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Article

Comprehensive Analysis of GRAS Gene Family and Their Expression under GA3, Drought Stress and ABA Treatment in Larix kaempferi

by
Miaomiao Ma
,
Lu Li
,
Xuhui Wang
,
Chunyan Zhang
,
Solme Pak
and
Chenghao Li
*
State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China
*
Author to whom correspondence should be addressed.
Forests 2022, 13(9), 1424; https://doi.org/10.3390/f13091424
Submission received: 28 July 2022 / Revised: 26 August 2022 / Accepted: 2 September 2022 / Published: 5 September 2022
(This article belongs to the Special Issue Forest-Tree Gene Regulation in Response to Abiotic and Biotic Stress)
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Abstract

:
The GRAS family transcription factors play important roles in regulating plant growth and responses to abiotic stress, which can be utilized to breed novel plants with improved abiotic stress resistance. However, the GRAS gene family has been largely unexplored for tree species, particularly for Larix kaempferi, which has high economic and ecological values, challenging practices for breeding abiotic stress-resistant L. kaempferi. In order to improve the stress resistance by regulating the transcription factors in L. kaempferi, we identified 11 GRAS genes in L. kaempferi and preliminarily characterized them through comprehensive analyses of phylogenetic relationships, conserved motifs, promoter cis-elements, and expression patterns, as well as protein interaction network prediction. The phylogenetic analysis showed that the LkGRAS family proteins were classified into four subfamilies, including DELLA, HAM, SCL, and PAT1, among which the SCL subfamily was the largest one. Conserved motif analysis revealed many putative motifs such as LHRI-VHIID-LHRII-PFYRE-SAW at C-terminals of the LkGRAS proteins; we discovered a unique motif of the LkGRAS genes. Promoter cis-acting element analysis exhibited several putative elements associated with abiotic stresses and phytohormones; the abscisic acid-responsive elements (ABRE) and G-box are the most enriched elements in the promoters. Through expression profiles of LkGRAS genes in different tissues and under drought-stress and phytohormones (GA3 and ABA) treatments, it was demonstrated that LkGRAS genes are most active in the needles, and they rapidly respond to environmental cues such as drought-stress and phytohormone treatments within 24 h. Protein interaction network prediction analysis revealed that LkGRAS proteins interact with various proteins, among which examples are the typical GA, ABA, and drought-stress signaling factors. Taken together, our work identifies the novel LkGRAS gene family in L. kaempferi and provides preliminary information for further in-depth functional characterization studies and practices of breeding stress-resistant L. kaempferi.

1. Introduction

The GRAS gene family encodes a large transcription factor (TF) family crucial for plant growth, development, and responses to environmental stresses. Its name “GRAS” was derived from three TFs including GAI (Gibberellic Acid Insensitive), RGA (Repressor of GAI), and SCR (Scarecrow) which are the typical members of GRAS TFs [1]. The GRAS domain is conserved throughout the GRAS TFs at the carboxyl (C)-terminus, which mainly includes the five motifs, namely, LHR I (Leucine Heptapide Repeat I), LHR II, VHIID, PFYRE, and SAW [2], while they have a high degree of variability at the amino (N)-terminus [3]. It is currently known that the GRAS gene family consists of seven to 16 subfamilies, and the number depends on the plant species; seven in Arabidopsis thaliana [4], eight in Oryza sativa [3], 11 in Citrus sinensis [5], 13 in Ricinus communis [6], and 16 in Medicago truncatula [7].
The GRAS genes play significant roles in plant growth, development, and defense responses to various biotic and abiotic stresses, as well as phytohormone signaling and symbiosis formation. Their expression has been observed in various plant organs and tissues, including needle, stem, root, fruit, coleoptile, radicle, anther, and silk [8], and vary according to developmental stages and environmental conditions [9,10], suggesting their roles in plant development and response to environmental cues. DELLA, DLT, HAM, PAT1, LAS, LISCL, SCR, SCL3, SHR, and SCL4/7 are typical subfamilies of GRAS proteins [11] that have been implicated in plant development as follows. In A. thaliana, DELLA is a central regulator that plays a major role in regulating GA signal [12], and HAM is involved in chlorophyll synthesis, the proliferation of meristem cells, and polar organization [4,13,14]. PAT1 is a putative component of the phytochrome A signaling pathway [4], while the LAS subfamily increases inflorescence number [15], shortens flowering time [6], and promotes flowering induction [16] and lateral bud growth [17,18]. LlSCL regulates the pre-meiotic phase of anthers and promotes microspore genesis [19], and SCL3 integrates the gibberellin acid (GA) pathway [12]. The SHR and SCR complex participates in controlling plant organ development [20,21]. In addition, several GRAS genes are known to be associated with plant responses to abiotic stresses. In tobacco, GRAS1 was induced by various stresses, which then increasing the level of reactive oxygen species [15]. Overexpression of PAT1 enhanced tolerance to abiotic stress in Arabidopsis [22]. The SCL4/7 subfamily members in rapeseed enhanced tolerance against drought and salt stresses [23]. GRAS6-silenced tomato plants showed increased sensitivity to drought stress [20]. By regulating the expression of the stress-related gene, GRAS23 has been demonstrated to enhance resistance against drought and oxidative stress in rice by regulating several stress-related genes [24]. In tomato, the GRAS40 gene is essential to regulate the activation of abiotic stress-inducible promoters and auxin and gibberellin signaling [25].
L. kaempferi is an important fast-growing native tree species in northern China that has high economic and ecological value. L. kaempferi belongs to a conifer species, generally called larch trees, with great value for wood production and ecological afforestation. Larch trees constitute forests in large areas of China, Eastern Europe, and Western North America. Among larch trees, L. kaempferi has several superiorities over others; it grows faster at the juvenile stage, has longer, fibrous, denser wood, and can adapt more easily to the environment than other larch trees. Thus, L. kaempferi is now recognized as an important tree species for various economical uses, such as timber and pulp production and papermaking, as well as afforestation and ornamental purposes. The problem is that recent climate-change-derived abiotic stresses such as drought are severely challenging afforestation practices of L. kaempferi, which calls for breeding novel L. kaempferi varieties with improved abiotic stress resistance. The GRAS gene family is a candidate gene family that can be utilized to breed novel L. kaempferi varieties with improved abiotic stress resistance. However, the GRAS gene family has not yet been largely explored in L. kaempferi, probably due to the unavailability of L. kaempferi genome information. The whole genome of L. kaempferi was recently sequenced [26] and it is, therefore, possible to perform genome-wide identification analysis for important TFs such as the GRAS TFs.
In this study, we, for the first time, identified the GRAS gene family in the L. kaempferi whole genome and then performed comprehensive analyses. In total, we identified 11 GRAS genes from the L. kaempferi whole genome and analyzed the evolutionary relationship, conserved motifs, and promoter cis-elements. We further analyzed the expression pattern of LkGRAS genes in different organs and tissues, including the root, stem, and needles in L. kaempferi. We also analyzed the expression of the GRAS genes under GA3, ABA, and drought treatments. Finally, we predicted the protein interaction network of LkGRAS proteins. This study provides a comprehensive overview of the L. kaempferi GRAS gene family as well as a preliminary basis for further in-depth research on the roles of LkGRAS factors in regulating L. kaempferi responses to phytohormone and abiotic stresses. More importantly, this study provides valuable information for further studies of L. kaempferi to improve stress resistance by regulating transcription factors.

