A bamboo 'PeSAPK4-PeMYB99-PeTIP4-3' regulatory model involved in water transport.

Water plays crucial roles in expeditious growth and osmotic stress of bamboo. Nevertheless, the molecular mechanism of water transport remains unclear. In this study, an aquaporin gene, PeTIP4-3, was identified through a joint analysis of root pressure and transcriptomic data in moso bamboo (Phyllostachys edulis). PeTIP4-3 was highly expressed in shoots, especially in the vascular bundle sheath cells. Overexpression of PeTIP4-3 could increase drought and salt tolerance in transgenic yeast and rice. A co-expression pattern of PeSAPK4, PeMYB99 and PeTIP4-3 was revealed by WGCNA. PeMYB99 exhibited an ability to independently bind to and activate PeTIP4-3, which augmented tolerance to drought and salt stress. PeSAPK4 could interact with and phosphorylate PeMYB99 in vivo and in vitro, wherein they synergistically accelerated PeTIP4-3 transcription. Overexpression of PeMYB99 and PeSAPK4 also conferred drought and salt tolerance in transgenic rice. Further ABA treatment analysis indicated that PeSAPK4 enhanced water transport in response to stress via ABA signaling. Collectively, an ABA-mediated cascade of PeSAPK4-PeMYB99-PeTIP4-3 is proposed, which governs water transport in moso bamboo.

Regulatory networks of senescence-associated gene-transcription factors promote degradation in Moso bamboo shoots.

Bamboo cultivation, particularly Moso bamboo (Phyllostachys edulis), holds significant economic importance in various regions worldwide. Bamboo shoot degradation (BSD) severely affects productivity and economic viability. However, despite its agricultural consequences, the molecular mechanisms underlying BSD remain unclear. Consequently, we explored the dynamic changes of BSD through anatomy, physiology and the transcriptome. Our findings reveal ruptured protoxylem cells, reduced cell wall thickness and the accumulation of sucrose and reactive oxygen species (ROS) during BSD. Transcriptomic analysis underscored the importance of genes related to plant hormone signal transduction, sugar metabolism and ROS homoeostasis in this process. Furthermore, BSD appears to be driven by the coexpression regulatory network of senescence-associated gene transcription factors (SAG-TFs), specifically PeSAG39, PeWRKY22 and PeWRKY75, primarily located in the protoxylem of vascular bundles. Yeast one-hybrid and dual-luciferase assays demonstrated that PeWRKY22 and PeWRKY75 activate PeSAG39 expression by binding to its promoter. This study advanced our understanding of the molecular regulatory mechanisms governing BSD, offering a valuable reference for enhancing Moso bamboo forest productivity.

Unveiling the Biological Function of Phyllostachys edulis FBA6 (PeFBA6) through the Identification of the Fructose-1,6-Bisphosphate Aldolase Gene.

Fructose-1,6-bisphosphate aldolase (FBA) is a pivotal enzyme in various metabolic pathways, including glycolysis, gluconeogenesis, and the Calvin cycle. It plays a critical role in CO2 fixation. Building on previous studies on the FBA gene family in Moso bamboo, our study revealed the biological function of PeFBA6. To identify CSN5 candidate genes, this study conducted a yeast two-hybrid library screening experiment. Subsequently, the interaction between CSN5 and PeFBA6 was verified using yeast two-hybrid and LCI experiments. This investigation uncovered evidence that FBA may undergo deubiquitination to maintain glycolytic stability. To further assess the function of PeFBA6, it was overexpressed in rice. Various parameters were determined, including the light response curve, CO2 response curve, and the levels of glucose, fructose, sucrose, and starch in the leaves of overexpressing rice. The results demonstrated that overexpressed rice exhibited a higher saturation light intensity, net photosynthetic rate, maximum carboxylation rate, respiration rate, and increased levels of glucose, fructose, and starch than wild-type rice. These findings indicated that PeFBA6 not only enhanced the photoprotection ability of rice but also improved the photosynthetic carbon metabolism. Overall, this study enhanced our understanding of the function of FBA and revealed the biological function of PeFBA6, thereby providing a foundation for the development of excellent carbon fixation bamboo varieties through breeding.

