Identification of drought-responsive microRNAs in tomato using high-throughput sequencing

Drought is a major abiotic stress affecting crop productivity and quality. As a class of noncoding RNA, microRNA (miRNA) plays important roles in plant growth, development, and stress response. However, their response and roles in tomato drought stress is largely unknown. Here, by using high-throughput sequencing, we compared the miRNA profiles before and after drought treatment in two tomato genotypes: M82, a drought-sensitive cultivated tomato (Solanum lycopersicum), and IL2-5, a drought-tolerant introgression line derived from M82 and the tomato wild species S. pennellii (LA0716). A total of 108 conserved and 208 novel miRNAs were identified, among them, 32 and 68 were significantly changed in expression after stress. Further, 1936 putative target genes were predicted for those differentially-expressed miRNAs. Gene ontology and pathway analysis showed that many of the target genes were involved in stress resistance, such as genes in GO terms including response to stress, defense response, response to stimulus, phosphorylation, and signal transduction. Our results suggested that miRNAs play an essential role in the drought response of tomato. This work will help to further characterize specific miRNAs functioning in drought tolerance.     

Differential expression of candidate genes for lignin biosynthesis under drought stress in maize leaves

In order to provide information for the development of molecular selection markers for drought tolerance improvement, the methods of prometric analysis, quantitative real-time PCR and field evaluation were employed for the identification of the differential expression of candidate genes under drought stress in maize. At seventeen, twenty-four and forty-eight hours of polyethylene glycol-simulated drought stress at the seventh leaf stage, leaf samples were collected from two drought-tolerant inbred lines for prometric analysis by two-dimensional electrophoresis and peptide mass fingerprinting. Fifty-eight proteins out of more than 500 were found in response to drought stress. Three drought-induced spots 2506, 3507 and 4506 showed sequence similarity with cinnamyl alcohol dehydrogenase, cytochrome protein 96A8 and S-adenosyl-L-methionine synthase, respectively. The expression of two key enzymes to lignin biosynthesis was quantified by quantitative real-time PCR among three drought-tolerant and one drought-sensitive inbred lines under drought stress and well-watered control conditions. After a decrease at the beginning of drought stress, the expression of cinnamyl alcohol dehydrogenase and caffeateO-methyltransferase recovered at twenty-four hours of the drought stress in the three drought-tolerant lines, but not in the drought-sensitive lines. Leaf lignin content, anthesis-silking interval and grain weight per plant were investigated with six inbred lines of varying drought tolerance under drought stress and well-watered control. Drought tolerance coefficients of these three characters were calculated and the correlation coefficients among these drought tolerance coefficients were estimated. Significant difference in leaf lignin content was found among the inbred lines and in response to drought stress. Close correlations were observed between the drought tolerant coefficients for leaf lignin content and grain weight per plant, and between the drought tolerant coefficients for leaf lignin content and anthesis-silking interval. These results indicate that leaf lignin content is a useful index for evaluation of drought tolerance in maize. Molecular selection markers can be developed on the basis of differential expression of the candidate genes and applied to maize improvement for drought tolerance.     

Cloning and characterization of miRNAs from maize seedling roots under low phosphorus stress

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition in plants and animals. In this study, a small RNA library was constructed to identify conserved miRNAs as well as novel miRNAs in maize seedling roots under low level phosphorus stress. Twelve miRNAs were identified by high throughput sequencing of the library and subsequent analysis, two belong to conserved miRNA families (miRNA399b and miRNA156), and the remaining ten are novel and one of latter is conserved in gramineous species. Based on sequence homology, we predicted 125 potential target genes of these miRNAs and then expression patterns of 7 miRNAs were validated by semi-RT-PCR analysis. MiRNA399b, Zma-miR3, and their target genes (Zmpt1 and Zmpt2) were analyzed by real-time PCR. It is shown that both miRNA399b and Zma-miR3 are induced by low phosphorus stress and regulated by their target genes (Zmpt1 and Zmpt2). Moreover, Zma-miR3, regulated by two maize inorganic phosphate transporters as a newly identified miRNAs, would likely be directly involved in phosphate homeostasis, so was miRNA399b in Arabidopsis and rice. These results indicate that both conserved and maize-specific miRNAs play important roles in stress responses and other physiological processes correlated with phosphate starvation, regulated by their target genes. Identification of these differentially expressed miRNAs will facilitate us to uncover the molecular mechanisms underlying the progression of maize seedling roots development under low level phosphorus stress.     

