Litchi Flowering is Regulated by Expression of Short Vegetative Phase Genes

in Journal of the American Society for Horticultural Science

Short vegetative phase (SVP), a MADS-domain transcription factor, was shown to act as a repressor of flowering in arabidopsis (Arabidopsis thaliana). Although the role of SVPs in flowering is well characterized in the model plant arabidopsis, little is known in evergreen woody litchi (Litchi chinensis). In this study, three litchi SVP homologs (LcSVP1, LcSVP2, and LcSVP3) were cloned, and the bioinformatic analysis of the LcSVPs was carried out to identify their molecular characteristics. Their expression patterns in the apical meristem (AM) during the transition from vegetative to reproductive phase were studied under natural flowering inductive conditions. Also, brassinosteroid (BR) treatment under low temperature conditions was performed to elucidate the role of LcSVPs in the BR-regulated flowering. The results showed that LcSVPs belonged to the MADS superfamily. LcSVP relative expression levels in AMs of the early- and late-flowering cultivars showed decreasing trends with the transition from vegetative to reproductive growth. Under low temperature condition, relative expression levels of LcSVP1, LcSVP2, and LcSVP3 in AMs or panicle primordia showed decreasing trends, whereas those in the AMs of the BR-treated trees remained at relatively high levels. Relative expression analysis of the litchi homolog, flowering locus t 1 (LcFT1), showed that the BR-treated leaves had lower relative expression level than nontreated control leaves. The findings suggest that LcSVPs act as repressors involved in flowering in natural conditions and the BR-regulated flowering.

Abstract

Short vegetative phase (SVP), a MADS-domain transcription factor, was shown to act as a repressor of flowering in arabidopsis (Arabidopsis thaliana). Although the role of SVPs in flowering is well characterized in the model plant arabidopsis, little is known in evergreen woody litchi (Litchi chinensis). In this study, three litchi SVP homologs (LcSVP1, LcSVP2, and LcSVP3) were cloned, and the bioinformatic analysis of the LcSVPs was carried out to identify their molecular characteristics. Their expression patterns in the apical meristem (AM) during the transition from vegetative to reproductive phase were studied under natural flowering inductive conditions. Also, brassinosteroid (BR) treatment under low temperature conditions was performed to elucidate the role of LcSVPs in the BR-regulated flowering. The results showed that LcSVPs belonged to the MADS superfamily. LcSVP relative expression levels in AMs of the early- and late-flowering cultivars showed decreasing trends with the transition from vegetative to reproductive growth. Under low temperature condition, relative expression levels of LcSVP1, LcSVP2, and LcSVP3 in AMs or panicle primordia showed decreasing trends, whereas those in the AMs of the BR-treated trees remained at relatively high levels. Relative expression analysis of the litchi homolog, flowering locus t 1 (LcFT1), showed that the BR-treated leaves had lower relative expression level than nontreated control leaves. The findings suggest that LcSVPs act as repressors involved in flowering in natural conditions and the BR-regulated flowering.

Flowering plants undergo a transition from vegetative to reproductive growth. SVP, a MADS-domain transcription factor, was demonstrated to act as a repressor for flowering in arabidopsis (Hartmann et al., 2000) where it controls flowering by perceiving signals related to thermosensory, autonomous, and gibberelin (GA) pathways (Lee et al., 2007; Li et al., 2008). To maintain plants in a vegetative phase, SVP represses the expression of flowering locus t (FT) and twin sister of FT (TSF) in the phloem and suppressor of overexpression of constans 1 (SOC1) in the shoot apical meristem by directly binding to CArG boxes in FT and SOC1 (Jang et al., 2009; Li et al., 2008). Although the role of SVP in flowering is well known in the model plant arabidopsis (Li et al., 2008, 2010), little is known in evergreen woody plants.

Litchi is a Sapindaceae evergreen woody plant. It is also an important fruit tree that is widely cultivated in southeast Asia. Flowering in litchi is triggered by low temperatures and enhanced by drought in autumn and winter (Chen and Huang, 2005; Menzel and Simpson 1988). However, irregular flowering is a serious problem affecting litchi production (Lu et al., 2014). During the past nearly 20 years, warm winters associated with global warming have resulted in poor litchi flowering. Hence, it is important to understand the genetics of flowering to find ways through its regulation. We have previously shown that reactive oxygen species (ROS) promotes flowering (Zhou et al., 2012). We then performed RNA-sequencing and identified a ROS-responsive LcSVP in AMs of litchi and found that it might act as a floral repressor involved in the ROS-regulated flowering in litchi (Lu et al., 2017). However, the role of LcSVPs in litchi flowering still needs further investigation.

Plant hormones are very important in flowering regulation (Campos-Rivero et al., 2017). The plant hormone signals are perceived and transmitted to the nuclear by series signal transduction components to regulate the expression of flowering related genes, such as constants (CO), flowering locus c (FLC), SVP, FT, and SOC1 (Conti, 2017; Li et al., 2010; Martínez et al., 2004). In litchi, abscisic acid was shown to promote flowering, whereas GAs inhibit flowering (Chen et al., 2014; Cui et al., 2013). Brassinosteroids are a group of polyhydroxylated plant steroid hormones that are ubiquitously present throughout the plant kingdom and play pivotal roles in plant growth and development with regulatory functions during cell elongation, cell division, vascular differentiation, and biotic and abiotic stress responses (Clouse and Sasse, 1998; Nolan et al., 2017). In arabidopsis, BR biosynthetic det2 mutants exhibited delayed flowering time (Li et al., 2010). In rice (Oryza sativa), SVP-group MADS-box proteins are shown as negative regulators of BR response (Lee et al., 2008), demonstrating the role of SVP in BR response in herbaceous plants. However, little is known about their roles in the flowering of evergreen woody litchi.

In the present study, three litchi homologs LcSVP1, LcSVP2, and LcSVP3 were cloned, and the bioinformatic analysis of the LcSVPs was carried out to identify their molecular characteristics. Their expressions in the AM during the transition from vegetative to reproductive phase were studied, and BR treatment under inductive low temperature was performed to elucidate the role of LcSVPs in litchi flowering.

Materials and Methods

Plant materials and growth conditions.