A new biotechnology for in-planta gene editing and its application in promoting flavonoid biosynthesis in bamboo leaves.

BACKGROUND: Bamboo is a perennial and renewable biomass forest resource and its leaf flavonoid is an antioxidant for biological and pharmacological research. The established genetic transformation and gene editing systems in bamboo are significantly limited by the dependence on bamboo regeneration capability. The way to improve the flavonoid content in bamboo leaves through biotechnology is still not feasible. RESULTS: Here, we developed an in-planta, Agrobacterium-mediated gene expression method for exogenous genes via wounding and vacuum in bamboo. We demonstrated that the RUBY served as a reporter efficiently expressed in bamboo leaves and shoots, albeit unable to integrate into the chromosome. We have also developed a gene editing system by creating an in situ mutant of the bamboo violaxanthin de-epoxidase (PeVDE) gene in bamboo leaves, with lower NPQ values under the fluorometer, which can serve as a native reporter for gene editing. Furthermore, the bamboo leaves with increased flavonoid content were achieved by knocking out the cinnamoyl-CoA reductase genes. CONCLUSIONS: Our method can be applied for the functional characterization of novel genes in a short time and is helpful for bamboo leaf flavonoid biotechnology breeding in the future.

Identification and characterization of CircRNA-associated CeRNA networks in moso bamboo under nitrogen stress.

BACKGROUND: Nitrogen is a macronutrient element for plant growth and development. Circular RNAs (circRNAs) serve as pivotal regulators for the coordination between nutrient supply and plant demand. Moso bamboo (Phyllostachys edulis) is an excellent plant with fast growth, and the mechanism of the circRNA-target module in response to nitrogen remains unclear. RESULTS: Deep small RNA sequencing results of moso bamboo seedlings under different concentrations of KNO3 (N0 = 0 mM, N6 = 6 mM, N18 = 18 mM) were used to identify circRNAs. A total of 549 circRNAs were obtained, of which 309 were generated from corresponding parental coding genes including 66 new ones. A total of 536 circRNA-parent genes were unevenly distributed in 24 scaffolds and were associated with root growth and development. Furthermore, 52 differentially expressed circRNAs (DECs) were obtained, including 24, 33 and 15 DECs from three comparisons of N0 vs. N6, N0 vs. N18 and N6 vs. N18, respectively. Based on integrative analyses of the identified DECs, differentially expressed mRNAs (DEGs), and miRNAs (DEMs), a competitive endogenous RNA (ceRNA) network was constructed, including five DECs, eight DEMs and 32 DEGs. A regulatory module of PeSca_6:12,316,320|12,372,905-novel_miR156-PH02Gene35622 was further verified by qPCR and dual-luciferase reporter assays. CONCLUSION: The results indicated that circRNAs could participate in multiple biological processes as miRNA sponges, including organ nitrogen compound biosynthesis and metabolic process regulation in moso bamboo. Our results provide valuable information for further study of circRNAs in moso bamboo under fluctuating nitrogen conditions.

Development of dual-visible reporter assays to determine the DNA-protein interaction.

The application of DNA-protein interaction reporter assays for relational or ratiometric measurements within an experimental system is popular in biological research. However, the existing reporter-based interaction assays always require special equipment, expensive chemicals, and a complicated operation. Here, we developed a DNA-protein interaction technology integrating two visible reporters, RUBY and UV-visible GFP (eYGFPuv), which allows the expression of the cassette reporter contained cis-acting DNA element (DE) fused upstream of TATA box and RUBY, and a constitutive promoter regulating eYGFPuv in the same construct. The interaction of transcription factor (TF) and the DE can be detected by co-expressed the cassette reporter and TF in tobacco leaves where the cassette reporter alone serves as a control. We also revealed that eight function-unknown bamboo AP2/ERFs interacted with the DE of ANT-AP2R1R2 (ABE), DRE (DBE), GCC-box (EBE), and RAV1 binding element (RBE), respectively, which are consistent with the results by dual-luciferase reporter assays. Thus, the dual-visible reporters offer a convenient, visible, and cost-saving alternative to other existing techniques for DNA-protein interaction in plants.

