A β-ketoacyl-CoA Synthase OsCUT1 Confers Increased Drought Tolerance in Rice

Gao Xiuying, Zhang Ye, Zhang Hongsheng, Huang Ji

Research Paper

A β-ketoacyl-CoA Synthase OsCUT1 Confers Increased Drought Tolerance in Rice

Gao Xiuying1, 2, #, Zhang Ye3, #, Zhang Hongsheng1, 2, Huang Ji1, 2

(; These authors contributed equally to this work)

Drought stress is one of the major environmental factors affecting crop growth and productivity. Cuticular wax plays essential roles in protecting plants from environmental stress via forming a hydrophobic barrier on leaf epidermis. In this study, we analyzed nine members (OsCUT1?OsCUT9) of β-ketoacyl-CoA synthase, the rate-limiting key enzyme for cuticular wax synthesis in rice by homology search and domain prediction. The expression levels of OsCUT genes under different abiotic stresses were investigated anddown-regulated by abiotic stress was selected for further function validation. Compared to the wild type, overexpression of() exhibited significantly increased drought resistance. Epicuticular wax was increased on the leaf surface ofand the chlorophyll leaching experiment showed that the cuticular permeability was decreased in theplants. Moreover, overexpression ofdidn’t result in the significant changes of major agronomic traits. In total, these results suggested thatis a promising gene for engineering rice plants with enhanced drought tolerance.

rice; β-ketoacyl-CoA synthase; drought tolerance; cuticular wax;

Rice (L.) is one of the most important cereal crops worldwide and feeds more than half of the global population. As we know, rice productivity is limited by many environmental challenges, such as drought, freeze and salinity. Therefore, plants have evolved various mechanisms to adapt changes at the morphological, physiological and molecular levels. For drought resistance responses, several distinct mechanisms including adjustment of aerial development, maintenance of root growth, water uptake, osmo- protectants and antioxidants. Notably, cuticular wax accumulation is closely related to drought responses (Suh et al, 2005).

The cuticle is known as a hydrophobic layer that covers most primary aerial organs to protect plants from drought stress via preventing non-stomatal water loss. The cuticle is synthesized in epidermal cells and consists of cutin and wax (Kunst and Samuels, 2009; Buschhaus and Jetter, 2012). The cutin acts as the primary structural polymer, is comprised of C16 and C18 hydroxy and epoxy-hydroxy fatty acid monomers (Samuels et al, 2008). Cuticular wax can be divided into intracuticular wax and epicuticular wax. Both of them are typically composed of very long-chain fatty acids (VLCFAs), including aldehydes, alkanes, ketones, primary and secondary alcohols and esters, which are synthesized from C18 carbon chains by elongation reactions (Shepherd and Griffiths, 2006; Lee and Suh, 2013; Yeats and Rose, 2013). Firstly, the biosynthesis of wax occurs within the plastid of epidermal cells where the C16 and C18 fatty acids are produced. Then, these fatty acids are converted to CoA thioesters via the long-chain acyl-coenzyme A synthase for the generation of VLCFA precursors. The extension of C16 and C18 fatty acids to VLCFA precursors occurs on the endoplasmic reticulum (ER). Next, the formation of VLCFAs is carried out by the enzymes of the fatty acid elongase (FAE) complex. The FAE complex includes four enzymes: a β-ketoacyl-CoA synthase (KCS), a β-ketoacyl-CoA reductase (KCR), a 3-hydroxyacyl- CoA dehydratase (HCD) and-2,3-enoyl-CoA reductase (ECR). These enzymes catalyze the sequential reactions: condensation, dehydration and reduction, respectively. During the fatty acid elongation cycle, KCS catalyzes the condensation of VLCFAs, which is the first step in elongation process (Haslam and Kunst, 2013).

