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Bamboo with its remarkable growth rate and economic significance, offers an ideal system to investigate the molecular basis of organogenesis in rapidly growing plants, particular in monocots, where gene regulatory networks governing the maintenance and differentiation of shoot apical and intercalary meristems remain a subject of controversy. We employed both spatial and single-nucleus transcriptome sequencing on 10× platform to precisely dissect the gene functions in various tissues and early developmental stages of bamboo shoots. Our comprehensive analysis reveals distinct cell trajectories during shoot development, uncovering critical genes and pathways involved in procambium differentiation, intercalary meristem formation, and vascular tissue development. Spatial and temporal expression patterns of key regulatory genes, particularly those related to hormone signaling and lipid metabolism, strongly support the hypothesis that intercalary meristem origin from surrounded parenchyma cells. Specific gene expressions in intercalary meristem exhibit regular and dispersed distribution pattern, offering clues for understanding the intricate molecular mechanisms that drive the rapid growth of bamboo shoots. The single-nucleus and spatial transcriptome analysis reveal a comprehensive landscape of gene activity, enhancing the understanding of the molecular architecture of organogenesis and providing valuable resources for future genomic and genetic studies relying on identities of specific cell types., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Bamboo, renowned as the fastest-growing plant globally, matures within an astonishingly short period of 40-50 days from shoots, reaching heights of 10-20 meters. Moreover, it can be harvested for various uses within 3-5 years. Bamboo exhibits exceptional mechanical properties, characterized by high hardness and flexibility, largely attributed to its lignin content. Phenylalanine ammonia-lyase (PAL) catalyzes the crucial initial step in lignin biosynthesis, but its precise role in bamboo lignification processes remains elusive. Thus, elucidating the functions of PAL genes in bamboo lignification processes is imperative for understanding its rapid growth and mechanical strength. Here, we systematically identified and classified PAL genes in Moso bamboo, ensuring nomenclature consistency across prior studies. Subsequently, we evaluated PAL gene expression profiles using publicly available transcriptome data. The downregulation of PePALs expression in Moso bamboo through in planta gene editing resulted in a decrease in PAL activity and a subsequent reduction in lignin content. In contrast, overexpression of PePAL led to enhanced PAL activity and an increase in lignin content. These findings highlight the critical role of PAL in the lignin biosynthesis process of Moso bamboo, which will help to unravel the mechanism underpinning bamboo's rapid growth and mechanical strength, with a specific emphasis on elucidating the functions of PAL genes., (© 2024 Scandinavian Plant Physiology Society.)

Background: Woody bamboos are the only diverse large perennial grasses in mesic-wet forests and are widely distributed in the understory and canopy. The functional trait variations and trade-offs in this taxon remain unclear due to woody bamboo syndromes (represented by lignified culm of composed internodes and nodes). Here, we examined the effects of heritable legacy and occurrence site climates on functional trait variations in leaf and culm across 77 woody bamboo species in a common garden. We explored the trade-offs among leaf functional traits, the connection between leaf nitrogen (N), phosphorus (P) concentrations and functional niche traits, and the correlation of functional traits between leaves and culms., Results: The Bayesian mixed models reveal that the combined effects of heritable legacy (phylogenetic distances and other evolutionary processes) and occurrence site climates accounted for 55.10-90.89% of the total variation among species for each studied trait. The standardized major axis analysis identified trade-offs among leaf functional traits in woody bamboo consistent with the global leaf economics spectrum; however, compared to non-bamboo species, the woody bamboo exhibited lower leaf mass per area but higher N, P concentrations and assimilation, dark respiration rates. The canonical correlation analysis demonstrated a positive correlation (ρ = 0.57, P-value < 0.001) between leaf N, P concentrations and morphophysiology traits. The phylogenetic principal components and trait network analyses indicated that leaf and culm traits were clustered separately, with leaf assimilation and respiration rates associated with culm ground diameter., Conclusion: Our study confirms the applicability of the leaf economics spectrum and the biogeochemical niche in woody bamboo taxa, improves the understanding of woody bamboo leaf and culm functional trait variations and trade-offs, and broadens the taxonomic units considered in plant functional trait studies, which contributes to our comprehensive understanding of terrestrial forest ecosystems., (© 2024. The Author(s).)

A cleft lip, with or without a cleft palate, is a common birth defect caused by environmental factors or genetic mutations. Environmental factors, such as pharmaceutical exposure in pregnant women, are known to induce cleft lip, with or without cleft palate in the child. This study aimed to investigate the protective effect of Sasa veitchii extract (SE) on phenytoin-induced inhibition of cell proliferation in human lip mesenchymal cells (KD cells) and human embryonic palatal mesenchymal cells (HEPM cells). We demonstrated that cell proliferation was inhibited by phenytoin in a dose-dependent manner in both KD and HEPM cells. Co-treatment with SE restored phenytoin-induced toxicity in KD cells but did not protect HEPM cells against phenytoin-induced toxicity. Several microRNAs (miR-27b, miR-133b, miR-205, miR-497-5p, and miR-655-3p) is reported to associate with cell proliferation in KD cells. We measured the seven kinds of microRNAs (miR27b-3p, miR-27b-5p, miR-133b, miR-205-3p, miR-205-5p, miR-497-5p, and miR-655-3p) and found that SE suppressed miR-27b-5p induced by phenytoin in KD cells. Furthermore, co-treatment with SE enhanced the expression of miR-27b-5p downstream genes (PAX9, RARA, and SUMO1). These results suggest that SE protects phenytoin-induced cell proliferation inhibition by modulating miR-27b-5p.

Key Message: PeTCP10 can be induced by salt stresses and play important regulation roles in salt stresses response in transgenic Arabidopsis. Salt stress is one of the major adverse environmental factors that affect normal plant development and growth. PeTCP10, a Class I TCP member, was markedly expressed in moso bamboo mature leaf, root and stem under normal conditions and also induced by salt stress. Overexpressed PeTCP10 was found to enhance salt tolerance of transgenic Arabidopsis at the vegetative growth stage. It was also found capable to increase relative water content, while decreasing relative electrolyte leakage and Na + accumulation of transgenic Arabidopsis versus wild-type (WT) plants at high-salt conditions. In addition, it improved antioxidant capacity of transgenic Arabidopsis plants by promoting catalase activity and enhanced their H 2 O 2 tolerance. In contrast to WT plants, transcriptome analysis demonstrated that multiple genes related to abscisic acid, salt and H 2 O 2 response were induced after NaCl treatment in transgenic plants. Meanwhile, overexpressed PeTCP10 improved the tolerance of abscisic acid. Moreover, luciferase reporter assay results showed that PeTCP10 is able to directly activate the expression of BT2 in transgenic plants. In contrary, the germination rates of transgenic plants were significantly lower than those of WT plants under high-NaCl conditions. Both primary root length and survival rate at the seedling stage are also found lower in transgenic plants than in WT plants. It is concluded that overexpressed PeTCP10 enhances salt stress tolerance of transgenic plants at the vegetative growth stage, and it also improves salt sensitiveness in both germination and seedling stages. These research results will contribute to further understand the functions of TCPs in abiotic stress response., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Heat shock transcription factors (HSFs) are central elements in the regulatory network that controls plant heat stress response. They are involved in multiple transcriptional regulatory pathways and play important roles in heat stress signaling and responses to a variety of other stresses. We identified 41 members of the HSF gene family in moso bamboo, which were distributed non-uniformly across its 19 chromosomes. Phylogenetic analysis showed that the moso bamboo HSF genes could be divided into three major subfamilies; HSFs from the same subfamily shared relatively conserved gene structures and sequences and encoded similar amino acids. All HSF genes contained HSF signature domains. Subcellular localization prediction indicated that about 80% of the HSF proteins were located in the nucleus, consistent with the results of GO enrichment analysis. A large number of stress response-associated cis-regulatory elements were identified in the HSF upstream promoter sequences. Synteny analysis indicated that the HSFs in the moso bamboo genome had greater collinearity with those of rice and maize than with those of Arabidopsis and pepper. Numerous segmental duplicates were found in the moso bamboo HSF gene family. Transcriptome data indicated that the expression of a number of PeHsfs differed in response to exogenous gibberellin (GA) and naphthalene acetic acid (NAA). A number of HSF genes were highly expressed in the panicles and in young shoots, suggesting that they may have functions in reproductive growth and the early development of rapidly-growing shoots. This study provides fundamental information on members of the bamboo HSF gene family and lays a foundation for further study of their biological functions in the regulation of plant responses to adversity., (© 2021. The Author(s).)