Identification of the 14-3-3 Gene Family in Bamboo and Characterization of Pe14-3-3b Reveals Its Potential Role in Promoting Growth.

The 14-3-3 protein family plays an important role in regulating plant growth and development. The genes of the 14-3-3 family have been reported in multiple species. However, little is known about the 14-3-3 gene family in bamboo. In this study, a total of 58 genes belonging to the 14-3-3 family were identified in three representative bamboo species, i.e., Olyra latifolia, Phyllostachys edulis, and Bonia amplexicaulis, whose encoding proteins were grouped into ε and non-ε groups by phylogeny analysis with 14-3-3 proteins from Arabidopsis thaliana and Oryza sativa. The 14-3-3s had diverse gene structures and motif characteristics among the three bamboo species. Collinearity analysis suggested that the genes of the 14-3-3 family in bamboo had undergone a strong purification selection during evolution. Tissue-specific expression analysis showed the expression of Pe14-3-3s varied in different tissues of P. edulis, suggesting that they had functional diversity during growth and development. Co-expression analysis showed that four Pe14-3-3s co-expressed positively with eight ribosomal genes. Yeast two-hybrid (Y2H) assays showed that Pe14-3-3b/d could interact with Pe_ribosome-1/5/6, and qPCR results demonstrated that Pe14-3-3b/d and Pe_ribosome-1/5/6 had similar expression trends with the increase in shoot height, which further confirmed that they would work together to participate in the shoot growth and development of bamboo. Additionally, the transgenic Arabidopsis plants overexpressing Pe14-3-3b had longer roots, a larger stem diameter, an earlier bolting time and a faster growth rate than wild-type Arabidopsis, indicating that Pe14-3-3b acted as a growth promoter. Our results provide comprehensive information on 14-3-3 genes in bamboo and highlight Pe14-3-3b as a potential target for bamboo improvement.

Identification of KFB Family in Moso Bamboo Reveals the Potential Function of PeKFB9 Involved in Stress Response and Lignin Polymerization.

The Kelch repeat F-box (KFB) protein is an important E3 ubiquitin ligase that has been demonstrated to perform an important post-translational regulatory role in plants by mediating multiple biological processes. Despite their importance, KFBs have not yet been identified and characterized in bamboo. In this study, 19 PeKFBs were identified with F-box and Kelch domains; genes encoding these PeKFBs were unevenly distributed across 12 chromosomes of moso bamboo. Phylogenetic analysis indicated that the PeKFBs were divided into eight subclades based on similar gene structures and highly conserved motifs. A tissue-specific gene expression analysis showed that the PeKFBs were differentially expressed in various tissues of moso bamboo. All the promoters of the PeKFBs contained stress-related cis-elements, which was supported by the differentially expression of PeKFBs of moso bamboo under drought and cold stresses. Sixteen proteins were screened from the moso bamboo shoots' cDNA library using PeKFB9 as a bait through a yeast two-hybrid (Y2H) assay. Moreover, PeKFB9 physically interacted with PeSKP1-like-1 and PePRX72-1, which mediated the activity of peroxidase in proteolytic turnover. Taken together, these findings improved our understanding of PeKFBs, especially in response to stresses, and laid a foundation for revealing the molecular mechanism of PeKFB9 in regulating lignin polymerization by degrading peroxidase.

Multifaceted analyses reveal carbohydrate metabolism mainly affecting the quality of postharvest bamboo shoots.

