salt tolerance and the beneficial effect of Si in decreasing Na⁺ accumulation. These results indicate that PAs and ACC are involved in Si‐induced salt tolerance in sorghum and provide evidence that Si plays an active role in mediating salt tolerance.     

Exogenous application of spermine and putrescine mitigate adversities of drought stress in wheat by protecting membranes and chloroplast ultra-structure

Polyamines (PAs) are positively charged molecules known to mitigate drought stress; however, little is known about their mechanism of alleviating drought stress. We investigated the effects of PAs exogenously applied as a seed primer and as a foliar spray on the growth, membrane stability (MS), electrolyte leakage (EL), Na+ and K+ cations, reactive oxygen species (ROS), catalase (CAT; EC 1.11.1.6) and guaiacol peroxidase (GPX; EC 1.11.1.7) activity and chloroplast ultra-structure in wheat (Triticum aestivum L.; cv. Sakha-94) under drought stress. Three PA solutions, namely, putrescine, spermine and a mixture of the two (Mix), were each applied at a concentration of 100 µM. Our study demonstrated that the retardation of chlorophyll loss and elevation of Rubisco levels were involved in PA-enhanced growth under drought stress. These relationships were mainly reflected in elevated fresh weight and dry weight in response to foliar spraying with all PA solutions and seed priming with the Mix solution. The elevated growth seemed to be due to increased photosynthetic pigments, protein and Rubisco. In contrast, drought decreased growth, photosynthetic pigments, protein and Rubisco. MS was enhanced by PAs applied as a seed primer or foliar spray, as shown by clear reductions in EL %, malondialdehyde (MDA) content and the Na+/K+ ratio as well as reduced ROS markers and elevated CAT (but not GPX) activity. Further study showed that the Mix solution of PAs, applied either during seed priming or as a foliar spray, improved chloroplast ultra-structure, suggesting that improvements in Rubisco and photosynthetic pigments were involved in PA maintenance of chloroplast stability. Therefore, the present study showed that elevated CAT activity is the main mechanism through which PAs reduce ROS and MDA, thereby improving MS and protecting mesophyll cells structurally and functionally under drought stress in wheat.     

H_2S对干旱胁迫下白三叶幼苗抗氧化防御能力的影响

以‘拉丁诺’白三叶(Trifolium repens cv.‘Ladino’)为试验材料,研究外源H2S处理对PEG6 000(聚乙二醇)模拟干旱胁迫下白三叶叶片相对含水量(RWC)、膜脂过氧化、活性氧成分、抗氧化酶、抗坏血酸-谷胱甘肽循环代谢和非酶抗氧化物质的影响,以揭示H_2S调控白三叶抗旱性的生理机制。结果显示:(1)0.2 mmol/L的外源NaHS(H_2S供体)能显著提高干旱胁迫下白三叶的叶片相对含水量,维持显著较低的电解质渗透率(EL)和丙二醛(MDA)含量。(2)与直接干旱胁迫相比,干旱胁迫下外源添加NaHS处理的白三叶叶片内超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性显著增强,抗坏血酸-谷胱甘肽循环代谢中关键酶抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)、单脱水抗坏血酸还原酶(MDHAR)和谷胱甘肽还原酶(GR)活性及其抗氧化中间产物抗坏血酸(AsA)、谷胱甘肽(GSH)含量也显著提高。(3)叶片类黄酮、总酚和原花青素的含量在一定的胁迫时间范围内亦显著增加,并伴随着活性氧成分O_2~(-·)产生速率和H_2O_2水平降低。研究认为,外源H2S能通过促进干旱胁迫下白三叶体内的多重抗氧化防御能力来提高其幼苗的抗旱性。     

Starch biosynthesis contributes to the maintenance of photosynthesis and leaf growth under drought stress in maize

To understand the growth response to drought, we performed a proteomics study in the leaf growth zone of maize (Zea mays L.) seedlings and functionally characterized the role of starch biosynthesis in the regulation of growth, photosynthesis and antioxidant capacity, using the shrunken-2 mutant (sh2), defective in ADP-glucose pyrophosphorylase. Drought altered the abundance of 284 proteins overrepresented for photosynthesis, amino acid, sugar and starch metabolism, and redox-regulation. Changes in protein levels correlated with enzyme activities (increased ATP synthase, cysteine synthase, starch synthase, RuBisCo, peroxiredoxin, glutaredoxin, thioredoxin and decreased triosephosphate isomerase, ferredoxin, cellulose synthase activities, respectively) and metabolite concentrations (increased ATP, cysteine, glycine, serine, starch, proline and decreased cellulose levels). The sh2 mutant showed a reduced increase of starch levels under drought conditions, leading to soluble sugar starvation at the end of the night and correlating with an inhibition of leaf growth rates. Increased RuBisCo activity and pigment concentrations observed in WT, in response to drought, were lacking in the mutant, which suffered more oxidative damage and recovered more slowly after re-watering. These results demonstrate that starch biosynthesis contributes to maintaining leaf growth under drought stress and facilitates enhanced carbon acquisition upon recovery.     