Key Message: PePIP2;7, a leaf-specific aquaporin gene in bamboo, is upregulated under abiotic stresses. Overexpressing PePIP2;7 confers abiotic stresses tolerance in transgenic Arabidopsis plant and yeast. Aquaporins (AQPs) participate in the regulation of water balance in plants. However, the function of AQPs in bamboo remains unclear. Here, PePIP2;7 was identified as a leaf-specific aquaporin gene in moso bamboo based on the expression analysis of transcriptome data and PCR. In situ hybridization further indicated that PePIP2;7 was mainly expressed in mesophyll cells of mature leaves, while in immature leaves it was dominant in blade edge cells followed by mesophyll cells. Interestingly, PePIP2;7 was strongly expressed in the mesophyll cells near bulliform cells of immature leaves, suggesting that PePIP2;7 might function in water transport and contribute to leaf unfolding. The transient expression assay showed that PePIP2;7 was a plasma membrane intrinsic protein. Furthermore, PePIP2;7 was upregulated under abiotic stresses such as high light, drought, and NaCl. Compared with Col-0, transgenic Arabidopsis plants overexpressing PePIP2;7 had better seed germination rate, longer taproot length, higher SOD activity, and lower MDA content under abiotic stresses. Besides, yeasts expressing PePIP2;7 also had higher tolerance to stress compared to the control. Taken together, our results show that PePIP2;7 is leaf-specific and involved in stress response, which provides new insights into aquaporin function in bamboo.

The present study used soils contaminated with Fusarium oxysporum f. sp. capsici (CCS) and CCS amended with bamboo biochar (CCS + BC) to grow the pepper variety Qujiao No.1. The physiological performance, and transcriptome and metabolome profiling in leaf (L) and fruit (F) of Qujiao No.1 were conducted. Application of biochar improved soil properties, pepper plant nutrition and increased activities of enzymes related to pest/disease resistance, leading to superior physiological performance and lesser F. wilt disease incidence than plants from CCS. Most of the differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were involved in protein processing in endoplasmic reticulum (fruit), plant pathogen interaction (fruit), photosynthesis (leaf), phenylpropanoid biosynthesis (both tissues) and metabolic pathways (both tissues). Biochar improved plant photosynthesis, enhanced the immune system, energy production and increased stress signaling pathways. Overall, our results provide evidence of a number of pathways induced by biochar in pepper regulating its response to F. wilt disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bamboos, member of the family Poaceae, represent many interesting features with respect to their fast and extended vegetative growth, unusual, yet divergent flowering time across species, and impact of sudden, large scale flowering on forest ecology. However, not many studies have been conducted at the molecular level to characterize important genes that regulate vegetative and flowering habit in bamboo. In this study, two bamboo FD genes, BtFD1 and BtFD2, which are members of the florigen activation complex (FAC) have been identified by sequence and phylogenetic analyses. Sequence comparisons identified one important amino acid, which was located in the DNA-binding basic region and was altered between BtFD1 and BtFD2 (Ala146 of BtFD1 vs. Leu100 of BtFD2). Electrophoretic mobility shift assay revealed that this alteration had resulted into ten times higher binding efficiency of BtFD1 than BtFD2 to its target ACGT motif present at the promoter of the APETALA1 gene. Expression analyses in different tissues and seasons indicated the involvement of BtFD1 in flower and vegetative development, while BtFD2 was very lowly expressed throughout all the tissues and conditions studied. Finally, a tenfold increase of the AtAP1 transcript level by p35S::BtFD1 Arabidopsis plants compared to wild type confirms a positively regulatory role of BtFD1 towards flowering. However, constitutive expression of BtFD1 had led to dwarfisms and apparent reduction in the length of flowering stalk and numbers of flowers/plant, whereas no visible phenotype was observed for BtFD2 overexpression. This signifies that timely expression of BtFD1 may be critical to perform its programmed developmental role in planta.

The plant microbiota play a key role in plant productivity, nutrient uptake, resistance to stress and flowering. The flowering of moso bamboo has been a focus of study. The mechanism of flowering is related to nutrient uptake, temperature, hormone balance and regulation of key genes. However, the connection between microbiota of moso bamboo and its flowering is unknown. In this study, samples of rhizosphere soil, rhizomes, roots and leaves of flowering and nonflowering plants were collected, and 16S rRNA amplicon Illumina sequencing was utilized to separate the bacterial communities associated with different flowering stages of moso bamboo. We identified 5442 OTUs, and the number of rhizosphere soil OTUs was much higher than those of other samples. Principal component analysis (PCA) and hierarchical clustering (Bray Curtis dis) analysis revealed that the bacterial microorganisms related to rhizosphere soil and endophytic tissues of moso bamboo differed significantly from those in bulk soil and rhizobacterial and endosphere microbiomes. In addition, the PCA analyses of root and rhizosphere soil revealed different structures of microbial communities between bamboo that is flowering and not flowering. Through the analysis of core microorganisms, it was found that Flavobacterium, Bacillus and Stenotrophomonas played an important role in the absorption of N elements, which may affect the flowering time of moso bamboo. Our results delineate the complex host-microbe interactions of this plant. We also discuss the potential influence of bacterial microbiome in flowering, which can provide a basis for the development and utilization of moso bamboo.

Bamboo, a fast-growing non-timber forest plant with many uses, is a valuable species for green development. However, bamboo flowering is very infrequent, extending, in general, for up to 120 years. Ecologically, bamboo species are generally better adapted to various environments than other grasses. Therefore, the species deserves a special status in what could be called Ecological Bioeconomy. An understanding of the genetic processes of bamboo can help us sustainably develop and manage bamboo forests. Transposable elements (TEs), jumping genes or transposons, are major genetic elements in plant genomes. The rapid development of the bamboo reference genome, at the chromosome level, reveals that TEs occupy over 63.24% of the genome. This is higher than found in rice, Brachypodium, and sorghum. The bamboo genome contains diverse families of TEs, which play a significant role in bamboo's biological processes including growth and development. TEs provide important clues for understanding the evolution of the bamboo genome. In this chapter, we briefly describe the current status of research on TEs in the bamboo genome, their regulation, and transposition mechanisms. Perspectives for future research are also provided.

