Hrh-miRn458 Regulates Oil Biosynthesis of Sea Buckthorn via Targeting Transcription Factor WRI1

doi:10.11983/CBB22004

Abstract

Abstract: Transcription factor WRINKLED 1 (WRI1) plays important regulatory role in oil biosynthesis. Most studies focused on the regulation mechanism of its transcription, translation, and downstream target genes. Very few reports on its post transcriptional regulation. To explore the relationship between hrh-miRn458 and transcription factor WRI1 of sea buckthorn, and analyze their expression dynamics in oil biosynthesis of pulp and seeds, the bioinformatic method was used to predict the binding sites in the CDS region of the candidate target gene WRI1 by hrh-miRn458 mature sequence. Double luciferase reporter system and RNA pull down assay were used to verify whether hrh-miRn458 could target the CDS region of WRI1. The expression of hrh-miRn458 and WRI1 were analyzed using real-time fluorescent PCR technology in developing sea buckthorn pulp and seeds. The results showed that the positions between 309-327 of the CDS region of WRI1 gene were complementary to 15 bases of the mature hrh-miRn458 sequence. The vector of pmirGLO-WRI1-WT+hrh-miRn458 mimics significantly inhibited the luciferase activity (P<0.001), and RNA pull down assay further verified that hrh-miRn458 could pull down WRI1 in the system. In pulp and seeds of different developing stages, the expression level of hrh-miRn458 decreased at first and then increased, but the expression level of its target gene WRI1 first increased and then decreased. At the same developmental stage, the hrh-miRn458 expression level in pulp was less than that of seeds, but the expression level of its target gene WRI1 in pulp was more than that of seeds. Thus, sea buckthorn hrh-miRn458 may target the WRI1, and there is negatively regulative relationship between them. These data provide scientific references for further understanding the seed oil biosynthesis mechanism of sea buckthorn and breeding for high oil varieties.

Key words: sea buckthorn, oil biosynthesis, hrh-miRn458, transcription factor WRI1, qRT-PCR

Yu Miao, Ruan Chengjiang, Ding Jian, Li Jingbin, Lu Shunguang, Wen Xiufeng. Hrh-miRn458 Regulates Oil Biosynthesis of Sea Buckthorn via Targeting Transcription Factor WRI1[J]. Chinese Bulletin of Botany, 2022, 57(5): 635-648.

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.chinbullbotany.com/EN/10.11983/CBB22004

https://www.chinbullbotany.com/EN/Y2022/V57/I5/635

Figures/Tables 7

Table 1 Primers for qRT-PCR

Gene name	Primer sequence (5′→3′)
UBQ5-F	GGCGAGTTTGACCTTCTTCTT
UBQ5-R	CCACCTTGTTCTTCGTCTCC
U6-F	GGAACGATACAGAGAAGATTAGC
U6-R	TGGAACGCTTCACGAATTTGCG
WRI1-F	AACGCTCATGGAAGAACAATAATC
WRI1-R	GCCTTGTGACTCCTTTGTAAATC
hrh-miRn458-F	GAGGAAGGAGAAGTCGAG

Figure 1 Phenotype of fruits and seeds of Za56 (A) and their oil contents (B) at different developmental stages Different lowercase letters indicate significant differences of oil content between pulp and seeds at the same developmental stage at the level of 0.05. daa: Days after anthesis. Bar=3 mm

Figure 2 Precursor secondary structure of sea buckthorn hrh-miRn458 and binding sites in the targeted WRI1 gene (A) Precursor secondary structure, red indicates segments corresponding to the mature hrh-miRn458; (B) Binding sites in targeted WRI1 gene, the bases at both ends of the vertical line represent the target sites.

Figure 3 Sequence alignment of mature sequence of sea buckthorn hrh-miRn458 precursor (A) and phylogenetic relationship of HrWRI1 with its homologous genes in plants (B)

Figure 4 Sequences identification of pmirGLO-WRI1-WT/MUT+hrh-miRn458 and activity analysis of dual luciferase (A) Sequences of PCR products of pmirGLO-WRI1-WT/MUT+hrh-miRn458 from plasmid, yellow letters indicated mutation sequence; (B) Identification of pmirGLO-WRI1-WT/MUT plasmid digestion (a: Plasmid digested by XhoI enzyme; b: Plasmid DNA of WT; c: Plasmid digested by NheI enzyme; d: Plasmid DNA of MUT); (C) Activity analysis of dual luciferase (the numbers of 1, 2, 3 and 4 represents plasmid pmirGLO-WRI1-WT+mimics NC, pmirGLO-WRI1-WT+hrh-miRn458 mimics, pmirGLO-WRI1-MUT+mimics NC and pmirGLO-WRI1-MUT+hrh-miRn458 mimics, respectively.). *** Significant difference at the level of P<0.001; ns means no significant difference (P>0.05).

