Comprehensive Evolution and Expression anaLysis of PHOSPHATE 1 Gene Family in Allotetraploid Brassica napus and Its Diploid Ancestors.

The members of PHOSPHATE 1 (PHO1) family play important roles in plant phosphate (Pi) transport and adaptation to Pi deficiency. The functions of PHO1 family proteins have been reported in several plant species, with the exception of Brassica species. Here, we identified 23, 23, and 44 putative PHO1 family genes in Brassica rapa, Brassica oleracea, and Brassica napus by whole genome analysis, respectively. The phylogenetic analysis divided PHO1 family proteins into eight groups, which represented the orthologous relationships among PHO1 members. The gene structure and the conserved motif analysis indicated that the most PHO1 family genes had similar gene structures and the PHO1 proteins shared mutual conserved motifs. The chromosome distribution analysis showed that the majority of BnPHO1 family genes distributed analogously at chromosomes with BrPHO1 and BoPHO1 family genes. The data showed that PHO1 family genes were highly conserved during evolution from diploid to tetraploid. Furthermore, the expression analysis showed that PHO1 family genes had different expression patterns in plant tissues, suggesting the diversity of gene functions in Brassica species. Meanwhile, the expression analysis also revealed that some PHO1 family genes were significantly responsive to Pi deficiency, suggesting that PHO1 family genes play critical roles in Pi uptake and homeostasis under low Pi stress. Altogether, the characteristics of PHO1 family genes provide a reliable groundwork for further dissecting their functions in Brassica species.

CdWRKY2-mediated sucrose biosynthesis and CBF-signalling pathways coordinately contribute to cold tolerance in bermudagrass.

Bermudagrass (Cynodon dactylon) is one of the most widely cultivated warm-season turfgrass species around the world. Cold stress has been a key environmental factor that adversely affects the growth, development, and geographical distribution of bermudagrass; however, the underlying mechanism of bermudagrass responsive to cold stress remains largely unexplored. Here, we identified a cold-induced WRKY transcription factor CdWRKY2 from bermudagrass and demonstrated its function in cold stress response. Overexpression of CdWRKY2 enhanced cold tolerance in transgenic Arabidopsis and bermudagrass hairy roots, while knocking down CdWRKY2 expression via virus-induced gene silencing increased cold susceptibility. RNA sequencing showed that overexpression of CdWRKY2 in Arabidopsis activated the expression of genes involved in sucrose synthesis and metabolism, including sucrose synthase 1 (AtSUS1) and sucrose phosphate synthase 2F (AtSPS2F). CdSPS1, the homology gene of AtSPS2F in bermudagrass, was subsequently proven to be the direct target of CdWRKY2 by yeast one-hybrid, electrophoretic mobility shift assay, and transient expression analysis. As expected, overexpression of CdSPS1 conferred cold tolerance in transgenic Arabidopsis plants, whereas silencing CdSPS1 expression enhanced cold sensitivity in bermudagrass. Besides, CdCBF1 whose expression was dramatically up-regulated in CdWRKY2-overexpressing bermudagrass hairy roots but down-regulated in CdWRKY2-silencing bermudagrass both under normal and cold stress conditions was confirmed as another target of CdWRKY2. Collectively, this study reveals that CdWRKY2 is a positive regulator in cold stress by targeting CdSPS1 and CdCBF1 promoters and activating their expression to coordinately mediate sucrose biosynthesis and CBF-signalling pathway, which provides valuable information for breeding cold-resistant bermudagrass through gene manipulation.

Transcriptomic analysis of short-term heat stress response in Pinellia ternata provided novel insights into the improved thermotolerance by spermidine and melatonin.

