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Bamboo is one of the fastest-growing plants commonly used in food, fibre, paper, biofuel, ornamental and medicinal industries. Natural hybridization in bamboo is rare due to its long vegetative period followed by gregarious flowering and death of the entire population. In the current study, a new bamboo species, Bambusa changningensis, shows intermediate characteristics of Dendrocalamus farinosus and B. rigida morphologically, but it is unknown whether B. changningensis is a natural hybrid. Moreover, B. changningensis has been identified as a superior variety of Sichuan Province with high pulping yield, fibre length and width. Therefore, we analyzed the morphological characteristics, DNA markers, DNA barcoding and chloroplast genomes to identify the hybrid origin of B. changningensis and possible maternal parent. We have developed the transcriptomic data for B. changningensis and mined the SSR loci. The putative parental lines and hybrid were screened for 64 SSR makers and identified that SSR14, SSR28, SSR31 and SSR34 markers showed both alleles of the parental species in B. changningensis, proving heterozygosity. Sequencing nuclear gene GBSSI partial regions and phylogenetic analysis also confirm the hybrid nature of B. changningensis. Further, we have generated the complete chloroplast genome sequence (139505 bp) of B. changningensis. By analyzing the cp genomes of both parents and B. changningensis, we identified that B. rigida might be the female parent. In conclusion, our study identified that B. changningensis is a natural hybrid, providing evidence for bamboo’s natural hybridization. This is the first report on confirming a natural bamboo hybrid and its parents through SSR and chloroplast genome sequence.

Bamboo is an important model plant to study the molecular mechanisms of rapid shoot growth and flowering once in a lifetime. However, bamboo research about protein functional characterization is largely lagged behind, mainly due to the lack of gene transformation platforms. In this study, a protoplast transient gene expression system in moso bamboo has been first established. Using this reliable and efficient system, we have generated a set of multicolored fluorescent markers based on the targeting sequences from endogenous proteins, which have been validated by their comparative localization with Arabidopsis organelle markers, in a combination with pharmaceutical treatments. Moreover, we further demonstrated the power of this multicolor marker set for rapid, combinatorial analysis of the subcellular localization of uncharacterized proteins, which may play potential functions in moso bamboo flowering and fast growth of shoots. Finally, this protoplast transient gene expression system has been elucidated for functional analysis in protein–protein interaction by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. Taken together, in combination with the set of moso bamboo organelle markers, the protoplast transient gene expression system could be used for subcellular localization and functional study of unknown proteins in bamboo and will definitely promote rapid progress in diverse areas of research in bamboo plants.

Bamboo are woody grass species containing important economic and ecological values. Lei bamboo (Phyllostachys violascens) is a kind of shoot-producing bamboo species with the highest economic yield per unit area. However, identifying different varieties of Lei bamboo based on morphological characteristics is difficult. Microsatellites play an important role in plant identification and genetic diversity analysis and are superior to other molecular markers. In this study, we identified 18,356 expressed sequence tag-simple sequence repeat (EST-SSR) loci in Lei bamboo transcriptome data. A total of 11,264 primer pairs were successfully designed from unigenes of all EST-SSR loci, and 96 primer pairs were randomly selected and synthesized. A total of 54 primer pairs were used for classifying 16 Lei bamboo varieties and 10 different Phyllostachys species. The number of polymorphism alleles among the 54 primer pairs ranged from 3 to 12 for P. violascens varieties and 3 to 20 for Phyllostachys. The phylogenetic tree based on polymorphism alleles successfully distinguished 16 P. violascens varieties and 10 Phyllostachys species. Our study provides abundant EST-SSR resources that are useful for genetic diversity analysis and molecular verification of bamboo and suggests that SSR markers developed from Lei bamboo are more efficient and reliable than ISSR, SRAP or AFLP markers.

