Genome-Wide Identification and Analysis of MYB Transcription Factor Family in Hibiscus hamabo.

MYB transcription factors constitute one of the largest gene families in plants and play essential roles in the regulation of plant growth, responses to stress, and a wide variety of physiological and biochemical processes. In this study, 204 MYB proteins (HhMYBs) were identified in the Hibiscus hamabo Sieb. et Zucc (H. hamabo) genome and systematically analyzed based on their genomic sequence and transcriptomic data. The candidate HhMYB proteins and MYBs of Arabidopsis thaliana were divided into 28 subfamilies based on the analysis of their phylogenetic relationships and their motif patterns. Expression analysis using RNA-seq and quantitative real-time PCR (qRT-PCR) indicated that most HhMYBs are differentially regulated under drought and salt stresses. qRT-PCR analysis of seven selected HhMYBs suggested that the HhMYB family may have regulatory roles in the responses to stress and hormones. This study provides a framework for a more comprehensive analysis of the role of MYBs in the response to abiotic stress in H. hamabo.

IlAP2, an AP2/ERF Superfamily Gene, Mediates Cadmium Tolerance by Interacting with IlMT2a in Iris lactea var. chinensis.

Cadmium (Cd) stress has a major impact on ecosystems, so it is important to find suitable Cd-tolerant plants while elucidating the responsible molecular mechanism for phytoremediation to manage Cd soil contamination. Iris lactea var. chinensis is an ornamental perennial groundcover plant with strong tolerance to Cd. Previous studies found that IlAP2, an AP2/ERF superfamily gene, may be an interacting partner of the metallothionein gene IlMT2a, which plays a key role in Cd tolerance. To study the role of IlAP2 in regulating Cd tolerance in I. lactea, we analyzed its regulation function and mechanism based on a yeast two-hybrid assay, a bimolecular fluorescence complementation test, quantitative real-time PCR, transgenics and transcriptome sequencing. The results showed that IlAP2 interacts with IlMT2a and may cooperate with other transcription factors to regulate genes involved in signal transduction and plant hormones, leading to reduced Cd toxicity by hindering Cd transport. These findings provide insights into the mechanism of IlAP2-mediated stress responses to Cd and important gene resources for improving plant stress tolerance in phytoremediation.

Complete chloroplast genome sequence of Kosteletzkya pentacarpos.

Kosteletzkya pentacarpos is a promising plant being developed as a salt-tolerant biofuel crop that also the ability to fix heavy metals. Here, high-throughput sequencing technology was used to sequence and assemble the chloroplast genome of K. pentacarpos. The full length of the chloroplast genome is 161,777 bp, comprising a large single-copy region of 90,019 bp, a small single-copy region of 18,978 bp, and a pair of inverted repeats of 26,390 bp. A total of 113 genes were annotated, including 79 protein-coding, 30 transfer RNA, and 4 ribosomal RNA genes. Phylogenetic analysis based on whole chloroplast genome sequences showed that K. pentacarpos has a close relationship with Abelmoschus in Malvaceae. This study increases the available genomic information on K. pentacarpos, and provides a basis for the rational exploitation and utilization of germplasm resources.

The genome of hibiscus hamabo reveals its adaptation to saline and waterlogged habitat.

Hibiscus hamabo is a semi-mangrove species with strong tolerance to salt and waterlogging stress. However, the molecular basis and mechanisms that underlie this strong adaptability to harsh environments remain poorly understood. Here, we assembled a high-quality, chromosome-level genome of this semi-mangrove plant and analyzed its transcriptome under different stress treatments to reveal regulatory responses and mechanisms. Our analyses suggested that H. hamabo has undergone two recent successive polyploidy events, a whole-genome duplication followed by a whole-genome triplication, resulting in an unusually large gene number (107 309 genes). Comparison of the H. hamabo genome with that of its close relative Hibiscus cannabinus, which has not experienced a recent WGT, indicated that genes associated with high stress resistance have been preferentially preserved in the H. hamabo genome, suggesting an underlying association between polyploidy and stronger stress resistance. Transcriptomic data indicated that genes in the roots and leaves responded differently to stress. In roots, genes that regulate ion channels involved in biosynthetic and metabolic processes responded quickly to adjust the ion concentration and provide metabolic products to protect root cells, whereas no such rapid response was observed from genes in leaves. Using co-expression networks, potential stress resistance genes were identified for use in future functional investigations. The genome sequence, along with several transcriptome datasets, provide insights into genome evolution and the mechanism of salt and waterlogging tolerance in H. hamabo, suggesting the importance of polyploidization for environmental adaptation.

