Integrated metabolomics and transcriptomics reveal that HhERF9 positively regulates salt tolerance in Hibiscus hamabo Siebold & Zuccarini.

Hibiscus hamabo Siebold & Zuccarini is one of the few semi-mangrove plants in the genus Hibiscus that can survive in saline-alkali soil and flooded land, but the mechanism underlying its adaptation to salt soil remains unknown. Here, to uncover this unsolved mystery, we characterized the changes in the accumulation of specific metabolites under salt stress in H. hamabo by integrating physiological, metabolic, and transcriptomic data, and found that osmotic adjustment and abscisic acid (ABA) is highly associated with the salt stress response. Further, a weighted gene co-expression network analysis was performed on the root transcriptome data, which identified three key candidate transcription factors responsive to salt stress. Among them, the expression HhERF9 was significantly upregulated under salt stress and ABA treatment and was involved in regulating the expression of genes related to the salt stress response. Further research indicated that HhERF9 enhances the accumulation of proline and soluble sugars by regulating the expression of genes such as NHX2 and P5CS. These findings provide a reference for improving H. hamabo through targeted genetic engineering and lay a theoretical foundation for its future promotion and cultivation in saline-alkali areas.

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.

ADH Gene Cloning and Identification of Flooding-Responsive Genes in Taxodium distichum (L.) Rich.

As a flooding-tolerant tree species, Taxodium distichum has been utilized in afforestation projects and proven to have important value in flooding areas. Alcohol dehydrogenase (ADH), which participates in ethanol fermentation, is essential for tolerance to the anaerobic conditions caused by flooding. In a comprehensive analysis of the ADH gene family in T. distichum, TdADHs were cloned on the basis of whole-genome sequencing, and then bioinformatic analysis, subcellular localization, and gene expression level analysis under flooding were conducted. The results show that the putative protein sequences of 15 cloned genes contained seven TdADHs and eight TdADH-like genes (one Class III ADH included) that were divided into five clades. All the sequences had an ADH_N domain, and except for TdADH-likeE2, all the other genes had an ADH_zinc_N domain. Moreover, the TdADHs in clades A, B, C, and D had a similar motif composition. Additionally, the number of TdADH amino acids ranged from 277 to 403, with an average of 370.13. Subcellular localization showed that, except for TdADH-likeD3, which was not expressed in the nucleus, the other genes were predominantly expressed in both the nucleus and cytosol. TdADH-likeC2 was significantly upregulated in all three organs (roots, stems, and leaves), and TdADHA3 was also highly upregulated under 24 h flooding treatment; the two genes might play key roles in ethanol fermentation and flooding tolerance. These findings offer a comprehensive understanding of TdADHs and could provide a foundation for the molecular breeding of T. distichum and current research on the molecular mechanisms driving flooding tolerance.

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.

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.

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.

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.

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.