Litchi trees were cultivated in the experimental orchard of South China Agricultural University (Guangzhou, China) (lat. 23°9′40″N, long. 113°21′18″E). To determine the LcSVP expression patterns, 12-year-old air-layered trees of early-flowering ‘Sanyuehong’ and late-flowering ‘Nuomici’ grown in open fields were selected. From Sept. 2015 to Feb. 2016, the trees were subjected to chilling by interrupted cold waves as the natural flowering inductive condition (Fig. 1). Samples were collected at five stages of floral formation according to Huang and Chen (2005). After the terminal shoots had just matured, the AMs of ‘Sanyuehong’ were collected on 15 Sept. 2015 as prefloral induction stage (PFId). Then AMs were collected on 14 Oct. as floral induction stage (FId). On 11 Nov. when panicle primordia of ‘Sanyuehong’ emerged and could be recognized as “whitish millets,” the floral initiation stage (FIn), the panicle primordia were collected. After the FIn, panicle primordia continued to develop and were collected on 9 Dec. and 30 Dec. as the panicle developmental stage (PD). Similarly, samples of ‘Nuomici’ were collected on 15 Sept. as PFId stage, 11 Nov. and 9 Dec. as FId, 31 Jan. 2016 as FIn when the panicle primordia emerged, and 21 Feb. as PD as described by Lu et al. (2017). All the tissues were collected from three replicated trees and stored at −80 °C for RNA extraction and gene expression analysis.

Fig. 1.
Fig. 1.

Diurnal maximum temperature, diurnal average temperature, and diurnal minimum temperature changes in Autumn and Winter 2015–16.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 2; 10.21273/JASHS04316-17

For the study of LcSVP expression in different tissues, 12-year-old ‘Nuomic’ litchi trees subjected to winter chilling for floral induction were selected. Roots, stems, mature leaves, flowers, and buds were collected from three replicated trees from March to May, the flowering and fruiting season of litchi. Specifically, vegetative buds were picked up from new shoots, which could no longer produce floral buds at the season. All the tissues were frozen in liquid nitrogen and stored at −80 °C for RNA extraction and gene expression analysis.

For BR treatment, 5-year-old air-layered ‘Huaizhi’ litchi trees were grown in 30-L pots filled with loam, mushroom cinder, and coconut chaff (v:v:v, 3:1:1). Once the terminal shoots of the trees matured, 16 trees (1–1.5 m height, ≈100 terminal shoots per tree) with the same phenological stage were selected for the experiment. The trees were transferred to a growth chamber made from polycarbonate sheet and grown under natural irradiance (transmittance of 92%), a 12-h photoperiod, a relative humidity of 75% to 85%, and 18/13 °C day/night temperatures as inductive temperature (IT) for flowering. The trees were divided into two groups. One group was treated with 1 μM 24-epibrassinolide (EBL), a member of the BR family, at 0, 10, 20, 30, and 40 d during IT treatment, and the other group was sprayed with water as the IT control at the same time. Either the EBL or water was applied as foliar spray to runoff (≈200 mL per plant) using a pressure sprayer. Then AMs or panicle primordia were collected at 0 d (PFId), 30 d (FId), 60 d (FIn), and 80 d (PD) time points. Each sample was a mix of the AMs or panicle primordia from two trees, stored at −80 °C for RNA extraction and LcSVP expression analysis. At 0 d (PFId), 15 d (FId), 30 d (FId), 40 d (FId), 50 d (FId), 60 d (FIn), and 75 d (PD) time points, leaves of the terminal shoots from three replicated BR-treated or control trees were also collected and stored at −80 °C for RNA extraction and LcFT1 expression analysis. At the 80-d time point, flowering conditions of the BR-treated and control trees were calculated from six replicated trees. Proportion of flowering trees was calculated as the proportion of the flowering trees to the total tested trees. Proportion of flowering terminal shoots was calculated as the proportion of the flowering terminal shoots to the total terminal shoots in one tree.

RNA extraction.

Total RNA was extracted using the Plant Total RNA Isolation Kit (Huayueyang, Beijing, China) according to the manufacturer’s instruction and predigested with DNase I to eliminate the genomic DNA. The DNase-treated RNA concentration was determined using a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Wilmington, DE). The RNA integrity was visualized by 1.2% agarose gel electrophoresis.

Cloning and bioinformatic analysis of LcSVPs.

The primers of LcSVP1-F1/R1, LcSVP2-F1/R1, and LcSVP3-F1/R1 specific to LcSVP1, LcSVP2, and LcSVP3, respectively (Table 1), were designed according to our transcriptome data (Lu et al., 2017). The PCR products were isolated and ligated into the pMD18-T vector for T/A cloning (Takara, Dalian, China), sequenced by Sangon Biotech Co. (Shanghai, China). The sequence of LcSVP1, LcSVP1, and LcSVP3 was analyzed using BLAST program on the website of the National Center for Biotechnology Information (NCBI, Bethesda, MD). Conserved domain prediction was performed by CDD (NCBI). The sequences of the three LcSVPs were aligned with those of longan (Dimocarpus longan), which belongs to the same family of Sapindaceae, and the model plant arabidopsis. The sequence alignment was performed using ClustalX 1.83 program (Thompson et al., 1997). The amino acid sequence was deduced by DNAMAN (Lynnon Corp., Vaudreuil, QC, Canada). The deduced amino acid sequences of the three LcSVPs and the eight top hit sequences from NCBI were used to construct a phylogenetic tree using the neighbor joining (NJ) method of the MEGA software [version 6.0 (Tamura et al., 2013)]. The secondary structure of LcSVPs was predicted by SOPMA (Geourjon and Deleage, 1995). Phyre version 2.0 (Kelley et al., 2015) was used to predict the LcSVPs tertiary structure.

Table 1.

Primer sequences for cloning and quantitative real-time polymerase chain reaction (qRT-PCR).

Table 1.

Quantitative real-time polymerase chain reaction (RT-PCR) analysis.

First-strand cDNA was synthetized by using reverse transcriptase M-MLV [RNase H-(Takara)] from 1 μg of extracted total RNA. As shown in Table 1, the quantitative real-time polymerase chain reaction (qRT-PCR) primers LcSVP1-F2/R2 (length of amplicon 100 bp) , LcSVP2-F2/R2 (length of amplicon 171 bp), LcSVP3-F2/R2 (length of amplicon 95 bp), and LcFT-F/R (length of amplicon 229 bp) were designed using Primer 5.0 software (Premier Biosoft, Palo Alto, CA) and synthesized by Sangon Biotech Co. The litchi homolog β-actin (accession number HQ588865.1) was used as the reference gene because of its stable expression (Wei et al., 2012). The qRT-PCR was performed according to the method described by Yang et al. (2017) on a real-time PCR machine (CFX; Bio-Rad Laboratories, Hercules, CA). The qPCRs were run as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 30 s in 96-well optical reaction plates (Bio-Rad Laboratories). Each qRT-PCR analysis was performed in triplicate. The transcript quantification of the genes was performed in relation to Actin and they were calculated by 2−ΔΔCT method (Livak and Schmittgen, 2001).

Statistical analysis.

