Comprehensive genome-wide analysis of the DREB gene family in Moso bamboo (Phyllostachys edulis): evidence for the role of PeDREB28 in plant abiotic stress response.

Dehydration response element binding (DREB) proteins are vital for plant abiotic stress responses, but the understanding of DREBs in bamboo, an important sustainable non-timber forest product, is limited. Here we conducted a comprehensive genome-wide analysis of the DREB gene family in Moso bamboo, representing the most important running bamboo species in Asia. In total, 44 PeDREBs were identified, and information on their gene structures, protein motifs, phylogenetic relationships, and stress-related cis-regulatory elements (CREs) was provided. Based on the bioinformatical analysis, we further analyzed PeDREBs from the A5 group and found that four of five PeDREB transcripts were induced by salt, drought, and cold stresses, and their proteins could bind to stress-related CREs. Among these, PeDREB28 was selected as a promising candidate for further functional characterization. PeDREB28 is localized in nucleus, has transcriptional activation activity, and could bind to the DRE- and coupling element 1- (CE1) CREs. Overexpression of PeDREB28 in Arabidopsis and bamboo improved plant abiotic stress tolerance. Transcriptomic analysis showed that broad changes due to the overexpression of PeDREB28. Furthermore, 628 genes that may act as the direct PeDREB28 downstream genes were identified by combining DAP-seq and RNA-seq analysis. Moreover, we confirmed that PeDREB28 could bind to the promoter of pyrabactin-resistance-like gene (DlaPYL3), which is a homolog of abscisic acid receptor in Arabidopsis, and activates its expression. In summary, our study provides important insights into the DREB gene family in Moso bamboo, and contributes to their functional verification and genetic engineering applications in the future.

Auxin response factors fine-tune lignin biosynthesis in response to mechanical bending in bamboo.

Lignin contributes to plant mechanical properties during bending loads. Meanwhile, phytohormone auxin controls various plant biological processes. However, the mechanism of auxin's role in bending-induced lignin biosynthesis was unclear, especially in bamboo, celebrated for its excellent deformation stability. Here, we reported that auxin response factors (ARF) 3 and ARF6 from Moso bamboo (Phyllostachys edulis (Carrière) J. Houz) directly regulate lignin biosynthesis pathway genes, and affect lignin biosynthesis in bamboo. Auxin and lignin exhibited asymmetric distribution patterns, and auxin promoted lignin biosynthesis in response to bending loads in bamboo. Employing transcriptomic and weighted gene co-expression network analysis approach, we discovered that expression patterns of ARF3 and ARF6 strongly correlated with lignin biosynthesis genes. ARF3 and ARF6 directly bind to the promoter regions of 4-coumarate: coenzyme A ligase (4CL3, 4CL7, and 4CL9) or caffeoyl-CoA O-methyltransferase (CCoAOMT2) genes, pivotal to lignin biosynthesis, and activate their expressions. Notably, the efficacy of this binding hinges on auxin levels. Alternation of the expressions of ARF3 and ARF6 substantially altered lignin accumulation in transgenic bamboo. Collectively, our study shed light on bamboo lignification genetics. Auxin signaling could directly modulate lignin biosynthesis genes to impact plant lignin content.

The transcriptional dynamics during de novo shoot organogenesis of Ma bamboo (Dendrocalamus latiflorus Munro): implication of the contributions of the abiotic stress response in this process.

De novo shoot organogenesis is an important biotechnological tool for fundamental studies in plant. However, it is difficult in most bamboo species, and the genetic control of this highly dynamic and complicated regeneration process remains unclear. In this study, based on an in-depth analysis at the cellular level, the shoot organogenesis from calli of Ma bamboo (Dendrocalamus latiflorus Munro) was divided into five stages. Subsequently, single-molecule long-read isoform sequencing of tissue samples pooled from all five stages was performed to generate a full-length transcript landscape. A total of 83 971 transcripts, including 73 209 high-quality full-length transcripts, were captured, which served as an annotation reference for the subsequent RNA sequencing analysis. Time-course transcriptome analysis of samples at the abovementioned five stages was conducted to investigate the global gene expression atlas showing genome-wide expression of transcripts during the course of bamboo shoot organogenesis. K-means clustering analysis and stage-specific transcript identification revealed important dynamically expressed transcription regulators that function in bamboo shoot organogenesis. The majority of abiotic stress-responsive genes altered their expression levels during this process, and further experiments demonstrated that exogenous application of moderate but not severe abiotic stress increased the shoot regeneration efficiency. In summary, our study provides an overview of the genetic flow dynamics during bamboo shoot organogenesis. Full-length cDNA sequences generated in this study can serve as a valuable resource for fundamental and applied research in bamboo in the future.