Bamboo shoot is one of nutritious vegetables in China. However, the edible quality of fresh bamboo shoots deteriorates easily after harvest. Here, morphological, physiological, transcriptomic and microRNA sequencing analyses were conducted to investigate the postharvest characteristics of moso bamboo (Phyllostachys edulis) shoots. Rapid decreases of soluble sugars, structural polysaccharides and hydrolyzed tannins, and increases of lignin and condensed tannins were observed in the postharvest bamboo shoots. Differentially expressed genes (DEGs) and miRNAs with opposite trends were mainly enriched in structural polysaccharide metabolism, starch and sucrose metabolism and glycolysis pathways, which were consistent with the changes of carbohydrates. A co-expression network of carbohydrate metabolism was constructed, which was verified by qPCR and yeast one-hybrid (Y1H) assay. Furthermore, the function of one hub glycosyltransferase gene was validated in Arabidopsis, which confirmed that it was involved in xylan biosynthesis. These results are of great significance for revealing the carbohydrate metabolism mechanisms of postharvest bamboo shoots and provide a potential candidate gene for molecular breeding related to xylan in the future.

Comprehensive Analyses of Simple Sequence Repeat (SSR) in Bamboo Genomes and Development of SSR Markers with Peroxidase Genes.

Simple sequence repeats (SSRs) are one of the most important molecular markers, which are widespread in plants. Bamboos are important forest resources worldwide. Here, the comprehensive identification and comparative analysis of SSRs were performed in three woody and two herbaceous bamboo species. Altogether 567,175 perfect SSRs and 71,141 compound SSRs were identified from 5737.8 Mb genome sequences of five bamboo species. Di-nucleotide SSRs were the most predominant type, with an average of ~50,152.2 per species. Most SSRs were located in intergenic regions, while those located in genic regions were relatively less. Moreover, the results of annotation distribution indicated that terms with P450, peroxidase and ATP-binding cassette transporter related to lignin biosynthesis might play important roles in woody and herbaceous bamboos under the mediation of SSRs. Furthermore, the peroxidase gene family consisted of a large number of genes containing SSRs was selected for the evolutionary relationship analysis and SSR markers development. Fifteen SSR markers derived from peroxidase family genes of Phyllostachys edulis were identified as polymorphic in 34 accessions belonging to seven genera in Bambusoideae. These results provided a comprehensive insight of SSR markers into bamboo genomes, which would facilitate bamboo research related to comparative genomics, evolution and marker-assisted selection.

Integrative analyses of morphology, physiology, and transcriptional expression profiling reveal miRNAs involved in culm color in bamboo.

Culm color variation is an interesting phenomenon that contributes to the breeding of new varieties of ornamental plants during domestication. De-domesticated variation is considered ideal for identifying and interpreting the molecular mechanisms of plant mutations. However, the variation in culm color of bamboo remains unknown. In the present study, yellow and green culms generated from the same rhizome of Phyllostachys vivax cv. Aureocaulis (P. vivax) were used to elucidate the molecular mechanism of culm color formation. Phenotypic and physiological data showed that environmental suitability was higher in green culms than in yellow culms. High-throughput sequencing analysis showed 295 differentially expressed genes (DEGs) and 22 differentially expressed miRNAs (DEMs) in two different colored bamboo culms. There were 103 DEM-DEG interaction pairs, of which a representative "miRNA-mRNA" regulatory module involved in photosynthesis and pigment metabolism was formed by 14 DEM-DEG pairs. The interaction of the three key pairs was validated by qPCR and dual-luciferase assays. This study provides new insights into the molecular mechanism of miRNAs involved in P. vivax culm color formation, which provides evidence for plant de-domestication and is helpful for revealing the evolutionary mechanism of bamboo.

PeVDE, a violaxanthin de-epoxidase gene from moso bamboo, confers photoprotection ability in transgenic Arabidopsis under high light.

