Targeted Mutagenesis of NAC Transcription Factor Gene, OsNAC041, Leading to Salt Sensitivity in Rice

doi:10.1016/j.rsci.2018.12.005

Abstract

Abstract:

Salinity is a major abiotic stress factor that seriously affects plant growth. Many genes are involved in the response to salt stress with various metabolism pathways. A number of plant transcription factor family genes have been found to be involved in the salt stress response, and NAM, ATAF and CUC (NAC) transcription factors are thought to act as active regulators during abiotic stress, especially salt stress. In this study, we detected a rice NAC transcription factor coding gene, OsNAC041, and confirmed that it influenced the germination of seeds under salt stress and salt tolerance of plants. OsNAC041 was primarily expressed in the leaves and located in the nucleus. Furthermore, the CRISPR/Cas9 method was used to obtain a targeted osnac041 mutant, of which the plant height was higher than that of the wild-type, showing increased salt sensitivity. Moreover, RNA-seq analysis revealed a number of differentially expressed genes (DEGs) involved in several important signaling pathways in the osnac041 mutant. Subsequently, Kyoto Encyclopedia of Genes and Genomes annotation also revealed differential expression of DEGs associated with mitogen-activated protein kinase signaling, peroxisome, eukaryotic- type ABC transporters, photosynthesis and plant hormones, which are involved in stress-related signaling pathways. Overall, our study suggested that OsNAC041 was involved in the salt stress response in rice. These findings not only provide empirical evidence of OsNAC041 function, but also provide new insight into its potential application in rice resistance breeding.

Key words: rice, NAC transcription factor, CRISPR, Cas9, RNA-seq, salinity

Bo Wang, Zhaohui Zhong, Huanhuan Zhang, Xia Wang, Binglin Liu, Lijia Yang, Xiangyan Han, Deshui Yu, Xuelian Zheng, Chunguo Wang, Wenqin Song, Chengbin Chen, Yong Zhang. Targeted Mutagenesis of NAC Transcription Factor Gene, OsNAC041, Leading to Salt Sensitivity in Rice[J]. Rice Science, 2019, 26(2): 98-108.

Figures/Tables 16

Fig. 1. Expression profile and subcellular location analysis of OsNAC041.A, Quantitative real-time PCR (qRT-PCR) analysis of OsNAC041 expression in samples of root, stem and leaf from two-week-old wild-type (WT) plants. B, qRT-PCR analysis of OsNAC041 expression patterns in WT plants before (NaCl-) and after (NaCl+) salt stress. C, Nuclear localization of OsNAC041 protein in the rice protoplast. NLS, Nuclear localization signal. Scale bar = 20 µm. Data represent Mean ± SE (n = 3). **, Significance at the 0.01 level.

Fig. 2. Targeted mutagenesis of the OsNAC041 locus using CRISPR-Cas9 system.A, Design of sgRNA sites for the OsNAC041 exons. B, Single-strand conformation polymorphism analysis of 10 independent OsNAC041- sgRNA01 T0 lines. M, Marker; WT, Wild-type. C, Sanger sequencing of the target site in the OsNAC041-sgRNA01 T0 lines. D, Phenotypic analysis of OsNAC041 T1 mutant lines under normal condition.

Fig. 3. OsNAC041 targeted mutants showed the salt-sensitive phenotype.A, Germination of OsNAC041 T1 mutant and wild-type (WT) seeds grown on 1/2 MS medium supplemented with NaCl (150 mmol/L). B, Phenotypic analysis of OsNAC041 T1 mutant lines under salt stress. C, Growth of OsNAC041 T1 mutant lines in plugs. D, Levels of O2- and H2O2 in WT and OsNAC041 T1 mutant lines subjected to salt stress. Salt-stressed leaf samples were incubated in nitro-blue tetrazolium (NBT) or diaminobenzidine (DAB) solution. E, Chlorophyll content after 15-day salt stress. F, H2O2 content after 15-day salt stress. G, O2- production rate after 15-day salt stress. H, Malondialdehyde (MDA) content after 15-day salt stress. I, Catalase (CAT) activity after 15-day salt stress. J, Peroxidase (POD) activity after 15-day salt stress. K, Superoxide dismutase (SOD) activity after 15-day salt stress. Data represent Mean ± SE (n = 3). **, Significant at the 0.01 level.

