R3-MYB proteins OsTCL1 and OsTCL2 modulate seed germination via dual pathways in rice

Yong Yi, Chn Lin, Xueyn Peng, Meishn Zhng, Jiming Wu, Chunmei Meng, Shengcho Ge,Yunfeng Liu,*, Yun Su,*

a State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University,Nanning 530004,Guangxi, China

b Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China

Keywords:ABA Drought stress Phospholipase Salt stress Rice

ABSTRACT Salt and drought stress are common abiotic factors that exert a detrimental influence on seed germination, potentially leading to significantly impaired growth and production in rice.Gaining a comprehensive understanding of the molecular response of seeds to abiotic stress during the germination is of paramount importance.In the present study, we identified two R3-MYB genes in rice, namely OsTCL1 and OsTCL2, and characterized their roles in regulating seed germination under salt and drought stress.Plants with tcl1 and tcl2 mutant alleles exhibited delayed seed germination,particularly under stress conditions.The tcl1 tcl2 double mutant showed an even more pronounced reduction in germination during initial stages of germination,thereby indicating a redundant regulatory function of OsTCL1 and OsTCL2 in seed germination under abiotic stresses.Furthermore,we demonstrated that the transcript levels of several phospholipase D (PLD) genes were downregulated in the tcl1 tcl2 mutant, resulting in a decreased level of the phosphatidic acid (PA) product.Application of 1-butanol, a competitive substrate inhibitor of PLD-dependent production of PA,attenuated the stress response of the tcl1 tcl2 mutant.This suggests that OsTCL1 and OsTCL2 partially modulate seed germination through the PLD-PA signaling pathway.Moreover,there were alterations in the expression of genes involved in abscisic acid(ABA)biosynthesis,metabolism and signaling transduction in the double mutant.These changes affected the endogenous ABA level and ABA response, thereby influencing seed germination.Application of both 1-butanol and ABA synthesis inhibitor sodium tungstate(Na2WO4)nearly eliminated the differences in stress response between wild type and the tcl1 tcl2 mutant.This indicates that OsTCL1 and OsTCL2 synergistically coordinate seed germination under abiotic stresses through both the PLD-PA signaling and ABA-mediated pathways.

1.Introduction

Rice(Oryza sativa)holds significant importance as a staple food source for humans,and its production plays a crucial role in ensuring food security [1,2].The successful initiation of seed germination forms the fundamental basis for rice production and greatly influences the vigor and development of rice seedlings.The ambient environmental conditions, particularly humidity and soil salt concentrations, are critical factors influencing seed germination[3,4].Abiotic stresses can cause prolonged seed dormancy,reduced germination rates, weakened growth of seedling roots and shoots,and accelerated senescence of roots,ultimately leading to compromised crop growth and yield[5,6].The regulation of seed germination under abiotic stress involves a complex molecular network composed of various proteins and signaling molecules.Previous studies established the tight connection between seed germination and plant hormones.Notably, abscisic acid (ABA) serves as a key hormone that orchestrates the balance between seed dormancy and germination [7–9].ABA accumulates during seed maturation to cause seed dormancy and prevent premature germination.However, when environmental conditions become favorable, the seed ABA level decreases, initiating germination [8,10].Consequently,crucial genes involved in ABA synthesis,degradation and signaling transduction were identified as significant players in seed development and germination [11].These genes include ABA-responsive genes such as PYL, PP2C, OsbZIP10, OsABI1, OREB1, LEA3, RAB21 and SnRK2 [12–17], as well as ABA synthesis genes OsNCED and OsABA8 [12,18,19].In contrast, gibberellic acid (GA) exhibits an antagonistic effect on seed germination as it promotes seed germination[20].Other plant hormones such as cytokinin(CK)and ethylene (ETH) also facilitate seed germination by counteracting the effects of ABA, whereas auxin acts synergistically with ABA in inducing seed dormancy [21].In addition, salicylic acid (SA) and jasmonic acid (JA) affect seed germination by modulating the ABA or GA signaling pathways [22–24].

