Overexpression of Lolium multiflorum LmMYB1 Enhances Drought Tolerance in Transgenic Arabidopsis

Lolium multiflorum is one of the world-famous forage grasses with rich biomass, fast growth rate and good nutritional quality. However, its growth and forage yield are often affected by drought, which is a major natural disaster all over the world. MYB transcription factors have some specific roles in response to drought stress, such as regulation of stomatal development and density, control of cell wall and root development. However, the biological function of MYB in L. multiflorum remains unclear. Previously, we elucidated the role of LmMYB1 in enhancing osmotic stress resistance in Saccharomyces cerevisiae. Here, this study elucidates the biological function of LmMYB1 in enhancing plant drought tolerance through an ABA-dependent pathway involving the regulation of cell wall development and stomatal density. After drought stress and ABA stress, the expression of LmMYB1 in L. multiflorum was significantly increased. Overexpression of LmMYB1 increased the survival rate of Arabidopsis thaliana under drought stress. Under drought conditions, expression levels of drought-responsive genes such as AtRD22, AtRAB and AtAREB were up-regulated in OE compared with those in WT. Further observation showed that the stomatal density of OE was reduced, which was associated with the up-regulated expression of cell wall-related pathway genes in the RNA-Seq results. In conclusion, this study confirmed the biological function of LmMYB1 in improving drought tolerance by mediating cell wall development through the ABA-dependent pathway and thereby affecting stomatal density.


Introduction
Drought, as a widespread environmental factor in nature [1][2][3], has become one of the most critical factors restricting the yield of forage grass [4,5].Lolium multiflorum is a world-famous cold season forage grass, which has the advantages of fast growth, rich biomass and high nutritional value.It has become the main forage grass for green feed production in winter [6,7].However, its growth and development are often affected by drought, especially seasonal drought in winter.The results of studies on materials with extreme differences in drought tolerance of L. multiflorum show that the metabolic pathways such as sucrose synthesis, root development and ABA signal transduction are involved in the response of drought stress [8,9], and the gene family of MYB, NAC and WRKY is also a response to drought stress [10,11].The changes in L. multiflorum under drought stress are well understood, but the mechanisms by which key drought tolerance genes regulate these phenotypic changes remain unclear.
Plants respond to drought stress in a variety of ways.In order to cope with oxidative stress caused by drought stress, their own enzymatic scavenging system will be used for defense [12,13].On the other hand, changes in leaf structure and morphology also play an important role in coping with drought.The stomata of plants make them more adaptable to the changeable climate and environment [13][14][15].Studies have shown that xerophytes improve their adaptability to water deficit by sinking the stomatal surface of the leaf epidermis under drought conditions, increasing stomatal density and decreasing stomatal diameter [16,17].However, some scholars have come to a different conclusion that the smaller the stomatal density, the stronger the drought resistance, because when the stomatal density decreases, the gas exchange resistance increases, leading to a reduction in transpiration and finally the improvement of drought resistance [18].
Transcription factors (TF) play an important role in plant stress response [19,20].In our previous study, we found that many TF families, such as MYB, NAC, etc., are involved in drought response of L. multiflorum under drought stress (Figure S1) [11].MYB, as one of the most active TF families in the leaf tissue of L. multiflorum under drought stress, has a variety of functions [21].The conserved domain of the MYB gene family is generally located in the N-terminal and consists of the conserved helix-turn-helix protein-DNA binding region (W-X 19 -W-X 19 -W and F/I-X 18 -W-X 18 -W) consisting of 51-52 amino acids [22].
The MYB gene family regulates cell wall, root and stomatal development [23].For example, AtMYB46 and RrMYB18 promote the biosynthesis of the cell wall by up-regulating the expression of cell wall-associated genes [24]; AtMYB96 as ABA signal response genes, can activate cuticular wax biosynthesis [25,26]; AtMYB41 can direct ectopic suberin synthesis [27]; and MdSIMYB1 regulates the expression of auxin response genes to promote root growth [28].It was found that AtMYB60, as a transcriptional suppressor, was involved in regulating the development of guard cells and stomatal movement.After the knockout of AtMYB60, it was found that stomatal size was reduced and drought tolerance was enhanced [29].Interestingly, AtMYB61 regulated stomatal size in contrast to AtMYB60, and overexpression of AtMYB61 reduced stomatal size [30].For MYB to regulate stomatal development, only two genes were found.AtMYB124 and AtMYB88 negatively control the expression of genes associated with stomatal development, but positively regulate the expression of genes related to stress conditions [31,32].However, it is still unclear how the MYB gene family enhances stress resistance in L. multiflorum.
Our research group has previously analyzed the MYB gene family of L. multiflorum, and conducted preliminary verification of candidate genes through subcellular localization and overexpression verification of S. cerevisiae, and found that LmMYB1 is located in the nucleus and can improve the resistance of S. cerevisiae to osmotic stress [11].In order to further study the physiological and molecular mechanisms of LmMYB1 under drought stress, this study overexpressed LmMYB1 in A. thaliana.The results showed that overexpression of LmMYB1 improved the drought tolerance of transgenic A. thaliana plants by participating in cell wall biological processes and reducing stomatal density.

