Identification of peach growth regulating-factors and their expression under UVB


 Background: Growth-regulating factors (GRFs) are one of the most important plant-specific transcription factors with vital roles in multiple biological processes. GRFs have been identified in a variety of plant species, but a handful of research has addressed GRF genes in peach (Prunus persica).Results: Here, we report 46 members of the GRF family in four Rosaceae, divided into six subfamilies according to phylogeny, gene structure, and motif composition. We detected three collinear gene pairs generated from peach by whole-genome duplication or segmental duplication, but no tandem repeats were detected. Expression pattern analysis found that most PpGRFs were preferentially expressed in young tissues, At the sametime, multiple types cis-elements were observed in PpGRF promoters, and PpGRFs could positively respond to ultraviolet B-rays (UVB) and gibberellin (GA)treatments at the transcriptional level. Also, the content of GA3 and indole-3-acetic acid (IAA) changed significantly after UVB irradiation, indicating that GRFs might be involved in new shoot development in peach.Conclusions: This study identified 10 GRF genes in the peach genome and systematically analyzed their properties, thereby providing a foundation for researchers to have a better understanding of this gene family in peach. PpGRF 3, 4, 5, 6, 7, 9, and 10 positive responses to UVB and GA3 signals indicate that they can serve as candidate functional genes to further study how tree potential is regulated in peach.


Background
Many transcription factors (TFs) have been identi ed through evolutionary analyses in peach, including AP2,WRKY, MYB, TCP, NAC, NF-Y, and SPL TFs [1][2][3][4][5][6]. However, the roles of GRFs remain unknown in peach. GRFs are highly conserved in plants, which belongs to a small family of transcription factors that. The rst identi ed GRF gene was described in rice, and was shown to mediate gibberellin-induced stem growth regulation in deepwater rice [7]. evolutionally conserved QLQ and WRC domains are contained in the GRF proteins' N-terminal region [7][8][9]. The amino acid sequence of the QLQ (Gln, Leu, Gln) domain is somewhat similar to the protein-protein interaction domain of SWI2/SNF2 [10]. The QLQ domain is thought to help GRF proteins to interact with growth regulators (GIFs) to regulate plant growth and development [8,11]. The WRC (Trp, Arg, Cys) domain is a plant-speci c domain capable of binding to DNA and has a nuclear localization signal and a zinc nger motif [7,8]. Also, most GRF proteins have transactivation activity in the C-terminal region and share some less conserved motifs, such as the GGPL (Gly, Gly, Pro, Leu), TQL (Thr, Gln, Leu), and FFD structure domains [11,12].
Leaf and cotyledon sizes are increased in Arabidopsis lines overexpressing AtGRF1-3. This nding suggests that the AtGRF1-3 genes have roles in leaf and cotyledon development [8]. In the initial study, only the AtGRF1/2/3 triple mutant had smaller, narrower leaves, and shorter petioles compared to the wild type; however, later reports also demonstrated the rst pair of AtGRF3 single-gene mutants. The mean size of leaves was also reduced by 15%, and these changes in organ size resulting from increased GRF expression are due to increased cell size rather than increased cell numbers [8,17].
Interestingly, in contrast to AtGRF1 and AtGRF2, the overexpression of AtGRF5 showed a larger leaf area due to proliferation of cells. Also, the biochemical analysis showed that GRF and GIF combine to form a transcription complex in vivo. By means of a positive feedback regulatory loop, it might be a effective way for the GRF-GIF complex to activate its own transcription, thereby regulating the increase in the number of cells and ultimately controlling leaf size [14,15,[17][18][19]. Overexpression of TCPs (TCP4) resulted in reduced transcription levels of GIF, suggesting that they play an intermediary role in the GIF-GRF-miR396 regulatory network [20,21].
It has been shown that miRNA396 directly inhibits the expression of GRFs in various plant species through post-transcriptional regulation [11,23]. The heterologous expression of Ath-miR396a (from Arabidopsis thaliana) in tobacco causes a reduction in the transcription level of the GRF gene in tobacco, accompanied by similar narrow-leaf phenotypes, such as reduced leaf size. Also, Ath miR396a overexpression affects ower development relative to wild type, for inatance, increasing the number of petals, anthers, and carpels, and producing shortened (curved) stamens [24]. Similarly, ectopic expression of Ptc-miR396c (from Populus trichocarpa) resulted in changes in tobacco growth and ower development, accompanied by a decrease in the abundance of NtGRF transcripts [24]. Interestingly, not all GRFs are miR396 target genes. AtGRF1-4 and AtGRF7-9 are miR396 target genes in Arabidopsis, but overexpression of miR396 also inhibits AtGRF6 expression [25,26]. MiR396 expression is caused by various abiotic stresses, such as high salinity, low temperature, drought stress, and UVB. Several reports indicate that UVB leaf growth inhibition requires GRF1 activity, while miR396/GRF exerts it has an in uence on organ growth by regulating (at least partially) the expression of GA metabolic genes [25,[27][28][29].
UVB radiation treatment of Arabidopsis causes the accumulation of DNA damage and limits leaf expansion by inhibiting cell division in proliferating tissues [30]. Reduced maize leaf growth under UVB irradiation is the result of reduced cell production and a shortened growth zone [29]. This inhibition is regulated, at least in part, by transcription factors of the GRF family, some of which are probably regulated by miR396, which modi es GA levels [29]. Soybean plant morphology is substantially altered in response to shade stress. At the same time, under shade stress conditions, the GmGRF9 and GmGRF17 expression levels were strongly suppressed, and GmGRF5 was up-regulated [31].
Peach trees are native to China, where they have a cultivation history of about 3000 years [32]. Because peach has a relatively small genome and high economic and nutritional value, it has become the focus of research [33]. The protected cultivation of peach trees is more economical than traditional cultivation, but it limits the growth of fruit trees, yield, and fruit avor. Among various environmental factors, UVB radiation is an important factor, causing abiotic stress that potentially in uences the biological processes of plants [33][34][35]. Considering greenhouse light conditions, more in-deep researches which are connected with the molecular and metabolic mechanisms under UVB irradiance are now needed.
In this study, a total of 46 GRFs were characterized. We analyzed the phylogenetic relationships among GRF genes, their protein motifs, and gene structure across multiple species, including Arabidopsis thaliana, Maize (Zea mays L.), soybean (Glycine max), rice (Oryza sativa), poplar (Populus trichocarpa), and four Rosaceae fruit species. Moreover, we validated gene expression patterns by transcriptome analysis and quantitative real-time PCR (qRT-PCR) in peach, and identi ed several candidate GRF genes closely associated with peach. These results will contribute to the further researches of their molecular functions.

