11 publications mentioning vvi-MIR156g

Open access articles that are associated with the species Vitis vinifera and mention the gene name MIR156g. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1

While the majority of the grape SBP-box genes lacking a miR156/157 target site were expressed ubiquitously and constitutively, most genes bearing a miR156/157 target site exhibited distinct expression patterns, possibly due to the inhibitory role of the microRNA.

Our experimental results, which were consistent with the expression patterns of miR156/157 -targeted genes in tomato and rice [21], [23], indicated that the miR156/157 -targeted grape SBP-box genes were generally expressed in a similar fashion to the VvmiR156f gene in all tissues tested (Fig. 6).

Furthermore, the majority of these 12 grape SBP-box genes with miR156/157 target sites also exhibited the highest levels of expression in the early stages of fruit development, which gradually decreased or even vanished during the fruit ripening process.

In general, the expression patterns of the 18 SBP-box genes could be classified into two types according to the presence or lack, of a miR156/157 target site (Fig. 6).

In contrast to the grape SBP-box genes discussed above, Group 1 and Group 5 genes did not contain a miR156/157 target site and were all expressed ubiquitously and constitutively, with little or no variation in any of the tissues analyzed (Fig. 6).

In the case of genes lacking a miR156/157 target site, including VvSBP4, VvSBP5, VvSBP6, VvSBP7, VvSBP14 and VvSBP17, there tended to be little or no variation in expression in any of the tissues tested.

These results indicate that grape genes from these two groups may have functions that are distinct from the miR156/157 -targeted SBP-box genes in Groups 2, 3 and 4. Grape SBP-box Genes are Responsive to Abiotic and Biotic StressesTranscriptional control of stress-responsive genes is a crucial means by which plants respond to a range of abiotic and biotic stresses and research carried out in recent years has been productive in identifying transcription factors that are important for regulating these types of responses [60].

These results indicate that grape genes from these two groups may have functions that are distinct from the miR156/157 -targeted SBP-box genes in Groups 2, 3 and 4. Transcriptional control of stress-responsive genes is a crucial means by which plants respond to a range of abiotic and biotic stresses and research carried out in recent years has been productive in identifying transcription factors that are important for regulating these types of responses [60].

In contrast, genes containing a miR156/157 target site, including VvSBP1, VvSBP2, VvSBP3, VvSBP8, VvSBP9, VvSBP10, VvSBP11, VvSBP12, VvSBP13, VvSBP15, VvSBP16 and VvSBP18, were expressed at relatively higher levels in leaves, stems and tendrils compared to the reproductive tissues analyzed.

Indeed, 12 of 18 grape SBP-box genes contained a miR156/157 target site in the V. vinifera genome (Fig. 1).

The miR156/157 target sites are denoted by blue vertical lines.

In rice, for instance, 11 of the 19 SBP-box genes have been revealed to be putative targets of OsmiR156 [21], while 10 of 15 SBP-box gene family members in tomato were found to carry putative miR156/ 157-response elements [23].

Interestingly, the miR156/157 -targeted SBP-like genes, including sequences from rice, Arabidopsis and grape, were distributed into only three of the subgroups (Groups 2, 3 and 4).

Of the twelve grape SBP genes containing a miR156/157 target site, Group 4 members (with the exception of VvSBP4) bore this site within their 3′ UTRs (Fig. 3B), which is similar to AtSPL3, AtSPL4 and AtSPL5 in Arabidopsis.

As a gene family encoding transcription factors, more than half of the SBP-box genes identified to date have been found to be targeted by miR156/ 157.

To date, miR156/157 target sites were found in 10 Arabidopsis [57], 11 rice [21] and 10 tomato [23] SBP-box genes.

In most cases, miR156/157-regulated SBP-box genes tend to play a role in the control of phase change and reproductive development [58], [59].

The three groups of SBP-box genes discussed above (Groups 2, 3, and 4), with the exception of SBP4, all contain a miR156/157 target site.

Furthermore, both the locations of miR156/157 target sites and composition of encoded SBP domains were compared in each of the grape SBP genes to gain further insight into their evolutionary relationship with one another.

59Schwab R (2012b) Roles of miR156 and miR172 in Reproductive Development.

SBP-box genes from Arabidopsis, rice, tomato, and grape that contain complementary sequences for miR156/157 are marked with an asterisk.

0059358.g001 Figure 1Alignment of miR156/157 complementary sequences within grape SBP genes.

