Identification of miR390-TAS3-ARF pathway in response to salt stress in Helianthus tuberosus L.

MicroRNA390 (miR390), an ancient and highly conserved miRNA family in land plants, plays multiple roles in plant growth, development and stress responses. In this study, we isolated and identified MIR390, miR390, TAS3a/b/c, tasiARF-1/2/3 (trans-acting small interfering RNAs influencing Auxin Response Factors) and ARF2/3/4 in Jerusalem artichoke (Helianthus tuberosus L.). Treatment with 100 mM NaCl induced expression of miR390, increased cleavage of TAS3, produced high levels of tasiARFs, and subsequently enhanced cleavage of ARF3/4, which was most likely associated with salt tolerance of the plants. In contrast, treatment with 300 mM NaCl inhibited expression of miR390, attenuated cleavage of TAS3, produced a small amount of tasiARFs, and reduced cleavage of ARF3/4. We proposed that ARF2, one of the targets of tasiARFs, induced under salinity was likely to play an active role in salt tolerance of Jerusalem artichoke. The study of the miR390-TAS3-ARF model in Jerusalem artichoke may broaden our understanding of salt tolerance mechanisms, and provides a theoretical support for further genetic identification and breeding crops with increased tolerance to salt stress.

Identification of a group of XTHs genes responding to heavy metal mercury, salinity and drought stresses in Medicago truncatula.

Xyloglucan endotransglucosylase/hydrolases (XTH) are one of the key enzymes regulating cell wall construction, extension and metabolism. In the study, 44 XTH protein genes from Medicago truncatula genome were identified using bioinformatics, microarray and RT-PCR. Each XTH was showed to possess a highly conserved domain ((D/N)-E-(I/L/F/V)-D-(F/I/L)-E-(F/L)-L-G), and most of XTHs possess four Cys in the C terminal region, which suggests the potential for generating disulfide bonds. Based on the XTH protein sequences, these XTHscan be classified into three major families and each family can be subdivided into more groups. Examination of the genomic location of XTH genes on M. truncatula chromosomes showed that the evolutional expansion of the genes was possibly attributed to localized gene duplications. To investigate the possible involvement of the XTHs responding to heavy metals and other abiotic stresses, the XTH genes were exposed to heavy metal (Hg or Cu), salt and drought stresses. There were 28, 21 and 21 MtXTH genes found to respond to HgCl2, salt and drought stresses, respectively, but their expression were different under the stresses. Some of the XTH genes were well confirmed by quantitative RT-PCR (qRT-PCR). We further specified expression of a XTH gene Medtr4g128580 (MtXTH3) under different environmental stresses, and showed that MtXTH3 was induced by Hg exposure. These results indicated that a group of MtXTHs could be differentially expressed under the environmental stresses.

A cotton miRNA is involved in regulation of plant response to salt stress.

The present study functionally identified a new microRNA (microRNA ovual line 5, miRNVL5) with its target gene GhCHR from cotton (Gossypium hirsutum). The sequence of miRNVL5 precursor is 104 nt long, with a well developed secondary structure. GhCHR contains two DC1 and three PHD Cys/His-rich domains, suggesting that GhCHR encodes a zinc-finger domain-containing transcription factor. miRNVL5 and GhCHR express at various developmental stages of cotton. Under salt stress (50-400 mM NaCl), miRNVL5 expression was repressed, with concomitant high expression of GhCHR in cotton seedlings. Ectopic expression of GhCHR in Arabidopsis conferred salt stress tolerance by reducing Na(+) accumulation in plants and improving primary root growth and biomass. Interestingly, Arabidopsis constitutively expressing miRNVL5 showed hypersensitivity to salt stress. A GhCHR orthorlous gene At2g44380 from Arabidopsis that can be cleaved by miRNVL5 was identified by degradome sequencing, but no confidential miRNVL5 homologs in Arabidopsis have been identified. Microarray analysis of miRNVL5 transgenic Arabidopsis showed six downstream genes (CBF1, CBF2, CBF3, ERF4, AT3G22920, and AT3G49200), which were induced by salt stress in wild-type but repressed in miRNVL5-expressing Arabidopsis. These results indicate that miRNVL5 is involved in regulation of plant response to salt stress.

The F-box family genes as key elements in response to salt, heavy mental, and drought stresses in Medicago truncatula.

F-box protein is a subunit of Skp1-Rbx1-Cul1-F-box protein (SCF) complex with typically conserved F-box motifs of approximately 40 amino acids and is one of the largest protein families in eukaryotes. F-box proteins play critical roles in selective and specific protein degradation through the 26S proteasome. In this study, we bioinformatically identified 972 putative F-box proteins from Medicago truncatula genome. Our analysis showed that in addition to the conserved motif, the F-box proteins have several other functional domains in their C-terminal regions (e.g., LRRs, Kelch, FBA, and PP2), some of which were found to be M. truncatula species-specific. By phylogenetic analysis of the F-box motifs, these proteins can be classified into three major families, and each family can be further grouped into more subgroups. Analysis of the genomic distribution of F-box genes on M. truncatula chromosomes revealed that the evolutional expansion of F-box genes in M. truncatula was probably due to localized gene duplications. To investigate the possible response of the F-box genes to abiotic stresses, both publicly available and customer-prepared microarrays were analyzed. Most of the F-box protein genes can be responding to salt and heavy metal stresses. Real-time PCR analysis confirmed that some of the F-box protein genes containing heat, drought, salicylic acid, and abscisic acid responsive cis-elements were able to respond to the abiotic stresses.

