Cloning and Functional Analysis of PtoYABBY2 and PtoYABBY12 in Populus tomentosa

doi:10.13560/j.cnki.biotech.bull.1985.2025-0122

Abstract

Abstract:

Objective To explore the role of PtoYABBY2 and PtoYABBY12 genes in the flowering process and leaf development of Populus tomentosa, providing valuable reference genes for molecular design and breeding of P. tomentosa. Method PtoYABBY2 and PtoYABBY12 were cloned by homologous cloning, and bioinformatics, phylogeny, expression pattern and subcellular localization of them were analyzed. The function of these two genes was investigated by allogeneic transformation of Arabidopsis. Result PtoYABBY2 and PtoYABBY12 had complete YABBY and C2C2 domains, both belong to the YAB2 subclass of the YABBY gene family, and their proteins are located in the nucleus. PtoYABBY2 and PtoYABBY12 showed different expression patterns during the development of male and female flower buds in P. tomentosa. Two genes were expressed in the roots, stems, young leaves and mature leaves of P. tomentosa, with the high expression in mature leaves. Compared with wild-type Arabidopsis, the transgenic plants of PtoYABBY2 and PtoYABBY12 both showed obvious early-flowering phenotypes, but no significant changes in leaf morphology, inflorescence structure and floral organ morphology. In the transgenic lines, the relative expressions of flowering-promoting genes AtFT, AtSOC1, AtFUL and AtLFY were higher than those in wild-type Arabidopsis, while the relative expression of flowering-repressing gene AtFLC was significantly lower than that in wild-type Arabidopsis. Conclusion PtoYABBY2 and PtoYABBY12 play a key role in the flowering process, which can positively promote early flowering of plants.

Key words: Populus tomentosa, YABBY, flowering time, expression pattern, functional research

ZHANG Chao-chao, HAN Kai-yuan, WANG Tong, CHEN Zhong. Cloning and Functional Analysis of PtoYABBY2 and PtoYABBY12 in Populus tomentosa[J]. Biotechnology Bulletin, 2025, 41(9): 256-264.

Figures/Tables 9

Table 1 Primer sequence

引物名称 Primer name	正向引物 Forward primer （5′‒3′）	反向引物 Reverse primer （5′‒3′）	用途 Purpose
PtoYAB2	ATGCATCACATAAACGAAAGGAAA	TCACAGGGAGCCATTGATCTCGTA	扩增PCR
PtoYAB12	ATGTCTCTAGACATTGCTTCTGAACG	TTATGCATCCATGCCGGTG	扩增PCR
PtoYAB2-qPCR	CTTTGGACTGAAGCTGGACA	TCACAGGGAGCCATTGAT	RT-qPCR
PtoYAB12-qPCR	TCCCAGTCTTCTTCCTCGGG	GTTCTTGGCTGCATTGCTGA
PtACTIN	TATCTCCTCTGTCTCCGACT	GCATCATCACCTGCAAAC
AtFT （AT1G65480）	CTTGGCAGGCAAACAGTGTATGCAC	GCCACTCTCCCTCTGACAATTGTAGA
AtFUL （AT5G60910）	TTGCAAGATCACAACAATTCGCTTCT	GAGAGTTTGGTTCCGTCAACGACGAT
AtSOC1 （AT2G45660）	AGCTGCAGAAAACGAGAAGCTCTCTG	GGGCTACTCTCTTCATCACCTCTTCC
AtLFY （AT5G61850）	ACGCCGTCATTTGCTACTCT	CTTTCTCCGTCTCTGCTGCT
AtFLC （AT5G10140）	CTAGCCAGATGGAGAATAATCATCATG	TTAAGGTGGCTAATTAAGTAGTGGGAG
AtACTIN2 （AT3G18780）	AGTGGTCGTACAACCGGTATTGT	GATGGCATGGAGGAAGAGAGAAAC