2. Materials and Methods

2.1. Genome-Wide Identification and Phylogenetic Analysis of LkGRAS Genes

The genomic DNA, CDS, and protein sequences of L. kaempferi were obtained from NCBI (http://www.ncbi.nlm.nih.gov/) (accessed on 11 September 2021). Whole GRAS family members were searched in L. kaempferi using profile hidden Markov models (HMM); the GRAS binding domain (PF03514) was queried in the Pfam database (http://pfam.xfam.org/) (accessed on 11 September 2021) and then used to search all putative L. kaempferi GRAS protein members with the HMMER3 package. Redundant sequences were manually detected and eliminated, and then the remaining sequences were examined to confirm whether the GRAS binding domain is conserved throughout the sequences using the online programs CDD (https://www.ncbi.nlm.nih.gov/cdd) (accessed on 11 September 2021), Pfam (http://pfam.xfam.org/) (accessed on 11 September 2021), and SMART (http://smart.embl-heidelberg.de/) (accessed on 11 September 2021). The L. kaempferi GRAS protein sequences were aligned and visualized using EMBL-EBI (https://www.ebi.ac.uk/Tools/services/web/tool/) (accessed on 23 December 2021) and Jalview. We set the basic options, including “annotations, format, and color”. The physical and chemical properties of the L. kaempferi GRAS proteins were analyzed using the ExPASy proteomics server (http://web.expasy.org/protparam/) (accessed on 23 December 2021) to analyze the characteristics of the GRAS proteins.
The amino acid sequences of GRAS proteins in A. thaliana and O. sativa were downloaded from Phytozome (Phytozome v12.1: Home) and then aligned using Clustal X (version 2.0) and Bioedit (version 7.2.5) with a gap opening penalty and gap extension penalty of 10 and 0.1, respectively. Molecular features and phylogenetic relationships between the GRAS genes of L. kaempferi, A. thaliana, and O. sativa were analyzed using MEGA software (v7.0) with the maximum likelihood method parameters as the Poisson model, partial deletion (95%), and 500 bootstrap replications [27].

2.2. Conserved Motif and Promoter Cis-Element Analysis of LkGRAS Genes

Conserved motifs in the LkGRAS genes were investigated using MEME (Multiple Em for Motif Elicitation program 5.1.1; http://meme-suite.org/tools/meme) (accessed on 23 December 2021) with the following parameters: the maximum number of motifs was set to 15, and the optimum motif width was set to 6 to 50 residues [28]. The Pfam and SMART tools were used to perform each structural motif annotation.
The sequences of LkGRAS genes were downloaded from the L. kaempferi genome database in NCBI, and their promoters, 2000 bp upstream of the translation start site, were identified. Then, putative cis-elements were searched throughout the promoters using the online database PlantCARE [29].

