Genome-wide Characterization of GRAS Transcription Factors and Their Potential Roles in Development and Drought Resilience in Rose (Rosa chinensis)

In the past few years, plant-specic GRAS transcription factors (TFs) were reported to play an essential role in regulating several biological processes, such as plant growth and development, phytochrome signal, arbuscular mycorrhiza (AM) symbiosis, environmental stress responses. GRAS genes have been thoroughly studied in several plant species, but unexplored in Rosa chinensis (rose). In this study, 59 rose GRAS genes (RcGRAS) were identied. Phylogenetic analyses grouped RcGRAS genes into 17 subfamilies, of which subfamily Rc2 was Rosaceae family-specic. Gene structure analyses showed that most of the RcGRAS genes were intronless and were relatively conserved. Cis-element analyses suggested that RcGRAS genes may involve in distinct biological processes and responsive to diverse abiotic stresses. Most of the genes were localized in the nucleus, except for a few in the cytoplasm. Gene expression analysis was also performed in various tissues, during gibberellin (GA) and drought stress treatment. The expression patterns of RcGRAS genes during GA treatment and in response to drought stresses suggested the potential functions of these genes in regulating stress and hormone responses. In summary, a comprehensive exploration of the rose GRAS gene family was performed, and the generated information can be utilized for further functional-based studies on this family.

However, a combined phylogenetic analysis using eight different angiosperm species, regrouped GRAS genes into 17 different subfamilies (Cenci and Rouard, 2017).
Several GRAS TFs have functionally characterized in plants for their functional role during growth, developmental and signal transduction processes. For instance, two GRAS genes SCR and SHR form complex and play a key role in asymmetric cell division and radial root patterning by differential expression in the quiescent centre and stelar tissue (Nakajima et al., 2001;Sabatini et al., 2003). The SCR and SHR genes were also involved in osmotic and salinity stress regulation (Bolle, 2016). DELLA, a major class of GRAS genes play a substantial role in GA signaling in a negative manner (Achard and Genschik, 2009;Davière and Achard, 2013;Fukazawa et al., 2014). GAI and RGA are degraded via the E3 SCFSLY complex in the presence of GA. Degradation of DELLA activates the downstream genes positively involved in plant growth. DELLA gain of function mutant showed GA insensitivity and resulted into dwarf phenotype and vice-versa (Cheng et al., 2004;Wild et al., 2012). In addition to GA signalling, DELLAs were also involved in abscisic acid (ABA), auxin, brassinosteroids (BRs) and cytokinins (CKs) signalling, thereby regulating a range of growth and developmental processes in plants (Davière and Achard, 2016;Dolgikh et al., 2019). Genes including HAM, LAS, and MOC1 are reported to develop and maintain shoot apical and axillary meristem (Greb et al., 2003;Stuurman et al., 2002). In rice, MOC1 is mainly expressed in the axillary buds and regulates tillering process (Tong et al., 2009). Similarly, another gene in rice, GS6 belongs to DLT subfamily of GRAS TFs negatively regulates grain size (Sun et al., 2013). GRAS genes also found to be involved in AM symbiotic association and phytochrome A signal transduction. For example, RAM1 (GRAS gene) was found as a master regulator for AM symbiotic association (Hartmann et al., 2019;Pimprikar et al., 2016). Phosphorylate CYCLOPS interact with RAM1 as well as DELLA to induce hyphopodia formation. More recently, GRAS TF, NSP2 (AhNSP2) was shown to control the nodulation in cultivated peanuts (Peng et al., 2021).The PAT1 and SCL21 GRAS TF members considered positive regulators of phytochrome A signal transduction in plants (Bolle et al., 2000;Muntha et al., 2019;Torres-Galea et al., 2013).
Rose, the most important ornamental crop, has high demand in different industries like perfumery, cosmetics and pharma, etc. Being small size genome (560Mb) and diverse ploidy (2x-10x), the rose has been considered as a model plant for evolutionary and polyploidization studies (Zhang et al., 2013). After the release of whole genome sequences (Nakamura et al., 2018;Raymond et al., 2018;Saint-Oyant et al., 2018), a genome-wide study for identifying important gene families have been taken place in rose in the last two years. Some important examples include the identi cation of AP2/ERF , WRKY (X. , Rdr1 (Menz et al., 2020), R2R3-MYB (Han et al., 2019) gene families and their potential role in different biological process i.e., growth and development, phenylpropanoid pathway, abiotic stress tolerance, and tolerance against biotic stresses like Botrytis, gray mold and black spot. However, a comprehensive study of rose GRAS genes is still lacking.
This study identi ed 59 GRAS genes in rose using the latest rose genome database (Saint-Oyant et al., 2018). We performed phylogenetic, conversed motif, gene structural (exon/introns) and cis-acting regulatory elements analyses of RcGRAS genes. The comparative analysis for the GRAS genes from rose, Arabidopsis and rice was also conducted. Furthermore, expression pro les of the RcGRAS genes were investigated in different tissues and responses to GA and drought stress treatments using qRT-PCR. Altogether, our study provided some insights that could be used in future functional validation of RcGRAS genes and may be bene cial for rose improvement.