Drepanostachyum falcatum (Nees) Keng f. is one of the most widely distributed shrubby bamboo species in the temperate region of northwest (NW) Himalayas. Along with the other three temperate bamboo species, namely Yushania anceps , Thamnocalamus spathiflorus and Himalayacalamus falconeri , commonly called as 'ringal', and utilized for making various articles of household and commercial purpose by local artisans. Despite huge ecological and socio-economic importance, they are least studied and lacks baseline genetic information. In this study, ~10 Gb genome sequence data with 70.68 million reads were generated for D. falcatum , through genome skimming approach based on high throughput next-generation sequencing technology with Illumina protocol. The high-quality reads were de novo assembled into 31,997 contigs, which comprised 1943 microsatellite repeats. The dinucleotide and trinucleotide repeats were most abundantly distributed in the genome with 52.95 and 41.17%, respectively. Depending on the sufficient flanking sequence, only 1123 repeats were successfully tagged with primer pairs and these sites were designated as sequence-tagged microsatellite (STMS) markers. Further, a subset of 106 STMS markers were validated through PCR amplification; 77 marker loci were successfully amplified, and 48 of these showed polymorphism. Same set of marker loci were also tested for their cross-amplification in other three temperate bamboo species of the NW Himalayas, which revealed good level of transferability (27-48%) but lesser polymorphism (4-12%). In addition, the genomewide in silico cross-amplification revealed poor cross-transferability in other bamboo taxa representing four different phylogenetic lineages, namely Phyllostachys edulis (10.2%), Bonia amplexicaulis (3.03%), Guadua angustifolia (1.60%), Olyra latifolia (0.89%) and Raddia guianensis (0.36%). Ten polymorphic markers were further used to estimate the measures of genetic diversity in two natural populations, which revealed high genetic diversity (polymorphic information content, PIC = 0.889; expected heterozygosity, H e = 0.756) and low genetic differentiation ( F ST =0.061; Nm = 5.445). To the best of our knowledge, this is one of the pioneer studies carried out for the development of genomic STMS markers through genome skimming approach in Indian bamboo species. The marker information generated here is novel and of paramount importance for future genetic studies in D. falcatum as well as other temperate bamboo species through cross-transferability.

Uridine diphosphate glucose dehydrogenases (UGDHs) are critical for synthesizing many nucleotide sugars and help promote the carbohydrate metabolism related to cell wall synthesis. In plants, UGDHs are encoded by a small gene family. Genome-wide analyses of these genes have been conducted in Glycine max and Arabidopsis thaliana, however, the UGDH gene family has not been comprehensively and systematically investigated in moso bamboo (Phyllostachys edulis), which is a special woody grass monocotyledonous species. In this study, we identified nine putative PeUGDH genes. Furthermore, analysis of gene duplication events and divergences revealed that the expansion of the PeUGDH family was mainly due to segmental and tandem duplications approximately 4.76-83.16 million years ago. An examination of tissue-specific PeUGDH expression indicated that more than 77% of the genes were predominantly expressed in the stem. Based on relative expression levels among PeUGDH members in different tissues in moso bamboo, PeUGDH4 was selected for detailed analysis. The results of subcellular localization indicated that PeUGDH4-GFP fusion proteins was observed to be localized in the cytoplasm. The ectopic overexpression of PeUGDH4 in Arabidopsis significantly increased the contents of hemicellulose and soluble sugar, suggesting that PeUGDH4 acts as a key enzyme involved in bamboo cell wall synthesis.

Moso bamboo (Phyllostachysheterocycla (Carr.) Mitford cv. Pubescens) is an economically valuable plant in bamboo production areas of southern China, for which the management mode is crucial for improving the comprehensive benefits of bamboo forest stands. In this respect, mixed forested areas of bamboo and broad-leaved tree species can provide sound ecological management of bamboo in forestry operations. To further this goal, an outstanding question is to better understand the spatial distribution of soil bacterial communities in relation to the proportion of mixed in bamboo and broad-leaved forest. We analyzed soil bacterial community diversity and composition along a proportional gradient of 0-40% mixed-ratio (as represented by the width and size of the broad-leaved tree crown over the plot area) of bamboo and broad-leaved forest in Tianbao Yan Nature Reserve using the highthroughputsequencing of the 16S rRNA gene.Specifically, the sampling plots for the mixed proportions were divided according to the percentage of summed projected area of live broadleaf tree crowns. The main broad-leaved species in the five mixed ratio plots are the same. Each plot was 20 m × 20 m in size, and a total of 15 plots were established, three per forest ratio class. From each plot, soil samples were taken at the surface (0-10 cm depth) in December 2017. Our analysis revealed that soil bacterial diversity community structure and dominant flora changed under different mixing ratios of bamboo and broad-leaved trees. In the stand with a mixed ratio of 10-20%, the bacterial diversity index is higher; however, the diversity was lowest in the 20-30% stands. Among the 20-30% forest soil, Acidobacteria (Solibacteria, Solibacteriales, Acidobacteriales) was more abundant than in soils from other mixed-ratio stands.Redundancy analysis showed that mixed forest stand structure, soil pH, organic carbon, total nitrogen, and soil moisture all contributed to shaping the bacterial community structure. Changes in microbial communities were associated with species diversity in tree layers, availability of soil nutrients (SOC and TN), and changes in soil physical properties (MS, pH). Together, these empirical results suggest that different mixing ratios in the bamboo-broad-leaved mixed forest could influence the soil bacterial community structure indirectly, specifically by affecting the soil physical and chemical properties of the forest.

C-glycosylated flavones (CGFs) are promising candidates as anti-nociceptive compounds. The leaves of bamboo and related crops in the grass family are a largely unexploited bioresource with a wide array of CGFs. We report here pathway-specific enzymes including C-glycosyltransferases (CGTs) and P450 hydroxylases from cereal crops and bamboo species accumulating abundant CGFs. Mining of CGTs and engineering of P450s that decorate the flavonoid skeleton allowed the production of desired CGFs (with yield of 20-40 mg/L) in an Escherichia coli cell factory. We further explored the antinociceptive activity of major CGFs in mice models and identified isoorientin as the most potent, with both neuroanalgesic and anti-inflammatory effects superior to clinical drugs such as rotundine and aspirin. Our discovery of the pain-alleviating flavonoids elicited from bamboo and crop leaves establishes this previously underutilized source, and sheds light on the pathway and pharmacological mechanisms of the compounds.

Bambusapervariabilis × Dendrocalamopsisgrandis, a fast-growing and easily propagated bamboo species, has been extensively planted in the southern China, resulting in huge ecological benefits. In recent years, it was found that the pathogenic fungus Arthrinium phaeospermum caused the death of a large amount of bamboo. In this study, the transcriptome of B. pervariabilis × D. grandis, induced by inactivated protein AP-toxin from A. phaeospermum was sequenced and analyzed, to reveal the resistance mechanism induced by biotic agents of B. pervariabilis × D. grandis against A. phaeospermum at the gene level. Transcriptome sequencing was performed by Illumina HiSeq 2000 in order to analyze the differentially expressed genes (DEGs) of B. pervariabilis × D. grandis in response to different treatment conditions. In total, 201,875,606 clean reads were obtained, and the percentage of Q30 bases in each sample was more than 94.21%. There were 6398 DEGs in the D-J group (inoculation with a pathogenic spore suspension after three days of AP-toxin induction) compared to the S-J group (inoculation with a pathogenic spore suspension after inoculation of sterile water for three days) with 3297 up-regulated and 3101 down-regulated genes. For the D-S group (inoculation with sterile water after inoculation of AP-toxin for three days), there were 2032 DEGs in comparison to the S-S group (inoculation with sterile water only), with 1035 up-regulated genes and 997 down-regulated genes. These identified genes were mainly involved in lignin and phytoprotein synthesis, tetrapyrrole synthesis, redox reactions, photosynthesis, and other processes. The fluorescence quantitative results showed that 22 pairs of primer amplification products were up-regulated and 7 were down-regulated. The rate of similarity between these results and the sequencing results of the transcription group was 100%, which confirmed the authenticity of the transcriptome sequencing results. Redox proteins, phenylalanine ammonia lyase, and S-adenosine-L-methionine synthetase, among others, were highly expressed; these results may indicate the level of disease resistance of the bamboo. These results provide a foundation for the further exploration of resistance genes and their functions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Moso bamboo is well-known for its rapid-growth shoots and widespread rhizomes. However, the regulatory genes of these two processes are largely unexplored. GATA transcription factors regulate many developmental processes, but their roles in moso bamboo height control and rhizome development remains unexplored. Here, thirty-one bamboo GATA factors (PeGATAs) were identified, which are evolutionarily closer to rice than Arabidopsis, and their gene expression patterns were analyzed in bamboo development and phytohormone response with bioinformatics and molecular methods. Interestingly, PeGATAs could only be classified into three groups. Phytohormone responsive cis-elements were found in PeGATA promoters and the expression profiles showed that PeGATA genes might respond to gibberellin acid and abscisic acid but not to auxin at the transcriptional level. Furthermore, PeGATA genes have a tissue-specific expression pattern in bamboo rhizomes. Interestingly, most PeGATA genes were down-regulated during the rapid-growth of bamboo shoots. In addition, over-expressing one of the PeGATA genes, PeGATA26, significantly repressed the primary root length and plant height of transgenic Arabidopsis plants, which may be achieved by promoting the gibberellin acid turnover. Overall, our results provide insight into the function of GATA transcription factors in bamboo, and into genetic resources for engineering plant height., Competing Interests: The authors declare no conflict of interest.