In, 21 genes are predicted to encode KCS. AtKCS18 catalyzes the biosynthesis of C20 and C22 VLCFAs for lipid storage, while AtKCS2 and AtKCS20 function redundantly in the VLCFA elongation up to C22 (James et al, 1995; Franke et al, 2009; Lee et al, 2009). AtKCS5 and AtKCS6/CUT1 are involved in the elongation of fatty acyl-CoAs longer than C24 VLCFAs for producing cuticular wax (Millar et al, 1999; Fiebig et al, 2000; Hooker et al, 2002). In addition, AtKCS10 and AtKCS13 can affect the morphological structure and stress tolerance in plants (Yephremov et al, 1999; Gray et al, 2000; Pruitt et al, 2000; Joubès et al, 2008). Rice cuticular wax contains predominantly C28?C32 VLCFAs, and thus the wax biosynthesis in rice is associated with many KCS genes. Several wax biosynthesis genes have previously been identified in rice by the characterization of wax crystal-sparse leaf mutants.encodes a KCS that is involved in the biosynthesis of cuticular wax on rice leaves, and the VLCFA precursors of C20?C24 of total wax are reduced on leaf ofplants (Yu et al, 2008)./encodes a very long-chain acyl-CoA reductase relating to the elongation of VLCFAs, which is a homolog ofgenes (). The amount of wax decreases tremendously inmutant (Qin et al, 2011; Mao et al, 2012).encodes a KCR that affects cuticular wax production in VLCFA elongation in rice (Gan et al, 2016).OsWSL4/OsKCS6 is predicted to encode a KCS that is homologous to, which physically interacts with CER2- like protein, catalyzing the elongation of VLCFAs longer than C22 (Gan et al, 2017; Wang et al, 2017).is predicted to encode a cytochrome P450 family member CYP96B5, which is involved in the formation of epidermal wax crystals on rice leaf affecting drought sensitivity. Overexpression ofincreases C29 primary alcohol and decreases alkane, whereas-knockout mutant plants indicate the opposite function (Zhang D et al, 2020).

Table 1. Details of nine CUT proteins in rice.

MSU ID is from http://rice.uga.edu/index.shtml and RAP ID is from https://rapdb.dna.affrc.go.jp. ORF, Open reading fragment.

In this study, we analyzed the chromosome distribution, conservation and expression patterns of rice CUT gene family members. Next, we focusedfor further functional analysis. The subcellular localization showed that OsCUT1 locates in ER, which was consistent with the property of KCS proteins. Overexpression ofincreased wax synthesis and drought tolerance without yield penalty, suggesting a promising gene target for engineering rice plants with increased drought resistance.

Results

OsCUT gene family members in rice

Through homology search and domain prediction, nine genes encoding specific CUT proteins were ultimately identified in the rice genome, which were named as?(Table 1). The lengths of the proteins encoded by these genes were between 274 and 543 aa and the molecular weight ranged from 30.3 to 60.1 kDa. The chromosome mapping showed that OsCUT genes were distributed on chromosomes 1, 2, 3, 6 and 10, but the distribution was uneven. Among them, chromosome 6 contained the largest number of CUTgenes (Fig. S1). To study the phylogenetic relationship of rice CUT proteins, a phylogenic tree was constructed using the CUT protein sequences of five different species (rice, maize, peanut, sorghum and). According to the topology of the evolutionary tree, these proteins can be grouped into 10 subgroups (Groups α, β, γ, δ, ε, θ, κ, ι, ξ and η). The κ group contains the largest number of rice CUT proteins, a total of six, followed by the ι group, which contains two rice CUT proteins. According to the phylogenetic relationship of the protein sequences, the functions of plant CUT proteins with known functions can be used to predict the functions of rice CUT proteins (Fig. 1). These results indicated that CUT genes in rice might derive from gene duplication and involve novel functions, which are more complicated than.

