A Vacuolar Processing Enzyme RsVPE1 Gene of Radish Is Involved in Floral Bud Abortion under Heat Stress

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.

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.

Introduction
Floral bud abortion (FBA) is an adverse biological phenomenon affecting radish (Raphalus sativus L.) reproduction, which is commonly associated with radish breeding practices and the creation of radish germplasm resources. During FBA, the floral bud first stops growing and then gradually turns from green to yellow, until it finally dries out completely ( Figure 1). Plants exhibiting FBA are often eliminated during artificial breeding practices or lost naturally, even if they have good economic characteristics or specific traits, which is ultimately harmful for the selection of new radish cultivars and conservation of biodiversity. FBA is a widespread phenomenon in plant reproduction and evolution, having been observed in Cucurbita texana [1], dwarf bean [2], cowpea [3], rape [4], Chinese cabbage [5], radish [6,7], and the model plant Arabidopsis thaliana [8]. FBA in plants, including C. texana, cowpea, dwarf beans and A. thaliana, is most commonly thought to result from abiotic stress. Despite the widespread occurrence of FBA, there is limited knowledge about its molecular mechanism, with only a few preliminary studies published to date. Zhang et al. analyzed gene expression during 10 continuous developmental periods of FBA in Chinese cabbage using cDNA-AFLP, and found 72 novel expressed sequence tags (ESTs) related to the aborted buds [5]. Jia et al. evaluated the aborted and normal buds of radish and obtained 107 differentially expressed fragments related to FBA. During FBA in radish, characteristics of programmed cell death (PCD) were detected at the DNA level [6,7]. Numerous reports indicate that proteases with caspase-like activity exist in plants and mediate processes of cell death during development and stress responses [9,10]. Previous work has described the vacuole-localized cysteine proteases called vacuolar processing enzymes (VPEs), which were originally discovered in the maturation of seed storage proteins [11]. Phylogenic, as well as expression and sequence analysis of VPEs have indicated that they are classified into the three following categories: seed type, seed coat type, and vegetative type [12,13]. Two vegetative type VPE proteins have been identified in Arabidopsis, namely αVPE and γVPE, both of which are mainly expressed in vegetative tissues. The mRNA expression of these genes is up-regulated in response to various abiotic stresses, such as wounding, senescence, and treatment with hormones including jasmonic acids, ethylene, and salicylic acid [14][15][16]. Furthermore, it was reported that a functional VPE protein acts as a critical factor initiating hypersensitive cell death in response to infection with tobacco mosaic virus (TMV) and Pseudomonas syringae pv. phaseolicola in tobacco [17]. Studies have provided evidence that AtγVPE exhibiting a caspase-1 activity is a key molecule in toxin-induced cell death [18]. TDF72, a transcript-derived fragment obtained by cDNA-AFLP in our previous study, was up-regulated in the aborted buds and exhibited 89% sequence homology with the AtγVPE gene, suggesting that VPE has a role in FBA, since VPE has been known to link with PCD. Previous research into plant VPEs has focused mainly on plant senescence and pathogen-induced hypersensitive cell death, but its role in FBA is poorly understood.
In this study, TDF72 was used to analyze the role of VPE in FBA, by isolation of the VPE gene RsVPE1 from radish flower buds. Expression of the RsVPE1 gene was analyzed in different tissues of radish plants. In addition, to examine the effect increased RsVPE1 expression on FBA, RsVPE1 was overexpressed under the CaMV 35S promoter in transgenic Arabidopsis thaliana plants.

Molecular Cloning and Sequence Analysis of RsVPE1
To investigate the role of VPEs in FBA, the RsVPE1 gene was identified and characterized. The complete 5' and 3' regions of the putative RsVPE1 gene were isolated by rapid amplification of cDNA ends (RACE) from radish flower buds. Based on the sequence of TDF72, the 3'-cDNA end and 5'-cDNA end were determined by RACE to be 937 bp and 1030 bp in length, respectively. The full-length cDNA of the RsVPE1 was 1825 bp, containing the start codon ATG, the stop codon TAG, a    Subsequently, to evaluate the molecular evolutionary relationships of RsVPE1 against other VPEs, a phylogenic tree was constructed. RsVPE1 exhibited the closest relationship with AtγVPE ( Figure 4), belonging to a novel group of cysteine proteinases and is up-regulated in association with various types of cell death and under stress conditions [12,[14][15][16][17]. Although the result showed that the RsVPE1 gene shared 90% identity with AtγVPE, there are differences in the predicted protein sequences of these two genes. The predicted protein of RsVPE1 is made of 489 amino acid residues. However, the predicted protein of AtγVPE is consisted of 494 amino acid residues. Such differences would be reflected in the functions of these vegetative type VPEs.