Expression Profile of LmMYB1 Induced by ABA and Drought Stress
According to the previous transcriptomic study, we found that LmMYB1 and genes related to the ABA biosynthetic pathway were strongly induced by drought stress in L. multiflorum (Figure 2A) [11].In order to reveal the biological function of LmMYB1, we analyzed its expression pattern under drought stress and ABA treatment on 'Chuannong No.1′.As is shown in Figure 2, LmMYB1 was up-regulated 3.57 times after 12 h of drought stress, and 3.21 times after 24 h of ABA treatment, respectively (Figure 2).Altogether, LmMYB1 potentially responds to drought and ABA.Thus, LmMYB1 is selected as a candidate gene for further functional studies.

Expression Profile of LmMYB1 Induced by ABA and Drought Stress
According to the previous transcriptomic study, we found that LmMYB1 and genes related to the ABA biosynthetic pathway were strongly induced by drought stress in L. multiflorum (Figure 2A) [11].In order to reveal the biological function of LmMYB1, we analyzed its expression pattern under drought stress and ABA treatment on 'Chuannong No.1' As is shown in Figure 2, LmMYB1 was up-regulated 3.57 times after 12 h of drought stress, and 3.21 times after 24 h of ABA treatment, respectively (Figure 2).Altogether, Lm-MYB1 potentially responds to drought and ABA.Thus, LmMYB1 is selected as a candidate gene for further functional studies.

Overexpression of LmMYB1 Could Enhance Abiotic Stress Tolerance
In order to study the function of LmMYB1 under drought stress, we constructed LmMYB1 overexpressed Arabidopsis driven by the 35S promoter (Figure S2) and converted it to Arabidopsis by Agrobacterium infection.Then, homozygous lines of OE1-OE9 were obtained by hygromycin screening and self-cross for three generations.Among them, the relative expression level of LmMYB1 in OE1, OE4 and OE9 was the highest, which was about 8, 11 and 10 times higher than that of the lowest lines (Figure S3).Therefore, these three lines were selected for subsequent experiments.

Overexpression of LmMYB1 Could Enhance Abiotic Stress Tolerance
In order to study the function of LmMYB1 under drought stress, we constructed LmMYB1 overexpressed Arabidopsis driven by the 35S promoter (Figure S2) and converted it to Arabidopsis by Agrobacterium infection.Then, homozygous lines of OE1-OE9 were obtained by hygromycin screening and self-cross for three generations..Among them, the relative expression level of LmMYB1 in OE1, OE4 and OE9 was the highest, which was about 8, 11 and 10 times higher than that of the lowest lines (Figure S3).Therefore, these three lines were selected for subsequent experiments.
To further verify the function of this gene, Arabidopsis seedlings of similar sizes, WT, OE1, OE4, and OE9, were grown on 1/2 MS plates under different conditions.After one week, there was no significant difference between WT and OE plants on the normal condition (control) plate (Figure 3A).However, under drought stress (200 mM mannitol) and salt stress (100 mM NaCl) conditions, the OE plants exhibited significantly richer aboveground fresh weight than the WT (Figure 3B).Under drought stress (200 mM mannitol) and ABA stress (5 µM), the OE plants showed significantly longer root length than the WT (Figure 3C).In conclusion, exogenous expression of LmMYB1 can improve the tolerance of Arabidopsis seedlings to drought stress, salt stress and ABA stress.To further verify the function of this gene, Arabidopsis seedlings of similar sizes, WT, OE1, OE4, and OE9, were grown on 1/2 MS plates under different conditions.After one week, there was no significant difference between WT and OE plants on the normal condition (control) plate (Figure 3A).However, under drought stress (200 mM mannitol) and salt stress (100 mM NaCl) conditions, the OE plants exhibited significantly richer aboveground fresh weight than the WT (Figure 3B).Under drought stress (200 mM mannitol) and ABA stress (5 µM), the OE plants showed significantly longer root length than the WT (Figure 3C).In conclusion, exogenous expression of LmMYB1 can improve the tolerance of Arabidopsis seedlings to drought stress, salt stress and ABA stress.

LmMYB1-Activated ROS Clearing System Enhances Drought Tolerance
To further study the resistance of OE Arabidopsis adult plants to drought stress, a potted natural drought experiment was conducted in this study.The results showed that, after 14 days without water, the WT rosette leaves turned yellow and wilted, and some died.Some rosette leaves of the OE lines turned yellow but most of them were only restricted in growth (Figure 4A).After resuming watering, it was observed that most of the OE lines recovered, while only a small part of the WT did (Figure 4A,B).Under drought stress, on the one hand, the OE lines have significantly lower MDA content and electrolyte leakage (EL) (Figure 4C,D), indicating that WT plant cells are more seriously damaged by drought stress.On the other hand, the OE lines showed significantly more active antioxidant enzyme activity, indicating that the OE strain had a stronger ability to clear the negative effects of drought stress (Figure 4E-G).Taken together, overexpression of LmMYB1 can reduce oxidative damage under drought by reducing MDA, EL and increasing antioxidant enzyme activity of Arabidopsis adult plants.

