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.

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.

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 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.

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.

Genome-Wide Analysis of the AP2/ERF Transcription Factors Family and the Expression Patterns of DREB Genes in Moso Bamboo (Phyllostachys edulis).

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.

Characterization of the floral transcriptome of Moso bamboo (Phyllostachys edulis) at different flowering developmental stages by transcriptome sequencing and RNA-seq analysis.

BACKGROUND: As an arborescent and perennial plant, Moso bamboo (Phyllostachys edulis (Carrière) J. Houzeau, synonym Phyllostachys heterocycla Carrière) is characterized by its infrequent sexual reproduction with flowering intervals ranging from several to more than a hundred years. However, little bamboo genomic research has been conducted on this due to a variety of reasons. Here, for the first time, we investigated the transcriptome of developing flowers in Moso bamboo by using high-throughput Illumina GAII sequencing and mapping short reads to the Moso bamboo genome and reference genes. We performed RNA-seq analysis on four important stages of flower development, and obtained extensive gene and transcript abundance data for the floral transcriptome of this key bamboo species. RESULTS: We constructed a cDNA library using equal amounts of RNA from Moso bamboo leaf samples from non-flowering plants (CK) and mixed flower samples (F) of four flower development stages. We generated more than 67 million reads from each of the CK and F samples. About 70% of the reads could be uniquely mapped to the Moso bamboo genome and the reference genes. Genes detected at each stage were categorized to putative functional categories based on their expression patterns. The analysis of RNA-seq data of bamboo flowering tissues at different developmental stages reveals key gene expression properties during the flower development of bamboo. CONCLUSION: We showed that a combination of transcriptome sequencing and RNA-seq analysis was a powerful approach to identifying candidate genes related to floral transition and flower development in bamboo species. The results give a better insight into the mechanisms of Moso bamboo flowering and ageing. This transcriptomic data also provides an important gene resource for improving breeding for Moso bamboo.

Transcriptome sequencing and analysis of the fast growing shoots of moso bamboo (Phyllostachys edulis).

BACKGROUND: The moso bamboo, a large woody bamboo with the highest ecological, economic, and cultural value of all bamboos, has one of the highest growth speeds in the world. Genetic research into moso bamboo has been scarce, partly because of the lack of previous genomic resources. In the present study, for the first time, we performed de novo transcriptome sequencing and mapped to the moso bamboo genomic resources (reference genome and genes) to produce a comprehensive dataset for the fast growing shoots of moso bamboo. RESULTS: The fast growing shoots mixed with six different heights and culms after leaf expansion of moso bamboo transcriptome were sequenced using the Illumina HiSeq™ 2000 sequencing platform, respectively. More than 80 million reads including 65,045,670 and 68,431,884 clean reads were produced in the two libraries. More than 81% of the reads were matched to the reference genome, and nearly 50% of the reads were matched to the reference genes. The genes with log 2 ratio > 2 or < -2 (P<0.001) were characterized as the most differentially expressed genes. 6,076 up-regulated and 4,613 down-regulated genes were classified into functional categories. Candidate genes which mainly involved transcript factors, plant hormones, cell cycle regulation, cell wall metabolism and cell morphogenesis genes were further analyzed and they may form a network that regulates the fast growth of moso bamboo shoots. CONCLUSION: Firstly, our data provides the most comprehensive transcriptomic resource for moso bamboo to date. Candidate genes have been identified and they are potentially involved in the growth and development of moso bamboo. The results give a better insight into the mechanisms of moso bamboo shoots rapid growth and provide gene resources for improving plant growth.