Figure 5 qRT-PCR detection of hrh-miRn458 and WRI1 expression (A) Expressions of hrh-miRn458 enriched by probe and NC groups; (B) Expression of WRI1 enriched by probe and NC groups. * and *** indicated significant differences of expression of hrh-miRn458 and WRI1 between NC and probe groups at the level of 0.01 and 0.001, respectively.

Figure 6 Expression of hrh-miRn458 and WRI1 in sea buckthorn seeds and pulp at different developmental stages (A) Expression of hrh-miRn458 and WRI1 in sea buckthorn seed; (B) Expression of hrh-miRn458 and WRI1 in sea buckthorn pulp. Different lowercase letters indicate significant differences of oil content between hrh-miRn458 and WRI1 of seeds and pulp at the same developmental stage at the level of 0.05. daa: Days after anthesis

References 65

[1]	丁健, 阮成江, 关莹, 单金友 (2017a). 沙棘果肉和种子发育过程中含油量及脂肪酸组成的对比分析. 中国油脂 42(5), 140-144.
[2]	丁健, 阮成江, 关莹, 吴立仁, 单金友 (2017b). 沙棘种子高积累碳十八不饱和脂肪酸的多基因协同调控机制. 西北植物学报 37, 1080-1089.
[3]	杜维, 丁健, 阮成江 (2018). 沙棘果实发育过程中内源激素水平的动态变化. 植物学报 53, 219-226.
[4]	金龙飞, 周丽霞, 曹红星, 杨耀东 (2022). WRI1调控植物油脂合成的研究进展. 中国油料作物学报 44, 687-698.
[5]	李晓康, 刘红芳, 刘婧琳, 郑明, 华玮 (2018). 油菜miR156基因家族及其靶基因生物信息学分析及鉴定. 中国油料作物学报 40, 163-173.
[6]	廉永善, 陈学林 (1996). 沙棘属植物的系统分类. 沙棘 (1), 15-24.
[7]	刘华, 于芮芦, 龚静云, 王建军, 李东, 陈明训 (2021). 谷子SiMYB76基因抑制种子油脂积累的功能分析. 西北农业学报 30, 838-847.
[8]	王丽丽, 赵韩生, 孙化雨, 董丽莉, 娄永峰, 高志民 (2015). 胁迫条件下毛竹miR164b及其靶基因PeNAC1表达研究. 林业科学研究 28, 605-611.
[9]	王田幸子, 朱峥, 陈悦, 刘玉晴, 燕高伟, 徐珊, 张彤, 马金姣, 窦世娟, 李莉云, 刘国振 (2021). 水稻OsWRKY42是Xa21介导的抗白叶枯病途径新元件. 植物学报 56, 687-698.
[10]	吴丹丹, 陈永坤, 杨宇, 孔春艳, 龚明 (2021). 小桐子半胱氨酸蛋白酶家族和相应miRNAs的鉴定及其对低温锻炼的响应. 植物学报 56, 544-558.
[11]	吴玉, 沈永宝, 史锋厚 (2019). 调控植物种子发育的转录因子研究进展. 生物技术通报 35(11), 150-159. DOI
[12]	尹丹丹, 李珊珊, 吴倩, 冯成庸, 李冰, 王倩玉, 王亮生, 徐文忠 (2018). 我国6种主要木本油料作物的研究进展. 植物学报 53, 110-125.
[13]	张翠桔, 莫蓓莘, 陈雪梅, 崔洁 (2020). 植物miRNA作用方式的分子机制研究进展. 生物技术通报 36(7), 1-14. DOI
[14]	张莞晨, 阮成江, 丁健, 刘祾悦, 蔡爽, 熊朝伟, 吴波 (2018). 沙棘非种子组织和种子油脂合成积累的源汇基因协同表达. 分子植物育种 16, 5561-5566.
[15]	An D, Kim H, Ju S, Go YS, Kim HY, Suh MC (2017). Expression of Camelina WRINKLED1 isoforms rescue the seed phenotype of the Arabidopsis wri1 mutant and increase the triacylglycerol content in tobacco leaves. Front Plant Sci 8, 34.
[16]	Belide S, Petrie JR, Shrestha P, Singh SP (2012). Modification of seed oil composition in Arabidopsis by artificial microRNA-mediated gene silencing. Front Plant Sci 3, 168.
[17]	Cheng K, Pan YF, Liu LM, Zhang HQ, Zhang YM (2021). Integrated transcriptomic and bioinformatics analyses reveal the molecular mechanisms for the differences in seed oil and starch content between Glycine max and Cicer arietinum. Front Plant Sci 12, 743680.
[18]	Ding J, Ruan CJ, Guan Y, Krishna P (2018). Identification of microRNAs involved in lipid biosynthesis and seed size in developing sea buckthorn seeds using high-throughput sequencing. Sci Rep 8, 4022.
[19]	Ding J, Ruan CJ, Guan Y, Li H, Du W, Lu SG, Wen XF, Tang K, Chen Y (2022). Nontargeted metabolomic and multigene expression analyses reveal the mechanism of oil biosynthesis in sea buckthorn berry pulp rich in palmitoleic acid. Food Chem 374, 131719.
[20]	Divi UK, Zhou XR, Wang PH, Butlin J, Zhang DM, Liu Q, Vanhercke T, Petrie JR, Talbot M, White RG, Taylor JM, Larkin P, Singh SP (2016). Deep sequencing of the fruit transcriptome and lipid accumulation in a non-seed tissue of Chinese tallow, a potential biofuel crop. Plant Cell Physiol 57, 125-137.
