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Mowing, as a common grassland utilization strategy, affects nutrient status in soil by plant biomass removal. Phosphorus (P) cycle plays an important role in determining grassland productivity. However, few studies have addressed the impacts of mowing on P cycling in grassland ecosystems. Here, we investigated the effects of various mowing regimes on soil P fractions and P accumulation in plants and litters. We specifically explored the mechanisms by which mowing regulates ecosystem P cycling by linking aboveground community with soil properties. Our results showed that mowing increased soil dissolvable P concentrations, which probably met the demand for P absorption and utilization by plants, thus contributing to an increased P accumulation by plants. Mowing promoted grassland P cycling by a reciprocal relationship between plants and microbes. Short-term mowing enhanced P cycling mainly through increased root exudation-evoked the extracellular enzyme activity of microbes rather than the alternations in microbial biomass and community composition. Long-term mowing increased P cycling mainly by promoting carbon allocation to roots, thereby leading to greater microbial metabolic activity. Although mowing-stimulation of organic P mineralization lasted for 15 consecutive years, mowing did not result in soil P depletion. These results demonstrate that P removal by mowing will not necessarily lead to soil P limitation. Our findings would advance the knowledge on soil P dynamic under mowing and contribute to resource-efficient grassland management.

Belowground plant traits play important roles in plant diversity loss driven by atmospheric nitrogen (N) deposition. However, the way N enrichment shapes plant microhabitats by patterning belowground traits and finally determines aboveground responses is poorly understood. Here, we investigated the rhizosheath trait of 74 plant species in seven N-addition-simulation experiments across multiple grassland ecosystems in China. We found that rhizosheath formation differed among plant functional groups and contributed to changes in plant community composition induced by N enrichment. Compared with forb species, grass and sedge species exhibited more distinct rhizosheaths; moreover, grasses and sedges expanded their rhizosheaths with increasing N-addition rate which allowed them to colonize belowground habitats. Grasses also shaped a different microenvironment around their roots compared with forbs by affecting the physicochemical, biological and stress-avoiding properties of their rhizosphere soil. Rhizosheaths act as a “biofilm-like shield” by accumulating of protective compounds, carboxylic anions and polysaccharides, determined by both plants and microorganisms. This enhanced the tolerance of grasses and sedges to stresses induced by N enrichment. Conversely, forbs lacked the protective rhizosheaths which renders their roots sensitive to stresses induced by N enrichment, thus contributing to their disappearance under N-enriched conditions. This study uncovers the processes by which belowground facilitation and trait matching affects aboveground responses under conditions of N enrichment, which advances our mechanistic understanding of the contribution of competitive exclusion and environmental tolerance to plant diversity loss caused by N deposition.

Atmospheric nitrogen (N) deposition, climatic variables and anthropogenic management affect grassland-ecosystem stability by driving changes in plant community and loss of plant diversity. Determination of their influences on plant composition and diversity is essential for predicting ecosystem response to future global changes and for making policies of sustainable grassland management. We evaluated the relative contributions of atmospheric N deposition, climatic variables and grassland management to plant diversity and N accumulation by temporally monitoring the changes in plant community and N status in a temperate grassland ecosystem. Ambient low-level N deposition combined with long-term grassland enclosure led to increases in above-ground biomass and abundance of grasses and sedges, and decreases in those of forbs. The dominant species of perennial forbs and grasses also displayed divergence in their biomass and abundance over time. Nitrogen deposition made greater contributions to the divergent changes in perennial forbs and grasses. However, air temperature and precipitation fluctuations mainly contributed to the annual fluctuations in annual and biennial species. Long-term enclosure aggravated the cumulative effects of soil inorganic N on plant-diversity loss while mowing had opposite effects on the N accumulation. Synthesis and applications. Ambient low-level N deposition can drive loss of plant diversity due to N accumulation in grassland ecosystem under long-time enclosure. Therefore, appropriate grassland management, such as rationally grazing and/or mowing, may be effective practices in terms of maintenance of plant biodiversity under the scenario of enhanced atmospheric N deposition.

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.

Aims Despite extensive studies on effects of elevated CO2 concentration ([CO2]e) on plant growth, few studies have investigated the responses of native grassland plant species to [CO2]e in terms of nutrient acquisition.