Reported data of flowering condition are the means of the six replicates, and those of the qRT-PCR are the means of the three replicates. Statistical analyses were performed by variance using SPSS (version 19.0; IBM Corp., Armonk, NY). The differences among treatment means were evaluated by using Duncan’s multiple range test at a 0.05 P level, and those between the treatment and the control were evaluated by using Student’s test at a 0.05 P level. se was calculated by using Microsoft Excel 2010 (Microsoft, Redmond, WA).

Results

Cloning and characterization of LcSVPs.

We identified a 684-bp-long open reading frame (ORF) of LcSVP1 with 227 deduced amino acids, a 642-bp-long ORF of LcSVP2 with 213 deduced amino acids, and a 681-bp-long ORF of LcSVP3 with 226 deduced amino acids (Supplemental Fig. 1). NCBI BLASTp results showed that LcSVP1, LcSVP2, and LcSVP3 belonged to the MADS superfamily (Supplemental Fig. 2). Alignment of predicted protein sequences showed that the MADS box and K box among the SVPs share high similarity (Fig. 2). The predicted secondary structure indicated that LcSVP1 contained 119 alpha helices, 16 beta turns, and 92 random coils. LcSVP2 contained 117 alpha helices, 16 beta turns, and 80 random coils. LcSVP3 contained 120 alpha helices, 16 beta turns, and 90 random coils (Supplemental Fig. 3). Tertiary structure prediction indicated that the structures of LcSVP1 and LcSVP3 are similar to each other, whereas that of LcSVP2 is quite different from the other two (Supplemental Fig. 4). Phylogenetic analysis indicated that the three genes belonged to two branches. LcSVP1 and LcSVP3 were clustered in one branch, in which the LcSPV1 shared 100% identity with longan SVP1 (DlSVP1). LcSVP2 and DlSVP2 which shared 100% identity were clustered in another branch (Fig. 3).

Fig. 2.
Fig. 2.

Alignment of predicted protein sequences of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, LcSVP3, DlSVP1, DlSVP1, and AtSVP (Arabidopsis thaliana: AtSVP; Litchi chinensis: LcSVP1, LcSVP2, LcSVP1; Dimocarpus longan: DlSVP1, DlSVP1).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 2; 10.21273/JASHS04316-17

Fig. 3.
Fig. 3.

Phylogenetic tree of short vegetative phase (SVP) genes. The tree was constructed by using MEGA software [version 6.0 (Tamura et al., 2013)] and neighbor joining method with 1000 bootstrap replicates [Litchi chinensis: LcSVP1 (Unigene0029062), LcSVP2 (Unigene0078101), LcSVP3 (Unigene0079789); Populus euphratica: PeJ (XP_011016930.1); Populus tomentosa: PtMADS box transcription factor (AAR92206.1); Theobroma cacao: TcMADS box transcription factor (EOY15444.1), TcSVP (XP_017981093.1), TcSVP (XP_007042844.2); Ricinus communis: RcSVP isoform X1 (XP_015571455.1); Shorea beccariana: SbSVP (BAN89455.1); Citrus trifoliate: CtAGL24 (APB02632.1); Citrus sinensis: CsSVP (XP_006472470.1), CsJ-like isoform X2 (XP_006487503.1), CsJ-like isoform X1 (XP_006487501.1); Nelumbo nucifera: NnSVP isoform X2 (XP_010254525.1), NnJ-like isoform X2 (XP_010277489.1); Populus euphratica: PeSVP (XP011021843.1); Ziziphus jujuba: ZjJ (XP_015890560.1); Jatropha curcas: JcSVP (XP_012081656.1); Vitis vinifera: VvSVP (XP_019073897.1); Malus domestica: MdJ-like (AOA32867.1); Dimocarpus longan: DlSVP1 (AIY25020.1), DlSVP2 (AIY25021.1); Juglans regia: JrJ isoform X2 (XP_018806751.1); Actinidia deliciosa: AdSVP1 (AFA37963.1); Actinidia chinensis: AcSVP4 (AFA37970.1)].

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 2; 10.21273/JASHS04316-17

LcSVPs expression patterns in different tissues.

To determine the transcriptional expression patterns of LcSVPs in the vegetative and reproductive tissues, roots, stems, flowers, leaves, vegetative buds, and fruit of ‘Nuomici’ were separately collected. Expression levels of LcSVP1, LcSVP2, and LcSVP3 in the specific tissues were determined by using qRT-PCR. As shown in Fig. 4, LcSVP1 is strongly expressed in vegetative buds, but less expressed in roots, stems, leaves, flowers, and fruit. The relative expression of LcSVP1 in buds was 57.2- and 13.3-fold higher than that in roots and fruit, respectively. LcSVP2 was strongly expressed in vegetative buds and leaves, but less expressed in roots, stems, flowers, and fruit. Relative expression level of LcSVP1 in leaves and buds was 9.9- and 9.5-fold higher than that in flowers, respectively. No transcription of LcSVP2 could be determined in fruit tissues. Relative expression of LcSVP3 in buds was significantly higher than that in roots, stems, leaves, flowers, and fruit. On the whole, LcSVPs were strongly expressed in vegetative buds, but less expressed in roots, flowers, and fruit, indicating that LcSVPs expression was suppressed in roots and the reproductive tissues of flowers and fruit.

Fig. 4.
Fig. 4.

Expression patterns of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, and LcSVP3 in different tissues of ‘Nuomici’ litchi. (A) Images of the roots, stems, flowers, fruit, buds, and leaves. (B) Relative expression of the LcSVP1, LcSVP2, and LcSVP3. Data are means of three replicates and the bars represent ses. Different lowercase letters above column means indicate significance at P < 0.05 according to Duncan’s multiple rage test using SPSS (version 19.0; IBM Corp.).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 2; 10.21273/JASHS04316-17

LcSVPs expression patterns in different cultivars under natural flowering inductive conditions.

To find out whether the LcSVPs transcription was suppressed during the transition from vegetative to reproductive growth, the AMs or panicle primordia were collected from the early-flowering ‘Sanyuehong’ and the late-flowering ‘Nuomici’ in autumn and winter, during which the temperature decreased, and the trees were subjected to chilling by interrupted cold waves (Fig. 1). The litchi trees were therefore subjected to natural chilling conditions for floral induction. The transformation from vegetative buds to reproductive floral buds happened in this period. On 11 Sept. 2015, panicle prmirodia emerged in the early flowering ‘Sanyuehong’ (Fig. 5A), whereas on 31 Jan. 2016, panicle prmirodia emerged in the late-flowering ‘Nuomici’ (Fig. 5B). Relative expression levels of LcSVP1, LcSVP2, and LcSVP3 in these tissues were determined from PFId to PD. The results indicated that relative expression levels of these three LcSVPs in both cultivars showed decreasing trends with the transition from vegetative to reproductive growth (Fig. 5C).