Plant materials, gibberellin and stress treatments
The rose genotype belong to Rosa chinensis were used in this study. Uniform growing plants in plastic pots containing a 1:2:1 soil/peat/sand mix and in greenhouse conditions (photoperiod =14h light/10hdark and temperature = 22 ± 2°C) were used for expression pro ling of RcGRAS genes. The leaf, stem and ower buds samples were collected from two pots (considered as two biological replication) for tissue-speci c expression patterns analysis under normal conditions. Gibberellin (GA) treatment was given according to Randoux et al. (Randoux et al., 2012) with slight modi cations. Rose plants were sprayed once with GA 3 (30 μM) and maintained for one-week and then plants were pruned. Subsequently, the pruned plants were sprayed with GA 3 (30 μM) three times a week (on alternative days) for 34 d. After treatment, the leaf, ower bud, and stem tissues were collected (two biological replications). Drought stress (DS) was imposed according to Niu and Rodriguez (Niu and Rodriguez, 2009), the irrigation was withheld for 30 days. The DS was determined through visual stress sign (wilting). The leaf tissues were collected (two biological replications) from control and DS treated plants. All samples were collected in liquid nitrogen and stored at −80 °C till further use.

Identi cation of GRAS family genes in rose
To identify the rose GRAS family genes, hidden markov model (HMM) pro le of the GRAS domain (PF03514) was downloaded from the Pfam database (Mistry et al., 2021). The HMM le was used to examine all protein sequences in the rose genome using Rosa chinensis Homozygous Genome v2.0 (Saint-Oyant et al., 2018). Domain composition analysis was performed using NCBI-conserved domain database (Marchler-Bauer et al., 2012) and SMART server (Schultz et al., 2000). Molecular weight (MW), isoelectric point (pI), and an amino acid number of rose GRAS proteins were determined using the ExPasy program (http://www.expasy.org/).

Exon-Intron and conserved motif analysis
The gene structures of rose GRAS TFs were illustrated using TBtools (Chen et al., 2020) with the GFF3 le of the rose genome. MEME v5.1.1 suite (Bailey et al., 2009) was used to identify conserved motifs of rose GRAS proteins with default parameters and the maximum number of motifs was 10. The MEME-motifs and Seq Logos were visualized on TBtools.

Phylogenetic analysis and classi cation of RcGRAS genes
The GRAS protein sequences of rose (59), Arabidopsis (33) available at TAIR database (https://www.arabidopsis.org/), rice (52) available at Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/), strawberry (54) available at GDR database ( https://www.rosaceae.org/projects/strawberry_genome/tools ) were aligned using MUSCLE program available at MEGA vX (Stecher et al., 2020). The phylogenetic tree was built with MEGA vX software's help using Poisson model, pair-wise deletion, and neighbour Joining (NJ) algorithm with 1000 bootstraps. The RcGRAS proteins were also divided into different sub-families based on their grouping with characterized Arabidopsis and rice GRAS proteins.

Cis-acting regulatory elements (CARE) analysis of RcGRASs
The upstream 2 kb sequences of the RcGRAS genes were fetched from the Rosa chinensis Homozygous Genome v2.0 database. The fetched sequences were then submitted to the PlantCARE server (Lescot et al., 2002) to identify the conserved CAREs.