Phospholipase Ds (PLDs) play indispensable roles in maintaining plasma membrane stability and lipid homeostasis, and are involved in various physiological and developmental events in plants, such as seed germination, vegetative growth, pollen tube elongation, and leaf senescence [25,26].PLDs are essential for response to abiotic stress and interact with ABA in mediating stress reactions[27–30].For example,under salt and drought conditions,CsPLDα is induced in cucumber and enhances tolerance to high osmotic stress,alleviating the inhibitory effects of ABA on seed germination and seedling growth [27].Tobacco PLDα modulates stomata closure during ABA treatment,contributing to water conservation and drought resistance [28].In Arabidopsis, AtPLDα1 participates in ABA signaling by repressing the function of ABI1,a negative regulator of ABA response[29].Transcriptomic analysis identified seven rice PLD genes (OsPLDα1, OsPLDα4, OsPLDα5,OsPLDα6,OsPLDδ1,OsPLDφ and OsPLDζ1)that exhibited differential expressions under salt, cold, and drought stress [31].During seed germination, the activity of rice OsPLDβ1 was promoted and activated Stress-Activated Protein Kinase(SAPK)to induce ABA signaling transduction.Suppression of OsPLDβ1 expression reduced the sensitivity of seeds to exogenous ABA [30].The functions of PLDs are predominantly mediated through their product, phosphatidic acid (PA), which serves as an intracellular signaling molecule.PA is rapidly produced in response to various biotic and abiotic stresses [32].As a secondary lipid signaling messenger PA physically interacts with a number of target proteins involved in abiotic stress response and plant hormone-mediated pathways [33], including components of the ABA signaling network, such as ABI1 [29],RbohD/F [34], RGS1 [35] and SPHK1/2 [36].

R3-MYB is a subfamily of MYB transcription factors that feature a single or partial MYB repeat sequence in their proteins that are referred to as 1R-MYB and 1/2R-MYB,respectively [37,38].Understanding of the biological functions of R3-MYB in rice compared with other MYB proteins is limited.Two R3-MYB members, TRIPTYCHON (TRY) and CAPRICE (CPC) in Arabidopsis have been relatively well studied and shown to function in various physiological processes, including cell differentiation, plant growth, and flowering determination [39–41].Interestingly, studies on soybean have shown that homologs of CPC and TRY,namely GmMYB73,induce expression of several PLDs(PLDα1,PLDβ1,PLDζ1 and PLDγ1) [42], suggesting involvement of CPC and TRY in plant stress responses by regulating PLD expression.One rice R3-MYB protein, TRICHOMELESS1 (OsTCL1), a homolog of Arabidopsis CPC and TRY, was shown to regulate trichome and root hair formation when ectopically expressed in Arabidopsis, but not in rice [43].

In this study, we investigated the biological functions of two rice R3-MYB genes, OsTCL1 and OsTCL2, and elucidated their roles in regulating seed germination under abiotic stress conditions.Mutations in OsTCL1 and OsTCL2 led to a delay in seed germination under salt and drought stress.This delay was attributed,in part,to a decrease in PLD activity and a reduction in cellular PA levels in tcl1 tcl2 double mutants.These alterations in PLD-PA signaling may affect response to ABA, a hormone known to play a critical role in regulation of seed germination.We further demonstrated that OsTCL1 and OsTCL2 influence endogenous ABA levels and ABA signaling transduction by regulating the transcription of ABA synthesis and degradation genes, as well as genes involved in the ABA signaling transduction pathway.Collectively,our findings suggest that OsTCL1 and OsTCL2 are involved in seed germination by participating in both the PLD-PA signaling pathway and ABA-mediated signaling pathway.These insights expand our understanding of the functional role of R3-MYB proteins in the seed germination and provide valuable genetic resources for improving the rate of seed germination in crop species.

2.Materials and methods

2.1.Plant materials and plasmids

The rice material used in this study was Oryza sativa L.spp.japonica cv.Nipponbare.Rice lines with mutant genes tcl1 and tcl2 were generated by the CRISPR/Cas9 system using vector pYLgRNA-OsU6a which was kindly gifted by Dr.Yaoguang Liu,South China Agricultural University.The mutation sites were identified by sequencing and confirmed by PCR and following restriction enzymatic digestion.

2.2.Measurement of grain agronomic traits

Rice plants were grown in a paddy filed at Guangxi University under natural conditions with regular field management,including irrigation, fertilizer application and pest control.Grains were collected when plants were fully mature and subject to agronomic trait measurements including grain length, grain width, 1000-grain weight and grain chalkiness using an automatic MICROTEK ScanMaker i800 Plus (Shanghai Zhongjing Technology Co., LTD.,Shanghai, China) seed testing system.All measurements were repeated three times.