To better understand the structure of the rice CUT genes, the exon-intron structure of the rice CUTgenes was analyzed using the annotation information of the rice reference genome. Genes had different structures and varied in their lengths of introns. The MEME online prediction tool was used to identify the conserved motifs in the rice CUT proteins. A total of 10 conserved sequences were identified (Table S1). The results showed that the rice CUT proteins contained at least 5 conserved motifs, and most rice CUT proteins contained all the 10 conserved motifs (Fig. 2-A). Notably, four motifs (motifs 5, 6, 7 and 10) were not contained in OsCUT7 (LOC_Os06g15170) and OsCUT9 (LOC_Os10g28060), which was consistent with phylogenic tree, indicating that those two OsCUT proteins are differentiated than the others in function.

-regulatory elements in the promotor regions play important roles in the plant response to stress. Using the PlantCARE database, we identified 14 putative-regulatory elements in 2 000 bp upstream of these OsCUT genes, including ABRE (ABA- responsive elements), TGACG motifs, CGTCA motifs (which are involved in methyl jasmonate response), LTRs (low-temperature-responsiveelements), MYB (MYB-binding sites) and TCA elements. The elements associated with the highest number of stress response elements within the OsCUT gene family are ABRE, LTRs, CGTCA motifs, TGACG motifs, TCA elements and TC-rich repeats (Table S2). Most of the proteins in sub- family contained similar types and numbers of conserved motifs (Fig. 2-B). The types and the numbers of stress-related elements contained in each OsCUTgene promoter differed, indicating that the members of the OsCUT gene family can respond differently to various stresses.

Fig. 1. Phylogenetic tree of CUT family.

The phylogenetic tree was constructed by MEGAX using the maximum likelihood method. Different subfamilies are highlighted with different colors.

Expression and intracellular localization of OsCUT1

Wax production in aerial parts of the plants is critical for drought tolerance. To elucidate the roles of OsCUT gene family in stress resistance, we firstly analyzed their transcriptional levels under drought stress usingthe published transcriptome data by Wei et al (2017), which have been deposited in Gene Expression Omnibus in NCBI under accession number GSE83378. The differentially expressed genes were identified by comparison between well-watered and drought stress conditions. Then, we analyzed the expression levels of?, and found that onlywas down-regulated in two drought sensitive varieties (Fig. S2). According to the expression profiling of the CUT genes, we chosefor further analysis. Next, we analyzed the transcriptional levels ofunder 20% PEG6000 treatment by qRT-PCR, and found thatwas significantly down-regulated with drought stress (Fig. 3). Moreover, we also analyzed whetheris regulated by 100 mmol/L NaCl, 0.1 mmol/L H2O2, 0.1 mmol/L abscisic acid (ABA), cold or heat treatment by qRT-PCR (Fig. 3). The expression level ofincreased firstly and then decreased under the H2O2treatment, and thetranscript levels decreased significantly when plants were exposed to NaCl, ABA, 42 oC and 4 oC treatments.

Fig. 2. Gene structure, conserved motif and predicted-regulatory element in OsCUT genes.

A, Distributions of conserved motifs in OsCUT genes. Ten putative motifs are indicated in different colored boxes. The sequence information of the 10 motifs was in Table S1. The right figure shows exon/intron organization of OsCUT genes. Green boxes represent exons and black lines with the same length represent introns. The upstream/downstream regions of OsCUT genes are indicated with yellow boxes. The length of exons can be inferred by the scale at the bottom. UTR, Untranslated region; CDS, Coding sequence.

B, Predicted-regulatory element in OsCUT promoters. The sequence information of the 11 motifs was in Table S2. ARE, Elements for the anaerobic induction; LTR, Low-temperature-responsive element; ABRE, ABA-responsive element; MYB, MYB-binding site.

Then, we analyzed the expression ofin different tissues of rice with reverse transcription semi-quantitative PCR and qRT-PCR. The results demonstrated thathad a relatively high expression level in young panicles and leaves (Fig. 4-A and -B). When expressed under the control of the nativepromoter in a wild type (WT) background, the GUS protein activity was found to be presented in aerial parts of seedlings and total panicles (Fig. 4-C). To further investigate the subcellular location of OsCUT1 protein, we introduced the green fluorescent protein (GFP)-OsCUT1 fusion construct and the ER marker ER-mCherry into rice protoplast cells. The results showed that the GFP signal completely co-localized with the ER marker, indicating that OsCUT1 protein is located in the ER (Fig. 4-D).