RNA isolation and qRT-PCR analysis
Total RNA was isolated using PureLink TM RNA Mini Kit (Invitrogen) as per manufacturer protocol. The Verso cDNA Synthesis Kit (Thermo Scienti c) was used to convert total RNA to cDNA. Quantitative RT-PCR (qRT-PCR) was conducted using SYBR green master mix (Applied Biosystems) in StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, USA). The primers of the selected RcGRAS genes were designed using Primer Express ® Software ver3.0 (Applied Biosystems) and listed in Table S2. The housekeeping gene RcActin was used as internal control. Each reaction was conducted with three technical replications. Relative expression of genes were measured using the 2 −ΔΔCt method (Livak and Schmittgen, 2001

Genes encoding GRAS transcription factors (TFs) in the rose genome
A total of 59 genes that encoding GRAS TFs were identi ed from the rose genome. The basic descriptions of all RcGRAS TFs were listed in Table 1 and Table S3, including the gene nomenclature, chromosomal position, coding sequence (CDS) length, the protein length, the protein MW, theoretical pI, conserved domains, the predicted subcellular localization. All of these 59 RcGRAS proteins had a conserved GRAS domain (PF03514); however, three RcGRAS members (RcGRAS7, RcGRAS23 and RcGRAS25) also possessed a DELLA domain (PF12041). The protein length and molecular mass of the RcGRAS TFs showed enormous variations, with lengths ranged from 160 to 832 amino acids, molecular weights ranged from 19.08 to 93.79 kDa and isoelectric points ranged from 4.63 to 9.62 (Table 1). We named these GRAS candidates genes from RcGRAS1 to RcGRAS59 based on their coordinate position (from top to bottom) on seven different rose chromosomes.

Phylogenetic and syntenic analyses of GRAS genes
To identify the evolutionary relationships of the GRAS TFs in rose and to classify them into different subfamilies, a total of 198 full-length GRAS proteins, comprising 59 from rose, 33 from Arabidopsis, 52 from rice, 54 from strawberry, were used to construct a phylogenetic tree (Fig. 2). The GRAS proteins of the four species were grouped into 17 different subfamilies. Out of 17, 16 subfamilies (PAT1, LISCL, SCL3, SCL32, DELLA, SCR, DLT, HAM, LAS, SCL4/7, SHR, SCLA, RAD1, RAM1, NSP1, NSP2) were designated according to previous studies (Cenci and Rouard, 2017;Sidhu et al., 2020;Tian et al., 2004). A remaining Rosaceae-speci c subfamily (contains only rose and strawberry GRAS proteins) which is not grouped into any of the earlier reported subfamilies was named as Rc2 subfamily. It suggests that Rc2 subfamily had been gained in the rose and strawberry lineage after divergence from the most recent common ancestors (Arabidopsis and rice). Further analysis revealed that LISCL was the largest subfamily which accounted for 29% (58) of the total GRAS genes, while RAM1 and SCLA were the smallest subfamilies and each consist of three members (Fig. 2). It was also observed that in all clades strawberry GRAS were closer to rose GRAS proteins than other two species (Arabidopsis and rice) used in this study.
To further understand the evolutionary traces for the GRAS genes, we identi ed orthologous genes between rose and Arabidopsis and rose and rice. Total 26 and 20 GRAS orthologous gene pairs were identi ed between rose/Arabidopsis and rose/rice, respectively ( Fig. 3 and Table S4). We observed that each Arabidopsis GRAS gene had one to three rose orthologous genes, for instance AT5G59450 showed an ortholog relationship with the three rose genes (RcGRAS12, RcGRAS14 and RcGRAS38), it showed that some GRAS TFs in rose underwent duplication events. More number of orthologous genes between rose/Arabidopsis as compared to rose/rice emphasized the close relationship of rose and Arabidopsis. We also calculated K a /K s (non-synonymous substitution/synonymous substitution) to examine the molecular evolution of orthologous GRAS genes (Table S4). We found that most of the orthologous gene pairs had K a /K s < 1, indicating that the GRAS genes underwent purifying selection during the evolution.