2.3.Stress treatments

To analyze the germination rates under various stress treatments,seeds were cultured for 5 d on standard 1/2 MS solid medium containing 0 mmol L-1and 150 mmol L-1NaCl,250 mmol L-1mannitol,or 5 μmol L-1ABA with germination rate recorded every day.For the physiological experiments, the plants were cultivated in a bottomless 96-well plate floated on a container filled with 1/4 MS nutrient solution.The plants at the four-leaf stage,were transferred to 1/4 MS nutrient solution containing 150 mmol L-1NaCl(treated for 5 d and rehydrated for 10 d)or 250 mmol L-1mannitol(treated for 4–7 d and rehydrated for 10–12 d) for salt or drought stress treatment,after which survival rates were counted.A whole plant with green leaves and capable of regenerating shoots was considered a surviving plant.Tissue samples for assays of physiological parameters were obtained at various times after treatment.To evaluate the influence of seed germination of WT and the tcl1 tcl2 mutant by endogenous levels of PA and ABA under various conditions, 0.1% (v/v) 1-butanol (2-butanol as control) and 100 μmol L-1sodium tungstate were applied, respectively.

2.4.Seed germination assay and statistics

To measure seed germination rate, germinated seeds were counted every 24 h after sowing.Seedlings with at least 1 mm of radical was considered germinated.At least three independent experimental repeats were performed.Germination rate equals the number of germinated seeds/total seed number×100%.Significance differences between two groups of data were assessed using Student’s-t tests (*, P < 0.05; **, P < 0.01).

2.5.RNA extraction and qRT-PCR

Total RNA from various tissues including embryos,roots,stems,leaves and panicles was extracted using a TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) extraction kit.The RNA was reverse transcribed into cDNA using a StarScript II RT mix with a gDNA remover kit (GenStar).Quantitative RT-PCR (qRT-PCR) was performed using a TB Green Premix Ex Taq II (Tli RNase H plus)(Takara) kit on a Roche Light Cycler 96 instrument (Roche, Mannheim, Germany).Each PCR was conducted using three independent biological replicates.Transcript levels of target genes were normalized to those of OsUbi1.All primers used for qRT-PCR are listed in Table S1.

2.6.Membrane lipid extraction and analysis

Membrane lipids were extracted from embryos after 5 days of germination and analyzed as described previously [44].Briefly,more than 5 mg of fresh rice embryo material was collected and immediately put in preheated isopropanol(75°C)with 0.01%butylated hydroxytoluene for 15 min to prevent phospholipase activity.Afterwards, the tissues were extracted five times by chloroform/methanol (2:1 v/v), followed by additional washes with 1 mol L-1KCl and water, respectively.The lipids were evaporated under nitrogen gas and redissolved in chloroform based on the dry weight of embryo samples.The lipid samples were then subject to liquid chromatography and tandem mass spectrometry (multiplereaction-monitoring mode) analysis using a QTRAP 6500 mass spectrometer(ABSciex)coupled with a liquid chromatography system (1290 UPLC Infinity II, Agilent).Lipidomic data analysis was performed using MultiQuant and PeakView software (ABSciex) by comparing the peak area of target lipid species to those of internal standards.The values are presented as molar percentage (mol%).Five biological replicates of each genotype were analyzed.

2.7.PLD activity measurements

0.1 g of 2-day-old embryos after gemination were collected and ground thoroughly in liquid nitrogen.1 mL of extraction buffer(Solarbio,PLD Assay Kit)was added to the sample and mixed well.The supernatant was collected after centrifugation at 13,000 g at 4°C.The following procedures were as described in the kit manual.In this assay, PLD hydrolyzes phosphatidylcholine to choline,which is determined using choline oxidase and resulting in a colorimetric (570 nm) / fluorometric (λex = 530 nm/ λem = 585 nm)product that is proportional to the sample PLD activity.The reaction was performed in buffer (50 mmol L-1Tris-HCl and 5 mmol L-1CaCl2, pH 7.4) at 25 °C for 30 min.

2.8.Plant hormone measurements

Sterilized rice seeds were germinated and grown on 1/2 MS medium with or without 5 μmol L-1ABA for five days.The embryos were collected and grounded into powder in liquid nitrogen on day 5 after germination, followed by hormone extraction and detection.Cytokinins, including CZ and TZ, IAA and ACC were measured at Zhejiang Provincial Key Laboratory of Biotechnology on Specialty Economic Plants at Zhejiang Normal University.GA1,GA5, GA7 were measured at the National Centre for Plant Gene Research(Beijing, China),Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (Beijing, China).

2.9.RNA sequencing

Three biological leaf samples were collected prior to flowering from healthy WT and tcl1 tcl2 mutant plants grown in the field.RNA samples were extracted with Trizol reagent.cDNA library construction and subsequent sequencing were performed by BGI Genomics Company, Shenzhen, Guangdong.The raw sequencing data were quality checked prior to analysis and filtered by SPAPnuke software to remove reads containing adaptor sequences or more than 5%of unknown nucleotides.Afterwards,the clean reads were aligned with reference genome using HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts).Differentially expressed genes(DEGs)were analyzed using Cufflinks v.2.1.1 with a P value < 0.01.