Fig. 4. Targeted knockout of OsNAC041 resulted in global gene expression changes in rice.A, Number of differentially expressed genes (DEGs) in the wild-type (WT) and osnac041 (A/A) T1 mutant lines, based on expression profiles obtained by RNA-Seq. B, Clustering analysis of DEGs in WT and osnac041 T1 mutant lines. Targeted knockout of osnac041 resulted in global changes compared with the WT both with and without salt stress. The color scale corresponds to log2(FPKM) values of the genes. C, Gene ontology classification of DEGs in the following two comparisons: WT and osnac041 T1 mutant lines under normal and salt stress conditions. x-axis shows user selected GO terms; y-axis shows the percentages of genes (number of a particular gene divided by total gene number).

Fig. 5. Transcriptome analysis of genes systemically regulated in the wild-type (WT) and osnac041 T1 mutant lines in response to salt stress.A, Photosynthesis-related genes. B, Peroxisome-related genes. C, Water deprivation-related genes. D, Mitogen-activated protein kinase signal-related genes. E, Eukaryotic-type ABC transporter-related genes. F, Plant hormone regulatory pathway-related genes. Log2(Fold change, FC) of differentially expressed genes in WT and osnac041 T1 mutant lines before (NaCl-) and after (NaCl+) salt stress is defined as [-1.5 > log(FC) > 1.5] with false discovery rate (FDR) < 0.05, and the top 30 most significant differences are shown in the heat-map.

Supplemental Table 1 Result of the NLS-mapper predictions of the location of OsNAC041 expression.

Predicted bipartite NLS position	Sequence	Score
17	DLPGFRFHPTEEELLDFYLSRVVLGKKLHFNI	2.4
37	RVVLGKKLHFNIIGTLNIYRHDPWDLPGMAKI	2.4
37	RVVLGKKLHFNIIGTLNIYRHDPWDLPGMAKIGE	2.1
91	RTTERGFWKATGSDRAIRSSGDPKRVIGLKKTLV	2.2
91	RTTERGFWKATGSDRAIRSSGDPKRVIGLKKTLVFY	2.9
115	RVIGLKKTLVFYQGRAPRGTKTDWVMNEYRLPD	2
141	NEYRLPDYGAARAAAPPPKEDMVLCKIYR	2.9
161	DMVLCKIYRKATPLKELEQRASAMEEMQRGS	2.4
NLS, Nuclear localization signal.

Supplemental Table 2 Oligonucleotides used in this study

Name	Primer sequence
OsActin-RT-F	AGCTGCGGGTATCCATGAGA
OsActin-RT-R	GCAATGCCAGGGAACATAGTG
4324705-RT-F	GTTCTTCGTCCAGGCCATCGTCA
4324705-RT-R	GTAGGCCCATGCGTTGTTGTTGA
4324706-RT-F	GTCTTCGTCAGTGGCAACCTC
4324706-RT-R	GAAACATCTGGCTGAACCTGAG
4324707-RT-F	GGTCGCGTAGTTCAAGTGGG
4324707-RT-R	TCGCCTGACATTATTTCACCAA
4324708-RT-F	TGGACTCGCTGGACCAGAAC
4324708-RT-R	CGGTGTAGGGCTGGAACG
4324709-RT-F	GCAGACGCAGACCAAGAAGAA
4324709-RT-R	GGCGGCAGAGGAAAGAGC
4324710-RT-F	GCCACCATCGAGAAGACGAAGCG
4324710-RT-R	AGCAGGCCGAACTTGCCGTAG
4324711-RT-F	GTGGTTCGGTTCGCCAAGGTG
4324711-RT-R	ATCTCGTCCAGGGCGTAGTTGT
4324712-RT-F	TTGAAGGGATAGGAAGGACC
4324712-RT-R	AGACAGAGTCAGAAGGGAAG
4324713-RT-F	ACTGGAGGAATGTGGGACTGGC
4324713-RT-R	TCACCGACCCGAACAGCAAA
4324714-RT-F	CGCAGCTCCGACAACATCT
4324714-RT-R	GCTGGGTGACACTCTCTCTACAAG
OsNAC041-sgRNA01-F	GTGTGCGTAGGGAGACAGGCAGTGC
OsNAC041-sgRNA01-R	AAACGCACTGCCTGTCTCCCTACGC
NAC041-F	GCAAAATGATCGGAATGAAGTG
NAC041-R	GCGAAGACCAAAAGATTGATTG

