Min-Gui Zhao

Nitric Oxide Synthase-Dependent Nitric Oxide Production Is Associated with Salt Tolerance in Arabidopsis

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.

Nitric Reductase-Dependent Nitric Oxide Production Is Involved in Cold Acclimation and Freezing Tolerance in Arabidopsis

Nitric oxide (NO) is an important signaling molecule involved in many physiological processes in plants. We evaluated the role of NO in cold acclimation and freezing tolerance using Arabidopsis (Arabidopsis thaliana) wild type and mutants nia1nia2 (for nitrate reductase [NR]-defective double mutant) and Atnoa1/rif1 (for nitric oxide associated1/resistant to inhibition by fosmidomycin1) that exhibit defects in NR and reduced NO production, respectively. Cold acclimation induced an increase in endogenous NO production in wild-type and Atnoa1/rif1 leaves, while endogenous NO level in nia1nia2 leaves was lower than in wild-type ones and was little changed during cold acclimation. Cold acclimation stimulated NR activity and induced up-regulation of NIA1 gene expression. In contrast, cold acclimation reduced the quantity of NOA1/RIF1 protein and inhibited NO synthase (NOS) activity. These results indicate that up-regulation of NR-dependent NO synthesis underpins cold acclimation-induced NO production. Seedlings of nia1nia2 were less tolerant to freezing than wild-type plants. Pharmacological studies using NR inhibitor, NO scavenger, and NO donor showed that NR-dependent NO level was positively correlated with freezing tolerance. Furthermore, cold acclimation up- and down-regulated expression of P5CS1 and ProDH genes, respectively, resulting in enhanced accumulation of proline (Pro) in wild-type plants. The stimulation of Pro accumulation by cold acclimation was reduced by NR inhibitor and NO scavenger, while Pro accumulation by cold acclimation was not affected by the NOS inhibitor. In contrast to wild-type plants, cold acclimation up-regulated ProDH gene expression in nia1nia2 plants, leading to less accumulation in nia1nia2 plants than in wild-type plants. These findings demonstrate that NR-dependent NO production plays an important role in cold acclimation-induced increase in freezing tolerance by modulating Pro accumulation in Arabidopsis.

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

BackgroundMicroRNAs (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.ResultsTwo 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.ConclusionsWe 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.

Nitric oxide protects against oxidative stress under heat stress in the calluses from two ecotypes of reed

Calluses from two ecotypes of reed (Phragmites communis Trin.) plant (dune reed [DR] and swamp reed [SR]), which show different sensitivity to heat stress, were used to study plant acclimations to heat stress. SR callus suffered more oxidative damage than DR callus, and DR callus maintained higher relative growth rate and cell viability and lower ion leakage than SR callus under heat stress. Application of two nitric oxide donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP), dramatically alleviated heat stress induced ion leakage increase, growth suppression and cell viability decrease in both calluses under heat stress. H2O2 and MDA contents were decreased and the activities of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase increased in both calluses in the presence of NO donors under heat stress. The potassium salt of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific NO scavenger, arrested NO donors mediated protective effects. Moreover, measurement of the rate of NO release showed that NO production increased significantly in DR callus while kept stable in SR callus under heat stress. Pretreatment with cPTIO had no effect on SR callus under heat stress while it significantly enhanced oxidative stress in DR callus compared with that of heat stress alone. These results suggest that NO can effectively protect both calluses from oxidative stress induced by heat stress and that NO might act as a signal in activating active oxygen scavenging enzymes under heat stress and thus confer DR callus thermotolerance.

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 EL50 (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 EL50 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.

Identification and characterization of long non-coding RNAs involved in osmotic and salt stress in Medicago truncatula using genome-wide high-throughput sequencing

BackgroundLong non-coding RNAs (lncRNAs) have been shown to play crucially regulatory roles in diverse biological processes involving complex mechanisms. However, information regarding the number, sequences, characteristics and potential functions of lncRNAs in plants is so far overly limited.ResultsUsing high-throughput sequencing and bioinformatics analysis, we identified a total of 23,324 putative lncRNAs from control, osmotic stress- and salt stress-treated leaf and root samples of Medicago truncatula, a model legume species. Out of these lncRNAs, 7,863 and 5,561 lncRNAs were identified from osmotic stress-treated leaf and root samples, respectively. While, 7,361 and 7,874 lncRNAs were identified from salt stress-treated leaf and root samples, respectively. To reveal their potential functions, we analyzed Gene Ontology (GO) terms of genes that overlap with or are neighbors of the stress-responsive lncRNAs. Enrichments in GO terms in biological processes such as signal transduction, energy synthesis, molecule metabolism, detoxification, transcription and translation were found.ConclusionsLncRNAs are likely involved in regulating plant’s responses and adaptation to osmotic and salt stresses in complex regulatory networks with protein-coding genes. These findings are of importance for our understanding of the potential roles of lncRNAs in responses of plants in general and M. truncatula in particular to abiotic stresses.

