Abstract: Heat stress disturbs cellular homeostasis, thus usually impairs the yield of flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee). MicroRNAs (miRNAs) play a significant role in plant responses to different stresses by modulating gene expression at the post-transcriptional level. However, the roles that miRNAs and their target genes may play in heat tolerance of flowering Chinese cabbage remain poorly characterized. The current study sequenced six small RNA libraries generated from the leave tissues of flowering Chinese cabbage at 0, 6 and 12 h after 38 °C heat treatment, and identified 49 putative novel miRNAs and 43 known miRNAs that differentially expressed between heat-tolerant and heat-sensitive flowering Chinese cabbage. Among them, 14 novel and 9 known miRNA differentially expressed only in the heat-tolerant genotype under heat-stress, therefore, their target genes including disease resistance protein TAO1-like, RPS6 and reticuline oxidase-like protein etc. may play important roles in enhancing heat-tolerance. Go ontology revealed that targets of these differentially expressed miRNAs may play key roles in responses to temperature stimulus, cell part, cellular process, cell, membrane, biological regulation, binding and catalytic activities. Furthermore, KEGG pathway analysis identified their important functions in signal transduction, environmental adaptation, global and overview maps, as well as in stress adaptation and in MAPK signaling pathway such as cell death. These findings provide insight into the functions of the miRNAs in heat stress tolerance of flowering Chinese cabbage.
Abstract: Brassica crops face a combination of different abiotic and biotic stresses in the field that can reduce plant growth and development by affecting biochemical and morpho-physiological processes. Emerging evidence suggests that non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and long ncRNAs (lncRNAs), play a significant role in the modulation of gene expression in response to plant stresses. Recent advances in computational and experimental approaches are of great interest for identifying and functionally characterizing ncRNAs. While progress in this field is limited, numerous ncRNAs involved in the regulation of gene expression in response to stress have been reported in Brassica. In this review, we summarize the modes of action and functions of stress-related miRNAs and lncRNAs in Brassica as well as the approaches used to identify ncRNAs.
Abstract: Calmodulin (CaM) regulates plant disease responses through its downstream calmodulin-binding proteins (CaMBPs) often by affecting the biosynthesis or signaling of phytohormones, such as jasmonic acid (JA) and salicylic acid. However, how these CaMBPs mediate plant hormones and other stress resistance-related signaling remains largely unknown. In this study, we conducted analyses in Arabidopsis (Arabidopsis thaliana) on the functions of AtIQM1 (IQ-Motif Containing Protein1), a Ca2+-independent CaMBP, in JA biosynthesis and defense against the necrotrophic pathogen Botrytis cinerea using molecular, biochemical, and genetic analyses. IQM1 directly interacted with and promoted CATALASE2 (CAT2) expression and CAT2 enzyme activity and indirectly increased the activity of the JA biosynthetic enzymes ACX2 and ACX3 through CAT2, thereby positively regulating JA content and B. cinerea resistance. In addition, in vitro assays showed that in the presence of CaM5, IQM1 further enhanced the activity of CAT2, suggesting that CaM5 may affect the activity of CAT2 by combining with IQM1 in the absence of Ca2+. Our data indicate that IQM1 is a key regulatory factor in signaling of plant disease responses mediated by JA. The study also provides new insights that CaMBP may play a critical role in the cross talk of multiple signaling pathways in the context of plant defense processes.
Abstract: Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.
Abstract: Delta-1-pyrroline-5-carboxylate synthase gene1 (P5CS1) is the key gene involved in the biosynthesis of proline and is significantly induced by drought stress. The exploration of genetic variation in HvP5CS1 may facilitate a better understanding of the mechanism of drought adaptation in barley. In the current study, 41 polymorphisms including 16 single nucleotide polymorphisms (SNPs) and 25 insertions/deletions (indels) were detected in HvP5CS1 among 287 barley (Hordeum vulgare L.) accessions collected worldwide, with 13 distinct haplotypes identified in the barley collection. Five polymorphisms in HvP5CS1 were significantly (P < 0.001) associated with drought tolerance related traits in barley. the phenotypic variation of a given trait explained by each associated polymorphism ranged from 4.43% to 9.81%. two sequence variations that were significantly (p < 0.0001) associated with grain yield had marginally significant positive tajima’s d values in the sliding window, so they might have been selected for environmental adaptation. meanwhile, two haplotypes hvp5cs1_h1 and hvp5cs1_h4, which contained desired alleles of the two variations mentioned above, were significantly (p < 0.001) associated with drought tolerance related traits, and explained 5.00~11.89% of the phenotypic variations. these variations associated with drought tolerance related traits can be used as potential markers for improving drought tolerance in barley.