LmMYB1-Activated ROS Clearing System Enhances Drought Tolerance
To further study the resistance of OE Arabidopsis adult plants to drought stress, a potted natural drought experiment was conducted in this study.The results showed that, after 14 days without water, the WT rosette leaves turned yellow and wilted, and some died.Some rosette leaves of the OE lines turned yellow but most of them were only restricted in growth (Figure 4A).After resuming watering, it was observed that most of the OE lines recovered, while only a small part of the WT did (Figure 4A,B).Under drought stress, on the one hand, the OE lines have significantly lower MDA content and electrolyte leakage (EL) (Figure 4C,D), indicating that WT plant cells are more seriously damaged by drought stress.On the other hand, the OE lines showed significantly more active antioxidant enzyme activity, indicating that the OE strain had a stronger ability to clear the negative effects of drought stress (Figure 4E-G).Taken together, overexpression of LmMYB1 can reduce oxidative damage under drought by reducing MDA, EL and increasing antioxidant enzyme activity of Arabidopsis adult plants.

LmMYB1 Enhances the Expression of Drought-Related Genes
Drought response involves multiple pathways, such as the proline synthesis pathway, ABA dependent pathway, dehydration response pathway, etc.Many genes are involved in these processes, and these genes often act as marker genes to detect whether the pathway is responsive.To analyze the effect of LmMYB1 overexpression on Arabidopsis stressresponse genes, the expression levels of six genes (AtRAB18, AtAREB, AtRD22, AtCOR47, AtCOR15A and AtDREB) were determined after drought using qRT-PCR.As shown in Figure 5, all genes were up-regulated under drought stress.Meanwhile, under drought stress, the expression level of ABA-dependent-pathway-related genes (AtRAB18, AtAREB and AtRD22) in overexpressed LmMYB1 lines was significantly higher (p < 0.05) than that in the WT after drought stress, suggesting that LmMYB1 may be dependent on the ABA-dependent pathway for drought response (Figure 5).Stress-related genes (AtCOR47, AtCOR15A and AtDREB) were also significantly up-regulated, suggesting that LmMYB1 may regulate plant response to drought stress through the pathways corresponding to these genes.

LmMYB1 Enhances the Expression of Drought-Related Genes
Drought response involves multiple pathways, such as the proline synthesis pathway, ABA dependent pathway, dehydration response pathway, etc.Many genes are involved in these processes, and these genes often act as marker genes to detect whether the pathway is responsive.To analyze the effect of LmMYB1 overexpression on Arabidopsis stress-response genes, the expression levels of six genes (AtRAB18, AtAREB, AtRD22, AtCOR47, AtCOR15A and AtDREB) were determined after drought using qRT-PCR.As AtAREB and AtRD22) in overexpressed LmMYB1 lines was significantly higher (p < 0.05) than that in the WT after drought stress, suggesting that LmMYB1 may be dependent on the ABA-dependent pathway for drought response (Figure 5).Stress-related genes (AtCOR47, AtCOR15A and AtDREB) were also significantly up-regulated, suggesting that LmMYB1 may regulate plant response to drought stress through the pathways corresponding to these genes.

Transcriptomic Analysis of OE vs. WT under Drought Stress
In order to explore the molecular mechanism of overexpression of LmMYB1 to improve drought resistance, RNA-Seq analysis was performed on transgenic and wild-type A. thaliana under drought stress.Compared with the WT, 143 genes were up-regulated and 195 genes down-regulated in LmMYB1-OE plants (Figure S4).The term of up-regulated gene enrichment is mostly related to cell wall pathways, such as the Glucuronoxylan metabolic process, Xylan biosynthetic process, Xylan metabolic process, cell wall macromolecule biosynthetic process, etc., which specifies the stronger activity of genes related to cell wall pathways in LmMYB1-OE plants than the WT (Figure 6A,C).Most of the terms in which down-regulated expression genes were enriched were related to responses to other stresses (Figure 6B,C).The results showed that, under drought stress, overexpression of LmMYB1 in A. thaliana tended to mobilize cell wall-related pathways in response to drought, while reducing the response to other stresses, which should be a strategy for LmMYB1 to cope with drought stress as an environmental stress factor.
To confirm the RNA-Seq results, 11 DEGs from different categories were picked for qRT-PCR analysis (Figure 6D).Among the five cell wall pathway-related genes (PRR1, IRX12, CTL2, KIN1 and XTH6), except XTH6, all the others were up-regulated in LmMYB1-OE lines under drought stress.Similarly, the expression of antioxidant oxidase and JA signal response-related genes were also up-regulated in LmMYB1-OEs in response to drought treatment.Taken together, these results strongly demonstrated that LmMYB1 mediated drought tolerance by affecting cell way-related pathways, antioxidant oxidase activity and JA signal response.