?genes were alsoselected to test their responsiveness to drought and salt stresses in rice by qRT-PCR analysis.,,andwere down-regulated after 20% PEG6000 treatment. The expression ofpeaked at 1 h and then decreased under drought treatment.andshowed the similar trends, as they peaked at 3 h after drought stress and then decreased from 6 to 48 h. The expression ofshowed no obvious change under drought stress (Fig. S3). For salt treatment, the expression levels of,anddecreased after 100 mmol/L NaCl treatment. The expression of,andincreased at the early stage of the salt treatment and then decreased after treatment. However,did not change significantly after salt treatment, and the expression ofdecreased from 12 to 48 h (Fig. S4). These results indicated that there were different responses and regulatory mechanisms for the CUT family members under various abiotic stress conditions.

Fig. 3.Expression patterns ofafter 20% PEG6000, 100 mmol/L NaCl, 0.1 mmol/L H2O2, 0.1 mmol/L abscisic acid (ABA), 4 oC and 42 oC treatments.

The relative expression ofin different periods was verified by qRT-PCR. Total RNAs were extracted from rice seedlings of two-week-old plants.gene was used as a control. The transcript level at 0 h was defined as ‘1’. Data were Mean ± SE (= 3), and compared by the Student’s-test. *,< 0.05; **,< 0.01.

Fig. 4.Spatial expression pattern and subcellular location of OsCUT1.

A,expression in different tissues by reverse transcription semi-quantitative PCR.gene was used as a control.

B,expression in different tissues by qRT-PCR.gene was used as a control. Data were Mean ± SE (= 3), and compared by the Student’s-test. **,< 0.01.

C, GUS expression patterns of:transgenic rice plants. GUS activity was detected in different tissues: glume (a), root (b), lamina joint (c), stem (d), sheath (e) and leaf (f).

D, Co-expression of OsCUT1-GFP fusion protein and ER-mCherry fusion protein in rice protoplasts was imaged by a confocal microscopy with a Zeiss LSM780 fitted with green (OsCUT1-GFP) and red filters (ER-mCherry). Scale bars, 5 μm.

R, Root; S, Stem; L, Leaf; LS, Leaf sheath; P1, Panicle (～3 cm); P2, Panicle (～8 cm); P3, Panicle (～12 cm); GUS, β-Glucuronidase; GFP, Green fluorescent protein; ER, Endoplasmic reticulum.

Overexpression of OsCUT1 led to cuticular wax accumulation

To further analyze the function of, the full lengthcDNA under the control of the CaMV 35S promoter was over- expressed in rice, and several independent transgenic lines were obtained (Figs. 5-A and S5). Over- expression of() led to no markedly alterations in plant height, panicle length, 1000-grain weight and spikelet fertility compared to the WT Zhonghua 11 (ZH11) (Fig. 5-B to -E). We further checked whether the drought tolerance ofwas due to the wax accumulation. Through the scanning electron microscopy (SEM) analysis, it was observed that the matured leaf surface ofplants was covered with a dense layer of wax crystals (Fig. 5-F). Compared to the over- expression plants, fewer wax crystals were observed on the surfaces of WT (Fig. 5-G). Then, we analyzed the agronomic traits of, and found thatalso increased 1000-grain weight and wax content (Fig. S6).

Overexpression of OsCUT1 increased drought tolerance

Under the normal growth conditions, we did not observe phenotypic differences between these overexpression lines and WT. After recovery from the drought stress treatment, the survival rates of theplants were significantly higher than that of WT (Fig. 6-A to -C). To investigate whether the whole-plant drought sensitivity of theplants is altered, thechlorophyll leaching assay and the whole-plant drought- sensitivity assay were conducted. The chlorophyll leaching assay showed that chlorophyll leaching from theleaves was slower than that from the WT leaves (Fig. 6-D), indicating a decrease of cuticular permeability in theplants. As an indicator of drought sensitivity at the whole-plant level, the water-loss assay of detached leaves was also performed to compare the water retention capacity of theand WT plants (Fig. 6-E). The results indicated that the water loss rate of detached leaves from theplants was significantly lower than that from the WT plants at various time points. These results showed thatconfers high drought tolerance in rice.