: 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 PeHB s had diverse molecular functions, with 19 PeHB s being annotated as having xylem development, xylem, and phloem pattern formation functions. Co-expression network analysis showed that 10 of the 19 PeHB s 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 PeHB s except PeHB017 were expressed in at least one of the seven tissues of moso bamboo, and 90 PeHB s were expressed in all the tissues. The qRT-PCR results of the 19 PeHB s 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 PeHB s in moso bamboo, which will be helpful for future functional research on PeHB s with positive regulatory roles in the process of lignification.

NAC (NAM, ATAF, and CUC) transcription factors (TFs) are implicated in the transcriptional regulation of diverse processes and have been characterized in a number of plant species. However, NAC TFs are still not well understood in bamboo, especially their potential association with the secondary cell wall (SCW). Here, 94 PeNAC s were identified and characterized in moso bamboo ( Phyllostachys edulis ). Based on their gene structures and conserved motifs, the PeNACs were divided into 11 groups according to their homologs in Arabidopsis . PeNAC s were expressed variously in different tissues of moso bamboo, suggesting their functional diversity. Fifteen PeNAC s associated with the SCW were selected for co-expression analysis and validation. It was predicted that 396 genes were co-expressed with the 15 PeNAC s, in which 16 and 55 genes were involved in the lignin catabolic process and cellulose biosynthetic process respectively. As the degree of lignification in the growing bamboo shoots increased, all 15 PeNAC s were upregulated with a trend of rising first and then decreasing except PeNAC37 , which increased continuously. These results indicated that these PeNAC s might play important roles in SCW biosynthesis and lignification in bamboo shoots. Seven of 15 PeNAC s had been found positively co-expressed with seven PeMYB s, and they had similar expression patterns with those of the PeMYB s in bamboo shoots. The targeted sites of miR164 were found in 16 PeNAC s, of which three PeNAC s associated with SCW were validated to have an opposite expression trend to that of miR164 in growing bamboo shoots. In addition, three PeNAC s were selected and verified to have self-activation activities. These results provide comprehensive information of the NAC gene family in moso bamboo, which will be helpful for further functional studies of PeNAC s to reveal the molecular regulatory mechanisms of bamboo wood property., Competing Interests: The authors declare no conflict of interest. The funding sponsors contributed reagents/materials/analysis tools in this paper.

Moso bamboo (Phyllostachys edulis) is one of the fastest growing species with a maximum growth rate of 1 m/day. However, the regulator genes for this explosive growth phenomenon have not been functionally studied. Here, we found that Moso bamboo GSK3/shaggy-like kinase 1 (PeGSK1) acts as a negative regulator of cell growth. Over-expression of PeGSK1 in Arabidopsis showed significant growth arrest phenotypes, including dwarfism, small leaves, reduced cell length, and disturbed cell elongation of petiole. Furthermore, Overexpression of PeGSK1 fully inhibited the longer hypocotyl phenotype of Arabidopsis atgsk1 mutants. In addition, PeGSK1-overexpressing lines were resistant to exogenous BR treatment and PeGSK1 interacted with the brassinosteroid signal transduction key regulator BZR1. The BZR1-dependent cell growth genes were down-regulated in PeGSK1-overexpressing lines. These results indicated that PeGSK1 is functionally similar to AtGSK1 and inhibited cell growth via the brassinosteroid signaling pathway. Importantly, PeGSK1 also interacted with PeBZR1, and the expression pattern of PeGSK1 was negatively correlated with the internode elongation of bamboo, indicating that PeGSK1 is involved in the cell growth of bamboo. In summary, our results provide insight into the role of brassinosteroids in the rapid-growth of bamboo culms and identifying target genes for the genetic manipulation of plant height., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The basic leucine zipper (bZIP) transcription factor (TF) family is one of the largest gene families, and play crucial roles in many processes, including stress responses, hormone effects. The TF family also participates in plant growth and development. However, limited information is available for these genes in moso bamboo ( Phyllostachys edulis ), one of the most important non-timber forest products in the world. In the present study, 154 putative PhebZIP genes were identified in the moso bamboo genome. The phylogenetic analyses indicate that the PhebZIP gene proteins classify into 9 subfamilies and the gene structures and conserved motifs that analyses identified among all PhebZIP proteins suggested a high group-specificity. Microsynteny and evolutionary patterns analyses of the non-synonymous (Ka) and synonymous (Ks) substitution rates and their ratios indicated that paralogous pairs of PhebZIP genes in moso bamboo underwent a large-scale genome duplication event that occurred 7-15 million years ago (MYA). According to promoter sequence analysis, we further selected 18 genes which contain the higher number of cis -regulatory elements for expression analysis. The result showed that these genes are extensively involved in GA-, ABA- and MeJA-responses, with possibly different mechanisms. The tissue-specific expression profiles of PhebZIP genes in five plant tissues/organs/developmental stages suggested that these genes are involved in moso bamboo organ development, especially seed development. Subcellular localization and transactivation activity analysis showed that PhebZIP47 and PhebZIP126 were localized in the nucleus and PhebZIP47 with no transcriptional activation in yeast. Our research provides a comprehensive understanding of PhebZIP genes and may aid in the selection of appropriate candidate genes for further cloning and functional analysis in moso bamboo growth and development, and improve their resistance to stress during their life.

Auxin is an important hormone in many plant developmental processes. In this study, the auxin/indole acetic acid (Aux/IAA) gene family was comprehensively identified using Dendrocalamus sinicus transcriptome data. A total of 26 Aux/IAA genes (DsIAA1-DsIAA26) were mined using four conserved Aux/IAA family motifs (PF02309). They encoded hydrophilic proteins, including one or two nuclear localisation signals. The D. sinicus Aux/IAA proteins were classified into two groups, including seven sister-gene pairs based on their phylogenetic relationships. A phylogenetic tree generated by aligning 108 predicted protein sequences of 26 DsIAAs, 43 PhIAAs (Phyllostachys heterocycla), 29 AtIAAs (Arabidopsis), 31 OsIAAs (Oryza sativa) and 22 PtIAAs (Populus) revealed nine major groups. Among them, four groups, including 96 IAA proteins of all five species, suggested that the genes originated before divergence of monocots and dicots. The expression profiling in different tissues showed that most of the DsIAAs preferentially expressed in leaves and shoots, suggesting their important roles in the development of leaves and shoots in D. sinicus. Continuously high expression of DsIAA3, DsIAA4, DsIAA15, and DsIAA20 may be important for regulating shoot development in D. sinicus. These results provide useful information for further research into the function of Aux/IAA genes in woody sympodial bamboos.

