Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (20): 3875-3884.doi: 10.3864/j.issn.0578-1752.2022.20.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Function of FCS-Like Zinc-Finger Protein OsFLZ18 in Regulating Rice Flowering Time

MA YaMei(),ZHANG ShaoHong,ZHAO JunLiang(),LIU Bin()   

  1. Rice Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Key Laboratory of New Technology in Rice Breeding/Guangdong Rice Engineering Laboratory, Guangzhou 510640
  • Received:2022-05-20 Accepted:2022-06-23 Online:2022-10-16 Published:2022-10-24
  • Contact: JunLiang ZHAO,Bin LIU E-mail:mayamei@gdaas.cn;zhao_junliang@gdaas.cn;lbgz1009@163.com

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Abstract:

【Objective】Flowering time is an important agronomic trait which determines the yield and regional adaptability of rice, but the underlining molecular regulatory mechanism need further study. FCS-like Zinc finger proteins (FLZs) are a class of plant specific regulatory proteins which play essential roles in plant growth and stress response, but their functions in regulating flowering time have not been reported. This study aims to investigate the potential function of FLZ proteins in rice flowering time control. The finding will broaden our understanding on the molecular regulatory mechanism of rice flowering time.and provide new theoretical basis and gene resource for rice breeding. 【Method】Based on the target sequences published in RGAP database, OsFLZ18 overexpression vector and CRISPR-Cas9 vector were generated and introduced into Japonica variety Nipponbare by Agrobacterium tumefaciens-mediated genetic transformation assay. Homozygous CRISPR knockout mutants were screened by PCR and sequencing analyses. The quantitative real-time PCR (qRT-PCR) assay was used to examine the spatial-temporal expression and diurnal rhythmic expression of OsFLZ18, as well as the effects of OsFLZ18 on the transcription of several known flowering time-related genes. Yeast two-hybrid assay (Y2H) was used to test the interaction between OsFLZ18 and the flowering time-related regulatory proteins.【Result】OsFLZ18 was ubiquitously expressed in various rice tissues, with the highest expression level in 14 day-old seedling, followed by leaf sheaths and leaf blades at the tillering stage, and stem and young panicles at reproductive stages. The OsFLZ18-CRISPR vector was constructed and transformed into Nipponbare. Two independent homozygous OE lines (OE-2, OE-3) with higher OsFLZ18 expression level and two homozygous mutants (CRISPR-21, CRISPR-25) were selected for further study. Phenotypic observation showed that the OE lines flowered later than the wild-type plants under both natural long-day and short-day conditions in Guangzhou, while the CRISPR lines had no obvious differences in heading date when compared to the wild-type plants. The expression levels of Ehd1, Hd3a and RFT1 were significantly decreased in OE-2 plants compared with those in the wild-type plants under artificial short-day conditions, but no significant difference in the expression level of Hd1 was observed between them. The results of Y2H experiment showed that OsFLZ18 interacted with OsMADS51, a positive regulator of rice flowering time. Furthermore, OsFLZ18 exhibits a diurnal rhythmic expression profile, showing lower expression levels in the daytime and higher expression levels at night with a peak at midnight. 【Conclusion】Overexpression of OsFLZ18 delays rice flowering time.

Key words: rice, flowering time, FLZ, OsMADS51

Table 1

Primers used in this study"

引物名称 Primer name 引物序列 Primer sequence (5′-3′) 用途 Purpose
FLZ18-1-F CGCCTGCTTCCTCTGCAAGCgttttagagctagaaat 基因敲除载体
Gene editing
FLZ18-1-R GCTTGCAGAGGAAGCAGGCGCggcagccaagccagca
FLZ18-2-F CGTTCTGCAGCGACGACTGCgttttagagctagaaat
FLZ18-2-R GCAGTCGTCGCTGCAGAACGCaacacaagcggcagc
CRISPR-F AACCTGCCGTTAGATATGAT 靶点检测引物
Target detection primer
CRISPR-R AACATTCAGAGACAAACCAC
FLZ18-F CAGTGAATTCCACCCGATGATGGAGTCGAGGTACGTCA 酵母双杂载体
Y2H
FLZ18-R TATCGATGCCCACCCTCACACAGTGCCAGACACGG
MADS51-F CAGTGAATTCCACCCGATGCCCCCCCCCCCCCCCC
MADS51-R TATCGATGCCCACCCTCATGCACTTCCTTCCTCCT
FLZ18-qF ATGATGGAGTCGAGGTACGT qRT-PCR引物
Primers used for qRT-PCR
FLZ18-qR AGGTAGTGGTAATCGCCGTC
Hd3a-qF GAACTTCAACACCAAGGACTTC
Hd3a-qR TCAATTGTCTGAACCTGCAATG
Ehd1-qF TGGTTGGAAATCTCGAAAAACC
Ehd1-qR TCTCACCTCATTTTCTAGAGGC
RFT1-qF TCCGGCTAGCTTCATAAGTTAG
RFT1-qR CATAGCTGACACTGAGGTTAGT
Hd1-qF GTGGTACCTTCACAGATCACAA
Hd1-qR CTGTTGCTGATGGAATCTGTGTA
Ubq-qF ACCACTTCGACCGCCACTACT
Ubq-qR ACGCCTAAGCCTGCTGGTT