Fig. 5.
Fig. 5.

Morphology of the buds in ‘Sanyuehong’ (A) and ‘Nuomici’ (B) litchi from the prefloral induction stage (PFId) to the panicle developmental stage (PD), and expression patterns of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, and LcSVP3 in apical meristems (AMs) or panicle primordia of ‘Sanyuehong’ and ‘Nuomici’ in the nature-controlled flowering condition (C). Trees of the early flowering ‘Sanyuehong’ and the late-flowering ‘Nuomici’ grown in open fields were selected. AMs or panicle primordia of ‘Sanyuehong’ were collected on 15 Sept. 2015 as PFId, 14 Oct. as floral induction stage (FId), Nov. 11 as floral initiation stage (FIn), and 9 Dec. and 30 Dec. as the PD. Those of the ‘Nuomici’ were collected on 15 Sept. as PFId stage, 11 Nov. and 9 Dec. as FId, 31 Jan. 2016 as FIn, and 21 Feb. as PD. Data are means of three replicates and the bars represent ses.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 2; 10.21273/JASHS04316-17

LcSVPs expression patterns under BR-regulated flowering condition.

To further study the LcSVPs expression patterns by BR treatment, potted trees were sprayed with BR or water in a growth chamber under floral IT. After 80 d of treatment, flowering phenotype of the trees was determined. As shown in Table 2, the proportion of flowering trees of the BR treatment was lower than that of the control, and the proportion of flowering terminal shoots of the BR-treated trees was significantly lower than that of the control one. The results suggested that BR inhibited flowering of litchi under IT conditions. We further determined the LcSVP relative expression levels. As shown in Fig. 6, on the whole, relative expression levels of LcSVP1, LcSVP2, and LcSVP3 in AMs or panicle primordia of the control trees showed decreasing trends, whereas those of the BR-treated trees remained at relatively high levels. Relative expression analysis of LcFT1 showed that the BR-treated leaves had lower levels than the control ones. For example, at 40- and 60-d time points, relative expression of LcFT1 in control leaves was 42.9- and 27.3-fold higher than that in BR-treated leaves, respectively (Fig. 7).

Table 2.

Effects of brassinosteroid (BR) on the flowering of ‘Huaizhi’ litchi trees under inductive temperatures (n = 6).

Table 2.
Fig. 6.
Fig. 6.

Expression patterns of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, and LcSVP3 in apical meristems (AMs) or panicle primordia of the brassinosteroid-treated litchi trees under controlled inductive temperature (IT) conditions. Potted litchi trees were transferred to a growth chamber at 18/13 °C [day/night temperature (12/12 h day/night)] as IT for flowering. The trees were sprayed with 1 μM 24-epibrassinolide (EBL), or water as control at 0, 10, 20, 30, and 40 d of IT treatment. Data are means of three replicates and the bars represent ses.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 2; 10.21273/JASHS04316-17

Fig. 7.
Fig. 7.

Expression patterns of the litchi homolog flowering locus t 1 (LcFT1) in leaves of the brassinosteroid-treated trees under controlled inductive temperature (IT) condition. Trees were transferred to a growth chamber at 18/13 °C [day/night temperature (12/12 h day/night)] as IT for flowering. The trees were sprayed with 1 μM 24-epibrassinolide (EBL), or water as control at 0, 10, 20, 30, and 40 d of IT treatment. Data are means of three replicates and the bars represent ses.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 2; 10.21273/JASHS04316-17

Discussion

Flowering is a genetically controlled process during the transition from vegetative to reproductive development. Stress is simply defined as a condition in which the vegetative growth of plants is suppressed (Takeno, 2012). Many of the environmental conditions inducing flowering can be considered as stress conditions. The stressed plants do not need to wait for the arrival of a season when photoperiodic conditions are suitable for flowering, and such precocious flowering may be important for species preservation (Takeno, 2012). Under stressful conditions, ROS accumulates and functions as signals regulating plant growth and development (Ahanger et al., 2017). Flowering in litchi is triggered by low temperatures and enhanced by drought stress in autumn and winter (Chen and Huang, 2005; Menzel and Simpson, 1988). These two inductive factors can be regarded as stress conditions that can suppress vegetative growth of litchi. We have previously shown that ROS promotes flowering (Zhou et al., 2012), and identified ROS-responsive LcSVP1 in AMs of litchi. Our previous data showed that LcSVP1 might be involved in the ROS-controlled floral transition (Lu et al., 2017). We hypothesized that the LcSVPs might act as repressors in litchi flowering. Hence, in the present study, we clone the SVP homologs identified from our RNA-seq data set (Lu et al., 2014, 2017). We found that LcSVP1, LcSVP2, and LcSVP3 belonged to the MADS superfamily.

MADS-domain transcription factors have been identified in plants and were proved to be involved in flowering (Masiero et al., 2011). MADS-domain factors activate or repress transcription by directly binding to short sequences called CArG boxes that correspond to a 10 nucleotide sequence, CC(A/T)6GG, present in the regulatory sequences of target genes (de Folter and Angenent, 2006). In arabidopsis, SVP binds to CArG boxes in FT and SOC1 and acts as a floral repressor (Jang et al., 2009; Li et al., 2008). In kiwifruit (Actinidia sp.), the woody perennial vines, SVP-like genes may have distinct roles during bud dormancy and flowering (Wu et al., 2012). In woody tree peony (Paeonia suffruticosa), PsSVP can promote vegetative growth and suppress flowering (Wang et al., 2014). In the present study, we found that the LcSVPs were strongly expressed in vegetative buds and leaves, but least expressed in flowers and fruit, indicating that LcSVPs relative expression was suppressed in the reproductive tissues. Also, those levels in both the early flowering ‘Sanyuehong’ and the late-flowering ‘Nuomici’ showed decreasing trends with the transition from vegetative to reproductive growth under the floral inductive condition, suggesting that LcSVPs might act as repressors in litchi flowering, similar to those of the arabidopsis, kiwifruit, and tree peony (Jang et al., 2009; Wang et al., 2014; Wu et al., 2012).