2.3. Plant Materials and Treatments

Mature seeds of L. kaempferi were collected from 60-year-old trees in Qing Shan national Larch seed orchard in Heilongjiang province (the geographical coordinates are 133°53′28″–133°58′05″ E and 46°38′56″–46°44′20″ N) and stored at −20 °C. The seeds were sown in plastic pots (11 × 11 cm) containing a grit/soil mixture (1:3 ratio), and 30 days later, seedlings were transferred to 15 cm pots (one plant per pot) containing a grit/soil mixture (1:1 ratio). The seedlings were cultured for five months under a 16 h/8 h light/dark photoperiod, 150 µmol m−2 s−1 light intensity, 70% relative humidity [30], and the soil water content was kept at ≥70% field capacity [31].
We sampled roots, stem, and needles, respectively, before treatments to determine the tissue-specific expression pattern. For the ABA and GA treatment, the solution containing 100 µM ABA or 100 µM GA3 was prepared and sprayed on needles of the L. kaempferi seedlings [32]. The needles were then collected at 0, 6, 12, and 24 h after treatment [32] for further RNA extraction. In addition, for drought-stress treatment, watering was stopped, and soil moisture contents were temporally measured by the gravimetric method [26]. The degree of drought stress was determined by the soil moisture contents as follows: 70%–80% (CK, non-drought), 50%–60% (mild drought, MD), and 20%–35% (severe drought, SD) of the maximum field water capacity [33]. Temporal change in soil water contents is shown in Figure S1 and the needles were sampled at 6, 9, and 12 d for further RNA extraction. The plants at 0 days were used as control. All the treatments were sampled with three biological repeats for each seedling. The needles were carefully sampled and frozen immediately in liquid nitrogen, stored at −80 °C until RNA extraction.

2.4. RNA Extraction and Gene Expression Analysis by qRT-PCR

Total RNA was extracted from the needles using the CTAB (cetyltrimethylammonium bromide) method [34] and then reverse-transcribed to cDNA by Hi Script® II Q Select RT Super Mix for qRT-PCR. The genome DNA was eliminated by gDNA Wiper Mix. The primers were designed and checked for LkGRAS genes using the NCBI primer designing tool (https://www.ncbi.nlm.nih.gov/tools) (accessed on 23 December 2021). The qRT-PCR was performed to determine transcript levels of LkGRAS genes using SYBR Premix Ex Taq II (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The whole-genome sequencing of GRAS genes in the L. kaempferi gene (Whole Genome Shotgun (WGS): INSDC: WOXR00000000.2) was used as target genes. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal control gene [30]. The 2−∆∆Ct method was used to calculate the relative gene expression levels. All LkGRAS gene-specific primers used for qRT-PCR are listed in Table S1.
The qRT-PCR data were tabulated and loaded by HEML to generate a heat-map. We set “canvas” and “space” to resize the heat map. We also determined the position of the X and Y axes, meanwhile selecting “column and row” to generate the branch network. We set “note” to adjust the basic setting of the font, including size and color. In the end, we set “logarithmic 2” in the option of “statistics” and exported the image.

2.5. Protein Interaction Network Analysis

The STRING (version 11.0; https://string-db.org/cgi/input.pl) (accessed on 23 December 2021) database was employed to predict the protein interaction network of LkGRAS proteins; prediction was performed using amino acid sequence of LkGRAS proteins as query and Arabidopsis thaliana as the “organism”. The basic settings included “evidence” and “text-mining, experiments, databases, co-expression, neighborhood, gene fusion and co-occurrence”. The minimum required interaction score was set as medium confidence of 0.4.

2.6. Statistical Analysis of Data

The experimental data were analyzed by one-way analysis of variance (ANOVA) method using SPSS software (version 20, IBM, Chicago, IL, USA) to evaluate significant differences between the control and each treatment. Significant differences were defined as * p < 0.05 and ** p < 0.01.

3. Results

3.1. Identification of GRAS Genes Family in L. kaempferi

To determine the information of the GRAS family member in L. kaempferi, we identified 11 GRAS genes in L. kaempferi genome using HMM profile of the GRAS binding domain (PF03514) as a query and then analyzed their basic information as follows. Domain search analysis using SMART and Pfam databases demonstrated that all encoded LkGRAS proteins possess GRAS domains. We named these genes from LkGRAS1 to LkGRAS11 (Table 1). The number of protein lengths, molecular weight, grand average of hydrophilicity (GRAVY), and isoelectric points are shown in Table 1. The length of GRAS proteins in L. kaempferi is between 223 and 730 amino acids, and the molecular weights are from 25.25 kDa to 86.22 kDa. The predicted theoretical point (pI) value varies from 5.12 to 7.07. GRAVY values of all LkGRAS proteins are below zero, ranging from −0.533 to −0.075, suggesting that LkGRAS proteins belong to the hydrophilic protein group. The instability index for most LkGRAS proteins is greater than 40, indicating that most LkGRAS proteins are unstable. Only three LkGRAS proteins have a stable index from 37.84 to 39.67. The aliphatic index of all LkGRAS proteins ranged from 71.97 to 91.82. The research showed that the aliphatic index usually shows the domination of aliphatic side chains to indicate thermal stability [35].
Figure 1 shows the multiple sequence alignments of the GRAS gene family members of L. kaempferi. In the multiple sequence alignments outcome, the blue color and its intensity represent conserved domains and their homology degrees; darker color means a higher homology level. There are four conserved domains, including LHR (C1), PFYRE (C2), VHIID (C3), and SAW (C4).

