BeMADS1 is a key to delivery MADSs into nucleus in reproductive tissues-De novo characterization of Bambusa edulis transcriptome and study of MADS genes in bamboo floral development

Abstract

Background: The bamboo Bambusa edulis has a long juvenile phase in situ, but can be induced to flower during in vitro tissue culture, providing a readily available source of material for studies on reproductive biology and flowering. In this report, in vitro-derived reproductive and vegetative materials of B. edulis were harvested and used to generate transcriptome databases by use of two sequencing platforms: Illumina and 454. Combination of the two datasets resulted in high transcriptome quality and increased length of the sequence reads. In plants, many MADS genes control flower development, and the ABCDE model has been developed to explain how the genes function together to create the different whorls within a flower.

Results: As a case study, published floral development-related OsMADS proteins from rice were used to search the B. edulis transcriptome datasets, identifying 16 B. edulis MADS (BeMADS). The BeMADS gene expression levels were determined qRT-PCR and in situ hybridization. Most BeMADS genes were highly expressed in flowers, with the exception of BeMADS34. The expression patterns of these genes were most similar to the rice homologs, except BeMADS18 and BeMADS34, and were highly similar to the floral development ABCDE model in rice. Transient expression of MADS-GFP proteins showed that only BeMADS1 entered leaf nucleus. BeMADS18, BeMADS4, and BeMADS1 were located in the lemma nucleus. When co-transformed with BeMADS1, BeMADS15, 16, 13, 21, 6, and 7 translocated to nucleus in lemmas, indicating that BeMADS1 is a key factor for subcellular localization of other BeMADS.

Conclusion: Our study provides abundant B. edulis transcriptome data and offers comprehensive sequence resources. The results, molecular materials and overall strategy reported here can be used for future gene identification and for further reproductive studies in the economically important crop of bamboo.

Figure 1

Overview of sequence reads and assembly of the three B. edulis transcriptomes. The length distribution of the contigs obtained from de novo assembly of high-quality, clean reads from NGS data across three datasets, namely sequence data from Roche 454, Illumina, and Hybrid transcriptome. Panel A shows the lengths of all contigs from each dataset. Panel B: shows the contig lengths for only those contigs that had BLASTX hits in the NCBI protein database.

Figure 2

Assignment of COG and GO classifications to B. edulis unigenes across three transcriptome datasets. A. COG functional classification of the B. edulis transcriptome. The graph shows the percentage of the whole dataset that was annotated within any one COG function.A total of 9,347 (for 454 dataset), 29,654 (for Illumina dataset) and 6,158 (for Hybrid dataset) unigenes showed significant homologies to genes in the COG protein database and were distributed into 25 COG categories (A-Z, except X). B. GO classification of the B. edulis transcriptome. The graph shows the percentage of the whole dataset that was annotated within any one GO sub-category. A total of 15,916 unigenes from 454 dataset were distributed into 36 GO sub-categories (functional groups), 38,740 unigenes from Illumina dataset were distributed into 41 sub-categories, and 10,866 unigenes from Hybrid dataset were distributed into 34 sub-categories.

Figure 3

Phylogenetic tree based on amino acid sequences of MIKC-type MADS-box genes. 60 MIKC-type MADS-box genes were used: 16 from Bambusa edulis, 16 from rice (Oryza sativa), 10 from maize (Zea mays), and 18 from wheat (Triticum aestivum L.). Deduced full-length amino acid sequences were used for the alignments. The phylogenetic tree was constructed by the neighbor-joining method and evaluated by bootstrap analysis (MEGA version 4.0). Numbers on major branches indicate bootstrap percentage for 1,000 replicates. Six Arabidopsis sequences of the FLC subfamily were used as outgroups. Proteins from B. edulis were highlighted with red boxes. The three grass clades of FUL1, FUL2, and FUL3 within the AP1 subfamily and the two major clades of the SEP subfamily are labeled on the right. The five grass clades within the SEP subfamily are indicated by numbers showing their respective name according to previous studies [41], namely 1: LHS1/OsMADS1, 2: OsMADS5, 3: OsMADS34, 4: OsMADS7/45, 5: OsMADS8/24. Subfamilies of the plant MIKC-type genes and the functional classification according to the A/B/C/D/E classes are indicated at the right margin.

Figure 4

Developmental stage, organ and tissue-specific expression patterns of BeMADS genes. B. edulis RNA was extracted from different in vitro tissues and subjected to cDNA synthesis: R: roots; L: leaves; S: stems; F: flowers; 1–5: young to old florets, see Additional file 5; and the floral organs Le: lemma; Pa: palea; Lo: lodicules; An: anther; and Pi: pistil. Quantitative RT-PCR was undertaken using the primers in Additional file 6. The B. edulis tubulin gene was used as the internal control. The color intensity is related to the expression level, with darker indicating higher expression. The colors represent the classes of the gene from Figure 3: A: green, B: orange, C: blue, D: grey, E: pink.

Figure 5

In situ localization of BeMADS1 and BeMADS2 transcripts in early floral bud of B. edulis. Longitudinal sections were hybridized with DIG-labeled antisense and sense probes. Left: Hybridization signals of antisense (upper) and sense (lower) probe of BeMADS1. Right: Hybridization signals of antisense (upper) and sense (lower) probe of BeMADS2. The signals detected from sense probe were used as negative control. Pa: palea; Lo: lodicules; An: anther. Bar = 100 μm

Figure 6

Subcellular localization of BeMADS fused with fluorescent proteins in B. edulis lemmas and leaves. A. Plasmids harboring a YFP fusion with different BeMADS proteins (yellow signals, the number indicates the gene name) driven by the 35S promoter were transiently expressed in B. edulis lemma. These plasmids were delivered by particle bombardment. The NLS domain of VirD2 fused with mCherry was used as the nuclear marker (in red color). Bar = 20 μm. B. The subcellular localizations of YFP fusions of BeMADS18, 4 and 1 delivered by particle bombardment into leaf or lemma (yellow signals, Numbers indicate the BeMADS). Red: nuclear marker, VirD2-mCherry signals. Leaf: using leaves as the materials for transient expression. Bar as above.

Figure 7

Nuclear localization of BeMADS proteins during co-transformation with BeMADS1. Lemmas were used as material for transient transformation by particle bombardment. The tested YFP-BeMADS (numbers in left columns) were co-transformed with BeMADS1-CFP and VirD2-mCherry (nuclear marker). The micrographs in the left column are from Figure 5 and show the localizations of the BeMADS-YFP proteins in B. edulis leaves without co-expression of BeMADS1. Bar = 20 μm.