Plants employ an array of photoprotection mechanisms to alleviate the harmful effects of high light intensity. The violaxanthin cycle, which is associated with non-photochemical quenching (NPQ), involves violaxanthin de-epoxidase (VDE), and zeaxanthin epoxidase (ZEP) and is one of the most rapid and efficient mechanisms protecting plants under high light intensity. Woody bamboo is a class of economically and ecologically important evergreen grass species widely distributed in tropical and subtropical areas. However, the function of VDE in bamboo has not yet been elucidated. In this study, we found that high light intensity increased NPQ and stimulated the de-epoxidation of violaxanthin cycle components in moso bamboo (Phyllostachys edulis), whereas, samples treated with the VDE inhibitor (dithiothreitol) exhibited lower NPQ capacity, suggesting that violaxanthin cycle plays an important role in the photoprotection of bamboo. Further analysis showed that not only high light intensity but also extreme temperatures (4 and 42°C) and drought stress upregulated the expression of PeVDE in bamboo leaves, indicating that PeVDE is induced by multiple abiotic stresses. Overexpression of PeVDE under the control of the CaMV 35S promoter in Arabidopsis mutant npq1 mutant could rescue its NPQ, indicating that PeVDE functions in dissipating the excess absorbed light energy as thermal energy in bamboo. Moreover, compared with wild-type (Col-0) plants, the transgenic plants overexpressing PeVDE displayed enhanced photoprotection ability, higher NPQ capacity, slower decline in the maximum quantum yield of photosystem II (F v /F m ) under high light intensity, and faster recovery under optimal conditions. These results suggest that PeVDE positively regulates the response to high light intensity in bamboo plants growing in the natural environment, which could improve their photoprotection ability through the violaxanthin cycle and NPQ.

An Integrated Regulatory Network of mRNAs, microRNAs, and lncRNAs Involved in Nitrogen Metabolism of Moso Bamboo.

Nitrogen is a key macronutrient essential for plant growth and development, and its availability has a strong influence on biological processes. Nitrogen fertilizer has been widely applied in bamboo forests in recent decades; however, the mechanism of nitrogen metabolism in bamboo is not fully elucidated. Here, we characterized the morphological, physiological, and transcriptome changes of moso bamboo in response to different schemes for nitrogen addition to illuminate the regulation mechanism of nitrogen metabolism. The appropriate addition of nitrogen improved the chlorophyll content and Pn (net photosynthetic rate) of leaves, the nitrogen and ammonium contents of the seedling roots, the biomass of the whole seedling, the number of lateral roots, and the activity of enzymes involved in nitrogen metabolism in the roots. Based on the whole transcriptome data of the roots, a total of 8,632 differentially expressed mRNAs (DEGs) were identified under different nitrogen additions, such as 52 nitrate transporter genes, 6 nitrate reductase genes, 2 nitrite reductase genes, 2 glutamine synthase genes, 2 glutamate synthase genes (GOGAT), 3 glutamate dehydrogenase genes, and 431 TFs belonging to 23 families. Meanwhile, 123 differentially expressed miRNAs (DEMs) and 396 differentially expressed lncRNAs (DELs) were characterized as nitrogen responsive, respectively. Furthermore, 94 DEM-DEG pairs and 23 DEL-DEG pairs involved in nitrogen metabolism were identified. Finally, a predicted regulatory network of nitrogen metabolism was initially constructed, which included 17 nitrogen metabolic pathway genes, 15 TFs, 4 miRNAs, and 10 lncRNAs by conjoint analysis of DEGs, DEMs, and DELs and their regulatory relationships, which was supported by RNA-seq data and qPCR results. The lncRNA-miRNA-mRNA network provides new insights into the regulation mechanism of nitrogen metabolism in bamboo, which facilitates further genetic improvement for bamboo to adapt to the fluctuating nitrogen environment.

