Morphological and physiological changes of grape berries in response to GA application
To investigate the effects of GA on the grape berry and seed development, the morphological and physiological variation of grape cv. ‘Rosario Bianco’ was observed and examined. GA treatment conspicuously induced seedless berries with nearly 99% seedless ratio, while GA-untreated control plants had no seedless berries (Table 1 and Fig. 1). In addition, GA treatment distinctly affected the fruit shape index of grape berries, which increased from 0.26 to 0.43 along with the development of grape berries. GA-treated grape berries exhibited the longer fruit brush in contrast with control ones, and the smaller diameter of fruit brush relative to control plants (Table 1 and Fig.1). These morphological changes reflected that some physiological and molecular responsive to GA might be involved in the modulation of grape berry development.
Identification of VvmiR166s in grape cv. ‘Rosario Bianco’ by miR-RACE and their homologous sequences comparison with those in miRBase
miR-RACE technology was employed to verify VvmiR166a/b/c/d/e/f/g/h precise sequences in grape cv. ‘Rosario Bianco’. Our results demonstrated that the cloned sequences of all VvmiR166 family members were identical in length (21nt), and VvmiR166a/b were different from the validated miRNAs in miRbase 21.0 (Fig. 2). The sequence analysis showed that VvmiR166a exhibited two more bases “TC” at the 5’-end, while VvmiR166a had two more bases “TC” at the 3’-end (Fig. 2A). However, the identified sequences of the remaining members VvmiR166c/d/e/f/g/h were similar with those of homologous sequences in miRBase 21.0 (Fig. 2A). These results indicated that there is diversification as well as conservation in the sequences of miR166 family members. The determination of VvmiR166s precise sequences could provide significant base for prediction of their target genes, which contributed in recognizing the VvmiR166s roles in grapes. In contrast with the similarity of the mature sequences between VvmiR166 family members, their precursor sequences were less similar based on the alignment analysis. These eight VvmiRNA precursor sequences (VvMIRNAs) were divided into two groups, including VvMIR166a/b and VvMIR166c/d/e/f/g/h with 50% and 36% similarity, respectively (Fig. 2B, red box regions); while all VvmiR166s possessed about 90% identity at their mature sequences, confirming VvmiR166s had the higher conservation than corresponding VvMIR166s.
Analysis of sequences complementary degree and potential cleavage roles between VvmiR166s and their predicated target genes
The precise sequences of VvmiR166s were used as queries to search their target genes from grape mRNA database (http://genomes.cribi.unipd.it/DATA/V2/V2/). In total, four genes VvHOX32, VvREV, VvReloluta-Like, and VvHB15 were predicted as target genes for each member of the VvmiR166 family, and all target regions of VvmiR166 family members were located in the coding sequences (CDS) of their target genes (Table 2). Further functional annotation of target genes showed that VvHOX32, VvREV, VvReloluta-Like, and VvHB15 belong to the HD-ZIP III class of HD-Zip TF family (Table3). Analyzing the complementary degree of VvmiR166a/b/c/d/e/f/g/h and VvHB15, VvREV, VvReloluta-Like and VvHOX32 target genes, the result depicted that VvmiR166a/b displayed one mismatch base withVvHB15, VvREV, VvReloluta-Like and VvHOX32, while VvmiR166c/d/e/f/g/h and target genes had three mismatch bases (Fig. 3), indicating that VvmiR166a/b might have the stronger cleavage roles on these four target genes than VvmiR166c-h. Interestingly, we also revealed that all these mismatched positions between VvmiR166s and their targets almost occurred at both ends of VvmiR166s sequences. The result of high conservation at the sites with mismatch bases between miR160s and target genes was in line with other reports(Li and Mao 2007; Cuperus et al. 2011; Floyd and Bowman 2004), suggesting that this consistency might be one of the reasons for the conservation of miRNA-targeted modes within different plant species.
