Genome-wide identification and analysis of NPR family genes in Brassica juncea var. tumida.

Nonexpressor of pathogenesis-related (NPR) genes are bona fide transcription cofactors in the signal transduction pathway of salicylic acid (SA) and play critical regulatory roles in plant immunity. However, the NPR family genes in Brassica juncea var. tumida have not yet been comprehensively identified and analyzed as of yet. In the present study, NPR genes in B. juncea var. tumida seedlings were identified, and the tissue-specific expression patterns of NPR genes in the seedling were analyzed under salt stress (200 mM) treatment and infection by Plasmodiophora brassicae. A total of 19 NPR family genes clustering into six separate groups were identified in the genome of B. juncea var. tumida. These BjuNPR family genes were located in 11 of 18 chromosomes of B. juncea var. tumida and each possessed 1-5 exons. The BjuNPR family members had similar protein structures and conserved motifs. The BjuNPR genes exhibited tissue-specific expression patterns in the root, stem, leaf, flower and pod. Some BjuNPR genes were sensitive to salt stress and showed up-regulated or down-regulated expression patterns and most BjuNPR genes were up-regulated upon infection by P. brassicae. This study provides a foundation for further research into BjuNPR genes regulation in plant growth, development, and abiotic stress tolerance.

Genome-wide identification and expression analysis of jasmonate ZIM domain gene family in tuber mustard (Brassica juncea var. tumida).

Tuber mustard, which is the raw material of Fuling pickle, is a crop with great economic value. However, during growth and development, tuber mustard is frequently attacked by the pathogen Plasmodiophora brassicae and frequently experiences salinity stress. Jasmonic acid (JA) is a hormone related to plant resistance to biotic and abiotic stress. Jasmonate ZIM domain proteins (JAZs) are crucial components of the JA signaling pathway and play important roles in plant responses to biotic and abiotic stress. To date, no information is available about the characteristics of the JAZ family genes in tuber mustard. Here, 38 BjJAZ genes were identified in the whole genome of tuber mustard. The BjJAZ genes are located on 17 of 18 chromosomes in the tuber mustard genome. The gene structures and protein motifs of the BjJAZ genes are conserved between tuber mustard and Arabidopsis. The results of qRT-PCR analysis showed that BjuA030800 was specifically expressed in root, and BjuA007483 was specifically expressed in leaf. In addition, 13 BjJAZ genes were transiently induced by P. brassicae at 12 h, and 7 BjJAZ genes were induced by salt stress from 12 to 24 h. These results provide valuable information for further studies on the role of BjJAZ genes in the regulation of plant growth and development and in the response to biotic and abiotic stress.

Genome-wide identification and gene expression analysis of SOS family genes in tuber mustard (Brassica juncea var. tumida).

The Salt Overly Sensitive (SOS) pathway in Arabidopsis thaliana plays important roles in maintaining appropriate ion homeostasis in the cytoplasm and regulating plant tolerance to salinity. However, little is known about the details regarding SOS family genes in the tuber mustard crop (Brassica juncea var. tumida). Here, 12 BjSOS family genes were identified in the B. juncea var. tumida genome including two homologous genes of SOS1, one and three homologs of SOS2 and SOS3, two homologs of SOS4, two homologs of SOS5 and two homologs of SOS6, respectively. The results of conserved motif analysis showed that these SOS homologs contained similar protein structures. By analyzing the cis-elements in the promoters of those BjSOS genes, several hormone- and stress-related cis-elements were found. The results of gene expression analysis showed that the homologous genes were induced by abiotic stress and pathogen. These findings indicate that BjSOS genes play crucial roles in the plant response to biotic and abiotic stresses. This study provides valuable information for further investigations of BjSOS genes in tuber mustard.

Genome-Wide Identification and Gene Expression Analysis of ABA Receptor Family Genes in Brassica juncea var. tumida.