Transcriptomic Analysis of OE vs. WT under Drought Stress
In order to explore the molecular mechanism of overexpression of LmMYB1 to improve drought resistance, RNA-Seq analysis was performed on transgenic and wild-type A. thaliana under drought stress.Compared with the WT, 143 genes were up-regulated and 195 genes down-regulated in LmMYB1-OE plants (Figure S4).The term of up-regulated gene enrichment is mostly related to cell wall pathways, such as the Glucuronoxylan metabolic process, Xylan biosynthetic process, Xylan metabolic process, cell wall macromolecule biosynthetic process, etc., which specifies the stronger activity of genes related to cell wall pathways in LmMYB1-OE plants than the WT (Figure 6A,C).Most of the terms in which down-regulated expression genes were enriched were related to responses to other stresses (Figure 6B,C).The results showed that, under drought stress, overexpression of LmMYB1 in A. thaliana tended to mobilize cell wall-related pathways in response to drought, while reducing the response to other stresses, which should be a strategy for LmMYB1 to cope with drought stress as an environmental stress factor.
To confirm the RNA-Seq results, 11 DEGs from different categories were picked for qRT-PCR analysis (Figure 6D).Among the five cell wall pathway-related genes (PRR1, IRX12, CTL2, KIN1 and XTH6), except XTH6, all the others were up-regulated in LmMYB1-OE lines under drought stress.Similarly, the expression of antioxidant oxidase and JA signal response-related genes were also up-regulated in LmMYB1-OEs in response to drought treatment.Taken together, these results strongly demonstrated that LmMYB1 mediated drought tolerance by affecting cell way-related pathways, antioxidant oxidase activity and JA signal response.
To further explore the phenotypic effects of the genetic changes, the epidermis of the WT and OE lines were observed by optical microscope.Three biological replicates were set for the WT and OE lines, and fixed sections of rosette leaves in the same position were selected for each replicate.For each section, 10 photos of visual fields were randomly collected under visual fields (10 × 40) to calculate stomatal density.Interestingly, the results showed that the average stomatal density of the WT, OE1 and OE4 per field was 6.267, 5.200 and 4.933.OE lines significantly decreased leaf stomatal density by 17.02% (n = 30, p = 0.0026) and 21.29% (n = 30, p = 0.0002) compared with the WT (Figure 7).These results further suggest that LmMYB1 could enhance drought tolerance of A. thaliana by reducing stomatal density.Unfortunately, analysis of the transcriptome data found no significant differences in genes related to stomatal development and its signal transduction regulatory pathways between WT and OE (Figure S3).To further explore the phenotypic effects of the genetic changes, the epidermis of the WT and OE lines were observed by optical microscope.Three biological replicates were set for the WT and OE lines, and fixed sections of rosette leaves in the same position were selected for each replicate.For each section, 10 photos of visual fields were randomly collected under visual fields (10 × 40) to calculate stomatal density.Interestingly, the results 5.200 and 4.933.OE lines significantly decreased leaf stomatal density by 17.02% (n = 30, p = 0.0026) and 21.29% (n = 30, p = 0.0002) compared with the WT (Figure 7).These results further suggest that LmMYB1 could enhance drought tolerance of A. thaliana by reducing stomatal density.Unfortunately, analysis of the transcriptome data found no significant differences in genes related to stomatal development and its signal transduction regulatory pathways between WT and OE (Figure S3).

Discussion
Drought tolerance is a complex and inherent trait of plants that responds to drought stress at multiple levels [33].At one level, changes in transcriptional regulation involve thousands of genes, and transcription factors play a pivotal role in this process [34].The MYB family is one of the largest transcription factor families and shows specific roles in response to abiotic stress [23,35,36].This study systematically characterized the molecular functions of LmMYB1 in A. thaliana in response to drought stress.As a transcription factor, LmMYB1 increases the expression of downstream stress-related genes and ABA-dependent genes.At the same time, LmMYB1 improves ROS clearing by increasing antioxidant enzyme activity, and improves osmoprotection by decreasing MDA and EL.Further, we observed that LmMYB1 caused a decrease in the epidermal stomatal density of Arabidopsis.The results provided the foundation for further clarification of the stress tolerance mechanism and molecular breeding of L. multiflorum.
As an important plant hormone, abscisic acid (ABA) plays a key role in regulating plant responses to abiotic stresses [37].Studies have shown that ABA is involved in plant responses to various stresses such as drought, salt, and high temperature [38].This study was studied the LmMYB1 from L. multiflorum, which was found to be in the HTH conservative structure domain, which was induced by drought and ABA in L. multiflorum (Figure 2).At the same time, the drought stress treatment increased the expression of AtRD22, AtRAB18 and AtAREB in the transgenic LmMYB1 Arabidopsis (Figure 5).The root growth of the transgenic Arabidopsis seedlings was significantly better than the wild type