Analysis on motifs of promoters of VvMIR166s and their potential target genes
The promoter plays the key role in the initiation of transcription process, and its sequence contained some essential functional components (motifs), indicating the potential functions of genes. Here, we analyzed the motif elements of VvMIR166s and VvHB15, VvRevoluta, VvRevoluta-like, and VvHOX32. To conveniently depict the traits of these gene promoters, all motifs were classified into five different types, including light, hormone, tissue-specific, circadian and stress responsive elements. As shown in Fig. 4A, VvMIR166s almost has five kinds of cis-elements expect for VvMIR166e/f without circadian responsive elements, and among these five cis-elements, the number of light-related elements is the largest, accounting for more than 1/3 of all elements contained, which might derive from the fact that the light is necessary for photosynthesis in all green plants. Contrarily, all four target genes of VvmiR166s have the similar motif elements, of which VvREV-like and VvHOX32contained 5 types of cis-elements above, while VvREV and VvHB15 had 4 out of 5 kinds of cis-elements without circadian-related genes, implying their potential functions in these aspects mentioned above.
Our earlier work revealed that VvmiR166 family could conspicuously respond to GA(Han et al. 2014), and thus, we further analyzed the motifs related to hormones for the promoters of VvMIR166s and target genes. As shown in Fig. 4B, the promoters of VvMIR166s and target genes consist of motifs responsive to hormones like GA, abscisic acid (ABA), Ethylene(ET), methyl jasmonate (MeJA), salicylic acid(SA) and auxin. Interestingly, the promoters of VvMIR166a/b/c/d/e/g/h and VvHB15 contained cis-elements responsive to GA. SA responsive elements were predicted in the promoters of VvMIR166a/b/c/d/e//h and VvHB15. Motifs responsive to ABA were identified in the promoter regions of VvMIR166a/c and VvHOX32, while those responsive to MeJA were observed in the promoters of VvMIR166b/e and VvHB15.None of the promoters of VvMIR166family memebers possessed motifs responsive to auxin, while target genes VvRevoluta, VvRevoluta-like, and VvHOX32 had auxin-related motifs. These results suggested that VvmiR166 family and target geneVvHB15 might be mainly involved in the modulation of GA and SA, while GA and SA played the significant roles during grape berry and seed development, and thus we deduced VvmiR166 family and VvHB15 might involve in the regulation of grape berry and seed development by responding to GA and SA signal. On the other hand, we also observed that the diverse members of the VvmiR166 family might possess the redundancy and complementary of functions in responding hormones. The promoters of VvMIR166s and their target genes exhibited the diverse traits in the types and numbers of motifs.
Clone, sequence analysis and sub-cellular localization of target genes of VvmiR166s
Based on the predicted target mRNA sequences of VvmiR166s, we cloned and identified the sequences of VvHOX32, VvREV and VvHB15 in the berries of grape cv. ‘Rosario Bianco’. VvREV had an open reading frame (ORF) of 2532 bp encoding a protein of 844 amino acid residues, and the ORF of VvHOX32 was 2535 bp encoding a protein of 845 amino acid residues, while VvHB15 possessed 2520 bp ORF encoding a protein of 840 amino acid residues (Fig. 5A). These three proteins all comprised of four conserved domains, including homeo domain(HD), leucine-zipper domain (Zip), steroidogenic acute regulatory protein-related lipid transfer domain (START), and MEKHLA domain (Fig. 5A). We further compared the amino acid sequences of our VvHOX32, VvREV, and VvHB15 proteins encoding genes with their homologous proteins derived from Arabidopsis thaliana, Nicotiana tabacum, Fragaria vesca ssp. Vesca and Prunuspersica (Fig. 5B), and the phylogenetic tree was constructed using the MEGA software V.7 with the neighbor-joining algorithm (Fig. 5C). The Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn/index.php) was also employed to analyze the intron, exon and up/downstream UTRs.The targrt proteins VvREV, VvHOX32, and VvHB15 belong to HD-Zip III family, and their phylogenetic analysis divided them into the three subgroups. VvHB15 had the closest relationship to the PtHB15, while VvHOX32 was highly similar to NtHOX32, but VvREV located at one small branch far from other homologous genes in other plant species (Fig. 5C), indicating that the various members of same gene family might be involve in diverse evolution process. Further analysis of sequence structure revealed that the extra and intron number of these three target genes are similar, while they are different in the distribution in chromosomes. VvREV, VvHOX32 and VvHB15 exhibited longer introns in length and looser structures of their sequence in contrast with homologous genes of other five plant species, implying that the divergence during their evolution process (Fig. 5D). Sub-cellular localization analysis of VvREV, VvHOX32 and VvHB15 with the 35S-VvREV/VvHOX32/VvHB15:GFP and 35S-GFP being transiently expressed in tobacco leaf epidermal cells showed that the fluorescence in the tobacco cell trans-formed with 35S-VvREV:GFP was localized both in the cytoplasm and in the nucleus; 35S-VvHOX32:GFP was detected exclusively in cytoplasm, while 35S-VvHB15:GFP was just localized in the nucleus (Fig. 6). The divergence in their Sub-cellular localization implied the difference of their potential functions.