Brassinosteroids (BRs) are a group of plant steroid hormones that play crucial roles in a range of plant growth and development processes. BR action includes active BR formation by a complex biosynthesis process and driving BR biological function through signal transduction. Although the characterization of several BR action-related genes has been conducted in a few model plants, systematic information about these genes in bamboo is still lacking. We identified 64 genes related to BR action from the genome of moso bamboo (Phyllostachys edulis), including twenty that participated in BR biosynthesis and forty-four involved in BR signal transduction. The characteristics of all these candidate genes were identified by bioinformatics methods, including the gene structures, basic physical and chemical properties of proteins, conserved domains and evolutionary relationships. Based on the transcriptome data, the candidate genes demonstrated different expression patterns, which were further validated by qRT-PCR using templates from bamboo shoots with different heights. Thirty-four positive and three negative co-expression modules were identified by 44 candidate genes in the newly emerging bamboo shoot. The gene expression patterns and co-expression modules of BR action-related genes in bamboo shoots indicated that they might function to promote bamboo growth through BR biosynthesis and signal transduction processes. This study provides the first step towards the cloning and functional dissection of the role of BR action-related genes in moso bamboo, which also presents an excellent opportunity for genetic engineering using the candidate genes to improve bamboo quantity and quality.

Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants., (© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Moso bamboo (Phyllostachys edulis) is a well-known bamboo species of high economic value in the textile industry due to its rapid growth. Phytohormones, which are master regulators of growth and development, serve as important endogenous signals. However, the mechanisms through which phytohormones regulate growth in moso bamboo remain unknown to date., Results: Here, we reported that exogenous gibberellins (GA) applications resulted in a significantly increased internode length and lignin condensation. Transcriptome sequencing revealed that photosynthesis-related genes were enriched in the GA-repressed gene class, which was consistent with the decrease in leaf chlorophyll concentrations and the lower rate of photosynthesis following GA treatment. Exogenous GA applications on seedlings are relatively easy to perform, thus we used 4-week-old whole seedlings of bamboo for GA- treatment followed by high throughput sequencing. In this study, we identified 932 cis-nature antisense transcripts (cis-NATs), and 22,196 alternative splicing (AS) events in total. Among them, 42 cis-nature antisense transcripts (cis-NATs) and 442 AS events were differentially expressed upon exposure to exogenous GA 3 , suggesting that post-transcriptional regulation might be also involved in the GA 3 response. Targets of differential expression of cis-NATs included genes involved in hormone receptor, photosynthesis and cell wall biogenesis. For example, LAC4 and its corresponding cis-NATs were GA 3 -induced, and may be involved in the accumulation of lignin, thus affecting cell wall composition., Conclusions: This study provides novel insights illustrating how GA alters post-transcriptional regulation and will shed light on the underlying mechanism of growth modulated by GA in moso bamboo.

The Dof transcription factor (TF) family belongs to a class of plant-specific TFs and is involved in plant growth, development, and response to abiotic stresses. However, there are only very limited reports on the characterization of Dof TFs in moso bamboo (Phyllostachys edulis). In the present research, PheDof TFs showed specific expression profiles based on RNA-seq data analyses. The co-expression network indicated that PheDof12, PheDof14, and PheDof16 might play vital roles during flower development. Cis-regulatory element analysis of these PheDof genes suggested diverse functions. Expression patterns of 12 selected genes from seven different classes under three abiotic stresses (cold, salt, and drought) are further investigated by quantitative real-time PCR. This work will provide useful information for functional analysis and regulation mechanisms of Dof TFs in moso bamboo.

A central aim of this microbial ecology research was to investigate the mechanisms shaping the assembly of soil microbial communities. Despite the importance of bacterial and fungal mediation of carbon cycling in forest ecosystems, knowledge concerning their distribution patterns and underlying mechanisms remains insufficient. Here, soils were sampled from six bamboo forests across the main planting area of Moso bamboo in southern China. The bacterial and fungal diversities were assessed by sequencing 16S rRNA and ITS gene amplicons, respectively, with an Illumina MiSeq. Based on structural equation modelling, dispersal limitation had strongest impact on bacterial beta diversity, while the mean annual precipitation had a smaller impact by directly or indirectly mediating the soil organic carbon density. However, only the mean annual temperature and precipitation played direct roles in fungal beta diversity. Moreover, the co-occurrence network analyses revealed a possibly much higher network connectivity in the fungal network than in the bacteria. With less dispersal limitation, stronger environmental selection and a potentially more connected network, the fungal community had more important roles in the soil carbon metabolisms in bamboo forests. Fungal beta diversity and the clustering coefficient explained approximately 14.4% and 6.1% of the variation in the carbon metabolic profiles among sites, respectively, but that of bacteria only explained approximately 1.7% and 1.8%, respectively. This study explored soil microbial spatial patterns along with the underlying mechanisms of dispersal limitation, selection and connectivity of ecological networks, thus providing novel insights into the study of the distinct functional traits of different microbial taxa., (© 2017 John Wiley & Sons Ltd.)

Bamboo and rattan are widely grown for manufacturing, horticulture, and agroforestry. Bamboo and rattan production might help reduce poverty, boost economic growth, mitigate climate change, and protect the natural environment. Despite progress in research, sufficient molecular and genomic resources to study these species are lacking. We launched the Genome Atlas of Bamboo and Rattan (GABR) project, a comprehensive, coordinated international effort to accelerate understanding of bamboo and rattan genetics through genome analysis. GABR includes 2 core subprojects: Bamboo-T1K (Transcriptomes of 1000 Bamboos) and Rattan-G5 (Genomes of 5 Rattans), and several other subprojects. Here we describe the organization, directions, and status of GABR., (© The Authors 2017. Published by Oxford University Press.)

The unavailability of the reproductive structure and unpredictability of vegetative characters for the identification and phylogenetic study of bamboo prompted the application of molecular techniques for greater resolution and consensus. We first employed internal transcribed spacer (ITS1, 5.8S rRNA and ITS2) sequences to construct the phylogenetic tree of 21 tropical bamboo species. While the sequence alone could grossly reconstruct the traditional phylogeny amongst the 21-tropical species studied, some anomalies were encountered that prompted a further refinement of the phylogenetic analyses. Therefore, we integrated the secondary structure of the ITS sequences to derive individual sequence-structure matrix to gain more resolution on the phylogenetic reconstruction. The results showed that ITS sequence-structure is the reliable alternative to the conventional phenotypic method for the identification of bamboo species. The best-fit topology obtained by the sequence-structure based phylogeny over the sole sequence based one underscores closer clustering of all the studied Bambusa species (Sub-tribe Bambusinae), while Melocanna baccifera, which belongs to Sub-Tribe Melocanneae, disjointedly clustered as an out-group within the consensus phylogenetic tree. In this study, we demonstrated the dependability of the combined (ITS sequence+structure-based) approach over the only sequence-based analysis for phylogenetic relationship assessment of bamboo.

The moso bamboo genome contains the two structurally intact and thus potentially functional mariner-like elements Ppmar1 and Ppmar2. Both elements contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase gene. Here we investigated whether Ppmar1 is functional in yeast (Saccharomyces cerevisiae). We have designed a two-component system consisting of a transposase expression cassette and a non-autonomous transposon on two separate plasmids. We demonstrate that the Ppmar1 transposase Pptpase1 catalyses excision of the non-autonomous Ppmar1NA element from the plasmid and reintegration at TA dinucleotide sequences in the yeast chromosomes. In addition, we generated 14 hyperactive Ppmar1 transposase variants by systematic single amino acid substitutions. The most active transposase variant, S171A, induces 10-fold more frequent Ppmar1NA excisions in yeast than the wild type transposase. The Ppmar1 transposon is a promising tool for insertion mutagenesis in moso bamboo and may be used in other plants as an alternative to the established transposon tagging systems., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ty3-gypsy long-terminal repeat retroelements are ubiquitously found in many plant genomes. This study reports the occurrence of heterogeneous Ty3-gypsy retroelements in four representative bamboo species: Phyllostachys heterocycla (Carr.) Mitford cv. pubescens, P. heterocycla (Carr.) Mitford cv. heterocycla, Dendrocalamopsis oldhami, and Pleioblastus fortunei. Using degenerate oligonucleotide primers corresponding to the conserved domains of reverse transcriptase (rt) genes of Ty3-gypsy retroelements, 165 distinct sequences were amplified from genomic DNA. The length of the nucleotide sequences varied from 366 to 438 bp. The sequences demonstrated a high heterogeneity, with homology ranging from 52.2 to 99.8%. A phylogenetic tree was constructed, including Arabidopsis thaliana and Oryza sativa. Bamboo Ty3-gypsy sequences formed three distinct retroelement clusters (gypsy I-III). Further analysis indicated that there were not only nearly identical Ty3-gypsy retroelements found in distantly related species, but also highly diverse Ty3-gypsy retroelements observed in closely related species. The results of this study provide genetic and evolutionary information about the bamboo genome that could contribute to further studies of repetitive elements in bamboo as well as in other species.