3.Results

3.1.Expression patterns of OsTCL1 and OsTCL2 during developmental stages and in response to stress conditions

To characterize the physiological roles of R3-MYB proteins in rice we specifically selected genes OsTCL1 and OsTCL2 for the study due to the highest homology to Arabidopsis R3-MYB proteins[43].We initially examined the expression patterns of OsTCL1 and OsTCL2 in various tissues at different developmental stages,including embryos,roots,stems,leaves,leaf sheaths,panicles,and filling grains.Transcripts of both OsTCL1 and OsTCL2 were present in all tested tissues, indicating ubiquitous expression.Specifically,OsTCL1 exhibited high expression levels in embryos and leaf tissues across developmental stages, whereas OsTCL2 was predominantly expressed in embryos, leaves of young seedling and at tillering stages, as well as panicles (Fig.1A, B).Investigation of expression levels of OsTCL1 and OsTCL2 under salt treatment and drought conditions induced by mannitol over a period of 36 h showed that both treatments resulted in suppressed expression of both genes(Fig.1C–F).This observation suggests that OsTCL1 and OsTCL2 are involved in the response to abiotic stress.

3.2.Impact of OsTCL1 and OsTCL2 on seed germination under abiotic stress

The CRISPR/Cas9 system was used to generate tcl1 and tcl2 mutants to investigate the potential roles of OsTCL1 and OsTCL2 in plant stress response.Twelve mutants for each gene were categorized into four and five mutation types, respectively (Fig.S1A).For subsequent experiments, we used single tcl1 (#2) and tcl2 (#3) mutant lines (Fig.S1B, C), with each having a single nucleotide insertion in the sequence,resulting in a shifted open reading frame but maintaining intact gene transcription (Fig.S1D).Additionally, we selected a double tcl1 tcl2 mutant from the progeny of a cross of the two single mutants.Despite the reported role of R3-MYB in Arabidopsis in regulating trichome and root hair formation as well as flowering time,the growth phenotype of the rice tcl1 tcl2 double mutant was similar to the WT (Fig.S1E), consistent with a previous report [43].

To test the phenotypes of the tcl1,tcl2 and tcl1 tcl2 mutants under abiotic stress, we subjected the WT and mutant plants to salt and drought stresses.After salt treatment of 150 mmol L-1NaCl to 2-week-old seedlings of WT,tcl1,tcl2 and tcl1 tcl2 lines for 10 days followed by a 10-day recovery period,we observed no differences in survival rates or growth status between WT and mutant plants.Similarly,no discernible differences were observed between the plants subjected to drought treatment for 5 d followed by a 5-day recovery period (Fig.S2).These results indicated that OsTCL1 and OsTCL2 played no significant role in response to stress during the seedling stage.

Fig.1.Expression patterns of OsTCL1 and OsTCL2 and stress response of tcl1, tcl2 and tcl1 tcl2 mutants.Transcript levels of OsTCL1 (A) and OsTCL2 (B) in various tissues at different developmental stages were determined by qRT-PCR.The OsUbi1 gene was used as a reference control.Embryo materials were obtained at day 5 post germination.Root, stem, leaf, and leaf sheath samples were collected from 2-week-old seedlings and plants at the tillering stage; Samples of the same vegetative tissues, panicles, and filling grains were collected during the reproductive phase.The OsUbi1 gene was used as the reference control.Expression levels were analyzed under salt and mannitol treatments (C–F).The germination phenotype (G) of seedlings after 5 days on plates and germination rate (H) of WT, tcl1, tcl2 and tcl1 tcl2 mutants were evaluated under normal and stress conditions.Scale bar, 1 cm.Data are means ± SE values (n ≥20) from three independent experiments.(*, P < 0.05; **, P < 0.01).

Examination of the grain traits indicated that the tcl1 and tcl1 tcl2 mutants had shorter grains and tcl2 and tcl1 tcl2 mutant had wider grains than the WT(Fig.S3A,D).The tcl1 and tcl2 mutants had opposite effects on 1000-grains weight and grain chalkiness;the tcl1 tcl2 mutant had slightly increased 1000-grain weight but similar grain chalkiness to WT (Fig.S3C, D).Hence, the results suggested that OsTCL1 and OsTCL2 influenced grain traits.Considering the relatively high expression levels of OsTCL1 and OsTCL2 in seed embryos as well as their effects on grain traits(Fig.1A,B),we investigated their function during germination.Under normal conditions, the germination rates of the tcl1, tcl2 and tcl1 tcl2 mutants were lower than the WT during the first couple of days but reached a similar level at day 5.In contrast, when subjected to salt and mannitol treatments,significantly fewer seeds from all three mutants germinated compared with the WT; with lowest germination recorded for the double mutant (Fig.1G, H).This suggested roles of OsTCL1 and OsTCL2 in the seed germination under abiotic stress and functional redundancy between OsTCL1 and OsTCL2.We therefore primarily used the tcl1 tcl2 mutant in subsequent experiments.