Verification of VvmiR166s-guided cleavage roles on their target mRNAs in grapes
The detection of the cleaved products of target genes for miRNAs is necessary to study the miRNAs regulation mechanism on their target genes. miRNAs negatively regulate the target genes through miRNA-directed cleavage within the region of complementary or interfering with translation. Most of the plant miRNAs have been shown to guide cleavage of their target genes. To examine whether the three predicted targets ofVvmiR166s can also be cleaved, our modified RLM-RACE technology and developed PPM-RACE procedure were used to map the cleavage sites in predicted target genes from grapevines. All the anticipated RLM-RACE products (3’-end cleavage mRNAs) showing distinct bands on agarose gel were isolated and sequenced, and the 3’-end cleavage mRNA sequences could be mapped into VvHOX32, VvREV, and VvHB15.Thus these three genes could be validated as the target genes ofVvmiR166s, and their cleavage sites mainly happened at the 9th site from 5’-end ofVvmiR166s at the binding sites, which was similar to previous reports (Fig. 7). Subsequently, PPM-RACE was further employed to verify these cleavage roles and cleavage sites. The results showed that PPM-RACE products (5’-end cleavage mRNAs) were also mapped into VvHOX32, VvREV, and VvHB15, and their cleavage sites also mainly occurred at the 9th position. The consistency of cleavage sites of RLM-RACE and PPM-RACE further confirmed these genes to be the true targets ofVvmiR166s, and their negatively regulatory roles of VvmiR166s on these three target genes.
Spatio-temporal Expression patterns of VvmiR166s and their target mRNAs VvHOX32, VvREV, and VvHB15 during diverse grape berry tissues development
Expression profiles of miRNAs in various tissues not only confirm the existence of the miRNAs in the organisms but also provide clues about their physiological functions. To recognize the potential functions of VvmiR166s, qRT-PCR analysis was employed to detect the expression levels of VvMIR166s genes (VvmiR166s precursors) and their targets (the primer in Table.4) in the various tissues of grape berries for a more efficient characterization of VvmiR166s and their target genes. The results of qRT-PCR clearly indicated that VvmiR166s were expressed ubiquitously in three types of grape berry tissues (peel, flesh, and seed), showing some tissue and/or stage-specific patterns. As shown in Fig. 8, almost all VvmiR166s possessed a typical character of the lowest expression in grapevine stone hardening stage (46 DAF), while exhibited the ubiquitous expression level in other stages. For instance, VvmiR166d had the high expressions in the seeds of the remaining stages, followed by VvmiR166a, VvmiR166g and VvmiR166h, indicating these members might involve in the modulation of the development of the special stages. The expression of VvmiR166b/c/e exhibited the“V”mode from the 32DAF to 80DAF during the development of grape berries, the lowest expression level was detected at 46DAF in the corresponding tissues, suggesting that regulatory roles of VvmiR166b/c/e might possess the dynamic variation during grape berry development. Unlike the above two expression modes, VvmiR166f had the high expression levels in the seed and flesh at the 32DAF, while exhibited the low expression levels at the other stages, indicating VvmiR166f might mainly involve in the modulation of the special stage of grape berry development.
To further gain insight into the roles of VvmiR166s in grape berries, the expression levels of their target genes were analyzed. As shown in Fig. 9, three target genes of VvREV, VvHB15, and VvHOX32 exhibited the lowest expression levels in flesh tissues at various development stages of grape berries. VvREV and VvHB15 possessed the highest expression levels in the seed tissues at various development stages, while VvHOX32 had the higher expression levels in the peel tissues at different stages than the flesh and peel tissues. As described above, these three target genes belong to HD-Zip III family involved in modulation of vascular and xylem formation, we deduced that they might involve in regulating vascular and xylem formation of seed stones and peels of grape, and highly expressed in the whole seeds and peels.