GRAS family is one of plant specific transcription factors and plays diverse roles in the regulation of plant growth and development as well as in the plant disease resistance and abiotic stress responses. However, the investigation of GRAS family and multi-tissue gene expression profiles still remains unavailable in bamboo (Phyllostachys edulis). Here, we applied RNA-Seq analysis to monitor global transcriptional changes and investigate expression patterns in the five tissues of Ph. edulis, and analyzed a large-scale transcriptional events and patterns. Moreover, the tissue-specific genes and DEGs in different tissues were detected. For example, DEGs in panicle and leaf tissues were abundant in photosynthesis, glutathione, porphyrin and chlorophyll metabolism, whereas those in shoot and rhizome were majority in glycerophospholipid metabolism. In the portion of Ph. edulis GRAS (PeGRAS) analyses, we performed the analysis of phylogenetic, gene structure, conserved motifs, and analyzed the expression profiles of PeGRASs in response to high light and made a co-expression analysis. Additionally, the expression profiles of PeGRASs were validated using quantitative real-time PCR. Thus, PeGRASs based on dynamics profiles of gene expression is helpful in uncovering the specific biological functions which might be of critical values for bioengineering to improve bamboo breeding in future.

Dendrocalamus sinicus is the world's largest bamboo species with strong woody culms, and known for its fast-growing culms. As an economic bamboo species, it was popularized for multi-functional applications including furniture, construction, and industrial paper pulp. To comprehensively elucidate the molecular processes involved in its culm elongation, Illumina paired-end sequencing was conducted. About 65.08 million high-quality reads were produced, and assembled into 81,744 unigenes with an average length of 723 bp. A total of 64,338 (79%) unigenes were annotated for their functions, of which, 56,587 were annotated in the NCBI non-redundant protein database and 35,262 were annotated in the Swiss-Prot database. Also, 42,508 and 21,009 annotated unigenes were allocated to gene ontology (GO) categories and clusters of orthologous groups (COG), respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 33,920 unigenes were assigned to 128 KEGG pathways. Meanwhile, 8,553 simple sequence repeats (SSRs) and 81,534 single-nucleotide polymorphism (SNPs) were identified, respectively. Additionally, 388 transcripts encoding lignin biosynthesis were detected, among which, 27 transcripts encoding Shikimate O-hydroxycinnamoyltransferase (HCT) specifically expressed in D. sinicus when compared to other bamboo species and rice. The phylogenetic relationship between D. sinicus and other plants was analyzed, suggesting functional diversity of HCT unigenes in D. sinicus. We conjectured that HCT might lead to the high lignin content and giant culm. Given that the leaves are not yet formed and culm is covered with sheaths during culm elongation, the existence of photosynthesis of bamboo culm is usually neglected. Surprisedly, 109 transcripts encoding photosynthesis were identified, including photosystem I and II, cytochrome b6/f complex, photosynthetic electron transport and F-type ATPase, and 24 transcripts were characterized as antenna proteins that regarded as the main tool for capturing light of plants, implying stem photosynthesis plays a key role during culm elongation due to the unavailability of its leaf. By real-time quantitative PCR, the expression level of 6 unigenes was detected. The results showed the expression level of all genes accorded with the transcriptome data, which confirm the reliability of the transcriptome data. As we know, this is the first study underline the D. sinicus transcriptome, which will deepen the understanding of the molecular mechanisms of culm development. The results may help variety improvement and resource utilization of bamboos.

Key Message: PeSNAC1 , a stress-related NAC1 from Phyllostachys edulis , was characterized. Ectopic expression in Arabidopsis indicated that PeSNAC1 together with ped -miR164b participated in the regulation of organ boundaries and stress tolerance. NAC (NAM, ATAF1/2 and CUC2) participates in many different processes regulating plant growth, development, and stress response. A total of 125 NAC genes have been predicted in moso bamboo (Phyllostachys edulis), but their roles are poorly understood. PeSNAC1 targeted by ped-miR164b was focused for further study. The cleavage of PeSNAC1 mRNA guided by ped-miR164b was validated using RLM-5' RACE. Tissue-specific expression analysis demonstrated that ped-miR164b had a declining trend from root, sheath, leaf, to that of stem, which was opposite to that of PeSNAC1. Transgenic Arabidopsis plants overexpressing either PeSNAC1 (OX-PeSNAC1) or, ped-miR164b (OX-ped-miR164b) driven by the CaMV35S promoter were generated. OX-ped-miR164b plants showed similar phenotype of cuc2 mutants whose growth was seriously suppressed. Compared with Col-0, sense OX-PeSNAC1 plants grew rapidly and flowered earlier, whereas antisense plants grew slowly and exhibited delayed flowering. Sense OX-PeSNAC1 plants had the greatest number of lateral roots, while antisense OX-PeSNAC1 and OX-ped-miR164b plants had fewer lateral roots than Col-0. Under NaCl and PEG6000 stresses, survival rates were higher and F v/F m values declined more slowly in sense OX-PeSNAC1 plants than in Col-0, with lower survival rates and a more rapid decrease in F v/F m values conversely observed in antisense OX-PeSNAC1 and OX-ped-miR164b plants. These findings indicated that ped-miR164b-targeted PeSNAC1 may play key roles in plant development and tolerance to salinity and drought stresses.

Bamboo is one of the important plant for pulp, paper and charcoal industries. After China, India is the second largest bamboo reserve in Asia. Around the globe, wide genetic diversity of bamboo is present which serves as the base for selection and improvement. DNA based molecular markers appears to be a striking substitute for systematic assessment of the genetic diversity in conservation and genetic improvement of plants. DNA based molecular markers such as RAPD and ISSR were used to assess the genetic diversity in 13 bamboo genotypes. Total 120 RAPD and 63 ISSR primers were tested, of which only 42 polymorphic primers (30 RAPD and 12 ISSR), gave reproducible amplification profile and were used in this study. 30 RAPD primers yielded total 645 amplified fragments, of which 623 were polymorphic, and 20.76 polymorphic bands per primer were observed across 13 genotypes. 12 ISSR primers produced 246 amplified fragments, of which 241 were polymorphic, and 20.08 polymorphic bands per primer was observed across 13 different genotypes. The Jaccard's coefficient of RAPD, ISSR and pooled RAPD and ISSR dendrograms ranged from 0.26 to 0.83, 0.23 to 0.86 and 0.26 to 0.84 respectively. The present study found the large genetic diversity present between different elite genotypes of bamboo. Such investigation can deliver a well understanding of the available genotypes, which might be further exploited for the paper industry.

Bamboo, a fast-growing grass, has a higher strength-to-weight ratio than steel and concrete. The unique properties of bamboo come from the natural composite structure of fibers that consists mainly of cellulose microfibrils in a matrix of intertwined hemicellulose and lignin called lignin-carbohydrate complex (LCC). Here, we have used atomistic simulations to study the mechanical properties of and adhesive interactions between the materials in bamboo fibers. With this aim, we have developed molecular models of lignin, hemicellulose and LCC structures to study the elastic moduli and the adhesion energies between these materials and cellulose microfibril faces. Good agreement was observed between the simulation results and experimental data. It was also shown that the hemicellulose model has stronger mechanical properties than lignin while lignin exhibits greater tendency to adhere to cellulose microfibrils. The study suggests that the abundance of hydrogen bonds in hemicellulose chains is responsible for improving the mechanical behavior of LCC. The strong van der Waals forces between lignin molecules and cellulose microfibril is responsible for higher adhesion energy between LCC and cellulose microfibrils. We also found out that the amorphous regions of cellulose microfibrils are the weakest interfaces in bamboo fibrils. Hence, they determine the fibril strength.

