Bamboo is a very important forest resource. However, the prolonged vegetative stages and uncertainty of flowering brings difficulties in bamboo flowers sampling. Until now, the flowering mechanism of bamboo is still unclear. In this study, three successive stages of flowering buds and the corresponding vegetative buds non-flowering stage from Lei bamboo Phyllostachys violascens were collected for transcriptome analysis using Illumina RNA-Seq method. A total of differentially expressed genes DEGs were determined. Additionally, a total of 25 MADS-box unigenes were identified.
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In this study, three successive stages of flowering buds and the corresponding vegetative buds non-flowering stage from Lei bamboo Phyllostachys violascens were collected for transcriptome analysis using Illumina RNA-Seq method.
We generated about million clean reads from the above samples, and , unigenes were acquired with N50 of bp. A total of differentially expressed genes DEGs were determined. Additionally, a total of 25 MADS-box unigenes were identified. This transcriptome data presents fundamental information about the genes and pathways involved in flower induction and development of Lei bamboo.
Moreover, a critical sampling method is provided which could be benefit for bamboo flowering mechanism study. In plant kingdom, flowering is necessary for the transition from vegetative stage to reproductive stage as one of the most important process. Investigation into Arabidopsis thaliana reveals that the phenomenon of the flowering is controlled by diverse environmental and endogenous factors, such as temperature, light signals, day length and plant hormones [ 1 — 4 ]. For example, plant endogenous hormones Gibberellin GA could promote flower development and low temperature will cause flowering delay in Arabidopsis [ 5 , 6 ].
Additionally, the ABCDE model genes are widely used for understanding floral development [ 7 ], and the MADS-box genes are well known as ABC model factors to control the floral organ identity during flower development [ 8 ].
Bamboo is one of the most important non-timber forest resources in the world. Its fast growth and strong nitrogen fixation capacity have received much attention in economy and ecology. However, the prolong vegetative period and the death of bamboo after flowering, lead to great economic and ecological losses [ 12 ]. The uncertainty of bamboo flowering time increased the difficulty of sampling, further inhibiting the study of flower induction to bamboo.
Fortunately, the completion of moso bamboo Phyllostachys edulis draft genome sequencing is beneficial to screen the flowering related genes. For example, the transcription factors play an important role in gene expression, thereby influencing the floral transition, the PheMADS14 and PheMADS4 obtained from moso bamboo may play vital regulatory roles in flower development [ 13 — 16 ].
Lei bamboo Phyllostachys violascens , widely distributed over south of China, has high economic value because of its bamboo shoots edibility. The income for intensively managed Lei bamboo is about 20 times higher than that of rice [ 17 ]. During flowering, however, shoot production decreases sharply. Different from moso bamboo flowering gregariously with 60— years period [ 18 ], Lei bamboo flowers sporadically [ 19 ].
It suggests a similar but not identical flowering mechanism between moso bamboo and Lei bamboo. However, the flowering mechanism in Lei bamboo is still poorly understood.
Next-generation sequencing [ 23 ] technologies such as the Illumina Solexa, Roche , and ABI SOLID platforms have revolutionized biological research by providing genomic and transcriptome data rapidly and inexpensively [ 24 ].
The goal of this study was to obtain a complete set of assembled unigenes and transcripts for Lei bamboo and to identify flowering-related genes. Our data will be useful for Lei bamboo flowering study and provides a critical sampling method to study flowering mechanism in bamboos. After observing flowering characteristics of Lei bamboo for more than 10 years, we began to learn and pre-judge before flowering occurs with a certain probability [ 26 ].
Flowering buds and vegetative buds were collected at about 2 pm from expected flowering and non-flowering plants from the same rhizome, at different stages: March 8th and 29th and April 12th, The expected bud samples confirmed after bamboo flowering were used for transcriptome sequencing. Vegetative buds TV from a non-flowering bamboo plant growing on the same rhizome of the flowering bamboo plant were both sampled at the same time Fig.
The reproductive and vegetative bud samples of Lei bamboo. Raw data raw reads in the fastq format were first processed by using in-house perl scripts. The analysis of data output quality. GC content: the GC ratio of the total base number. Expression level of genes is calculated by using RPKM method [ 28 , 29 ]. For each sequenced library, the read counts were adjusted by using the edgeR program package with one scaling normalized factor.
The DEGseq R package was used for differential expression analyses. We used NCBI blast 2. Blast2GO v2. Multiple sequence alignment was performed using the full-length protein sequences in the program Clustal W. The Actin was used as reference gene. Assembly of the clean reads produced ,, unigenes with sequence length range of —16, bp.
A total of 2. According to the GO analysis Fig. These pathway assignments provide valuable information for investigating specific biochemical and development processes.
