Comparative Physiology and Transcriptome Analysis Provides Insights into the Regulatory Mechanism of Albinotic Bambusa oldhamii.

Albinism is a unique problem encountered in tissue culture experiments, but the underlying mechanism remains unclear in most bamboo species. In this study, we identified the putative regulatory genes in an albino mutant of Bambusa oldhamii using comparative physiology and transcriptome analysis. The degeneration of chloroplasts, low chlorophyll (Chl) content and reduced photosynthetic capacity were observed in albinotic B. oldhamii compared to normal lines. A total of 6191 unigenes were identified that were clearly differentially expressed between albino and normal lines by transcriptome sequencing. Most genes related to chloroplast development (such as Psa, Psb) and pigment biosynthesis (such as LHC, GUN4, ZEP) were downregulated significantly in albinotic lines, which might be responsible for the albino phenotype. Moreover, some transcription factors (TFs) such as PIF and GLK1 were identified to be involved in chloroplast development and Chl synthesis, indicating the involvement of putative regulatory pathways PIF-LHC and GLK1-LHC/Psa/Psb in albinotic B. oldhamii. Finally, the downregulation of some stress responsive TFs (like ICE1 and EREB1) suggested a reduction in stress resistance of albinotic B. oldhamii. The above findings provided new insights into the molecular mechanism of albinism in bamboo.

Molecular identification of Bambusa changningensis is the natural bamboo hybrid of B. rigida × Dendrocalamus farinosus.

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.

Network of miR396-mRNA in Tissue Differentiation in Moso Bamboo (Phyllostachys edulis).

MiR396 plays an essential role in various developmental processes. However, the miR396-mRNA molecular network in bamboo vascular tissue differentiation during primary thickening has not been elucidated. Here, we revealed that three of the five members from the miR396 family were overexpressed in the underground thickening shoots collected from Moso bamboo. Furthermore, the predicted target genes were up/down-regulated in the early (S2), middle (S3) and late (S4) developmental samples. Mechanistically, we found that several of the genes encoding protein kinases (PKs), growth-regulating factors (GRF), transcription factors (TFs), and transcription regulators (TRs) were the potential targets of miR396 members. Moreover, we identified QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) d omains in five PeGRF homologs and a Lipase_3 domain and a K_trans domain in another two potential targets, where the cleavage targets were identified via degradome sequencing (p < 0.05). The sequence alignment indicated many mutations in the precursor sequence of miR396d between Moso bamboo and rice. Our dual-luciferase assay revealed that ped-miR396d-5p binds to a PeGRF6 homolog. Thus, the miR396-GRF module was associated with Moso bamboo shoot development. Fluorescence in situ hybridization localized miR396 in the vascular tissues of the leaves, stems, and roots of pot Moso bamboo seedlings at the age of two months. Collectively, these experiments revealed that miR396 functions as a regulator of vascular tissue differentiation in Moso bamboo. Additionally, we propose that miR396 members are targets for bamboo improvement and breeding.

Overexpression of PvSVP1, an SVP-like gene of bamboo, causes early flowering and abnormal floral organs in Arabidopsis and rice.

