A vacuolar processing enzyme RsVPE1 gene of radish is involved in floral bud abortion under heat stress

Abstract

Radish floral bud abortion (FBA) is an adverse biological phenomenon that occurs during reproduction. Although FBA is a frequent occurrence, its molecular mechanism remains unknown. A transcript-derived fragment (TDF72), which was obtained by cDNA amplified fragment length polymorphism (cDNA-AFLP), was up-regulated in the aborted buds and exhibited 89% sequence homology with the AtγVPE gene. In this study, TDF72 was used to clarify the role of VPE in FBA by isolation of the VPE gene RsVPE1 from radish flower buds. The full-length genomic DNA was 2346 bp including nine exons and eight introns. The full-length cDNA was 1825 bp, containing a complete open reading frame (ORF) of 1470 bp, which encoded a predicted protein containing 489 amino acid residues, with a calculated molecular mass of 53.735 kDa. Expression analysis demonstrated that RsVPE1 was expressed in all tested organs of radish at different levels. Highest expression was detected in aborted flower buds, suggesting that RsVPE1 has a role in FBA. In order to analyze the role of RsVPE1 in FBA, RsVPE1 was overexpressed in transgenic Arabidopsis thaliana plants. Aborted flower buds appeared in transgenic plants subjected to heat stress. In addition, RsVPE1 expression in the transgenic plants reached a maximum when subjected to heat stress for 24 h and increased by 2.1-fold to 2.8-fold in three homozygous transgenic lines. These results indicated that RsVPE1 led to FBA when its expression levels exceeded a particular threshold, and provided evidence for the involvement of RsVPE1 in promoting FBA under heat stress.

Figure 1

Floral bud abortion (FBA) of radish.

Figure 2

Schematic diagram and nucleotide sequence of RsVPE1. (a) Schematic diagram of the genomic RsVPE1 sequence. Exons are indicated by hollow arrows followed by their names and sizes. Orange and blue boxes indicate the exons and introns, respectively; (b) Schematic diagram of the full-length RsVPE1 cDNA sequence. The numbers on the left are the nucleotide and amino acid positions.

Figure 3

Comparison of the predicted protein sequence of RsVPE1 with other vacuolar processing enzyme (VPE) proteins. The accession numbers of these proteins in the GenBank database are as follows: AtαVPE (NP_180165.1), AtβVPE (NP_176458.1), AtγVPE (NP_195020.1), CsVPE (XP_004142919.1), GmVPE (XP_003525979.1), MtVPE (XP_003603121.1), OsVPE (NP_0010565054.1), RcVPE (XP_002516472.1), SbVPE (XP_002448237.1), VvVPE (XP_002276759.1), ZmβVPE (ACG47915). At, Arabidopsis thaliana; Cs, Cucumis sativus; Gm, Glycine max; Mt, Medicago truncatula; Os, Oryza sativa; Rc, Ricinus communis; Rs, Raphalus sativus; Sb, sorghum bicolor; Vv, Vitis vinifera; Zm, Zea mays. Black indicates identical amino acids among the different species. Pink and blue represent similar amino acids.

Figure 4

Phylogenic tree of VPE proteins. The rooted gene tree (majority-rule consensus from 1000 bootstrap replicates) resulted from heuristic searching in Mega5.0. Bootstrap values are indicated at each branch node. At, Arabidopsis thaliana; Cs, Cucumis sativus; Gm, Glycine max; Mt, Medicago truncatula; Os, Oryza sativa; Rc, Ricinus communis; Rs, Raphalus sativus; Sb, sorghum bicolor; Vv, Vitis vinifera; Zm, Zea mays. Bootstrap values are indicated at each branch node. RsVPE1 was marked with red box. GenBank accession numbers are in parentheses after each species and protein name. Scale bar indicates similarity coefficient.

Figure 5

Expression analysis of RsVPE1 gene in radish plants. RsVPE1 expression in (a) radish plants and (b) the four parts of radish flower buds. NFB, normal flower buds; AFB, aborted flower buds; L, leaves; FS, flower stalks; YS, young siliques; NSE, sepals of normal flower buds; NPE, petals of normal flower buds; NST, stamens of normal flower buds; NPI, pistils of normal flower buds; ASE, sepals of aborted flower buds; APE, petals of aborted flower buds; AST, stamens of aborted flower buds; API, pistils of aborted flower buds. Error bars represent the mean ± SD of three independent biological replicates.

Figure 6

FBA in transgenic Arabidopsis under heat stress. (a) The image of homozygous transgenic Arabidopsis line and wild-type plant on the eighth day after heat stress; (b) Local amplification of (a). TL78(ck), transgenic plants at normal temperature; TL78(HT), transgenic plant on the eighth day after treated with 32 ± 0.5 °C for 36 h; WT(ck), Col-0 of Arabidopsis thaliana at normal temperature; WT(HT), Col-0 of Arabidopsis thaliana on the eighth day after treated with 32 ± 0.5 °C for 36 h.

Figure 7

The number of aborted flower buds on the primary inflorescence of Arabidopsis plants subjected to heat stress. WT, Col-0 of Arabidopsis thaliana; TL3, TL78, TL83 are homozygous transgenic lines. Error bars represent the mean ± SD of three independent biological replicates.

Figure 8

The expression of RsVPE1 gene in transgenic Arabidopsis flower buds. (a) RsVPE1 expression in transgenic plants under normal condition. The transcript level of RsVPE1 in TL3 was used as the calibrator; (b) RsVPE1 expression in transgenic plants subjected to heat stress. The transcript levels of RsVPE1 in three transgenic lines at 0 h heat stress were used as the calibrators, respectively. TL3, TL78, TL83 are homozygous transgenic lines. Error bars represent the mean ± SD of three independent biological replicates.