Supplemental Fig. 1 Validation of the RNA-seq results by quantitative real-time PCR.

Supplemental Fig. 2 Venn diagram analysis of the differentially expressed genes.

Supplemental Fig. 3 Alignment between OsNAC041 and AtNAC036.

Supplemental Fig. 4 Phylogenetic tree analysis of OsNAC041.

Supplemental Fig. 5 Analysis of photosynthesis-related differentially expressed genes and photosynthesis-antenna proteins.

Supplemental Fig. 6 Analysis of peroxisome-related differentially expressed genes.

Supplemental Fig. 7 Analysis of MAPK signaling-related differentially expressed genes.

Supplemental Fig. 8 Analysis of eukaryotic-type ABC transporter-related differentially expressed genes.

Supplemental Fig. 9 Analysis of plant hormone signal transduction-related differentially expressed genes.

References 56

[1]	Aida M, Ishida T, Fukaki H, Fujisawa H, Tasaka M.1997. Genes involved in organ separation in Arabidopsis: An analysis of the cup-shaped cotyledon mutant. Plant Cell, 9(6): 841-857.
[2]	Archer E K.2014. American society of plant biologists: Position statement on the education of young children about plants.Cbe-Life Sci Educ, 13(4): 575-576.
[3]	Cabello J V, Lodeyro A F, Zurbriggen M D.2014. Novel perspectives for the engineering of abiotic stress tolerance in plants.Curr Opin Biotech, 26: 62-70.
[4]	Chen X, Wang Y F, Lv B, Li J, Luo L Q, Lu S C, Zhang X, Ma H, Ming F.2014. The NAC family transcription factor OsNAP confers abiotic stress response through the ABA pathway. Plant Cell Physiol, 55(3): 604-619.
[5]	Depuydt S, Hardtke C S.2011. Hormone signalling crosstalk in plant growth regulation.Curr Biol, 21(9): 365-373.
[6]	Doczi R, Okresz L, Romero A E, Paccanaro A, Bogre L.2012. Exploring the evolutionary path of plant MAPK networks.Trends Plant Sci, 17(9): 518-525.
[7]	Dolferus R.2014. To grow or not to grow: A stressful decision for plants.Plant Sci, 229: 247-261.
[8]	Duan M, Zhang R X, Zhu F G, Zhang Z Q, Gou L M, Wen J Q, Dong J L, Wang T.2017. A lipid-anchored NAC transcription factor is translocated into the nucleus and activates glyoxalase I expression during drought stress.Plant Cell, 29(7): 1748-1772.
[9]	Ernst H A, Olsen A N, Larsen S, lo Leggio L.2004. Structure of the conserved domain of ANAC, a member of the NAC family of transcription factors.EMBO Rep, 5(3): 297-303.
[10]	Fang Y J, Liao K F, Du H, Xu Y, Song H Z, Li X H, Xiong L Z.2015. A stress-responsive NAC transcription factor SNAC3 confers heat and drought tolerance through modulation of reactive oxygen species in rice. J Exp Bot, 66: 6803-6817.
[11]	Francisco R M, Regalado A, Ageorges A, Burla B J, Bassin B, Eisenach C, Zarrouk O, Vialet S, Marlin T, Chaves M M, Martinoia E, Nagya R.2013. ABCC1, an ATP binding cassette protein from grape berry, transports anthocyanidin 3-O-glucosides. Plant Cell, 25(5): 1840-1854.
[12]	Hayashi H, Sakamoto A, Alia, Murata N.1998. Enhancement of stress tolerance by gene-engineering of betaine accumulation in plants.Photosynth: Mech Eff: 2419-2424.
[13]	Hu H H, Dai M Q, Yao J L, Xiao B Z, Li X H, Zhang Q F, Xiong L Z.2006. Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice.Proc Natl Acad Sci USA, 103: 12987-12992.