3.2. Phylogenetic Analysis of L. kaempferi GRAS Proteins

To investigate the evolutionary relationships and classification of the GRAS family in L. kaempferi, 37 A. thaliana, 63 O. sativa, and 11 LkGRAS proteins were used to construct a phylogenetic tree with the neighbor-joining (NJ) method in MEGA7.0 (Figure 2). According to the two clusterings and the relationship with A. thaliana and O. sativa, the GRAS proteins were classified into eight subfamilies (LISCL, RGL, PAT1, SCR, HAM, SCL3, SCL4/7, and DELLA). There are eight LkGRAS proteins belonging to the SCL (4) and PAT (4) subfamilies, while the other three proteins belong to the DELLA (1) and HAM (2). LkGRAS2, −5, −6, −7, −8, −9 proteins were clustered with the OsGRAS proteins, whereas LkGRAS1, −3, −4, −10, −11 were clustered with AtGRAS proteins. This indicates that the function of OsGRAS and AtGRAS proteins may provide a reliable reference to LKGRAS proteins.

3.3. Conserved Motifs of LkGRAS Proteins

The motifs analysis contributes to comprehensively understand the conserved characteristics of LkGRAS proteins and analyze structure in their conserved domain. We further confirmed the conserved motifs of LkGRAS proteins using MEME. In total, 15 distinct motifs were detected and named motif 1 to motif 15 (Figure 3). Since the structures and functions of the LkGRAS are not recognized completely, the motifs were defined based on sequence conservation. As per the previous research in GRAS domains characterization analysis, the LHRI-VHIID-LHRII-PFYRE-SAW structure domain determined the arrangements of motifs [1]. Motif 5 was highly conserved at the outermost part of C-terminal regions except for LkGRAS9 and LkGRAS10. The motifs were distributed mostly in the C-terminal. There were 10 motifs (motifs 1, 2, 3, 4, 5, 7, 8, 10, 12, and 14) in the C-terminal, while the remaining motifs (including motifs 6, 9, 11, 13, and 15) were at the N-terminal. Our results showed that conserved GRAS domains, including LHRI, VHIID, LHRII, PFYRE, and SAW domains (previously discovered by Pysh et al., 1999), included motif 1 (in VHIID domain), motif 2 (in PRYRE and SAW domains), motif 4 (in LHRII domain), motifs 5 and 6 (in LHRI domain), motif 5 (in SAW domain), and motif 7 (in PRYRE domain) (Figure S2). The motif 3 and motif 8 to motif 15 were not found to form a structure in certain domains in LkGRAS proteins, but they were still an indispensable part of the conserved structure domain [36].

3.4. Promoter Cis-Element Analysis

To understand possible regulation mechanisms of the LkGRAS genes, we analyzed the promoters of LkGRAS genes using PlantCARE and identified nine putative stress-related and phytohormone-related cis-elements (Figure S3). They include drought-inducibility elements (MBS) and low-temperature responsive elements (LTR), stress- and defense-responsive elements (TC-rich repeats elements), CGTCA/TGACG (MeJA-responsive elements), TCA-element (salicylic-acid-responsive elements), TGA-element (auxin-responsive elements), ABRE elements (abscisic-acid-responsive elements), and TA-rich repeats TC-box (gibberellin-responsive elements), as well as Box4 and G-box (light-responsive elements) (Table 2). The presence of these various stress- and phytohormone- responsive cis-elements suggested putative roles of LkGRAS genes in plant growth, development, and responses to abiotic stresses.

3.5. Tissue-Specific Expression Pattern of LkGRAS Genes

Tissue-specific expression profile for the genes belonging to a plant gene family reflects their tissue-specific functions. To determine tissue-specific expression profile of LkGRAS genes, we performed qPCR to analyze LkGRAS gene expression patterns in roots, stems, and needles at the same developmental stages and then generated a heat map (Figure 4) using the qPCR data. Expression levels of 11 LkGRAS genes were different to each other in the same tissue. In addition, different tissues exhibited different expression levels of LkGRAS genes. Most LkGRAS genes were weakly expressed in root tissues except the LkGRAS10, while they showed much higher expression levels in needle and stem tissues. In addition, the LkGRAS10 showed the highest expression level among the LkGRAS genes in roots and needles, as well as high expression level in stem tissue. Taken together, we demonstrated that LkGRAS genes are expressed in mostly needle, and among them, LkGRAS10 showed relatively high expression levels in all kinds of tissues tested here.