Transcriptional profiles of primary metabolism and signal transduction-related genes in response to water stress in field-grown sunflower genotypes using a thematic cDNA microarray

A sunflower cDNA microarray containing about 800 clones covering major metabolic and signal transduction pathways was used to study gene expression profiles in leaves and embryos of drought-tolerant and -sensitive genotypes subjected to water-deficit stress under field conditions. Using two-step ANOVA normalization and analysis models, we identified 409 differentially expressed genes among genotypes, water treatment and organs. The majority of the cDNA clones differentially expressed under water stress was found to display opposite gene expression profiles in drought-tolerant genotype compared to drought-sensitive genotype. These dissimilarities suggest that the difference between tolerant and non-tolerant plants seems to be associated with changes in qualitative but not quantitative mRNA expression. Comparing leaves and embryos, 82 cDNA clones showing organ-specific variation in gene expression levels were identified in response to water stress across genotypes. Genes related to amino acids and carbohydrates metabolisms, and signal transduction were induced in embryos and repressed in leaves; suggesting that vegetative and reproductive organs respond differentially to water stress. Adaptive mechanisms controlling water deficit tolerance are proposed and discussed.     

Genome-Wide Functional Analysis of Cotton (Gossypium hirsutum) in Response to Drought

Cotton is one of the most important crops for its natural textile fibers in the world. However, it often suffered from drought stress during its growth and development, resulting in a drastic reduction in cotton productivity. Therefore, study on molecular mechanism of cotton drought-tolerance is very important for increasing cotton production. To investigate molecular mechanism of cotton drought-resistance, we employed RNA-Seq technology to identify differentially expressed genes in the leaves of two different cultivars (drought-resistant cultivar J-13 and drought-sensitive cultivar Lu-6) of cotton. The results indicated that there are about 13.38% to 18.75% of all the unigenes differentially expressed in drought-resistant sample and drought-sensitive control, and the number of differentially expressed genes was increased along with prolonged drought treatment. DEG (differentially expression gene) analysis showed that the normal biophysical profiles of cotton (cultivar J-13) were affected by drought stress, and some cellular metabolic processes (including photosynthesis) were inhibited in cotton under drought conditions. Furthermore, the experimental data revealed that there were significant differences in expression levels of the genes related to abscisic acid signaling, ethylene signaling and jasmonic acid signaling pathways between drought-resistant cultivar J-13 and drought-sensitive cultivar Lu-6, implying that these signaling pathways may participate in cotton response and tolerance to drought stress.     

High-Throughput MicroRNA and mRNA Sequencing Reveals that MicroRNAs May Be Involved in Pectinesterase-Mediated Cold Resistance in Potato

Since potato cultivars are sensitive to low temperature, cold injury severely affects the geographical distribution and yield of potato. Although some miRNAs have been identified in response to cold stress in plants, there is no report about the role of miRNAs in the response to cold stress in potato. Here, via high throughput sequencing, we described the profiling of cold stress response to miRNA and mRNA in potato. Two small RNA and six mRNA libraries were constructed and sequenced. 296 known and 211 novel miRNAs were identified, in which 34 miRNAs in Cold Group (CG) had the higher expression quantity than which in Normal Group (NG) and 32 in CG had lower expression quantity than which in NG. 3068 differentially expressed genes were detected between NG and CG, in which 1400 genes were up-regulated and 1668 genes were down-regulated. The metabolism pathway of starch and sucrose (ko00500) is the common KEGG pathway in differentially expressed miRNA and mRNA. In this pathway, StuPME21575 and StuPME42971 are pectinesterase which mainly catalyzes the pectin-forming pectate, which are controlled by stu-miR6023 and stu-novel-miR42365. As the potato suffering cold stress, these two miRNAs expression levels became higher, but their target genes expression levels were just opposite and this result is the same with qRT-PCR.     

