Deciphering the regulatory role of PheSnRK genes in Moso bamboo: insights into hormonal, energy, and stress responses.

The SnRK (sucrose non-fermentation-related protein kinase) plays an important role in regulating various signals in plants. However, as an important bamboo shoot and wood species, the response mechanism of PheSnRK in Phyllostachys edulis to hormones, low energy and stress remains unclear. In this paper, we focused on the structure, expression, and response of SnRK to hormones and sugars. In this study, we identified 75 PheSnRK genes from the Moso bamboo genome, which can be divided into three groups according to the evolutionary relationship. Cis-element analysis has shown that the PheSnRK gene can respond to various hormones, light, and stress. The PheSnRK2.9 proteins were localized in the nucleus and cytoplasm. Transgenic experiments showed that overexpression of PheSnRK2.9 inhibited root development, the plants were salt-tolerant and exhibited slowed starch consumption in Arabidopsis in the dark. The results of yeast one-hybrid and dual luciferase assay showed that PheIAAs and PheNACs can regulate PheSnRK2.9 gene expression by binding to the promoter of PheSnRK2.9. This study provided a comprehensive understanding of PheSnRK genes of Moso bamboo, which provides valuable information for further research on energy regulation mechanism and stress response during the growth and development of Moso bamboo.

Chromosomal-level genome and metabolome analyses of highly heterozygous allohexaploid Dendrocalamus brandisii elucidate shoot quality and developmental characteristics.

Dendrocalamus brandisii (Munro) Kurz is a sympodial bamboo species with inimitable taste and flavorful shoots. Its rapid growth and use as high-quality material make this bamboo species highly valued for both food processing and wood applications. However, genome information for D. brandisii is lacking, primarily due to its polyploidy and large genome size. Here, we assembled a high-quality genome for hexaploid D. brandisii, which comprises 70 chromosomes with a total size of 2,756 Mb, using long-read HiFi sequencing. Furthermore, we accurately separated the genome into its three constituent subgenomes. We used Oxford Nanopore Technologies long reads to construct a transcriptomic dataset covering 15 tissues for gene annotation to complement our genome assembly, revealing differential gene expression and post-transcriptional regulation. By integrating metabolome analysis, we unveiled that well-balanced lignin formation, as well as abundant flavonoid and fructose contents, contribute to the superior quality of D. brandisii shoots. Integrating genomic, transcriptomic, and metabolomic datasets provided a solid foundation for enhancing bamboo shoot quality and developing efficient gene-editing techniques. This study should facilitate research on D. brandisii and enhance its use as a food source and wood material by providing crucial genomic resources.

Single-cell transcriptome atlas reveals spatiotemporal developmental trajectories in the basal roots of moso bamboo (Phyllostachys edulis).

Roots are essential for plant growth and development. Bamboo is a large Poaceae perennial with 1642 species worldwide. However, little is known about the transcriptional atlas that underpins root cell-type differentiation. Here, we set up a modified protocol for protoplast preparation and report single-cell transcriptomes of 14 279 filtered single cells derived from the basal root tips of moso bamboo. We identified four cell types and defined new cell-type-specific marker genes for the basal root. We reconstructed the developmental trajectories of the root cap, epidermis, and ground tissues and elucidated critical factors regulating cell fate determination. According to in situ hybridization and pseudotime trajectory analysis, the root cap and epidermis originated from a common initial cell lineage, revealing the particularity of bamboo basal root development. We further identified key regulatory factors for the differentiation of these cells and indicated divergent root developmental pathways between moso bamboo and rice. Additionally, PheWOX13a and PheWOX13b ectopically expressed in Arabidopsis inhibited primary root and lateral root growth and regulated the growth and development of the root cap, which was different from WOX13 orthologs in Arabidopsis. Taken together, our results offer an important resource for investigating the mechanism of root cell differentiation and root system architecture in perennial woody species of Bambusoideae.

GA20ox Family Genes Mediate Gibberellin and Auxin Crosstalk in Moso bamboo (Phyllostachys edulis).