Molecular dissection of mercury-responsive transcriptome and sense/antisense genes in Medicago truncatula.

We described a newly developed approach, namely next-generation tag sequencing, to identify global gene transcripts and complexity regulated by heavy metals in Medicago truncatula. Two cDNA libraries were generated from M. truncatula seedlings: treated and non-treated with the toxic heavy metal mercury Hg(II). With the large number of read-mapped genes generated, we observed that most of the genes were differentially expressed between the two libraries. In addition, several classes of new transcripts including transcription factors, antisense transcripts, and stress responsive genes were detected. The forty genes most altered in expression levels were associated with tolerance to environmental stress and secondary metabolism. Validation of genes by quantitative RT-PCR confirmed the results from deep-sequencing. Most of genes coding for metal transporters, sulfate metabolism, and cell wall solidification were significantly altered by Hg exposure. We also examined altered expression ratios of sense and antisense (S-AS) transcripts between the two libraries. By analyzing strand-specific information of read sequences, S-AS transcripts were found to be enriched with metal treatment. The transcriptome sequences were analyzed further with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) and showed diverse biological functions and metabolic pathways under the metal stress.

Genome-wide identification of Brassica napus microRNAs and their targets in response to cadmium.

MicroRNAs (miRNAs) are a distinct class of small RNAs in plants that not only regulate biological processes but also regulate response to environmental stresses. The toxic heavy metal cadmium (Cd) induces expression of several miRNAs in rapeseed (Brassica napus), but it is not known on a genome-wide scale how the expression of miRNAs and their target genes, is regulated by Cd. In this study, four small RNA libraries and four degradome libraries were constructed from Cd-treated and non-Cd-treated roots and shoots of B. napus seedlings. Using high-throughput sequencing, the study identified 84 conserved and non-conserved miRNAs (belonging to 37 miRNA families) from Cd-treated and non-treated B. napus, including 19 miRNA members that were not identified before. Some of the miRNAs were validated by RNA gel blotting. Most of the identified miRNAs were found to be differentially expressed in roots/shoots or regulated by Cd exposure. The study simultaneously identified 802 targets for the 37 (24 conserved and 13 non-conserved) miRNA families, from which there are 200, 537, and 65 targets, belonging to categories I, II, and III, respectively. In category I alone, many novel targets for miRNAs were identified and shown to be involved in plant response to Cd.

Molecular dissection of atrazine-responsive transcriptome and gene networks in rice by high-throughput sequencing.

The residue of atrazine (a herbicide) has become hazards in environments due to its intensive use. However, its molecular toxicity to on plants and human beings is not fully understood. In this study, we performed high-throughput sequencing of atrazine-exposed rice (Oryza sativa) to analyze global expression and complexity of genes in the crop. Four libraries were constructed from shoots and roots with or without atrazine exposure. We sequenced 5,751,861, 5,790,013, 5,375,999 and 6,039,618 clean tags that corresponded to 220,806, 111,301, 248,802 and 114,338 distinct tags for Root-Atr (root control, atrazine-free), Shoot-Atr (shoot control, atrazine-free), Root+Atr (root treated with atrazine) and Shoot+Atr (shoot treated with atrazine) libraries, respectively. Mapping the clean tags to gene databases generated 18,833-21,007 annotated genes for each library. Most of annotated genes were differentially expressed among the libraries. The most 40 differentially expressed genes were associated with resistance to environmental stress, degradation of xenobiotics and molecular metabolism. Validation of gene expression by quantitative RT-PCR confirmed the deep-sequencing results. The transcriptome sequences were further subjected to Gene Orthology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and showed modified biological functions and metabolic pathways. Our results not only highlight the transcriptional complexity in rice with atrazine but also represent a major improvement for analyzing transcriptional changes on a large scale in xenobiotics-responsive toxicology.

Genome-wide identification of Medicago truncatula microRNAs and their targets reveals their differential regulation by heavy metal.