3.3.OsTCL1 and OsTCL2 partially function in seed germination under stress via the PLD-PA pathway

To elucidate the mechanism by which OsTCL1 and OsTCL2 affect seed germination under stress,we first explored the expression of PLD genes in the tcl1 tcl2 mutant during stress treatments.PLDs were reported to respond to abiotic stress in rice [31] and were found to be targets of R3-MYB protein in soybean [42].The expression levels of most tested PLD genes except OsPLDα5 and OsPLDφ decreased in the tcl1 tcl2 mutant (Fig.2A) and total PLD activity of 5-day-old tcl1 tcl2 seedlings was significantly lower than the WT(Fig.2B).To further assess if these changes altered the content of secondary signaling messenger PA, we profiled the membrane lipids in 5-day-old WT and tcl1 tcl2 seedlings.The results demonstrated that the molar percentages of most membrane lipids were not significantly different between the WT and tcl1 tcl2 mutant although the PA level in the mutant tended to be lower(Fig.2C).However, some PA species, such as 32:0, 34:1, 36:6,36:4 and 36:3, were significantly lower in the mutant (Fig.2D),suggesting these decreased PA species levels were associated with changes in PLD expression level and activity.

To understand whether OsTCL1 and OsTCL2 rely on the PLD-PA pathway to regulate plant stress response,we applied 1-butanol,a competitive substrate inhibitor of PLD-dependent production of PA[45,46],to test the effect on seed germination under NaCl and mannitol treatments.Even though tcl1 tcl2 seeds under stress still revealed slower germination than WT in the presence of 1-butanol, the difference was much less pronounced than that in the absence of 1-butanol (Fig.2E, F), suggesting that a portion of the tcl1 tcl2 response to stress was mediated through modulation of endogenous PA levels catalyzed by PLDs.

3.4.OsTCL1 and OsTCL2 affect rice seed germination in response to exogenous ABA

Given the close crosstalk between the PLD-PA pathway and ABA signaling transduction in plant stress response[47],we speculated that OsTCL1 and OsTCL2 may be responsive to exogenous ABA.To test this, we measured the expression of OsTCL1 and OsTCL2 in the presence of exogenous ABA.Transcription of OsTCL1 and OsTCL2 was suppressed during ABA treatment, although the transcript level of OsTCL2 returned to the normal level after 24 h of treatment (Fig.S4A, B).

We then examined the effect of applied ABA on germination of tcl1 tcl2 mutant seeds.Like responses observed using salt and drought treatments, germination of tcl1 tcl2 mutant seeds displayed increased sensitivity to ABA relative to the WT (Fig.3A,B).Young tcl1 tcl2 mutant seedlings also showed higher sensitivity to ABA treatment than the WT (Fig.S4C, D), although they responded similarly to the WT under salt and drought conditions.These findings suggest that ABA may have a greater impact on young rice seedlings than salt and drought stress, thus rendering tcl1 tcl2 seedlings more responsive to ABA.

3.5.OsTCL1 and OsTCL2 respond partially to exogenous ABA through the PLD-PA pathway during seed germination

ABA acts as a negative regulator of seed germination; elevated PA levels were shown to enhance ABA perception and thereby suppress germination.We investigated whether OsTCL1 and OsTCL2 respond to exogenous ABA via the PLD-PA route.We applied 1-butanol, the inhibitor of the PLD-dependent production of PA, to assess the sensitivity of tcl1 tcl2 mutant plants to ABA.Even in the presence of 1-butonal,the tcl1 tcl2 mutant remained more sensitive to 2 μmol L-1or 5 μmol L-1ABA compared with WT.However, the extent of sensitivity to ABA was significantly reduced compared to no 1-butanol application (Fig.3C, D), a result similar to the effects of salt and mannitol treatment.The same experiment performed with 2-butonal showed that 2-butonal had little effect on seed germination (Fig.S5).Hence, the fact that the addition of 1-butanol did not abolish the difference between WT and the tcl1 tcl2 mutant suggested OsTCL1 and OsTCL2 perceived ABA only partially through the PLD-PA pathway and that other signaling factors must also be involved.

