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.

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.

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.

Identification and Evolution of the WUSCHEL-Related Homeobox Protein Family in Bambusoideae.

Bamboos (Bambusoideae) are fast-growing species due to their rapid growth rate and ability to reproduce annually via cloned buds produced on the rhizome. WUSCHEL-related homeobox (WOX) genes have been reported to regulate shoot apical meristem organization, lateral organ formation, cambium and vascular proliferation, and so on, but have rarely been studied in bamboos. In this study, the WOXs of both herbaceous bamboo species (12 OlaWOXs and nine RguWOXs) and woody bamboo species (18 GanWOXs, 27 PheWOXs, and 26 BamWOXs) were identified and categorized into three clades based on their phylogenetic relationship-ancient, intermediate, or WUS clade. Polyploidy is the major driver of the expansion of the bamboo WOX family. Eight conserved domains, besides the homeodomain, were identified by comparatively analyzing the WOXs of dicot and monocot species. Intensive purifying selection pressure in the coding region of specific domains explained the functional similarity of WOXs between different species. For Bambusoideae WOXs, polyploidy is the major driver of the expansion of the WOX family. Stronger purifying selection was found in orthologous WOXs of Bambusoideae, especially for WOX4s and WOX5s, which are conserved not only at the translational levels, but also at the genome level. Several conserved cis-acting elements were discovered at similar position in the promoters of the orthologous WOXs. For example, AP2/ERF protein-binding elements and B3 protein-binding elements were found in the promoters of the bamboo WOX4, while MYB protein-binding elements and Dof protein-binding elements were found in the promoters of bamboo WOX5, and MADS protein-binding sites was found in the promoters of bamboo WUS, WOX3, and WOX9. These conserved positions may play an important role in regulating the expression of bamboo WOXs. Our work provides insight into the origin and evolution of bamboo WOXs, and will facilitate functional investigations of the clonal propagation of bamboos.

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.

Integrated mRNA, MicroRNA Transcriptome and Degradome Analyses Provide Insights into Stamen Development in Moso Bamboo.

A flower is an essential organ for sexual reproduction in flowering plants, which has been extensively studied in model plants. In this study, we used transcriptomic, small RNA and degradome analyses to characterize key microRNAs (miRNAs) and their targets in floral organs of moso bamboo. In total, we identified 13,051 differentially expressed genes and 109 known miRNAs from 26 miRNA families. We aligned the miRNAs to known miRNA databases and revealed some conserved as well as novel miRNAs. Sixteen conserved miRNAs were specifically and highly expressed in stamens, including miRNA159 and miRNA166. In situ hybridization shows that miRNA159 plays a key role in the regulation of stamen development, and the expression levels of its targets PheMYB98 and PheMYB42 were low. Furthermore, Phe-MIRNA159 partially recovers phenotypes of mir159ab double mutant. Overexpression of Phe-MIR159 could cause failure in anther dehisce, and the mature pollens could not be dispersed and further reduce fertility in Arabidopsis. Semi-thin section result shows that anther endothelial layer of Phe-MIRNA159 overexpressing lines is lack of secondary thickening, resulting in limited force for anther opening. Phe-miR159 may regulate the expression of genes related to secondary thickening through negative regulation of AtMYB33, affecting the anther dehiscence. Taken together, this study provides insights regarding molecular networks underlying floral organs development of moso bamboo.

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.

Multifaceted Role of PheDof12-1 in the Regulation of Flowering Time and Abiotic Stress Responses in Moso Bamboo (Phyllostachys edulis).

DNA binding with one finger (Dof) proteins, forming an important transcriptional factor family, are involved in gene transcriptional regulation, development, stress responses, and flowering responses in annual plants. However, knowledge of Dofs in perennial and erratically flowering moso bamboo is limited. In view of this, a Dof gene, PheDof12-1, was isolated from moso bamboo. PheDof12-1 is located in the nucleus and has the highest expression in palea and the lowest in bract. Moreover, PheDof12-1 expression is high in flowering leaves, then declines during flower development. The transcription level of PheDof12-1 is highly induced by cold, drought, salt, and gibberellin A3 (GA3) stresses. The functional characteristics of PheDof are researched for the first time in Arabidopsis, and the results show that transgenic Arabidopsis overexpressing PheDof12-1 shows early flowering under long-day (LD) conditions but there is no effect on flowering time under short-day (SD) conditions; the transcription levels of FT, SOC1, and AGL24 are upregulated; and FLC and SVP are downregulated. PheDof12-1 exhibits a strong diurnal rhythm, inhibited by light treatment and induced in dark. Yeast one-hybrid (Y1H) assay shows that PheDof12-1 can bind to the promoter sequence of PheCOL4. Taken together, these results indicate that PheDof12-1 might be involved in abiotic stress and flowering time, which makes it an important candidate gene for studying the molecular regulation mechanisms of moso bamboo flowering.

The R2R3MYB Gene Family in Phyllostachys edulis: Genome-Wide Analysis and Identification of Stress or Development-Related R2R3MYBs.