[21]	Donaire L, Pedrola L, de la Rosa R, Llave C (2011). High-throughput sequencing of RNA silencing-associated small RNAs in olive (Olea europaea L.). PLoS One 6, e27-916.
[22]	Du W, Xiong CW, Ding J, Nybom H, Ruan CJ, Guo H (2019). Tandem mass tag based quantitative proteomics of developing sea buckthorn berries reveals candidate proteins related to lipid metabolism. J Proteome Res 18, 1958-1969.
[23]	Fan G, Liu Y, Du H, Kuang TT, Zhang Y (2020). Identification of drought-responsive miRNAs in Hippophae tibetana using high-throughput sequencing. 3 Biotech 10, 53.
[24]	Fatima T, Snyder CL, Schroeder WR, Cram D, Datla R, Wishart D, Weselake RJ, Krishna P (2012). Fatty acid composition of developing sea buckthorn (Hippophae rham- noides L.) berry and the transcriptome of the mature seed. PLoS One 7, e34099.
[25]	Focks N, Benning C (1998). Wrinkled1: a novel, low-seed- oil mutant of Arabidopsis with a deficiency in the seed- specific regulation of carbohydrate metabolism. Plant Physiol 118, 91-101.
[26]	Gao GR, Lv ZR, Zhang GY, Li JY, Zhang JG, He CY (2021). An ABA-flavonoid relationship contributes to the differences in drought resistance between different sea buckthorn subspecies. Tree Physiol 41, 744-755.
[27]	Gao LC, Wang YF, Zhu ZY, Chen H, Sun RH, Zheng YS, Li DD (2019). EgmiR5179 from the mesocarp of oil palm (Elaeis guineensis Jacq.) regulates oil accumulation by targeting NAD transporter 1. Ind Crops Prod 137, 126-136.
[28]	Ge Y, Zang XP, Yang Y, Wang T, Ma WH (2021). In-depth analysis of potential PaAP2/ERF transcription factor related to fatty acid accumulation in avocado (Persea ameri- cana Mill.) and functional characterization of two PaAP2/ ERF genes in transgenic tomato. Plant Physiol Biochem 158, 308-320.
[29]	Ji HY, Liu DT, Yang ZL (2021). High oil accumulation in tuber of yellow nutsedge compared to purple nutsedge is associated with more abundant expression of genes involved in fatty acid synthesis and triacylglycerol storage. Biotechnol Biofuels 14, 54.
[30]	Ji XJ, Mao X, Hao QT, Liu BL, Xue JA, Li RZ (2018). Splice variants of the castor WRI1 gene upregulate fatty acid and oil biosynthesis when expressed in tobacco leaves. Int J Mol Sci 19, 146.
[31]	Kong Q, Singh SK, Mantyla JJ, Pattanaik S, Guo L, Yuan L, Benning C, Ma W (2020). TEOSINTE BRANCHED1/ CYCLOIDEA/PROLIFERATING CELL FACTOR 4 interacts with WRINKLED1 to mediate seed oil biosynthesis. Plant Physiol 184, 658-665.
[32]	Kong Q, Yuan L, Ma W (2019). WRINKLED1, a ‘master regulator’ in transcriptional control of plant oil biosynthesis. Plants 8, 238.
[33]	Lee HG, Kim H, Suh MC, Kim HU, Seo PJ (2018). The MYB96 transcription factor regulates triacylglycerol accumulation by activating DGAT1 and PDAT1 expression in Arabidopsis seeds. Plant Cell Physiol 59, 1432-1442.
[34]	Li D, Jin CY, Duan SW, Zhu YN, Qi SH, Liu KG, Gao CH, Ma HL, Zhang M, Liao YC, Chen MX (2017). MYB89 transcription factor represses seed oil accumulation. Plant Physiol 173, 1211-1225.
[35]	Li JB, Ding J, Yu X, Li H, Ruan CJ (2019). Identification and expression analysis of critical microRNA-transcription factor regulatory modules related to seed development and oil accumulation in developing Hippophae rhamnoides seeds. Ind Crop Prod 137, 33-42.