Fig. 1

Spatiotemporal expression pattern of OsFLZ18 by qRT-PCR assay"

Fig. 2

Diagram of the OE vector and molecular characterization of OsFLZ18-CRISPR knockout mutants A: Diagram of pUBQ10:OsFLZ18:GFP vector; B: Mutation sites in OsFLZ18 by the CRISPR-Cas9 gene editing system. The protospacer adjacent motif (PAM) site is depicted as underline. Inserted nucleotides are shown in red, and deleted nucleotides are depicted as dashes"

Fig. 3

Heading dates of OsFLZ18 transgenic rice plants A: Phenotypic comparison of OsFLZ18 transgenic and WT plants under natural SD conditions (GuangZhou, Aug-Nov); B: Flowering times of OsFLZ18 transgenic plants and Nip. **: P<0.01"

Fig. 4

Expression pattern of key regulatory genes involved in flowering by qRT-PCR assay The open and filled bars at the bottom represent light and dark periods, respectively; ZT: Zeitgeber time; Nip: Nippobare; OE-2: OsFLZ18-OE line"

Fig. 5

Verification the interaction between OsFLZ18 and OsMADS51 by yeast two-hybrid assay"

Fig. 6

Diurnal rhythmic expression of OsFLZ18 A: Diurnal rhythmic expression of OsFLZ18 under ASDs; B: Diurnal rhythmic expression of OsFLZ18 under LDs. Data is downloaded from the Rice Expression Profile Database (https://ricexpro.dna.affrc.go.jp/). D: Daytime; N: Night"