Motif con gurations and gene structure analysis of RcGRAS Family
Motif composition analysis of RcGRAS proteins showed that most of the motifs were located C-terminus as compare to N-terminus (Fig. 4). In general, it was also observed that RcGRAS protein belongs to a similar subfamily had similar motif composition and orders. For instance, the LISCL subfamily contained all the ten types of motifs. Motifs 3, 9 and 10 were not found in HAM, motifs 3 and 10 were not found in NSP2 and LAS, motifs 8 and 10 were not found in DLT, motifs 6 and 7 were not found in RAD1 and motifs 2 and 9 were not found in Rc2 (Fig. 4 and Table S5). Further, motifs 1, 4, and 5 were found in all the 17 GRAS subfamilies, advocating signi cant roles in the GRAS genes' conserved function in rose. The variations in motif number and localizations on GRAS subfamilies suggested that GRAS proteins' function likely diverged during its expansion in plants.
Exon-intron pattern analysis of rose GRASs showed that RcGRAS genes have one to three exons. Most of the RcGRAS (48, 81.3%) do not have introns and only 11 (18.7 %) genes with 1-2 introns (Fig. 4). Generally, members of the same subfamily had similar gene structures and domains. For example, RcGRAS7, RcGRAS23 and RcGRAS25 belonged to the DELLA subfamily, and each of them consists of single exon and DELLA domain ( Fig. 4 and Table S3).

Cis-acting regulatory elements (CARE) in GRAS genes
The cis-acting regulatory elements (CAREs) play an important role in the transcriptional regulation of the genes. We also explored for the CAREs in the upstream sequences (2 kb) of identi ed RcGRAS genes. In total 47 types of CAREs were identi ed in the promotor region of RcGRAS genes (Fig. 5). These CAREs were classi ed into four categories according to their biological function, including (1) stress response elements, (2) development-related elements, (3) light-responsive elements, and (4) hormone response elements. Among these CAREs, the stress-responsive elements were most abundant (1175) and included ARE, CAAT, DRE-core, F-box, LTR, MBS, TC-rich repeats, W box, GC-motif. Stress-responsive elements were followed by light response (571), hormone-related (541) and development-related (74) elements (Fig. 5).
These results indicated that RcGRAS genes diversely respond to various abiotic and biotic stresses and might regulate different biological processes.

NLS prediction and sub-cellular localization
Generally, the intracellular distribution of TFs dynamically trades between the nucleus and cytoplasm. The NLS at the C-terminal of various TFs are required to regulate this localization (Heerklotz et al., 2001). The NLS prediction in the GRAS TFs revealed that out of 59 rose GRAS TFs, 31 members have either bipartite or monoparite or both types of NLS (Table S3). For instance, 25 GRAS TFs were predicted with bipartite NLS. Monopartite NLS was predicted in ve GRAS TFs (RcGRAS24, RcGRAS25, RcGRAS26, RcGRAS24, RcGRAS58 and RcGRAS59). One GRAS TFs (RcGRAS56) was predicted with both NLSs (Table S3). Further, subcellular prediction of rose GRAS TFs showed that most of these were con ned into the nucleus or cytoplasm. Forty-four RcGRAS genes were located in the nuclei and the remaining 15 were located in the cytoplasm (Table S3).

MicroRNA target analysis
According to earlier studies, GRAS TF members are regulated by miRNA171 in plants (Guo et al., 2019;Ma et al., 2014). Thus, we searched for putative target sites of miR171 on rose GRAS TFs. Our results showed that two RcGRAS genes (RcGRAS4 and RcGRAS57) belonging to HAM subfamily has targeted by miR171 ( Fig. 1 and Fig. S1).

Tissue-speci c expression of RcGRAS genes
Expression pro ling of total 22 GRAS genes were conducted on three different tissues including leaf, stem and ower bud. These genes belonged to 13 different subfamilies of GRAS. We observed different expressions the genes belonged to the same sub-family (Fig. 6). For instance, three genes (RcGRAS57, RcGRAs9, RcGRAS4) of subfamily HAM showed a different pattern of expression in three tissues. No signi cant differences were observed in expression for gene RcGRAS57 in leaf, stem and ower bud, however, RcGRAS9 signi cantly upregulated in stem than the leaf and bud, and in the case of RcGRAS4 signi cant downregulation was observed in ower bud as a comparison to leaf. Similarly, in case of DELLA, three genes (RcGRAS7, RcGRAS23, RcGRAS25) showed different expression patterns in three tissues. No expression difference was observed for RcGRAS23; however, RcGRAS7 and RcGRAS25 signi cantly down-and up-regulated in stem tissues respectively. RcGRAS30 of SCL3 subfamily showed 20-and 18-times higher expression in stem and ower bud compared to leaf, respectively; however, RcGRAS1 of SCL3 signi cantly down-regulated in stem and ower bud (Fig. 6).