3.6. Expression Analysis of LkGRAS Genes under GA3, ABA Treatment, and Drought Stress

The presence of various stress- and phytohormone-responsive cis-elements suggested involvement of LkGRAS genes in plant growth, development, and responses to abiotic stresses. To examine whether the LkGRAS genes take part in the abiotic stress and phytohormone response, we performed qPCR to analyze the expression level of LkGRAS genes in needles of L. kaempferi plants subjected to GA3 (100 µM), ABA (100 µM) treatment, and drought stress. Fold change > 2 was considered as significantly differentially expressed genes. Firstly, we analyzed the LkGRAS gene expression under GA3 treatment. As shown in Figure 5, all LkGRAS genes showed responses to exogenous GA3 treatment with diverse expression profiles; nine LkGRAS genes were upregulated, among which the expression levels of LkGRAS4, 5, and 7 shown were very significant. LkGRAS6 and 10 did not show a significant response to GA3 treatment (no more than twofold). Duration of GA3 treatment also differentially influenced the expression pattern of the LkGRAS genes. LkGRAS1, 3, and 8 were upregulated and reached a peak at 6 h, and LkGRAS2, 4, 5, and 7 at 12 h. The expression levels of LkGRAS9 and 11 consistently increased for 24 h. LkGRAS4, 5, and 7 had the highest expression levels among 11 LkGRAS genes in response to GA3 treatment. Then, we analyzed the LkGRAS gene expression under drought stress. Except for LkGRAS1, 3, 8, and 9, the other LkGRAS genes showed significant response to drought stress (Figure 6). LkGRAS5, 6, and 10 were initially upregulated (at 6 d after drought treatment), and then declined gradually later. LkGRAS2, 4, 7, and 11 showed upregulation and reached a peak at 9 d after treatment. Finally, we analyzed the LkGRAS genes expression under ABA treatment. The LkGRAS genes were sensitive to ABA treatment except for LkGRAS3, 6, 8, and 9 (Figure 7). Though the LkGRAS genes showed different expression levels, they had a similar expression tendency under ABA treatment. Notably, the LkGRAS genes were significantly induced at various points in time under ABA treatment. The expression level was upregulated and reached a peak at 6 h, then downregulated later. Nearly all genes were in line with this trend, but the LkGRAS3, 6, 8, and 9 always showed dramatically downregulated expression levels. The expression level of LkGRAS3, 6, and 8 were upregulated no more than twofold and showed lower expression levels together with LkGRAS9, while the expression levels of the other LkGRAS genes (LkGRAS2, 4, 5, 7, 10, and 11) compared to them were significant, and the expression levels of LkGRAS4, 5, 7, and 11 were very significant.
Collectively, the results showed that these LkGRAS genes responded to at least one kind of treatment. For instance, there were nine LkGRAS genes upregulated in the GA3 treatment (LkGRAS1, 2, 3, 4, 5, 7, 8, 9, 10, and 11) in which the LkGRAS1, 2, 4, 5, 7, 10 and 11 were upregulated in the ABA treatment. Apart from these LkGRAS genes, the LkGRAS3 and 9 also showed opposite expression results. Moreover, among the six drought-inducible genes (LkGRAS4, 5, 6, 7, 10, and 11), five were all upregulated by ABA (LkGRAS4, 5, 7, 10, and 11), and three by GA3 (LkGRAS4, 5, and 7). Meanwhile, the expression levels of LkGRAS4, 5, 7, 10, and 11 in ABA were consistent with those in drought, and LkGRAS4 and 7 exhibited significantly positive responses to all three kinds of treatments.

3.7. Protein Interaction Network of LkGRAS Proteins

Proteins hardly implement their functions independently, but interact with other proteins to regulate cellular biological processes and prediction of the knowledge of protein–protein interactions (PPIs); therefore, they can untangle the cellular behaviors and functionality of the proteins. To identify the relationship of LkGRAS proteins with other proteins, we predicted the protein interaction network for LkGRAS proteins using STRING. Each LkGRAS protein sequence could obtain more than one network, and only the networks with the highest scores are shown in Figure 8. The networks revealed that LkGRAS proteins within a subfamily interact with the same proteins. For example, LkGRAS1 and LkGRAS2 of the PAT1 subfamily interact with SCL28, while LkGRAS1 and LkGRAS10 of the same subfamily interact with WAK. LkGRAS6 and LkGRAS7 of the SCL subfamily interact with MYB87, whereas LkGRAS3 and LkGRAS11 of the same subfamily interact with GID1. LkGRAS8 and LkGRAS9 of the HAM subfamily interact with WOX4. It seems that the proteins in a subfamily have highly similar motif alignments and therefore share the same protein targets to interact with each other. In addition, there are several GA, ABA, and drought-stress-related proteins, including SCL28/30, JAZ1, GID1, SLY1, GA3Ox1, PIF3, XBAT35, WDR55, and AT5G67411, among the interacting proteins, implying the interactions between them and LkGRAS proteins under GA, ABA, and drought-stress treatment.