Identi cation of GRFs in Rosaceae
The GRF Hidden Markov Model (HMM) con guration le (PF08879 and PF08880) was used to identify GRF members. Furthermore, the SMART tool was used to verify the existence of two characteristic conserved domains (QLQ and WRC) in the candidate genes. After manually removing the incomplete sequences from peach (Prunus persica), European pear (Pyrus communis), strawberry (Fragaria vesca), and apple (Malus domestica), respectively, 46 GRF genes were identi ed and named according to their position on the chromosome. The GRFs were unevenly distributed across the chromosomes of the four species. The 46 predicted GRF proteins ranged from 187 to 843 amino acid residues in length, and their relative molecular mass varied from 20.70 to 94.27 kDa (Table 1).
To better explore the evolutionary relationships and predict the function of GRF proteins, we investigated the exon-intron patterns and motif characteristics of GRFs. We found some structural features among clades or subclades according to the alignment and motif results. As shown in Fig. 2, a total of 10 conserved motifs of Rosaceae GRFs were found using the MEME online software. Each subgroup has three to nine conserved motifs, and the motif composition is similar within subgroups [36][37][38][39][40]. All of the GRF family members contained motif 1 or motif 2. Based on such a gene structural analysis, we determined that motif 1 and motif 2 correspond to the WRC and QLQ domains, respectively. Some motifs occur only in certain speci c subgroups, which possibly contribute to functional diversity. For instance, motif 6 is unique to subgroup I, and motif 5 is unique to subgroup II, while motifs 3, 4, 7, 9, and 10 are concentrated in group III. Motifs 3, 4, and 9 are speci c to group IV. Motifs 3 and 4 are speci c to group VI. We also investigated the structure of GRF gene to further describe their evolutionary trajectory. All GRFs contained conserved QLQ and WRC domains in their N-terminal regions. However, group V has two WRC domains. These results indicate that they contain different exons numbers, varying from 3 to 12. The gene structures were similar or the same in each subgroup. Overall, phylogenetic relationships are strongly supported by gene structure and motif characteristics.

