Genomic insights of the WRKY genes in kenaf (Hibiscus cannabinus L.) reveal that HcWRKY44 improves the plant's tolerance to the salinity stress.

The WRKY transcription factors (TFs) are among the most diverse TF families of plants. They are implicated in various processes related to plant growth and stress response. Kenaf (Hibiscus cannabinus L.), an important fiber crop, has many applications, including the phytoremediation of saline-alkaline soil. However, the roles of WRKY TFs in kenaf are rarely studied. In the present study, 46 kenaf WRKY genes were genome-widely identified and characterized by gene structure, phylogeny and expression pattern analysis. Furthermore, the HcWRKY44 gene was functionally characterized in Arabidopsis under salinity and drought stresses. HcWRKY44 is a nuclear-localized protein that is positively induced by salinity and drought, with roots showing maximum accumulation of its transcripts. Under NaCl and abscisic acid (ABA) stress conditions, plants overexpressing HcWRKY44 had higher germination rates, better root growth and increased survival than control plants; however, it did not improve the ability to withstand drought stress. Moreover, ABA signaling genes (ABI1, ABI2, and ABI5), ABA-responsive genes (ABF4, RD29B, COR15A, COR47, and RD22), stress-related genes (STZ, P5CS, and KIN1), and ionic homeostasis-related genes (SOS1, AHA1, AHA2, and HKT1) were positively induced in HcWRKY44 transgenic plants under NaCl treatment. These results suggest that HcWRKY44 improved plant's tolerance to salt stress but not osmotic stress through an ABA-mediated pathway. In summary, this study provides provided comprehensive information about HcWRKY genes and revealed that HcWRKY44 is involved in salinity tolerance and ABA signaling.

Integrated Methylome and Transcriptome Analyses Reveal the Molecular Mechanism by Which DNA Methylation Regulates Kenaf Flowering.

DNA methylation regulates key biological processes in plants. In this study, kenaf seedlings were pretreated with the DNA methylation inhibitor 5-azacytidine (5-azaC) (at concentrations of 0, 100, 200, 400, and 600 µM), and the results showed that pretreatment with 200 µM 5-azaC promoted flowering most effectively. To elucidate the underlying mechanism, phytohormone, adenosine triphosphate (ATP), and starch contents were determined, and genome-wide DNA methylation and transcriptome analyses were performed on anthers pretreated with 200 µM 5-azaC (5-azaC200) or with no 5-azaC (control conditions; 5-azaC0). Biochemical analysis revealed that 5-azaC pretreatment significantly reduced indoleacetic acid (IAA) and gibberellic acid (GA) contents and significantly increased abscisic acid (ABA) and ATP contents. The starch contents significantly increased in response to 200 and 600 µM 5-azaC. Further genome-wide DNA methylation analysis revealed 451 differentially methylated genes (DMGs) with 209 up- and 242 downregulated genes. Transcriptome analysis showed 3,986 differentially expressed genes (DEGs), with 2,171 up- and 1,815 downregulated genes. Integrated genome-wide DNA methylation and transcriptome analyses revealed 72 genes that were both differentially methylated and differentially expressed. These genes, which included ARFs, PP2C, starch synthase, FLC, PIF1, AGL80, and WRKY32, are involved mainly in plant hormone signal transduction, starch and sucrose metabolism, and flowering regulation and may be involved in early flowering. This study serves as a reference and theoretical basis for kenaf production and provides insights into the effects of DNA methylation on plant growth and development.

A comprehensive integrated transcriptome and metabolome analyses to reveal key genes and essential metabolic pathways involved in CMS in kenaf.