To further study the role of LcSVPs in litchi flowering, their relative expression levels were determined under hormonal regulation. We sprayed potted litchi trees with the plant steroid hormone 24-EBL, a member of the BR family, and placed them under flowering ITs (18/13 °C) in the growth chamber. To our surprise, BR-inhibited flowering under inductive low temperature treatment, which is contrary from what were reported in herbaceous rice and arabidopsis (Lee et al., 2008; Li et al., 2010). BRs are regarded as growth-promoting hormones (Khripach et al., 2000). It has been proved that they can promote biosynthesis of GA, another growth-promoting hormone (Unterholzner et al., 2015). BR-regulated transcription factor BES1 binds to a regulatory element in promoters of GA biosynthesis genes to control their relative expression in a BR-induced manner (Unterholzner et al., 2015). GAs promote flowering in arabidopsis (Blazquez et al. 1998). In contrast, GAs inhibit flowering in litchi (Chen et al., 2014), which is evidenced by the fact that exogenous application of paclobutrazol, a GA biosynthesis inhibitor, promotes flowering (Ray and Ruby, 2004) and is a practical method for litchi production. Similar results were reported in citrus (Citrus sp.), a group of evergreen woody trees (Goldschmidt et al., 1997). Based on the contrasting effects of GA on flowering mechanism between the evergreen woody tree litchi and the herbaceous arabidopsis, it is speculated that BR has diverse effects on flowering of the two species. In our present study, reduced flowering was observed in BR-treated trees, of which relative expression levels of LcSVPs remained higher than the control trees. In addition, relative expression level of LcFT1 whose encoding protein is homologous to the arabidopsis florigen (Corbesier et al., 2007; Ding et al., 2015;Yang et al., 2007) was lower in the BR-treated leaves than that in the control ones. In litchi, two FT homologue genes LcFT1 and LcFT2 were identified. However, low temperature can only induce LcFT1 expression in leaves. Overexpression of LcFT1 in tobacco and arabidopsis induces precocious flowering (Ding et al., 2015). It is suggested that LcFT1 expression is positively correlated with litchi flowering. Hence, in the present study, we determined the relative expression of LcFT1. In accordance with the poor flowering in the BR-treated trees, a lower expression of LcFT1 in the BR-treated trees was found compared with the control trees. The expression of LcSVPs under BR regulation further suggested that LcSVPs might act as floral repressors through regulating the transcription of LcFT1 expression. Future study should be focused on the relationship between LcSVPs and LcFT1.

In conclusion, we cloned three LcSVP genes, and they were strongly expressed in buds, but less expressed in flowers and fruit. LcSVP relative expression levels in both of the early- and late-flowering cultivars showed decreasing trends with the transition from vegetative to reproductive growth. Relative expression levels of LcSVP1, LcSVP2, and LcSVP3 in AMs or panicle primordia of the control trees showed decreasing trends with the transition from vegetative to reproductive growth, whereas those in AMs of the BR-treated trees remained at relatively high levels. LcSVPs might act as floral repressors involved in flowering in natural conditions and the BR-regulated flowering.

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  • CuiZ.ZhouB.ZhangZ.HuZ.2013Abscisic acid promotes flowering and enhances LcAP1 expression in Litchi chinensis SonnS. Afr. J. Bot.887679

    • Search Google Scholar
    • Export Citation
  • ChenH.B.HuangH.B.2005Low temperature requirements for floral induction in lycheeActa Hort.665195202

  • ChenP.A.LeeC.L.RoanS.F.ChenI.Z.2014Effects of GA3 application on the inflorescence and yield of ‘Yu Her Pau’ litchiScientia Hort.1714550

    • Search Google Scholar
    • Export Citation
  • ContiL.2017Hormonal control of the floral transition: Can one catch them all?Dev. Biol.430288301

  • ClouseS.D.SasseJ.M.1998Brassinosteroids: Essential regulators of plant growth and developmentAnnu. Rev. Plant Physiol. Plant Mol. Biol.49427451

    • Search Google Scholar
    • Export Citation
  • CorbesierL.VincentC.JangS.H.FornaraF.FanQ.SearleI.GiakountisA.FarronaS.GissotL.TurnbullC.CouplandG.2007FT protein movement contributes to long-distance signaling in floral induction of ArabidopsisScience31610301033

    • Search Google Scholar
    • Export Citation
  • de FolterS.AngenentG.C.2006Trans meets cis in MADS scienceTrends Plant Sci.11224231

  • DingF.ZhangS.W.ChenH.B.SuZ.X.ZhangR.XiaoQ.S.LiH.L.2015Promoter difference of LcFT1 is a leading cause of natural variation of flowering timing in different litchi cultivars (Litchi chinensis Sonn.)Plant Sci.241128137

    • Search Google Scholar
    • Export Citation
  • GeourjonC.DeleageG.1995SOPMA: Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignmentsBioinformatics11681684

    • Search Google Scholar
    • Export Citation
  • GoldschmidtE.E.TamimM.GorenR.1997Gibberellins and flowering in citrus and other fruit trees: A critical analysisActa Hort.463201208

  • HartmannU.HohmannS.NettesheimK.WismanE.SaedlerH.HuijserP.2000Molecular cloning of SVP: A negative regulator of the floral transition in ArabidopsisPlant J.21351360

    • Search Google Scholar
    • Export Citation
  • HuangH.B.ChenH.B.2005A phase approach towards floral formation in lycheeActa Hort.665185194

  • JangS.TortiS.CouplandG.2009Genetic and spatial interactions between FT, TSF and SVP during the early stages of floral induction in ArabidopsisPlant J.60614625

    • Search Google Scholar
    • Export Citation
  • KelleyL.A.MezulisS.YatesC.M.WassM.N.SternbergM.J.E.2015The Phyre2 web portal for protein modeling, prediction and analysisNat. Protoc.10845858

    • Search Google Scholar
    • Export Citation
  • KhripachV.A.ZhabinskiiV.N.de GrootA.E.2000Twenty years of brassinosteroids: Steroidal plant hormones warrant better crops for the XXI centuryAnn. Bot.86441447

    • Search Google Scholar
    • Export Citation
  • LeeJ.H.YooS.J.ParkS.H.HwangI.LeeJ.S.AhnJ.H.2007Role of SVP in the control of flowering time by ambient temperature in ArabidopsisGenes Dev.21397402

    • Search Google Scholar
    • Export Citation
  • LeeS.ChoiS.C.AnG.2008Rice SVP-group MADS-box proteins, OsMADS22 and OsMADS55, are negative regulators of brassinosteroid responsesPlant J.5493105

    • Search Google Scholar
    • Export Citation
  • LiD.LiuC.ShenL.WuY.ChenH.RobertsonM.HelliwellC.A.ItoT.MeyerowitzE.YuH.2008A repressor complex governs the integration of flowering signals in ArabidopsisDev. Cell15110120

    • Search Google Scholar
    • Export Citation
  • LiJ.H.LiY.H.ChenS.Y.AnL.Z.2010Involvement of brassinosteroid signals in the floral-induction network of ArabidopsisJ. Expt. Bot.6142214230