Chromosomal localization and collinearity analyses of GRF genes
It's random for the distribution of the GRF genes in the four Rosaceae genomes. Unlike in previous studies, in the four Rosaceae, some chromosomes do not have a GRF gene. In M. domestica and P. communis, the GRF genes are mainly found on chromosomes 2 and 15. For P. persica and F. vesca, the GRF genes were principally distributed on chromosomes 2 and 7, respectively. Additionally, two GRF genes (MdGRF15 and MdGRF16) could not be mapped to any chromosome in the M. domestica genomes (Fig. 3).
Subsequently, in order to further and deeper infer the phylogenetic relationship between peach and other Rosaceae plants, the collinear relationships between three Rosaceae plants and peach ( Fig. 3) was analyzed. We found that nine FvGRF genes were collinear with PpGRF genes, followed by MdGRFs (14) and PcGRFs (10). Moreover, the collinear relationship in the peach genome was also examined to elucidate the evolution and origin dynamics of PpGRF genes. Three pairs of PpGRF genes (PpGRF01/08, PpGRF03/07, PpbZIP05/10) have a collinear relationship and were generated by whole-genome duplication (WGD) or segmental duplication ( Fig. 3 and Additional File 1: Supplementary Table S1). Furthermore, to determine the selection constraints on the duplicated PpGRF genes, we calculated the non-synonymous/synonymous substitution ratio (Ka/Ks) for each pair of duplicated genes. The Ka/Ks ratios of most PpGRF pairs were less than 1, the evidence shows that these PpGRFs had undergone purifying selection processes (Additional File 1: Supplementary Table S1).
UVB effect on peach leaf growth During the whole groeing period, the peach trees were treated with 1.44 Kj m − 2 d − 1 UVB radiation [34]. It can be seen from Fig. 4  Prediction of the miR396 target site and how its expression changes following UVB exposure Differently to Arabidopsis, all GRFs in peach have a sequence that is partially complementary to miR396, which is located at the WRC domain. To delve deeper into the evolutionary relationship among GRFs, we analyzed the WRC structures of all GRFs in the four Rosaceae. According to Fig. 6, we found that all GRFs of the four Rosaceae contain this part, which is complementary to the miR396 share a bulge at position 7 (counting from the 5 end of the miRNA). From this point of view, the evolution of GRFs in Rosaceae is highly conservative. We have identi ed a series of differentially expressed miRNAs that have been predicted to be responsive to low-dose UVB. To identify the expression pro les of miRNA396, we utilized transcriptome data of Illumina RNA-Seq reads that were generated and analyzed by Li et al [34]. We found that, in peach, miR396 was down-regulated after UVB irradiation, but this did not reach statistical signi cance. Also, we isolated another mirR319, which regulates TCP4, induces miR396, and represses GRF activity, which was also non-signi cantly downregulated following UVB irradiation [20] (Additional