KEY MESSAGE: Numbers of critical genes and pathways were found from the levels of transcriptome and metabolome, which were useful information for understanding of kenaf CMS mechanism. Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants that leads to the inability to produce or release functional pollen. However, there is lack of comprehensive studies to reveal the molecular basis of CMS occurrence in kenaf. Herein, we performed transcriptome and UPLC-MS-based metabolome analyses in the anthers of a CMS (UG93A) and its maintainer (UG93B) to sort out essential genes and metabolites responding to CMS in kenaf. Transcriptome characterized 7769 differentially expressed genes (DEGs) between these two materials, and pathway enrichment analysis indicated that these DEGs were involved mainly in pentose and glucuronate interconversions, starch and sucrose metabolism, taurine and hypotaurine metabolism. In the metabolome assay, a total of 116 significantly different metabolites (SDMs) were identified between the CMS and its maintainer line, and these SDMs were involved in eight KEGG pathways, including flavone and flavonol biosynthesis, glycerophospholipid metabolism, flavonoid biosynthesis, glycosylphosphatidylinositol-anchor biosynthesi. Integrated analyses of transcriptome and metabolome showed that 50 genes had strong correlation coefficient values (R2 > 0.9) with ten metabolites enriched in six pathways; notably, most genes and metabolites of flavonoid biosynthesis pathways and flavone and flavonol biosynthesis pathways involved in flavonoids biosynthetic pathways were downregulated in CMS compared to those in maintainer. Taken together, the decreased accumulation of flavonoids resulted from the compromised biosynthesis pathways coupled with energy deficiency in the anthers may contribute largely to CMS in UG93A of kenaf.

The genome of kenaf (Hibiscus cannabinus L.) provides insights into bast fibre and leaf shape biogenesis.

Kenaf is an annual crop that is widely cultivated as a source of bast (phloem) fibres, the phytoremediation of heavy metal-contaminated farmlands and textile-relevant compounds. Leaf shape played a unique role in kenaf improvement, due to the inheritance as a single locus and the association with fibre development in typical lobed-leaf varieties. Here we report a high-quality genome assembly and annotation for var. 'Fuhong 952' with 1078 Mbp genome and 66 004 protein-coding genes integrating single-molecule real-time sequencing, a high-density genetic map and high-throughput chromosome conformation capture techniques. Gene mapping assists the identification of a homeobox transcription factor LATE MERISTEM IDENTITY 1 (HcLMI1) gene controlling lobed-leaf. Virus-induced gene silencing (VIGS) of HcLMI1 in a lobed-leaf variety was critical to induce round (entire)-like leaf formation. Candidate genes involved in cell wall formation were found in quantitative trait loci (QTL) for fibre yield and quality-related traits. Comparative genomic and transcriptome analyses revealed key genes involved in bast fibre formation, among which there are twice as many cellulose synthase A (CesA) genes due to a recent whole-genome duplication after divergence from Gossypium. Population genomic analysis showed two recent population bottlenecks in kenaf, suggesting domestication and improvement process have led to an increase in fibre biogenesis and yield. This chromosome-scale genome provides an important framework and toolkit for sequence-directed genetic improvement of fibre crops.

Molecular cloning and subcellular localization of six HDACs and their roles in response to salt and drought stress in kenaf (Hibiscus cannabinus L.).

BACKGROUND: Histone acetylation is an important epigenetic modification that regulates gene activity in response to stress. Histone acetylation levels are reversibly regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The imperative roles of HDACs in gene transcription, transcriptional regulation, growth and responses to stressful environment have been widely investigated in Arabidopsis. However, data regarding HDACs in kenaf crop has not been disclosed yet. RESULTS: In this study, six HDACs genes (HcHDA2, HcHDA6, HcHDA8, HcHDA9, HcHDA19, and HcSRT2) were isolated and characterized. Phylogenetic tree revealed that these HcHDACs shared high degree of sequence homology with those of Gossypium arboreum. Subcellular localization analysis showed that GFP-tagged HcHDA2 and HcHDA8 were predominantly localized in the nucleus, HcHDA6 and HcHDA19 in nucleus and cytosol. The HcHDA9 was found in both nucleus and plasma membranes. Real-time quantitative PCR showed that the six HcHDACs genes were expressed with distinct expression patterns across plant tissues. Furthermore, we determined differential accumulation of HcHDACs transcripts under salt and drought treatments, indicating that these enzymes may participate in the biological process under stress in kenaf. Finally, we showed that the levels of histone H3 and H4 acetylation were modulated by salt and drought stress in kenaf. CONCLUSIONS: We have isolated and characterized six HDACs genes from kenaf. These data showed that HDACs are imperative players for growth and development as well abiotic stress responses in kenaf.