Chromosomal distribution of SBP and miR156 genes, as well as synteny of SBP-box genes in grape.

Expansion Patterns and Distribution of Grape SBP and miR156 Genes in the Grape Genome.

Expansion Patterns and Distribution of Grape SBP and miR156 Genes in the Grape GenomeAccording to available annotation information, the 18 grape SBP genes were dispersed on all grape chromosomes except for chromosomes 2, 3, 6, 9, 13 and 16.

Alignment of miR156/157 complementary sequences within grape SBP genes.

Although SBP-box genes have been identified in numerous plants including green algae, moss, silver birch, snapdragon, Arabidopsis, rice and maize, there is little information concerning SBP-box genes, or the corresponding miR156/157, function in grapevine.

SBP-box genes that contained complementary sequences for miR156/157 are marked with an asterisk.

Although grapevine (V. vinifera) is one of the most important perennial fruit crops worldwide, there is little information concerning SBP-box gene, or the corresponding miR156/157, function in this species [24].

SBP and miR156 genes are indicated by vertical orange and black lines, respectively.

0059358.g004 Figure 4Chromosomal distribution of SBP and miR156 genes, as well as synteny of SBP-box genes in grape.

In addition, in accordance with findings in other species, we also found that 12 of the 18 grape SBP genes identified in this study contained sequences that were complementary to miR156/157, with a maximum of one to three mismatches to the mature VvmiR156/157 sequences (Fig. 1).

Previously, nine members of the miR156/157 family, termed VvmiR156a to VvmiR156i, which are highly conserved in plants and are thought to interact with numerous SBP-box genes, were identified in the V. vinifera genome [53]– [56].

This provides yet another example of the mutual relationship between miR156/157 and SBP-box genes.

Eighteen SBP-box gene family members were identified in Vitis vinifera, twelve of which bore sequences that were complementary to miRNA156/157.

2

Putative targets of conserved miRNA families, such as miR156, miR159, miR171 and miR399, identified in this study, correspond to targets found in numerous plant species, including several grapevine cultivars, while the predicted functions of these targets were also similar with previous findings [26, 27, 58, 60, 68– 70] (S6 File).

The Arabidopsis AtSPL9, can positively regulate the expression of miR172, demonstrating the presence of a miR156-AtSPL9-miR172 regulatory cascade [85].

It has been shown that down-regulation of miR156 results in an increase in SPLs that promote juvenile to adult phase transition and flowering through activation of miR172 and MADS box genes in Arabidopsis [83, 84].

It was suggested that expression changes of miR156 and miR172 may lead to development of green leaf-like structures instead of flowers, also referred to as phyllody, as well as flower sterility in phytoplasma-infected red date and mulberry [81, 82].

The modification of leaf morphology due to regulation of SBP-box transcripts by miR156 overexpression was demonstrated in phytoplasma-infected Mexican lime trees, mulberry, and red date [52, 81, 82].

The differential expression of conserved miRNA families (vvi-miR156, miR159, vvi-miR160, vvi-miR171, vvi-miR172, vvi-miR319), known to be involved in different aspects of plant development [18], make these potential candidates that play a role in the interactions leading to symptoms associated with GY.

Likewise, miR156-overexpression in poplar (Populus spp. )

Expression analysis in this study, however, revealed a significant decrease in abundance of certain vvi-miR156 members in the phytoplasma-infected samples, which cannot be explained at this point.

The miR156/157 family, which is highly conserved in plants, can target numerous members of the SBP-box genes in V. vinifera.

The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis.

Differential expression of vvi-miR156 and vvi-miR172, leading to restricted phase transition, may lead to symptoms associated with GY such as abnormal leaf shape and size, as well as downward curling of leaves and flower abortion.

Studies on Arabidopsis and rice showed that cleavage of squamosa-promoter binding-like (SPL) proteins, due to miR156 overexpression, give rise to plants that are smaller, show delayed flowering and loss of apical dominance, initiate growth of more leaves with shorter plastochrons (in Arabidopsis) and causes reduced panicle size (in rice) [78, 79].

It was proposed that the miR156-SPL-miR172 regulatory pathway was activated in mulberry in response to phytoplasma infection [81].

3

Combining target prediction and expression profile, we shed light on the functional role of some miRNAs adding insight on inflorescence and fruit development, reinforcing, among others, the role of the miR156/miR172 regulatory circuit in both inflorescence and berry development and suggesting a role of miRNAs in regulating hormonal -driven fruit maturation and inflorescence development.