Analysis of cis-elements in the promoter sequences of PpGRF genes
To further explore the involvement of the PpGRF genes in light responsiveness, hormone signaling pathways, plant growth and development, their promoter sequences were analysed using PlantCARE software [42]. All cis-elements in the promoter regions of the PpGRF gene family members are shown in Fig. 7. There are at least six commonly occurring light-responsive elements (LREs): AE-box, ATCT-motif, Gbox, GT1-motif, GATA-motif and I-box, which have been demonstrated to be essential for the regulation of light mediated transcriptional activity [43][44][45]. The results indicated that all 10 PpGRF promoter regions contained two or more LREs among which G-box is the most abundant element (Additional File 3: Supplementary Table S3). In this area we quali cated many other important potential cis-elements, such as GARE motif and P-box (GA responsive element), a CAT-box (cis-acting regulatory element related to meristem expression), AuxRR-core and TGA-element (auxin-responsive element), an RY element (seedspeci c regulation) and a TC-rich repeats (defense and stress responsiveness) [46][47][48][49][50][51]. GARE, P-box, CATbox, AuxRR-core, TGA-element, RY element and TC-rich repeats were distributed within 3, 3, 7, 2, 3, 1 and 5 PpGRF promoter regions, respectively (Additional File 4: Supplementary Fig S1).

Expression pattern of GRF genes in peach
Although we identi ed GRF genes in the peach genome, the functions of these genes remain largely unknown. To sum up, GRFs were highly expressed in growing tissues [8]. Next, we studied the patterns of expression about the PpGRFs in root, stem, leaves, the shoot tip and hypocotyl. Except for PpGRF8, these genes were differentially expressed in different tissues (data not shown). Most of these were upregulated, especially in the shoot tip and root (Fig. 8). For example, the expression levels of PpGRF2, 3, 4, 5, 6, 7, 9, and 10 were the highest in the shoot tip. However, PpGRF1 showed relatively strong expression in the root. Of the analyzed genes, PpGRF3, PpGRF9, and PpGRF10 were more preferentially expressed in the shoot tip; more than 20 times greater than that in other tissues. To further investigate the potential functions of PpGRFs, we also evaluated the effects of GA 3 and UVB on the expression of the PpGRF gene ( Fig. 9). In abiotic stresses, over half of the total genes were up-regulated, with PpGRF1, 4, 5, 6, 7, and 10 being up-regulated after GA 3 treatment; PpGRF2, 3, 4, 5, 6, and 7 were up-regulated after UVB treatment.
On the other hand, several PpGRF genes (including PpGRF2, PpGRF6, and PpGRF7) were suddenly downregulated at 9 h after GA 3 treatment (Fig. 9). Also, PpGRF5, PpGRF9, and PpGRF10 were suddenly downregulated at 6 h after UVB treatment. Based on the expression patterns of PpGRFs, we propose that they likely have multiple functions in regulating growth and responsiveness to abiotic stresses.