The AP2/ERF transcription factor family, one of the largest families unique to plants, performs a significant role in terms of regulation of growth and development, and responses to biotic and abiotic stresses. Moso bamboo (Phyllostachys edulis) is a fast-growing non-timber forest species with the highest ecological, economic and social values of all bamboos in Asia. The draft genome of moso bamboo and the available genomes of other plants provide great opportunities to research global information on the AP2/ERF family in moso bamboo. In total, 116 AP2/ERF transcription factors were identified in moso bamboo. The phylogeny analyses indicated that the 116 AP2/ERF genes could be divided into three subfamilies: AP2, RAV and ERF; and the ERF subfamily genes were divided into 11 groups. The gene structures, exons/introns and conserved motifs of the PeAP2/ERF genes were analyzed. Analysis of the evolutionary patterns and divergence showed the PeAP2/ERF genes underwent a large-scale event around 15 million years ago (MYA) and the division time of AP2/ERF family genes between rice and moso bamboo was 15-23 MYA. We surveyed the putative promoter regions of the PeDREBs and showed that largely stress-related cis-elements existed in these genes. Further analysis of expression patterns of PeDREBs revealed that the most were strongly induced by drought, low-temperature and/or high salinity stresses in roots and, in contrast, most PeDREB genes had negative functions in leaves under the same respective stresses. In this study there were two main interesting points: there were fewer members of the PeDREB subfamily in moso bamboo than in other plants and there were differences in DREB gene expression profiles between leaves and roots triggered in response to abiotic stress. The information produced from this study may be valuable in overcoming challenges in cultivating moso bamboo.

Morphology-based taxonomy via exiguously reproductive organ has severely limitation on bamboo taxonomy, mainly owing to infrequent and unpredictable flowering events of bamboo. Here, we present the first genome-wide analysis and application of microsatellites based on the genome of moso bamboo (Phyllostachys edulis) to assist bamboo taxonomy. Of identified 127,593 microsatellite repeat-motifs, the primers of 1,451 microsatellites were designed and 1,098 markers were physically mapped on the genome of moso bamboo. A total of 917 markers were successfully validated in 9 accessions with ~39.8% polymorphic potential. Retrieved from validated microsatellite markers, 23 markers were selected for polymorphic analysis among 78 accessions and 64 alleles were detected with an average of 2.78 alleles per primers. The cluster result indicated the majority of the accessions were consistent with their current taxonomic classification, confirming the suitability and effectiveness of the developed microsatellite markers. The variations of microsatellite marker in different species were confirmed by sequencing and in silico comparative genome mapping were investigated. Lastly, a bamboo microsatellites database (http://www.bamboogdb.org/ssr) was implemented to browse and search large information of bamboo microsatellites. Consequently, our results of microsatellite marker development are valuable for assisting bamboo taxonomy and investigating genomic studies in bamboo and related grass species.

Amplified fragment length polymorphism (AFLP) fingerprints were used to reveal genotypic diversity of dwarf bamboo (Bashania fangiana) clonal populations with two different genet ages (≤30 years versus >70 years) at Wolong National Natural Reserve, Sichuan province, China. We generated AFLP fingerprints for 96 leaf samples, collected at 30 m intervals in the two populations, using ten selective primer pairs. A total of 92 genotypes were identified from the both populations. The mean proportion of distinguishable genotypes (G/N) was 0.9583 (0.9375 to 0.9792) and Simpson's index of diversity (D) was 0.9982 (0.9973 to 0.9991). So, two B. fangiana populations were multiclonal and highly diverse. The largest single clone may occur over a distance of about 30 m. Our results demonstrated that the genotypic diversity and genet density of B. fangiana clonal population did not change significantly (47 versus 45) with genet aging and low partitioned genetic differentiation was between the two populations (Gst = 0.0571). The analysis of molecular variance consistently showed that a large proportion of the genetic variation (87.79%) existed among the individuals within populations, whereas only 12.21% were found among populations. In addition, the high level of genotypic diversity in the two populations implies that the further works were needed to investigate the reasons for the poor seed set in B. fangiana after flowering.

MicroRNAs (miRNAs), a class of non-coding small endogenous RNAs of approximately 22 nucleotides, regulate gene expression at the post-transcriptional levels by targeting mRNAs for degradation or by inhibiting protein translation. Thousands of miRNAs have been identified in many species. However, there is no information available concerning miRNAs in ma bamboo (Dendrocalamus latiflorus), one of the most important non-timber forest products, which has essential ecological roles in forests. To identify miRNAs in D. latiflorus, a small RNA library was constructed from leaf tissues. Using next generation high-throughput sequencing technology and bioinformatics analysis, we obtained 11,513,607 raw sequence reads and identified 84 conserved miRNAs (54 mature miRNAs and 30 star miRNAs) belonging to 17 families, and 81 novel miRNAs (76 mature miRNAs and five star miRNAs) in D. latiflorus. One hundred and sixty-two potential targets were identified for the 81 novel bamboo miRNAs. Several targets for the novel miRNAs are transcription factors that play important roles in plant development. Among the novel miRNAs, 30 were selected and their expression profiles in response to different light conditions were validated by qRT-PCR. This study provides the first large-scale cloning and characterization of miRNAs in D. latiflorus. Eighty-four conserved and 81 novel miRNAs were identified in D. latiflorus. Our results present a broad survey of bamboo miRNAs based on experimental and bioinformatics analysis. Although it will be necessary to validate the functions of miRNAs by further experimental research, these results represent a starting point for future research on D. latiflorus and related species.

Dendrocalamus membranaceus Munro is a woody bamboo with a high economic and ecological value that often occurs as natural stands, such as in the large-scale forested areas of China's Yunnan Province. Due to its overexploitation, the habitat of D. membranaceus in Yunnan has been dramatically reduced, and the quality of the stands has declined. As a preliminary analysis in considering the effective protection for these germplasm resources, we assessed the genetic diversity of 12 natural populations in Yunnan, using inter-simple sequence repeat (ISSR) markers. From 10 ISSR primers, we generated 155 bands, of which 153 were polymorphic (98.71%). Compared with other species in the genus, this species demonstrated a greater genetic diversity (S = 0.349) and lower genetic differentiation (G(ST) = 0.252). Our analysis of molecular variance revealed that the genetic differentiation among the populations is significant. A large proportion of the genetic variation (78.95%) resides among the individuals within populations, whereas only 21.05% are found among populations. Mantel tests indicated no significant correlation between genetic and geographic distances among the populations. Given the low sexual reproducibility and characteristics of monocarpic plants, we recommend implementing in situ conservation measures for all of the D. membranaceus populations in Yunnan and collecting sufficient samples for ex situ conservation. Furthermore, the conservation area should be extended to its main natural habitats, the Lancang-Mekong River Valley.

We estimated the outcrossing rates at small-scale flowering sites of an endemic dwarf bamboo species, Sasa cernua. The multi-locus estimation of the outcrossing rate of the dwarf bamboo population was 0.148 (SD 0.118). Two culms with the highest outcrossing rates had heterozygous genotypes at one locus, but other culms in the locus were homozygotes. Five culms with high outcrossing rates bore 2-17% seeds with homozygous genotypes. Due to predominant selfing, the overall inbreeding coefficient of seeds was high, although it declined in seedlings. This suggests that selection against inbred progenies began early in the establishment process in the natural habitat.