Moso bamboo (Phyllostachys edulis) is one of the fastest growing plants. Gibberellin (GA) is a key phytohormone regulating growth, but there are few studies on the growth of Moso bamboo regulated by GA. The gibberellin 20 oxidase (GA20ox) gene family was targeted in this study. Chromosomal distribution and collinearity analysis identified 10 GA20ox genes evenly distributed on chromosomes, and the family genes were relatively conservative in evolution. The genetic relationship of GA20ox genes had been confirmed to be closest in different genera of plants in a phylogenetic and selective pressure analysis between Moso bamboo and rice. About 1/3 GA20ox genes experienced positive selective pressure with segmental duplication being the main driver of gene family expansion. Analysis of expression patterns revealed that only six PheGA20ox genes were expressed in different organs of shoot development and flowers, that there was redundancy in gene function. Underground organs were not the main site of GA synthesis in Moso bamboo, and floral organs are involved in the GA biosynthesis process. The auxin signaling factor PheARF47 was located upstream of PheGA20ox3 and PheGA20ox6 genes, where PheARF47 regulated PheGA20ox3 through cis-P box elements and cis-AuxRR elements, based on the result that promoter analysis combined with yeast one-hybrid and dual luciferase detection analysis identified. Overall, we identified the evolutionary pattern of PheGA20ox genes in Moso bamboo and the possible major synthesis sites of GA, screened for key genes in the crosstalk between auxin and GA, and laid the foundation for further exploration of the synergistic regulation of growth by GA and auxin in Moso bamboo.

Phytohormone Crosstalk of Cytokinin Biosynthesis and Signaling Family Genes in Moso Bamboo (Phyllostachys edulis).

Cytokinin is widely involved in the regulation of plant growth, but its pathway-related genes have not been reported in Moso bamboo. In this study, a total of 129 candidate sequences were identified by bioinformatic methods. These included 15 IPT family genes, 19 LOG family genes, 22 HK family genes, 11 HP family genes and 62 RR family genes. Phylogenetic analysis revealed that the cytokinin pathway was closely related to rice, and evolutionary pattern analysis found that most of the genes have syntenic relationship with rice-related genes. The Moso bamboo cytokinin pathway was evolutionarily conservative and mainly underwent purifying selection, and that gene family expansion was mainly due to whole-gene duplication events. Analysis of transcriptome data revealed a tissue-specific expression pattern of Moso bamboo cytokinin family genes, with auxin and gibberellin response patterns. Analysis of co-expression patterns at the developmental stages of Moso bamboo shoots revealed the existence of a phytohormone co-expression pattern centered on cytokinin signaling genes. The auxin signaling factor PheARF52 was identified by yeast one-hybrid assay as regulating the PheRR3 gene through a P-box element in the PheRR3 promoter region. Auxin and cytokinin signaling crosstalk to regulate Moso bamboo growth. Overall, we systematically identified and analyzed key gene families of the cytokinin pathway in Moso bamboo and obtained key factors for auxin and cytokinin crosstalk, laying the foundation for the study of hormone regulation in Moso bamboo.

Plasticity in the Morphology of Growing Bamboo: A Bayesian Analysis of Exogenous Treatment Effects on Plant Height, Internode Length, and Internode Numbers.

Sucrose (Suc) and gibberellin (GA) can promote the elongation of certain internodes in bamboo. However, there is a lack of field studies to support these findings and no evidence concerning how Suc and GA promote the plant height of bamboo by regulating the internode elongation and number. We investigated the plant height, the length of each internode, and the total number of internodes of Moso bamboo (Phyllostachys edulis) under exogenous Suc, GA, and control group (CTRL) treatments in the field and analyzed how Suc and GA affected the height of Moso bamboo by promoting the internode length and number. The lengths of the 10th-50th internodes were significantly increased under the exogenous Suc and GA treatments, and the number of internodes was significantly increased by the exogenous Suc treatment. The increased effect of Suc and GA exogenous treatment on the proportion of longer internodes showed a weakening trend near the plant height of 15-16 m compared with the CTRL, suggesting that these exogenous treatments may be more effective in regions where bamboo growth is suboptimal. This study demonstrated that both the exogenous Suc and GA treatments could promote internode elongation of Moso bamboo in the field. The exogenous GA treatment had a stronger effect on internode elongation, and the exogenous Suc treatment had a stronger effect on increasing the internode numbers. The increase in plant height by the exogenous Suc and GA treatments was promoted by the co-elongation of most internodes or the increase in the proportion of longer internodes.