Bamboo is a nontimber woody plant featuring a long vegetative stage and uncertain flowering time. Therefore, the genes belonging to flowering repressors might be essential in regulating the transition from the vegetative to reproductive stage in bamboo. The Short Vegetative Phase ( SVP) gene plays a pivotal role in floral transition and development. However, little is known about the bamboo SVP homologues. In this study, Phyllostachys violascens PvSVP1 is isolated by analysis of the P. edulis transcriptome database. Phylogenetic analysis shows that PvSVP1 is closely related to OsMADS55 (rice SVP homolog). PvSVP1 is ubiquitously expressed in various tissues, predominantly in vegetative tissues. To investigate the function of PvSVP1, PvSVP1 is overexpressed in Arabidopsis and rice under the influence of the 35S promoter. Overexpression of PvSVP1 in Arabidopsis causes early flowering and produces abnormal petals and sepals. Quantitative real-time PCR reveals that overexpression in Arabidopsis produces an early flowering phenotype by downregulating FLC and upregulating FT and produces abnormal floral organs by upregulating AP1, AP3 and PI expressions. Simultaneously, overexpression of PvSVP1 in rice alters the expressions of flowering-related genes such as Hd3a, RFT1, OsMADS56 and Ghd7 and promotes flowering under field conditions. In addition, PvSVP1 may be a nuclear protein which interacts with PvVRN1 and PvMADS56 on the yeast two-hybrid and BiFC systems. Our study suggests that PvSVP1 may play a vital role in flowering time and development by interacting with PvVRN1 and PvMADS56 in the nucleus. Furthermore, this study paves the way toward understanding the complex flowering process of bamboo.

Complete Chloroplast Genome Features of Dendrocalamusfarinosus and Its Comparison and Evolutionary Analysis with Other Bambusoideae Species.

Dendrocalamus farinosus is one of the essential bamboo species mainly used for food and timber in the southwestern region of China. In this study, the complete chloroplast (cp) genome of D. farinosus is sequenced, assembled, and the phylogenetic relationship analyzed. The cp genome has a circular and quadripartite structure, has a total length of 139,499 bp and contains 132 genes: 89 protein-coding genes, eight rRNAs and 35 tRNAs. The repeat analyses showed that three types of repeats (palindromic, forward and reverse) are present in the genome. A total of 51 simple sequence repeats are identified in the cp genome. The comparative analysis between different species belonging to Dendrocalamus revealed that although the cp genomes are conserved, many differences exist between the genomes. The analysis shows that the non-coding regions were more divergent than the coding regions, and the inverted repeat regions are more conserved than the single-copy regions. Moreover, these results also indicate that rpoC2 may be used to distinguish between different bamboo species. Phylogenetic analysis results supported that D. farinosus was closely related to D. latiflorus. Furthermore, these bamboo species' geographical distribution and rhizome types indicate two evolutionary pathways: one is from the tropics to the alpine zone, and the other is from the tropics to the warm temperate zone. Our study will be helpful in the determination of the cp genome sequences of D. farinosus, and provides new molecular data to understand the Bambusoideae evolution.

Genome-wide identification and evolution of WNK kinases in Bambusoideae and transcriptional profiling during abiotic stress in Phyllostachys edulis.

With-no-lysine (WNK) kinases play vital roles in abiotic stress response, circadian rhythms, and regulation of flowering time in rice, Arabidopsis, and Glycine max. However, there are no previous reports of WNKs in the Bambusoideae, although genome sequences are available for diploid, tetraploid, and hexaploid bamboo species. In the present study, we identified 41 WNK genes in five bamboo species and analysed gene evolution, phylogenetic relationship, physical and chemical properties, cis-elements, and conserved motifs. We predicted the structure of PeWNK proteins of moso bamboo and determined the exposed, buried, structural and functional amino acids. Real-time qPCR analysis revealed that PeWNK5, PeWNK7, PeWNK8, and PeWNK11 genes are involved in circadian rhythms. Analysis of gene expression of different organs at different developmental stages revealed that PeWNK genes are tissue-specific. Analysis of various abiotic stress transcriptome data (drought, salt, SA, and ABA) revealed significant gene expression levels in all PeWNKs except PeWNK11. In particular, PeWNK8 and PeWNK9 were significantly down- and up-regulated, respectively, after abiotic stress treatment. A co-expression network of PeWNK genes also showed that PeWNK2, PeWNK4, PeWNK7, and PeWNK8 were co-expressed with transcriptional regulators related to abiotic stress. In conclusion, our study identified the PeWNKs of moso bamboo involved in circadian rhythms and abiotic stress response. In addition, this study serves as a guide for future functional genomic studies of the WNK genes of the Bambusoideae.