DISCUSSION

The OsCUT gene family comprises the genes that encode KCS in rice. At present, the structure and function of the members of the CUT family have been analyzed in many plant species, such asand cotton, however, this information has been less reported in rice. In this study, by clustering with OsCUT1 protein, rice CUT proteins were divided into three groups. The OsCUT family genes had a large number of stress response elements, including elements that respond to low temperature, drought, mechanical damage and hormones, and it has been shown that stress-related elements in the promoter regions of these genes under adverse conditions can improve gene transcription and enhance the resistance to adverse conditions. These elements related to ABREs, CGTCA motifs, TGACG motifs and MYB motifs.

Fig. 5.Phenotypes ofover- expressionplants.

A, Morphology of() and wild type Zhonghua 11 (ZH11) plants at the reproductive stage. Scale bars are 20 cm and 5 cm for the left and right figures, respectively.

B?E, Quantification of plant height (B), panicle length (C), 1000-grain weight (D) and spikelet fertility (E) of theplants () compared with wild type ZH11. For quantification of plant height, panicle length and spikelet fertility, data were Mean ± SE (= 10). For quantification of 1000-grain weight, data were Mean ± SE (= 3). All data were compared by the Student’s-test.

F, Scanning electron microscopy of epicuticular wax on leaf surface ofand wild type ZH11. Scale bars, 10 μm.

G,transgenic plants in ZH11 background show significantly increased wax crystals on leaf surface compared to ZH11. Data were Mean ± SE (= 3), and compared by the Student’s-test. **,< 0.01.

Fig. 6.Effects of drought stress and altered cuticular permeability inoverexpression plants ().

A, Two-month-old wild type (WT) Zhonghua 11 (ZH11) andtransgenic plants before drought stress and after re-watering for 14 d. Scale bars, 10 cm.

B, Three-week-old WT andtransgenic plants before drought stress and after re-watering for 14 d.

C, Survival rates of WT andtransgenic plants for drought stress and re-watering for 14 d.

D, Ratios of total chlorophyll extracted from WT and.

E, Water loss rates in WT andtransgenic leaves.

Data were Mean ± SE (= 3), and compared by the Student’s-test. *,< 0.05; **,< 0.01.

The KCS gene family is related to the content and composition of epidermal wax, and the members actively participate in many physiological reactions during plant growth and development, especially plant stress resistance. At present, KCS genes have been studied in many plant species, such as, cotton, soybean, rice and apple (Joubès et al, 2008). In, the content of VLCFAs in the/mutants are significantly decreased (James et al, 1995).is involved in the VLCFAs synthesis in epidermal cells and mainly expressed in flowers and young leaves (Yephremov et al, 1999; Pruitt et al, 2000; Joubès et al, 2008)./is the key gene whose encoded protein is involved in the waxy synthesis of stem and pollen epidermis (Hooker et al, 2002). In rice, overexpression of the wax synthesis geneincreases wax crystals and enhances drought resistance in plants (Islam et al, 2009).encodes OsNDPK2, which regulates the response to abiotic stress, such as salt tolerance (Ye et al, 2016).andshow similar functions in the biosynthesis of epidermis wax and root suberin (Lee and Suh, 2013). In cotton, thehomolog gene mutation causes lower content of VLCFAs, which inhibits the cotton fiber elongation (Qin et al, 2007). In apple, it was found that(Qi et al, 2018),(Zhong et al, 2020),(Zhang et al, 2020a) and(Zhang et al, 2020b) are all involved in the wax synthesis.