Antioxidant response to drought in red and white clover

Antioxidant response to drought in red (Trifolium pratense L., cv. ??Start??) and white clover (Trifolium repens L, cv. ??Haifa?? and cv. ??Debut??) grown as soil cultures was evaluated in water-deprived and recovered plants. Drought provoked oxidative stress in leaves confirmed by the considerable changes in electrolyte leakage, malondialdehyde, hydrogen peroxides and proline contents. Immunoblot of ??-1-pyrroline-5-carboxylate synthetase (P5CS), which catalyzes the first two steps in proline biosynthesis, revealed strong induction of the enzyme in red clover plants submitted to drought. Water-deprived white clover plants exhibited distinct P5CS profiles. This was related to different drought tolerance of the studied T. repens cultivars. Isoenzyme analyses of superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) demonstrated certain differences in antioxidant defence among the tested varieties. It was confirmed that MnSOD (in both T. repens and T pratense) and FeSOD (in T. repens) isoforms were the most affected by drought. The red clover cultivar ??Start?? exhibited the lowest FeSOD and POX activities which could contribute to its poor performance under water deprivation.     

Involvement of polyamines in the drought resistance of rice

This study investigated whether and how polyamines (PAs) in rice (Oryza sativa L.) plants are involved in drought resistance. Six rice cultivars differing in drought resistance were used and subjected to well-watered and water-stressed treatments during their reproductive period. The activities of arginine decarboxylase, S-adenosyl-L-methionine decarboxylase, and spermidine (Spd) synthase in the leaves were significantly enhanced by water stress, in good agreement with the increase in putrescine (Put), Spd, and spermine (Spm) contents there. The increased contents of free Spd, free Spm, and insoluble-conjugated Put under water stress were significantly correlated with the yield maintenance ratio (the ratio of grain yield under water-stressed conditions to grain yield under well-watered conditions) of the cultivars. Free Put at an early stage of water stress positively, whereas at a later stage negatively, correlated with the yield maintenance ratio. No significant differences were observed in soluble-conjugated PAs and insoluble-conjugated Spd and Spm among the cultivars. Free PAs showed significant accumulation when leaf water potentials reached -0.51 MPa to -0.62 MPa for the drought-resistant cultivars and -0.70 MPa to -0.84 MPa for the drought-susceptible ones. The results suggest that rice has a large capacity to enhance PA biosynthesis in leaves in response to water stress. The role of PAs in plant defence to water stress varies with PA forms and stress stages. In adapting to drought it would be good for rice to have the physiological traits of higher levels of free Spd/free Spm and insoluble-conjugated Put, as well as early accumulation of free PAs, under water stress.     

Physiological Evaluation of Drought Stress Tolerance and Recovery in Cauliflower (Brassica oleracea L.) Seedlings Treated with Methyl Jasmonate and Coronatine

Coronatine (COR) is a chlorosis-inducing phytotoxin that mimics some biological activities of methyl jasmonate (MeJA). Although MeJA has been reported to alleviate drought stress, it is unclear if COR has the same ability. Our objective was to determine the influence of exogenously applied MeJA and COR on the growth and metabolism of cauliflower seedlings under drought stress and recovery. Both MeJA and COR enhanced the growth and accumulation of dry matter in cauliflower seedlings during drought-stressed and rewatering conditions. Treatment with MeJA or COR enhanced tolerance of drought stress through increased accumulation of chlorophyll and net photosynthetic rate. Enzymatic (superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, and glutathione reductase) and nonenzymatic antioxidant (proline and soluble sugar) systems were activated, and lipid peroxidant (malondialdehyde and hydrogen peroxide) was suppressed by MeJA and COR under drought stress. MeJA and COR also increased leaf relative water content and endogenous abscisic acid level under drought-stressed conditions. After rewatering, the contents of leaf water, chlorophyll, abscisic acid, and photosynthetic characteristics as well as enzymatic and nonenzymatic antioxidant systems showed nearly complete recovery. Both MeJA and COR can alleviate the adverse effects of drought stress and enhance the ability for water stress resistance through promotion of defense-related metabolism in cauliflower seedlings.     