Identification and expression analysis of the glycosyltransferase GT43 family members in bamboo reveal their potential function in xylan biosynthesis during rapid growth.

BACKGROUND: Xylan is one of the most abundant hemicelluloses and can crosslink cellulose and lignin to increase the stability of cell walls. A number of genes encoding glycosyltransferases play vital roles in xylan biosynthesis in plants, such as those of the GT43 family. However, little is known about glycosyltransferases in bamboo, especially woody bamboo which is a good substitute for timber. RESULTS: A total of 17 GT43 genes (PeGT43-1 ~ PeGT43-17) were identified in the genome of moso bamboo (Phyllostachys edulis), which belong to three subfamilies with specific motifs. The phylogenetic and collinearity analyses showed that PeGT43s may have undergone gene duplication, as a result of collinearity found in 12 pairs of PeGT43s, and between 17 PeGT43s and 10 OsGT43s. A set of cis-acting elements such as hormones, abiotic stress response and MYB binding elements were found in the promoter of PeGT43s. PeGT43s were expressed differently in 26 tissues, among which the highest expression level was found in the shoots, especially in the rapid elongation zone and nodes. The genes coexpressed with PeGT43s were annotated as associated with polysaccharide metabolism and cell wall biosynthesis. qRT-PCR results showed that the coexpressed genes had similar expression patterns with a significant increase in 4.0 m shoots and a peak in 6.0 m shoots during fast growth. In addition, the xylan content and structural polysaccharide staining intensity in bamboo shoots showed a strong positive correlation with the expression of PeGT43s. Yeast one-hybrid assays demonstrated that PeMYB35 could recognize the 5' UTR/promoter of PeGT43-5 by binding to the SMRE cis-elements. CONCLUSIONS: PeGT43s were found to be adapted to the requirement of xylan biosynthesis during rapid cell elongation and cell wall accumulation, as evidenced by the expression profile of PeGT43s and the rate of xylan accumulation in bamboo shoots. Yeast one-hybrid analysis suggested that PeMYB35 might be involved in xylan biosynthesis by regulating the expression of PeGT43-5 by binding to its 5' UTR/promoter. Our study provides a comprehensive understanding of PeGT43s in moso bamboo and lays a foundation for further functional analysis of PeGT43s for xylan biosynthesis during rapid growth.

A regulatory network driving shoot lignification in rapidly growing bamboo.

Woody bamboo is environmentally friendly, abundant, and an alternative to conventional timber. Degree of lignification and lignin content and deposition affect timber properties. However, the lignification regulatory network in monocots is poorly understood. To elucidate the regulatory mechanism of lignification in moso bamboo (Phyllostachys edulis), we conducted integrated analyses using transcriptome, small RNA, and degradome sequencing followed by experimental verification. The lignification degree and lignin content increased with increased bamboo shoot height, whereas phenylalanine ammonia-lyase and Laccase activities first increased and then decreased with shoot growth. Moreover, we identified 11,504 differentially expressed genes (DEGs) in different portions of the 13th internodes of different height shoots; most DEGs associated with cell wall and lignin biosynthesis were upregulated, whereas some DEGs related to cell growth were downregulated. We identified a total of 1,502 miRNAs, of which 687 were differentially expressed. Additionally, in silico and degradome analyses indicated that 5,756 genes were targeted by 691 miRNAs. We constructed a regulatory network of lignification, including 11 miRNAs, 22 transcription factors, and 36 enzyme genes, in moso bamboo. Furthermore, PeLAC20 overexpression increased lignin content in transgenic Arabidopsis (Arabidopsis thaliana) plants. Finally, we proposed a reliable miRNA-mediated "MYB-PeLAC20" module for lignin monomer polymerization. Our findings provide definite insights into the genetic regulation of bamboo lignification. In addition to providing a platform for understanding related mechanisms in other monocots, these insights could be used to develop strategies to improve bamboo timber properties.