We adopted a deep sequencing approach developed by Solexa (Illumina Inc., San Diego, CA, USA) to investigate global expression and complexity of microRNAs (miRNAs) and their targets from Medicago truncatula. Two small RNA libraries and two degradome libraries were constructed from mercury (Hg)-treated and Hg-free M. truncatula seedlings. For miRNAs, each library generated 18.5-18.6 million short sequences, resulting in 10.2-10.8 million clean reads. At least 52 new miRNA candidates with ≈ 21 nucleotides are perfectly matched to the M. truncatula genome. Statistical analysis on transcript abundance of the new candidate miRNAs revealed that most of them were differentially regulated by the heavy metal mercury Hg(II), with 12 miRNAs being specifically induced by Hg exposure. Additionally, we identified 201 individual miRNAs representing 63 known M. truncatula miRNA families, including 12 new conserved and one non-conserved miRNAs that have not been described before. Finally, 130 targets for 58 known (37 conserved and 21 non-conserved) miRNA families and 37 targets for 18 new M. truncatula-specific candidate miRNA families were identified by high-throughput degradome sequencing approach.

Bioinformatic identification and expression analysis of new microRNAs from Medicago truncatula.

MicroRNAs (miRNAs) are a class of non-protein coding small RNAs that regulate gene post-transcriptional expression in plants and animals. Although more than 1280 miRNAs in plant kingdom have been discovered thus far, only a handful of plant miRNAs has been intensively identified. The legume Medicago truncatula is a model system widely used to investigate gene transcription or post-transcriptional processes for arbuscular mycorrhiza development. In this study, we used a bioinformatics approach for ESTs (Expressed Sequence Tags)- and GSS (Genomic Survey Sequences)-wide prediction of novel miRNAs in M. truncatula. A total of 38 potential miRNAs were detected following a range of filtering criteria. After removal of 12 overlapping miRNAs that have already been deposited in miRNA Registry (Release 11.0), 26 miRNAs belonging to 15 families were found to be new. Using the newly identified miRNA sequences, we were able to further blast the M. truncatula mRNA database and detected 16 potential targets of miRNAs. Many miRNA targeted genes were predicted to encode transcription factors that regulate plant development, signaling, and metabolism. To verify the prediction of the miRNAs in M. truncatula, the qRT-PCR-based assay was performed. Our results showed that eight of the miRNA families displayed different patterns of expression in tissues. Additionally, expression of these miRNAs was up- or down-regulated by heavy metals (Hg, Cd, and Al). The above results implied that some of the miRNAs are involved in the regulation of development and plant response to heavy metal stress.

Biological detection and analysis of mercury toxicity to alfalfa (Medicago sativa) plants.

Mercury has become one of the major causes of toxic metal pollution in agricultural lands. Accumulation of mercury by plants may disrupt many cellular functions and block growth and development. To assess mercury toxicity, we performed an experiment focusing on the responses of alfalfa (Medicago sativa) to Hg(2+)-induced oxidative stress. Alfalfa plants were treated with 0-40microM HgCl(2) for 7d. The concentrations of Hg(2+) were positively correlated with the generation of O2- and H(2)O(2) in leaves. Treatment with Hg(2+) increased the activities of NADH oxidase and lipoxygenase (LOX) and damaged the biomembrane lipids. To understand biochemical responses under Hg stress, activities of several antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) were assayed. Analysis of SOD activity by non-denaturing polyacrylamide gel electrophoresis revealed five isoforms in leaves, but they showed different patterns. Also, eight isoenzymes of APX and seven of POD in leaves were detected. However, only one isoform of CAT was visualized. The total activities of APX, POD and CAT were generally enhanced. We also measured several antioxidative metabolites such as ascorbate and glutathione (GSH), and found that both differentially accumulated in leaves. These results indicate that the increased levels of O2- and H(2)O(2) under Hg stress were closely linked to the improved capacity of antioxidant enzymes. The data not only provide the important information for better understanding of the toxic and tolerance mechanisms, but as well can be used as a bio-indicator for soil contamination by Hg.

Metabolic adaptations to mercury-induced oxidative stress in roots of Medicago sativa L.

Alfalfa (Medicago sativa) roots were treated with mercuric ions in a concentration- and time-dependent manner, and lipid peroxidation was studied biochemically as well as histochemically along with other physiological responses. Histochemical staining with Schiff's reagent and Evans blue revealed that the peroxidation of membrane lipids and loss of plasma membrane integrity in Hg-treated roots occurred in the meristem and the elongation zone. The histochemical observations were supported by the quantitative determinations of thiobarbituric acid reactive substances (TBARS). However, under the mercuric ions stress, the alfalfa plants showed no significant alteration of hydrogen peroxide in roots. Analysis of lipoxygenase activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) showed that there were two isoforms in the root of alfalfa plants, but they showed quite different patterns under the Hg exposure. Also, using non-denaturing PAGE, activities of superoxide dismutase (SOD) and peroxidase (POD) were determined in roots after treatment with Hg ions. The total activities of SOD and POD increased in roots after Hg treatment of roots. Activity of ascorbate peroxides (APX) was stimulated at relatively high concentration of Hg (40microM), and after prolonged Hg exposure (20microM, 24h). In contrast, glutathione reductase activity was depressed at higher concentrations of Hg (10-20microM). Treatments of seedlings with 10-40microM Hg decreased the ascorbate and glutathione amounts but increased total non-protein thiols. The above results indicated that Hg exerted its toxic effect on the root growth of alfalfa by induction of oxidative stress.