The synthesis of VLCFAs is related with the fatty acid elongation enzymes, in which KCSs play essential roles in catalyzing the first reaction of fatty acid elongation and determining the chain length of products. The metabolic function of KCS is complex as the large number of annotated KCS genes in plants. Although the functions of several rice KCS proteins have been researched, we cannot deduce other KCS proteins by homology analysis alone. Nine OsCUT proteins were found via homology search and FAE1- CUT1-RppA domain prediction, and the genes were named as?. As leaves are the primary organs affected by environmental stress, alternation of wax accumulation in plant leaves is an essential physiological process. To elucidate the molecular mechanisms underlying abiotic stress-induced wax synthesis, the transcript levels of wax-related genes were analyzed under drought, salt and cold treatments. Transcript level changes of nine OsCUT genes under drought and salt treatments clearly suggested that different OsCUT genes show different responses to stress. The expression ofdecreased sharply under both drought and salt treatments, we thus focusedfor further research. It was also found thatgene was induced by ABA. The application of ABA to plants partially mimicked the effect of stress conditions. These results indicated that thegene may be regulated by substantially diverse regulatory systems under different stresses (Fig. S7).

Compared to WT, theplants showed decreased chlorophyll leaching, increased water loss rate, and enhanced sensitivity to drought. As we know, the C16- and C18-CoA are synthesized by plastids in the ER membrane by a fatty acid elongase complex, including KCS, KCR, β-ketoacyl-CoA dehydratase and ECR (Joubès et al, 2008). In this study, it has been found that OsCUT1 was also localized in ER.

The cuticle biosynthesis regulation is complex and involves signaling networks associated with different abiotic and biotic stress responses. In conclusion, the CUT genes in rice were systematically analyzed for their sequence and expression diversification in this study. Overexpression ofresulted in an increased cuticular wax accumulation on the leaf surface of the transgenic rice and significantly decreased sensitivity to drought stress. Genetic modification of this gene may have great potential for improving drought resistance of rice.

methods

Identification of CUT genes in rice genome

Rice genomic data were downloaded from the Ensemble database (http://asia.Ensembl.org/index.html). Using the OsCUT1 protein sequence as the query sequence, BlastP was used to search the CUT family genes from the whole genome database of rice. The NCBI CDD (https://www.ncbi.nlm.nih.gov/Structure/ bwrpsb/bwrpsb.cgi), Pfam database (https://xfam.org) and SMART database (http://smart.embl-heidelberg.de/) were used to confirm the conserved domains.

Phylogenetic analysis of CUT proteins

Phylogenetic analysis was performed using the full length sequences of CUT amino acids from maize, peanut, sorghum, andcombined with the sequence of the newly identified OsCUT proteins. Multiple sequences were aligned with Muscle by the MEGAX software and the 1000 bootstrap tests were used to construct an unrooted neighbor-joining phylogenetic tree. OsCUTs and KCS sequences used in phylogenetic tree analysis were listed in Data Set S1.

Gene structure and motif analyses

Gene structure analysis was performed using the Gene Structure Display Server (http://gsds.cbi.-pku.edu.cn/) program, with the default settings. Motifs within the CUT proteins were identified using MEME (http://meme-suite.org/), with the default settings [motif width: between 6 and 50 (inclusive)]. The maximum number of motifs was 10.

Analysis of cis-regulatory elements in promoter regions

The 2-kb upstream sequences of the OsCUTgene translation initiation codon were downloaded from the Phytozome database(http://phytozome-next.jgi.doe.gov). Using the PlantCare database (http://bioinforma-tics.psb.ugent.be/webtools/plantcare/html/),-regulatory elements in the 2-kb upstream regions were subsequently predicted.