Growth,ROS Markers,Antioxidant Enzymes,Osmotic Regulators and Metabolic Changes in Tartary Buckwheat Subjected to Short Drought

Tartary buckwheat (Fagopyrum tataricum) is an important pseudocereal feed crop with medicinal and nutritional value. Drought is one of the main causes of reduced growth and yield in these plants. We investigated the growth, physiological, and metabolic responses of the widely promoted Tartary buckwheat variety Chuan Qiao No. 1 to polyethylene glycol (PEG)-mediated drought stress. Drought significantly decreased shoot length, shoot biomass and relative water content. Root length, malondialdehyde content, electrolyte leakage, activities of superoxide dismutase, peroxidase, catalase and amylase, and contents of soluble sugar, soluble protein and proline were increased by PEG-mediated drought. Untargeted metabolomics analysis identified 32 core metabolites in seedlings subjected to PEG-mediated drought, 16 of which increased—including quercetin, isovitexin, cyanidin 3-O-beta-D-glucoside, L-arginine, and glycerophosphocholine, while the other 16 decreased—including 3-methoxytyramine, 2, 6-diaminopimelic acid, citric acid, UDP-alpha-D-glucose, adenosine, keto-D-fructose. The 32 core metabolites were enriched in 29 metabolic pathways, including lysine biosynthesis, citrate (TCA) cycle, anthocyanin biosynthesis, and aminoacyl-tRNA biosynthesis. Among them, taurine and hypotaurine metabolism, flavor and flavor biosynthesis, indole alkaline biosynthesis, and alanine, aspartate and glutamate metabolism were the four main metabolic pathways affected by drought. Our findings provide new insights into the physiological and metabolic response mechanisms of Tartary buckwheat to drought stress.     

Pyrrolizidine alkaloid biosynthesis in Phalaenopsis orchids: developmental expression of alkaloid-specific homospermidine synthase in root tips and young flower buds

Pyrrolizidine alkaloids (PAs) are typical compounds of plant secondary metabolism and are believed to be part of the plant's chemical defense. Within the monocotyledonous plants, PAs have been described in only a few genera, mainly orchids, including Phalaenopsis. Because phylogenetic analyses suggest an independent origin of PA biosynthesis within the monocot lineage, we have analyzed the developmentally regulated expression of homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis, at the cell level. HSS is expressed in the tips of aerial roots exclusively in mitotically active cells. Raphide crystal idioblasts present within the root apical meristem do not show HSS expression. In addition, young flower buds, but not mature flowers, express HSS and have been shown by tracer feeding experiments to be able to catalyze PAs. This second site of PA biosynthesis ensures high concentrations of PAs in the reproductive structures of the Phalaenopsis flower, even after the flower opens. Thus, in spite of its identical function in PA biosynthesis, HSS shows in Phalaenopsis a completely different spatial and developmental expression pattern in comparison to other PA-producing species. These results show that the proverbial diversity of plant secondary metabolism is not just a matter of structural diversity, but is also multifaceted in terms of pathway regulation and expression.     

Putrescine promotes changes in the endogenous polyamine levels and proteomic profiles to regulate organogenesis in <Emphasis Type="Italic">Cedrela fissilis</Emphasis> Vellozo (Meliaceae)

Improvements to in vitro organogenesis are essential for optimizing shoot development and understanding basic physiological processes. The addition of polyamines (PAs) to the culture medium has been used to modulate organogenesis in plants, and this work evaluated the effects of exogenous PAs on direct organogenesis from apical and cotyledonary nodal Cedrela fissilis explants as well as the effects of putrescine (Put) on endogenous PA levels and variations in protein abundance. The effects of exogenous Put, spermidine, and spermine at 0, 0.5, 1, 2.5, or 5 mM on shoot development were tested. The comparison of the tested PAs to the control treatment revealed that 2.5 mM Put significantly increased the length of shoots from cotyledonary nodal explants, which are more sensitive than apical nodal explants, and treatment with 2.5 mM Put significantly increased the endogenous total free-PA and free-Put levels in shoots compared with the control (no Put). A comparative proteomic analysis of shoots indicated that 2.5 mM Put significantly changed the abundance of proteins, primarily metabolic and cellular proteins associated with stress and energy processes such as cell division. These results show that Put functions in endogenous PA metabolism and alters protein abundance, thereby contributing to shoot development in C. fissilis.     

The effects of nor-spermidine and dicyclohexylamine on in vitro potato development and free polyamines content

The in vitro micropropagation of potato (Solanum tuberosum L., var. Spunta) on media containing nor-spermidine (nor-SPD), a natural polaymine (PA) or dicyclohexlamine (DCHA), a spermidine synthesis inhibitor was studied to test their effects on plantlet growth and on the level of free cellular polyamines. The triamine nor-SPD, inhibited spermidine synthesis and substantially reduced root growth. DCHA strongly inhibited potato growth but surprisingly the free spermidine level seemed unaffected. These data suggest that dicyclohexylamine acts on the growth and on the development of plants by mechanisms unrelated to polyamine metabolism. Conversely, nor-spermidine was effective in reducing cellular spermidine content and seems to be a spermidine biosynthesis inhibitor in plants.     