    • Search Google Scholar
    • Export Citation
  • LivakK.J.SchmittgenT.D.2001Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT methodMethods25402408

    • Search Google Scholar
    • Export Citation
  • LuX.KimH.ZhongS.ChenH.HuZ.ZhouB.2014De novo transcriptome assembly for rudimentary leaves in Litchi chinesis Sonn. and identification of differentially expressed genes in response to reactive oxygen speciesBMC Genomics15805

    • Search Google Scholar
    • Export Citation
  • LuX.LiJ.ChenH.HuJ.LiuP.ZhouB.2017RNA-seq analysis of apical meristem reveals integrative regulatory network of ROS and chilling potentially related to flowering in Litchi chinensisSci. Rpt.710183

    • Search Google Scholar
    • Export Citation
  • MartínezC.PonsE.PratsG.LeónJ.2004Salicylic acid regulated flowering time and link defence responses and reproductive developmentPlant J.37209217

    • Search Google Scholar
    • Export Citation
  • MasieroS.ColomboL.GriniP.E.SchnittgerA.KaterM.M.2011The emerging importance of type I MADS box transcription factors for plant reproductionPlant Cell23865872

    • Search Google Scholar
    • Export Citation
  • MenzelC.M.SimpsonD.X.1988Effect of temperature on growth and flowering of litchi (Litchi chinensis Sonn.) cultivarsJ. Hort. Sci.63349360

    • Search Google Scholar
    • Export Citation
  • NolanT.ChenJ.YinY.2017Cross-talk of brassinosteroid signaling in controlling growth and stress responsesBiochem. J.47426412661

  • RayP.K.RubyR.2004Effect of paclobutrazol on flowering in “China” litchi (Litchi chinensis Sonn.)Indian J. Plant. Physiol.9208211

  • TakenoK.2012Stress-induced flowering p. 331–345. In: P. Ahmad and M.N.V. Prasad (eds.). Abiotic stress responses in plants. Springer New York NY

  • TamuraK.StecherG.PetersonD.FilipskiA.KumarS.2013MEGA6: Molecular evolutionary genetics analysis version 6.0Mol. Biol. Evol.3027252729

    • Search Google Scholar
    • Export Citation
  • ThompsonJ.D.GibsonT.J.PlewniakF.JeanmouginF.HigginsD.G.1997The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis toolsNucl. Acids Res.2548764882

    • Search Google Scholar
    • Export Citation
  • UnterholznerS.J.RozhonW.PapacekM.CiomasJ.LangeT.KugleK.G.MayerK.F.SiebererT.PoppenbergerB.2015Brassinosteroids are master regulators of gibberellin biosynthesis in ArabidopsisPlant Cell2722612272

    • Search Google Scholar
    • Export Citation
  • WangS.XueJ.AhmadiN.HollowayP.ZhuF.RenX.ZhangX.2014Molecular characterization and expression patterns of PsSVP genes reveal distinct roles in flower bud abortion and flowering in tree peony (Paeonia suffruticosa)Can. J. Plant Sci.9411811193

    • Search Google Scholar
    • Export Citation
  • WeiY.Z.LaiB.HuF.C.LiX.J.HuG.B.WangH.C.2012Cloning and stability analysis of reference genes for expression studies by quantitative real-time PCR in litchiJ. South China Agr. Univ.33303306

    • Search Google Scholar
    • Export Citation
  • WuR.M.WaltonE.F.RichardsonA.C.WoodM.HellensR.Varkonyi-GasicE.2012Conservation and divergence of four kiwifruit SVP-like MADS-box genes suggest distinct roles in kiwifruit bud dormancy and floweringJ. Expt. Bot.63797807

    • Search Google Scholar
    • Export Citation
  • YangH.F.LuX.Y.ChenH.B.WangC.C.ZhouB.Y.2017Low temperature-induced leaf senescence and the expression of senescence-related genes in the panicles of Litchi chinensisBiol. Plant.61315322

    • Search Google Scholar
    • Export Citation
  • YangY.J.KlejnotJ.YuX.H.LiuX.M.LinC.T.2007Florigen (II): It is a mobile proteinJ. Integr. Plant Biol.4916651669

  • ZhouB.LiN.ZhangZ.HuangX.ChenH.HuZ.PangX.LiuW.LuY.2012Hydrogen peroxide and nitric oxide promote reproductive growth in Litchi chinensisBiol. Plant.56321329

    • Search Google Scholar
    • Export Citation
Supplemental Fig. 1.
Supplemental Fig. 1.

Deduced amino acid sequence of the short vegetative phase (SVP) homologs (LcSVPs) cDNA in litchi.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 2; 10.21273/JASHS04316-17

Supplemental Fig. 2.
Supplemental Fig. 2.

NCBI BLASTp results of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, and LcSVP3 in litchi.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 2; 10.21273/JASHS04316-17

Supplemental Fig. 3.
Supplemental Fig. 3.

Prediction of the secondary structure of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, and LcSVP3 in litchi. H = alpha helice; E = beta turn; C = random coil.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 2; 10.21273/JASHS04316-17

Supplemental Fig. 4.
Supplemental Fig. 4.

Tertiary structure prediction of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, and LcSVP3 in litchi.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 2; 10.21273/JASHS04316-17

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Contributor Notes

This work was supported by the National Natural Science Foundation (project no. 31572080 and 31772249) and the China Agricultural Industry Project (project no. CARS-33).

Corresponding author. E-mail: zhoubiyan@scau.edu.cn.

  • View in gallery

    Diurnal maximum temperature, diurnal average temperature, and diurnal minimum temperature changes in Autumn and Winter 2015–16.

  • View in gallery

    Alignment of predicted protein sequences of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, LcSVP3, DlSVP1, DlSVP1, and AtSVP (Arabidopsis thaliana: AtSVP; Litchi chinensis: LcSVP1, LcSVP2, LcSVP1; Dimocarpus longan: DlSVP1, DlSVP1).