Over the years, the wetlands covered by Sphagnum in Bibai, Japan have been turning into areas of aridity, resulting in an invasion of Sasa into the bogs. Yet little is known about the methane-cycling microorganisms in such environments. In this study, the methanotrophic, methanogenic, and archaeal community structures within these two types of wetland vegetation were studied by phylogenetic analysis targeting particulate methane monooxygenase (pmoA), methyl coenzyme M reductase (mcrA), and the archaeal 16S rRNA gene. The pmoA library indicated that Methylomonas and Methylocystis predominated in the Sphagnum-covered and Sasa-invaded areas, respectively. The mcrA and 16S rRNA libraries indicated that Methanoregula were abundant methanogens in the Sphagnum-covered area. In the Sasa-invaded area, by contrast, mcrA genes were not detected, and no 16S rRNA clones were affiliated with previously known methanogens. Because the Sasa-invaded area still produced methane, of the various uncultured populations detected, novel euryarchaeotal lineages are candidate methane producers.

Ty1-copia retroelements have been found in all major plants and are largely responsible for the huge differences in the genome size. In this study we isolated and sequenced Ty1-copia reverse transcriptase (rt) gene fragments from 44 representative species of bamboo and nine cultivars or forms of Phyllostachys pubescens. Phylogenetic analysis of 72 distinct Ty1-copia rt sequences showed that Ty1-copia retroelements were widespread, diverse and abundant in these species of Bambusoideae subfamily. In addition, a molecular phylogeny of the species of the Bambusoideae subfamily was established by using the internal transcribed spacer sequences of nuclear ribosomal DNA (ITS) sequences. The comparison between ITS- and Ty1-copia rt- based trees is obviously incongruent. The results suggested either the existence of horizontal transfer events between phylogenetically distant species, or an ancestral Ty1-copia retroelement polymorphism followed by different evolution and stochastic losses.

Dwarf bamboos in the genus Sasa are believed to be long-lived, synchronously flowering, and monocarpic plants. However, the monocarpy of dwarf bamboo has not been confirmed, because whether all ramets within one genet flower at the same time cannot be determined without differentiating the genetic structure among ramets. This study aims to evaluate the reproductive traits of Sasa pubiculmis by verifying the monocarpy and physiological integration between flowering ramets and non-flowering ramets during a 4-year flowering period. One genotypically identified genet, which covered an area of approximately 3 ha, had both flowering and non-flowering patches of ramets during the 4-year flowering period (2004-2007). A fraction of the flowering genet remained non-flowering during the 4 years of observation, and did not die after mass flowering. Flowering ramets were physically connected to non-flowering ramets via rhizomes, and assimilated (13)C was allocated from non-flowering ramets to flowering ramets. Consequently, we clarified that this dwarf bamboo potentially has polycarpic reproductive traits rather than monocarpic, and a genet can keep rhizomes and non-flowering patches alive to sustain the organism after mass flowering.

Dwarf bamboo species are monocarpic. They flower simultaneously and die after several decades. The type of flowering in the genus Sasa ranges from sporadic to gregarious. In order to determine whether or not the sporadic flowering of dwarf bamboo is fixed genetically, we investigated the distribution of clones using eight microsatellite (SSR) loci in a sporadic flowering patch of Sasa cernua Makino, a major dwarf bamboo species found in central Hokkaido. In May 2006, flowering occurred on 60.5% of living culms in a 1600 m(2) patch. We established a 50 x 10 m study plot in this patch and noted 1267 clumps consisting of 2529 living culms. We investigated all 1267 clumps and identified six multilocus genotypes as clones using five variable SSR loci. All flowering clumps belonged to the same clone. On the other hand, non-flowering vegetative clumps were also discovered to be of the same clone. These data suggest that all flowering culms originated from a single clone of a sporadic flowering patch of S. cernua. Clonal analysis for investigation of sporadic flowering of S. cernua revealed that only a portion of a clone flowers and dies instead of the whole clone.

Simple sequence repeat (SSR) markers are valuable tools for many purposes such as phylogenetic, fingerprinting, and molecular breeding studies. However, only a few SSR markers are known and available in bamboo species of the tropics (Bambusa spp.). Considering that grass genomes have co-evolved and share large-scale synteny, theoretically it should be possible to use the genome sequence based SSR markers of field crops such as rice (Oryza sativa) and sugarcane (Saccharum spp.) for genome analysis in bamboo. To test this, 98 mapped SSR primers representing 12 linkage groups of rice and 20 EST-derived sugarcane SSR primers were evaluated for transferability to 23 bamboo species. Of the tested markers, 44 (44.9%) rice and 15 (75%) sugarcane SSR primers showed repeatable amplification in at least one species of bamboo and thus were successfully utilized for phylogenetic and genetic diversity analyses. Transferred SSR primers revealed complex amplification patterns in bamboo, with an average of 9.62 fragments per primer, indicating a high level of polyploidy and genetic variability in bamboo. Forty-two of these primers (34 rice and 8 sugarcane SSR primers) detected an average of 2.12 unique fragments per primer and thus could be exploited for species identification. Six bamboo SSR primers exhibited cross transferability, to varying degrees, to different bamboo species. The genetic similarity coefficient indicated a high level of divergence at the species level (73%). However, a relatively low level of diversity was observed within species (25% in 20 accessions of Dendrocalamus hamiltonii). Further, cluster analysis revealed that the major grouping was in accordance with the taxonomical classification of bamboo. Thus, the rice and sugarcane SSRs can be utilized for phylogenetic and genetic diversity studies in bamboo.

Moso bamboo (Phyllostachys pubescens) is one of the world's most important bamboo species. It has the largest area of all planted bamboo--over two-thirds of the total bamboo forest area--and the highest economic value in China. Moso bamboo is a tetraploid (4x=48) and a special member of the grasses family. Although several genomes have been sequenced or are being sequenced in the grasses family, we know little about the genome of the bambusoids (bamboos). In this study, the moso bamboo genome size was estimated to be about 2034 Mb by flow cytometry (FCM), using maize (cv. B73) and rice (cv. Nipponbare) as internal references. The rice genome has been sequenced and the maize genome is being sequenced. We found that the size of the moso bamboo genome was similar to that of maize but significantly larger than that of rice. To determine whether the bamboo genome had a high proportion of repeat elements, similar to that of the maize genome, approximately 1000 genome survey sequences (GSS) were generated. Sequence analysis showed that the proportion of repeat elements was 23.3% for the bamboo genome, which is significantly lower than that of the maize genome (65.7%). The bamboo repeat elements were mainly Gypsy/DIRS1 and Ty1/Copia LTR retrotransposons (14.7%), with a few DNA transposons. However, more genomic sequences are needed to confirm the above results due to several factors, such as the limitation of our GSS data. This study is the first to investigate sequence composition of the bamboo genome. Our results are valuable for future genome research of moso and other bamboos.

Kuma-zasa is Japanese folk medicine derived from plants of genus Sasa, family Bambusaceae. Although the plants of origin of Kuma-zasa were reported to be Sasa palmata, S. senanensis, S. yahikoensis, and S. kurilensis, authentication of those plants was difficult because of similarity in morphology. Several methods for the classification of genus Sasa are available, but none involve a genetic approach. Here, we performed the genetic profiling of genus Sasa, including the four species used medicinally. Thirteen sequences were observed in chloroplast DNA intron between rbcL and ORF106 and partial ORF106 regions of 34 specimens of 16 Sasa species and one specimen of Phyllostachys pubescens. We observed differences in alignment in this region among the specimens. The analyzed lengths varied from 759 to 821 bp depending on the specimen. There were nine base substitutions, eight successive thymines or adenines, and one to three repeat units of 31 bp. Moreover, we could not find species-specific alignment: different alignments were observed in specimens of the same species, while the same alignment was observed in specimens of different species. In the phylogenetic tree reconstructed by maximum parsimony analysis, medicinally used species did not form a cluster, although most of them were positioned close to each other. The genetic profiling of Sasa species would be of use in determining the botanical origin of the herbal medicine derived from the leaves of Sasa plants.