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Seed germination is an elaborate process during life cycle and regulated by numerous intrinsic and environmental cues. The role of amino acids in seed germination under salt stress has been reported, however the underlying mechanisms by which amino acids mitigate salt stress-induced seed germination remain largely elusive. Here, we reported that seed germination of Arabidopsis thaliana wild-type was suppressed by exposure to salt stress, and that the inhibition was markedly alleviated by exogenous application of those amino acids including Gly, Cys, Ser and Met. Calcium channel blocker La 3+ suppressed the mitigating effect of those amino acids on seed germination under salt stress. We further demonstrated that germination of wild-type seeds was suppressed by the glutamate receptor (GLR) antagonist 6,7-dinitriquinoxaline-2,3-dinoe (DNQX) under salt stress and the alleviative effects of amino acid on seed germination under salt stress were suppressed by DNQX. In addition, mutation of clade 3 GLRs led to lower seed germination rates of atglr3.4 and atglr3.7 mutants than those of wild-type seeds when exposed to NaCl and the mitigating effects of amino acids on seed germination rates of atglr3.4 and atglr3.7 were less effective than those of wild-type seeds. Moreover, seed germination of atglr3.4-1 , atglr3.4-2 and atglr3.7 mutants were less sensitive to CaCl 2 and DNQX than wild-type under salt stress. Taken together, these findings highlight the involvement of GLR-dependent Ca 2+ influx in alleviation of salt stress-induced inhibition of seed germination by exogenous amino acids.

Medicago truncatula is a model legume species with a diverse genetic diversity. To explore whether different ecotypes of Medicago truncatula differ in their response to sulfur deficiency, the effects of sulfur (S) deficiency on the two ecotypes (A17 and R108) were investigated. Sulfur deficiency stimulated lateral root initiation in the root apical region of both ecotypes, but did not affect their overall biomass. The S deficiency-induced changes in root morphology were more distinct in R108 plants than in A17 plants. Exposure to S deficiency led to a greater reduction in leaves and roots of R108 than those of A17 plants. The concentrations of majority of other mineral nutrients were increased in A17 plants, while they were mainly reduced in R108 plants under S-deficient conditions. A greater reduction in R108 plants than in A17 plants was found under S-deficient conditions. An accumulation of anthocyanin in R108 was observed under S-deficient conditions. These results indicate that the ecotype R1...

Seed germination is sensitive to salt stress. ABA and Ca2+ are involved in the regulation of seed germination under salt stress. Ca2+ influx mediated by glutamate receptors (GLRs) plays important roles in many physiological processes in plants. Here, we investigated the correlation of GLRs, Ca2+ and ABA during seed germination in response to salt stress by using Arabidopsis thaliana wild-type and T-DNA insertion knockout mutants of glutamate receptor homolog3.4. We demonstrated that atglr3.4-1 and atglr3.4-2 mutants were more sensitive to NaCl during seed germination and post-germination growth than wild-type plants. Treatments of wild-type seedlings with NaCl evoked a marked elevation in cytosolic Ca2+ activity ([Ca2+]cyt), and the elevation was inhibited by antagonists of GLRs, while the NaCl-induced elevation in [Ca2+]cyt was impaired in atglr3.4-1 and atglr3.4-2 mutants. Moreover, the mutants exhibited a lower expression of SOS3, SOS2 and SOS1, and greater accumulation of Na+ than wild-type seeds in the presence of NaCl. Mutation of AtGLR3.4 rendered the mutants more sensitive to ABA, while overexpression of AtGLR3.4 made the transgenic lines more tolerant to ABA in terms of seed germination. However, there was no difference in ABA content between atglr3.4 mutants and wild-type seeds, accompanied by lower expression of ABI3 and ABI4 in atglr3.4 mutants when challenged with NaCl. These results demonstrate that AtGLR3.4-mediated Ca2+ influx may be involved in the regulation of seed germination under salt stress by modulating Na+ accumulation through the SOS pathway.