Analysis of Tissue-Specific RsVPE1 Expression in Radish Plants
Real-time PCR was performed to determine the tissue-specific expression of RsVPE1 in radish plants. RsVPE1 transcripts were produced in all the analyzed radish tissues including leaves, flower stalks, flower buds (normal and aborted) and young siliques ( Figure 5a). However, the expression levels of RsVPE1 gene varied in different tissues. The highest RsVPE1 expression was detected in aborted flower buds, which indicates that RsVPE1 plays an important role in FBA. Furthermore, the RsVPE1 gene expression in different organs of radish flower buds were analyzed ( Figure 5b). Interestingly, the levels of RsVPE1 transcripts were higher in the different organs of aborted flower buds than in the corresponding organs of normal flower buds, with the highest expression levels in the sepals and the lowest expression levels in the pistils. It has been reported that the cells of the sepal, petal and anther in aborted flower buds exhibited obvious degeneration, resulting in abnormality of flower bud development. However, degeneration of pistil tissue was not observed in aborted flower buds [7]. In this study, RsVPE1 expression levels in the pistils were the lowest among the four parts of flower bud, suggesting that RsVPE1 almost does not affect the development of pistils.

RsVPE1-Overexpressing in Transgenic Arabidopsis Plants
To further investigate the role of RsVPE1 in plants, we generated transgenic Arabidopsis expressing the RsVPE1 cDNA under the control of the CaMV 35S promoter, and eight homozygous transgenic lines were obtained by antibiotic resistance screening and PCR confirmation. Three independent homozygous transgenic lines (TL3, TL78 and TL83) were randomly selected for further analysis. The expression level was checked by real-time PCR analysis. To examine the possible phenotype of homozygous transgenic lines, T 3 progeny of the RsVPE1-overexpressed lines and the Columbia ecotype (Col-0) plants were grown in the greenhouse under normal conditions. Compared with Col-0 plants, no obvious morphological or developmental abnormalities were observed in transgenic plants when they were grown in artificial soil at normal temperatures in the greenhouse.

FBA in RsVPE1-Overexpressing Transgenic Arabidopsis Plants under Heat Stress
Col-0 had been used as a model of heat-tolerance research in Arabidopsis accessions and reported that aborted flower buds were not observed in plants exposed to 32 °C or 34 °C, regardless of the duration of the exposure [8]. Therefore, transgenic plants were subjected to 32 ± 0.5 °C for 36 h in our study. In accordance with the observations reported by Warner et al. [8], aborted flower buds were not found in Col-0. However, significant phenotypic changes, in the form of aborted flower buds, were observed in transgenic Arabidopsis plants in response to heat stress. There were 13.1 to 16.4 aborted flower buds in three transgenic lines. FBA began to display on the third day after heat stress. All aborted flower buds appeared on the eighth day after heat stress ( Figures 6 and 7). Based on the flower bud ages and positions, we could speculate that FBA occurred in the flower buds of stage 9 to 12. Although the expression of RsVPE1 from the constitutive 35S-promoter should not be markedly affected by environmental conditions, RsVPE1 expression levels of three transgenic lines (TL3, TL78 and TL83) reached maximums at 24 h heat stress and were shown 2.1-fold, 2.8-fold and 2.5-fold increase compared with their expression levels at 0 h, respectively (Figure 8b). In addition, RsVPE1 expressed in transgenic lines under normal condition (Figure 8a), but aborted flower buds were not observed ( Figure 7). These results suggested that RsVPE1 gene and heat stress were important to the occurrences of FBA in transgenic Arabidopsis plants. Perhaps, heat stress promoted the RsVPE1 expression to reach a particular threshold for FBA, or the heat stress induced other metabolisms, which assisted RsVPE1 to cause FBA in transgenic Arabidopsis plants. Furthermore, studies have provided evidence that there were typical characteristics of PCD during FBA in radish and vegetative type VPEs acted as a key factor initiating in PCD [17,18]. As Figure 4 shown, RsVPE1 is a member of the vegetative type of VPEs. It could be assumed that RsVPE1 expression was up-regulated to a level that is sufficient to promote programmed cell death (PCD), leading to FBA.