Heat stress has been a major environmental factor limiting the growth and development of Pinellia ternata which is an important Chinese traditional medicine. It has been reported that spermidine (SPD) and melatonin (MLT) play pivotal roles in modulating heat stress response (HSR). However, the roles of SPD and MLT in HSR of P. ternata, and the potential mechanism is still unknown. Here, exogenous SPD and MLT treatments alleviated heat-induced damages in P. ternata, which was supported by the increased chlorophyll content, OJIP curve, and relative water content, and the decreased malondialdehyde and electrolyte leakage. Then, RNA sequencing between CK (control) and Heat (1 h of heat treatment) was conducted to analyze how genes were in response to short-term heat stress in P. ternata. A total of 14,243 (7870 up- and 6373 down-regulated) unigenes were differentially expressed after 1 h of heat treatment. Bioinformatics analysis revealed heat-responsive genes mainly included heat shock proteins (HSPs), ribosomal proteins, ROS-scavenging enzymes, genes involved in calcium signaling, hormone signaling transduction, photosynthesis, pathogen resistance, and transcription factors such as heat stress transcription factors (HSFs), NACs, WRKYs, and bZIPs. Among them, PtABI5, PtNAC042, PtZIP17, PtSOD1, PtHSF30, PtHSFB2b, PtERF095, PtWRKY75, PtGST1, PtHSP23.2, PtHSP70, and PtLHC1 were significantly regulated by SPD or MLT treatment with same or different trends under heat stress condition, indicating that exogenous application of MLT and SPD might enhance heat tolerance in P. ternata through regulating these genes but may with different regulatory patterns. These findings contributed to the identification of potential genes involved in short-term HSR and the improved thermotolerance by MLT and SPD in P. ternata, which provided important clues for improving thermotolerance of P. ternata.

The ARF7 and ARF19 Transcription Factors Positively Regulate PHOSPHATE STARVATION RESPONSE1 in Arabidopsis Roots.

PHOSPHATE STARVATION RESPONSE1 (PHR1) is a key regulatory component of the response to phosphate (Pi) starvation. However, the regulation of PHR1 in this response remains poorly understood. Here, we report that PHR1 is a target of the transcription factors AUXIN RESPONSE FACTOR7 (ARF7) and ARF19 and is positively regulated by auxin signaling in Arabidopsis (Arabidopsis thaliana) roots. PHR1 expression was induced by exogenous auxin and suppressed by auxin transport inhibitors in Arabidopsis roots. In the PHR1 promoter, three auxin-response elements, which are bound directly by ARF7 and ARF19, were shown to be essential for PHR1 expression. The arf7, arf19, and arf7 arf19 mutants showed down-regulated expression of PHR1 and downstream Pi starvation-induced genes in roots; they also exhibited defective Pi uptake in roots and overaccumulation of anthocyanin in shoots. The induction of lateral root formation in response to low Pi and to exogenous auxin was decreased in the phr1 mutant, whereas the expression of LATERAL ORGAN BOUNDARIES-DOMAIN16 (LBD16) and LBD29 was not changed significantly. PHR1 acted independently of LBD16 and LBD29 in the regulation of lateral root formation in response to low Pi. Under low-Pi conditions, lateral root impairment in the arf7 arf19 mutant was partially rescued by constitutive expression of PHR1, demonstrating that reduced PHR1 expression contributed to the arf7 arf19 phenotype. In addition to PHR1, other genes encoding MYB-CC members also were targets of ARF7 and ARF19. Our work thus reveals a mechanism coordinating auxin signaling and the PHR1 regulon in Arabidopsis responses to Pi deficiency.

The B3 gene family in Medicago truncatula: Genome-wide identification and the response to salt stress.

The B3 family genes constitute a pivotal group of transcription factors that assume diverse roles in the growth, development, and response to both biotic and abiotic stresses in plants. Medicago truncatula is a diploid plant with a relatively small genome, adopted as a model species for legumes genetics and functional genomic research. In this study, 173 B3 genes were identified in the M. truncatula genome, and classified into seven subgroups by phylogenetic analysis. Collinearity analysis revealed that 18 MtB3 gene pairs arose from segmented replication events. Analysis of expression patterns disclosed that 61 MtB3s exhibited a spectrum of expression profiles across various tissues and in the response to salt stress, indicating their potential involvement in salt stress signaling response. Among these genes, MtB3-53 exhibited tissue-specific differential expression and demonstrated a rapid response to salt stress induction. Overexpression of MtB3-53 gene in Arabidopsis improves salt stress tolerance by increasing plant biomass and chlorophyll content, while reducing leaf cell membrane damage. Moreover, salt treatment resulted in more up-regulation of AtABF1, AtABI3, AtHKT1, AtKIN1, AtNHX1, and AtRD29A in MtB3-53 transgenic Arabidopsis plants compared to the wild type, providing evidences that MtB3-53 enhances plant salt tolerance not only by modulating ion homeostasis but also by stimulating the production of antioxidants, which leads to the alleviation of cellular damage caused by salt stress. In conclusion, this study provides a fundamental basis for future investigations into the B3 gene family and its capacity to regulate plant responses to environmental stressors.