Fig. 1 Cloning of PtoYAB2 and PtoYAB12

Fig. 2 Multiple sequence alignment of YAB2 and YAB12 from different species

Fig. 3 Phylogenetic tree of multispecies

Fig. 4 Subcellular localization of PtoYAB2 and PtoYAB12

Fig. 5 Expressions of PtoYAB2 and PtoYAB12 in male and female flower buds and different tissues of P. tomentosaS1‒S8 refer to the flower buds at the eight key developmental stages of flower bud development (S1 flower induction stage, S2 flower primordium formation stage, S3 organogenesis stage, S4 elongation stage, S5 archespore formation stage, S6 and S7 dormancy stage, and S8 archespore genesis stage); R, S, L and OL refer to the root, stem, young leaf and mature leaf respectively. ♀ indicates female flower buds and ♂ indicates male flower buds. Different lowercase letters indicate significant differences at the 0.05 level. The same below

Fig. 6 Identification and phenotypic analysis of transgenic A. thalianaA: Positive identification of transgenic Arabidopsis thaliana. B: Comparison of flowering time between transgenic A. thaliana and wild type. C: Comparison of leaf phenotypes between transgenic A. thaliana and wild type. D: Comparison of indeterminate inflorescences and floral organs between transgenic A. thaliana and wild type. YAB2-6, YAB2-8, and YAB2-18 are PtoYAB2 transgenic A. thaliana lines, and YAB12-7, YAB12-8, and YAB12-10 are PtoYAB12 transgenic A. thaliana lines, the same below. The scale of B and C is 1 cm, the scale of D from top to bottom is 1 cm and 1mm

Table 2 Bolting flowering time of wild-type and transgenic A. thaliana

株系 Line	光照条件 Light condition	植株数 Number of plants	开花时间 Time of anthesis （d）
WT （Col）	LD	10	20.33±0.36
YAB2-6	LD	11	16.00±0.25**
YAB2-8	LD	12	16.50±0.36**
YAB2-18	LD	10	16.00±0.37**
YAB12-7	LD	12	16.16±0.37**
YAB12-8	LD	12	16.00±0.30**
YAB12-10	LD	10	15.75±0.25**

Fig. 7 Analysis of changes in endogenous gene expression in transgenic A. thaliana