Homo- and Hetero-Dimers of CAD Enzymes Regulate Lignification and Abiotic Stress Response in Moso Bamboo.

Lignin biosynthesis enzymes form complexes for metabolic channelling during lignification and these enzymes also play an essential role in biotic and abiotic stress response. Cinnamyl alcohol dehydrogenase (CAD) is a vital enzyme that catalyses the reduction of aldehydes to alcohols, which is the final step in the lignin biosynthesis pathway. In the present study, we identified 49 CAD enzymes in five Bambusoideae species and analysed their phylogenetic relationships and conserved domains. Expression analysis of Moso bamboo PheCAD genes in several developmental tissues and stages revealed that among the PheCAD genes, PheCAD2 has the highest expression level and is expressed in many tissues and PheCAD1, PheCAD6, PheCAD8 and PheCAD12 were also expressed in most of the tissues studied. Co-expression analysis identified that the PheCAD2 positively correlates with most lignin biosynthesis enzymes, indicating that PheCAD2 might be the key enzyme involved in lignin biosynthesis. Further, more than 35% of the co-expressed genes with PheCADs were involved in biotic or abiotic stress responses. Abiotic stress transcriptomic data (SA, ABA, drought, and salt) analysis identified that PheCAD2, PheCAD3 and PheCAD5 genes were highly upregulated, confirming their involvement in abiotic stress response. Through yeast two-hybrid analysis, we found that PheCAD1, PheCAD2 and PheCAD8 form homo-dimers. Interestingly, BiFC and pull-down experiments identified that these enzymes form both homo- and hetero- dimers. These data suggest that PheCAD genes are involved in abiotic stress response and PheCAD2 might be a key lignin biosynthesis pathway enzyme. Moreover, this is the first report to show that three PheCAD enzymes form complexes and that the formation of PheCAD homo- and hetero- dimers might be tissue specific.

Analysis of mitochondrial recombination in the male sterile Brassica juncea cybrid Og1 and identification of the molecular basis of fertility reversion.

KEY MESSAGE: Recombinations between the parental genomes produced a novel mitochondrial genome in the cytoplasmic male sterile Brassica juncea cybrid Og1. A mitochondrial stoichiometric shift greatly reduced the molecule containing male-sterility-inducing orf138 gene leading to reversion to male fertility. An improved, chlorosis-corrected, cytoplasmic male sterile Brassica juncea cybrid Og1 derived from Ogura cytoplasm shows frequent reversion to male fertility. To determine the nature of mitochondrial recombination in the cybrid and to uncover the molecular mechanism of male fertility reversion, we sequenced the mitochondrial genomes of Og1, its isonuclear parental lines (OgRLM and Brassica juncea RLM198) and the revertant line (Og1-rt). Assembly of Og1 mitochondrial genome gave two circular molecules, Og1a (250.999 kbp) and Og1b (96.185 kbp) sharing two large direct repeat regions capable of recombining to form a single circular molecule. Og1a contains all essential mitochondrial genes, but the male-sterility-causing orf138 was uniquely present in Og1b along with 16 other complete or partial genes already represented in Og1a. Eleven and four recombinations between the parental mitochondrial genomes produced the Og1a and the Og1b molecules, respectively. Five genes were duplicated within Og1a, of which trnfM was inherited from both the parents while the other four genes, atp4, cox1 nad4L and trnM, were inherited from RLM198. RFLP analysis revealed that orf138-containing molecules were less abundant than Og1a in the male-sterile plants while og1b bearing molecules were undetectable in the revertant line. However, orf138 transcripts were amplified in RT-PCR and were also detected in northern blots revealing that Og1b molecules are not completely lost in the revertant plants. This is the first report where the mitochondrial genome of a cybrid is compared with its actual parents. The findings are discussed in the light of previous reports on mitochondrial genome recombination in cybrids.

PeSNAC-1 a NAC transcription factor from moso bamboo (Phyllostachys edulis) confers tolerance to salinity and drought stress in transgenic rice.