3.6.OsTCL1 and OsTCL2 regulate endogenous ABA level and ABA signaling response

Having observed the involvement OsTCL1 and OsTCL2 in response to exogenous ABA, we proceeded to study their impact on ABA signaling response by examining transcript levels of key genes involved in the ABA signaling pathway during germination of tcl1 tcl2 mutant seeds.Expression of genes OsPP2C51, OsPYL9,OsLEA3, OsRAB21 and OsbZIP10 differed between WT and tcl1 tcl2 mutant(Fig.4A).In addition,the transcription level of ABA synthesis gene OsNCED3 was higher in the tcl1 tcl2mutant, whereas the expression of ABA degradation gene OsABA8ox-1 was lower(Fig.4B), indicating that OsTCL1 and OsTCL2 affected the endogenous ABA level.For confirmation, we measured ABA levels in WT and tcl1 tcl2 mutant plants.The ABA level was significantly higher in the mutant than the WT,regardless of added 1-butonal(Fig.4C),suggesting that the increased ABA level in the tcl1 tcl2 mutant was independent of PA.We also measured ABA levels in 2-day-old seedlings of WT and tcl1 tcl2 mutant under ABA and mannitol treatments and found that tcl1 tcl2 mutant seedlings still exhibited elevated ABA levels compared with the WT (Fig.S6A).

To assess the extent to which the endogenous ABA level determines germination of tcl1 tcl2 seeds under stress, we applied the ABA synthesis inhibitor, sodium tungstate (Na2WO4), along with stress treatments.Although Na2WO4slightly alleviated the difference in germination between the WT and tcl1 tcl2 mutant seeds during the treatments, the latter germinated more slowly than the WT (Fig.5), indicating that OsTCL1 and OsTCL2 partially regulate seed germination through modulation of endogenous ABA levels.We also measured PLD expression levels and enzymatic activities when Na2WO4was applied to both genotypes and found no effect of Na2WO4treatment (Figs.2,S6B,C),demonstrating that the impact of OsTCL1 and OsTCL2 on PLD-PA pathway was independent of variation in endogenous ABA level.

Since the PLD-PA pathway and endogenous ABA levels are downstream targets of OsTCL1 and OsTCL2, we sought to test the effect of combinations of 1-butonal and Na2WO4on the germination of tcl1 tcl2 mutant seeds.Application of both inhibitors almost abolished the difference between WT and tcl1 tcl2 mutant regardless of ABA,salt or mannitol treatments(Fig.6), indicating that OsTCL1 and OsTCL2 regulate seed germination under stress conditions via both the PLD-PA and the endogenous ABA pathways.

3.7.Broader impact of OsTCL1 and OsTCL2 on other plant hormone pathways

Fig.2.Effect of OsTCL1 and OsTCL2 on the PLD-PA pathway.Relative expression levels of multiple PLD genes(A),overall PLD activity(B),membrane lipid profiling(C),and PA species levels (D)were assessed in 5-day-old seedlings of WT and tcl1 tcl2 mutant.The germination phenotypes (E) and germination rates(F) of 5-day-old WT and tcl1 tcl2 mutant seedlings grown on plates under normal and stress conditions in the presence and absence of 1-butanol.Scale bar,1 cm.The data are means±SE values(n ≥20)from three independent experiments.(*, P < 0.05; **, P < 0.01).

Since the PLD-PA pathway is involved in multiple molecular signaling networks, including plant hormone responses such as ABA,GA, ETH, CK, JA, and auxin, we suspected that OsTCL1 and OsTCL2 could also influence other hormone pathways.To investigate this,we performed RNA-seq analysis of WT and tcl1 tcl2 mutant to identify differentially expressed genes (DEGs) at a global transcriptional scale, particularly those involved in plant hormonemediated processes.Among the 945 down-regulated and 1382 up-regulated genes, a subset belonged to plant hormone signaling transduction (Fig.7A–C).We measured the levels of various plant hormones in WT and tcl1 tcl2 plants in the presence and absence of exogenous ABA, including GAs (GA1, GA5, GA7), CKs (cis-Zeatin, CZ and trans-Zeatin, TZ), auxin (IAA), and the immediate precursor of ethylene (1-aminocyclopropane-l-carboxylic acid,ACC).All except IAA exhibited lower levels in the tcl1 tcl2 mutant compared with the WT, regardless of ABA application.Under normal conditions,IAA levels in WT and the tcl1 tcl2 mutant were similar, but with exogenous ABA, its level was higher in the latter(Fig.7D).Moreover,critical genes involved in other plant hormone signaling transduction showed differential expression patterns between WT and tcl1 tcl2 mutant (Fig.S7), indicating that OsTCL1 and OsTCL2 regulate plant response to abiotic stress by coordinating various endogenous plant hormones.