Production of purple Ma bamboo (Dendrocalamus latiflorus Munro) with enhanced drought and cold stress tolerance by engineering anthocyanin biosynthesis.

MAIN CONCLUSION: Overexpression of the leaf color (Lc) gene in Ma bamboo substantially increased the accumulation level of anthocyanin, and improved plant tolerance to cold and drought stresses, probably due to the increased antioxidant capacity. Most bamboos, including Ma bamboo (Dendrocalamus latiflorus Munro), are naturally evergreen and sensitive to cold and drought stresses, while it's nearly impossible to make improvements through conventual breeding due to their long and irregular flowering habit. Moreover, few studies have reported bamboo germplasm innovation through genetic engineering as bamboo genetic transformation remains difficult. In this study, we have upregulated anthocyanin biosynthesis in Ma bamboo, to generate non-green Ma bamboo with increased abiotic stress tolerance. By overexpressing the maize Lc gene, a bHLH transcription activator involved in the anthocyanin biosynthesis in Ma bamboo, we generated purple bamboos with increased anthocyanin levels including cyanidin-3-O-rutinoside, peonidin 3-O-rutinoside, and an unknown cyanidin pentaglycoside derivative. The expression levels of 9 anthocyanin biosynthesis genes were up-regulated. Overexpression of the Lc gene improved the plant tolerance to cold and drought stress, probably due to increased antioxidant capacity. The levels of the cold- and drought-related phytohormone jasmonic acid in the transgenic plants were also enhanced, which may also contribute to the plant stress-tolerant phenotypes. High anthocyanin accumulation level did not affect plant growth. Transcriptomic analysis showed higher expressions of genes involved in the flavonoid pathway in Lc transgenic bamboos compared with those in wild-type ones. The anthocyanin-rich bamboos generated here provide an example of ornamental and multiple agronomic trait improvements by genetic engineering in this important grass species.

Genome-wide analysis and transcriptomic profiling of the auxin biosynthesis, transport and signaling family genes in moso bamboo (Phyllostachys heterocycla).

BACKGROUND: Auxin is essential for plant growth and development. Although substantial progress has been made in understanding auxin pathways in model plants such as Arabidopsis and rice, little is known in moso bamboo which is famous for its fast growth resulting from the rapid cell elongation and division. RESULTS: Here we showed that exogenous auxin has strong effects on crown and primary roots. Genes involved in auxin action, including 13 YUCCA (YUC) genes involved in auxin synthesis, 14 PIN-FORMED/PIN-like (PIN/PILS) and 7 AUXIN1/LIKE-AUX1 (AUX1/LAX) members involved in auxin transport, 10 auxin receptors (AFB) involved in auxin perception, 43 auxin/indole-3-aceticacid (AUX/IAA) genes, and 41 auxin response factors (ARF) involved in auxin signaling were identified through genome-wide analysis. Phylogenetic analysis of these genes from Arabidopsis, Oryza sativa and bamboo revealed that auxin biosynthesis, transport, and signaling pathways are conserved in these species. A comprehensive study of auxin-responsive genes using RNA sequencing technology was performed, and the results also supported that moso bamboo shared a conserved regulatory mechanism for the expression of auxin pathway genes; meanwhile it harbors its own specific properties. CONCLUSIONS: In summary, we generated an overview of the auxin pathway in bamboo, which provides information for uncovering the precise roles of auxin pathway in this important species in the future.

Uncovering the miRNA-mediated regulatory network involved in Ma bamboo (Dendrocalamus latiflorus) de novo shoot organogenesis.

Bamboo is an important non-timber forest product and is well-known for its reluctance to regenerate. Recently we have established a de novo shoot organogenesis (DNSO) protocol in Ma bamboo (Dendrocalamus latiflorus) and revealed the transcriptomic dynamics during Ma bamboo regeneration, which suggested the potential roles of Ma bamboo microRNAs (DlamiRNAs) in this process. However, how DlamiRNAs regulate bamboo DNSO is poorly understood. Here we performed integrated analysis with sRNAome, degradome, and transcriptome sequencing by using samples covering the four stages of the bamboo DNSO process. A total of 727 DlamiRNAs showed differential expression during the bamboo DNSO process, and the core DlamiRNA-DlamRNA- mediated regulatory networks for bamboo DNSO were constructed. Based on the results, DlamiR156 was selected for further functional characterization of its potential roles in bamboo DNSO. Transgenic bamboos with increased DlamiR156 levels exhibited an enhancement in their regeneration efficiency. Conversely, when DlamiR156 levels were downregulated, the regeneration efficiencies of transgenic bamboos decreased. Our findings show that the DlamiRNA-mediated regulatory pathways are significant in the process of bamboo regeneration and will contribute to our understanding of the molecular mechanisms governing plant organogenesis in a more comprehensive manner.