The MYB transcription factor (TF) is one of the largest gene families in plants and involved to multiple biological processes. However, little is known about the MYB family and its functional role in the genome of moso bamboo. In the present study, a total of 114 R2R3MYB genes were first identified from moso bamboo genome and full-length non-chimeric (FLNC) reads. Phylogenetic analysis coupled with gene structure analysis and motif determination resulted in the division of these PheR2R3MYBs into 17 subgroups. The position of eight proteins along an external branch in the phylogenetic tree suggested their relatively ancient origin. The genes in this group were all substituted by (Met, M)/(Arg, R) at conservative W residues in both R2 and R3 repeats, and half were found to possess no transcriptional activation activity. The analysis of evolutionary patterns and divergence suggests that the expansion of PheMYBs was mainly attributable to whole genome duplication (WGD) under different selection pressures. Expressional analysis based on microarray and qRT-PCR data performed diverse expression patterns of R2R3MYBs in response to both various abiotic stimuli and flower development. Furthermore, the co-expression analysis of R2R3MYBs suggested an intricate interplay of growth- and stress-related responses. Finally, we found a hub gene, PheMYB4, was involved in a complex proteins interaction network. Further functional analysis indicated that ectopic overexpression of its homologous gene, PheMYB4-1, could increase tolerance to cold treatment and sensitivity to drought and salt treatment of transgenic Arabidopsis seedlings. These findings provide comprehensive insights into the MYB family members in moso bamboo and offer candidate MYB genes for further studies on their roles in stress resistance.

Characterization of moso bamboo (Phyllostachys edulis) Dof transcription factors in floral development and abiotic stress responses.

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.

The association of hormone signalling genes, transcription and changes in shoot anatomy during moso bamboo growth.

Moso bamboo is a large, woody bamboo with the highest ecological, economic and cultural value of all the bamboo types and accounts for up to 70% of the total area of bamboo grown. However, the spatiotemporal variation role of moso bamboo shoot during growth period is still unclear. We found that the bamboo shoot growth can be divided into three distinct periods, including winter growth, early growth and late growth based on gene expression and anatomy. In the early growth period, lateral buds germinated from the top of the bamboo joint in the shoot tip. Intercalary meristems grew vigorously during the winter growth period and early growth period, but in the late growth period, mitosis in the intercalary meristems decreased. The expression of cell cycle-associated genes and the quantity of differentially expressed genes were higher in early growth than those in late growth, appearing to be influenced by hormonal concentrations. Gene expression analysis indicates that hormone signalling genes play key roles in shoot growth, while auxin signalling genes play a central role. In situ hybridization analyses illustrate how auxin signalling genes regulate apical dominance, meristem maintenance and lateral bud development. Our study provides a vivid picture of the dynamic changes in anatomy and gene expression during shoot growth in moso bamboo, and how hormone signalling-associated genes participate in moso bamboo shoot growth.

Characterization and expression analysis of the WRKY gene family in moso bamboo.

The WRKY family of transcription factors (TFs) is one of the ten largest families of TFs in higher plants and has been implicated in multiple biological processes. Here, we identified 121 WRKY TFs in moso bamboo, including five novel members that were not annotated in the Phyllostachys edulis genomic database. Estimation of the divergence time of paralogous gene pairs revealed an important role of the recent whole-genome duplication in the expansion of the WRKY family. Expression analysis based on quantitative reverse-transcription polymerase chain reaction (qRT-PCR) data revealed that a large number of PheWRKY genes varied significantly under cold or drought stress treatments, which could be defined as abiotic stress-responsive genes. The overexpression of PheWRKY72-2 in Arabidopsis resulted in a decreased sensitivity to drought stress during early seedling growth. PheWRKY72-2 may enhance plant tolerance to stress by functioning as a positive regulator of stoma closure. Our study provides a theoretical foundation and some experimental evidence for further functional verification of the PheWRKY family of TFs.

Analysis of MADS-Box Gene Family Reveals Conservation in Floral Organ ABCDE Model of Moso Bamboo (Phyllostachys edulis).

Mini chromosome maintenance 1, agamous, deficiens, and serum response factor (MADS)-box genes are transcription factors which play fundamental roles in flower development and regulation of floral organ identity. However, till date, identification and functions of MADS-box genes remain largely unclear in Phyllostachys edulis. In view of this, we performed a whole-genome survey and identified 34 MADS-box genes in P. edulis, and based on phylogeny, they were classified as MIKCC, MIKC∗, Mα, and Mß. The detailed analysis about gene structure and motifs, phylogenetic classification, comparison of gene divergence and duplication are provided. Interestingly, expression patterns for most genes were found similar to those of Arabidopsis and rice, indicating that the well-established ABCDE model can be applied to P. edulis. Moreover, we overexpressed PheMADS15, an AP1-like gene, in Arabidopsis, and found that the transgenic plants have early flowering phenotype, suggesting that PheMADS15 might be a regulator of flowering transition in P. edulis. Taken together, this study provides not only insightful comprehension but also useful information for understanding the functions of MADS-box genes in P. edulis.