Abscisic acid (ABA) plays important roles in multiple physiological processes, such as plant response to stresses and plant development. The ABA receptors pyrabactin resistance (PYR)/ PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) play a crucial role in ABA perception and signaling. However, little is known about the details regarding PYL family genes in Brassica juncea var. tumida. Here, 25 PYL family genes were identified in B. juncea var. tumida genome, including BjuPYL3, BjuPYL4s, BjuPYL5s, BjuPYL6s, BjuPYL7s, BjuPYL8s, BjuPYL10s, BjuPYL11s, and BjuPYL13. The results of phylogenic analysis and gene structure showed that the PYL family genes performed similar gene characteristics. By analyzing cis-elements in the promoters of those BjuPYLs, several hormone and stress related cis-elements were found. The results of gene expression analysis showed that the ABA receptor homologous genes were induced by abiotic and biotic stress. The tissue-specific gene expression patterns of BjuPYLs also suggested those genes might regulate the stem swelling during plant growth. These findings indicate that BjuPYLs are involved in plant response to stresses and organ development. This study provides valuable information for further functional investigations of PYL family genes in B. juncea var. tumida.

Infection of Plasmodiophora brassicae changes the fungal endophyte community of tumourous stem mustard roots as revealed by high-throughput sequencing and culture-dependent methods.

Diverse fungal endophytes live in plants and are shaped by some abiotic and biotic stresses. Plant disease as particular biotic stress possibly gives an impact on the communities of fungal endophytes. In this study, clubroot disease caused by an obligate biotroph protist, Plasmodiophora brassicae, was considered to analyze its influence on the fungal endophyte community using an internal transcribed spacer (ITS) through high-throughput sequencing and culture-dependent methods. The results showed that the diversity of the endophyte community in the healthy roots was much higher than the clubroots. Ascomycota was the dominant group of endophytes (Phoma, Mortierella, Penicillium, etc.) in the healthy roots while P. brassicae was the dominant taxon in the clubroots. Hierarchical clustering, principal component analysis (PCA), principal coordinates analysis (PCoA) and analysis of similarities (ANOSIM) indicated significant differences between the endophyte communities in the healthy roots and clubroots. Linear discriminant analysis effect size (LefSe) analysis showed that the dominant genera could be regarded as potential biomarkers. The endophyte community in the healthy roots had a more complex network compared with the clubroots. Also, many plant pathogenic Fusarium were isolated from the clubroots by the culture-dependent method. The outcome of this study illustrates that P. brassicae infection may change the fungal endophyte community associated with the roots of tumourous stem mustard and facilitates the entry of soil pathogen into the roots.

Genome-Wide Analysis of Auxin Receptor Family Genes in Brassica juncea var. tumida.

Transport inhibitor response 1/auxin signaling f-box proteins (TIR1/AFBs) play important roles in the process of plant growth and development as auxin receptors. To date, no information has been available about the characteristics of the TIR1/AFB gene family in Brassica juncea var. tumida. In this study, 18 TIR1/AFB genes were identified and could be clustered into six groups. The genes are located in 11 of 18 chromosomes in the genome of B. juncea var. tumida, and similar gene structures are found for each of those genes. Several cis-elements related to plant response to phytohormones, biotic stresses, and abiotic stresses are found in the promoter of BjuTIR1/AFB genes. The results of qPCR analysis show that most genes have differential patterns of expression among six tissues, with the expression levels of some of the genes repressed by salt stress treatment. Some of the genes are also responsive to pathogen Plasmodiophora brassicae treatment. This study provides valuable information for further studies as to the role of BjuTIR1/AFB genes in the regulation of plant growth, development, and response to abiotic stress.

The Soybean GmNARK Affects ABA and Salt Responses in Transgenic Arabidopsis thaliana.

GmNARK (Glycine max nodule autoregulation receptor kinase) is the homolog of Arabidopsis thaliana CLAVATA1 (CLV1) and one of the most important regulators in the process of AON (Autoregulation of Nodulation), a process that restricts excessive nodule numbers in soybean. However, except for the function in AON, little is known about this gene. Here, we report that GmNARK plays important roles in process of plant response to abiotic stresses. Bioinformatic analysis and subcellular localization experiment results showed that GmNARK was a putative receptor like kinase and located at membrane. The promoter of GmNARK contains manifold cis regulatory elements that are responsive to hormone and stresses. Gene transcript expression pattern analysis in soybean revealed GmNARK was induced by ABA and NaCl treatment in both shoot and root. Overexpression of GmNARK in Arabidopsis resulted in higher sensitivity to ABA and salt treatment during seed germination and greening stages. We also checked the expression levels of some ABA response genes in the transgenic lines; the results showed that the transcript level of all the ABA response genes were much higher than that of wild type under ABA treatment. Our results revealed a novel role of GmNARK in response to abiotic stresses during plant growth and development.