Discussion
Growth regulators are one of the most important plant-speci c transcription factors ,they play a vital role in plant growth and development, especially in regulating organ size [14,18,52]. The GRF gene families in model plants (Arabidopsis, rice, soybean, and poplar) have been identi ed and described in detail, but little is known about the GRFs in Rosaceae plants [8,9,11,39]. In order to have a better understanding of the characteristics and functions of PpGRFs, we studied PpGRF family members by bioinformatics assay with other three Rosaceae plants. Meanwhile, by performing qRT-PCR analysis, plant morphology analysis and hormone content determination to analyze the function of PpGRF. In the current study, we identi ed 46 GRF members in four representative Rosaceae plants, and found that the number of GRFs in apples is relatively large, perhaps due to a genome-wide replication event in the apple genome [53,54]. Phylogenetic analysis showed that the four species of Rosaceae GRF could be divided into six groups (I-VI) (Fig. 1), which is consistent with the classi cation of Arabidopsis, soybean, and rice GRFs [8,9,11]. The homologous pairs of GRF proteins in the four Rosaceae plants are more common. According to the topology of the phylogenetic tree, we found that some ancestral GRFs existed before the evolutionary divergence of the Rosaceae. By analyzing the GRF domains, we found that all of the speculated GRFs contain QLQ and WRC domains, and the VI subfamily contains two WRC domains. Similar results also exist in the Arabidopsis genome but not in the rice genome, suggesting that a major expansion occurred after the eudicots diverged from the monocots and before the separation of Rosaceae (Additional File 5, Supplementary Table S4). The structures of GRFs belonging to different subgroups in intron and exon structures showed low homology. GRFs of the same subfamily have the same or similar genetic structure, especially the number and length of exons. Also, based on our MEME analysis, each subfamily contains distinct motif arrangements. The differences in these characteristics of subfamilies suggest that GRF members are functionally diverse, which also supports the classi cations used in this study [8,9,11,39]. The expansion of the GRF family has been achieved mainly through gene duplication, especially largescale duplication (WGD or segment duplication) [11,39]. In this study, three pairs of PpGRF genes were distributed in duplication blocks, which means large-scale, repeats that promote the ampli cation of the GRF gene family in peach. We also found that the Ka/Ks ratios of all the PpGRFs gene pairs were less than 1, indicating that they have experienced strong purifying selection.
The excessive vigor of fruit trees will lead to the growth of new shoot, which is not conducive to the storage of assimilates, ower bud differentiation, or the balance between vegetative and reproductive growth [55]. This is one of the critical prevention and control goals in fruit tree cultivation management technology.
Here we found that during the growth period of new shoot, UVB supplementary light treatment can signi cantly inhibit the growth of new shoot, but has little effect on the changes in the thickness of new shoot. Perhaps UVB inhibits the proliferation of cells in developing leaves. The shoot tip is the growth point of the extension of the new shoot, and its hormone content determines the growth of the new shoot. UVB treatment can signi cantly reduce the content of IAA and increase the content of GA 3 in the shoot tips. Therefore, we infer that the levels of GA 3 and IAA in the shoot tips inhibit the growth of the new shoot to a certain extent, and thus regulate tree potential. Studies in maize indicate that the inhibitory effect of UVBs on growth is mediated by GRF, some of which might be regulated by miR396, thereby modifying GA levels in the leaf growth zone [29,30,56]. To further verify the above hypothesis, we tested the expression of miR396. Under UVB treatment, miR396 maintained a low level of expression in the function leaves and was slightly down-regulated. Unlike in Arabidopsis, miR396 was not highly expressed in the leaf primordia, and then it accumulates gradually with the development of Arabidopsis leaves [26].
At the same time, mirR319 expression was also down-regulated (although not reaching statistical signi cance). Probably we test the miRNA in the mature leaves, Most of the GRF genes are strongly expressed in the active growth and development tissues, such as shoot tip, root and ower bud, but the mature stem and leaf tissues are weakly erpressed [8,18].
We further explored the expression patterns of GRFs. First, qRT-PCR was used to detect the expression of PpGRFs in various tissues. The spatial expression pattern of PpGRF gene has some important similarities with other plants. Several studies have reported that GRFs transcription levels decrease as organs age. The expression levels of PpGRF2, 3, 4, 5, 6, 7, 9, and 10 in the shoot tip meristem was higher than that in mature tissues, suggesting that these genes play a crucial part in the elongation of the new shoot [57]. PpGRF1 was more highly-expressed in root than in the other tissues, indicating that PpGRFs is also important in root growth [58][59][60]. A well-documented effect of UVB exposure on plants is the reduction of biomass, and miR396/GRFs exerts its effect on organ growth at least in part by regulating the expression of genes related to GA metabolism [25,27,28]. Our study demonstrates that PpGRFs can widely respond to UVB and GA 3 treatments at transcriptional levels, with almost half of the members showing induction and a few showing repression.
Notably, most PpGRFs showed a response peak after 9 h of GA 3 treatment, but the response peak after UVB treatment appeared after 6 h. The analysis of cis-elements in the PpGRF promoter sequences found that some members contained multiple types of light responsive elements and GA responsive element, implying the involvement of PpGRFs in different light response and hormone signaling pathways. This shows that there are differences in how GRFs participate in these two signaling pathways. There is much evidence indicating the role of GRFs in various biological processes through interacting with different clients, such as hormone pathways, stomatal behavior, reactive oxygen species balance, and ion transport [61][62][63][64][65]. In Arabidopsis and maize, UVB radiation inhibits leaf growth by decreasing the expression of GRFs [29,30,56]. This ultimately leads to a reduced expression of GA biosynthetic genes and increased levels of catabolic transcripts [26,29,30,56]. In addition, the PpGRF promoter regions contain various types of cis-elements, which is helpful to explore the upstream genes of the PpGRFs involved in regulating growth and responsiveness to abiotic stresses. Overall, further study of the function of PpGRFs can reveal the behaviours of PpGRFs during regulate tree growth and development, providing real and reliable information for further studying of the biological functions of PpGRFs.