  • View in gallery

    Phylogenetic tree of short vegetative phase (SVP) genes. The tree was constructed by using MEGA software [version 6.0 (Tamura et al., 2013)] and neighbor joining method with 1000 bootstrap replicates [Litchi chinensis: LcSVP1 (Unigene0029062), LcSVP2 (Unigene0078101), LcSVP3 (Unigene0079789); Populus euphratica: PeJ (XP_011016930.1); Populus tomentosa: PtMADS box transcription factor (AAR92206.1); Theobroma cacao: TcMADS box transcription factor (EOY15444.1), TcSVP (XP_017981093.1), TcSVP (XP_007042844.2); Ricinus communis: RcSVP isoform X1 (XP_015571455.1); Shorea beccariana: SbSVP (BAN89455.1); Citrus trifoliate: CtAGL24 (APB02632.1); Citrus sinensis: CsSVP (XP_006472470.1), CsJ-like isoform X2 (XP_006487503.1), CsJ-like isoform X1 (XP_006487501.1); Nelumbo nucifera: NnSVP isoform X2 (XP_010254525.1), NnJ-like isoform X2 (XP_010277489.1); Populus euphratica: PeSVP (XP011021843.1); Ziziphus jujuba: ZjJ (XP_015890560.1); Jatropha curcas: JcSVP (XP_012081656.1); Vitis vinifera: VvSVP (XP_019073897.1); Malus domestica: MdJ-like (AOA32867.1); Dimocarpus longan: DlSVP1 (AIY25020.1), DlSVP2 (AIY25021.1); Juglans regia: JrJ isoform X2 (XP_018806751.1); Actinidia deliciosa: AdSVP1 (AFA37963.1); Actinidia chinensis: AcSVP4 (AFA37970.1)].

  • View in gallery

    Expression patterns of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, and LcSVP3 in different tissues of ‘Nuomici’ litchi. (A) Images of the roots, stems, flowers, fruit, buds, and leaves. (B) Relative expression of the LcSVP1, LcSVP2, and LcSVP3. Data are means of three replicates and the bars represent ses. Different lowercase letters above column means indicate significance at P < 0.05 according to Duncan’s multiple rage test using SPSS (version 19.0; IBM Corp.).

  • View in gallery

    Morphology of the buds in ‘Sanyuehong’ (A) and ‘Nuomici’ (B) litchi from the prefloral induction stage (PFId) to the panicle developmental stage (PD), and expression patterns of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, and LcSVP3 in apical meristems (AMs) or panicle primordia of ‘Sanyuehong’ and ‘Nuomici’ in the nature-controlled flowering condition (C). Trees of the early flowering ‘Sanyuehong’ and the late-flowering ‘Nuomici’ grown in open fields were selected. AMs or panicle primordia of ‘Sanyuehong’ were collected on 15 Sept. 2015 as PFId, 14 Oct. as floral induction stage (FId), Nov. 11 as floral initiation stage (FIn), and 9 Dec. and 30 Dec. as the PD. Those of the ‘Nuomici’ were collected on 15 Sept. as PFId stage, 11 Nov. and 9 Dec. as FId, 31 Jan. 2016 as FIn, and 21 Feb. as PD. Data are means of three replicates and the bars represent ses.

  • View in gallery

    Expression patterns of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, and LcSVP3 in apical meristems (AMs) or panicle primordia of the brassinosteroid-treated litchi trees under controlled inductive temperature (IT) conditions. Potted litchi trees were transferred to a growth chamber at 18/13 °C [day/night temperature (12/12 h day/night)] as IT for flowering. The trees were sprayed with 1 μM 24-epibrassinolide (EBL), or water as control at 0, 10, 20, 30, and 40 d of IT treatment. Data are means of three replicates and the bars represent ses.

  • View in gallery

    Expression patterns of the litchi homolog flowering locus t 1 (LcFT1) in leaves of the brassinosteroid-treated trees under controlled inductive temperature (IT) condition. Trees were transferred to a growth chamber at 18/13 °C [day/night temperature (12/12 h day/night)] as IT for flowering. The trees were sprayed with 1 μM 24-epibrassinolide (EBL), or water as control at 0, 10, 20, 30, and 40 d of IT treatment. Data are means of three replicates and the bars represent ses.

  • View in gallery

    Deduced amino acid sequence of the short vegetative phase (SVP) homologs (LcSVPs) cDNA in litchi.

  • View in gallery

    NCBI BLASTp results of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, and LcSVP3 in litchi.

  • View in gallery

    Prediction of the secondary structure of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, and LcSVP3 in litchi. H = alpha helice; E = beta turn; C = random coil.

  • View in gallery

    Tertiary structure prediction of the short vegetative phase (SVP) homologs LcSVP1, LcSVP2, and LcSVP3 in litchi.

  • AhangerM.A.AkramN.A.AshrafM.AlyemeniM.N.WijayaL.AhmadP.2017Signal transduction and biotechnology in response to environmental stressesBiol. Plant.61401416

    • Search Google Scholar
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  • BlazquezM.A.GreenR.NilssonO.SussmanM.R.WeigelD.1998Gibberellins promote flowering of arabidopsis by activating the LEAFY promoterPlant Cell10791800

    • Search Google Scholar
    • Export Citation
  • Campos-RiveroG.Osorio-MontalvoP.Sanchez-BorgesR.Us-CamasR.Duarte-AkeF.De-la-PenaC.2017Plant hormone signaling in flowering: An epigenetic point of viewJ. Plant Physiol.2141627

    • Search Google Scholar
    • Export Citation
  • CuiZ.ZhouB.ZhangZ.HuZ.2013Abscisic acid promotes flowering and enhances LcAP1 expression in Litchi chinensis SonnS. Afr. J. Bot.887679

    • Search Google Scholar
    • Export Citation
  • ChenH.B.HuangH.B.2005Low temperature requirements for floral induction in lycheeActa Hort.665195202

  • ChenP.A.LeeC.L.RoanS.F.ChenI.Z.2014Effects of GA3 application on the inflorescence and yield of ‘Yu Her Pau’ litchiScientia Hort.1714550

    • Search Google Scholar
    • Export Citation
  • ContiL.2017Hormonal control of the floral transition: Can one catch them all?Dev. Biol.430288301

  • ClouseS.D.SasseJ.M.1998Brassinosteroids: Essential regulators of plant growth and developmentAnnu. Rev. Plant Physiol. Plant Mol. Biol.49427451

    • Search Google Scholar
    • Export Citation
  • CorbesierL.VincentC.JangS.H.FornaraF.FanQ.SearleI.GiakountisA.FarronaS.GissotL.TurnbullC.CouplandG.2007FT protein movement contributes to long-distance signaling in floral induction of ArabidopsisScience31610301033

    • Search Google Scholar
    • Export Citation
  • de FolterS.AngenentG.C.2006Trans meets cis in MADS scienceTrends Plant Sci.11224231

  • DingF.ZhangS.W.ChenH.B.SuZ.X.ZhangR.XiaoQ.S.LiH.L.2015Promoter difference of LcFT1 is a leading cause of natural variation of flowering timing in different litchi cultivars (Litchi chinensis Sonn.)Plant Sci.241128137

    • Search Google Scholar
    • Export Citation
  • GeourjonC.DeleageG.1995SOPMA: Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignmentsBioinformatics11681684