GmTIR1/GmAFB3-based auxin perception regulated by miR393 modulates soybean nodulation.

Auxins play important roles in the nodulation of legumes. However, the mechanism by which auxin signaling regulates root nodulation is largely unknown. In particular, the role of auxin receptors and their regulation in determinate nodule development remains elusive. We checked the expression pattern of the auxin receptor GmTIR1/GmAFB3 genes in soybean. We analyzed the functions of GmTIR1/AFB3 in the regulation of rhizobial infection and nodule number, and also tested the functions of miR393 during nodulation and its relationship with GmTIR1/AFB3. The results showed that GmTIR1 and GmAFB3 genes exhibit diverse expression patterns during nodulation and overexpression of GmTIR1 genes significantly increased inflection foci and eventual nodule number. GmTIR1/AFB3 genes were post-transcriptionally cleaved by miR393 family and knock-down of the miR393 family members significantly increased rhizobial infection and the nodule number. Overexpression of the mutated form of GmTIR1C at the miR393 cleavage site that is resistant to miR393 cleavage led to a further increase in the number of infection foci and nodules, suggesting that miR393s modulate nodulation by directly targeting GmTIR1C. This study demonstrated that GmTIR1- and GmAFB3-mediated auxin signaling, that is spatio-temporally regulated by miR393, plays a crucial role in determinate nodule development in soybean.

Soybean miR172c targets the repressive AP2 transcription factor NNC1 to activate ENOD40 expression and regulate nodule initiation.

MicroRNAs are noncoding RNAs that act as master regulators to modulate various biological processes by posttranscriptionally repressing their target genes. Repression of their target mRNA(s) can modulate signaling cascades and subsequent cellular events. Recently, a role for miR172 in soybean (Glycine max) nodulation has been described; however, the molecular mechanism through which miR172 acts to regulate nodulation has yet to be explored. Here, we demonstrate that soybean miR172c modulates both rhizobium infection and nodule organogenesis. miR172c was induced in soybean roots inoculated with either compatible Bradyrhizobium japonicum or lipooligosaccharide Nod factor and was highly upregulated during nodule development. Reduced activity and overexpression of miR172c caused dramatic changes in nodule initiation and nodule number. We show that soybean miR172c regulates nodule formation by repressing its target gene, Nodule Number Control1, which encodes a protein that directly targets the promoter of the early nodulin gene, ENOD40. Interestingly, transcriptional levels of miR172c were regulated by both Nod Factor Receptor1α/5α-mediated activation and by autoregulation of nodulation-mediated inhibition. Thus, we established a direct link between miR172c and the Nod factor signaling pathway in addition to adding a new layer to the precise nodulation regulation mechanism of soybean.

Identification and Dynamic Regulation of microRNAs Involved in Salt Stress Responses in Functional Soybean Nodules by High-Throughput Sequencing.

Both symbiosis between legumes and rhizobia and nitrogen fixation in functional nodules are dramatically affected by salt stress. Better understanding of the molecular mechanisms that regulate the salt tolerance of functional nodules is essential for genetic improvement of nitrogen fixation efficiency. microRNAs (miRNAs) have been implicated in stress responses in many plants and in symbiotic nitrogen fixation (SNF) in soybean. However, the dynamic regulation of miRNAs in functioning nodules during salt stress response remains unknown. We performed deep sequencing of miRNAs to understand the miRNA expression profile in normal or salt stressed-soybean mature nodules. We identified 110 known miRNAs belonging to 61 miRNA families and 128 novel miRNAs belonging to 64 miRNA families. Among them, 104 miRNAs were dramatically differentially expressed (>2-fold or detected only in one library) during salt stress. qRT-PCR analysis of eight miRNAs confirmed that these miRNAs were dynamically regulated in response to salt stress in functional soybean nodules. These data significantly increase the number of miRNAs known to be expressed in soybean nodules, and revealed for the first time a dynamic regulation of miRNAs during salt stress in functional nodules. The findings suggest great potential for miRNAs in functional soybean nodules during salt stress.