Phenotyping of transgenic plants

The cDNA ofwas cloned into pCAMBIA1301S under the control of CaMV 35S promoter. The construct was transformed into rice Zhonghua 11 (L. subsp.) following the standard rice transformation protocol. Transgenic and WT seeds were germinated on Murashige and Skoog (MS) medium for stress testing. Drought resistance at the reproductive stage was evaluated under drought stress conditions in a refined paddy field facilitated with a movable rain-off shelter. For chlorophyll leaching assay, the third leaf from the top was sampled from each tiller at the heading stage and the leaf was cut into segments (～3 cm) and immersed into 30 mL of 80% ethanol at room temperature in the dark. The chlorophyll concentration was quantified with a spectrophotometer (Alpha-1860, LASPEC, China) at wavelengths of 663 and 645 nm by using the standard method (Lolle et al, 1997). SEM was used to study surfaces of matured leaves ofand WT plants. Fragments of flag leaves were air dried. Epicuticle wax were imaged on a HITACHI scanning electron microscope (HITACHI, Japan) for examination (Gan et al, 2017).

Plant growth and treatments

For detecting the transcript levels of OsCUT genes, rice plants of Nipponbare (L. spp.) were grown in the greenhouse with a 14 h light/10 h dark cycle. Two-week-old seedlings were treated with abiotic and chemical stresses. Abiotic treatments were conducted as previously reported (Yuan et al, 2018). Drought stress was applied in 20% PEG6000 solution and plant leaves were sampled at 0 to 48 h after treatment. For cold and heat stresses, seedlings were transferred into a growth chamber at 4oC or 42 oC and sampled at 0 to 48 h. The seedlings were submersed in 100 mmol/L NaCl solution for salt stress and sampled at 0 to 48 h after treatment. Chemical treatment was conducted by spraying leaves with 0.1 mmol/L H2O2or ABA and sampled at 0 to 48 h.

Total RNA isolation and RT-qPCR analysis

Total RNA was extracted with a High Purity Total RNA Rapid Extraction Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized using HiScript?II Q RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme, Nanjing, China). qRT-PCR was performed using AceQ?qPCR SYBR Green Master Mix Kit (Vazyme, Nanjing, China) and Roche 480 Real-Time PCR System following the manufacturer’s instructions. The ricegene () was used as an internal control and for the normalization in the analysis. The relative gene expression levels were calculated using the 2?ΔΔCtmethod. The data were presented as the Mean ± SE of three replicates. The primers for qRT-PCR were listed in Table S3.

Transient expression assay in rice protoplast

To determine the subcellular localization of OsCUT1, the full- lengthcoding sequence was fused with GFP in the pAN580 vector to produce the OsCUT1-GFP fusion protein in plants. Rice protoplasts were isolated from 2-week-old rice seedlings and transfected with 10 μg plasmid DNA by the PEG-mediated transformation method. The GFP signal was visualized using a confocal laser scanning microscope (LSM780, Zeiss, Germany) after incubation at 26 oC for 12 h. The PCR primers used for transient expression assay were listed in Table S3.

ACKNOWLEDGEMENTS

This study was supported by the National Natural Science Foundation of China (Grant Nos. 32071918 and 32000227). We thank Jiangsu Collaborative Innovation Center and Cyrus Tang Seed Innovation Center, Nanjing Agricultural University, China for Modern Crop Production.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

Data Set S1. OsCUTs and KCS sequences used in phylogenetic tree analysis.

Fig. S1. Chromosomal distribution of rice OsCUT genes.

Fig. S2. Transcription analyses ofin young panicles of Khaumeo and Nipponbare varieties under drought stress.

Fig. S3. Expression patterns ofgenes after 20% PEG6000 treatment.

Fig. S4. Expression patterns ofgenes after salt treatment.

Fig. S5. Identification oftransgenic lines with reverse transcription semi-quantitative PCR assay.

Fig. S6. Quantification of plant height, panicle length, 1000- grain weight, spikelet fertility and wax content ofplants compared with wild type.

Fig. S7.Possible working model for OsCUT1.

Table S1. Conserved motifs in OsCUT protein.

Table S2. Predicted-regulatory elements in OsCUT promoters.

Table S3. Primers used in this study.

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24 August 2021;

13 December 2021

s:Huang Ji(huangji@njau.edu.cn); Zhang Hongsheng (hszhang@njau.edu.cn)

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