Genome-Wide Identification of Lettuce GRAS Gene Family Reveals That LsGRAS13 Is Negative Regulator of Thermally Induced Bolting in Lactuca sativa

Lettuce is susceptible to high-temperature stress during cultivation, which leads to bolting and affecting yield. However plant-specific transcription factors known as GRAS proteins play an important role in regulating plant growth development and abiotic stress responses. In this study, the whole LsGRAS genome of lettuce was identified. The results showed that 59 LsGRAS genes were distributed unevenly across nine chromosomes, 100% of which were located in the nucleus. Phylogenetically classified into nine conserved subfamilies. Chromosome localization and gene structure analysis suggested that duplication events and a large number of intronless genes may be the reason for the massive expansion of the LsGRAS gene family. To investigate the expression profiles of these genes in lettuce, we analyzed the transcription levels of all 59 LsGRAS genes in RNA-Seq data under high-temperature treatment with exogenous melatonin. We found that the expression level of LsGRAS13 was higher on 6, 9, 15, 18, and 27 days under high-temperature treatment with melatonin compared to the same treatment days without melatonin. Functional assays revealed that silencing LsGRAS13 resulted in accelerated bolting of lettuce, whereas the flower bud differentiation rate was faster in LsGRAS13 -silenced plants than in control plants. In this study, the LsGRAS gene was comprehensively annotated and analyzed. Meanwhile, the expression pattern of the LsGRAS gene under high-temperature treatment was deeply explored, which is of great significance for the response mechanism of plants to high-temperature stress and the improvement of high-temperature stress resistance of lettuce, and provides valuable information and candidate genes.


Problem
Lactuca (Lactuca sativa L.) is a kind of vegetable mainly cultivated in fields or facilities, but it is easy to suffer from high temperatures during cultivation, leading to the bolting of lettuce (Chen et al., 2017;Fukuda et al., 2011). High-temperature conditions can accelerate the process of bolting plants and limit the sale of lettuce (Han et al., 2021). Unfortunately, lettuce yields are susceptible to a variety of abiotic and biological stresses, such as high salinity, drought, high/low temperatures, and pathogen infections (Borowski et al., 2014;Boter et al., 2019). These pressures have led to severe declines in lettuce production in many parts of the world and limited its cultivation in different geographical areas Huot et al., 2014). Considerable research work has been carried out to gain insight into the biochemical, molecular, and cellular responses of lettuce plants to abiotic and biological stresses (Vanstraelen et al., 2012). Many lettuce transcription factor families, such as WKRY (Du et al., 2022), R2R3-MYB (Park et al., 2022), GRF (Zhang et al., 2021), and MADS (Ning et al., 2019), have been investigated and studied. However, there have been relatively few comprehensive studies on the GRAS gene family in lettuce. Because the GRAS gene plays an important role in plant development and physiological processes, it is urgent to explore and analyze related genes to fill in the research gap. GRAS gene family is a family of genes that play a regulatory role in plants, including a series of transcription factors. These genes play a key role in regulating growth and development, signaling, and stress response in plants. Although the function of other transcription factor families in lettuce has been studied, the GRAS gene family is relatively limited. By studying the GRAS gene family in lettuce, we can better understand the function of these genes in the growth and development of lettuce and its stress response. This will help us design more effective lettuce breeding strategies to improve the adaptability and stress resistance of lettuce.

