Metal ions steer the duality in microbial community recovery from nitrogen enrichment by shaping functional groups.

Atmospheric nitrogen (N) deposition has been substantially reduced due to declines in the reactive N emission in major regions of the world. Nevertheless, the impact of reduced N deposition on soil microbial communities and the mechanisms by which they are regulated remain largely unknown. Here, we examined the effects of N addition and cessation of N addition on plant and soil microbial communities through a 17-year field experiment in a temperate grassland. We found that extreme N input did not irreversibly disrupt the ecosystem, but ceasing high levels of N addition led to greater resilience in bacterial and fungal communities. Fungi exhibited diminished resilience compared to bacteria due to their heightened reliance on changes in plant communities. Neither bacterial nor fungal diversity fully recovered to their original states. Their sensitivity and resilience were mainly steered by toxic metal ions and soil pH differentially regulating on functional taxa. Specifically, beneficial symbiotic microbes such as N-fixing bacteria and arbuscular mycorrhizal fungi experienced detrimental effects from toxic metal ions and lower pH, hindering their recovery. The bacterial functional groups involved in carbon decomposition, and ericoid mycorrhizal and saprotrophic fungi were positively influenced by soil metals, and demonstrated gradual recovery. These findings could advance our mechanistic understanding of microbial community dynamics under ongoing global changes, thereby informing management strategies to mitigate the adverse effects of N enrichment on soil function.

Seeds exhibit the most stable elemental composition with nitrogen addition in an Inner Mongolian grassland.

Variation in biomass elemental composition of grassland plants may have important implications for ecosystem functioning in response to global change. However, relevant studies have mostly focused on variation of nitrogen (N) and phosphorus (P) concentrations in plant leaves, while few studies have evaluated other elements and plant organs of grassland species. Here, we examined the effects of N addition on multi-element concentrations, and analyzed their patterns across different organs (leaf, stem, root and seed) of five plant species in a steppe community of the Inner Mongolian grassland. Our results showed that seeds exhibited the most stable elemental composition with N addition, and that manganese (Mn) and iron (Fe) concentrations were substantially more variable than macro-elements in response to N addition. In particular, we identified a set of significant negative relationships between elemental concentrations and their corresponding CVs (coefficients of variation) for all plant organs as a whole and for each individual organ. We further found that changes in soil pH and the availability of soil nutrients contributed mostly to variation in the biomass elemental composition of major plants in this community. These findings are important for accurately assessing the effects of N deposition on the biochemical cycling of nutrient elements in grassland ecosystems, and provide critical clues for developing effective approaches to adaptively managing grassland resources as well as mitigating the impact of global change on the dryland ecosystems in the Mongolia Plateau.

Mowing accelerates phosphorus cycling without depleting soil phosphorus pool.

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.

An integrated belowground trait-based understanding of nitrogen-driven plant diversity loss.

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 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 the accumulation 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 affect 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.

Glutamate Receptor Homolog3.4 is Involved in Regulation of Seed Germination Under Salt Stress in Arabidopsis.

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.

A novel soil manganese mechanism drives plant species loss with increased nitrogen deposition in a temperate steppe.

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.

CIPK23 is involved in iron acquisition of Arabidopsis by affecting ferric chelate reductase activity.

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.

Rhizosphere bacterial communities of dominant steppe plants shift in response to a gradient of simulated nitrogen deposition.

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.

Genome variations account for different response to three mineral elements between Medicago truncatula ecotypes Jemalong A17 and R108.

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.

Medicago truncatula ecotypes A17 and R108 differed in their response to iron deficiency.

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.

Ethylene negatively regulates aluminium-induced malate efflux from wheat roots and tobacco cells transformed with TaALMT1.

An important mechanism for Al(3+) 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 Al(3+)-activated efflux of malate was investigated using Al(3+)-tolerant wheat genotype ET8, which has high expression of TaALMT1. Exposure of ET8 plants to Al(3+) enhanced ethylene evolution in root apices. Treatment with the ethylene synthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene gas suppressed Al(3+)-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 Al(3+) 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. Al(3+)-induced inhibition of root elongation was ameliorated by pretreatment with AIB. In addition, ethylene donor (Ethrel) also inhibited Al(3+)-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 Al(3+)-induced malate efflux by targeting TaALMT1-mediated malate efflux by an unknown mechanism.

Systemic regulation of sulfur homeostasis in Medicago truncatula.

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.

Wheat genotypes differing in aluminum tolerance differ in their growth response to CO2 enrichment in acid soils.

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.

A Medicago truncatula EF-hand family gene, MtCaMP1, is involved in drought and salt stress tolerance.

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.

Expression of a Medicago falcata small GTPase gene, MfARL1 enhanced tolerance to salt stress in Arabidopsis thaliana.

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.

Identification of aluminum-responsive microRNAs in Medicago truncatula by genome-wide high-throughput sequencing.

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.

Identification of drought-responsive microRNAs in Medicago truncatula by genome-wide high-throughput sequencing.

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.

Comparative studies on tolerance of Medicago truncatula and Medicago falcata to freezing.

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.

Aluminium-induced inhibition of root elongation in Arabidopsis is mediated by ethylene and auxin.

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.

Ethylene is involved in nitrate-dependent root growth and branching in Arabidopsis thaliana.

*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.