Transcriptomic Analysis Provides Novel Insights into the Heat Stress-Induced Response in Codonopsis tangshen.

Codonopsis tangshen Oliv (C. tangshen) is a valuable traditional Chinese medicinal herb with tremendous health benefits. However, the growth and development of C. tangshen are seriously affected by high temperatures. Therefore, understanding the molecular responses of C. tangshen to high-temperature stress is imperative to improve its thermotolerance. Here, RNA-Seq analysis was performed to investigate the genome-wide transcriptional changes in C. tangshen in response to short-term heat stress. Heat stress significantly damages membrane stability and chlorophyll biosynthesis in C. tangshen, as evidenced by pronounced malonaldehyde (MDA), electrolyte leakage (EL), and reduced chlorophyll content. Transcriptome analysis showed that 2691 differentially expressed genes (DEGs) were identified, including 1809 upregulated and 882 downregulated. Functional annotations revealed that the DEGs were mainly related to heat shock proteins (HSPs), ROS-scavenging enzymes, calcium-dependent protein kinases (CDPK), HSP-HSP network, hormone signaling transduction pathway, and transcription factors such as bHLHs, bZIPs, MYBs, WRKYs, and NACs. These heat-responsive candidate genes and TFs could significantly regulate heat stress tolerance in C. tangshen. Overall, this study could provide new insights for understanding the underlying molecular mechanisms of thermotolerance in C. tangshen.

Genome-wide evolution and expression analysis of the MYB-CC gene family in Brassica spp.

The MYB-CC family is a subtype within the MYB superfamily. This family contains an MYB domain and a predicted coiled-coil (CC) domain. Several MYB-CC transcription factors are involved in the plant's adaptability to low phosphate (Pi) stress. We identified 30, 34, and 55 MYB-CC genes in Brassica rapa, Brassica oleracea, and Brassica napus, respectively. The MYB-CC genes were divided into nine groups based on phylogenetic analysis. The analysis of the chromosome distribution and gene structure revealed that most MYB-CC genes retained the same relative position on the chromosomes and had similar gene structures during allotetraploidy. Evolutionary analysis showed that the ancestral whole-genome triplication (WGT) and the recent allopolyploidy are critical for the expansion of the MYB-CC gene family. The expression patterns of MYB-CC genes were found to be diverse in different tissues of the three Brassica species. Furthermore, the gene expression analysis under low Pi stress revealed that MYB-CC genes may be related to low Pi stress responses. These results may increase our understanding of MYB-CC gene family diversification and provide the basis for further analysis of the specific functions of MYB-CC genes in Brassica species.

Transcriptome profiling analysis reveals the role of silique in controlling seed oil content in Brassica napus.

Seed oil content is an important agronomic trait in oilseed rape. However, the molecular mechanism of oil accumulation in rapeseeds is unclear so far. In this report, RNA sequencing technique (RNA-Seq) was performed to explore differentially expressed genes in siliques of two Brassica napus lines (HFA and LFA which contain high and low oil contents in seeds, respectively) at 15 and 25 days after pollination (DAP). The RNA-Seq results showed that 65746 and 66033 genes were detected in siliques of low oil content line at 15 and 25 DAP, and 65236 and 65211 genes were detected in siliques of high oil content line at 15 and 25 DAP, respectively. By comparative analysis, the differentially expressed genes (DEGs) were identified in siliques of these lines. The DEGs were involved in multiple pathways, including metabolic pathways, biosynthesis of secondary metabolic, photosynthesis, pyruvate metabolism, fatty metabolism, glycophospholipid metabolism, and DNA binding. Also, DEGs were related to photosynthesis, starch and sugar metabolism, pyruvate metabolism, and lipid metabolism at different developmental stage, resulting in the differential oil accumulation in seeds. Furthermore, RNA-Seq and qRT-PCR data revealed that some transcription factors positively regulate seed oil content. Thus, our data provide the valuable information for further exploring the molecular mechanism of lipid biosynthesis and oil accumulation in B. nupus.