Phylogeny, transcriptional profile, and auxin-induced phosphorylation modification characteristics of conserved PIN proteins in Moso bamboo (Phyllostachys edulis).

Auxin polar transport is an important way for auxin to exercise its function, and auxin plays an irreplaceable role in the rapid growth of Moso bamboo. We identified and performed the structural analysis of PIN-FORMED auxin efflux carriers in Moso bamboo and obtained a total of 23 PhePIN genes from five gene subfamilies. We also performed chromosome localization and intra- and inter-species synthesis analysis. Phylogenetic analyses of 216 PIN genes showed that PIN genes are relatively conserved in the evolution of the Bambusoideae and have undergone intra-family segment replication in Moso bamboo. The PIN genes' transcriptional patterns showed that the PIN1 subfamily plays a major regulatory role. PIN genes and auxin biosynthesis maintain a high degree of consistency in spatial and temporal distribution. Phosphoproteomics analysis identified many phosphorylated protein kinases that respond to auxin regulation through autophosphorylation and phosphorylation of PIN proteins. The protein interaction network showed that there is a plant hormone interaction regulatory network with PIN protein as the core. We provide a comprehensive PIN protein analysis that complements the auxin regulatory pathway in Moso bamboo and paves the way for further auxin regulatory studies in bamboo.

Photosynthesis, Phytohormone Signaling and Sugar Catabolism in the Culm Sheaths of Phyllostachys edulis.

Culm sheaths play an important role in supporting and protecting bamboo shoots during the growth and development period. The physiological and molecular functions of bamboo sheaths during the growth of bamboo shoots remain unclear. In this study, we investigated the morphological anatomy of culm sheaths, photosynthesis in sheath blades, storage and distribution of sugars, and the transcriptome of the sheath. Respiration in the base of the culm sheath was higher than that in the sheath blades; chloroplasts matured with the development of the sheath blades, the fluorescence efficiency Fv/Fm value increased from 0.3 to 0.82; and sucrose and hexose accumulated in the sheath blade and the culm sheath. The sucrose, glucose, and fructose contents of the middle sheath blades were 10.66, 5.73, and 8.84 mg/g FW, respectively. Starches accumulated in parenchymal cells close to vascular bundles. Genes related to the plant hormone signaling pathway and sugar catabolism were highly expressed in the culm sheath base. These findings provide a research basis for further understanding the possible role of bamboo sheaths in the growth and development of bamboo shoots.

Ultrastructure change and transcriptome analysis of GA3 treatment on seed germination of moso bamboo(Phyllostachys edulis).

Exploring the mechanism of Gibberellic acid (GA3) treatment on seed germination of moso bamboo can lay a foundation for its future breeding and research. In this study, the germination-related indicators (germination rate, germination potential, vigor index, respiration rate) with different content of GA3 treatment were measured, and the ultrastructure of moso bamboo seeds treated with low and high GA3 concentrations was observed during the germination process. In addition, the transcriptome data of the germination seeds, with and without GA3 treatment were analyzed. The results showed that the low GA3 concentration (10 mol/L) increased the germination rate, germination potential, vigor index and respiration rate, thus promoting the germination of moso bamboo seeds, but a high concentration of GA3 (50 mol/L) inhibited the seed germination. The low GA3 concentration accelerated the decomposition of starch and fat and promoted the vacuole formation of cells, but the high GA3 concentration damaged organelles and increased the endocytosis of cells. Compared with untreated moso bamboo seeds, the seeds had fewer genes expressed after GA3 treatment. Starch and carbon metabolism play a very important role in seed development and embryo viability, whether the seed is treated with GA3 or not. After hormone treatment, GID1 and DELLA-related genes homologous to rice genes is not expressed, but the expression of PIF4, PIF5, GA3ox2, GA2oxs, etc., were up-regulated.