Compared to the correlation of VvmiR166s and their targets at the expression levels, we revealed that VvmiR166f and VvHOX32 had the reverse expression modes during grape berry development, implying VvmiR166f might negatively regulate the expression of the VvHOX32. Besides this, the remaining VvmiR166 family members showed the negative correlation with these three target genes at their expression levels in the certain tissues at some special stages of grape berry development, like in seed and peel tissues at the 46DAF and 60DAF (Fig. 8 and Fig. 9). Considering the redundancy and complementary traits of function of miRNA, the total expression levels of all members of VvmiR166 family were detected to compare to each target gene,the results showed that except for VvHOX32 in peel tissues, VvmiR166shad negative correlations with three target genes at their expression levels in special periods of various tissues (See* marked in Fig. 10). For instance, there were negative relationships in some extent betweenVvmiR166sand VvHB15andVvREV in seed tissues during the later period of grape development (Fig. 10 * marked), while the expression modes of VvmiR166s and VvHOX32 were similar at the former stages (Fig. 10 * marked). In addition, VvmiR166s and target genes also showed opposite expression trend in peel and flesh tissues at multiple development stages (see Fig. 10 * marked). These results further presented some evidence to support the negative regulatory roles of VvmiR166s on three target genes in some special stages of grapes.
Modes of VvmiR166s and their target mRNAs responding to GA during the development of grape berries
GA plays the significant roles in grape berry development, and our previous study showed VvmiR166s could respond to exogenous GA(Han et al. 2014). In the present study, the modes of VvmiR166s and their target mRNAs responsive to GA in various tissues of berries during grape berry development were further investigated. The results showed that in seed tissues, the expression of VvmiR166a/b/f/g were down-regulated by GA in almost all stages, depressed at the lowest levels at 46DAF. The remaining members exhibited the diverse variation trends during diverse stages of seeds development, implying that GA might modulate seeds development mainly by inhibiting the expressions of VvmiR166a/b/f/g. In contrast, VvmiR166b/c/h was down-regulated by GA only at the former berry development stages in peel tissues, like 32DAF and 46DAF. The remaining members were up-regulated or exhibited on change in expression level under GA treatment in all stages. GA treatment reducedVvmiR166c/d/f/h expression at 32DAF in flesh tissues, while there was no significant effect of GA treatment on the remaining members in all stages (Fig. 11). All these results confirmed that VvmiR166 family possessed the tissue and stage-specific characters as well as diversification in responding to GA.
In contrast with VvmiR166s, their target genes exhibited the differential expression modes responsive to GA in grape berries (Fig. 12). The two interesting cases were observed among them, in one case, GA treatment induced VvHB15 expression at 46 DAF in seeds and peels, especially reverse to those of several members of VvmiR166 family in seeds, indicating GA manipulated seeds development possibly by repressing the expressions of VvmiR166to promote VvHB15 expression. Another case is that all these target genes were obviously up-regulated by GA at all stages in fresh tissues. These findings demonstrated that these target genes possessed diverse modes responsive to GA in grape fresh tissues with those in corresponding seeds and peels, and meanwhile suggesting VvmiR166 family might negatively modulate VvHB15expression in the special tissues of the certain stage grape berries by responding to GA. Our findings confirmed that VvmiR166 family possessed the characterization of stage/tissue-specific involved in the grape berry development in response to GA by mediating their target genes.