    • Search Google Scholar
    • Export Citation
  • GoldschmidtE.E.TamimM.GorenR.1997Gibberellins and flowering in citrus and other fruit trees: A critical analysisActa Hort.463201208

  • HartmannU.HohmannS.NettesheimK.WismanE.SaedlerH.HuijserP.2000Molecular cloning of SVP: A negative regulator of the floral transition in ArabidopsisPlant J.21351360

    • Search Google Scholar
    • Export Citation
  • HuangH.B.ChenH.B.2005A phase approach towards floral formation in lycheeActa Hort.665185194

  • JangS.TortiS.CouplandG.2009Genetic and spatial interactions between FT, TSF and SVP during the early stages of floral induction in ArabidopsisPlant J.60614625

    • Search Google Scholar
    • Export Citation
  • KelleyL.A.MezulisS.YatesC.M.WassM.N.SternbergM.J.E.2015The Phyre2 web portal for protein modeling, prediction and analysisNat. Protoc.10845858

    • Search Google Scholar
    • Export Citation
  • KhripachV.A.ZhabinskiiV.N.de GrootA.E.2000Twenty years of brassinosteroids: Steroidal plant hormones warrant better crops for the XXI centuryAnn. Bot.86441447

    • Search Google Scholar
    • Export Citation
  • LeeJ.H.YooS.J.ParkS.H.HwangI.LeeJ.S.AhnJ.H.2007Role of SVP in the control of flowering time by ambient temperature in ArabidopsisGenes Dev.21397402

    • Search Google Scholar
    • Export Citation
  • LeeS.ChoiS.C.AnG.2008Rice SVP-group MADS-box proteins, OsMADS22 and OsMADS55, are negative regulators of brassinosteroid responsesPlant J.5493105

    • Search Google Scholar
    • Export Citation
  • LiD.LiuC.ShenL.WuY.ChenH.RobertsonM.HelliwellC.A.ItoT.MeyerowitzE.YuH.2008A repressor complex governs the integration of flowering signals in ArabidopsisDev. Cell15110120

    • Search Google Scholar
    • Export Citation
  • LiJ.H.LiY.H.ChenS.Y.AnL.Z.2010Involvement of brassinosteroid signals in the floral-induction network of ArabidopsisJ. Expt. Bot.6142214230

    • Search Google Scholar
    • Export Citation
  • LivakK.J.SchmittgenT.D.2001Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT methodMethods25402408

    • Search Google Scholar
    • Export Citation
  • LuX.KimH.ZhongS.ChenH.HuZ.ZhouB.2014De novo transcriptome assembly for rudimentary leaves in Litchi chinesis Sonn. and identification of differentially expressed genes in response to reactive oxygen speciesBMC Genomics15805

    • Search Google Scholar
    • Export Citation
  • LuX.LiJ.ChenH.HuJ.LiuP.ZhouB.2017RNA-seq analysis of apical meristem reveals integrative regulatory network of ROS and chilling potentially related to flowering in Litchi chinensisSci. Rpt.710183

    • Search Google Scholar
    • Export Citation
  • MartínezC.PonsE.PratsG.LeónJ.2004Salicylic acid regulated flowering time and link defence responses and reproductive developmentPlant J.37209217

    • Search Google Scholar
    • Export Citation
  • MasieroS.ColomboL.GriniP.E.SchnittgerA.KaterM.M.2011The emerging importance of type I MADS box transcription factors for plant reproductionPlant Cell23865872

    • Search Google Scholar
    • Export Citation
  • MenzelC.M.SimpsonD.X.1988Effect of temperature on growth and flowering of litchi (Litchi chinensis Sonn.) cultivarsJ. Hort. Sci.63349360

    • Search Google Scholar
    • Export Citation
  • NolanT.ChenJ.YinY.2017Cross-talk of brassinosteroid signaling in controlling growth and stress responsesBiochem. J.47426412661

  • RayP.K.RubyR.2004Effect of paclobutrazol on flowering in “China” litchi (Litchi chinensis Sonn.)Indian J. Plant. Physiol.9208211

  • TakenoK.2012Stress-induced flowering p. 331–345. In: P. Ahmad and M.N.V. Prasad (eds.). Abiotic stress responses in plants. Springer New York NY

  • TamuraK.StecherG.PetersonD.FilipskiA.KumarS.2013MEGA6: Molecular evolutionary genetics analysis version 6.0Mol. Biol. Evol.3027252729

    • Search Google Scholar
    • Export Citation
  • ThompsonJ.D.GibsonT.J.PlewniakF.JeanmouginF.HigginsD.G.1997The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis toolsNucl. Acids Res.2548764882

    • Search Google Scholar
    • Export Citation
  • UnterholznerS.J.RozhonW.PapacekM.CiomasJ.LangeT.KugleK.G.MayerK.F.SiebererT.PoppenbergerB.2015Brassinosteroids are master regulators of gibberellin biosynthesis in ArabidopsisPlant Cell2722612272

    • Search Google Scholar
    • Export Citation
  • WangS.XueJ.AhmadiN.HollowayP.ZhuF.RenX.ZhangX.2014Molecular characterization and expression patterns of PsSVP genes reveal distinct roles in flower bud abortion and flowering in tree peony (Paeonia suffruticosa)Can. J. Plant Sci.9411811193

    • Search Google Scholar
    • Export Citation
  • WeiY.Z.LaiB.HuF.C.LiX.J.HuG.B.WangH.C.2012Cloning and stability analysis of reference genes for expression studies by quantitative real-time PCR in litchiJ. South China Agr. Univ.33303306

    • Search Google Scholar
    • Export Citation
  • WuR.M.WaltonE.F.RichardsonA.C.WoodM.HellensR.Varkonyi-GasicE.2012Conservation and divergence of four kiwifruit SVP-like MADS-box genes suggest distinct roles in kiwifruit bud dormancy and floweringJ. Expt. Bot.63797807

    • Search Google Scholar
    • Export Citation
  • YangH.F.LuX.Y.ChenH.B.WangC.C.ZhouB.Y.2017Low temperature-induced leaf senescence and the expression of senescence-related genes in the panicles of Litchi chinensisBiol. Plant.61315322

    • Search Google Scholar
    • Export Citation
  • YangY.J.KlejnotJ.YuX.H.LiuX.M.LinC.T.2007Florigen (II): It is a mobile proteinJ. Integr. Plant Biol.4916651669

  • ZhouB.LiN.ZhangZ.HuangX.ChenH.HuZ.PangX.LiuW.LuY.2012Hydrogen peroxide and nitric oxide promote reproductive growth in Litchi chinensisBiol. Plant.56321329

    • Search Google Scholar
    • Export Citation
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