This is because SPL, the target of miR156, is an elicitor of miR172 promoter region which, in turn, targets APETALA2.

SPL genes, targeted by vvi-miR156, decrease during berry ripening in grapevine [82] and, accordingly, we showed that vvi-miR156 is more expressed in the last stage of berry maturation.

Furthermore, in Arabidopsis, an over expression of miR156 determines a reduced SPL genes activity and, by a subsequent regulation of several genes, lead to an accumulation of anthocyanins [84], likely connected to DFR gene activation [85], a flavonoid biosynthetic gene.

From our data, miR156 family members target nine different SPL genes, six of which are negatively correlated to vvi-miR156 when considering their abundances (see Additional file 10).

As observed in other species [48– 50], in grapevine an increase in miR156 levels corresponds to a low expression of miR172 and viceversa.

In inflorescence, a well recognized role is played by miR156-miR172 regulation: miR156 decreases during inflorescence development while miR172 increases (Figs.   9A and 9B).

Panels A-D: miRNAs tested in the inflorescences/flowers samples; Panels E-H: miRNAs tested in the berries samplesIn inflorescences, the well-known regulatory circuit involving miR156 and miR172 is confirmed by both sequencing and RT-qPCR data (Figs.   9A and 9B).

Panels A-D: miRNAs tested in the inflorescences/flowers samples; Panels E-H: miRNAs tested in the berries samples In inflorescences, the well-known regulatory circuit involving miR156 and miR172 is confirmed by both sequencing and RT-qPCR data (Figs.   9A and 9B).

These data reinforce the idea vvi-miR156 may be responsible for inducing maturation programs in grapevine berries, in an ethylene independent manner, through SPL genes regulatory networks and anthocyanins accumulation, secondary metabolites accumulating in mature berries of red grapevine varieties.

First of all, as observed in inflorescences, miR156 and miR172 are inversely correlated, and seem to be actively involved in fruit maturation: vvi-miR156 shows a gradual increase during ripening (as confirmed by RT-qPCR Fig.   9E), with a corresponding decrease of vvi-miR172.

Some of the most conserved miRNAs reveal intriguing profiles: among others, miR156 and miR164 seem to be involved in berry maturation, in an ethylene independent manner.

4

For example, in GRSPaV-free grapevines, miR166 and miR396 were down-regulated by drought, similar to their reported expression in O. Sativa 37; miR168 was up-regulated in Arabidopsis thaliana, in agreement with its response to ABA-inducing stresses 16 and miR156, miR167 and miR397 were not affected by drought in grapevine, thus confirming their species-specific drought response 35 38.

For example, in infected grapevines, we observed that the regulation of some miRNAs and their targets involved in leaf development was influenced by virus infection (miR156, miR164, miR319, miR394, miR396; Fig. 2, Supplementary Fig. S4).

In Arabidopsis, Usami et al. 49 demonstrated that miR156 and SBP genes are involved in determining leaf cell number and size; overexpression of SBP genes with a deletion in the miR156 target site leads to an increase in cell number.

Relative expression levels of miR396, miR156, and miR164 and their respective targets in Grapevine rupestris stem pitting -associated virus(GRSPaV)-free and infected ‘Bosco’ leaves as determined by qRT-PCR.

Accordingly, we observed a similar regulation in response to an increase in VvSBP expression and low miR156 levels only in leaves of infected plants.

A negative correlation was found between miR156 and VvSBP expression in WW and SWS conditions.

The Squamosa promoter -binding protein gene (VvSBP, VIT_01s0010g03910) is a member of a plant-specific transcription factor family with a broad range of functions, and was previously demonstrated to be a target of miR156 24.

Our data suggest that the virus -induced changes in miRNA expression between WW and SWS conditions; in particular, the presence of GRSPaV, significantly affected the accumulation of miR156, miR164, miR166, miR394, miR396, and miR3639 in response to SWS (Fig. 2, Supplementary Fig. S4) and the relevance of this is discussed later.

5

In Arabidopsis, SPL5 belongs to the miR156/7 -targeted SPL subfamily as in grapevine (Figure S1) [24] and act as a positive regulator of juvenile-to-adult phase change transition and flowering in Arabidopsis [49], [50], regulated by SOC1 [34], while the miR156/7 non -targeted SPL8 gene is involved in pollen sac development [51].