4.Discussion

In this study, we have uncovered novel functions of R3-MYB genes OsTCL1 and OsTCL2 in regulating seed germination under salt and drought stress in rice.Our findings suggest that OsTCL1 and OsTCL2 redundantly manipulate the PLD-PA and ABA-mediated signaling pathways to facilitate response to stress.

A decrease in endogenous ABA levels in the tcl1 tcl2 mutant was attributed to altered expression of ABA synthesis and degradation genes.However, the precise molecular mechanism by which OsTCL1 and OsTCL2 influence expression of these genes remains unclear and warrants further investigation.Moreover, we cannot rule out the possibility of potential interactions between the PLD-PA pathway and ABA signal transduction.Given the alterations in endogenous ABA levels and other plant hormones (GAs,CZ, TZ and ACC) in the tcl1 tcl2 mutant under abiotic stress, it is uncertain whether OsTCL1 and OsTCL2 directly modulate ABA levels, subsequently affecting other plant hormone contents,whether there is a reciprocal relationship, or whether OsTCL1 and OsTCL2 directly impact individual plant hormones.Further studies are required to address these questions and gain a full understanding of the underlying mechanisms.

Fig.3.Response of WT and tcl1 tcl2 mutant plants to exogenous ABA.The phenotypic response(A)and germination rate(B)of WT and tcl1 tcl2 mutant plants were tested and compared in the presence of 2 μmol L-1 or 5 μmol L-1 of exogenous ABA.The germination of tcl1 tcl2 seeds was progressively slower than WT as the concentration of ABA increased.Co-treatment of ABA and 1-butanol alleviated the difference(C,D).Scale bar,1 cm.Images were recorded after 5 d.The data are means±SE values(n ≥20)from three independent experiments.

Fig.4.Effects of OsTCL1 and OsTCL2 on ABA biosynthesis and signal transduction.Transcript levels of genes involved in ABA signaling (OsPP2C51, OsPYL9, OsLEA3,OsRab21, and OsbZIP10) (A) and ABA metabolism (OsNCED3 and OSABA8ox-1) (B)were quantified using qRT-PCR in embryos of WT and tcl1 tcl2 mutant with and without a 5-day treatment of 5 μmol L-1 ABA.Gene OsUbi1 was used as the control.(C) ABA contents in WT and tcl1 tcl2 mutant embryos under normal and 1-butanol treatment conditions.Values are mean ± SE (n = 3) from three independent experiments.(*, P < 0.05; **, P < 0.01).

Mutation of OsTCL1 and OsTCL2 during seed germination led to upregulation of OsNCED3 expression while repressing OsABA8ox-1 expression,resulting in the accumulation of endogenous ABA content (Fig.4B).This indicates that knockout of OsTCL1 and OsTCL2 enhanced ABA biosynthesis and prolonged its presence.Moreover,the expression levels of OsTCL1 and OsTCL2 were initially decreased during early ABA treatment(Fig.S4A,B),suggesting that ABA suppressed expression of OsTCL1 and OsTCL2.When plants are subjected to abiotic stress, endogenous ABA synthesis is activated,enabling its association with ABA receptors to initiate signaling transduction and respond to adverse environments [48,49].Our results demonstrated that expression of OsTCL1 and OsTCL2 was inhibited under salt and mannitol treatments (Fig.1C–F), consistent with the response observed during ABA treatment.These findings indicate that, on one hand, ABA transiently suppresses expression of OsTCL1 and OsTCL2, while on the other hand, the OsTCL1 and OsTCL2 proteins inhibit endogenous ABA accumulation.Thus, we conclude that OsTCL1 and OsTCL2 function antagonistically to endogenous ABA levels.A prior study reported that ABA promotes PLD activity and enhances PA production [29].In contrast, even though ABA levels were increased in the tcl1 tcl2 mutant (Fig.S6A), the expression of PLD and levels of certain PA species were repressed(Fig.2A–D),further supporting the antagonistic relationship between OsTCL1 and OsTCL2, and ABA metabolism.Nevertheless, more evidence is required to establish a solid conclusion.

Compared with R2R3-MYB,1R-MYB is relatively less studied in plant response to abiotic stress.To date,only two rice 1R-MYB proteins, namely OsMYBS3 and OsMYB48-1, are reported to function in stress response.OsMYBS3 was observed to accumulate under cold stress, and its overexpression enhanced resistance to cold damage in rice [50].In contrast, the expression of OsMYB48-1 was induced by ABA, salt, drought, and cold treatments; plants overexpressing OsMYB48-1 exhibited hypersensitivity to exogenous ABA and accumulated higher levels of endogenous ABA under drought stress.Overexpression of OsMYB48-1 also upregulated expression of ABA synthesis genes OsNCED4 and OsNCED5, early ABA responding genes OsPP2C68 and OsRK1,and late ABA responding genes RAB21, OsLEA3, RAB16C and RAB16D.Upregulationmediated ABA synthesis and signaling transduction thereby enhances the resistance of rice to abiotic stresses [51].Here, we shed light on the roles of newly identified R3-MYB members in regulating seed germination under abiotic stress through dual signaling pathways.