14-3-3 Proteins are a family of highly conserved regulatory molecules expressed in all eukaryotic cells and regulate a diverse set of biological responses in plants. However, their functions in flowering of Phyllostachys violascens are poorly understood. In this study, four non-𝜀 Pv14-3-3 genes from P. violascens were identified and named PvGF14b, PvGF14c, PvGF14e, and PvGF14f. qRT-PCR analyses revealed that PvGF14b and PvGF14e exhibited widely expressed in all tested bamboo tissues. PvGF14b was highest expression in root and lowest in immature leaf. Whereas PvGF14c and PvGF14f showed tissue-specific expression. PvGF14c was mainly expressed in immature and mature leaves. PvGF14f was highest expression in mature leaves. These four genes were not significantly differentially expressed in mature leaf before bamboo flowering and during flower development. PvGF14b and PvGF14c were not induced by circadian rhythm. PvGF14c displayed subcellular localization in the cytoplasm and PvFT in nucleus and cytoplasm. Yeast two-hybrid screening and bimolecular fluorescence complementation confirmed the interaction between PvGF14c and PvFT. The overexpression of PvGF14b, PvGF14c, and PvGF14e significantly delayed flowering time in transgenic Arabidopsis under long-day condition. These findings suggested that at least three PvGF14 genes are involved in flowering and may act as a negative regulator of flowering by interacting with PvFT in bamboo.

Because of the long and unpredictable flowering period in bamboo, the molecular mechanism of bamboo flowering is unclear. Recent study showed that Arabidopsis PIN1-type parvulin 1 (Pin1At) is an important floral activator and regulates floral transition by facilitating the cis/trans isomerization of the phosphorylated Ser/Thr residues preceding proline motifs in suppressor of overexpression of CO 1 (SOC1) and agamous-like 24 (AGL24). Whether bamboo has a Pin1 homolog and whether it works in bamboo flowering are still unknown. In this study, we cloned PvPin1, a homolog of Pin1At, from Phyllostachys violascens (Bambusoideae). Bioinformatics analysis showed that PvPin1 is closely related to Pin1-like proteins in monocots. PvPin1 was widely expressed in all tested bamboo tissues, with the highest expression in young leaf and lowest in floral bud. Moreover, PvPin1 expression was high in leaves before bamboo flowering then declined during flower development. Overexpression of PvPin1 significantly delayed flowering time by downregulating SOC1 and AGL24 expression in Arabidopsis under greenhouse conditions and conferred a significantly late flowering phenotype by upregulating OsMADS56 in rice under field conditions. PvPin1 showed subcellular localization in both the nucleus and cytolemma. The 1500-bp sequence of the PvPin1 promoter was cloned, and cis-acting element prediction showed that ABRE and TGACG-motif elements, which responded to abscisic acid (ABA) and methyl jasmonate (MeJA), respectively, were characteristic of P. violascens in comparison with Arabidopsis. On promoter activity analysis, exogenous ABA and MeJA could significantly inhibit PvPin1 expression. These findings suggested that PvPin1 may be a repressor in flowering, and its delay of flowering time could be regulated by ABA and MeJA in bamboo.

Bamboo is one of the fastest-growing plants commonly used in food, fibre, paper, biofuel, ornamental and medicinal industries. Natural hybridization in bamboo is rare due to its long vegetative period followed by gregarious flowering and death of the entire population. In the current study, a new bamboo species, Bambusa changningensis, shows intermediate characteristics of Dendrocalamus farinosus and B. rigida morphologically, but it is unknown whether B. changningensis is a natural hybrid. Moreover, B. changningensis has been identified as a superior variety of Sichuan Province with high pulping yield, fibre length and width. Therefore, we analyzed the morphological characteristics, DNA markers, DNA barcoding and chloroplast genomes to identify the hybrid origin of B. changningensis and possible maternal parent. We have developed the transcriptomic data for B. changningensis and mined the SSR loci. The putative parental lines and hybrid were screened for 64 SSR makers and identified that SSR14, SSR28, SSR31 and SSR34 markers showed both alleles of the parental species in B. changningensis, proving heterozygosity. Sequencing nuclear gene GBSSI partial regions and phylogenetic analysis also confirm the hybrid nature of B. changningensis. Further, we have generated the complete chloroplast genome sequence (139505 bp) of B. changningensis. By analyzing the cp genomes of both parents and B. changningensis, we identified that B. rigida might be the female parent. In conclusion, our study identified that B. changningensis is a natural hybrid, providing evidence for bamboo's natural hybridization. This is the first report on confirming a natural bamboo hybrid and its parents through SSR and chloroplast genome sequence. [ABSTRACT FROM AUTHOR]