Abstract: Flowering Chinese cabbage is one of the most important vegetable crops in southern China. Genetic improvement of various agronomic traits in this crop is underway to meet high market demand in the region, but the progress is hampered by limited number of molecular markers available in this crop. This study aimed to develop EST-SSR markers from transcriptome sequences generated by next-generation sequencing. RNA-seq of eight cabbage samples identified 48,975 unigenes. Of these unigenes, 23,267 were annotated in 56 gene ontology (GO) categories, 6,033 were mapped to 131 KEGG pathways, and 7,825 were assigned to clusters of orthologous groups (COGs). From the unigenes, 8,165 EST-SSR loci were identified and 98.57% of them were 1–3 nucleotide repeats with 14.32%, 41.08% and 43.17% of mono-, di- and tri-nucleotide repeats, respectively. Fifty-eight types of motifs were identified with A/T, AG/CT, AT/AT, AC/GT, AAG/CTT and AGG/CCT the most abundant. The lengths of repeated nucleotide sequences in all SSR loci ranged from 12 to 60 bp, with most (88.51%) under 20 bp. Among 170 primer pairs were randomly selected from a total of 4,912 SSR primers we designed, 48 yielded unambiguously polymorphic bands with high reproducibility. Cluster analysis using 48 SSRs classified 34 flowering Chinese cabbage cultivars into three groups. A large number of EST-SSR markers identified in this study will facilitate marker-assisted selection in the breeding programs of flowering Chinese cabbage.
Abstract: Flowering Chinese cabbage is one of the most important vegetable crops in southern China. Genetic improvement of various agronomic traits in this crop is underway to meet high market demand in the region, but the progress is hampered by limited number of molecular markers available in this crop. This study aimed to develop EST-SSR markers from transcriptome sequences generated by next-generation sequencing. RNA-seq of eight cabbage samples identified 48,975 unigenes. Of these unigenes, 23,267 were annotated in 56 gene ontology (GO) categories, 6,033 were mapped to 131 KEGG pathways, and 7,825 were assigned to clusters of orthologous groups (COGs). From the unigenes, 8,165 EST-SSR loci were identified and 98.57% of them were 1–3 nucleotide repeats with 14.32%, 41.08% and 43.17% of mono-, di- and tri-nucleotide repeats, respectively. Fifty-eight types of motifs were identified with A/T, AG/CT, AT/AT, AC/GT, AAG/CTT and AGG/CCT the most abundant. The lengths of repeated nucleotide sequences in all SSR loci ranged from 12 to 60 bp, with most (88.51%) under 20 bp. Among 170 primer pairs were randomly selected from a total of 4,912 SSR primers we designed, 48 yielded unambiguously polymorphic bands with high reproducibility. Cluster analysis using 48 SSRs classified 34 flowering Chinese cabbage cultivars into three groups. A large number of EST-SSR markers identified in this study will facilitate marker-assisted selection in the breeding programs of flowering Chinese cabbage.
Abstract: The transition from vegetative to reproductive growth phase is a pivotal and complicated process in the life cycle of flowering plants which requires a comprehensive response to multiple environmental aspects and endogenous signals. In Arabidopsis, six regulatory flowering time pathways have been defined by their response to distinct cues, namely photoperiod, vernalization, gibberellin, temperature, autonomous and age pathways, respectively. Among these pathways, the autonomous flowering pathway accelerates flowering independently of day length by inhibiting the central flowering repressor FLC. FCA, FLD, FLK, FPA, FVE, FY and LD have been widely known to play crucial roles in this pathway. Recently, AGL28, CK2, DBP1, DRM1, DRM2, ESD4, HDA5, HDA6, PCFS4, PEP, PP2A-B’γ, PRMT5, PRMT10, PRP39-1, REF6, and SYP22 have also been shown to be involved in the autonomous flowering time pathway. This review mainly focuses on FLC RNA processing, chromatin modification of FLC, post-translational modification of FLC and other molecular mechanisms in the autonomous flowering pathway of Arabidopsis.
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