Conclusions
In this study, we identi ed 46 GRFs from four Rosaceae and investigated their phylogenetic classi cation, protein motifs, and gene structures. By further analysis, GRFs belonging to the same subgroup had similar gene structures and motif compositions. Chromosomal localization and collinearity analyses suggested that large-scale duplication contributed to the expansion of the PpGRF family. Physiological experiments and expression analyses have revealed the involvement of PpGRFs under UVB and GA 3 signaling and identi ed some important candidates for regulating the elongation of the new shoot, as well as in abiotic stress response. More research is needed to explore this hypothesis in more detail.This systematic study will advance the understanding of GRF-mediated signal cascades in regulating peach tree development and abiotic stress response. These results will provide a solid and reliable basis for further research on the GRFs in peach.

Plant material and treatments
The peach cultivars (Prunus persica var. nectarine Zhongyou No.5) used in this study were sourced from the city of Tai'an, China. All trees were planted for 7 years under standard horticultural practices and were completely productive. In the experiment, there were two treatment groups. The UVB treatments were carried out with 1.44 Kj m − 2 d − 1 UVB radiation, and the young leaves were sprayed with 100 µM of GA 3 for hormone treatment [16,34]. Leaf samples were collected at 3, 6, 9, and 24 h post-treatment. At each time point, we randomly collected 10 young leaves, which were immediately frozen in liquid nitrogen and stored at -80℃ until use. In the tissue expression pattern experiment, we collected samples of ve tissues, including root, stem, leaves, shoot tip, and hypocotyl, which were immediately frozen in liquid nitrogen and stored at -80℃ until use.

Sequence collection and identi cation
The four Rosaceae genome sequences were downloaded from the Genome Database for Rosaceae (GDR) (http://www.rosaceae.org/). To identify putative GRF genes from peach, apple, strawberry, and European pear, initially, the HMM pro les of the GRF domains (PF08879 and PF08880) downloaded from Pfam (http://pfam.xfam.org/) were used to search the database using HMMER3.0 (http://hmmer.janelia.org/). The NCBI CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and SMART (http://smart.embl-heidelberg.de/) were also used to determine the PF08879 and PF08880 domains of GRF, and sequences that lack similar integrity are deleted. Finally, they used the ExPASy proteomics server database to predicted their molecular weights and isoelectric points (http://expasy.org/).

Phylogenetic analysis and sequence alignment
We used full-length GRF protein sequences (from four Rosaceae species, Arabidopsis, soybean, rice, and poplar) to construct a phylogenetic tree. MEGA 7.0 software was employed to construct phylogenetic trees using the neighbor-joining (NJ) method, and the bootstrap test was replicated 1000 times [66]. The conservative motifs were predicted by the MEME online program (http://meme-suite.org/), in which the maximum value of the motif was set to 10. The intron-exon arrangements were determined by aligning its cDNA sequence with the genomic sequence by TBtools software package. Then, the TBtools software package was used to integrate these analyses [67].
Chromosomal location and collinearity analysis MCScanX (http://chibba.pgml.uga.edu/mcscan2/) was used to analyze the WGD/segmental, tandem, proximal, and dispersed duplications to investigate the mechanisms mediating GRF gene family evolution. Based on the statistics of MCScanX, the Ka, Ks and Ka/Ks of duplicated genes were calculated using the KaKs_Calculator 2.0 software [68]. TBtools was used to conduct local analysis of Synteny of four Rosaceae genomes and locate the genes to the chromosomal positions [67,69]. The RNA-seq data were obtained from our laboratory. The datasets supporting the conclusions of this article were included with in the article and its Supplementary les.

RNA extraction and quantitative RT-PCR analysis
Mechanism (ZR2018MC023). These fundings provided the nancial support to the research projects, but did not involve in project design, data collection, analysis, or preparation of the manuscript.