Transcript Profile of Flowering Regulatory Genes in VcFT-Overexpressing Blueberry Plants

Abstract

In order to identify genetic components in flowering pathways of highbush blueberry (Vaccinium corymbosum L.), a transcriptome reference composed of 254,396 transcripts and 179,853 gene contigs was developed by assembly of 72.7 million reads using Trinity. Using this transcriptome reference and a query of flowering pathway genes of herbaceous plants, we identified potential flowering pathway genes/transcripts of blueberry. Transcriptome analysis of flowering pathway genes was then conducted on leaf tissue samples of transgenic blueberry cv. Aurora (‘VcFT-Aurora’), which overexpresses a blueberry FLOWERING LOCUS T-like gene (VcFT). Sixty-one blueberry transcripts of 40 genes showed high similarities to 33 known flowering-related genes of herbaceous plants, of which 17 down-regulated and 16 up-regulated genes were identified in ‘VcFT-Aurora’. All down-regulated genes encoded transcription factors/enzymes upstream in the signaling pathway containing VcFT. A blueberry CONSTANS-LIKE 5-like (VcCOL5) gene was down-regulated and associated with five other differentially expressed (DE) genes in the photoperiod-mediated flowering pathway. Three down-regulated genes, i.e., a MADS-AFFECTING FLOWERING 2-like gene (VcMAF2), a MADS-AFFECTING FLOWERING 5-like gene (VcMAF5), and a VERNALIZATION1-like gene (VcVRN1), may function as integrators in place of FLOWERING LOCUS C (FLC) in the vernalization pathway. Because no CONSTAN1-like or FLOWERING LOCUS C-like genes were found in blueberry, VcCOL5 and VcMAF2/VcMAF5 or VRN1 might be the major integrator(s) in the photoperiod- and vernalization-mediated flowering pathway, respectively. The major down-stream genes of VcFT, i.e., SUPPRESSOR of Overexpression of Constans 1-like (VcSOC1), LEAFY-like (VcLFY), APETALA1-like (VcAP1), CAULIFLOWER 1-like (VcCAL1), and FRUITFULL-like (VcFUL) genes were present and showed high similarity to their orthologues in herbaceous plants. Moreover, overexpression of VcFT promoted expression of all of these VcFT downstream genes. These results suggest that VcFT’s down-stream genes appear conserved in blueberry.

Overexpression of blueberry FLOWERING LOCUS T is associated with changes in the expression of phytohormone-related genes in blueberry plants

“…Our quantitative RT–PCR results confirmed the upregulation (six genes) and downregulation (four genes) of all selected genes (Supplementary Figure S5). In addition to our previous confirmation of the selected DE flowering pathway genes, 39 our quantitative RT–PCR results gave more evidence to support the reliability of our RNA sequencing data.…”

“…Because VcSOC1 can interact with GA pathway genes and the ‘VcFT-Aurora’ plants showed retarded growth, we hypothesized that the VcFT -OX has a potential impact on retarded plant growth through the expression of either phytohormone genes or dwarf-related genes. Using the annotated RNA sequencing data of our previous samples, 39 in-depth analysis in this study revealed the overall effect of the VcFT -OX on blueberry gene networks (Figure 4). …”

“…14 In our recent transcriptome analysis of the VcFT -OX transgenic blueberry focusing on flowering pathway genes, 61 transcript contigs of 33 known flowering-related genes showed differential expression. 39 Of these DE flowering genes, both VcFT and VcSOC1 are major integrators of the flowering pathway. Because VcSOC1 can interact with GA pathway genes and the ‘VcFT-Aurora’ plants showed retarded growth, we hypothesized that the VcFT -OX has a potential impact on retarded plant growth through the expression of either phytohormone genes or dwarf-related genes.…”

“…39 Briefly, RNA sequencing reads of three biological replicates for each of the three non-transgenic ‘Aurora’ plants and three ‘VcFT-Aurora’ plants were analyzed. Two technical replicates were sequenced for each biological replicate.…”

“…39 However, the overall gene networks responding to overexpressing a blueberry ( Vaccinium corymbosum L.) FT gene ( VcFT ) (herein VcFT -OX) are not known. The aim of this study was to annotate the blueberry transcriptome reference by using a transcriptome assembly tool called Trinotate (https://trinotate.github.io), to identify the DE genes in the phytohormone or dwarfing-related pathways, and to develop the first gene network models in blueberry that show the potential interactions of all DE-expressed genes driven by the VcFT -OX.…”