The functional analysis of total unigenes in Lei bamboo. In order to better to analyze the flowering mechanism of Lei bamboo, we categorized the samples according to their development stages and analyzed the DEGs involved in TV and TF stages Fig. After removing the duplicated ones separately, a total of DEGs in vegetative buds and DEGs in flowering buds were finally identified. The venn diagram of differentially expressed genes DEGs in Lei bamboo. The expression profile suggests these plant hormones related DEGs should be involved in the regulating of Lei bamboo flower induction Fig.
It reflects that the flower induction of Lei bamboo possibly affected by environmental stress factors. Therefore, the genes involved in stress responsive and phytohormone should be essential for the process of Lei bamboo flower induction. The heatmap of the selected DEGs in Lei bamboo flowering a. In this stage, the floral organ began to mature. The heatmap of the selected DEGs involved in floral organ development of Lei bamboo.
The phylogenetic trees were constructed by using the NJ Fig. In the NJ and ML trees, a bit interior branches exist some minor differences, but in general, the classification results are very similar. It suggests that most proteins have similar origin in both trees. Here, we used NJ phylogenetic tree for further analysis.
According to the phylogenetic tree Fig. According to RNA-seq results, eight genes showed different patterns of expression in different samples Fig. As the randomness and uncertainty of bamboo flowering, how to obtain the accurate flower samples is the most crucial problem for bamboo flowering mechanism study. Here, the sampling time of bamboo flowering materials was earlier than that in most usual researches.
This method makes it better to identify the flower induction related genes in bamboo. It is not only beneficial to avoid interference from vegetative growth related genes in bamboo flowering mechanism study, but also helpful to identify the related genes involved in vegetative periods in Lei bamboo. Above all, our research will provide a new sampling method for studying the flowering mechanism of bamboo.
We obtained , assembled unigenes using RNA-seq, with DEGs identified between flowering and vegetative buds at the three stages of Lei bamboo.
Although the RNA-seq databases of certain bamboo species, such as moso bamboo, Ma bamboo Dendrocalamus latiflorus and Bambusa edulis [ 52 — 54 ], have been established, we provided transcriptome data in Lei bamboo for the first time. Finally, a number of DEGs were determined according to the expression profiles, indicating their functional roles in early flowering initiation and flower development of Lei bamboo.
Interestingly, the stress-responsive genes were also identified to be highly-expressed in moso bamboo, but the genes employed in typical flowering promotion pathways such as those in the photoperiod, gibberellins, ambient-temperature or light-quality pathways and floral pathway integrator FPI genes were expressed lowly in these floral tissues [ 52 ].
It is highly likely that the flowering of bamboo associated more with the environmental stress factors than photoperiod or other traditional pathways. Plant hormones play necessary role during flower development [47—51]. In previous study, the endogenous hormone concentrations were found to be changed with the development of flower bud morphological differentiation [ 55 ].
For higher plants, the anther and carpel developments were recognized as the most important stages of organogenesis regarding sexual reproduction processes [ 46 ]. Therefore, these DEGs might have essential roles in the sextual reproduction process of Lei bamboo. The family of MADS-box genes play important roles in plant flower development.
They are expressed regionally specific in particular flower tissues, during differentiation growth of the flower development. According to previous reports, MADS-box genes participate in floral organ development and flowering time regulation [ 58 ].
In rice, 34 MADS-box genes have been identified, and 15 of which regulate flower development [ 61 ]. In moso bamboo, 34 MADS-box genes were identified and the expression patterns of most genes were found to similar to Arabidopsis and rice [ 23 ].
Additionally, it is generally accepted that the C class genes AG control the development of stamen and pistil [ 45 ], and the D class genes are involved in ovule identity [ 11 ]. But in petunia, D genes are not essential to confer ovule identity, instead, this function is redundantly shared among all AG members, and D genes might participate in floral determinacy [ 62 ]. The E genes were known to participate in regulation of flower organ characteristics [ 63 ]. These genes play fundamental roles by interacting with other MADS-box genes products [ 47 , 64 , 65 ].
Whether this interaction is suitable for Lei bamboo needs to be further explored. We are thankful to Dr. XLin and XLi designed the experiments. YJ and QH analyzed data and wrote the manuscript. XLin revised and edited the manuscript.
All authors read and approved the paper. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Xuan Li, Email: nc. Xinchun Lin, Email: moc. Supplementary information accompanies this paper at Read article at publisher's site DOI :
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Grojinn Footnotes Funding This work was supported by grants from Beijing science and technology plan No. This number varied slightly depending on inclusion of undetectable CETPs that were located behind and in front of the lipo-protein particles. The results from the study of Ranalletta et al. In the control sample Fig.
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