Justification
GRAS proteins have been widely studied in the last decade and are plant-specific Ho-Plágaro et al., 2019;Fan et al., 2017;Liu et al., 2019;Sun et al., 2012). GRAS protein consists of 400~770 amino acid residues and can be divided into highly conserved carboxyl (C-) terminal region and variable amino (N-) terminal region (Bolle et al., 2004;Chen et al., 2019). The GRAS domain is mainly composed of five motifs at the carboxyl (C-) terminal, namely leucine heptapeptide Repeat sequence I (LHR I), leucine heptapeptide repeat sequence II (LHR II), VHIID, fire and SAW motif (Cenci et al., 2017). Notably, these five motifs play an important role in GRAS interactions with other proteins (Tian et al., 2004). In contrast, the N-terminal, although composed of chaotic domains involved in molecular recognition, exhibits a high degree of instability. The GRAS variable N-terminal can be folded and modified to form specific molecular binding structures (Sun et al., 2016). Based on this, GRAS protein is widely involved in many key processes of signal transduction, root radial elongation, axillary meristem formation, and stress response (Niu et al., 2017;Li et al., 2008;Jaiswal et al., 2022). The GRAS family has been reported to be endemic to more than 50 species of plant transcription factor (TF) (Bolle et al., 2004). To date, GRAS gene families have been identified and analyzed in more than 30 monocotyledon and dicotyledon plants, such as rice, maize, Arabidopsis, barley, rose, etc. Kumari et al., 2022). Previously, the Arabidopsis GRAS gene family was divided into eight subfamilies, including DELLA, SCL3, LAS, SCR, HAM, SHR, LISCL, and PAT1, based on conserved domains and functions (Hirsch et al., 2009;Liu et al., 2014;Sarwar et al., 2021).
Temperature plays an important role in the growth of lettuce, however, when the outside temperature reaches a certain level, the lettuce will experience heat stress. Heat stress (HS) is defined as an increase in temperature that exceeds a threshold level for a while and permanently affects plant growth and development (Wahid et al., 2020). It can disrupt the homeostasis of normal cells, leading to severe growth, developmental arrest, and even death of the organism. Although members of the CRAS gene family are important in helping plants resist heat stress, there has been relatively little research on the relationship between heat stress and the CRAS gene family.

Background
In this study, we identified 59 LsGRAS gene members from the lettuce genome and identified the phylogenetic relationships, gene structure, motif composition, chromosome location, and gene replication events of the LsGRAS members. In addition, we studied the expression pattern of the LsGRAS gene in lettuce under high-temperature stress and analyzed the cis-elements in the promoter region of the LsGRAS gene. Considering the importance of heat stress on gene expression in lettuce, we selected a representative LsGRAS13 gene for functional analysis. In conclusion, this study is of great significance for the LsGRAS gene to improve the high-temperature stress resistance of lettuce in the future.

Identification and Basic Information of LsGRAS
To identify GRAS genes in lettuce, the TAIR database (https://www.arabidopsis.org/) was used as a reference (Wang et al., 2020). After downloading the lettuce genome and genome annotation files from the Phytozome v12.1.6 database (https://phytozome.jgi.doe.gov/pz/portal.html), the CDS sequence and protein sequence of the CRBRx gene were extracted using TBtools (Wang et al., 2020), and homology retrieval was performed using BLASTp. To ensure the accuracy of the results, duplicate protein sequences were manually removed and an initial search was performed using BLAST conserved domain search method to identify protein sequences with domains. At the same time, the ExPASy tool was used to predict the molecular weight and isoelectric point of the protein sequence, which is very important for understanding the properties and functions of the protein. Finally, WOLF PSORT was used to predict the subcellular localization of the LsGRAS gene, which helps to reveal the position and function of the gene in the cell (Horton et al., 2007).

Phylogenetic Analyses and Classifications of the LsGRAS Proteins
By combining bioinformatics tools and image processing software, valuable information can be extracted from gene sequence data. The GRAS protein sequences of Arabidopsis and lettuce were compared using MEGA 7.0 software (https://www.megasoftware.net/), the Poisson model was used to estimate the evolutionary distance of the sequences, and the missing data processing method was used to deal with the missing data in the aligned sequences. To evaluate the reliability of the analysis results, 1000 times Bootstrap resampling was performed. The GRAS phylogenetic relationships of Arabidopsis and lettuce were obtained by constructing phylogenetic trees of GRAS proteins of Arabidopsis and lettuce using neighborhood linkage (NJ) (Xie et al., 2018). To present the results of the study, the phylogenetic tree images were modified and optimized using FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/) and Adobe Illustrator 2019 CC software to ensure clarity and readability.