Fig.5.Application of Na2WO4 alleviated the impact of OsTCL1 and OsTCL2 on seed germination.Phenotype of 6-day-old germinating seeds(A)and germination rates of WT and tcl1 tcl2 mutant(B)were assessed under normal and indicated stress treatments along with ABA synthesis inhibitor Na2WO4.Scale bar,1 cm.The values are means±SE(n ≥20) from three independent experiments.

Fig.6.Impaired effect of OsTCL1 and OsTCL2 on seed germination following dual application of 1-butanol and Na2WO4.Phenotypes of 5-day-old germinating seeds(A)and germination rates(B)of WT and tcl1 tcl2 mutant under normal and various stress treatments,in the presence of 1-butanol and Na2WO4 applications.Scale bar,1 cm.Values are means ± SE (n ≥20) from three independent experiments.

Transcripts of OsTCL1 and OsTCL2 were detected in various tissues, with relatively high levels in seed embryos, leaves and panicles (Fig.1A, B).However, the phenotypic impact of the tcl1 tcl2 double mutant was observed only in seed germination.The discrepancy between gene expression pattern and phenotypic stage is probably due to redundant functions of other R3-MYB members:the R3-MYB family in rice consists of multiple members [43].While OsTCL1 and OsTCL2 may play a dominate role during seed germination, other members of the family might exhibit greater activity in young seedlings and leaves.A second possibility is the effectiveness of treatments: the salt and drought treatments employed in our study could have had a significant impact on rice embryos but limited effects on young seedlings at later developmental stages, especially on tillering plants and panicles.

Differential expression of multiple PLDs, and overall PLD activity was attenuated in the tcl1 tcl2 mutant,the level of PLD product PA was not significantly altered possibly due to redundancy among the PLDs.Also, the dynamic regulation of PA is tightly controlled.Although PA can be generated by the PLC/DGK (phospholipase C/-diacylglycerol kinase)and LPAAT(lysophosphatidic acid acyltransferase)pathways in addition to PLDs,several PA degradation pathways operate to maintain its homeostasis.

Fig.7.RNA-seq analysis of WT and tcl1 tcl2 mutant.(A) Volcano plot depicting genes detected in WT and tcl1 tcl2 mutant.(B) Pie graph illustrating the distribution of upregulated and down-regulated genes in WT vs.tcl1 tcl2 mutant.(C) Bubble plot displaying the KEGG pathway enrichment analysis of differentially expressed genes (DEGs)involved in abiotic stress response in WT vs.tcl1 tcl2 mutant.(D)Quantification of various plant hormones in 5-day-old embryos of WT and tcl1 tcl2 mutant with and without exogenous ABA treatment.Values are means ± SE (n = 3) from three independent experiments.

5.Conclusions

Our findings provide valuable insights into the roles of rice R3-MYB proteins OsTCL1 and OsTCL2 in regulating seed germination under abiotic stress conditions.These proteins are involved in mediating the PLD-PA pathway and ABA signaling transduction.The study not only expands our understanding of the functions of 1R-MYB proteins in rice,but also offers genetic information that will contribute to the development of rice varieties with improved salt and drought tolerance.

CRediT authorship contribution statement

Yong Yi:Data curation, Investigation, Methodology.Chan Lin:Data curation,Investigation,Methodology.Xueyan Peng:Data curation, Investigation, Validation.Meishan Zhang:Formal analysis.Jiaming Wu:Investigation, Validation.Chunmei Meng:Resources.Shengchao Ge:Conceptualization.Yunfeng Liu:Conceptualization,Funding acquisition,Project administration,Writing–original draft.Yuan Su:Conceptualization,Funding acquisition,Writing–review&editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank Dr.Yaoguang Liu for the kind gift of the pYLgRNAOsU6a plasmid.This work was supported by the National Natural Science Foundation of China (31970603), Natural Science Foundation of Guangxi province (2019GXNSFDA185001), State Key Laboratory for Conservation and Utilization of Subtropical Agrobioresources (SKLCUSA-a202008 and SKLCUSA-a01), and Guangxi University-Bama Research Fund (20220006).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2023.10.004.