Iron deficiency is one of the major limiting factors affecting quality and production of crops in calcareous soils. Numerous signaling molecules and transcription factors have been demonstrated to play a regulatory role in adaptation of plants to iron deficiency. However, the mechanisms underlying the iron deficiency-induced physiological processes remain to be fully dissected. Here, we demonstrated that the protein kinase CIPK23 was involved in iron acquisition. Lesion of CIPK23 rendered Arabidopsis mutants hypersensitive to iron deficiency, as evidenced by stronger chlorosis in young leaves and lower iron concentration than wild-type plants under iron-deficient conditions by down-regulating ferric chelate reductase activity. We found that iron deficiency evoked an increase in cytosolic Ca(2+) concentration and the elevated Ca(2+) would bind to CBL1/CBL9, leading to activation of CIPK23. These novel findings highlight the involvement of calcium-dependent CBL-CIPK23 complexes in the regulation of iron acquisition. Moreover, mutation of CIPK23 led to changes in contents of mineral elements, suggesting that CBL-CIPK23 complexes could be as "nutritional sensors" to sense and regulate the mineral homeostasis in Arabisopsis.

Loss of plant diversity with increased anthropogenic nitrogen (N) deposition in grasslands has occurred globally. In most cases, competitive exclusion driven by preemption of light or space is invoked as a key mechanism. Here, we provide evidence from a 9-yr N-addition experiment for an alternative mechanism: differential sensitivity of forbs and grasses to increased soil manganese (Mn) levels. In Inner Mongolia steppes, increasing the N supply shifted plant community composition from grass-forb codominance (primarily Stipa krylovii and Artemisia frigida, respectively) to exclusive dominance by grass, with associated declines in overall species richness. Reduced abundance of forbs was linked to soil acidification that increased mobilization of soil Mn, with a 10-fold greater accumulation of Mn in forbs than in grasses. The enhanced accumulation of Mn in forbs was correlated with reduced photosynthetic rates and growth, and is consistent with the loss of forb species. Differential accumulation of Mn between forbs and grasses can be linked to fundamental differences between dicots and monocots in the biochemical pathways regulating metal transport. These findings provide a mechanistic explanation for N-induced species loss in temperate grasslands by linking metal mobilization in soil to differential metal acquisition and impacts on key functional groups in these ecosystems.

Enhanced deposition of atmospheric nitrogen (N) has profound impacts on ecosystem processes such as above-ground productivity and community structure in grasslands across the globe. But how N deposition affects below-ground processes of grasslands is less well known. Here, we evaluated the effects of chronic N amendment at a relatively low rate (20kgha(-1)year(-1)) on root traits (root productivity, root biomass, root/shoot ratio) in Inner Mongolia steppes by rhizotron and ingrowth core and soil monolith techniques at levels of individual species, functional groups and ecosystem. For 8years, N amendment suppressed above-ground net primary production (ANPP), photosynthetic rates and root biomass of forbs, but enhanced ANPP and root biomass of grasses. This led to an overall reduction in below-ground productivity of the grassland by 24-33%, while ANPP remained unchanged. Nitrogen amendment acidified soil and subsequently increased extractable soil manganese (Mn) concentration. Nitrogen amendment increased foliar Mn concentrations in forb, but not grass species, leading to a significant inhibition of photosynthetic rates in forb species.Synthesis. These findings highlight the importance of the differentiating responses of plant functional groups to long-term N deposition and the important consequences of these responses for below-ground productivity and long-term soil C sequestration.

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.

Aluminum (Al) toxicity is a major factor limiting plant growth in acid soils. Elevated atmospheric CO2 [CO2] enhances plant growth. However, there is no report on the effect of elevated [CO2] on growth of plant genotypes differing in Al tolerance grown in acid soils. We investigated the effect of short-term elevated [CO2] on growth of Al-tolerant (ET8) and Al-sensitive (ES8) wheat plants and malate exudation from root apices by growing them in acid soils under ambient [CO2] and elevated [CO2] using open-top chambers. Exposure of ET8 plants to elevated [CO2] enhanced root biomass only. In contrast, shoot biomass of ES8 was enhanced by elevated [CO2]. Given that exudation of malate to detoxify apoplastic Al is a mechanism for Al tolerance in wheat plants, ET8 plants exuded greater amounts of malate from root apices than ES8 plants under both ambient and elevated [CO2]. These results indicate that elevated [CO2] has no effect on malate exudation in both ET8 and ES8 plants. These novel findings have important implications for our understanding how plants respond to elevated [CO2] grown in unfavorable edaphic conditions in general and in acid soils in particular.

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.

MicroRNAs (miRNAs) play important roles in response of plants to biotic and abiotic stresses. Aluminum (Al) toxicity is a major factor limiting plant growth in acidic soils. However, there has been limited report on the involvement of miRNAs in response of plants to toxic Al(3+). To identify Al(3+)-responsive miRNAs at whole-genome level, high-throughput sequencing technology was used to sequence libraries constructed from root apices of the model legume plant Medicago truncatula treated with and without Al(3+). High-throughput sequencing of the control and two Al(3+)-treated libraries led to generation of 17.1, 14.1 and 17.4 M primary reads, respectively. We identified 326 known miRNAs and 21 new miRNAs. Among the miRNAs, expression of 23 miRNAs was responsive to Al(3+), and the majority of Al(3+)-responsive mRNAs was down-regulated. We further classified the Al(3+)-responsive miRNAs into three groups based on their expression patterns: rapid-responsive, late-responsive and sustained-responsive miRNAs. The majority of Al(3+)-responsive miRNAs belonged to the 'rapid-responsive' category, i.e. they were responsive to short-term, but not long-term Al(3+) treatment. The Al(3+)-responsive miRNAs were also verified by quantitative real-time PCR. The potential targets of the 21 new miRNAs were predicted to be involved in diverse cellular processes in plants, and their potential roles in Al(3+)-induced inhibition of root growth were discussed. These findings provide valuable information for functional characterization of miRNAs in Al(3+) toxicity and tolerance.