Gene structures and Conserved Motif, Gene Structure, Phylogenic Tree Analysis of LsGRAS
In this study, TBtools was used to describe the gene structure of the lettuce genome GFF3 file, and MEME v5.1.1 (http://meme-suite.org/tools/meme) was used to analyze the conserved motif of the LsGRAS protein (Chen et al., 2020). Meanwhile, the phylogenetic tree of lettuce and Arabidopsis was constructed by the NJ method.

LsGRAS Gene Chromosomal Locations, Duplications and Synteny Analyses
According to the genomic information of lettuce obtained in Phytozome, the distribution of LsGRAS genes on each chromosome was determined by analysis, and the chromosomal location map was drawn. At the same time, the repetition patterns of the LsGRAS gene were studied and the repetition map was generated to reveal the gene repetition events in the lettuce genome, and the gene density information of each chromosome or scaffold was calculated. The study was designed to explore synergistic relationships between the homologous LsGRAS gene in lettuce and other species. To further understand this problem and compare the homologous LsGRAS genes in lettuce and other species, genome data and gene annotation files of Arabidopsis (TAIR annotation release 10) and Solanum lycopersicum (V1.1) were downloaded, and TBtools software was used to draw and construct synchronous analysis graphs (Chen et al., 2020).

Transcriptome Analysis for Stress Treatment
We analyzed the LsGRAS gene transcriptome data in lettuce leaves under high-temperature stress. Specifically, we conducted experiments on lettuce plants, in which the experimental group received exogenous melatonin treatment with a concentration of 100 μmol L -1 ; The control group was treated with 0 μmol L -1 melatonin. We also subjected the plants to heat stress . To observe the phenotypic changes of leaves at different time points, leaves that showed significant changes on 6, 9, 15, 18, and 27 days were selected for further study. We analyzed these leaves using RNA-Seq technology to see how the genes changed at the transcriptional level. By analyzing transcriptome data, we identified transcriptional changes associated with the LsGRAS gene under these conditions.

Cis-Element Analyses of LsGRAS Genes
Identification and analysis of gene sequence is an important step to reveal biological function in research. The upstream 2000 bp sequence of the LsGRAS13 gene was identified and submitted to the PlantCARE website to predict the cis-element of the component promoter region of the gene (Chen et al., 2020). The identification process is based on previous research recommendations for promoter region length and aims to capture potential regulatory elements. By submitting this sequence to PlantCARE, the type, location and number of cis-elements in the LsGRAS13 gene sequence can be determined, and their functions in gene regulation can be inferred.

Construction and Infection of LsGRAS13 Virus-Induced Gene Silencing (VIGS) Vector
To clone the 291 bp gene fragment of LsGRAS13, lettuce cDNA was used. The cloned fragment and empty pTRV2 vector were digested with EcoRI and BmaHI enzymes to obtain the recombinant plasmid. The recombinant plasmid was transformed into Agrobacterium GV3101, and the infection buffer (containing 10 mM MgCl 2 , 10 mM MES, and 20 mM acetosyringone) was prepared. The experiment was divided into a blank control group (WT), a negative control group (pTRV2 + pTRV1), and an experimental group (TRV2-LsGRAS13). After treatment at high temperature (35 °C) for one week, plant morphological changes were observed, and new leaves growing after injection were randomly selected for qRT-PCR detection.