PheGRF4e initiated auxin signaling during moso bamboo shoot development.

BACKGROUND: As a ubiquitous acid-regulating protein family in eukaryotes, general regulatory factors (GRFs) are active in various life activities of plants. However, detailed investigations of the GRFs gene family in moso bamboo are scarce. METHODS AND RESULTS: Genome-wide characteristics of the GRF gene family in moso bamboo were analyzed using the moso bamboo genome. GRF phylogeny, gene structure, conserved domains, cis-element promoters, and gene expression were systematically analyzed. A total of 20 GRF gene family members were identified in the moso bamboo genome. These genes were divided into ε and non-ε groups. qRT-PCR (real-time quantitative reverse transcription polymerase chain reaction) showed that PheGRF genes responded to auxin and gibberellin treatment. To further study PheGRF gene functions, a yeast two-hybrid experiment was performed and verified by a bimolecular fluorescence complementation experiment. The results showed that PheGRF4e could interact with PheIAA30 (auxin/indole-3-acetic acid, an Aux/IAA family gene), and both were found to act mainly on the root tip meristem and vascular bundle cells of developing shoots by in situ hybridization assay. CONCLUSIONS: This study revealed that PheGRF genes were involved in hormone response during moso bamboo shoot development, and the possible regulatory functions of PheGRF genes were enriched by the fact that PheGRF4e initiated auxin signaling by binding to PheIAA30.

New Insights Into the Local Auxin Biosynthesis and Its Effects on the Rapid Growth of Moso Bamboo (Phyllostachys edulis).

Auxin plays a crucial regulatory role in higher plants, but systematic studies on the location of auxin local biosynthesis are rare in bamboo and other graminaceous plants. We studied moso bamboo (Phyllostachys edulis), which can grow up to 1 m/day and serves as a reference species for bamboo and other fast-growing species. We selected young tissues such as root tips, shoot tips, young culm sheaths, sheath blades, and internode divisions for local auxin biosynthesis site analysis. IAA immunofluorescence localization revealed that auxin was similarly distributed in different stages of 50-cm and 300-cm bamboo shoots. Shoot tips had the highest auxin content, and it may be the main site of auxin biosynthesis in the early stage of rapid growth. A total of 22 key genes in the YUCCA family for auxin biosynthesis were identified by genome-wide identification, and these had obvious tissue-specific and spatio-temporal expression patterns. In situ hybridization analysis revealed that the localization of YUCCA genes was highly consistent with the distribution of auxin. Six major auxin synthesis genes, PheYUC3-1, PheYUC6-1, PheYUC6-3, PheYUC9-1, PheYUC9-2, and PheYUC7-3, were obtained that may have regulatory roles in auxin accumulation during moso bamboo growth. Culm sheaths were found to serve as the main local sites of auxin biosynthesis and the auxin required for internode elongation may be achieved mainly by auxin transport.

Overexpression of PheNAC3 from moso bamboo promotes leaf senescence and enhances abiotic stress tolerance in Arabidopsis.