[14]	Hu H H, You J, Fang Y J, Zhu X Y, Qi Z Y, Xiong L Z.2008. Characterization of transcription factor gene SNAC2 conferring cold and salt tolerance in rice. Plant Mol Biol, 67: 169-181.
[15]	Hu H H, Xiong L Z.2014. Genetic engineering and breeding of drought-resistant crops.Annu Rev Plant Biol, 65(1): 715-741.
[16]	Jeong J S, Kim Y S, Redillas M C F R, Jang G, Jung H, Bang S W, Choi Y D, Ha S H, Reuzeau C, Kim J K.2013. OsNAC5 overexpression enlarges root diameter in rice plants leading to enhanced drought tolerance and increased grain yield in the field.Plant Biotechnol J, 11(1): 101-114.
[17]	Kato H, Motomura T, Komeda Y, Saito T, Kato A.2010. Overexpression of the NAC transcription factor family gene ANAC036 results in a dwarf phenotype in Arabidopsis thaliana. J Plant Physiol, 167(7): 571-577.
[18]	Kosugi S, Hasebe M, Tomita M, Yanagawa H.2009. Systematic identification of yeast cell cycle-dependent nucleocytoplasmic shuttling proteins by prediction of composite motifs.Proc Natl Acad Sci USA, 106: 10171-10176.
[19]	Kou L L, Hu H C, Ma L, Ke X N, Liu M Y, Lian W M, Jin K, Xie L J, Liu Q P.2018. Functional analysis of a copper/zinc SOD encoding gene in response to arsenite stress in rice.Chin J Rice Sci, 32(5): 437-444. (in Chinese with English abstract)
[20]	Kudo M, Kidokoro S, Yoshida T, Mizoi J, Todaka D, Fernie A R, Shinozaki K, Yamaguchi-Shinozaki K.2017. Double overexpression of DREB and PIF transcription factors improves drought stress tolerance and cell elongation in transgenic plants.Plant Biotechnol J, 15(4): 458-471.
[21]	Kunieda T, Mitsuda N, Ohme-Takagi M, Takeda S, Aida M, Tasaka M, Kondo M, Nishimura M, Hara-Nishimura I.2008. NAC family proteins NARS1/NAC2 and NARS2/NAM in the outer integument regulate embryogenesis in Arabidopsis. Plant Cell, 20(10): 2631-2642.
[22]	Lata C, Prasad M.2011. Role of DREBs in regulation of abiotic stress responses in plants.J Exp Bot, 62(14): 4731-4748.
[23]	Lee D K, Chung P J, Jeong J S, Jang G, Bang S W, Jung H, Kim Y S, Ha S H, Choi Y D, Kim J K.2017. The rice OsNAC6 transcription factor orchestrates multiple molecular mechanisms involving root structural adaptions and nicotianamine biosynthesis for drought tolerance. Plant Biotechnol J, 15(6): 754-764.
[24]	Lefevre F, Baijot A, Boutry M.2015. Plant ABC transporters: Time for biochemistry?Biochem Soc T, 43(5): 931-936.
[25]	Liang C Z, Wang Y Q, Zhu Y N, Tang J Y, Hu B, Liu L C, Ou S J, Wu H K, Sun X H, Chu J F, Chu C C.2014. OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice.Proc Natl Acad Sci USA, 111: 10013-10018.
[26]	Liu X M, Nguyen X C, Kim K E, Han H J, Yoo J, Lee K, Kim M C, Yun D J, Chung W S.2013. Phosphorylation of the zinc finger transcriptional regulator ZAT6 by MPK6 regulates Arabidopsis seed germination under salt and osmotic stress. Biochem Biophys Res Comm, 430(3): 1054-1059.
[27]	Ma C Q, Wang Y G, Gu D, Nan J D, Chen S X, Li H Y.2017. Overexpression of S-adenosyl-L-methionine synthetase 2 from sugar beet M14 increased Arabidopsis tolerance to salt and oxidative stress. Int J Mol Sci, 18(4): 847.