Identification of LsGRAS Genes in Lettuce
In this study, a total of 59 LsGRAS genes of lettuce were identified by a homology search of Arabidopsis GRAS sequences (Table 1). These genes are located at different locations on nine chromosomes ( Figure 1). Based on their chromosomal locations, the 59 LsGRAS genes were named LsGRAS1~LsGRAS59. At the same time, some basic characteristics of LsGRAS family members were analyzed, including open reading frame (ORF) length, isoelectric point (pI), protein molecular weight (MW), and predicted subcellular localization. The smallest protein is LsGRAS18 with 420 amino acids (aa), while the largest protein is LsGRAS34 with 802 aa. Their molecular weight varies from 47,516.82 to 87,207.15 Dalton (Da). pI values ranged from 4.9 to 8.6 (LsGRAS1 and LsGRAS6). All LsGRAS genes showed 100% nuclear localization based on predicted subcellular localization ( Table 1). The results of this study have important significance for the gene regulatory network of lettuce and the growth and development process of the plant. The analysis of the basic characteristics of the LsGRAS gene family provides more gene resources for the study.
The distribution and interrelationships of plant genes are of great significance for revealing gene function and evolution. The results showed that there were 59 LsGRAS genes distributed on nine chromosomes. Among these chromosomes, the number of LsGRAS genes on chromosome five was the most, reaching eleven, while the number of LsGRAS genes on chromosome four was the least, only four ( Figure 1). These results indicated that the distribution of LsGRAS gene on chromosomes was uneven. Interestingly, 17 genes were physically adjacent to at least one other LsGRAS gene. For example, LsGRAS2 and LsGRAS3, LsGRAS14 and LsGRAS15, LsGRAS31 and LsGRAS32, LsGRAS34 and LsGRAS35, LsGRAS39 and LsGRAS40, LsGRAS41~LsGRAS43, and LsGRAS55~LsGRAS58 are adjacent to each other. These adjacent LsGRAS genes account for about 50% of the total number of genes. These results suggest that there may be some functional or regulatory relationship between LsGRAS genes on chromosomes. Based on the discovery of its distribution and adjacency, we can propose some hypotheses to guide further research. For example, the increase in the number of LsGRAS genes on chromosome five may be related to the importance of this chromosome in plant growth and development, and adjacent LsGRAS genes may share common regulatory factors or functional modules to coordinate plant physiological responses through interaction. In addition, by comparing the expression patterns and functional characteristics of LsGRAS genes on other chromosomes, we can further reveal their diversity and evolutionary relationship.

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Transcriptome Analysis Under Exogenous Melatonin
To investigate the expression profiles of 59 LsGRAS members in lettuce under exogenous melatonin, we conducted RNA-Seq analysis using publicly available leaf transcriptome data from NCBI (Bio project PRJNA810911). The results showed that the expression levels of lettuce treated with high temperatures and exogenous melatonin were higher than those treated with non-exogenous melatonin at different periods, and members of the DELLA family may have played an important role ( Figure 5). Specific analysis results showed that the expression pattern of the LsGRAS13 gene was consistent, and the expression level of high-temperature melatonin treatment (HM) was higher than that of non-exogenous melatonin treatment at high temperature (H) on the 6, 9, 15, 18, and 27 day. In addition, the expression level of the LsGRAS52 gene was the highest. The expression level of HM treatment was higher than that of H treatment on the 6, 9, 15, and 18 days, but the expression level was opposite on the 27 day. In addition, five LsGRAS genes were not expressed in lettuce, while 15 LsGRAS genes were not sensitive to high-temperature stress. These results provide important clues for us to better understand the regulatory mechanism of exogenous melatonin and high temperature on the growth and development of lettuce.
To investigate the mechanism of the LsGRAS gene in plants, we selected the LsGRAS13 gene with high transcription level and significant change pattern for cis-element analysis based on the results of RNA-Seq analysis. We isolated 2000 bp upstream of the LsGRAS13 promoter start codon, identified this region, and screened out some important cis-elements (Table 3). These cis-components include GA-responsive element (P-box, TATC-box), light-responsive element (G-box, CTt-Motif, MRE), auxin response element (Box 4), MeJA-responsive element (CGTCA-motif), and salicylic-responsive element (TCA). These findings suggest that the LsGRAS13 gene plays a role in regulating light and hormone responses during plant growth and development.
Especially under high-temperature stress, the function of this gene is activated and involved in regulating plant growth and development. This study provides important clues to further reveal the function of the LsGRAS13 gene and its role in plant stress response and provides valuable information for genetic engineering and agricultural breeding research.