Discussion
Drought tolerance is a complex and inherent trait of plants that responds to drought stress at multiple levels [33].At one level, changes in transcriptional regulation involve thousands of genes, and transcription factors play a pivotal role in this process [34].The MYB family is one of the largest transcription factor families and shows specific roles in response to abiotic stress [23,35,36].This study systematically characterized the molecular functions of LmMYB1 in A. thaliana in response to drought stress.As a transcription factor, LmMYB1 increases the expression of downstream stress-related genes and ABA-dependent genes.At the same time, LmMYB1 improves ROS clearing by increasing antioxidant enzyme activity, and improves osmoprotection by decreasing MDA and EL.Further, we observed that LmMYB1 caused a decrease in the epidermal stomatal density of Arabidopsis.The results provided the foundation for further clarification of the stress tolerance mechanism and molecular breeding of L. multiflorum.
As an important plant hormone, abscisic acid (ABA) plays a key role in regulating plant responses to abiotic stresses [37].Studies have shown that ABA is involved in plant responses to various stresses such as drought, salt, and high temperature [38].This study was studied the LmMYB1 from L. multiflorum, which was found to be in the HTH conservative structure domain, which was induced by drought and ABA in L. multiflorum (Figure 2).At the same time, the drought stress treatment increased the expression of AtRD22, AtRAB18 and AtAREB in the transgenic LmMYB1 Arabidopsis (Figure 5).The root growth of the transgenic Arabidopsis seedlings was significantly better than the wild type (Figure 3A,C).These results show that LmMYB1 is involved in the plant's response to adversity through the ABA-dependent pathway (Figure 8).(Figure 3A,C).These results show that LmMYB1 is involved in the plant's response to adversity through the ABA-dependent pathway (Figure 8).As sessile organisms, plants cannot move and could make some morphological changes in the face of external stresses to resist them [39].Changes in leaf morphology include changes in leaf shape, epidermis and stomata [40][41][42].Based on the statistics of stomatal parameters and drought phenotypes of rice populations after radiation mutagenesis, researchers found that smaller stomata with lower stomatal density had higher resistance and higher seed yield under drought stress [43].Similar patterns have been found in grape [44] and carrot [45], where stomatal density is negatively correlated with plant drought tolerance.The findings in this study, that LmMYB1 overexpressed Arabidopsis has lower stomatal density and higher drought resistance, are also consistent with these studies just mentioned (Figure 7).As stomata are exchange channels for water and gas, it seems like a paradoxical and interesting question that overexpression of LmMYB1 reduces stomatal density conferring drought tolerance with little influence on normal growth (Figure 3B) rate.Since L. multiflorum is a kind of high-quality forage, LmMYB1 would be a valuable candidate gene conferring drought tolerance.We will validate its function in its native plants via overexpression assay and investigate detailed information and the mechanisms of LmMYB1-mediated stomatal density in the regulation of drought tolerance in future studies.
Stomatal density is affected by transcription factors, signal peptides and environmental factors.These internal and external factors can not only affect stomatal development independently, but also synergistically regulate the formation of stomata [46].The regulation process of stomatal density and distribution on the leaf surface is highly plastic, and there are many influencing factors [47][48][49].In addition to the identified genes directly regulating stomatal development (SPCH/MUTE/FAMA) [50], other potential factors may also contribute.Studies have shown that fucosylation-dependent dimerization of the cell wall pectic domain rhamnogalacturonan-II may be essential for stomatal development and leaf water retention [51].In addition, it has been found that galactosyltransferase regulates the synthesis and composition of pectin, thereby affecting stomatal development and dynamics [52].In this study, the RNA-Seq data revealed a significant enrichment of a large number of up-regulated genes related to cell wall biological pathways (Figure 6A,B), such as genes involved in the regulation of secondary cell wall biosynthesis, pinoresinol reductase catalyzes (PRR1) [53], irregular xylem12 (IRX12) [54] and chitinase-like2 protein (CTL2) [55], which play important roles in cell wall development.A recent study found that ZmIRX15A (irregular xylem 15A), a gene encoding the xylem deposition enzyme in maize, As sessile organisms, plants cannot move and could make some morphological changes in the face of external stresses to resist them [39].Changes in leaf morphology include changes in leaf shape, epidermis and stomata [40][41][42].Based on the statistics of stomatal parameters and drought phenotypes of rice populations after radiation mutagenesis, researchers found that smaller stomata with lower stomatal density had higher resistance and higher seed yield under drought stress [43].Similar patterns have been found in grape [44] and carrot [45], where stomatal density is negatively correlated with plant drought tolerance.The findings in this study, that LmMYB1 overexpressed Arabidopsis has lower stomatal density and higher drought resistance, are also consistent with these studies just mentioned (Figure 7).As stomata are exchange channels for water and gas, it seems like a paradoxical and interesting question that overexpression of LmMYB1 reduces stomatal density conferring drought tolerance with little influence on normal growth (Figure 3B) rate.Since L. multiflorum is a kind of high-quality forage, LmMYB1 would be a valuable candidate gene conferring drought tolerance.We will validate its function in its native plants via overexpression assay and investigate detailed information and the mechanisms of LmMYB1-mediated stomatal density in the regulation of drought tolerance in future studies.
Stomatal density is affected by transcription factors, signal peptides and environmental factors.These internal and external factors can not only affect stomatal development independently, but also synergistically regulate the formation of stomata [46].The regulation process of stomatal density and distribution on the leaf surface is highly plastic, and there are many influencing factors [47][48][49].In addition to the identified genes directly regulating stomatal development (SPCH/MUTE/FAMA) [50], other potential factors may also contribute.Studies have shown that fucosylation-dependent dimerization of the cell wall pectic domain rhamnogalacturonan-II may be essential for stomatal development and leaf water retention [51].In addition, it has been found that galactosyltransferase regulates the synthesis and composition of pectin, thereby affecting stomatal development and dynamics [52].In this study, the RNA-Seq data revealed a significant enrichment of a large number of up-regulated genes related to cell wall biological pathways (Figure 6A,B), such as genes involved in the regulation of secondary cell wall biosynthesis, pinoresinol reductase catalyzes (PRR1) [53], irregular xylem12 (IRX12) [54] and chitinase-like2 protein (CTL2) [55], which play important roles in cell wall development.A recent study found that ZmIRX15A (irregular xylem 15A), a gene encoding the xylem deposition enzyme in maize, negatively regulates stomatal density by changing cell wall composition [56], which is consistent with our observed results (Figure 6D).However, how LmMYB1 mediates downstream genes integrated in cell wall-related pathways in regulating drought tolerance remains unclear.Further studies on such themes would provide more knowledge of the MYB-regulated network.