4. Discussion

The GRAS gene family encodes plant-specific TFs, which play essential roles in various biological processes. To date, the GRAS gene family has been extensively reported in various plant species including A. thaliana [37], Brassica campestris [38], Brassica juncea [16], C. sinensis [5], Glycine max [39], Gossypium hirsutum L. [10], Ipomoea trifida [40], Juglans regia L. [41], Malus domestica [42], Manihot esculenta [43], M. truncatula [7], Nelumbo nucifera [44], O. sativa [3], Panax ginseng [45], Populus L. [46], R. communis [6], Solanum lycopersicum [47], Triticum aestivum [48], and Zea mays L. [49]. Notably, the GRAS gene family has been largely unexplored in tree species; only reported in cassava [43] and poplar [46]. In our work, we identified the GRAS gene family in L. kaempferi, which is an economically and ecologically important tree species in northeastern China, for the first time. Then, we performed comprehensive analyses including phylogenetic analysis, conserved motif, and promoter cis-element analyses, tissue-specific and phytohormone and abiotic stress-triggered expression profile analysis, as well as protein interaction network prediction analysis for the L. kaempferi GRAS gene family.
Genome-wide identification and phylogenetic analysis revealed that the LkGRAS gene family (abbreviation of LkGRAS gene family) includes 11 GRAS genes which are further classified into four main subfamilies: DELLA, HAM, SCL, and PAT1. Other subfamilies, such as DLT, LAS, LISCL, SCR, and SHR, are not found in the LkGRAS gene family; this would probably be due to incompleteness of L. kaempferi genome database or unique feature of the L. kaempferi species. The structure of LkGRAS genes further showed that they have highly conserved motifs at C-terminal regions; conserved motifs were arranged as LHRI-VHIID-LHRII-PFYRE-SAW at C-terminals, while their N-terminal regions showed high variability that may be associated with functional divergence among the LkGRAS proteins. All LkGRAS proteins except LkGRAS9 and 10 have the SAW motif in the C-terminal region, consistent with previous findings [4] that reported the presence of the SAW motif in the C-terminal region in the A. thaliana GRAS family. We also found that the LkGRAS proteins in the same subfamily have a similar motif arrangement in the C-terminal region. For example, the LkGRAS proteins of the PAT1 subfamily all have a motif5 and a motif7 arranged at the C-terminal region. In addition, the motif2 domain is present in both PAT1 and SCL subfamilies. It postulates that these LkGRAS genes might have similar functions in biological processes. In addition, promoter cis-element analysis indicated that the promoters of LkGRAS genes contain many cis-acting elements such as drought-inducibility elements (MBS) and low-temperature responsive elements (LTR), stress- and defense-responsive elements (TC-rich repeats elements), CGTCA/TGACG (MeJA-responsive elements), TCA-element (salicylic-acid-responsive elements), TGA-element (auxin-responsive elements), ABRE elements (abscisic-acid-responsive elements), and TA-rich repeats TC-box (gibberellin-responsive elements), as well as Box4 and G-box (light-responsive elements), suggesting the roles of GRAS TFs in the L. kaempferi response to environmental cues (drought, low temperature) and phytohormones (auxin, ABA, gibberellin, MeJA, and salicylic acid).
Due to the presence of putative stress and phytohormone-related cis-acting elements in the promoters of LkGRAS gene family members, the expression profiles of LkGRAS genes were investigated under drought, GA3, and ABA treatments. Before this, expression of the LkGRAS genes was examined in different tissues and it was demonstrated that the LkGRAS genes were highly expressed in needles. Then, expression of the LkGRAS genes in needles was further investigated under drought, GA3, and ABA treatments. Upon GA3 treatment, LkGRAS4, 5, and 7 showed relatively high expression compared to others. LkGRAS5 belongs to the DELLA family gene. The DELLA proteins are known as repressors of gibberellin response in plants [50]; DELLA proteins are essential components in the intracellular GA3 degradation system, negatively regulating GA3 signaling in Arabidopsis. Many previous studies reported that the GA-DELLA module is conserved and plays a central role in GA signaling in plants [51,52,53]. Upregulation of LkGRAS5 (DELLA subfamily) upon GA3 treatment in our work was consistent with the findings in the above previous studies. These findings also verified our result indirectly, that when we apply exogenous GA to L. kaempferi, the GA oxidases genes of the LkGRAS family will show high expression levels of degraded gibberellin. In addition, under ABA treatment, LkGRAS that belong to PAT1 and SCL subfamilies exhibited high expression, indicating that PAT1 and SCL subfamilies are associated with the ABA pathway. LkGRAS2, 4, 5, 7, and 11 showed relatively high expression levels compared to the other genes, and among them, LkGRAS5, 7, and 11 were expressed at the highest levels. The presence of ABRE elements in the promoters of LkGRAS5, 7, and 11 would be one of the putative reasons why they showed strong upregulation under ABA treatment. Moreover, the LkGRAS genes showed different expression patterns under GA3 and ABA. This might be due to the antagonistic roles of GA and ABA in plant growth and development [54]. In our work, LkGRAS2, 4, 5, 7, 10, and 11 showed higher expression levels under ABA treatment than under GA3 treatment (except for LkGRAS2 and 11), and LkGRAS3, 9, and 10 showed contrasting patterns of expression under GA3 and ABA treatments. These results implied that LkGRAS genes might be involved in the antagonistic effects of GA and ABA on plant growth and development. In addition to GA and ABA, plant response to drought stress is also known to be related to GRAS genes. Previous works revealed that the AtPAT1 subfamily of the GRAS family gene in Arabidopsis could increase the tolerance of the plant to abiotic stress, such as cold, drought, and salt [23,55]. The SCL subfamily was also demonstrated to participate in drought-stress response [24]. Consistently, our work also manifested that most of the LkGRAS genes responded to drought stress; among which LkGRAS4 belongs to the PAT1 subfamily and LkGRAS7 and 11 belong to the SCL subfamily. It can be inferred that the PAT1 and SCL subfamilies of GRAS genes in L. kaempferi are involved in drought-stress response. In addition, drought-stress-related cis-acting elements are present in promoters of the differentially expressed LkGRAS genes under drought stress. Among the drought-inducible genes, LkGRAS4, 7, 10, and 11 showed high expression levels in drought-stress and ABA treatment. In plants, signaling pathways of ABA and drought-stress response are interrelated with each other. It, therefore, appeared that drought and ABA treatments both induced the expression of LkGRAS4, 7, 10, and 11. LkGRAS4 and 7 also belong to the PAT1 subfamily, which showed a high expression level under GA and ABA treatments. Overall, expression profiles of the LkGRAS genes showed consistency with the prediction from the cis-acting elements in promoters of the LkGRAS genes. The LkGRAS genes with drought-inducibility elements showed high expression levels under drought stress. The highly induced LkGRAS genes under GA3 or ABA treatments also possess GA- or ABA-related cis-acting elements in their promoters. Each of the LkGRAS genes contains at least two cis-elements related to phytohormone or abiotic stress responsiveness.
Moreover, the protein interaction network of LkGRAS proteins was predicted using the STRING database, which could provide a supplementary understanding of orthologous proteins’ roles in biological processes [56]. We found that the LkGRAS proteins within the same subfamily revealed similar protein interaction networks. Among the interacting proteins, we found that several factors, such as SCL28, JAZ1, GID1, SLY1, GA3Ox1, PIF3, XBAT35, WDR55, and AT5G67411, have previously been known to be associated with GA, ABA, and drought-stress responses. SCL28, which is a GRAS type TF in A. thaliana [57] and is known to be involved in ABA-mediated stress responses [58], interacts with LkGRTAS1, 2, 5, and 7 proteins. JAZ1, WDR55, and XBAT35 are also the ABA response factors [59,60,61], which are predicted to interact with LkGRAS3, 4, and 5 proteins, respectively, in our study. In addition, JAZ1 and WDR55, which can regulate drought-stress responses through ABA pathways, interact with LkGRAS3 and 5, respectively. PIF3, which is previously known to enhance resistance to drought stress [62], interacts with LkGRAS3 and 11. In addition to ABA and drought-related factors, there are GA response factors such as GA3Ox1, GID1, and SLY1 among the total interacting factors. GA3Ox1, which is the enzyme for GA biosynthesis, interacts with LkGRAS3 and GID1, which is a gibberellin receptor protein [63,64] and interacts with LkGRAS11. SLY1, which is known to positively regulate GA signaling [65], interacts with both LkGRAS3 and 11.