Summary Exudation of malate is an important mechanism underlying tolerance of wheat to aluminium toxicity. Here we show that ethylene is involved in regulation of ALMT1-dependent malate efflux from wheat roots., An important mechanism for Al3+ tolerance in wheat is exudation of malate anions from the root apex through activation of malate-permeable TaALMT1 channels. Here, the effect of ethylene on Al3+-activated efflux of malate was investigated using Al3+-tolerant wheat genotype ET8, which has high expression of TaALMT1. Exposure of ET8 plants to Al3+ enhanced ethylene evolution in root apices. Treatment with the ethylene synthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene gas suppressed Al3+-induced malate efflux from root apices, whereas the intracellular malate concentrations in roots were not affected. Malate efflux from root apices was enhanced in the presence of Al3+ by two antagonists of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) and 2-aminoisobutyric acid (AIB). An increase in Al accumulation in root apices was observed when treated with ACC, whereas AVG and AIB suppressed Al accumulation in root apices. Al3+-induced inhibition of root elongation was ameliorated by pretreatment with AIB. In addition, ethylene donor (Ethrel) also inhibited Al3+-induced malate efflux from tobacco cells transformed with TaALMT1. ACC and the anion-channel blocker niflumate had a similar and non-additive effect on Al-induced malate efflux from root apices. Treatment of ET8 plants with ACC enhanced expression of TaALMT1, suggesting that the inhibitory effect of ethylene on Al-induced malate efflux is unlikely to occur at the transcriptional level. These findings indicate that ethylene may behave as a negative regulator of Al3+-induced malate efflux by targeting TaALMT1-mediated malate efflux by an unknown mechanism.

Sulfur (S) is an essential macronutrient for plants, and deficiency in soil S availability limits plant growth. Adaptive strategies have been evolved by plants to respond to S deficiency by coordinating systemic regulatory mechanism. A split-root experiment using legume model plant Medicago truncatula Gaertn. was conducted to investigate the systemic response to S deficiency. Plant growth, root morphology and S contents under varying conditions of S supply were determined, and the expression of genes encoding sulfate transporter (MtSULTRs) and MtAPR1 encoding an enzyme involved in S assimilation was monitored. Our results demonstrated that there was an apparent systemic response of M. truncatula to heterogeneous S supply in terms of root length, S contents, and S uptake and assimilation at the transcriptional level. When exposed to heterogeneous S supply, M. truncatula plants showed proliferation of lateral roots in S-rich medium and reduction in investment to S-depleted roots. Growth was stimulated with half-part of roots exposed to S-deficient medium. There were different expression patterns of MtSULTRs and MtAPR1 in response to heterogeneous S supply both in roots and shoots of M. truncatula. Expression of MtSULTR1.1 and MtSULTR1.3 was systemically responsive to S deficiency, leading to an enhancement of S uptake in roots exposed to S-sufficient medium. In addition, the response of S-deprived seedlings to re-supply of sulfate and Cys was also analyzed. It was shown that sulfate, but not Cys, may serve as a systemic signal to regulate the expression of genes associated with S absorption and assimilation in M. truncatula. These findings provide a comprehensive picture of systemic responses to S deficiency in leguminous species.

To understand the role of small GTPases in response to abiotic stress, we isolated a gene encoding a small GTPase, designated MfARL1, from a subtracted cDNA library in Medicago falcata, a native legume species in semi-arid grassland in northern China. The function of MfARL1 in response to salt stress was studied by expressing MfARL1 in Arabidopsis. Wild-type (WT) and transgenic plants constitutively expressing MfARL1 showed comparable phenotype when grown under control conditions. Germination of seeds expressing MfARL1 was less suppressed by salt stress than that of WT seeds. Transgenic seedlings had higher survival rate than WT seedlings under salt stress, suggesting that expression of MfARL1 confers tolerance to salt stress. The physiological and molecular mechanisms underlying these phenomena were elucidated. Salt stress led to a significant decrease in chlorophyll contents in WT plants, but not in transgenic plants. Transgenic plants accumulated less amounts of H(2)O(2) and malondialdehyde than their WT counterparts under salt stress, which can be accounted for by the higher catalase activities, lower activities of superoxide dismutase, and peroxidase in transgenic plants than in WT plants. Transgenic plants displayed lower Na(+)/K(+) ratio due to less accumulation of Na(+) than wild-type under salt stress conditions. The lower Na(+)/K(+) ratio may result from less accumulation of Na(+) due to reduced expression of AtHKT1 that encodes Na(+) transporter in transgenic plants under salt stress. These findings demonstrate that MfARL1 encodes a novel stress-responsive small GTPase that is involved in tolerance to salt stress.