Molecular cloning and subcellular localization of six HDACs and their roles in response to salt and drought stress in kenaf (Hibiscus cannabinus L)

BACKGROUND: Histone acetylation is an important epigenetic modification that regulates gene activity in response to stress. Histone acetylation levels are reversibly regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The imperative roles of HDACs in gene transcription, transcriptional regulation, growth and responses to stressful environment have been widely investigated in Arabidopsis. However, data regarding HDACs in kenaf crop has not been disclosed yet. RESULTS: In this study, six HDACs genes (HcHDA2, HcHDA6, HcHDA8, HcHDA9, HcHDA19, and HcSRT2) were isolated and characterized. Phylogenetic tree revealed that these HcHDACs shared high degree of sequence homology with those of Gossypium arboreum. Subcellular localization analysis showed that GFP-tagged HcHDA2 and HcHDA8 were predominantly localized in the nucleus, HcHDA6 and HcHDA19 in nucleus and cytosol. The HcHDA9 was found in both nucleus and plasma membranes. Real-time quantitative PCR showed that the six HcHDACs genes were expressed with distinct expression patterns across plant tissues. Furthermore, we determined differential accumulation of HcHDACs transcripts under salt and drought treatments, indicating that these enzymes may participate in the biological process under stress in kenaf. Finally, we showed that the levels of histone H3 and H4 acetylation were modulated by salt and drought stress in kenaf. CONCLUSIONS: We have isolated and characterized six HDACs genes from kenaf. These data showed that HDACs are imperative players for growth and development as well abiotic stress responses in kenaf.

Emulsifying conditions and processing parameters optimisation of kenaf seed oil-in-water nanoemulsions stabilised by ternary emulsifier mixtures.

Kenaf ( Hibiscus cannabinus L.) seed oil has been proven for its multi-pharmacological benefits; however, its poor water solubility and stability have limited its industrial applications. This study was aimed to further improve the stability of pre-developed kenaf seed oil-in-water nanoemulsions by using food-grade ternary emulsifiers. The effects of emulsifier concentration (1, 5, 10, 15% w/w), homogenisation pressure (16,000, 22,000, 28,000 psi), and homogenisation cycles (three, four, five cycles) were studied to produce high stability of kenaf seed oil-in-water nanoemulsions using high pressure homogeniser. Generally, results showed that the emulsifier concentration and homogenisation conditions had great effect ( p < 0.05) on the particle sizes, polydispersity index and hence the physical stability of nanoemulsions. Homogenisation parameters at 28,000 psi for three cycles produced the most stable homogeneous nanoemulsions that were below 130 nm, below 0.16, and above -40 mV of particle size, polydispersity index, and zeta potential, respectively. Field emission scanning electron microscopy micrograph showed that the optimised nanoemulsions had a good distribution within nano-range. The optimised nanoemulsions were proved to be physically stable for up to six weeks of storage at room temperature. The results from this study also provided valuable information in producing stable kenaf seed oil nanoemulsions for the future application in food and nutraceutical industries.

Reference Gene Selection for qRT-PCR Normalization Analysis in kenaf (Hibiscus cannabinus L.) under Abiotic Stress and Hormonal Stimuli.