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.

Summary • Aluminum (Al) is toxic to plants when solubilized into Al3+ in acidic soils, and becomes a major factor limiting plant growth. However, the primary cause for Al toxicity remains unknown. • Nitric oxide (NO) is an important signaling molecule modulating numerous physiological processes in plants. Here, we investigated the role of NO in Al toxicity to Hibiscus moscheutos. • Exposure of H. moscheutos to Al3+ led to a rapid inhibition of root elongation, and the inhibitory effect was alleviated by NO donor sodium nitroprusside (SNP). NO scavenger and inhibitors of NO synthase (NOS) and nitrate reductase had a similar inhibitory effect on root elongation. The inhibition of root elongation by these treatments was ameliorated by SNP. • Aluminum inhibited activity of NOS and reduced endogenous NO concentrations. The alleviation of inhibition of root elongation induced by Al, NO scavenger and NOS inhibitor was correlated with endogenous NO concentrations in root apical cells, suggesting that reduction of endogenous NO concentrations resulting from inhibition of NOS activity could underpin Al-induced arrest of root elongation in H. moscheutos.

We evaluated effects of 9-year simulated nitrogen (N) deposition on microbial composition and diversity in the rhizosphere of two dominant temperate grassland species: grass Stipa krylovii and forb Artemisia frigida. Microbiomes in S. krylovii and A. frigida rhizosphere differed, but changed consistently along the N gradient. These changes were correlated to N-induced shifts to plant community. Hence, as plant biomass changed, so did bacterial rhizosphere communities, a result consistent with the role that N fertilizer has been shown to play in altering plant-microbial mutualisms. A total of 23 bacterial phyla were detected in the two rhizospheric soils by pyrosequencing, with Proteobacteria, Acidobacteria, and Bacteroidetes dominating the sequences of all samples. Bacterioidetes and Proteobacteria tended to increase, while Acidobacteria declined with increase in N addition rates. TM7 increased >5-fold in the high N addition rates, especially in S. krylovii rhizosphere. Nitrogen addition also decreased diversity of OTUs (operational taxonomic units), Shannon and Chao1 indices of rhizospheric microbes regardless of plant species. These results suggest that there were both similar but also specific changes in microbial communities of temperate steppes due to N deposition. These findings would contribute to our mechanistic understanding of impacts of N deposition on grassland ecosystem by linking changes in plant traits to their rhizospheric microbes-mediated processes.

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 truncatula Gaertn is a model legume species with a wide genetic diversity. To evaluate the responses of the two M. truncatula ecotypes, the effect of Fe deficiency on ecotype A17 and ecotype R108, which have been widely used in physiological and molecular studies, was investigated. A greater reduction in shoot Fe concentration of R108 plants than that of A17 plants was observed under Fe-deficient conditions. Exposure to Fe-deficient medium led to a greater increase in ferric chelate reductase (FCR) activity in roots of A17 than those of R108 plants, while expression of genes encoding FCR in roots of A17 and R108 plants was similarly up-regulated by Fe deficiency. Exposure of A17 plants to Fe-deficient medium evoked an ethylene evolution from roots, while the same treatment had no effect on ethylene evolution from R108 roots. There was a significant increase in expression of MtIRT encoding a Fe transporter in A17, but not in R108 plants, upon exposure to Fe-deficient medium. Transcripts of MtFRD3 that is responsible for loading of iron chelator citrate into xylem were up-regulated by Fe deficiency in A17, but not in R108 plants. These results suggest that M. truncatula ecotypes A17 and R108 differed in their response and adaptation to Fe deficiency, and that ethylene may play an important role in regulation of greater tolerance of A17 plant to Fe deficiency. These findings provide important clues for further elucidation of molecular mechanism by which legume plants respond and adapt to low soil Fe availability.