5. Conclusions

In conclusion, we identified 11 GRAS family genes in L. kaempferi and analyzed their phylogenetic tree, conserved motifs, and promoter cis-elements. The 11 LkGRAS genes are classified into four subfamilies, including DELLA, HAM, SCL, and PAT1. The LkGRAS proteins all have conserved LHRI-VHIID-LHRII-PFYRE-SAW motifs at C-terminals and their promoters contain many cis-acting elements associated with abiotic stresses and phytohormones. In addition, we evaluated the expression patterns of LkGRAS genes in different tissues and under GA3, ABA, and drought-stress treatments using qRT-PCR. LkGRAS genes were mainly expressed in needles and were significantly induced upon exogenous treatment by phytohormones (GA3 and ABA) and drought stress. We also predicted the protein interaction network of LkGRAS proteins. Preliminary results of our work on the LkGRAS gene families provided knowledge that would be the basic information for further in-depth functional characterization of LkGRAS family genes in L. kaempferi.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f13091424/s1, Figure S1: Temporal change in the soil water contents under non-watered condition; Figure S2: Conserved GRAS domains (LHRI, VHIID, LHRII, PFYRE, and SAW domains); Figure S3: The nine putative stress-related and phytohormone-related cis-elements; Table S1: Primers for quantitative qRT-PCR.

Author Contributions

Conceptualization, M.M. and C.L.; experimental design: C.L.; material collection and performing the experiments: M.M., X.W., L.L. and C.Z.; data analysis: M.M.; software, M.M.; writing—original draft: M.M.; writing—review and editing: M.M., S.P. and C.L. All authors approved the final draft. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Genetically Modified Organisms Breeding Major Projects of China (Grant No. 2018ZX08020003).

Data Availability Statement

The data is available on request from the corresponding author.