Plants have developed numerous strategies to cope with phosphorus (P) deficiency resulting from low availability in soils. Evolution of ethylene and up-regulation of root secreted acid phosphatase activity are common for plants in response to P deficiency. To determine the role of ethylene in response of plants to P deficiency, we investigated the effects of ethylene precursor (1-amino cyclopropane-1-carboxylic acid, ACC) and ethylene synthesis antagonists (aminoethoxyvinylglycine AVG, cobalt, Co 2+ ) on P con

Aluminium (Al) is phytotoxic when solubilized into Al(3+) in acidic soils. One of the earliest and distinct symptoms of Al(3+) toxicity is inhibition of root elongation. To decipher the mechanism by which Al(3+) inhibits root elongation, the role of ethylene and auxin in Al(3+)-induced inhibition of root elongation in Arabidopsis thaliana was investigated using the wild type and mutants defective in ethylene signalling (etr1-3 and ein2-1) and auxin polar transport (aux1-7 and pin2). Exposure of wild-type Arabidopsis to AlCl(3) led to a marked inhibition of root elongation, and elicited a rapid ethylene evolution and enhanced activity of the ethylene reporter EBS:GUS in root apices. Root elongation in etr1-3 and ein2-1 mutants was less inhibited by Al(3+) than that in wild-type plants. Ethylene synthesis inhibitors, Co(2+) and aminoethoxyvinylglycine (AVG), and an antagonist of ethylene perception (Ag(+)) abolished the Al(3+)-induced inhibition of root elongation. There was less inhibition of root elongation by Al(3+) in aux1-7 and pin2 mutants than in the wild type. The auxin polar transport inhibitor, naphthylphthalamic acid (NPA), substantially alleviated the Al(3+)-induced inhibition of root elongation. The Al(3+) and ethylene synthesis precursor aminocyclopropane carboxylic acid (ACC) increased auxin reporter DR5:GUS activity in roots. The Al(3+)-induced increase in DR5:GUS activity was reduced by AVG, while the Al(3+)-induced increase in EBS:GUS activity was not altered by NPA. Al(3+) and ACC increased transcripts of AUX1 and PIN2, and this effect was no longer observed in the presence of AVG and Co(2+). These findings indicate that Al(3+)-induced ethylene production is likely to act as a signal to alter auxin distribution in roots by disrupting AUX1- and PIN2-mediated auxin polar transport, leading to arrest of root elongation.

†Background and Aims Root growth and development are closely dependent upon nitrate supply in the growth medium. To unravel the mechanism underlying dependence of root growth on nitrate, an examination was made of whether endogenous nitric oxide (NO) is involved in nitrate-dependent growth of primary roots in maize. †Methods Maize seedlings grown in varying concentrations of nitrate for 7 d were used to evaluate the effects on root elongation of a nitric oxide (NO) donor (sodium nitroprusside, SNP), a NO scavenger (methylene blue, MB), a nitric oxide synthase inhibitor (N v -nitro-L-arginine, L-NNA), H2O2, indole-3-acetic acid (IAA) and a nitric reducatse inhibitor (tungstate). The effects of these treatments on endogenous NO levels in maize root apical cells were investigated using a NO-specific fluorescent probe, 4, 5-diaminofluorescein diacetate (DAF-2DA) in association with a confocal microscopy. †Key Results Elongation of primary roots was negatively dependent on external concentrations of nitrate, and inhibition by high external nitrate was diminished when roots were treated with SNP and IAA. MB and L-NNA inhibited root elongation of plants grown in low-nitrate solution, but they had no effect on elongation of roots grown in highnitrate solution. Tungstate inhibited root elongation grown in both low- and high-nitrate solutions. Endogenous NO levels in root apices grown in high-nitrate solution were lower than those grown in low-nitrate solution. IAA and SNP markedly enhanced endogenous NO levels in root apices grown in high nitrate, but they had no effect on endogenous NO levels in root apical cells grown in low-nitrate solution. Tungstate induced a greater increase in the endogenous NO levels in root apical cells grown in low-nitrate solution than those grown in high-nitrate solution. †Conclusions Inhibition of root elongation in maize by high external nitrate is likely to result from a reduction of nitric oxide synthase-dependent endogenous NO levels in maize root apical cells.

Inhibition of root elongation by toxic aluminum (Al(3+)) occurs rapidly and is one of the most distinct and earliest symptoms of Al toxicity. To elucidate mechanism underlying Al(3+)-induced inhibition of root elongation, we investigated the involvement of ethylene in Al(3+)-induced inhibition of root elongation using the legume model plants Lotus japonicus and Medicago truncatula. Root elongation of L. japonicus and M. truncatula was rapidly inhibited by exposure to AlCl(3). A similar rapid inhibition of root elongation by the ethylene-releasing substance, ethephon, and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), was also observed. The Al(3+)-induced inhibition of root elongation was substantially ameliorated in the presence of antagonists of ethylene biosynthesis [Co(2+) and aminoethoxyvinylglycine (AVG)]. Al(3+) increased the activity of ACC oxidase (ACO), and induced a rapid evolution of ethylene from root apices and expression of genes of ACC synthase (ACS) and ACO. These findings suggest that induction of ethylene evolution resulting from up-regulation of ACS and ACO plays a critical role in Al(3+)-induced inhibition of root elongation.