Genome-wide analysis and expression characteristics of small auxin-up RNA (SAUR) genes in moso bamboo (Phyllostachys edulis).

Moso bamboo (Phyllostachys edulis) is well known for its rapid shoot growth. Auxin exerts pleiotropic effects on plant growth. The small auxin-up RNA (SAUR) genes are early auxin-responsive genes involved in plant growth. In total, 38 SAUR genes were identified in P. edulis (PheSAUR). A comprehensive overview of the PheSAUR gene family is presented, including the gene structures, phylogeny, and subcellular location predictions. A transcriptome analysis indicated that 37 (except PheSAUR18) of the PheSAUR genes were expressed during shoot growth process and that the PheSAUR genes were differentially expressed. Furthermore, quantitative real-time PCR analysis indicated that all of the PheSAUR genes could be induced in different tissues of seedlings and that 37 (except PheSAUR41) of the PheSAUR genes were up-regulated after indole-3-acetic acid (IAA) treatment. These results reveal a comprehensive overview of the PheSAUR gene family and may pave the way for deciphering their functions during bamboo development.

Main regulatory pathways, key genes and microRNAs involved in flower formation and development of moso bamboo (Phyllostachys edulis).

Moso bamboo is characterized by infrequent sexual reproduction and erratic flowering habit; however, the molecular biology of flower formation and development is not well studied in this species. We studied the molecular regulation mechanisms of moso bamboo development and flowering by selecting three key regulatory pathways: plant-pathogen interaction, plant hormone signal transduction and protein processing in endoplasmic reticulum at different stages of flowering in moso bamboo. We selected PheDof1, PheMADS14 and six microRNAs involved in the three pathways through KEGG pathway and cluster analysis. Subcellular localization, transcriptional activation, Western blotting, in situ hybridization and qRT-PCR were used to further investigate the expression patterns and regulatory roles of pivotal genes at different flower development stages. Differential expression patterns showed that PheDof1, PheMADS14 and six miRNAs may play vital regulatory roles in flower development and floral transition in moso bamboo. Our research paves way for further studies on metabolic regulatory networks and provides insight into the molecular regulation mechanisms of moso bamboo flowering and senescence.

Genome-wide analysis of shoot growth-associated alternative splicing in moso bamboo.

Alternative splicing (AS) significantly enhances transcriptome complexity and is differentially regulated in a wide variety of physiological processes in plants, including shoot growth. Presently, the functional implications and conservation of AS occurrences are not well understood in the moso bamboo genome. To analyze the global changes in AS during moso bamboo shoot growth, fast-growing shoots collected at seven different heights and culms after leaf expansion were sequenced using the Illumina HiSeq™ 2000 sequencing platform. It was found that approximately 60.74 % of all genes were alternatively spliced, with intron retention (IR) being the most frequent AS event (27.43 %). Statistical analysis demonstrated that variations of AS frequency and AS types were significantly correlated with changes in gene features and gene transcriptional level. According to the phylogenetic analysis of isoform expression data and AS frequency, the bamboo shoot growth could be divided into four different growth periods, including winter bamboo shoot (S1), early growth period (S2-S5), late growth period (S6 and S7), and mature period (CK). In addition, our data also showed that the winter bamboo shoot had the highest number of AS events. Twenty-six putative Serine/arginine-rich (SR) proteins were identified, producing a total of 109 transcripts. AS events were frequently and specifically regulated by SR splicing factors throughout shoot growth, resulting in changes to the original open reading frame (ORF) and subsequently changes to conserved domains. The AS product-isoforms showed regular expression change during the whole shoot growth period, thus influencing shoot growth. All together, these data indicate that AS events are adjusted to different growth stages, providing briefness and efficient means of gene regulation. This study will provide a very useful clue for future functional analyses.

Identification and characterization of microRNAs at different flowering developmental stages in moso bamboo (Phyllostachys edulis) by high-throughput sequencing.

Researching moso bamboo flowering has been difficult because of its unknown flowering interval and the rarity of florescent samples. To identify microRNAs (miRNAs) and study their expression patterns during the flower developmental process of moso bamboo, small RNAs from non-flowering leaves and four flower developmental periods were sequenced using Illumina technology. In total, 409 known miRNAs and 492 differentially expressed novel miRNAs were identified in moso bamboo. Of the known miRNAs that were differentially expressed between non-flowering and flowering samples, 64 were predicted to have a total of 308 targets. Among the miRNAs, seven known and five novel miRNAs were selected, as were four of their target genes, and their expression profiles were validated using qRT-PCR. The results indicated that the miRNA expression levels were negatively correlated with those of their targets. The research comprehensively revealed that the differentially expressed miRNAs and their targets participated in diverse biological pathways and played significant regulatory roles in moso bamboo flowering. The data provide a significant resource for understanding the molecular mechanisms in moso bamboo flowering and senescence, and serve as the primary foundation for further studies on metabolic regulatory networks that involve miRNAs.