Expression pro le of RcGRAS genes to exogenous GA treatment
Under GA treatment, downregulation was observed for most of the GRAS genes (Fig. 7A). We examine the expression of 11 GRAS genes under GA treatment in three tissues leaf, stem and ower bud. These genes belonged to seven sub-families. No expression was detected for GRAS genes belonging to four subfamilies, including DELLA, LISCL, HAM, and SCL32 in any tissue under exogenous GA treatment. In the case of RcGRAS1 (SCL3), signi cant downregulation was observed in stem and ower bud under GA treatment; however, GA treatment did not affect the RcGRAS1 expression in leaf. Different expression patters were observed in case of two genes (RcGRAS6 and RcGRAS16) of PAT1 sub-family. RcGRAS16 was signi cantly down regulated under GA treatment, however, RcGRAS6 was signi cantly upregulated in leaf, downregulated in stem under GA treatment. No effect of GA was observed in the expression of RcGRAS3 (Rc2) gene (Fig. 7A).

Discussions
GRAS genes are reported to play various role in plant growth and development, phytohormone and phytochrome signaling, AM symbiosis and stress responses (Bolle, 2004;Davière and Achard, 2016;Fode et al., 2008;Greb et al., 2003;Guo et al., 2019;Heckmann et al., 2006;. They are hence promising targets that could be used for plant and crops species improvement. GRAS gene family has been identi ed and characterised in more than 30 plant species (Table S1); however, associated studies on the rose GRAS gene family have not been performed. We identi ed and characterized 59 GRAS TF family members in the rose genome. All the identi ed RcGRAS genes were randomly distributed on seven rose chromosomes (Chr1-Chr7) (Fig. 1). Markedly, Chr5 had the highest number of genes (25 genes), which were mainly belong to LISCL (19), PAT1 (3), SCL3 (1), RAD1 (1) and LAS (1) subfamilies. The number of GRAS members in rose is higher than the Arabidopsis (33 members), cabbage (35 members) and sacred lotus (38 members), close to that in barrel clover (59), strawberry (54), and pepper (50), and less than that in mustard (88), rapeseed (92), soybean (117), cotton (150), apple (127) for detail see Supplementary Table S1. This variation in GRAS gene numbers might be associated with genome size or gene duplication events during the evolution of a particular plant species (Grimplet et al., 2016).
Interestingly, LlSCL subfamily members in rose were found in a higher number as compared to other members, in total 35.6 % (21 of 59 genes) of GRAS genes belongs to the LISCL subfamily. This number is signi cantly higher than plant species, such as cotton (13%), tea plant (19%), castor beans (15%), Arabidopsis (21%) and rice (20%) but similar to strawberry (35%) belongs to family Rosaceae. These observations indicated an apparent expansion of LlSCL subfamily during the Rosaceae family's evolution.
The phylogenetic analyses showed that the GRAS proteins of rose, Arabidopsis (model eudicot plant species, rice (model monocot plant species), and strawberry (model plant for Rosaceae family) were grouped into 17 different subfamilies (Fig. 2). Interestingly, three of these subfamilies (SCLA, RAM1, and RAD1), do not have members in Arabidopsis; however, in the case of rice, members of these three subfamilies were available. These results suggested that these three subfamilies existed before the divergence of dicots and monocots, however, somehow lost in Arabidopsis. We also found one subfamily Rc2, appeared to be a Rosaceae-speci c family, because GRAS members belong to Arabidopsis or rice were not present in this family. Similar speci c families were also reported in other plant species, such as Rc-GRAS in castor beans (Xu et al., 2016), Pt20 family in populus and tomato (Huang et al., 2015;Liu and Widmer, 2014), G-GRAS in cotton (Zhang et al., 2018), and Fve39 in strawberry . The species-speci c families may be evolved from common ancestors but after the divergence of monocot and eudicot. To further explore the evolutionary relationships of GRAS members, we also carried out the synteny analyses. We observed that the number of orthologous gene pairs between rose and Arabidopsis were more than that between rose and rice (Fig. 3). It further suggested that rose is genetically closer to Arabidopsis as compared to rice.
As earlier studies showed that the GRAS family in the plant has been evolved from the prokaryotes via horizontal gene transfer (HGT) along with gene duplications (Zhang et al., 2012). Results of gene structure analysis of RcGRAS genes showed that most (81%) of them were introless (Fig. 4), which is a typical feature in prokaryotes (Roy and Penny, 2007). It suggested that RcGRAS genes originated from the prokaryotes via HGT. Similar results were also observed in other species such as Arabidopsis (Tian et al., 2004), soybean (L. , orange , buckwheat (M. , maize (Guo et al., 2017) and cassava (Shan et al., 2020), where, most of the GRAS genes lack introns (60-80%) or have only single intron (30-40%). But, some of the LISCL members (5) in rose had introns suggested the recent evolution of LISCL subfamily in rose.
Further, our NLS prediction and subcellular localization analyses showed that most of the RcGRAS genes had either bipartite or monoparite or both, and are localized in the nucleus or cytoplasm (Table S3). These results are in agreement with earlier studies (Guo et al., 2019;Song et al., 2014;N. Wang et al., 2020) and suggested that the function of these GRAS genes may be conserved in plants.