Genome-wide study of the GRAS gene family in Hibiscus hamabo Sieb. et Zucc and analysis of HhGRAS14-induced drought and salt stress tolerance in Arabidopsis.

GRAS proteins are widely distributed plant-specific transcription factors. In this study, we identified 59 GRAS proteins (HhGRASs) from the genomic and transcriptomic datasets of Hibiscus hamabo Sieb. et Zucc. These proteins were phylogenetically divided into nine subfamilies. RNA-seq analysis revealed that most HhGRASs were expressed in response to abiotic stresses. Results from quantitative real-time PCR analysis of nine selected HhGRASs suggested that HhGRAS14 was significantly upregulated under multiple abiotic stresses; therefore, this gene was selected for further study. Silencing HhGRAS14 in H. hamabo reduced the tolerance to drought and salt stress, while overexpression in Arabidopsis thaliana significantly increased the tolerance to drought and salt and reduced the sensitivity to abscisic acid (ABA). In summary, we analyzed the GRAS family of proteins in semi-mangrove plants for the first time and identified a gene that responds to drought and salt stress, which provided the basis for a comprehensive analysis of GRAS genes and insight into the abiotic stress response mechanism in H. hamabo.

Genome-Wide Identification and Characterization of NAC Family in Hibiscus hamabo Sieb. et Zucc. under Various Abiotic Stresses.

NAC transcription factor is one of the largest plant gene families, participating in the regulation of plant biological and abiotic stresses. In this study, 182 NAC proteins (HhNACs) were identified based on genomic datasets of Hibiscus hamabo Sieb. et Zucc (H. hamabo). These proteins were divided into 19 subfamilies based on their phylogenetic relationship, motif pattern, and gene structure analysis. Expression analysis with RNA-seq revealed that most HhNACs were expressed in response to drought and salt stress. Research of quantitative real-time PCR analysis of nine selected HhNACs supported the transcriptome data's dependability and suggested that HhNAC54 was significantly upregulated under multiple abiotic stresses. Overexpression of HhNAC54 in Arabidopsis thaliana (A. thaliana) significantly increased its tolerance to salt. This study provides a basis for a comprehensive analysis of NAC transcription factor and insight into the abiotic stress response mechanism in H. hamabo.

The complete chloroplast genome sequence of Hibiscus coccineus.

Hibiscus coccineus is famous for its wide geographical distribution and the showy flowers of scarlet rose mallow. It belongs to the Malvaceae family and has greatly ornamental and ecological value. In this study, high-throughput sequencing and bioinformatics technology were used to assemble the complete chloroplast genome sequence, which will provide more genomic information for studying the genetic diversity and phylogenetic relationship. The full length of chloroplast genome is 160,280 bp, composed of a large single-copy (LSC) region of 89,121 bp, a small single-copy (SSC) region of 18,673 bp, and two inverted repeats (IRs) of 26,243 bp. A total of 113 genes were annotated, including 79 protein-coding genes, 30 tRNA, and four rRNA genes. Phylogenetic tree analysis revealed that the Hibiscus coccineus is closest to Hibiscus mutabilis in the Hibiscus L.

Genome-wide Analysis of Basic Helix-Loop-Helix Family Genes and Expression Analysis in Response to Drought and Salt Stresses in Hibiscus hamabo Sieb. et Zucc.

The basic helix-loop-helix (bHLH) family of transcription factors is one of the most significant and biggest in plants. It is involved in the regulation of both growth and development, as well as stress response. Numerous members of the bHLH family have been found and characterized in woody plants in recent years. However, no systematic study of the bHLH gene family has been published for Hibiscus hamabo Sieb. et Zucc. In this research, we identified 162 bHLH proteins (HhbHLHs) from the genomic and transcriptomic datasets of H. hamabo, which were phylogenetically divided into 19 subfamilies. According to a gene structural study, the number of exon-introns in HhbHLHs varied between zero and seventeen. MEME research revealed that the majority of HhbHLH proteins contained three conserved motifs, 1, 4, and 5. The examination of promoter cis-elements revealed that the majority of HhbHLH genes had several cis-elements involved in plant growth and development and abiotic stress responses. In addition, the overexpression of HhbHLH2 increased salt and drought stress tolerance in Arabidopsis.

Selection and validation of appropriate reference genes for RT-qPCR analysis of flowering stages and different genotypes of Iris germanica L.

