GhmiR858 Inhibits the Accumulation of Proanthocyanidins by Targeting GhTT2L in Cotton (Gossypium hirsutum).

Proanthocyanidins (PAs) are predominantly regulated at the transcriptional level by sophisticated regulatory networks. In cotton, the role of miRNAs as key regulatory factors at the post-transcriptional level is still unclear. Here, we demonstrated that GhmiR858 negatively regulates PA accumulation in cotton leaves and calli by targeting GhTT2L. Excessive expression of GhmiR858 restrained the expression of GhTT2L, resulting in a significant decrease in PA abundance. Conversely, a reduction in GhmiR858 activity upregulated GhTT2L, which increased PA accumulation. Additionally, GhTT2L was found to positively regulate PA accumulation in both cotton and Arabidopsis. Further analyses showed that GhTT2L interacted with transcription factor GhTTG1, which directly binds to the GhANR promoter, to facilitate its transcription. This study provides new information to guide future studies of the PA regulatory mechanisms affected by miRNAs as well as the breeding of novel varieties of colored cotton with rich PAs.

Cloning and functional analysis of GhDFR1, a key gene of flavonoid synthesis pathway in naturally colored cotton.

BACKGROUND: The naturally colored brown cotton fiber is the most widely used environmentally friendly textile material, which primarily contains proanthocyanidins and their derivatives. Many structural genes in the flavonoid synthesis pathway are known to improve the genetic resources of naturally colored cotton. Among them, DFR is a crucial late enzyme to synthesis both anthocyanins and proanthocyanidins in the plant flavonoid pathway. METHODS: The protein sequences of GhDFRs were analyzed using bioinformatic tools. The expression levels of GhDFRs in various tissues and organs of upland cotton Zongxu1 (ZX1), were analyzed by quantitative real-time PCR, and the expression pattern of GhDFR1 during fiber development of white cotton and brown cotton was analyzed further. The function of GhDFR1 in NCC ZX1 was preliminarily analyzed by virus induced gene silencing (VIGS) technology. RESULTS: Bioinformatic analysis revealed that GhDFRs sequences in upland cotton genome were extremely conserved. Furthermore, evolutionary tree analysis revealed that the functions of GhDFR1 and GhDFR2, and GhDFR3 and GhDFR4, presented different and shared some similarities. Our study showed GhDFR1 and GhDFR2 were specifically expressed in fibers, while GhDFR3 and GhDFR4 were specifically expressed in petals. GhDFR1 was exclusively expressed in brown cotton fiber at various stages of development and progressively increased with the growth of fiber, but the trend of expression in white cotton was quite the opposite. We silenced GhDFR1 expression in brown cotton fiber using VIGS technology, and observed the VIGS-interference plants. After reducing the expression level of GhDFR1, the period for significant GhDFR1 expression in the developing fibers changed, reducing the content of anthocyanins, and lightening the color of mature cotton fibers. CONCLUSION: GhDFR1 was preferentially expressed in brown cotton during fiber development. The timing of GhDFR1 expression for flavonoid synthesis altered, resulting in anthocyanin contents reduced and the fiber color of the GhDFR1i lines lightened. These findings showed the role of GhDFR1 in fiber coloration of NCC and provided a new candidate for NCC genetic improvement.

Cloning and characterization of the MYB transcription factor gene GhTT2 in Gossypium hirsutum.

As one of the important secondary metabolites, proanthocyanidins (PAs) are not only a defense mechanism for plants to cope with biotic and abiotic stresses, but also a key factor affecting the development and quality of plants. Although the biosynthetic and metabolic pathways of proanthocyanidins have been basically clarified in the model plants, the regulatory mechanism in cotton has not been fully elucidated. In this work, a transcription factor gene GhTT2 (transparent testa 2) was cloned from Gossypium hirsutum. Its gene structure, expression pattern, subcellular localization, and function were further analyzed. The results show that the GhTT2 has a typical MYB domain and is predominantly expressed in fibers. Its transcription level was negatively correlated with anthocyanin content. The GhTT2-GFP fusion protein is located in the nucleus. Moreover, yeast transformation results show that GhTT2 has obvious transcriptional activation characteristics. Furthermore, the content of proanthocyanidins in GhTT2-silenced cottons is significantly reduced, indicating that GhTT2 may be involved in regulation of the proanthocyanidins biosynthesis in Gossypium hirsutum. These results provide a reference for further elucidating the molecular mechanisms of MYB transcription factors involved in the regulation of the biosynthetic pathway of PAs.

Genome-Wide Identification of the NAC Transcription Factors in Gossypium hirsutum and Analysis of Their Responses to Verticillium wilt.

