Investigation of Regulatory Mechanism of Floral Integrators in Upland Cotton

doi:10.13304/j.nykjdb.2021.0924

Abstract

Abstract:

Flowering is the important stage for plant to produce sexual organs. Floral integrators integrate the environmental and endogenous signals to ultimately activate floral transition at the appropriate time， enabling the plants to reproductive success. Although genes floral integrators （FT， SOC1 and LFY） have been cloned in cotton， their evolutionary relationship in tetraploid cotton remains unknown. In this study， the homologous of flowering integrators were identified through comparing the released cotton genomes. The results revealed that FT and LFY were evolutionally conserved among diploid and tetraploid cottons， while SOC1 displayed sequence diversity. Specifically， cotton flowering integrators displayed unified expression in leaf and shoot apical meristem with a gradual increase during seedling development. The unreported sequences GhLFY-2， GhFT-2 and GhSOC1-5 were cloned and overexpressed in Arabidopsis， resulting in conserved function of promoting floral transition. However， the regulatory mechanism between the flowering integrators was peculiar in cotton. There was a conserved GhFT-2/GhSOC1-5/GhLFY-2 linear regulatory pathway and a direct pathway in which GhFT-1 and GhSOC1-1 regulate GhAP1 independent of GhLFY. The diversity of flowering time regulation might associate with adaptation of cotton to the environment.

Key words: upland cotton （Gossypium hirsutum L.）, floral integrator, flowering time regulation, regulatory mechanism

摘要：

开花是植物产生有性生殖器官的重要阶段，开花整合子整合来自环境和植物体内的信号，进而激活花芽分化，使植物在适宜的时间产生足够多的后代，保障生殖繁衍。棉花中开花整合子基因（FT、SOC1和LFY）已被克隆，但它们在棉花中的进化关系尚不清楚。参考陆地棉基因组，明确了棉属开花整合子同源基因数目，FT和LFY在棉属中进化具有保守性，而SOC1基因序列具有多样性。不同于其他物种，棉花开花整合子基因均在顶端分生组织和叶片中表达量较高，且随着棉苗的生长发育其表达量逐步升高。克隆了GhLFY-2、GhFT-2和GhSOC1-5基因，发现其促进开花的功能是保守的，但开花时间调控机制存在物种特异性。陆地棉中既存在保守的GhFT-2/GhSOC1-5/GhLFY-2线性调控路径，又进化出GhFT-1和GhSOC1-1直接调控GhAP1的路径，表明棉花开花时间调控机制的多样性可能与其对环境的适应性有关。

关键词: 陆地棉, 开花整合子, 开花时间调控, 调控机制

CLC Number:

S562

Liting CHEN, Yuanyuan YAN. Investigation of Regulatory Mechanism of Floral Integrators in Upland Cotton[J]. Journal of Agricultural Science and Technology, 2023, 25(6): 11-21.

陈丽婷, 阎媛媛. 陆地棉开花整合子调控机制解析[J]. 中国农业科技导报, 2023, 25(6): 11-21.

Figures/Tables 7

Table 1 Primer sequences in the study

引物名称 Primer name	引物序列 Primer sequence（5’-3’）	用途 Purpose
GhLFY-2-F（PstI）	TTCTGCAGCTTGTTTTCACTTCAAAAATGGACCCTGAG	基因克隆 Gene cloning
GhLFY-2-R（SmaI）	TTCCCGGGTTAGAACCCCATATGATCATGTCC
GhSOC1-4-F（EcoRI）	CCGAATTCGTTCCAAGTTCTTTGCTTGCCAAT
GhSOC1.4-R（BamHI）	TTGGATCCGCATGGGATTAACTTTGTTGTATC
GhFT-2-F（EcoRI）	TTGAATTCGCGATATCATGCCTAGAGATAGAGATCC
GhFT-2-R（XmaI）	TTCCCGGGTCATGTCCTACGGCCACCGGAT
GhLFY-2-RT-F	AATTCGAGGGTGGTGATGAT	qRT-PCR
GhLFY-2-RT-R	CTCCGTTACGATGAAAGGGT
GhSOC1-5-RT-F	GGCGCATAGAGAACGATACA
GhSOC1-5-RT-R	TTTGTATGCCGCCTATAACG
GhFT-2-RT-F	GGGATGATCTGAGGACCTTCT
GhFT-2-RT-R	CACCAGTTGTGGCTGGAATA
AtTUB2-F	ATCCGTGAAGAGTACCCAGAT
AtTUB2-R	AAGAACCATGCACTCATCAGC

Fig. 1 Sequence analysis of cotton floral integrator

Fig. 2 Phylogenetic analysis of plant floral integrator genesNote：Gh—Gossypium hirsutum； Gb—Gossypium barbadense； Ga—Gossypium arboreum； Gr—Gossypium raimondii； At—Arabidopsis thaliana； Pt—Populus tomentosa； Nt—Nicotiana tabacum； Vv—Vitis vinifera.