Lolium Multiflorum Material, Growth Condition and Treatment
The samples of L. multiflorum cv.'Chuannong No.1 for expression pattern analysis were planted in the growth chamber at Chengdu Campus of Sichuan Agricultural University.The diurnal cycle of the growth chamber was 14/10 h and 22/20 • C. Photons were 750 µmol m −2 •s −1 .The air humidity was 70%.The seeds of L. multiflorum cv.'Chuannong No.1 were disinfected and cultured in square pots (height 6 cm, length 25 cm, width 6 cm).The seeds were disinfected with 1% sodium hypochlorite solution for 10 min, washed with deionized water 3-5 times, and 150 seeds were evenly sown in each square basin.Water was used before germination, and 1/2 Hoagland solution was used after germination.When the seedlings were grown under normal conditions for 30 days, stress treatment was carried out.To simulate drought treatment, 15% PEG-6000 was used, and 30 µmol ABA was used to stimulate ABA treatment.Leaves were collected at 0 h, 3 h, 6 h, 12 h and 24 h after drought and ABA treatment.All tissues were stored in liquid nitrogen immediately after sampling and then at −80 • C.

Multiple Sequence Alignment and Bioinformation Analysis of the LmMYB1 Gene
The LmMYB1 gene sequence was extracted from transcriptome data [11], after cloning, to obtain the exact sequence.At the same time, other sequences of homologous proteins were downloaded from the National Center for Biotechnology Information (NCBI).DNA-MAN software, version 5.1.5(Lynnon BioSoft, San Ramon, CA, USA), was used to analyze the amino acid sequences under default parameters.A phylogenetic tree was subsequently constructed using the neighbor-joining method with MEGA software (version 11.0.10) with the following parameters: the bootstrap test method (1000 replicates) and pairwise deletion [57].

Generation of Transgenic Arabidopsis Plants
For Arabidopsis transformation, the CDS of LmMYB1 was cloned from the cDNA of L. multiflorum leaves and ligated into the PHG vector under the control of the CaMV 35S promoter.PHG-LmMYB1 was further transformed into Agrobacterium tumefaciens GV3101.Arabidopsis transformation was conducted with the floral dipping method with A. thaliana Col-0 [58].The positive transgenic plants were screened by Hygromycin, and at least 9 independent transgenic plants were obtained until the T3 generation for further analysis.

Evaluation of Drought Resistance in Transgenic Lines
The seeds of different lines were seeded in 1/2 MS medium and cultured vertically.When the root length was about 1 cm, they were transferred to 1/2 MS, 1/2 MS + 5 µM ABA, 1/2 MS + 200 mM mannitol and 1/2 MS + 100 mM NaCl in a sterile environment.The phenotypes were observed and photographed after stress, while above weight and root length of the seedlings were measured.
The WT and OE lines (OE1, OE4, OE9) with the same growth after 12 days of germination on 1/2 MS medium were transferred to a soil culture pot (the substrate was peat soil, perlite and vermiculite, the volume ratio was 8:1:1).After 3 weeks of cultivation under normal conditions, watering was stopped, and the phenotype was observed and photographed when the natural drought occurred.One week after rewatering, observation continued and photographs were taken again, and the survival rate was recorded.Drought stress-related physiological indicators including antioxidant enzymes (POD, SOD and CAT), MDA content and electrolyte leakage (EL) were measured before and after drought stress to identify the responses of different wild type and overexpression lines to drought stress.

Extraction of RNA and qRT-PCR Analysis
Total RNA was extracted according to the manufacturer's instructions for the Trelief ® RNAprep Pure Plant Kit (# TSP411, Tsingke Biotechnology Co., Ltd., Beijing, China).For the next step of reverse transcription to proceed smoothly, the RNA concentration should be above 200 ng/uL, and the OD260/OD280 value should be 1.8-2.0.Then, the first-strand cDNA was synthesized from 1000 ng RNA using SynScript ® III RT SuperMix for qPCR (#TSK314S, Tsingke Biotechnology Co., Ltd., Beijing, China).qRT-PCR was performed using an ArtiCan CEO SYBR qPCR Mix (#TSE401, Tsingke Biotechnology Co., Ltd., Beijing, China).The reaction solutions and qRT-PCR procedures were performed according to the kit's instructions.Finally, the qRT-PCR reaction was performed using CFX Connect™ Real-Time System (Bio-Rad, Hercules, CA, USA).LmHIS3 and LmeIF4A (Table S1) were used as the reference genes to analyze the expression patterns of LmMYB1 under drought and ABA in L. multiflorum [59].The specific primers used for the expressions of stress-related and ABA-signaling gene analysis are shown in Table S1.AtACT2 (Table S1) was used as the reference gene to analyze expression levels of marker genes.qRT-PCR data were analyzed using the 2 −∆∆Ct method [60].Three technical replicates of each sample were analyzed with qRT-PCR.