[1] SONG H Y, TRO S, IMAIZUMI T. Flowering time regulation: Photoperiod- and temperature-sensing in leaves. Trends in Plant Science, 2013, 18: 575-583.
doi: 10.1016/j.tplants.2013.05.003 pmid: 23790253
[2] 徐铨, 奥本裕, 王晓雪. 水稻开花期调控分子机理研究进展. 植物遗传资源学报, 2014, 15(1): 129-136.
XU Q, OKUMOTO Y, WANG X X. Research progress on regulatory molecular mechanisms of flowering time in rice. Journal of Plant Genetic Resources, 2014, 15(1): 129-136. (in Chinese)
[3] 王诗宇, 王志兴, 隋亚云, 黄河, 宋晓波, 郭素华. 光周期调控水稻开花期的分子机制研究进展. 北方水稻, 2020, 50(3): 49-52.
WANG S Y, WANG Z X, SUI Y Y, HUANG H, SONG X B, GUO S H. Research progress of the molecular mechanism of photoperiod control rice flowering. North Rice, 2020, 50(3): 49-52. (in Chinese)
[4] 王玉博, 王悦, 刘雄, 唐文帮. 水稻光周期调控开花的研究进展. 中国水稻科学, 2021, 35(3): 207-224.
doi: 10.16819/j.1001-7216.2021.0514
WANG Y B, WANG Y, LIU X, TANG W B. Research progress of photoperiod regulation in rice flowering. Chinese Journal of Rice Science, 2021, 35(3): 207-224. (in Chinese)
doi: 10.16819/j.1001-7216.2021.0514
[5] SHRESTHA R, GOMEZ-ARIZA J, BRAMBILLA V, FORNARA F. Molecular control of seasonal flowering in rice, Arabidopsis and temperate cereals. Annals Botany, 2014, 114(7): 1445-1458.
doi: 10.1093/aob/mcu032
[6] DOI K, IZAWA T, FUSE T, YAMANOUCHI U, KUBO T, SHIMATANI Z, YANO M, YOSHIMURA A. Ehd1, a B-type response regulator in rice, confers short-day promotion of flowering and controls FT-Iike gene expression independently of Hd1. Genes & Development, 2004, 18(8): 926-936.
doi: 10.1101/gad.1189604
[7] LIN H X, YAMAMOTO T, SASAKI T. Characterization and detection of epistatic interactions of three QTLs, Hd1, Hd2 and Hd3, controlling heading date in rice using nearly isogenic lines. Theoretical and Applied Genetics, 2000, 101(7): 1021-1028.
doi: 10.1007/s001220051576
[8] YANO M, KATAYOSE Y, ASHIKARI M, YAMANOUCHI U, MONNA L, FUSE T, BABA T, YAMAMOTO K, UMEHARA Y, NAGAMURA Y, SASAKI T. Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS. The Plant Cell, 2000, 12(12): 2473-2483.
doi: 10.1105/tpc.12.12.2473
[9] ISHIKAWA R, TAMAKI S, YOKOI S, INAGAKI N, SHINOMURA T, TAKANO M, SHIMAMOTO K. Suppression of the floral activator gene Hd3a is the principal cause of the night break effect in rice. The Plant Cell, 2005, 17(12): 3326-3336.
doi: 10.1105/tpc.105.037028
[10] KOMIYA R, IKEGAMI A, TAMAKI S, YOKOI S, SHIMAMOTO K. Hd3a and RFT1are essential for flowering in rice. Development, 2008, 135(4): 767-774.
doi: 10.1242/dev.008631
[11] NEMOTO Y, NONOUE Y, YANO M, IZAWA T. Hd1, a CONSTANS ortholog in rice, functions as an Ehd1 repressor through interaction with monocot-specific CCT-domain protein Ghd7. The Plant Journal, 2016, 86(3): 221-233.
doi: 10.1111/tpj.13168
[12] DU A, TIAN W, WEI M, YAN W, HE H, ZHOU D, HUANG X, LI S, OUYANG X. The DTH8-Hd1 module mediates day-length-dependent regulation of rice flowering. Molecular Plant, 2017, 10: 948-961.
doi: S1674-2052(17)30138-7 pmid: 28549969
[13] ZONG W B, REN D, HUANG M H, SUN K L, FENG J L, ZHAO J, XIAO D D, XIE W B, LIU S Q, ZHANG H, QIU R, TANG W J, YANG R Q, CHEN H Y, XIE X R, CHEN L T, LIU Y G, GUO J X. Strong photoperiod sensitivity is controlled by cooperation and competition among Hd1, Ghd7 and DTH8 in rice heading. New Phytologist, 2021, 229(3): 1635-1649.
doi: 10.1111/nph.16946 pmid: 33089895
[14] ZHAO J, CHEN H Y, REN D, TANG H W, QIU R, FENG J L, LONG Y M, NIU B X, CHEN D P, ZHONG T Y, LIU Y G, GUO J X. Ehd1) and Heading date 3a (Hd3a)RFT1) control differential heading and contribute to regional adaptation in rice (Oryza sativa). New Phytologist, 2015, 208(3): 936-948.
doi: 10.1111/nph.13503
[15] XUE W Y, XING Y Z, WENG X Y, TANG W J, WANG L, ZHOU H J, YU S B, XU C G, LI X H, ZHANG Q F. Natural variation in Ghd7is an important regulator of heading date and yield potential in rice. Nature Genetics, 2008, 40(6): 761-767.
doi: 10.1038/ng.143
[16] MATSUBARA K, YAMANOUCHI U, WANG Z X, MINOBE Y, IZAWA T, YANO M. Ehd2, a rice ortholog of the maize INDETERMINATE1gene, promotes flowering by up-regulating Ehd1. Plant Physiology, 2008, 148(3): 1425-1435.