Iris germanica L. is a perennial herbaceous plant that has been widely cultivated worldwide and is popular for its elegant and vibrantly colorful flowers. Selection of appropriate reference genes is the prerequisite for accurate normalization of target gene expression by quantitative real-time PCR. However, to date, the most suitable reference genes for flowering stages have not been elucidated in I. germanica. In this study, eight candidate reference genes were examined for the normalization of RT-qPCR in three I. germanica cultivars, and their stability were evaluated by four different algorithms (GeNorm, NormFinder, BestKeeper, and Ref-finder). The results revealed that IgUBC and IgGAPDH were the most stable reference genes in '00246' and 'Elizabeth', and IgTUB and IgUBC showed stable expression in '2010200'. IgUBC and IgGAPDH were the most stable in all samples, while IgUBQ showed the least stability. Finally, to validate the reliability of the selected reference genes, the expression patterns of IgFT (Flowering Locus T gene) was analyzed and emphasized the importance of appropriate reference gene selection. This work presented the first systematic study of reference genes selection during flower bud development and provided guidance to research of the molecular mechanisms of flowering stages in I. germanica.

Full-Length Transcriptome Sequencing and Comparative Transcriptome Analysis to Evaluate Drought and Salt Stress in Iris lactea var. chinensis.

Iris lactea var. chinensis (I. lactea var. chinensis) is a perennial herb halophyte with salt and drought tolerance. In this study, full-length transcripts of I. lactea var. chinensis were sequenced using the PacBio RSII sequencing platform. Moreover, the transcriptome was investigated under NaCl or polyethylene glycol (PEG) stress. Approximately 30.89 G subreads were generated and 31,195 unigenes were obtained by clustering the same isoforms by the PacBio RSII platform. A total of 15,466 differentially expressed genes (DEGs) were obtained under the two stresses using the Illumina platform. Among them, 9266 and 8390 DEGs were obtained under high concentrations of NaCl and PEG, respectively. In total, 3897 DEGs with the same expression pattern under the two stresses were obtained. The transcriptome expression profiles of I. lactea var. chinensis under NaCl or PEG stress obtained in this study may provide a resource for the same and different response mechanisms against different types of abiotic stress. Furthermore, the stress-related genes found in this study can provide data for future molecular breeding.

Screening and Identification of Host Proteins Interacting with Iris lactea var. chinensis Metallothionein IlMT2a by Yeast Two-Hybrid Assay.

Iris lactea var. chinensis (Fisch.) (I. lactea var. chinensis) is a well-known cadmium (Cd)-tolerant plant and we have previously shown that the metallothionein gene, IlMT2a, of the plant may be playing a key role in conferring the Cd tolerance. In this study, we have identified several proteins interacting with the IlMT2a by screening yeast two-hybrid library constructed from cDNAs isolated from Cd-treated I. lacteal var. chinensis plants. Putative functions of these proteins include those involved in photosynthesis, ROS scavenge, nutrient transport, and transcriptional regulation, to name a few. In particular, another metallothionein, which we assigned the name of IlMT3, was identified as an interacting partner of the IlMT2a. Unlike IlMT2a, it did not provide any significant protection against Cd toxicity in transgenic Arabidopsis thaliana L. (A. thaliana). To our knowledge, this is the first time ever reporting the interaction of two metallothionein proteins in plants. Learning the biological significance of the interaction between IlMT2a and IlMT3 would be the focus of future study and would be able to provide valuable insights into the understanding plant metallothionein's diverse and complex roles in coordinating many important cellular physiologies including stress responses, gene regulations, and energy metabolisms.

Transcriptome Analysis of Salt Stress in Hibiscus hamabo Sieb. et Zucc Based on Pacbio Full-Length Transcriptome Sequencing.

Hibiscus hamabo Sieb. et Zucc is an important semi-mangrove plant with great morphological features and strong salt resistance. In this study, by combining single molecule real time and next-generation sequencing technologies, we explored the transcriptomic changes in the roots of salt stressed H. hamabo. A total of 94,562 unigenes were obtained by clustering the same isoforms using the PacBio RSII platform, and 2269 differentially expressed genes were obtained under salt stress using the Illumina platform. There were 519 differentially expressed genes co-expressed at each treatment time point under salt stress, and these genes were found to be enriched in ion signal transduction and plant hormone signal transduction. We used Arabidopsis thaliana (L.) Heynh. transformation to confirm the function of the HhWRKY79 gene and discovered that overexpression enhanced salt tolerance. The full-length transcripts generated in this study provide a full characterization of the transcriptome of H. hamabo and may be useful in mining new salt stress-related genes specific to this species, while facilitating the understanding of the salt tolerance mechanisms.

Quantitative proteomic analysis reveals complex regulatory and metabolic response of Iris lactea Pall. var. chinensis to cadmium toxicity.