Plant Materials and Growth Conditions
Radish FBA material L9 was used in this study (Figure 1) (provided by the Chinese Cabbage Laboratory of the Northwest A&F University, Yangling, Shaanxi, China). Radish plants were planted in the greenhouse in spring.

Cloning and Sequence Analysis of RsVPE1 Gene
Genomic DNA was isolated from leaves using a modified CTAB protocol [19]. Total RNA was isolated from radish floral buds with TRIZOL™ according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The complete 5' and 3' regions of the putative RsVPE1 gene were isolated by RACE. Approximately 1 μg of total RNA was used initially for first-strand synthesis using the SMART™ RACE cDNA Amplification Kit according to the manufacturer's instructions (Taraka, Dalian, China). The gene-specific primers 5'GSP (5'-GATGAGTCCTGACCGACCCAGAACCTAC A-3') and 3'GSP (5'-CCCTCCACCTGAGTATGAAACTTGCTTAG-3') were used for 5'-RACE and 3'-RACE, respectively. The universal primers for 5'-RACE and 3'-RACE were provided in the kit. The full-length cDNA and gDNA of the RsVPE1 gene was obtained by PCR amplification using forward (5'-ATGCCACCTGTCCCCGTCGCCGTT-3') and reverse (5'-TTAAGCACTGAATCCACGGTGA-3') primers. The PCR fragments were cloned into the pMD18-T vector (Takara,) and sequenced by Sangon Biotech Co., Ltd. (Shanghai, China).
NCBI bioinformatics tools were used to compare and analyze the nucleotide and protein sequence data. Multi-sequence alignment was conducted using ClustalX version 1.83 with default parameters.
The phylogenic tree was built using the neighbor-joining (NJ) method using MEGA software (version 5.0) [20].

Tissue-Specific Expression Patterns of the RsVPE1 Gene in Radish
To analyze tissue-specific expression of the RsVPE1 gene in radish plants, leaves, flower stalks, flower buds, young siliques, sepals, petals, stamens and pistils of normal and aborted flower buds were collected from radish FBA material L9. Total RNA was extracted from samples using Trizol™ according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was obtained using the PrimeScript™ RT reagent Kit (Takara, Dalian, China). The EF-1-alpha gene (GO479260) (forward primer 5'-ATACCAGGCTTGAGCATACCG-3' and reverse primer 5'-GCCAAAGAGGCCATCAGACAA-3') was used as a loading control. The specific primer sequences used for expression analysis were as follows: 5'-TCCTGACCGACCCAGAACCTA-3' and 5'-ATACAGTGTTGCCTGGATGGAAG-3'. Real-time PCR reactions were repeated three times in a 20 μL reaction containing 10.0 μL SYBR Premix Ex Taq, 0.8 μL forward primer (10 μM), 0.8 μL reverse primer (10 μM), 2 μL diluted cDNA and sterile ddH2O with a Bio-Rad iQ5 instrument (Foster City, CA, USA). Thermal cycling parameters were as follows: 3 min at 95 °C and 40 cycles of 20 s at 95 °C, 30 s at 58 °C and 30 s at 72 °C. The relative mRNA levels of RsVPE1 were normalized to those of EF-1-alpha using the 2 −ΔΔCt method [21].

Genetic Transformation of RsVPE1 into Arabidopsis
The open reading frame (ORF) of RsVPE1 was cloned into the pCAMBIA2301-35S-Nos vector (Cambia, Canberra, Australia). The Col-0 of Arabidopsis thaliana was used in this study. The plants were grown under 14-h light/10-h dark conditions at 24 °C during the day and 20 °C at night (24/20 °C) for transformation. The constructs were transferred into Agrobacterium tumefaciens GV3101 and transformed into Col-0 using the method by Clough et al. [22]. The seeds of the T 0 generation were harvested and placed on MS [23] agar medium (0.8%) containing 50 μg·mL −1 kanamycin monosulfate (Km) after surface sterilized using a liquid method. Then the surviving transformants (T 1 ) were confirmed by PCR to amplify RsVPE1 gene and the NPTII gene. The confirmed transgenic plants were harvested individually. The T 2 seeds were placed on MS agar medium (0.8%) containing 50 μg·mL −1 Km and the transgenic lines with a 3:1 segregation ratio (resistant:sensitive) were selected to produce T 3 seeds. The T 3 lines displaying 100% Km resistance were considered to be homozygous for the transgenic gene and used for further experiments. The Col-0 was used as a control. All seeds of Col-0 and transgenic plants were collected at the same stage.