[36]	Liebsch D, Palatnik JF (2020). MicroRNA miR396, GRF transcription factors and GIF co-regulators: a conserved plant growth regulatory module with potential for breeding and biotechnology. Curr Opin Plant Biol 53, 31-42.
[37]	Liu JF, Luo QZ, Zhang XZ, Zhang Q, Cheng YQ (2020). Identification of vital candidate microRNA/mRNA pairs regulating ovule development using high-throughput sequencing in hazel. BMC Dev Biol 20, 13.
[38]	Ma W, Kong Q, Mantyla JJ, Yang Y, Ohlrogge JB, Benning C (2016). 14-3-3 protein mediates plant seed oil biosynthesis through interaction with AtWRI1. Plant J 88, 228-235.
[39]	Nasaruddin NM, Harikrishna K, Othman RY, Hoon LS, Harikrishna JA (2007). Computational prediction of microRNAs from oil palm (Elaeis guineensis Jacq.) expressed sequence tags. As-Pac J Mol Biol Biotechnol 15, 107-113.
[40]	Parreira JR, Cappuccio M, Balestrazzi A, Fevereiro P, Araújo SS (2021). MicroRNAs expression dynamics reveal post-transcriptional mechanisms regulating seed development in Phaseolus vulgaris L. Hortic Res 8, 18.
[41]	Qu J, Ye J, Geng YF, Sun YW, Gao SQ, Zhang BP, Chen W, Chua NH (2012). Dissecting functions of KATANIN and WRINKLED1 in cotton fiber development by virus-induced gene silencing. Plant Physiol 160, 738-748.
[42]	Rich MK, Vigneron N, Libourel C, Keller J, Xue L, Hajheidari M, Radhakrishnan GV, Le Ru A, Diop SI, Potente G, Conti E, Duijsings D, Batut A, Le Faouder P, Kodama K, Kyozuka J, Sallet E, Bécard G, Rodriguez-Franco M, Ott T, Bertrand-Michel J, Oldroyd GED, Szövényi P, Bucher M, Delaux PM (2021). Lipid exchanges drove the evolution of mutualism during plant terrestrialization. Science 372, 864-868.
[43]	Ruan CJ, Rumpunen K, Nybom H (2013). Advances in improvement of quality and resistance in a multipurpose crop: sea buckthorn. Crit Rev Biotechnol 33, 126-144.
[44]	Schommer C, Debernardi JM, Bresso EG, Rodriguez RE, Palatnik JF (2014). Repression of cell proliferation by miR319-regulated TCP4. Mol Plant 7, 1533-1544.
[45]	Song QX, Liu YF, Hu XY, Zhang WK, Ma B, Chen SY, Zhang JS (2011). Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing. BMC Plant Biol 11, 5.
[46]	Sun QF, Xue JY, Lin L, Liu DX, Wu J, Jiang JJ, Wang YP (2018). Overexpression of soybean transcription factors GmDof4 and GmDof11 significantly increase the oleic acid content in seed of Brassica napus L. Agronomy 8, 222.
[47]	Tang GY, Xu PL, Li PX, Zhu JQ, Chen GX, Shan L, Wan SB (2021). Cloning and functional characterization of seed-specific LEC1A promoter from peanut (Arachis hypogaea L.). PLoS One 16, e0242949.
[48]	Tang LL, You WX, Wang Q, Huang F, Shao CW (2022). MicroRNA ssa-mir-196a-4 deceases lgr8 expression in testis development of Chinese tongue sole (Cynoglossus semilaevis). Comp Biochem Physiol Part B: Biochem Mol Biol 258, 110695.
[49]	Wang L, Ruan CJ, Bao AM, Li H (2021a). Small RNA profiling for identification of microRNAs involved in regulation of seed development and lipid biosynthesis in yellowhorn. BMC Plant Biol 21, 464.
[50]	Wang YF, Zou JX, Zhao J, Zheng YS, Li DD (2021b). EgmiR5179 regulates lipid metabolism by targeting EgMADS16 in the mesocarp of oil palm (Elaeis guineensis). Front Plant Sci 12, 722596.