Kenaf (Hibiscus cannabinus L.), an environmental friendly and economic fiber crop, has a certain tolerance to abiotic stresses. Identification of reliable reference genes for transcript normalization of stress responsive genes expression by quantitative real-time PCR (qRT-PCR) is important for exploring the molecular mechanisms of plants response to abiotic stresses. In this study, nine candidate reference genes were cloned, and their expression stabilities were assessed in 132 abiotic stress and hormonal stimuli samples of kenaf using geNorm, NormFinder, and BestKeeper algorithms. Results revealed that HcPP2A (Protein phosphatase 2A) and HcACT7 (Actin 7) were the optimum reference genes across all samples; HcUBC (Ubiquitin-conjugating enzyme like protein) was the worst reference gene for transcript normalization. The reliability of the selected reference genes was further confirmed by evaluating the expression profile of HcWRKY28 gene at different stress durations. This work will benefit future studies on discovery of stress-tolerance genes and stress-signaling pathways in this important fiber crop.

Reference genes selection for transcript normalization in kenaf (Hibiscus cannabinus L.) under salinity and drought stress.

Kenaf (Hibiscus cannabinus) is an economic and ecological fiber crop but suffers severe losses in fiber yield and quality under the stressful conditions of excess salinity and drought. To explore the mechanisms by which kenaf responds to excess salinity and drought, gene expression was performed at the transcriptomic level using RNA-seq. Thus, it is crucial to have a suitable set of reference genes to normalize target gene expression in kenaf under different conditions using real-time quantitative reverse transcription-PCR (qRT-PCR). In this study, we selected 10 candidate reference genes from the kenaf transcriptome and assessed their expression stabilities by qRT-PCR in 14 NaCl- and PEG-treated samples using geNorm, NormFinder, and BestKeeper. The results indicated that TUBα and 18S rRNA were the optimum reference genes under conditions of excess salinity and drought in kenaf. Moreover, TUBα and 18S rRNA were used singly or in combination as reference genes to validate the expression levels of WRKY28 and WRKY32 in NaCl- and PEG-treated samples by qRT-PCR. The results further proved the reliability of the two selected reference genes. This work will benefit future studies on gene expression and lead to a better understanding of responses to excess salinity and drought in kenaf.

Expression of kenaf mitochondrial chimeric genes HM184 causes male sterility in transgenic tobacco plants.

Chimeric genes resulting from the rearrangement of a mitochondrial genome were generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). In the study, earlier we reported that identifying a 47 bp deletion at 3'- flanking of atp9 that was linked to male sterile cytoplasm in kenaf. The truncated fragment was fused with atp9, a mitochondrial transit signal (MTS) and/or GFP, comprised two chimeric genes MTS-HM184-GFP and MTS-HM184. The plant expression vector pBI121 containing chimeric genes were then introduced to tobacco plants by Agrobacterium-mediated T-DNA transformation. The result showed that certain transgenic plants were male sterility or semi-sterility, while some were not. The expression analysis further demonstrated that higher level of expression were showed in the sterility plants, while no expression or less expression in fertility plants, the levels of expression of semi-sterility were in between. And the sterile plant (containing MTS-HM184-GFP) had abnormal anther produced malformed/shriveled pollen grains stained negative that failed to germinate (0%), the corresponding fruits was shrunken, the semi-sterile plants having normal anther shape produced about 10-50% normal pollen grains, the corresponding fruits were not full, and the germination rate was 58%. Meanwhile these transgenic plants which altered on fertility were further analyzed in phenotype. As a result, the metamorphosis leaves were observed in the seedling stage, the plant height of transgenic plants was shorter than wild type. The growth duration of transgenic tobacco was delayed 30-45 days compared to the wild type. The copy numbers of target genes of transgenic tobacco were analyzed using the real-time quantitative method. The results showed that these transgenic plants targeting-expression in mitochondrial containing MTS-HM184-GFP had 1 copy and 2 copies, the other two plants containing MTS-HM184 both had 3 copies, but 0 copy in wild type. In summary, the two manual chimeric genes might be related to male sterility in kenaf.