The NAC family is one of the largest transcription factor families unique to plants, which regulates the growth and development, biotic and abiotic stress responses, and maturation and senescence in plants. In this study, PheNAC3, a NAC gene, was isolated and characterized from moso bamboo (Phyllostachys edulis). PheNAC3 belong to the NAC1 subgroup and has a conserved NAC domain on the N-terminus, which with 88.74% similarity to ONAC011 protein. PheNAC3 localized in the nucleus and exhibited transactivation activity. PheNAC3 was upregulated during the process of senescence of leaves and detected shoots. PheNAC3 was also induced by ABA, MeJA, NaCl and darkness, but it had no remarkable response to PEG and SA treatments. Overexpression of PheNAC3 could cause precocious senescence in Arabidopsis. Transgenic Arabidopsis displayed faster seed germination, better seedling growth, and a higher survival rate than the wild-type under salt or drought stress conditions. Moreover, AtSAG12 associated with senescence and AtRD29A and AtRD29b related to ABA were upregulated by PheNAC3 overexpression, but AtCAB was inhibited. These findings show that PheNAC3 may participate in leaf senescence and play critical roles in the salt and drought stress response.

Transcriptome profiling of postharvest shoots identifies PheNAP2- and PheNAP3-promoted shoot senescence.

The juvenile shoots of Phyllostachys edulis have been used as a food source for thousands of years, and it is recognized as a potential source of nutraceuticals. However, its rapid senescence restricts bamboo production and consumption, and the underlying molecular mechanisms of rapid shoot senescence remain largely unclear. In the present study, transcriptome profiling was employed to investigate the molecular regulation of postharvest senescence in shoots, along with physiological assays and anatomical dissections. Results revealed a distinct shift in expression postharvest, specifically transitions from cellular division and differentiation to the relocation of nutrients and programmed cell death. A number of regulatory and signaling factors were induced during postharvest senescence. Moreover, transcription factors, including NAM, ATAF and CUC (NAC) transcription factors, basic helix-loop-helix transcription factors, basic region/leucine zipper transcription factors, MYB transcription factors and WRKY transcription factors, were critical for shoot postharvest senescence, of which NACs were the most abundant. PheNAP2 and PheNAP3 were induced in postharvest shoots and found to promote leaf senescence in Arabidopsis by inducing the expression of AtSAG12 and AtSAG113. PheNAP2 and PheNAP3 could both restore the stay-green Arabidopsis nap to the wild-type phenotype either under normal growth condition or under abscisic acid treatment. Collectively, these results suggest that PheNAPs may promote shoot senescence. These findings provide a systematic view of shoot senescence and will inform future studies on the underlying molecular mechanisms responsible for shoot degradation during storage.

Expression Analysis and Regulation Network Identification of the CONSTANS-Like Gene Family in Moso Bamboo (Phyllostachys edulis) Under Photoperiod Treatments.

CONSTANS (CO)/CONSTANS-like (COL) genes that have been studied in annual model plants such as Arabidopsis thaliana and Oryza sativa play key roles in the photoperiodic flowering pathway. Moso bamboo is a perennial plant characterized by a long vegetative stage and flowers synchronously followed by widespread death. However, the characteristics of COL in moso bamboo remain unclear. In view of this, we performed a genome-wide identification and expression analysis of the COL gene family in moso bamboo. Fourteen nonredundant PheCOL genes were identified, and we analyzed gene structures, phylogeny, and subcellular location predictions. Phylogenetic analyses indicated that 14 PheCOLs could be clustered into three groups, and each clade was well supported by conserved intron/exon structures and motifs. A number of light-related and tissue-specific cis-elements were randomly distributed within the promoter sequences of the PheCOLs. The expression profiling of PheCOL genes in various tissues and developmental stages revealed that most of PheCOL genes were most highly expressed in the leaves and took part in moso bamboo flower development and rapid shoot growth. In addition, the transcription of PheCOLs exhibited a clear diurnal oscillation in both long-day and short-day conditions. Most of the PheCOL genes were inhibited under light treatment and upregulated in dark conditions. PheCOLs can interact with each other. Subcellular localization result showed that PheCOL14 encoded a cell membrane protein, and it bound to the promoter of PheCOL3. Taken together, the results of this study will be useful not only as they contribute to comprehensive information for further analyses of the molecular functions of the PheCOL gene family, but also will provide a theoretical foundation for the further construction of moso bamboo photoperiod regulation networks.