doi: 10.1104/pp.108.125542
[17] LEE Y S, JEONG D H, LEE D Y, YI J, RYU C H, KIM S L, JEONG H J, CHOI S C, JIN P, YANG J, CHO L H, CHOI H, AN G. OsCOL4 is a constitutive flowering repressor upstream of Ehd1and downstream of OsphyB. The Plant Journal, 2010, 63(1): 18-30.
[18] JIANG P F, WANG S L, ZHENG H, LI H, ZHANG F, SU Y H, XU Z T, LIN H Y, QIAN Q, DING Y. SIP1 participates in regulation of flowering time in rice by recruiting OsTrx1 to Ehd1. New Phytologist, 2018, 219: 422-435.
doi: 10.1111/nph.15122
[19] SHENG P, WU F Q, TAN J J, ZHANG H, MA W W, CHEN L P, WANG J C, WANG J, ZHU S S, GUO X P, WANG J L, ZHANG X, CHANG Z J, BAO Y Q, WU C Y, LIU C M, WAN J M. A CONSTANS-like transcriptional activator, OsCOL13, functions as a negative regulator of flowering downstream of OsphyB and upstream of Ehd1in rice. Plant Molecular Biology, 2016, 92(1/2): 209-222.
doi: 10.1007/s11103-016-0506-3
[20] ZHANG H, ZHU S S, LIU T Z, WANG C M, CHENG Z J, ZHANG X, CHEN L P, SHENG P, CAI M H, LI C N, WANG J C, ZHANG Z, CHAI J T, ZHOU L, LEI C L, GUO X P, WANG J L, WANG J, JIANG L, WU C Y, WAN J M. DELAYED HEADING DATE1 interacts with OsHAP5C/D, delays flowering time and enhances yield in rice. Plant Biotechnology Journal, 2019, 17(2): 531-539.
doi: 10.1111/pbi.12996 pmid: 30107076
[21] KIM S L, LEE S, KIM H J, NAM H G, AN G. OsMADS51is a short-day flowering promoter that functions upstream of Ehd1, OsMADS14, and Hd3a. Plant Physiology, 2007, 145(4): 1484-1494.
doi: 10.1104/pp.107.103291
[22] LAITY J H, LEE B M, WRIGHT P E. Zinc finger proteins: New insights into structural and functional diversity. Current Opinion in Structural Biology, 2001, 11(1): 39-46.
doi: 10.1016/s0959-440x(00)00167-6 pmid: 11179890
[23] MANNULLY C T, GOPAN N, LAXMI A. Comprehensive evolutionary and expression analysis of FCS-Like Zinc finger gene family yields insights into their origin, expansion and divergence. PLoS ONE, 2015, 10(8): e0134328.
doi: 10.1371/journal.pone.0134328
[24] JAMSHEER K, LAXMI A. Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress. Frontiers in Plant Sciences, 2015, 6: 746.
[25] CHEN X, ZHANG Z, VISSER R G F, BROEKGAARDEN C, VOSMAN B. Overexpression of IRM1 enhances resistance to aphids in Arabidopsis thaliana. PLoS ONE, 2013, 8(8): e70914.
doi: 10.1371/journal.pone.0070914
[26] HOU X N, LIANG Y Z, HE X L, SHEN Y Z, HUANG Z J. A novel ABA-responsive TaSRHP gene from wheat contributes to enhanced resistance to salt stress in Arabidopsis thaliana. Plant Molecular Biology, 2013, 31(4): 791-801.
[27] HE Y, GAN S. A novel zinc-finger protein with a proline-rich domain mediates ABA-regulated seed dormancy in Arabidopsis. Plant Molecular Biology, 2004, 54(1): 1-9.
doi: 10.1023/B:PLAN.0000028730.10834.e3
[28] CHEN X Q, LI X B, YANG C, YAN W, LIU C L, TANG X Y, GAO C J. Genome-wide identification and characterization of FCS-Like Zinc Finger (FLZ) Family Genes in maize (Zea mays) and functional analysis of ZmFLZ25in plant abscisic acid response. International Journal of Molecular Sciences, 2021, 22(7): 3529.
doi: 10.3390/ijms22073529
[29] MA Y M, ZHAO J L, FU H, YANG T F, DONG J F, YANG W, CHEN L, ZHOU L, WANG J, LIU B, ZHANG S H, EDWARDS D. Genome-wide identification, expression and functional analysis reveal the involvement of FCS-Like Zinc finger gene family in submergence response in rice. Rice, 2021, 14(1): 76.
doi: 10.1186/s12284-021-00519-3 pmid: 34417910
[30] HORI K, OGISO-TANAKA E, MATSUBARA K, YAMANOUCHI U, EBANA K, YANO M. Hd16, a gene for casein kinase I, is involved in the control of rice flowering time by modulating the day-length response. The Plant Journal, 2013, 76(1): 36-46.
doi: 10.1111/tpj.12268 pmid: 23789941
[31] LIVAK K, SCHMITTGEN T. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method. Methods, 2001, 25(4): 402-408.
doi: 10.1006/meth.2001.1262
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