Cold acclimation-induced freezing tolerance of Medicago truncatula seedlings is negatively regulated by ethylene

To evaluate the role of ethylene in cold acclimation and cold stress, freezing tolerance and characteristics associated with cold acclimation were investigated using legume model plant Medicago truncatula Gaertn Jemalong A17. There was a rapid suppression of ethylene production during cold acclimation in A17 plants. Ethylene level was negatively correlated with freezing tolerance as inhibition of ethylene biosynthesis by inhibitors of ethylene biosynthesis enhanced freezing tolerance, while exogenous application of ethylene reduced cold acclimation-induced freezing tolerance. The involvement of ethylene signaling in modulation of freezing tolerance and cold acclimation was further studied using ethylene-insensitive mutant sickle skl. Although skl mutant was more tolerant to freezing than its wild-type counterpart A17 plants, cold acclimation enhanced freezing tolerance in 17 plants, but not in skl mutant. Expression of several ethylene response genes including EIN3, EIN3/EIL and ERFs was suppressed in skl mutant compared to A17 plants under non-cold-acclimated conditions. Cold acclimation downregulated expression of EIN3, EIN3/EIL and ERFs in A17 plants, while expression patterns of these genes were relatively constant in skl mutant during cold acclimation. Cold acclimation-induced increases in transcription of MtCBFs and MtCAS15 were suppressed in skl mutant compared with A17 plants. These results suggest that MtSKL1 is required for perception of the change of ethylene level in M. truncatula plants for the full development of the cold acclimation response by suppressing expression of MtEIN3 and MtEIN3/EIL1, which in turn downregulates expression of MtERFs, leading to the enhanced tolerance of M. truncatula to freezing by upregulating MtCBFs and MtCAS15.

Ethylene-responsive miRNAs in roots of Medicago truncatula identified by high-throughput sequencing at whole genome level

Ethylene is one of the classical plant hormones with a diverse function in plant growth and development. Root elongation is sensitive to ethylene such that treatments with ethylene and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) inhibit root growth. MicroRNA as one type of endogenous, non-coding small RNAs, plays an important role in regulation of plant growth, development and hormonal signaling by affecting expression of target genes. However, there has been no detailed study to evaluate the role of microRNAs in mediation of ethylene-dependent physiological processes in plants. Medicago truncatula is a model plant widely used for investigation of molecular biology in legume species. In this study, we constructed two small RNA libraries from roots of M. truncatula treated with and without ACC. High-throughput sequencing was employed to sequence the small RNA libraries, and more than 30 M raw reads were obtained. We annotated 301 known miRNAs and identified 3 new miRNAs in the two libraries. Treatment of M. truncatula with 10 μM ACC led to changes in expression of 8 miRNAs. The targets of the ethylene-responsive miRNAs were predicted by bioinformatic approach. The potential role of the ethylene-responsive miRNAs in the ethylene-induced inhibition of root elongation is discussed. These results are useful for functional characterization of miRNAs in mediation of ethylene-dependent physiological processes in general and root elongation 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 gens 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 MtCaMP1exhibited 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.

Glucose-induced inhibition of seed germination in Lotus japonicus is alleviated by nitric oxide and spermine.

Seed germination is sensitive to glucose (Glc), nitric oxide (NO) and polyamine (PA). To elucidate whether cross-talk among Glc, NO and PAs occurs in mediation of seed germination, effects of Glc, NO and spermine on seed germination of Lotus japonicus were studied. Glc retarded seed germination in a concentration-dependent manner. NO donor sodium nitroprusside (SNP) alleviated Glc-induced inhibition of seed germination, whereas the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO) diminished the SNP-dependent alleviation of seed germination. These observations indicate that Glc may inhibit seed germination by interacting with NO signaling pathways. Exogenous spermine enhanced and the inhibitor of the spermine synthase, methylglyoxal-bis-guanyl hydrazone (MGBG), inhibited seed germination, respectively. Like SNP, spermine alleviated the Glc-induced inhibition of seed germination, whereas MGBG exaggerated the Glc-induced inhibition of seed germination. These results suggest that Glc may inhibit the spermine synthesis, leading to reductions in seed germination. NO scavenger and spermine synthase inhibitor diminished the SNP-induced alleviation of Glc-induced inhibition of seed germination. These findings reveal that both NO and spermine participate in the Glc-induced inhibition of seed germination in L. japonicus.