The NAC transcription factors (NACs) are among the largest plant-specific gene regulators and play essential roles in the transcriptional regulation of both biotic and abiotic stress responses. Verticillium wilt of cotton caused by Verticillium dahliae (V. dahliae) is a destructive soil-borne disease that severely decreases cotton yield and quality. Although NACs constitute a large family in upland cotton (G. hirsutum L.), there is little systematic investigation of the NACs' responsive to V. dahliae that has been reported. To further explore the key NACs in response to V. dahliae resistance and obtain a better comprehension of the molecular basis of the V. dahliae stress response in cotton, a genome-wide survey was performed in this study. To investigate the roles of GhNACs under V. dahliae induction in upland cotton, mRNA libraries were constructed from mocked and infected roots of upland cotton cultivars with the V. dahliae-sensitive cultivar "Jimian 11" (J11) and V. dahliae-tolerant cultivar "Zhongzhimian 2" (Z2). A total of 271 GhNACs were identified. Genome analysis showed GhNACs phylogenetically classified into 12 subfamilies and distributed across 26 chromosomes and 20 scaffolds. A comparative transcriptome analysis revealed 54 GhNACs were differentially expressed under V. dahliae stress, suggesting a potential role of these GhNACs in disease response. Additionally, one NAC090 homolog, GhNAC204, could be a positive regulator of cotton resistance to V. dahliae infection. These results give insight into the GhNAC gene family, identify GhNACs' responsiveness to V. dahliae infection, and provide potential molecular targets for future studies for improving V. dahliae resistance in cotton.

Integrative Analysis of Expression Profiles of mRNA and MicroRNA Provides Insights of Cotton Response to Verticillium dahliae.

Cotton Verticillium wilt, caused by the notorious fungal phytopathogen Verticillium dahliae (V. dahliae), is a destructive soil-borne vascular disease and severely decreases cotton yield and quality worldwide. Transcriptional and post-transcriptional regulation of genes responsive to V. dahliae are crucial for V. dahliae tolerance in plants. However, the specific microRNAs (miRNAs) and the miRNA/target gene crosstalk involved in cotton resistance to Verticillium wilt remain largely limited. To investigate the roles of regulatory RNAs under V. dahliae induction in upland cotton, mRNA and small RNA libraries were constructed from mocked and infected roots of two upland cotton cultivars with the V. dahliae-sensitive cultivar Jimian 11 (J11) and the V. dahliae-tolerant cultivar Zhongzhimian 2 (Z2). A comparative transcriptome analysis revealed 8330 transcripts were differentially expressed under V. dahliae stress and associated with several specific biological processes. Moreover, small RNA sequencing identified a total of 383 miRNAs, including 330 unique conserved miRNAs and 53 novel miRNAs. Analysis of the regulatory network involved in the response to V. dahliae stress revealed 31 differentially expressed miRNA−mRNA pairs, and the up-regulation of GhmiR395 and down-regulation of GhmiR165 were possibly involved in the response to V. dahliae by regulating sulfur assimilation through the GhmiR395-APS1/3 module and the establishment of the vascular pattern and secondary cell wall formation through GhmiR165-REV module, respectively. The integrative analysis of mRNA and miRNA expression profiles from upland cotton lays the foundation for further investigation of regulatory mechanisms of resistance to Verticillium wilt in cotton and other crops.

Dendrobium nobile Alkaloids Protects against H2O2-Induced Neuronal Injury by Suppressing JAK-STATs Pathway Activation in N2A Cells.

The purpose of this study was to investigate the preventive effect and mechanism of Dendrobium alkaloids (DNLA) on oxidative stress-related death in neuronal cells. Our results demonstrated that DNLA has a direct neuroprotective effect through oxidative stress in N2A cells induced by hydrogen peroxide (H2O2). CCK8, lactate dehydrogenase (LDH), intracellular Ca2+, intracellular reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were used to evaluate the mechanism of DNLA neutralization by H2O2-induced injury. Results presented in the paper indicate that treatment with DNLA (35 ng/mL) significantly attenuated decreases in cell viability, release of LDH, and apoptosis after H2O2-induced neuronal injury. Furthermore, DNLA significantly reduced intracellular Ca2+ up-regulation, ROS production, and inhibited mitochondrial depolarization. Moreover, DNLA treatment significantly downregulated expressions of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-6, nitric oxide synthase, janus kinase-signal transducer and activators of transcription (JAK-STATs) signaling in N2A cells, all of which were H2O2-induced. Taken together, our findings suggested that DNLA may inhibit the expression of pro-inflammatory and pro-apoptotic factors by blocking JAK-STATs signaling after oxidative stress injury. This research provides a potential experimental basis for further application of DNLA to prevent various human nervous system diseases caused by oxidative stress.