In addition, five SPL-related transcripts also showed expression in both tendril and inflorescences at later developmental stages (VvSPL1-L, VvSPL3-L, VvSPL12-L, VvSPL14-L and VvSPL7-L) These genes, except VvSPL3-L, do not show enough sequence complementarity with miR156 to be its potential targets.

Except the first one, they are all potential targets of grapevine miR156/7 (Figure S1) [24].

Some Arabidopsis counterparts of these genes (SPL12, SPL14 and SPL7) also belong to the miR156/7 non -targeted SPL subfamily and are the largest proteins in the family [27].

SPL genes in this cluster were VvSPL2-L1, VvSPL2- L2 and VvSPL4-L, all of them are potentially targeted by miR156 in grapevine (Figure S1) [24].

Sequences were aligned using MUSCLE software [63] and those SPL genes with more than 90% identity with miR156 sequence were identified as potentially targeted.

Grapevine miR156 sequences were obtained from miRBase (http://www.

Figure S1 Sequence similarities between VvSPL genes and Vvi-miR156.

6

Interestingly, cluster 1 contains transcripts homologous to SPL3, 4, 5 and 9, all belonging to the miR156/7 -targeted SPL subfamily, which act as positive regulators of juvenile-to-adult phase change transition and flowering in Arabidopsis [63, 64, 66] and are regulated by SOC1[67].

Arabidopsis SPL2 is also miR156 targeted and seems to be involved in lateral organ development within the reproductive phase [68].

The miR156/7 non -targeted SPL8 gene is involved in pollen sac development [69] and required for male fertility [70].

Ten of the 16 Arabipdopsis SPL genes are post-transcriptionally regulated by miR156, which incorporates endogenous age/development signals into vegetative phase transition and flowering [61, 62].

Most Arabidopsis counterparts of these genes belong to the miR156/7 non -targeted SPL subfamily (SPL1, 7, 12, 14 and 16) that comprises the larger proteins in the family [70].

7

In this study, the expression of six conserved miRNAs, including miR156, miR171, miR172, miR395, miR397, and miR398, were downregulated after cold stress, and there was no conserved miRNAs showed significant upregulated.

Several transcription factors were found in the target genes, including the squamosa promoter binding (SBP) protein (the putative target of vvi-miR156), APETALA2 (AP2, vvi-miR172), basic helix-loop-helix (bHLH, vvi-miR3640 [*]), GRAS (novel_mir_39), bZIP (novel_mir_4, novel_mir_42), and MYB-like binding protein (novel_mir_13).

The targets of conserved miRNAs, such as miR156, miR171, and miR172, had been investigated in several grapevine cultivars, and their functions were almost in accordance with the previous studies (Carra et al., 2009; Mica et al., 2010; Pantaleo et al., 2010; Wang et al., 2011a, 2012).

The medium miRNA families were miR395, miR156, miR166, and miR399, with 13, 9, 8, and 8 members, respectively.

The most abundant miRNA family was miR156, with 11,426,364 and 9,449,021 reads in the NCT and CT libraries, respectively (Table 1).

8

The best example of this phenomenon is the location of SNPs in the va-miR156 family, where 15 of the 20 SNPs created new mismatch sites from the match position upon which miRNAs bind their target genes, while the variant bases of the other five SNPs occurred in the original mismatch positions of miRNAs and the binding regions in their target genes.

Although the va-miR169 family had the highest number of miRNA members with SNPs, the va-miR166 family topped in the list of total number of SNPs, followed by va-miR156, va-miR167 and va-miR169 families (Figure 3).

9

For example, recent work has linked molecular pathways regulating the timing of shape changes throughout the shoot (miR156/172 and their targets) with leaf morphology (CUP‐SHAPED COTYLEDON (CUC)‐induced serrations) through a mediator (TCPs, TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORs) (Rubio‐Somoza et al., 2014).

10

miR156 and miR529 initiate TAS6, which targets an mRNA that encodes a zinc finger protein [11].

11

In some cases, the most commonly observed sequences were identical to at least one of the predicted mature sequences (notably: miR156, miR160, miR164, miR167, miR169, miR172, miR394, miR399) while for other families, the predominant mature miRNA sequenced exhibited small variations (shifts or differences of length of one or two bases) with respect to the predicted mature sequences (e. g. miR166, miR393, miR395, miR396, miR408).

For some families, thousands or even hundreds of thousands of short RNA sequences were recovered (miR156, miR164, miR166, miR167, miR172, miR393, miR396), while less than 1000 sequences corresponded to each of the remaining represented families.