[28]	Mentewab A, Stewart C N.2005. Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants. Nat Biotechnol, 23(9): 1177-1180.
[29]	Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R.2010. Reactive oxygen species homeostasis and signalling during drought and salinity stresses.Plant Cell Environ, 33(4): 453-467.
[30]	Munoz-Munoz J L, Garcia-Molina F, Garcia-Ruiz P A, Arribas E, Tudela J, Garcia-Canovas F, Rodriguez-Lopez J N.2009. Enzymatic and chemical oxidation of trihydroxylated phenols.Food Chem, 113(2): 435-444.
[31]	Nuruzzaman M, Sharoni A M, Satoh K, Moumeni A, Venuprasad R, Serraj R, Kumar A, Leung H, Attia K, Kikuchi S.2012. Comprehensive gene expression analysis of the NAC gene family under normal growth conditions, hormone treatment, and drought stress conditions in rice using near-isogenic lines (NILs) generated from crossing Aday selection (drought tolerant) and IR64.Mol Genet Genomics, 287(5): 389-410.
[32]	Peleg Z, Blumwald E.2011. Hormone balance and abiotic stress tolerance in crop plants.Curr Opin Plant Biol, 14(3): 290-295.
[33]	Petricka J J, Winter C M, Benfey P N.2012. Control of Arabidopsis root development. Annu Rev Plant Biol, 63: 563-590.
[34]	Pitzschke A, Datta S, Persak H.2014. Salt stress in Arabidopsis: Lipid transfer protein AZI1 and its control by mitogen-activated protein kinase MPK3. Mol Plant, 7(4): 722-738.
[35]	Rai G K, Rai N P, Rathaur S, Kumar S, Singh M.2013. Expression of rd29A::AtDREB1A/CBF3 in tomato alleviates drought induced oxidative stress by regulating key enzymatic and non- enzymatic antioxidants. Plant Physiol Biochem, 69: 90-100.
[36]	Redillas M C F R, Jeong J S, Kim Y S, Jung H, Bang S W, Choi Y D, Ha S H, Reuzeau C, Kim J K.2012. The overexpression of OsNAC9 alters the root architecture of rice plants enhancing drought resistance and grain yield under field conditions. Plant Biotechnol J, 10(7): 792-805.
[37]	Schmidt R, Mieulet D, Hubberten H M, Obata T, Hoefgen R, Fernie A R, Fisahn J, Segundo B S, Guiderdoni E, Schippers J H M, Mueller-Roeber B.2013. SALT-RESPONSIVE ERF1 regulates reactive oxygen species-dependent signaling during the initial response to salt stress in rice.Plant Cell, 25(6): 2115-2131.
[38]	Shen H S, Liu C T, Zhang Y, Meng X P, Zhou X, Chu C C, Wang X P.2012. OsWRKY30 is activated by MAP kinases to confer drought tolerance in rice.Plant Mol Biol, 80(3): 241-253.
[39]	Shen J B, Lv B, Luo L Q, He J M, Mao C J, Xi D D, Ming F.2017. The NAC-type transcription factor OsNAC2 regulates ABA-dependent genes and abiotic stress tolerance in rice. Sci Rep, 7: 40641.
[40]	Shi Y T, Ding Y L, Yang S H.2015. Cold signal transduction and its interplay with phytohormones during cold acclimation.Plant Cell Physiol, 56(1): 7-15.
[41]	Souer E, van Houwelingen A, Kloos D, Mol J, Koes R.1996. The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries.Cell, 85(2): 159-170.
[42]	Sousa R H V, Carvalho F E L, Ribeiro C W, Passaia G, Cunha J R, Lima-Melo Y, Margis-Pinheiro M, Silveira J A G.2015. Peroxisomal APX knockdown triggers antioxidant mechanisms favourable for coping with high photorespiratory H2O2 induced by CAT deficiency in rice.Plant Cell Environ, 38: 499-513.