RNA-Seq Analysis
The aboveground leaf of the wild type and overexpressed line (OE4) of A. thaliana were taken after 30 days of growth by natural drought after 6 days.Each sample consisted of three individual plants, with three biological replicates at each point.Total RNA was extracted for cDNA synthesis.The construction of the cDNA library, sequencing, and data filtering were performed by Gene Denovo (Gene Denovo Co., Ltd., Guangzhou, China).The differentially expressed genes (DEGs) were analyzed using DESeq2 (log2 fold changes ≥ 2, FDR < 0.001) [61].To understand which genes LmMYB1 affects and what function those genes have, enrichment analyses and gene ontology (GO) annotation were analyzed using TopGO and the online tool agriGO (http://bioinfo.cau.edu.cn/agriGO/,accessed on 15 September 2021) [62].To cluster and screen DEGs and determine which pathways are affected by LmMYB1, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed by cluster-Profiler [63].All the raw data generated are housed in the National Genome Sciences Data Center (CRA011179).

Measurement of Stomatal Density
Samples were taken at 30 d from Arabidopsis leaves to observe stomatal density.The clipped leaves were quickly placed in 2.5% glutaraldehyde (pH 7.2) for fixation.The largest leaf of 3 independent plants from each line was taken and stored in a refrigerator at 4 • C. The leaves were removed from the glutaraldehyde fixing solution, the leaf skin was wiped clean with absorbent cotton dipped in alcohol and a thin layer of nail polish was smeared on the skin.After drying, the nail polish film was peeled off with tweezers and placed on a slide so that it could be stretched naturally and observed under a microscope.A slice was made for each sample.Ten visual fields were randomly selected from the section, observed under an optical microscope (10 × 40), photographed, and the number of visual fields was counted and the average value calculated.

Statistical Analysis
In this study, the values obtained in the figures were expressed as the means.The error bars were based on standard deviation (SD) in figures.A one-way ANOVA followed by Tukey's multiple comparisons tests were used to determine the significant differences among groups.

Conclusions
In this study, the coding sequence of the LmMYB1 gene was cloned from L. multiflorum and functionally verified in Arabidopsis overexpression.The results showed that LmMYB1

Figure 1 .
Figure 1.Multi-sequence alignment and evolutionary relationship analysis of the LmMYB1 protein.(A) Secondary structure of the LmMYB1 protein.The green boxes represent conserved domains, and the red triangles represent conserved amino acids.(B) Comparison between the homology of the LmMYB1 protein and the MYB protein in other plants.The conserved regions of the amino acid sequence are marked with green boxes.R represents the conserved domain of MYB (W-X19-W-X19-W and F/I-X18-W-X18-W).The black background represents a 100% consistent sequence.Red and blue represent 75% and 50% respectively.(C) Phylogenetic tree analysis of LmMYB1 (noted with a red star) and homologous in other plants.

Figure 1 .
Figure 1.Multi-sequence alignment and evolutionary relationship analysis of the LmMYB1 protein.(A) Secondary structure of the LmMYB1 protein.The green boxes represent conserved domains, and the red triangles represent conserved amino acids.(B) Comparison between the homology of the LmMYB1 protein and the MYB protein in other plants.The conserved regions of the amino acid sequence are marked with green boxes.R represents the conserved domain of MYB (W-X 19 -W-X 19 -W and F/I-X 18 -W-X 18 -W).The black background represents a 100% consistent sequence.Red and blue represent 75% and 50% respectively.(C) Phylogenetic tree analysis of LmMYB1 (noted with a red star) and homologous in other plants.

Figure 2 .
Figure 2. Gene expression profile of L. multiflorum under abiotic stress.(A) Related genes of the ABA biosynthetic pathway and LmMYB1 under drought stress in the previous transcriptome data [11].The ordinate represents the fold change (CK vs. DR) of genes under drought stress.There are three biological replicates per treatment, with the average shown in the figure.The color from blue to red indicates low to high gene expression.The expression profile of LmMYB1's response to drought stress (B) and ABA treatment (C) in 'Chuannong No. 1′.Three biological repetitions per time point, three technical repetitions.The standard deviation is represented by the error bar.LmHIS3 and LmeIF4A were used as the reference genes and qRT-PCR data were analyzed using the 2 −∆∆Ct method.The asterisk indicates the difference compared to 0 h (* p < 0.05, ** p < 0.01).