Differential miRNA expression in maize ear subjected to shading tolerance

Maize is an important crop worldwide. Its grain yield is susceptible to decrease under conditions of abiotic stress, such as shade in subtropical and temperate zones. The genetic basis of shade tolerance has not been determined in maize. MicroRNAs (miRNAs) are known to play critical roles in plant stress responses, including responses to environmental stress; but shade-associated miRNAs have not previously been identified in maize. In this study, the shade-sensitive inbred line 502 was used to examine miRNA expression differences in maize ear, after a 10-day treatment of either shade or exposure to natural light. A total of 130 known miRNAs belonging to 21 families were identified, of which 45 miRNAs were differentially expressed between shaded and natural light treatments. Twelve novel miRNAs were also predicted. In total, 94 miRNAs were upregulated and 48 downregulated in plants exposed to shaded conditions, compared with those exposed to natural light. These differentially expressed miRNAs may participate in regulating hormone homeostasis, metabolism, development and flower timing. These results suggest that the decrease of maize yield under shaded conditions may partly be determined by the differential expression of shade-induced miRNAs.     

Analysis of miRNA-seq combined with gene expression profile reveals the complexity of salinity stress response in <Emphasis Type="Italic">Oryza sativa</Emphasis>

High salinity is a major abiotic stressor that affects crop productivity and quality. While proper seedling growth is critical for crop reproduction under high salinity stress. Nowadays, genes/miRNAs expression is used for studying salinity stress response in rice seedlings. However, analysis of miRNA combined with gene expression is rare. To this end, we used miRNA-seq and gene expression profile to ascertain 6335 genes (3276 genes up-regulated, 3059 genes down-regulated) and 126 miRNAs (47 miRNAs up-regulated, 79 miRNAs down-regulated) that respond to salinity stress in rice seedlings. We then used these 126 miRNAs (including the novel miRNA osa-Chr12_1506) to identify 121 differentially expressed predicted target genes. In addition, we identified 34 miRNA-target RNA pairs, consisting of 9 differentially expressed miRNAs with complementary expression patterns. Combined with previous studies, we proposed a simple model for the molecular mechanism of a 12-h salinity stress response in rice seedlings. The findings lead to a deeper understanding of the function of miRNAs and genes that respond to salinity, and contributed to the elucidation of the complex mechanisms activated by salinity stress.     

System analysis of microRNAs in the development and aluminium stress responses of the maize root system

MicroRNAs (miRNAs) are a class of regulatory small RNAs (sRNAs) that down‐regulate target genes through mRNA cleavage or translational inhibition. miRNA is known to play an important role in the root development and environmental responses in both the Arabidopsis and rice. However, little information is available to form a complete view of miRNAs in the development of the maize root system and Al stress responses in maize. Four sRNA libraries were generated and sequenced from the early developmental stage of primary roots (PRY), the later developmental stage of maize primary roots (PRO), seminal roots (SR) and crown roots (CR). Through integrative analysis, we identified 278 miRNAs (246 conserved and 32 novel ones) and found that the expression patterns of miRNAs differed dramatically in different maize roots. The potential targets of the identified conserved and novel miRNAs were also predicted. In addition, our data showed that CR is more resistant to Al stress compared with PR and SR, and the differentially expressed miRNAs are likely to play significant roles in different roots in response to environmental stress such as Al stress. Here, we demonstrate that the expression patterns of miRNAs are highly diversified in different maize roots. The differentially expressed miRNAs are correlated with both the development and environmental responses in the maize root. This study not only improves our knowledge about the roles of miRNAs in maize root development but also reveals the potential role of miRNAs in the environmental responses of different maize roots.