Nitric oxide (NO) has emerged as a key molecule involved in many physiological processes in plants. To characterize roles of NO in tolerance of Arabidopsis (Arabidopsis thaliana) to salt stress, effect of NaCl on Arabidopsis wild-type and mutant (Atnoa1) plants with an impaired in vivo NO synthase (NOS) activity and a reduced endogenous NO level was investigated. Atnoa1 mutant plants displayed a greater Na+ to K+ ratio in shoots than wild-type plants due to enhanced accumulation of Na+ and reduced accumulation of K+ when exposed to NaCl. Germination of Atnoa1 seeds was more sensitive to NaCl than that of wild-type seeds, and wild-type plants exhibited higher survival rates than Atnoa1 plants when grown under salt stress. Atnoa1 plants had higher levels of hydrogen peroxide than wild-type plants under both control and salt stress, suggesting that Atnoa1 is more vulnerable to salt and oxidative stress than wild-type plants. Treatments of wild-type plants with NOS inhibitor and NO scavenger reduced endogenous NO levels and enhanced NaCl-induced increase in Na+ to K+ ratio. Exposure of wild-type plants to NaCl inhibited NOS activity and reduced quantity of NOA1 protein, leading to a decrease in endogenous NO levels measured by NO-specific fluorescent probe. Treatment of Atnoa1 plants with NO donor sodium nitroprusside attenuated the NaCl-induced increase in Na+ to K+ ratio. Therefore, these findings provide direct evidence to support that disruption of NOS-dependent NO production is associated with salt tolerance in Arabidopsis.

The capacity of a plant to take up nitrate is a function of the activity of its nitrate-transporter systems and the size and architecture of its root system. It is unclear which of the two components, root system or nitrate-uptake system, is more important in nitrogen (N) acquisition under nitrogen-sufficiency conditions. Two maize (Zea mays L.) inbred lines (478 and Wu312) grown in nutrient solution in a controlled environment were compared for their N acquisition at 0.1, 0.5, 2.5, 5, and 10 mmol L−1 nitrate supply. Genotype 478 could take up more N than Wu312 at all nitrate concentrations, though the shoot biomass of the two genotypes was similar. Genotype 478 had a larger leaf area and longer root length. The specific N uptake rate of 478 (μmol N g−1 root. d−1) was lower than that of Wu312. In an independent nitrate-depletion experiment, the potential nitrate uptake rate of 478 was also lower than that of Wu312. No genotypic difference was found in photosynthesis rate. It was concluded that th...

Response of root system architecture to nutrient availability in soils is an essential way for plants to adapt to soil environments. Nitrate can affect root development either as a result of changes in the external concentration, or through changes in the internal nutrient status of the plant. Nevertheless, less is known about the physiological mechanisms. In the present study, two maize (Zea mays L.) inbred lines (478 and Wu312) were used to study a possible role of cytokinin in nitrate-mediated root growth in nutrient solutions. Root elongation of 478 was more sensitive to high nitrate supply than that of Wu312. Medium high nitrate (5 mM) inhibited root elongation in 478, while, root elongation in Wu312 was only inhibited at high NO 3 − supply (20 mM). Under high nitrate supply, the root elongation zone in 478 became swollen and the site of lateral root elongation was close towards the root tip. Both of the phenomena are typical of root growth induced by exogenous cytokinin treatments. Correspondingly, zeatin and zeatin nucleotide (Z + ZR) concentrations were increased at higher nitrate supply in 478, whereas they were constant in Wu312. Furthermore, exogenous cytokinin 6-benzylaminopurine (6-BA) completely reversed the stimulatory effect of low nitrate on root elongation. Therefore, it is supposed that the inhibitory effect of high concentration of nitrate on root elongation is, at least in part, mediated by increased cytokinin level in roots. High nitrate supply may have negative influences on root apex activity by affecting cytokinin metabolism so that root apical dominance is weakened and, therefore, root elongation is suppressed and lateral roots grow closer to the root apex. Nitrate suppressed lateral root elongation in Wu312 at concentration higher than 5 mM. In 478, however, this phenomenon was not significant even at 20 mM nitrate. Although exogenous 6-BA (20 nM) could suppress lateral root elongation as well, the inhibitory effect of high NO 3 − concentration of nitrate on lateral root growth cannot be explained by changes in endogenous cytokinin alone.