Effect of GA on the regulatory modes of VvmiR166s:VvHB15 during GA-induced grape seedless berry development
From the analysis above, it is revealed that VvmiR166:VvHB15 module might be one of key regulators amongst VvmiR166 family during GA-induced grape seedless berry development. To further recognize the effect of GA on regulatory modes of Vvmi166:VvHB15 pair, we analyzed the variation of GA manipulating on the expression correlation of VvmiR166s and VvHB15. Considering of the stage specificity of VvmiRNAs and target genes at their expression levels, the two special important developmental periods of 32DAF-46DAF (from young to stone hardening stage, called as stage I) and 60DAF-86DAF (from color turning to mature stage, called as stage II) were selected for investigation of the expression correlations betweenVvmiR166s and VvHB15. As Fig. 13 showed, VvmiR166s and VvHB15 had apparent positive correlations (r=1) at stage I of seed, peel and fresh tissues in control plants, while part members of VvmiR166 family and VvHB15exhibitednegativecorrelation(r=-1) at stage II of corresponding tissues (See the blue※). The result indicated that VvmiR166s might play roles at stage II of seed, peel and fresh tissues through negatively modulating the expression of VvHB15. Interestingly, VvmiR166 family members and VvHB15 expression possessed significant negative correlation (r=-1)under GA treatments in comparison with untreated control plants(See red ※), suggesting VvmiR166 family could be modulated by GA to negatively mediate the expression of VvHB15 mainly at stage I of grape seed and peel tissues. These results confirmed that GA could efficiently change the regulatory modes of VvmiR166s on VvHB15, where their negatively modulated roles occurred at from stage II (CK) to stage I(GA treatment).
On the other hand, we also revealed that during GA-induced grape seedless berry development, GA could up-regulated the expressionsofVvmiR166 members at seeds, leading to inhibition of VvHB15expression, while previous studies showed ArabidopsisAtHB15-OE could inhibit lignin synthesis(Boualem et al. 2008), thus we deduced VvmiR166s: VvAtHB15 pairs might be one of reasons that GA repressed grape seed stone development to induce grape seedless berries. This could provide the significant clue for further elucidating the molecular mechanism of GA-induced grape seedless berry.
Roles of VvmiR166s-mediated VvHB15 in modulation of lignin synthesis process
To recognize the potential function of VvmiR166s and VvHB15, we constructed their corresponding transient over-expression pCAMBIA1302 vector (VvMIR166s/VvHB15-OE) (Fig.14A), and the empty pCAMBIA1302 vector was used as the control. These vectors were transiently transformed into tobacco plants using an Agrobacterium-mediated method. After dark culture of 3 days, DNA was extracted, and specific primers were used for PCR detection. The precise sequences after gel electrophoresis and sequencing were identical to purpose ones (Fig.14B), confirming the transient over-expression of VvMIR166s/VvATHB15 in tobacco plants (Fig.14C).
In view of the potential functions of HD-ZIP III transcription factors in the modulation of lignin synthesis of mode plants(Boualem et al. 2008), we further investigated the effect of over-expression of VvMIR166s/VvHB15 on the expression levels of genes (NtPAL, NtCCR, NtCAD, NtLaccase, Nt4CL, Nt4CL, NtCOMT) involved in the lignin synthesis pathway. Results showed that among VvMIR166 family, VvMIR166h might be one of main regulators in this process, over-expression of VvMIR166hin tobacco plantsevidently up-regulated the expression of NtPAL4, NtCCR1, NtCOMT1 and NtCCoAOMT6, followed by VvMIR166b (markedly up-regulated those of VvLaccasse3 and VvLaccase17) and VvMIR166a (observably up-regulated that of VvCAD1), while over-expression of VvHB15 obviously down-regulated the expression of NtPAL4, NtCCR1 and NtCAD1(Fig. 14D), indicating VvMIR166s possessed the opposite roles toVvHB15 in modulation of lignin synthesis. Furthermore, the transient over-expression of VvmiR166s in Tobaccos obviously decreased the expressions of NtHB15 and NtHB15-likegenes, which were high conserved with VvHB15, especially complete consistent VvmiR166s-targeted regions in these three orthologous genes. Together with the modes of VvmiR166s and their targets responsive to GA, it was revealed that the potential roles of VvmiR166b/h during GA-induced grape seedless berry process might function to inhibit grape seed-stone development through the key signal pathway of “GA-VvmiR166h/b-VvHB15-lignin synthesis genes” (Fig.15). All these results confirmed that VvmiR166b/h might modulate the synthesis of lignin mainly by negatively regulating VvHB15 in grapes (Fig.14E), which was in line with previous studies by Boualem (Boualem et al. 2008).