Cadmium pollution has become a serious environmental problem. Iris lactea var. chinensis showed strong Cd tolerance and accumulation ability, which has significant potential to be applied for the phytoremediation of Cd-contaminated soil. However, the lack of molecular information on the mechanism of I. lactea response to Cd limited the improvement of phytoremediation efficiency. In this study, label-free proteomics analysis of Cd response in I. lactea showed that there were 163 and 196 differentially expressed proteins (DEPs) in the shoots and roots, respectively. Bioinformatics analysis indicated the DEPs responding to Cd stress mainly involved in signal transduction, ion transport, redox etc., and participate in the pathway of amino acid biosynthesis, lignin biosynthesis, glycerolipid metabolism and glutathione metabolism. Besides, differential expression of seven DEPs was validated via gene expression analysis. Finally, we found that a Cd-induced mannose-specific lectin (IlMSL) from I. lactea enhanced the Cd sensitivity and increased Cd accumulation in yeast. The results of this study will enhance our understanding of the molecular mechanism of Cd tolerance and accumulation in I. lactea and ultimately provide valuable resources for using Cd tolerant genes for developing efficient strategies for phytoremediation of Cd-contaminated soils or limiting Cd accumulation in food crops.

Phylogenetic and Transcription Analysis of Hibiscus hamabo Sieb. et Zucc. WRKY Transcription Factors.

WRKY transcription factors are known to play important roles in the regulation of various aspects of plant growth and development, including germination, stress resistance, and senescence. Nevertheless, there is little information about the WRKY genes in Hibiscus hamabo Sieb. et Zucc., an important semimangrove plant. In this study, HhWRKY genes in H. hamabo were identificated based on Illumina RNA-sequencing and isoform sequencing from salt-treated roots. Then phylogenetic analysis and conserved motif analysis of the WRKY family in H. hamabo and Arabidopsis thaliana were used to classify WRKY genes. Sixteen HhWRKY genes were selected from different groups to detect their expression patterns using real-time quantitative PCR in different organ (root, old leaf, tender leaf, receptacle, petal, or stamen) from 10-year-old H. hamabo plants grown in their natural environment and in seedlings with 8 to 10 true leaves challenged by phytohormone (salicylic acid, methyl jasmonate, or abscisic acid) and abiotic stress (drought, salt, or high temperature). As a result, the identified 78 HhWRKY genes were divided into two major groups and several subgroups based on their structural and phylogenetic features. Most transcripts of the selected 16 HhWRKY genes were more abundant in old than in tender leaves of H. hamabo. HhWRKY genes were regulated in reaction to abiotic stresses and phytohormone treatments and may participate in signaling networks to improve plant stress resistance. Some of HhWRKY genes behaved as would be predicted based on their homology with A. thaliana WRKY genes, but others showed divergent behavior. This systematic analysis lays the foundation for further identification of WRKY gene functions, with the aim of improving woody plants.

The SrWRKY71 transcription factor negatively regulates SrUGT76G1 expression in Stevia rebaudiana.

SrUGT76G1 is vital for the biosynthesis of rebaudioside A, D and M in Stevia rebaudiana Bertoni; however, its transcriptional regulatory mechanism remains unknown. In this study, the 2050-bp promoter region of SrUGT76G1 was isolated by the TAIL-PCR method, and sequence analysis revealed the presence of several W-box cis-elements, which are the recognition motifs of WRKY transcription factors. Furthermore, SrWRKY71, characterized by a typical WRKY domain and a C2H2 zinc finger-like motif, was identified as a putative transcriptional regulator of SrUGT76G1. The transcript of SrWRKY71 predominantly accumulated in leaves and was present at a lower level in stems, roots and flowers. The SrWRKY71-GFP fusion protein was specifically localized to the nucleus in tobacco epidermal cells. In addition, the N and C terminal regions of SrWRKY71 contributed to its transactivation activity. Y1H and EMSA assays validated that SrWRKY71 binds directly to W-box1 and W-box2 in the proximal promoter region of SrUGT76G1. Moreover, SrWRKY71 represses the expression level of SrUGT76G1 in both tobacco leaves and stevia callus. Taken together, the data in this study represent the first identification of an essential upstream transcription factor of SrUGT76G1 and provides new insight into the regulatory network of steviol glycoside biosynthesis in Stevia rebaudiana.

Efficient virus-induced gene silencing in Hibiscus hamabo Sieb. et Zucc. using tobacco rattle virus.