Fig. 3 Expression of floral integrators genes in Gossypium hirsutumA： Tissue-specific expression of flowering integrator of Gossypium hirsutum； B： Expression trends of flowering integrators in cotton leaves during seedling development； C： Expression trends of flowering integrators in cotton SAM during seedling development. R—Root； S—Stem； L—Leaf； TLS—True leaf stage

Fig. 4 Cotton flowering integrators promotes floweringA： Early flowering phenotype of transgenic plants； B： Comparison of gene expression and flowering time between WT and transgenic plants； C： Flowering time of WT and transgenic lines， ** indicates significant differences （P<0.01）； D： Solitary flowers produced in GhLFY-2 transgenic plants

Fig. 5 Expression of floral integrator genes in transgenic Arabidopsis plantsNote：A， B and C shows relative expression of flowering integrators in GhFT-2， GhSOC1-5 and GhLFY-2 transgenic plants， respectively.

Fig. 6 Model of cotton flowering regulatory mechanism

References 38

1	喻树迅,王寒涛,魏恒玲,等.棉花早熟性研究进展及其应用[J].棉花学报, 2017, 29(): 1-10.
	YU S X, WANG H T, WEI H L, et al.. Research progress and application of early maturity in upland cotton [J]. Cotton Sci., 2017, 29(S1): 1-10.
2	FORNARA F, MONTAIGU A D, COUPLAND G. SnapShot: control of flowering in Arabidopsis [J]. Cell, 2010, 141(3): 550-550.
3	WIGGE P. FT, a mobile developmental signal in plants [J]. Curr. Biol., 2011, 21(9): R374-R378.
4	KOBAYASHI Y, KAYA H, GOTO K, et al.. A pair of related genes with antagonistic roles in mediating flowering signals [J]. Science, 2000, 286(5446): 1960-1962.
5	ABE M, KOBAYASHI Y, YAMAMOTO S, et al.. FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex [J]. Science, 2005, 309(5737): 1052-1056.
6	WIGGE P A, MIN C K, JAEGER K E, et al.. Integration of spatial and temporal information during floral induction in Arabidopsis [J]. Science, 2005, 309(5737): 1056-1059.
7	TAMAKI S, MATSUO S, WONG H L, et al.. Hd3a protein is a mobile flowering signal in rice [J]. Science, 2007, 316(5827): 1033-1036.
8	MCGARRY R C, PREWITT S, AYRE B G. Overexpression of FT in cotton affects architecture but not floral organogenesis [J/OL]. Plant Signal Behav., 2013, 8(4):e23602 [2021-12-03]. .
9	GUO D, LI C, DONG R, et al.. Molecular cloning and functional analysis of the FLOWERING LOCUS T(FT) homolog GhFT1 from Gossypium hirsutum [J]. J. Integr. Plant Biol., 2015, 57(6): 522-533.
10	LI C, ZHANG Y, ZHANG K, et al.. Promoting flowering, lateral shoot outgrowth, leaf development, and flower abscission in tobacco plants overexpressing cotton FLOWERING LOCUS T (FT)-like gene GhFT1 [J/OL]. Front. Plant Sci., 2015, 6: 454 [2021-12-03]. .
11	BLÁZQUEZ M A, WEIGEL D. Integration of floral inductive signals in Arabidopsis [J]. Nature, 2000, 404(6780): 889-892.
12	YOO S K, CHUNG K S, KIM J, et al.. Constant activates suppressor of overexpression of constant 1 through flowering locus T to promote flowering in Arabidopsis [J]. Plant Physiol., 2005, 139(2): 770-778.
13	ZHANG X, WEI J, FAN S, et al.. Functional characterization of GhSOC1 and GhMADS42 homologs from upland cotton (Gossypium hirsutum L.) [J]. Plant Sci., 2016, 242: 178-186.
14	CERDÁN P D, CHORY J. Regulation of flowering time by light quality [J]. Nature, 2003, 423(6942):881-885.
15	SVEN E, HENRIK B, THOMAS M, et al.. GA4 is the active gibberellin in the regulation of LEAFY transcription and Arabidopsis floral initiation [J]. Plant Cell, 2006, 18(9): 2172-2181.
16	LILJEGREN S J, GUSTAFSON-BROWN C, PINYOPICH A, et al.. Interactions among APETALA1, LEAFY, and TERMINAL FLOWER1 specify meristem fate [J]. Plant Cell, 1999, 11(6): 1007-1018.
17	WAGNER D, SABLOWSKI R, MEYEROWITZT E M. Transcriptional activation of APETALA1 by LEAFY [J]. Science, 1999, 285(5427): 582-584.