The impact of genetic modified Ma bamboo on soil microbiome.

Evaluating the potential alteration of microbial communities is a vital step for biosafety of genetic modified plants. Recently, we have produced genetic modified Ma bamboo with increased cold and drought tolerance by anthocyanin accumulation. In this work, we aim to study the potential effects on microbial communities in rhizosphere soils during the cultivation of genetic modified bamboo. Rhizosphere and surrounding soil were collected at 3-month post-transplant. The amplicon (16S rDNA and ITS1) were sequenced for analysis of bacterial and fungal communities. Multiple software and database (Picrust2, FAPROTAX and FUNGulid) were applied to predict and compare the microbial functions involving basic metabolisms, nitrogen usage and presence of plant pathogens. There were no substantial change of the structure and abundance of rhizosphere soil microbial communities between genetic modified and wild type bamboo. For the surrounding soil, the bacterial biota α-diversity increased (chao1: 1,001 ± 80-1,276 ± 84, observed species: 787 ± 52-1,194 ± 137, PD whole tree: 75 ± 4-117 ± 18) and fungal biota α-diversity decreased (chao1: 187 ± 18-145 ± 10) in samples of genetic modified bamboo compared to those of wild type bamboo. The microbiota predicted functions did not change or had no negative alteration between genetic modified and wild type bamboo, in both rhizosphere and surrounding soils. As a conclusion, the growth of genetic modified bamboo had no substantial change on rhizosphere soil microbial communities, while minor alteration on bamboo surrounding soil microbial communities with no harmful effects. Moreover, the genetic modified bamboo had no negative effect on the predicted functions of microbiota in soil.

Overexpression of a Novel Arabidopsis Gene SUPA Leads to Various Morphological and Abiotic Stress Tolerance Alternations in Arabidopsis and Poplar.

With the development of sequencing technology, the availability of genome data is rapidly increasing, while functional annotation of genes largely lags behind. In Arabidopsis, the functions of nearly half of the proteins are unknown and this remains one of the main challenges in current biological research. In an attempt to identify novel and rapid abiotic stress responsive genes, a number of salt-up (SUP) regulated genes were isolated by analyzing the public transcriptomic data, and one of them, SUPA, was characterized in this study. The expression of SUPA transcripts was rapidly up-regulated by various abiotic stress factors (<15 min), and SUPA protein is mainly localized in the peroxisome. Overexpression of SUPA in Arabidopsis leads to the elevated accumulation of reactive oxygen species (ROS), strong morphological changes and alternations in abiotic stress tolerance. The transcriptome analysis showed changes in expression of genes involved in stress response and plant development. Interestingly, ectopic overexpression of SUPA in poplar leads to a dwarf phenotype with severely curved leaves and changes in the plant tolerance of abiotic stresses. Our study reinforces the potential roles of SUPA in normal plant growth and the abiotic stress response.

An Efficient Plant Regeneration and Transformation System of Ma Bamboo (Dendrocalamus latiflorus Munro) Started from Young Shoot as Explant.

Genetic engineering technology has been successfully used in many plant species, but is limited in woody plants, especially in bamboos. Ma bamboo (Dendrocalamus latiflorus Munro) is one of the most important bamboo species in Asia, and its genetic improvement was largely restricted by the lack of an efficient regeneration and transformation method. Here we reported a plantlet regeneration and Agrobacterium-mediated transformation protocol by using Ma bamboo young shoots as explants. Under our optimized conditions, embryogenic calluses were successfully induced from the excised young shoots on callus induction medium and rapidly grew on callus multiplication medium. Shoots and roots were regenerated on shoot induction medium and root induction medium, respectively, with high efficiency. An Agrobacterium-mediated genetic transformation protocol of Ma bamboo was established, verified by PCR and GUS staining. Furthermore, the maize Lc gene under the control of the ubiquitin promoter was successfully introduced into Ma bamboo genome and generated an anthocyanin over-accumulation phenotype. Our methods established here will facilitate the basic research as well as genetic breeding of this important bamboo species. Key achievements: A stable and high efficiency regeneration and Agrobacterium-mediated transformation protocol for Ma bamboo from vegetative organ is established.