Analysis of 427 genomes reveals moso bamboo population structure and genetic basis of property traits.

Moso bamboo (Phyllostachys edulis) is an economically and ecologically important nontimber forestry species. Further development of this species as a sustainable bamboo resource has been hindered by a lack of population genome information. Here, we report a moso bamboo genomic variation atlas of 5.45 million single-nucleotide polymorphisms (SNPs) from whole-genome resequencing of 427 individuals covering 15 representative geographic areas. We uncover low genetic diversity, high genotype heterozygosity, and genes under balancing selection underlying moso bamboo population adaptation. We infer its demographic history with one bottleneck and its recently small population without a rebound. We define five phylogenetic groups and infer that one group probably originated by a single-origin event from East China. Finally, we conduct genome-wide association analysis of nine important property-related traits to identify candidate genes, many of which are involved in cell wall, carbohydrate metabolism, and environmental adaptation. These results provide a foundation and resources for understanding moso bamboo evolution and the genetic mechanisms of agriculturally important traits.

Identification and Expression Analyses of Invertase Genes in Moso Bamboo Reveal Their Potential Drought Stress Functions.

Invertases (INVs) can irreversibly hydrolyze sucrose into fructose and glucose, which play principal roles in carbon metabolism and responses to various stresses in plants. However, little is known about the INV family in bamboos, especially their potential function in drought stress. In this study, 29 PeINVs were identified in moso bamboo (Phyllostachys edulis). They were clustered into alkaline/neutral invertase (NINV) and acid invertase (AINV) groups based on the gene structures, conserved motifs, and phylogenetic analysis results. The collinearity analysis showed nine segmental duplication pairs within PeINVs, and 25 pairs were detected between PeINVs and OsINVs. PeINVs may have undergone strong purification selection during evolution, and a variety of stress and phytohormone-related regulatory elements were found in the promoters of PeINVs. The tissue-specific expression analysis showed that PeINVs were differentially expressed in various moso bamboo tissues, which suggested that they showed functional diversity. Both the RNA-seq and quantitative real-time PCR results indicated that four PeINVs were significantly upregulated under drought stress. Co-expression network and Pearson's correlation coefficient analyses showed that these PeINVs co-expressed positively with sugar and water transport genes (SWTGs), and the changes were consistent with sugar content. Overall, we speculate that the identified PeINVs are spatiotemporally expressed, which enables them to participate in moso bamboo growth and development. Furthermore, PeINVs, together with SWTGs, also seem to play vital roles in the response to drought stress. These results provide a comprehensive information resource for PeINVs, which will facilitate further study of the molecular mechanism underlying PeINVs involvement in the response to drought stress in moso bamboo.

Refinement of bamboo genome annotations through integrative analyses of transcriptomic and epigenomic data.

Bamboo, one of the most crucial nontimber forest resources worldwide, has the capacity for rapid growth. In recent years, the genome of moso bamboo (Phyllostachys edulis) has been decoded, and a large amount of transcriptome data has been published. In this study, we generated the genome-wide profiles of the histone modification H3K4me3 in leaf, stem, and root tissues of bamboo. The trends in the distribution patterns were similar to those in rice. We developed a processing pipeline for predicting novel transcripts to refine the structural annotation of the genome using H3K4me3 ChIP-seq data and 29 RNA-seq datasets. As a result, 12,460 novel transcripts were predicted in the bamboo genome. Compared with the transcripts in the newly released version 2.0 of the bamboo genome, these novel transcripts are tissue-specific and shorter, and most have a single exon. Some representative novel transcripts were validated by semiquantitative RT-PCR and qRT-PCR analyses. Furthermore, we put these novel transcripts back into the ChIP-seq analysis pipeline and discovered that the percentages of H3K4me3 in genic elements were increased. Overall, this work integrated transcriptomic data and epigenomic data to refine the annotation of the genome in order to discover more functional genes and study bamboo growth and development, and the application of this predicted pipeline may help refine the structural annotation of the genome in other species.