By VIGS Silencing LsGRAS13 Can Delay Lettuce Bolting
To investigate the function of the LsGRAS13 gene, Agrobacterium pTRV2-LsGRAS13 containing a transient vector was first transformed into lettuce plants. The results showed that after two weeks of transient infection, the stem length of pTRV2-LsGRAS13 increased while no change was observed in the control group ( Figures 6A and  6B). To observe the growth of lettuce stem tips, the stem tips of WT, TRV2, and pTRV2-LsGRAS13 were paraffin selected, respectively, and it was found that flower bud differentiation of silencing pTRV2-LsGRAS13 was more significant than that of the control ( Figure 6A). Finally, qRT-PCR assay detected a significant down-regulation of the relative expression of TRV-LsGRAS13, which was more than three times that of control plants ( Figure 6C). These findings suggest that the VIGS technique effectively silenced the LsGRAS13 gene in lettuce and significantly elongated the stem length of the lettuce. The results showed that LsGRAS13 plays an important role in the bolting of lettuce and acts as a negative regulator of bolting.

Discuss
GRAS has lycopersic 2021). In t These resu family exp distributed (Figure 1), (Wang et (120. Vol. 15, No. 7; directly or indirectly reflect the similarities and differences in their functions. Through phylogenetic analysis and genetic structure research, we explore the evolution process of plant genes and predict and explain their functions. It is important to analyze the evolutionary relationship of genes among different species to reveal the common characteristics and differences among species. Studies have shown that GRAS members in dicotyledonous plants have better homology than those in monocotyledonous plants (Wang et al., 2020). In this study, the GRAS members of lettuce were analyzed and compared with one monocotyledon (Arabidopsis) and one dicotyledon (Solanum Lycopersicum). Surprisingly, the results are consistent with previous research that lettuce and Solanum Lycopersicum have the highest homology, meaning they may have retained some common features and functions for evolution. In Arabidopsis, however, the linear homology relationship between lettuce and Arabidopsis GRAS members was weak. This suggests that GRAS members may have undergone some special changes and adaptations during plant evolution and that such differences may be caused by evolutionary differences between species. In conclusion, we speculate that the common characteristics of GRAS members in different species may be closely related to their evolutionary differences. Further exploration of the intersection of GRAS members in different species is of great value to our understanding of the function and evolution of GRAS members.
Structural analysis is an important method to provide clues about the subgroups to which GRAS members belong, and can often reveal functional similarities between GRAS members within the same subfamily (Xu et al., 2016). In this study, DELLA proteins of lettuce (LsGRAS7, LsGRAS13, LsGRAS21, LsGRAS23, LsGRAS29, LsGRAS30, and LsGRAS52) were predicted to be correlated with AtGAI, AtRGA and AtRGL1 proteins. Meanwhile, the DELLA family consists of GAI, RGA, RGL1, RGL2, and RGL3 (Zhang et al., 2021). By analyzing the transcriptome data and 59 LsGRAS proteins, we found that LsGRAS13 showed some regular changes ( Figure 5). In addition, cis-acting element analysis showed that there were many light and hormone-related cis-acting elements in a certain LsGRAS gene under high-temperature treatment, suggesting that it may have a high correlation in these environmental conditions. P-box is an important component of the GA pathway, which binds to transcription factors and plays a role in response to GA-mediated osmotic stress signals (Yi et al., 2021). Previous studies have shown that DELLA proteins are a branch of the GRAS gene family and play a negative regulatory role in GA signaling as a possible transcriptional regulator (Waseem et al., 2022;Ito et al., 2021). Overall, the structural analysis provides us with clues about the subgroups to which GRAS members belong and reveals functional similarities between members within the same subfamily. DELLA proteins have shown potential importance in their association with AtGAI, AtRGA, and AtRGL1 proteins in lettuce and other plants. In addition, transcriptome data and cis-acting element analysis can be used to understand the regulation mechanism of the LsGRAS gene in high-temperature treatment and phytohormone response, which is of great significance for further understanding the function and regulation mechanism of the GRAS gene family.
In conclusion, through systematic mining and preliminary characterization of GRAS gene family in lettuce, we have taken the first step to exploring this gene family. However, further studies are still needed to uncover the role of the LsGRAS gene in other biological processes, as well as to explore the function and regulatory mechanisms of the entire gene family. This study provides new clues into the biology of lettuce plants.