Response of proteome and morphological structure to short‐term drought and subsequent recovery in Cucumis sativus leaves

Drought is the primary limitation to plant growth and yield in agricultural systems. Cucumber (Cucumis sativus) is one of the most important vegetables worldwide and has little tolerance for water deficit. To understand the drought stress response strategy of this plant, the responses of cucumber to short‐term drought and rewatering were determined in this study by morphological structure and proteomic analyses. The leaf relative water content was significantly decreased under drought, and the cell structure was altered, while rewatering obviously alleviated the symptoms of water shortage and cell damage. A total of 320 and 246 proteins exhibiting significant abundance changes in response to drought and recovery, respectively, were identified. Our proteome analysis showed that 63 co‐regulated proteins were shared between drought and rewatering, whereas most of the responsive proteins were unique. The proteome is adjusted through a sequence of regulatory processes including the biosynthesis of secondary metabolites and the glutathione metabolism pathway, which showed a high correlation between protein abundance profile and corresponding enzyme activity. Drought and recovery regulated different types of proteins, allowing plants to adapt to environmental stress or restore growth, respectively, which suggests that short‐term drought and recovery are almost fully uncoupled processes. As an important component of the antioxidant system in plants, glutathione metabolism may be one of the main strategies for regulating antioxidant capacity during drought recovery. Our results provide useful information for further analyses of drought adaptability in cucumber plants.     

Polyamines in yellow lupin (<Emphasis Type="Italic">Lupinus luteus</Emphasis> L.) tolerance to soil drought

Polyamines (PAs) are related to many physiological processes, including soil drought stress. Two yellow lupin ‘Morocco 4’ (drought tolerant) and ‘Taper’ (drought sensitive) were exposed to soil drought for 2 weeks. The half of the examined plants were additionally sprayed with a solution of polyamine biosynthesis inhibitor—dl-α-difluoromethylarginine (DFMA). Yellow lupin leaves showed a 19% increase and seeds a 54% decrease in the total PA contents. The seeds contained fourfold less PAs than the leaves under drought conditions. The highest amount of spermidine and lack of agmatine were found in the leaves, while in the seeds the highest content of spermine and the presence of agmatine was confirmed. The use of DFMA under drought conditions decreased the content of spermine in ‘Morocco 4’ and ‘Taper’ (41 and 19%, respectively) and spermidine in ‘Taper’ (by 13%), as well as reduced two out of three of the yield components. More tolerant ‘Morocco 4’, after DFMA treatment was characterized by a higher spermidine and spermine content and a smaller decrease in yield components compared to the less tolerant ‘Taper’. Simultaneously subjecting plants to soil drought and DFMA treatment caused in ‘Morocco 4’ a decline in the number of pods and seeds per plant and seeds dry weight per plant (64, 50 and 54%, respectively), while in ‘Taper’ a reduction of the number of pods per plant and seeds per pod (32 and 27%, respectively) was observed. These results confirm that PAs are involved in yellow lupin tolerance and may play a protective function under soil drought conditions.     

Higher accumulation of gamma-aminobutyric acid induced by salt stress through stimulating the activity of diamine oxidases in Glycine max (L.) Merr. roots.

Polyamines (PAs) are assumed to perform their functions through their oxidative product such as gamma-aminobutyric acid (GABA) formation. However, there is only limited information on the interrelation between PA degradation and GABA accumulation under salt stress. In order to reveal a quantitative correlation between PA oxidation and GABA accumulation, the effects of treatments with different NaCl concentrations, along with aminoguanidine (AG, a specific inhibitor of diamine oxidases (DAO; EC: 1.4.3.6)) and a recovery test from salt stress on endogenous free PAs, gamma-aminobutyric acid (GABA) accumulation and DAO activity were determined in roots of soybean [Glycine max (L.) Merr.] cultivar Suxie-1. The results showed that the levels of putrescine (Put), cadaverine (Cad), and spermidine (Spd) decreased significantly with increasing salt concentrations. This occurred because salt stress strongly promoted DAO activity to stimulate PA degradation. GABA accumulation increased with growing NaCl concentrations, about an 11- to 17-fold increase as compared with the control plants. AG treatment increased the accumulation of endogenous free PAs as a result of a strong retardation of DAO activity, but decreased GABA accumulation. The recovery for 6 days in 1/2 Hoagland solution from 100mM NaCl stress resulted in a decrease in DAO activity, a rebound of PA levels and a simultaneous reduction of GABA content. A close correlation was observed between the changes in DAO activity and GABA accumulation. The results indicated that higher GABA accumulation (about 39%) induced by salt stress could come from PA degradation, suggesting that PAs might perform their functions through GABA formation under salt stress.