[43]	Tang X, Zheng X L, Qi Y P, Zhang D W, Cheng Y, Tang A T, Voytas D F, Zhang Y.2016. A single transcript CRISPR-Cas9 system for efficient genome editing in plants.Mol Plant, 9(7): 1088-1091.
[44]	Tang X, Lowder L G, Zhang T, Malzahn A A, Zheng X L, Voytas D F, Zhong Z H, Chen Y Y, Ren Q R, Li Q, Kirkland E R, Zhang Y, Qi Y P.2017. A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants.Nat Plants, 3: 17108.
[45]	Theodoulou F L, Kerr I D.2015. ABC transporter research: Going strong 40 years on.Biochem Soc Trans, 43(5): 1033-1040.
[46]	Tian X J, Li X F, Zhou W J, Ren Y K, Wang Z Y, Liu Z Q, Tang J Q, Tong H N, Fang J, Bu Q Y.2017. Transcription factor OsWRKY53 positively regulates brassinosteroid signaling and plant architecture. Plant Physiol, 175(3): 1337-1349.
[47]	Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S.2006. Early infection of scutellum tissue with Agrobacterium allows high- speed transformation of rice. Plant J, 47: 969-976.
[48]	Wu Y C, Liu C L, Kuang J, Ge Q, Zhang Y, Wang Z Z.2014. Overexpression of SmLEA enhances salt and drought tolerance in Escherichia coli and Salvia miltiorrhiza. Protoplasma, 251: 1191-1199.
[49]	Xiong H Y, Yu J P, Miao J L, Li J J, Zhang H L, Wang X, Liu P L, Zhao Y, Jiang C H, Yin Z G, Li Y, Guo Y, Fu B Y, Wang W S, Li Z K, Ali J, Li Z C.2018. Natural variation in OsLG3 increases drought tolerance in rice by inducing ROS scavenging. Plant Physiol, 178(1): 451-467.
[50]	Xu Z Y, Kim S Y, Hyeon D Y, Kim D H, Dong T, Park Y, Jin J B, Joo S H, Kim S K, Hong J C, Hwang D, Hwang I.2013. The Arabidopsis NAC transcription factor ANAC096 cooperates with bZIP-type transcription factors in dehydration and osmotic stress responses. Plant Cell, 25(11): 4708-4724.
[51]	Zheng X N, Chen B, Lu G J, Han B.2009. Overexpression of a NAC transcription factor enhances rice drought and salt tolerance.Biochem Biophy Res Co, 379(4): 985-989.
[52]	Zheng X L, Yang S X, Zhang D W, Zhong Z H, Tang X, Deng K J, Zhou J P, Qi Y P, Zhang Y.2016. Effective screen of CRISPR/ Cas9-induced mutants in rice by single-strand conformation polymorphism.Plant Cell Rep, 35(7): 1545-1554.
[53]	Zhong Z H, Zhang Y X, You Q, Tang X, Ren Q R, Liu S S, Yang L J, Wang Y, Liu X P, Liu B L, Zhang T, Zheng X L, Le Y, Zhang Y, Qi Y P.2018. Plant genome editing using FnCpf1 and LbCpf1 nucleases at redefined and altered PAM sites.Mol Plant, 11(7): 999-1002.
[54]	Zhou J P, Deng K J, Cheng Y, Zhong Z H, Tian L, Tang X, Tang A T, Zheng X L, Zhang T, Qi Y P, Zhang Y.2017. CRISPR-Cas9 based genome editing reveals new insights into microRNA function and regulation in rice.Front Plant Sci, 8: 1598.
[55]	Zhou M Q, Xu M, Wu L H, Shen C, Ma H, Lin J.2014. CbCBF from Capsella bursa-pastoris enhances cold tolerance and restrains growth in Nicotiana tabacum by antagonizing with gibberellin and affecting cell cycle signaling. Plant Mol Biol, 85(3): 259-275.
[56]	Zhu J K.2002. Salt and drought stress signal transduction in plants.Annu Rev Plant Biol, 53: 247-273.