[51]	Wu B, Ruan CJ, Han P, Ruan D, Xiong CW, Ding J, Liu SH (2019). Comparative transcriptomic analysis of high- and low-oil Camellia oleifera reveals a coordinated mechanism for the regulation of upstream and downstream multigenes for high oleic acid accumulation. 3 Biotech 9, 257.
[52]	Wu B, Ruan CJ, Shah AH, Li DH, Li H, Ding J, Li JB, Du W (2022). Identification of miRNA-mRNA regulatory modules involved in lipid metabolism and seed development in a woody oil tree (Camellia oleifera). Cells 11, 71.
[53]	Yang Z, Liu XL, Li N, Du C, Wang K, Zhao CZ, Wang ZH, Hu YG, Zhang M (2019). WRINKLED1 homologs highly and functionally express in oil-rich endosperms of oat and Castor. Plant Sci 287, 110193.
[54]	Yang Z, Liu XL, Wang K, Li ZW, Jia QL, Zhao CZ, Zhang M (2022). ABA-INSENSITIVE 3 with or without FUSCA3 highly up-regulates lipid droplet proteins and activates oil accumulation. J Exp Bot 73, 2077-2092.
[55]	Ye J, Wang CM, Sun YW, Qu J, Mao HZ, Chua NH (2018). Overexpression of a transcription factor increases lipid content in a woody perennial Jatropha curcas. Front Plant Sci 9, 1479.
[56]	Yeap WC, Lee FC, Shabari Shan DK, Musa H, Appleton DR, Kulaveerasingam H (2017). WRI1-1, ABI5, NF-YA3 and NF-YC2 increase oil biosynthesis in coordination with hormonal signaling during fruit development in oil palm. Plant J 91, 97-113.
[57]	Yin DD, Li SS, Shu QY, Gu ZY, Wu Q, Feng CY, Xu WZ, Wang LS (2018). Identification of microRNAs and long non-coding RNAs involved in fatty acid biosynthesis in tree peony seeds. Gene 666, 72-82.
[58]	Zhai ZY, Liu H, Shanklin J (2017). Phosphorylation of WRINKLED1 by KIN10 results in its proteasomal degradation, providing a link between energy homeostasis and lipid biosynthesis. Plant Cell 29, 871-889.
[59]	Zhang GY, Chen DG, Zhang T, Duan AG, Zhang JG, He CY (2018a). Transcriptomic and functional analyses unveil the role of long non-coding RNAs in anthocyanin biosynthesis during sea buckthorn fruit ripening. DNA Res 25, 465-476.
[60]	Zhang GY, Diao SF, Zhang T, Chen DG, He CY, Zhang JG (2019). Identification and characterization of circular RNAs during the sea buckthorn fruit development. RNA Biol 16, 354-361.
[61]	Zhang GY, Duan AG, Zhang JG, He CY (2017). Genome- wide analysis of long non-coding RNAs at the mature stage of sea buckthorn (Hippophae rhamnoides Linn) fruit. Gene 596, 130-136.
[62]	Zhang K, Zhang XR, Cai ZQ, Zhou J, Cao R, Zhao Y, Chen ZG, Wang DH, Ruan W, Zhao Q, Liu GQ, Xue YC, Qin Y, Zhou B, Wu LG, Nilsen T, Zhou Y, Fu XD (2018b). A novel class of microRNA-recognition elements that function only within open reading frames. Nat Struct Mol Biol 25, 1019-1027.
[63]	Zhang T, Gao GR, Liu JJ, Yang GJ, Lv ZR, Zhang JG, He CY (2020). Transcripts and ABA-dependent signaling in response to drought stress in Hippophae rhamnoides L. Trees 34, 1033-1045.
[64]	Zhao XC, Yang GY, Liu XQ, Yu ZD, Peng SB (2020). Integrated analysis of seed microRNA and mRNA transcriptome reveals important functional genes and microRNA- targets in the process of walnut (Juglans regia) seed oil accumulation. Int J Mol Sci 21, 9093.
[65]	Zhao YT, Wang M, Fu SX, Yang WC, Qi CK, Wang XJ (2012). Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production- and development-correlated expression and new small RNA classes. Plant Physiol 158, 813-823.