Earlier, it was reported that miR171 post-transcriptionally regulates some HAM subfamily genes (Guo et al., 2019;Ma et al., 2014). Interestingly, in the present study, two RcGRAS genes (RcGRAS4 and RcGRAS57) belonging to HAM subfamily were found to have a putative binding site for miR171 (Fig. S1).
The miR171-GRAS module has been reported to involve in regulation meristem development and maintenance in plants (Fan et al., 2015;Huang et al., 2017). Our results also indicated that miR171-GRAS module might also be involved in meristem development and maintenance in rose.
The CAREs are involved in the regulation of several biological processes in plants, i.e., growth, development and stress responses (Biłas et al., 2016). Promoter analysis of RcGRAS genes revealed that the CAREs associated with stress (ARE, CAAT, GC-motif, MBS, DRE-core, F-box, LTR, TC-rich repeats, W box), light response (MRE, ACE, AE-box, ATCT-motif, GATA, I-box, CAG-motif, BoxII) and hormone-related (ABRE, CGTCA-motif, ERE, P-Box, TATC-box, TCA-element) were broadly existed (Fig. 5). The presence of a similar type of CAREs in the promoter region of GRAS genes in other plant species has also been documented Shan et al., 2020;. These results indicated that RcGRAS genes may also be involved in various stresses and regulation of different biological processes in rose.
To explore the expression patterns of RcGRAS genes in different tissues, we examined the relative expression of GRAS genes in leaf, stem, and ower bud, and identi ed the tissue speci c expression of GRAS genes, which suggested the possible involvement of GRAS gene in different growth and development processes in case of rose also. For instance, RcGRAS9 (HAM) showed signi cantly higher expression in stem tissue. HAM protein is well known for the maintenance of indeterminate shoot growth in owering plants (Engstrom, 2012). The interactions between HAM, Wuschel (WUS) and CLAVATA3 (CLV3) maintain the indeterminate stem growth (Engstrom, 2012). Further, DELLA proteins are the negative regulator of GA signaling. In presence of GA, DELLA degraded and resulted into activation of downstream genes involved in plant growth particularly stem elongation. It has been shown that DELLA form a complex with IDD (DELLA/IDD complex) and upregulate the SCL3 (a positive regulator of GA) (Yoshida et al., 2014). During present study, signi cantly downregulation of RcGRAS7 (DELLA) and upregulation of RcGRAS30 (SCL3) in stem and ower bud as comparison with the leaf were good concurrence with earlier reports, and suggested possible interaction of DELLA and SCL3 in case of rose also. In Arabidopsis, SHR/SCR regulatory complex has been characterized for the development of endodermis in shoot, root and leaves (Cui et al., 2007;Dhondt et al., 2010;Yoon et al., 2016). During the present study, the similar expression pattern of RcGRAS19 (SCR) and RcGRAS11 (SHR) in three tissues of rose also support the formation of a regulatory complex of SHR/SCR and their role in plant development and growth. During the present study, different expression patterns of GRAS genes of the same or other family suggested the multitasking role of GRAS genes in plant growth and development in the case of rose also.
GA is an important phytohormone that promotes seed germination, elongation of root and shoot, fruit ripening, etc. GRAS genes are well characterized to be involved in GA signaling in different plant species and act as a repressor of GA response in plants (Park et al., 2013;Tyler et al., 2004). Application of exogenous GA led to downregulation of GRAS genes particularly DELLA, subsequently activation of downstream genes involved in plant growth. In Brachypodium, the expression of most of the GRAS genes declined to zero under exogenous GA treatment (Niu et al., 2019); however, some GRAS genes showed different expression in different tissues. Similarly, in Citrus sinensis, most GRAS genes downregulated under GA treatment . In the present study, expression analysis of GRAS genes under GA treatment suggested the involvement of RcGRAS in GA regulatory pathways. Expression of GRAS genes of four sub-families, including DELLA, LISCL, HAM, and SCL32, declined to zero. This emphasized the important role of these GRAS genes in GA signaling and regulatory pathways. Some RcGRAS (RcGRAS1 and RcGRAS6) showed differential expression patterns in different tissue suggested the speci c role of GRAS genes in different tissues. Expression of the rose speci c GRAS gene (RcGRAS3) did not alter in any of the tissues, indicating that RcGRAS had some other role in plant growth than GA signaling.
Further, we have also observed a signi cant difference in RcGRAS genes' expression under drought stress, which suggested a promising role of GRAS genes in drought stress signaling in rose. Earlier studies also directed GRAS genes' involvement in various abiotic stresses (Liu et al., 2017;Mayrose et al., 2006;Xu et al., 2015). In Soybean, the majority of GRAS genes were up-regulated under drought stress; however, few (18 out of the 52) GRAS genes were repressed under drought stress suggested a promising and dynamic role of GRAS genes in providing stress tolerance. During the present study, surprisingly, none of the ve gene showed higher expression under drought stress. One possible reason might be that consideration of very few genes during the present study for expression pro ling under drought stress and we might lose the information of some potent genes which actually upregulate. Second, down-regulation of these four genes might play an important role in the activation of downstream genes for providing tolerance to plant under drought stress conditions.