18	HEMPEL F D, WEIGEL D, MANDEL M A, et al.. Floral determination and expression of floral regulatory genes in Arabidopsis [J]. Development, 1997, 124(19): 3845-3853.
19	BLAZQUEZ M A, SOOWAL L N, LEE I, et al.. LEAFY expression and flower initiation in Arabidopsis [J]. Development, 1997, 124(19): 3835-3844.
20	WEIGEL D, NILSSON O. A developmental switch sufficient for flower initiation in diverse plants [J]. Nature, 1995, 377(6549): 495-500.
21	AHEARN K P, JOHNSON H A, DETLEF W, et al.. NFL1, a Nicotiana tabacum LEAFY-like gene, controls meristem initiation and floral structure [J]. Plant Cell Physiol., 2001, 10: 1130-1139.
22	KYOZUKA J, KONISHI S, NEMOTO K, et al.. Down-regulation of RFL, the FLO/LFY homolog of rice, accompanied with panicle branch initiation [J]. Proc. Natl. Acad. Sci. USA, 1998, 95(5): 1979-1982.
23	LI J, FAN S L, SONG M Z, et al.. Cloning and characterization of a FLO/LFY ortholog in Gossypium hirsutum L [J]. Plant Cell Rep., 2013, 32(11): 1675-1686.
24	LEE J, OH M, PARK H, et al.. SOC1 translocated to the nucleus by interaction with AGL24 directly regulates LEAFY [J]. Plant J., 2008, 55(5): 832-843.
25	YAN Y, SHEN L, CHEN Y, et al.. A MYB-domain protein EFM mediates flowering responses to environmental cues in Arabidopsis [J]. Dev. Cell, 2014, 30(4): 437-448.
26	HIGUCHI Y, NARUMI T, ODA A, et al.. The gated induction system of a systemic floral inhibitor, antiflorigen, determines obligate short-day flowering in chrysanthemums [J]. Proc. Natl. Acad. Sci. USA, 2013, 110(42): 17137-17142.
27	SONG Q, ZHANG T, STELLY D M, et al.. Epigenomic and functional analyses reveal roles of epialleles in the loss of photoperiod sensitivity during domestication of allotetraploid cottons [J/OL]. Genome Biol., 2017, 18(1): 99 [2021-12-03]. .
28	MA Z, ZHANG Y, WU L . et al ..High-quality genome assembly and resequencing of modern cotton cultivars provide resources for crop improvement [J]. Nat. Genet., 2021, 53: 1385-1391.
29	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method [J]. Methods, 2001, 25(4): 402-408.
30	SARAH M N, SINARA A, GUSTAVO M A, et al.. Genome-wide analysis of the MADS-box gene family in polyploid cotton (Gossypium hirsutum) and in its diploid parental species (Gossypium arboreum and Gossypium raimondii) [J]. Plant Physiol. Bioch., 2018, 127: 169-184.
31	REN Z Y, YU D Q, YANG Z E, et al.. Genome-wide identification of the MIKC-Type MADS-box gene family in Gossypium hirsutum L. unravels their roles in flowering [J/OL]. Front. Plant Sci., 2017, 8: 384 [2021-12-03]. .
32	PATIRANAGE D, ASARE E, MALDONADO-TAIPE N, et al.. Haplotype variations of major flowering time genes in quinoa unveil their role in the adaptation to different environmental conditions [J]. Plant Cell Environ., 2021, 44(8): 2565-2579.
33	樊红娟,程雪敏,郑思,等.棉花GhFT基因的克隆及功能分析[J].分子植物育种, 2019, 18(17): 29-36.
	FAN H J, CHENG X M, ZHENG S, et al.. The cloning and function analysis of GhFT gene in Gossypium hirsutum [J]. Mol. Plant Breed., 2020, 18(17): 29-36.
34	JACK T. Molecular and genetic mechanisms of floral control [J]. Plant Cell, 2004, 16 (Supp. l): S1-S17.
35	MAIZEL A, BUSCH M A, TANAHASHI T, et al.. The floral regulator LEAFY evolves by substitutions in the DNA binding domain [J]. Science, 2005, 308(5719): 260-263.
36	KELLY A J, BONNLANDER M B, MEEKS-WAGNER D R. NFL, the tobacco homolog of FLORICAULA and LEAFY, is transcriptionally expressed in both vegetative and floral meristems [J]. Plant Cell, 1995, 7(2): 225-234.
37	HOFER J, TURNER L, HELLENS R, et al.. UNIFOLIATA regulates leaf and flower morphogenesis in pea [J]. Curr. Biol., 1997, 7(8): 581-587.
38	CARMONA M J, CUBAS P, MARTÍNEZ-ZAPATER J M. VFL, the grapevine FLORICAULA/LEAFY ortholog, is expressed in meristematic regions independently of their fate [J]. Plant Physiol., 2002, 130(1): 68-77.

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