Genome-wide analysis of laccase genes in moso bamboo highlights PeLAC10 involved in lignin biosynthesis and in response to abiotic stresses.

KEY MESSAGE: Twenty-three PeLACs have been identified in moso bamboo, overexpression of PeLAC10 increases the lignin content and confers drought and phenolic acid tolerance in transgenic Arabidopsis. Laccases (LACs) have multifunction involved in the processes of cell elongation, lignification and stress response in plants. However, the function of laccases in bamboo remain unclear. Here, a total of 23 laccase genes (PeLAC1-PeLAC23) were identified in moso bamboo (Phyllostachys edulis). The diverse gene structure and expression pattern of PeLACs suggested that their function should be spatiotemporal and complicated, which was supported by the expression profiles in different tissues of moso bamboo. Eighteen PeLACs were identified as the targets of ped-miR397. The putative ped-miR397-binding site in the coding region of PeLAC10 was further confirmed by RLM-5' RACE, indicating that PeLAC10 was regulated by ped-miR397 after transcription. With the increasing shoot height, the expression abundance of PeLAC10 was up-regulated and reached the maximum in 15 cm shoots, while that of ped-miR397 was relative lower and showed the minimum in 15 cm shoots. PeLAC10 was up-regulated obviously under both ABA (100 µmol L-1) and NaCl (400 mmol L-1) treatments, and it was down-regulated under the GA3 (100 µmol L-1) treatment. The transgenic Arabidopsis plants over-expressing PeLAC10 became slightly smaller and their petioles were shorter than those of Col-0. However, they had a stronger capacity in resistance to phenolic acids and drought besides higher lignin content in stems. These results indicated that overexpression of PeLAC10 was helpful to increase the content of lignin in transgenic Arabidopsis and improve the adaptability to phenolic acid and drought stresses.

Identification of Homeobox Genes Associated with Lignification and Their Expression Patterns in Bamboo Shoots.

: Homeobox (HB) genes play critical roles in regulating various aspects of plant growth and development. However, little is known about HB genes in bamboo. In this study, a total of 115 HB genes (PeHB001‒PeHB115) were identified from moso bamboo (Phyllostachys edulis) and grouped into 13 distinct classes (BEL, DDT, HD-ZIP I‒IV, KNOX, NDX, PHD, PINTOX, PLINC, SAWADEE, and WOX) based on the conserved domains and phylogenetic analysis. The number of members in the different classes ranged from 2 to 24, and they usually varied in terms of exon‒intron distribution pattern and length. There were 20 conserved motifs found in 115 PeHBs, with motif 1 being the most common. Gene ontology (GO) analysis showed that PeHBs had diverse molecular functions, with 19 PeHBs being annotated as having xylem development, xylem, and phloem pattern formation functions. Co-expression network analysis showed that 10 of the 19 PeHBs had co-expression correlations, and three members of the KNOX class were hub proteins that interacted with other transcription factors (TFs) such as MYB, bHLH, and OVATE, which were associated with lignin synthesis. Yeast two-hybridization results further proved that PeHB037 (BEL class) interacted with PeHB057 (KNOX class). Transcriptome expression profiling indicated that all PeHBs except PeHB017 were expressed in at least one of the seven tissues of moso bamboo, and 90 PeHBs were expressed in all the tissues. The qRT-PCR results of the 19 PeHBs showed that most of them were upregulated in shoots as the height increased. Moreover, a KNOX binding site was found in the promoters of the key genes involved in lignin synthesis such as Pe4CL, PeC3H, PeCCR, and PeCOMT, which had positive expression correlations with five KNOX genes. Similar results were found in winter bamboo shoots with prolonged storage time, which was consistent with the degree of lignification. These results provide basic data on PeHBs in moso bamboo, which will be helpful for future functional research on PeHBs with positive regulatory roles in the process of lignification.