Medicago falcata is a legume species that exhibits great capacity of tolerance to abiotic stresses. To elucidate the mechanism underlying tolerance of M. falcata to freezing, we compared the characteristics of M. falcata in response to cold acclimation and freezing with those of the legume model plant Medicago truncatula. M. falcata seedlings were more tolerant to freezing than M. truncatula, as evidenced by a lower value of EL(50) (temperature at which 50% electrolyte leakage after freezing) and greater survival rate for M. falcata than M. truncatula. Cold acclimation led to greater reduction in EL(50) for M. falcata than M. truncatula. Sucrose was the most abundant sugar in both M. falcta and M. truncatula, and a greater accumulation of sucrose and Pro in M. falcata than in M. truncatula during cold acclimation was observed. Cold acclimation induced small amounts of raffinose and stachyose in M. falcata, but not in M. truncatula. The activities of sucrose phosphate synthase and sucrose synthase were greater in M. falcata than in M. truncatula. In contrast, the activity of acid invertase was higher in M. truncatula than in M. falcata. There was an increase in transcript of CRT binding factor (CBF) upon exposure to low temperature in the two species. The low temperature-induced increase in transcript of CBF2 was much higher in M. truncatula than in M. falcata, while transcript of CBF3 in M. falcata was greater than that in M. truncatula. There were sustained increases in transcripts of cold acclimation specific (CAS), a downstream target of CBF, during cold acclimation and the increases were greater in M. falcata than in M. truncatula. These results demonstrate that accumulation of greater amounts of soluble sugars coupled with higher CBF3 and CAS transcript levels in M. falcata may play a role in conferring greater tolerance of M. falcata to freezing than that of M. truncatula.

*Here, we investigated the role of ethylene in high nitrate-induced change in root development in Arabidopsis thaliana using wild types and mutants defective in ethylene signaling (etr1, ein2) and nitrate transporters (chl1, nrt2.1). *The length and number of visible lateral roots (LRs) were reduced upon exposure of wild-type seedlings grown on low (0.1 mM) to high nitrate concentration (10 mM). There was a rapid burst of ethylene production upon exposure to high nitrate concentration. *Ethylene synthesis antagonists, cobalt (Co(2+)) and aminoethoxyvinylglycine (AVG), mitigated the inhibitory effect of high nitrate concentration on lateral root growth. The etr1-3 and ein2-1 mutants exhibited less reductions in LR length and number than wild-type plants in response to high nitrate concentration. Expression of nitrate transporters AtNRT1.1 and AtNRT2.1 was upregulated and downregulated in response to high nitrate concentration, respectively. A similar upregulation and downregulation of AtNRT1.1 and AtNRT2.1 was observed by ethylene synthesis precursor aminocyclopropane carboxylic acid (ACC) and AVG in low and high nitrate concentration, respectively. Expression of AtNRT1.1 and AtNRT2.1 became insensitive to high nitrate concentration in etr1-3 and ein2-1 plants. *These findings highlight the regulatory role that ethylene plays in high nitrate concentration-regulated LR development by modulating nitrate transporters.

Here, the effects of the ethylene-releasing compound, ethephon, and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), on ionic currents across plasma membranes and on the cytosolic Ca(2+) activity ([Ca(2+)](c)) of tobacco (Nicotiana tabacum) suspension cells were characterized using a patch-clamp technique and confocal laser scanning microscopy. Exposure of tobacco protoplasts to ethephon and ACC led to activation of a plasma membrane cation channel that was permeable to Ba(2+), Mg(2+) and Ca(2+), and inhibited by La(3+), Gd(3+) and Al(3+). The ethephon- and ACC-induced Ca(2+)-permeable channel was abolished by the antagonist of ethylene perception (1-metycyclopropene) and by the inhibitor of ACC synthase (aminovinylglycin), indicating that activation of the Ca(2+)-permeable channels results from ethylene. Ethephon elicited an increase in the [Ca(2+)](c) of tobacco suspension cells, as visualized by the Ca(2+)-sensitive probe Fluo-3 and confocal microscopy. The ethephon-induced elevation of [Ca(2+)](c) was markedly inhibited by Gd(3+) and BAPTA, suggesting that an influx of Ca(2+) underlies the elevation of [Ca(2+)](c). These results indicate that an elevation of [Ca(2+)](c), resulting from activation of the plasma membrane Ca(2+)-permeable channels by ethylene, is an essential component in ethylene signaling in plants.