BACKGROUND: Hibiscus hamabo Sieb. et Zucc. is a semi-mangrove plant used for the ecological restoration of saline-alkali land, coastal afforestation and urban landscaping. The genetic transformation H. hamabo is currently inefficient and laborious, restricting gene functional studies on this species. In plants, virus-induced gene silencing provides a pathway to rapidly and effectively create targeted gene knockouts for gene functional studies. METHODS: In this study, we tested the efficiency of a tobacco rattle virus vector in silencing the cloroplastos alterados 1 (CLA1) gene through agroinfiltration. RESULTS: The leaves of H. hamabo showed white streaks typical of CLA1 gene silencing three weeks after agroinfiltration. In agroinfiltrated H. hamabo plants, the CLA1 expression levels in leaves with white streaks were all significantly lower than those in leaves from mock-infected and control plants. CONCLUSIONS: The system presented here can efficiently silence genes in H. hamabo and may be a powerful tool for large-scale reverse-genetic analyses of gene functions in H. hamabo.

An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium 'Zhongshanshan'.

Adventitious root (AR) formation from cuttings is the primary manner for the commercial vegetative propagation of trees. Cuttings is also the main method for the vegetative reproduction of Taxodium 'Zhongshanshan', while knowledge of the molecular mechanisms regulating the processes is limited. Here, we used mRNA sequencing and an isobaric tag for relative and absolute quantitation-based quantitative proteomic (iTRAQ) analysis to measure changes in gene and protein expression levels during AR formation in Taxodium 'Zhongshanshan'. Three comparison groups were established to represent the three developmental stages in the AR formation process. At the transcript level, 4743 genes showed an expression difference in the comparison groups as detected by RNA sequencing. At the protein level, 4005 proteins differed in their relative abundance levels, as indicated by the quantitative proteomic analysis. A comparison of the transcriptome and proteome data revealed regulatory aspects of metabolism during AR formation and development. In summary, hormonal signal transduction is different at different developmental stages during AR formation. Other factors related to carbohydrate and energy metabolism and protein degradation and some transcription factor activity levels, were also correlated with AR formation. Studying the identified genes and proteins will provide further insights into the molecular mechanisms controlling AR formation.

Exogenous glutathione increased lead uptake and accumulation in Iris lactea var. chinensis exposed to excess lead.

Long- and short-term hydroponic experiments were conducted to study the effect of different concentrations of exogenous glutathione (GSH) on Pb uptake, translocation, and gene expresses in Iris lactea var. chinensis exposed to excess lead (Pb). Exogenous GSH remarkedly promoted Pb uptake and translocation in long-term (14 d) experiment, and the GSH-dose-dependent increases in shoot and root Pb contents existed obviously when GSH concentrations were lower than 800 mg·L-1. The fresh weight in gradual rise in plants was observed with the increase of exogenous GSH concentration. In short-term (24 h) experiment, Pb contents in roots under Pb with L-buthionine sulfoximine (BSO, a known inhibitor of GSH biosynthesis) treatments were significantly lower than that under Pb exposure alone. The transcript levels of three genes (Ilγ-ECS, IlGS, and IlPCS) involved in GSH synthesis and metabolism, showed no significant change in expression pattern except that upregulation after 24 h of treatment with Pb and GSH in comparison with that of the single Pb treatment. Further, the level of IlGS transcript after exposure for 4 h was much higher than that of Ilγ-ECS and IlPCS transcripts. All these results obtained here suggest that exogenous GSH can increase Pb accumulation, detoxification, and translocation to the shoot.

Iris lactea var. chinensis (Fisch.) cysteine-rich gene llCDT1 enhances cadmium tolerance in yeast cells and Arabidopsis thaliana.

IlCDT1, a cysteine-rich protein, was isolated from Iris lactea var. chinensis (Fisch.) (I. lactea var. chinensis). Its transcription was up-regulated by the exogenous application of Cd. The truncated IlCDT1 (25-54) containing 14 Cys residues confers Cd tolerance to yeast as the intact IlCDT1, indicating that Cys residues are required for Cd tolerance presumably by chelating Cd. When the gene was constitutively expressed in A. thaliana, root length of transgenic lines was longer than that of wild-type under 100 µM or 200 µM Cd stress. However, Cd absorption in wild-type was more than in two trangenic lines under 100 µM Cd exposure. IlCDT1 may directly bind Cd, through chelating Cd and avoiding the Cd uptake into the cells. Together, IlCDT1 may be a promising gene for the Cd tolerance improvement. SUMMARY: Cysteine-rich gene llCDT1 enhances cadmium tolerance in yeast cells and Arabidopsis thaliana.