Figure 2 .
Figure 2. Gene expression profile of L. multiflorum under abiotic stress.(A) Related genes of the ABA biosynthetic pathway and LmMYB1 under drought stress in the previous transcriptome data [11].The ordinate represents the fold change (CK vs. DR) of genes under drought stress.There are three biological replicates per treatment, with the average shown in the figure.The color from blue to red indicates low to high gene expression.The expression profile of LmMYB1's response to drought stress (B) and ABA treatment (C) in 'Chuannong No. 1'.Three biological repetitions per time point, three technical repetitions.The standard deviation is represented by the error bar.LmHIS3 and LmeIF4A were used as the reference genes and qRT-PCR data were analyzed using the 2 −∆∆Ct method.The asterisk indicates the difference compared to 0 h (* p < 0.05, ** p < 0.01).

Figure 4 .
Figure 4. Overexpression of LmMYB1 can improve drought tolerance in A. thaliana.(A) Phenotype of the WT and transgenic lines (OE1, OE4, OE9) under drought stress.(B) Survival rate of the WT and transgenic lines after re-watering.(C-G) Shows measurements of tolerance-related physiological parameters.Data are presented as mean and SD values of four independent experiments.Asterisks indicate significant difference (* p < 0.05, ** p < 0.01, by independent sample t-test) between the WT and OE lines.

Figure 4 .
Figure 4. Overexpression of LmMYB1 can improve drought tolerance in A. thaliana.(A) Phenotype of the WT and transgenic lines (OE1, OE4, OE9) under drought stress.(B) Survival rate of the WT and transgenic lines after re-watering.(C-G) Shows measurements of tolerance-related physiological parameters.Data are presented as mean and SD values of four independent experiments.Asterisks indicate significant difference (* p < 0.05, ** p < 0.01, by independent sample t-test) between the WT and OE lines.

Figure 5 .
Figure 5. Analysis of stress resistance gene expression in WT and overexpressed LmMYB1 under drought stress.The value is the average of three biological repetitions and three technical repetitions.The standard deviation is represented by the error bar.AtACT2 was used as the reference gene and qRT-PCR data were analyzed using the 2 −∆∆Ct method.The asterisk indicates the difference compared to WT (* p < 0.05, ** p < 0.01).

Figure 5 .
Figure 5. Analysis of stress resistance gene expression in WT and overexpressed LmMYB1 under drought stress.The value is the average of three biological repetitions and three technical repetitions.The standard deviation is represented by the error bar.AtACT2 was used as the reference gene and qRT-PCR data were analyzed using the 2 −∆∆Ct method.The asterisk indicates the difference compared to WT (* p < 0.05, ** p < 0.01).

16 Figure 6 .
Figure 6.GO enrichment analysis and core DEGs heat map of WT vs. OE gene.(A,B) The top ten terms enriched by up-regulated and down-regulated genes were shown, respectively.The vertical axis represents different biological processes, and the horizontal axis represents rich factors.The size of the dots represents the number of genes enriched.The color of the dots represents the -log10(Qvalue) of the event.(C) The heat map of core genes.The core gene is derived from the biological process enriched in the previous step.The right ordinate is the number and name of different genes.(D) Validation of transcriptome data of key genes.In qRT-PCR, each square is formed by three technical repetitions and AtACT2 was used as the reference gene.The right ordinate shows the name of the gene and its approximate functional range.The color from blue to red indicates low to high gene expression.

Figure 6 .
Figure 6.GO enrichment analysis and core DEGs heat map of WT vs. OE gene.(A,B) The top ten terms enriched by up-regulated and down-regulated genes were shown, respectively.The vertical axis represents different biological processes, and the horizontal axis represents rich factors.The size of the dots represents the number of genes enriched.The color of the dots represents the -log 10 (Qvalue) of the event.(C) The heat map of core genes.The core gene is derived from the biological process enriched in the previous step.The right ordinate is the number and name of different genes.(D) Validation of transcriptome data of key genes.In qRT-PCR, each square is formed by three technical repetitions and AtACT2 was used as the reference gene.The right ordinate shows the name of the gene and its approximate functional range.The color from blue to red indicates low to high gene expression.

Figure 7 .
Figure 7. Overexpression of LmMYB1 decreased stomatal density in A. thaliana.(A-C) Representation figure of stomatal density of the WT and transgenic lines (OE1, OE4).The red circle represents each stoma.Scale = 1 µm.(D) Statistics of stomatal density of the WT and transgenic lines (OE1 OE4).There were three biological replicates per line and 10 visual fields (10 × 40) were collected for each biological replicate.Asterisks indicate significant difference (** p < 0.01, *** p < 0.001, by independent sample t-test) between WT and OE lines.

Figure 7 .
Figure 7. Overexpression of LmMYB1 decreased stomatal density in A. thaliana.(A-C) Representation figure of stomatal density of the WT and transgenic lines (OE1, OE4).The red circle represents each stoma.Scale = 1 µm.(D) Statistics of stomatal density of the WT and transgenic lines (OE1, OE4).There were three biological replicates per line and 10 visual fields (10 × 40) were collected for each biological replicate.Asterisks indicate significant difference (** p < 0.01, *** p < 0.001, by independent sample t-test) between WT and OE lines.

Figure 8 .
Figure 8. Hypothetical model of the LmMYB1-regulated network in the drought response.

Figure 8 .
Figure 8. Hypothetical model of the LmMYB1-regulated network in the drought response.