Heat Stress on Transgenic Plants
The seeds of transgenic plants and Col-0 were fully germinated on MS agar medium for 10 days. The seedlings were then transferred from the medium to artificial soil containing peat, vermiculite and pearlites. To identify the effect of heat stress, transgenic and Col-0 plants were grown in the 24/20 °C chamber until the first flower opened. Ten plants of each type remained in the 24/20 °C chamber, while another ten plants of each type were transferred to an illumination growth chamber maintained at a constant air temperature (32 ± 0.5 °C) and relative humidity (82% ± 10%) under the same photoperiod and irradiance for 36 h. The plants were then moved back to the 24/20 °C chamber. The number of flower buds that failed to open (aborted floral buds) was determined on the eighth day after treated with 32 ± 0.5 °C for 36 h.

Analysis of RsVPE1 Gene Expression in Transgenic Arabidopsis Plants
To evaluate the expression levels of RsVPE1 gene in transgenic Arabidopsis plants under normal condition, flower buds were collected from Col-0 and three independent transgenic lines, then frozen in liquid nitrogen and stored at −80 °C for future use. In addition, to analyze the effects of heat stress on RsVPE1 expression levels, total RNA was extracted from Arabidopsis flower buds of different time points during heat stress using Trizol™ according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was obtained using the PrimeScript™ RT reagent Kit (Takara, Dalian, China). Real-time PCR was conducted using the first-strand cDNA templates to detect the expression level of RsVPE1 in Arabidopsis flower buds. The Arabidopsis AtACTIN gene (forward primer 5'-ACGTCGGAGACGAGGCACAG-3' and reverse primer 5'-AGCGGCGCTTCG GTTAGCAAT-3') was used as a loading control. The specific primer sequences used for expression analysis were as follows: 5'-TTCTTCCGCCCTCCCAACAAC-3' and 5'-ATCCGCCTGATGCCTGT AGTTC-3'. Real-time PCR reactions were repeated three times in a 20 μL reaction containing 10.0 μL SYBR Premix Ex Taq, 0.8 μL forward primer (10 μM), 0.8 μL reverse primer (10 μM), 2 μL diluted cDNA and sterile ddH2O with a Bio-Rad iQ5 instrument (Foster City, CA, USA). Thermal cycling parameters were as follows: 3 min at 95 °C and 40 cycles of 20 s at 95 °C, 30 s at 58 °C and 30 s at 72 °C. The relative expression levels of RsVPE1 were normalized to those of AtACTIN using the 2 −ΔΔCt method [21].

Conclusions
In conclusion, a VPE gene, named RsVPE1, was isolated from flower buds of radish. RsVPE1 transcript levels varied in different tissues of radish plants. The highest RsVPE1 expression was detected in aborted flower buds, which indicates that RsVPE1 plays an important role in FBA. Although RsVPE1 was expressed in three independent homozygous transgenic Arabidopsis lines, no obvious morphological or developmental abnormalities were observed in transgenic plants when these plants were grown in artificial soil at normal temperatures. However, aborted flower buds were observed in transgenic Arabidopsis plants in response to heat stress. In addition, RsVPE1 expression levels reached maximums at 24 h heat stress and were shown 2.1-fold to 2.8-fold increase compared with their expression levels at 0 h, respectively. These results implied that RsVPE1 gene and heat stress were important to the occurrences of FBA in transgenic Arabidopsis plants. The data also demonstrated that RsVPE1 played an important role in flower development of radish and transgenic Arabidopsis plants. When the expression level of RsVPE1 reaches a particular threshold, the development of flower buds is hindered. It could be assumed that RsVPE1 expression was up-regulated to a level that is sufficient to promote programmed cell death (PCD), leading to FBA.