NAC (NAM, AFAT and CUC) proteins play necessary roles in plant response to environmental stresses. However, the functional roles of NAC genes in moso bamboo (Phyllostachys edulis), an essential economic perennial woody bamboo species, are not well documented. In this study, we retrieved 152 PeNAC genes from the moso bamboo V2 genome, and PeSNAC-1 was isolated and functionally characterized. PeSNAC-1 was localized in the nucleus and had no transactivation activity in yeast. PeSNAC-1 extremely expressed in rhizome and young roots (0.1 and 0.5 cm) and was significantly induced by drought and salt treatments but repressed by abscisic acid (ABA), methyl jasmonate and high temperature (42 °C) in moso bamboo. Under water shortage and salinity conditions, survival ratios, Fv/Fm values, physiological indexes such as activities of superoxide dismutase, peroxidase and catalase and contents of malondialdehyde, H2O2 and proline were significantly higher in transgenic rice than the wild type, which suggests enhanced tolerance to drought and salt stress in PeSANC-1 overexpressed plants. Transcript levels of Na+/H+ antiporter and Na+ transporter genes (OsSOS1, OsNHX1 and OsHKT1;5), ABA signaling and biosynthesis genes (OsABI2, OsRAB16, OsPP2C68, OsLEA3-1, OsLEA3, OsNCED3, OsNCED4 and OsNCED5) and ABA-independent genes (OsDREB1A, OsDREB1B and OsDREB2A) were substantially higher in transgenic as compared with the wild type. Moreover, protein interaction analysis revealed that PeSNAC-1 could interact with stress responsive PeSNAC-2/4 and PeNAP-1/4/5 in both yeast and plant cells, which indicates a synergistic effect of those proteins in regulating the moso bamboo stress response. Our data demonstrate that PeSNAC-1 likely improved salt and drought stress tolerance via modulating gene regulation in both ABA-dependent and independent signaling pathways in transgenic rice. In addition, PeSNAC-1 functions as an important positive stress regulator in moso bamboo, participating in PeSNAC-1 and PeSNAC-2/4 or PeSNAC-1 and PeNAP-1/4/5 interaction networks.

An evolutionarily conserved mitochondrial orf108 is associated with cytoplasmic male sterility in different alloplasmic lines of Brassica juncea and induces male sterility in transgenic Arabidopsis thaliana.

Nuclear-mitochondrial gene interactions governing cytoplasmic male sterility (CMS) in angiosperms have been found to be unique to each system. Fertility restoration of three diverse alloplasmic CMS lines of Brassica juncea by a line carrying the fertility-restorer gene introgressed from Moricandia arvensis prompted this investigation to examine the molecular basis of CMS in these lines. Since previous studies had found altered atpA transcription associated with CMS in these lines, the atpA genes and transcripts of CMS, fertility-restored, and euplasmic lines were cloned and compared. atpA coding and downstream sequences were conserved among CMS and euplasmic lines but major differences were found in the 5' flanking sequences of atpA. A unique open reading frame (ORF), orf108, co-transcribed with atpA, was found in male sterile flowers of CMS lines carrying mitochondrial genomes of Diplotaxis berthautii, D. catholica, or D. erucoides. In presence of the restorer gene, the bicistronic orf108-atpA transcript was cleaved within orf108 to yield a monocistronic atpA transcript. Transgenic expression of orf108 with anther-specific Atprx18 promoter in Arabidopsis thaliana gave 50% pollen sterility, indicating that Orf108 is lethal at the gametophytic stage. Further, lack of transmission of orf108 to the progeny showed for the first time that mitochondrial ORFs could also cause female sterility. orf108 was found to be widely distributed among wild relatives of Brassica, indicating its ancient origin. This is the first report that shows that CMS lines of different origin and morphology could share common molecular basis. The gametic lethality of Orf108 offers a novel opportunity for transgene containment.