Conclusions
In total, we systematically identi ed 59 GRAS genes in rose using the recently available rose genome information. These RcGRAS genes were randomly distributed on seven rose chromosomes. The identi ed RcGRAS genes were further categorized into 17 subfamilies based on their phylogenetic grouping with Arabidopsis and rice. Gene structure and motif pattern analyses indicated the GRAS members belong to the same subfamily exhibited similarities, which may specify their parallel gene functions. It was also observed that most RcGRAS genes lack introns, suggesting that the RcGRAS gene structures were prominently conserved. Moreover, CARE and expression analyses suggested their prevalent role in regulations of plant growth and development, GA and drought stress signaling. This study could assist the future function validation of GRAS genes and be utilized in rose improvement programs.

Declarations
CRediT authorship contribution statement

Declaration of competing interest
The authors report no declarations of interest. Zhang, S., Li, X., Fan, S., Zhou, L., Wang, Y., 2020    The schematic represent the motif patterns of the RcGRAS proteins. Different colored boxes indicated the ten different MEME-motifs. The sequence information for each MEME-motif in the form of sequence LOGO was also provided.   Tissue-speci c expression pro les of 22 selected RcGRAS genes in leaf, stem and ower bud tissues. Yaxis represents the relative expression value. Bars represent the mean values of two biological and technical replicates ± SE. Lowercase alphabets represent signi cant difference (p <0.05, Student's t-test). Differential expression detected for RcGRAS genes in response to GA treatment (A) and drought stress treatment (B) in different rose plant tissues. Untreated tissue was considered as control (C) to determine relative fold change. Y-axis represents the relative expression value. Bars represent the mean values of two biological and technical replicates ± SE. Asterisks (*) marks on the bars represent signi cantly up-or down-regulation as compared to control (P < 0.05, Student's t-test). CL, leaf tissue of control sample; CS, stem tissue of control sample; CB, ower bud tissue of control sample; GAL, leaf tissue of GA treated sample; GAS, stem tissue of GA treated sample; GAB, ower bud tissue of GA treated sample; DS, leaf tissue of drought stress treated sample.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. SupplymenteryTables.docx