BACKGROUND: Calcium-binding proteins that contain EF-hand motifs have been reported to play important roles in transduction of signals associated with biotic and abiotic stresses. To functionally characterize genes of EF-hand family in response to abiotic stress, an MtCaMP1 gene belonging to EF-hand family from legume model plant Medicago truncatula was isolated and its function in response to drought and salt stress was investigated by expressing MtCaMP1 in Arabidopsis. METHODOLOGY/PRINCIPAL FINDINGS: Transgenic Arabidopsis seedlings expressing MtCaMP1 exhibited higher survival rate than wild-type seedlings under drought and salt stress, suggesting that expression of MtCaMP1 confers tolerance of Arabidopsis to drought and salt stress. The transgenic plants accumulated greater amounts of Pro due to up-regulation of P5CS1 and down-regulation of ProDH than wild-type plants under drought stress. There was a less accumulation of Na(+) in the transgenic plants than in WT plants due to reduced up-regulation of AtHKT1 and enhanced regulation of AtNHX1 in the transgenic plants compared to WT plants under salt stress. There was a reduced accumulation of H2O2 and malondialdehyde in the transgenic plants than in WT plants under both drought and salt stress. CONCLUSIONS/SIGNIFICANCE: The expression of MtCaMP1 in Arabidopsis enhanced tolerance of the transgenic plants to drought and salt stress by effective osmo-regulation due to greater accumulation of Pro and by minimizing toxic Na(+) accumulation, respectively. The enhanced accumulation of Pro and reduced accumulation of Na(+) under drought and salt stress would protect plants from water default and Na(+) toxicity, and alleviate the associated oxidative stress. These findings demonstrate that MtCaMP1 encodes a stress-responsive EF-hand protein that plays a regulatory role in response of plants to drought and salt stress.

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.

Background Resequencing can be used to identify genome variations underpinning many morphological and physiological phenotypes. Legume model plant Medicago truncatula ecotypes Jemalong A17 (J. A17) and R108 differ in their responses to mineral toxicity of aluminum and sodium, and mineral deficiency of iron in growth medium. The difference may result from their genome variations, but no experimental evidence supports this hypothesis. Results A total of 12,750 structure variations, 135,045 short insertions/deletions and 764,154 single nucleotide polymorphisms were identified by resequencing the genome of R108. The suppressed expression of MtAACT that encodes a putative aluminum-induced citrate efflux transporter by deletion of partial sequence of the second intron may account for the less aluminum-induced citrate exudation and greater accumulation of aluminum in roots of R108 than in roots of J. A17, thus rendering R108 more sensitive to aluminum toxicity. The higher expression-level of MtZpt2-1 encoding a TFIIIA-related transcription factor in J. A17 than R108 under conditions of salt stress can be explained by the greater number of stress-responsive elements in its promoter sequence, thus conferring J. A17 more tolerant to salt stress than R108 plants by activating the expression of downstream stress-responsive genes. YSLs (Yellow Stripe-Likes) are involved in long-distance transport of iron in plants. We found that an YSL gene was deleted in the genome of R108 plants, thus rendering R108 less tolerance to iron deficiency than J. A17 plants. Conclusions The deletion or change in several genes may account for the different responses of M. truncatula ecotypes J. A17 and R108 to mineral toxicity of aluminum and sodium as well as iron deficiency. Uncovering genome variations by resequencing is an effective method to identify different traits between species/ecotypes that are genetically related. These findings demonstrate that analyses of genome variations by resequencing can shed important light on differences in responses of M. truncatula ecotypes to abiotic stress in general and mineral stress in particular.

Background MicroRNAs (miRNAs) are small, endogenous RNAs that play important regulatory roles in development and stress response in plants by negatively affecting gene expression post-transcriptionally. Identification of miRNAs at the global genome-level by high-throughout sequencing is essential to functionally characterize miRNAs in plants. Drought is one of the common environmental stresses limiting plant growth and development. To understand the role of miRNAs in response of plants to drought stress, drought-responsive miRNAs were identified by high-throughput sequencing in a legume model plant, Medicago truncatula. Results Two hundreds eighty three and 293 known miRNAs were identified from the control and drought stress libraries, respectively. In addition, 238 potential candidate miRNAs were identified, and among them 14 new miRNAs and 15 new members of known miRNA families whose complementary miRNA*s were also detected. Both high-throughput sequencing and RT-qPCR confirmed that 22 members of 4 miRNA families were up-regulated and 10 members of 6 miRNA families were down-regulated in response to drought stress. Among the 29 new miRNAs/new members of known miRNA families, 8 miRNAs were responsive to drought stress with both 4 miRNAs being up- and down-regulated, respectively. The known and predicted targets of the drought-responsive miRNAs were found to be involved in diverse cellular processes in plants, including development, transcription, protein degradation, detoxification, nutrient status and cross adaptation. Conclusions We identified 32 known members of 10 miRNA families and 8 new miRNAs/new members of known miRNA families that were responsive to drought stress by high-throughput sequencing of small RNAs from M. truncatula. These findings are of importance for our understanding of the roles played by miRNAs in response of plants to abiotic stress in general and drought stress in particular.