Acknowledgments

We thank Jia Yang for proofreading the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Multiple sequence alignments of the L. kaempferi GRAS gene family members. Blue shading marks identical residues, light blue shading marks conserved residues. Positions of the basic region of the GRAS domain and conserved domains (C1–C4) are demarcated by lines above sequences.
Figure 1. Multiple sequence alignments of the L. kaempferi GRAS gene family members. Blue shading marks identical residues, light blue shading marks conserved residues. Positions of the basic region of the GRAS domain and conserved domains (C1–C4) are demarcated by lines above sequences.
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Figure 2. Phylogenetic analysis of the GRAS gene family members from L. kaempferi, O. sativa, and A. thaliana. Branches with less than 50% bootstrap support were collapsed. The phylogenetic tree was constructed using the maximum likelihood (ML) method of MEGA 7.0 with 500 bootstrap replicates.
Figure 2. Phylogenetic analysis of the GRAS gene family members from L. kaempferi, O. sativa, and A. thaliana. Branches with less than 50% bootstrap support were collapsed. The phylogenetic tree was constructed using the maximum likelihood (ML) method of MEGA 7.0 with 500 bootstrap replicates.
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Figure 3. Phylogenetic relationships and conserved motifs of LkGRAS proteins. Phylogenetic tree (A) of LkGRAS proteins was constructed by using the neighbor-joining (NJ) method with 1000 bootstrap replicates in MEGA 7.0, and conserved motifs (B) were obtained using MEME.
Figure 3. Phylogenetic relationships and conserved motifs of LkGRAS proteins. Phylogenetic tree (A) of LkGRAS proteins was constructed by using the neighbor-joining (NJ) method with 1000 bootstrap replicates in MEGA 7.0, and conserved motifs (B) were obtained using MEME.
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Figure 4. Tissue-specific expression pattern of LkGRAS genes. The relative gene expression levels were calculated using the 2−∆∆Ct method. Different colors represent different expression levels: blue, green, and red colors represent low, mild, and high expression levels, respectively.
Figure 4. Tissue-specific expression pattern of LkGRAS genes. The relative gene expression levels were calculated using the 2−∆∆Ct method. Different colors represent different expression levels: blue, green, and red colors represent low, mild, and high expression levels, respectively.
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Figure 5. The relative expression level of the LkGRAS genes in needles under GA3 treatment using qRT-PCR. Error bars represent the deviations from three biological replicates. The x-axis represents the time points after 100 µM GA3 treatment (* p < 0.05, ** p < 0.01).
Figure 5. The relative expression level of the LkGRAS genes in needles under GA3 treatment using qRT-PCR. Error bars represent the deviations from three biological replicates. The x-axis represents the time points after 100 µM GA3 treatment (* p < 0.05, ** p < 0.01).
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Figure 6. The relative expression levels of the LkGRAS genes in needles under drought stress using qRT-PCR. Error bars represent the deviations from three biological replicates. The x-axis represents the time points after drought stress (* p < 0.05, ** p < 0.01).
Figure 6. The relative expression levels of the LkGRAS genes in needles under drought stress using qRT-PCR. Error bars represent the deviations from three biological replicates. The x-axis represents the time points after drought stress (* p < 0.05, ** p < 0.01).
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Figure 7. The relative expression levels of the LkGRAS genes in needles under ABA treatment using qRT-PCR. Error bars represent the deviations from three biological replicates. The x-axis represents the time points after 100 µM ABA treatment (* p < 0.05, ** p < 0.01).
Figure 7. The relative expression levels of the LkGRAS genes in needles under ABA treatment using qRT-PCR. Error bars represent the deviations from three biological replicates. The x-axis represents the time points after 100 µM ABA treatment (* p < 0.05, ** p < 0.01).
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Figure 8. The predicted protein interaction network of LkGRAS proteins. (A–K) The potential protein interaction networks of each protein were predicted by the STRING database. Different colored lines represent different evidence of an interaction.
Figure 8. The predicted protein interaction network of LkGRAS proteins. (A–K) The potential protein interaction networks of each protein were predicted by the STRING database. Different colored lines represent different evidence of an interaction.
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Table
Table 1. Basic information of L. kaempferi GRAS family members.
Table 1. Basic information of L. kaempferi GRAS family members.
NameGene IDLengthMolecular Weight (kDa)Theoretical pIGRAVY Value
LkGRAS1Lk_f2p60_250961968.865.12−0.336
LkGRAS2Lk_f2p57_271472180.35 5.16 −0.533
LkGRAS3Lk_f2p39_201559464.40 5.65 −0.075
LkGRAS4Lk_f4p60_308169677.896.31−0.423
LkGRAS5Lk_f2p60_298773082.16 5.67 −0.459
LkGRAS6Lk_f2p49_155244750.46 6.10 −0.331
LkGRAS7Lk_f2p39_277578186.22 5.19 −0.358
LkGRAS8Lk_f2p16_268463471.625.58 −0.291
LkGRAS9Lk_f2p7_222147651.89 7.07 −0.233
LkGRAS10Lk_f2p60_299922825.776.23−0.258
LkGRAS11Lk_f2p49_114122325.25 5.66 −0.238
Table
Table 2. Cis-element analysis of promoter regions of LkGRAS genes.
Table 2. Cis-element analysis of promoter regions of LkGRAS genes.
NameMREMBSLTRABRETGATCABox4G-BoxCGTCATGACG
LkGRAS10001001000
LkGRAS22040023100
LkGRAS30003113244
LkGRAS41100020011
LkGRAS50102020211
LkGRAS60005000955
LkGRAS70015010644
LkGRAS80100100500
LkGRAS90213104000
LkGRAS100210130011
LkGRAS111003201133
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Ma, M.; Li, L.; Wang, X.; Zhang, C.; Pak, S.; Li, C. Comprehensive Analysis of GRAS Gene Family and Their Expression under GA3, Drought Stress and ABA Treatment in Larix kaempferi. Forests 2022, 13, 1424. https://doi.org/10.3390/f13091424

AMA Style

Ma M, Li L, Wang X, Zhang C, Pak S, Li C. Comprehensive Analysis of GRAS Gene Family and Their Expression under GA3, Drought Stress and ABA Treatment in Larix kaempferi. Forests. 2022; 13(9):1424. https://doi.org/10.3390/f13091424

Chicago/Turabian Style

Ma, Miaomiao, Lu Li, Xuhui Wang, Chunyan Zhang, Solme Pak, and Chenghao Li. 2022. "Comprehensive Analysis of GRAS Gene Family and Their Expression under GA3, Drought Stress and ABA Treatment in Larix kaempferi" Forests 13, no. 9: 1424. https://doi.org/10.3390/f13091424

APA Style

Ma, M., Li, L., Wang, X., Zhang, C., Pak, S., & Li, C. (2022). Comprehensive Analysis of GRAS Gene Family and Their Expression under GA3, Drought Stress and ABA Treatment in Larix kaempferi. Forests, 13(9), 1424. https://doi.org/10.3390/f13091424

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