References 29

[1]	Finet C, Floyd SK, Conway SJ, et al. Evolution of the YABBY gene family in seed plants [J]. Evol Dev, 2016, 18(2): 116-126.
[2]	Han HQ, Liu Y, Jiang MM, et al. Identification and expression analysis of YABBY family genes associated with fruit shape in tomato (Solanum lycopersicum L.) [J]. Genet Mol Res, 2015, 14(2): 7079-7091.
[3]	Zhao SP, Lu D, Yu TF, et al. Genome-wide analysis of the YABBY family in soybean and functional identification of GmYABBY10 involvement in high salt and drought stresses [J]. Plant Physiol Biochem, 2017, 119: 132-146.
[4]	Bonaccorso O, Lee JE, Puah L, et al. FILAMENTOUS FLOWER controls lateral organ development by acting as both an activator and a repressor [J]. BMC Plant Biol, 2012, 12: 176.
[5]	Bowman JL, Smyth DR. CRABS CLAW a gene that regulates carpel and nectary development in Arabidopsis, encodes a novel protein with zinc finger and helix-loop-helix domains [J]. Development, 1999, 126(11): 2387-2396.
[6]	Zhang XL, Yang ZP, Zhang J, et al. Ectopic expression of BraYAB1-702, a member of YABBY gene family in Chinese cabbage, causes leaf curling, inhibition of development of shoot apical meristem and flowering stage delaying in Arabidopsis thaliana [J]. Int J Mol Sci, 2013, 14(7): 14872-14891.
[7]	Zhang X, Ding L, Song AP, et al. Dwarf and robust plant regulates plant height via modulating gibberellin biosynthesis in Chrysanthemum [J]. Plant Physiol, 2022, 190(4): 2484-2500.
[8]	Huang ZJ, Van Houten J, Gonzalez G, et al. Genome-wide identification, phylogeny and expression analysis of SUN OFP and YABBY gene family in tomato [J]. Mol Genet Genomics, 2013, 288(3/4): 111-129.
[9]	Yang ZE, Gong Q, Wang LL, et al. Genome-wide study of YABBY genes in upland cotton and their expression patterns under different stresses [J]. Front Genet, 2018, 9: 33.
[10]	Han KY, Lai M, Zhao TY, et al. Plant YABBY transcription factors: a review of gene expression, biological functions, and prospects [J]. Crit Rev Biotechnol, 2025, 45(1): 214-235.
[11]	Yang H, Shi GX, Li X, et al. Overexpression of a soybean YABBY gene, GmFILa, causes leaf curling in Arabidopsis thaliana [J]. BMC Plant Biol, 2019, 19(1): 234.
[12]	Hou HL, Lin Y, Hou XL. Ectopic expression of a pak-choi YABBY gene, BcYAB3, causes leaf curvature and flowering stage delay in Arabidopsis thaliana [J]. Genes, 2020, 11(4): 370.
[13]	郭效诚. 蜡梅YABBY家族CpYABBY5-1与CpCRC功能研究 [D]. 重庆: 西南大学, 2023.
	Guo XC. Study on CpYABBY5-1 and CpCRC function of Chimonanthus praecox YABBY family [D]. Chongqing: Southwest University, 2023.
[14]	Sun XD, Guan YL, Hu XY. Isolation and characterization of IaYABBY2 gene from Incarvillea arguta [J]. Plant Mol Biol Report, 2014, 32(6): 1219-1227.
[15]	Liu SN, Li XF, Yang H, et al. Ectopic expression of BoYAB1, a member of YABBY gene family in Bambusa oldhamii, causes leaf curling and late flowering in Arabidopsis thaliana [J]. J Hortic Sci Biotechnol, 2020, 95(2): 169-174.
[16]	Ionescu IA, Møller BL, Sánchez-Pérez R. Chemical control of flowering time [J]. J Exp Bot, 2017, 68(3): 369-382.
[17]	Gaudinier A, Blackman BK. Evolutionary processes from the perspective of flowering time diversity [J]. New Phytol, 2020, 225(5): 1883-1898.
[18]	Zhang TP, Li CY, Li DX, et al. Roles of YABBY transcription factors in the modulation of morphogenesis, development, and phytohormone and stress responses in plants [J]. J Plant Res, 2020, 133(6): 751-763.
[19]	Li J, Chen TT, Gao K, et al. Unravelling the novel sex determination genotype with ‘ZY’ and a distinctive 2.15-2.95 Mb inversion among poplar species through haplotype-resolved genome assembly and comparative genomics analysis [J]. Mol Ecol Resour, 2024, 24(7): e14002.
[20]	Chen Z, Rao P, Yang XY, et al. A global view of transcriptome dynamics during male floral bud development in Populus tomentosa [J]. Sci Rep, 2018, 8(1): 722.
[21]	郝颖颖, 周明珠, 韩开元, 等. 毛白杨PtYABBY基因家族3个成员的克隆与表达分析 [J]. 北京林业大学学报, 2022, 44(1): 48-57.
	Hao YY, Zhou MZ, Han KY, et al. Cloning and expression analysis of three members of PtYABBYs in Populus tomentosa [J]. J Beijing For Univ, 2022, 44(1): 48-57.
[22]	Xiang J, Liu RQ, Li TM, et al. Isolation and characterization of two VpYABBY genes from wild Chinese Vitis pseudoreticulata [J]. Protoplasma, 2013, 250(6): 1315-1325.
[23]	Jang S, Hur J, Kim SJ, et al. Ectopic expression of OsYAB1 causes extra stamens and carpels in rice [J]. Plant Mol Biol, 2004, 56(1): 133-143.
[24]	van der Knaap E, Chakrabarti M, Chu YH, et al. What lies beyond the eye: the molecular mechanisms regulating tomato fruit weight and shape [J]. Front Plant Sci, 2014, 5: 227.
[25]	Shi TT, Zhou L, Ye YF, et al. Characterization of YABBY transcription factors in Osmanthus fragrans and functional analysis of OfYABBY12 in floral scent formation and leaf morphology [J]. BMC Plant Biol, 2024, 24(1): 589.
[26]	Helliwell CA, Wood CC, Robertson M, et al. The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex [J]. Plant J, 2006, 46(2): 183-192.
[27]	Shen LS, Zhang Y, Sawettalake N. A Molecular switch for FLOWERING LOCUS C activation determines flowering time in Arabidopsis [J]. Plant Cell, 2022, 34(2): 818-833.
[28]	Whittaker C, Dean C. The FLC locus: a platform for discoveries in epigenetics and adaptation [J]. Annu Rev Cell Dev Biol, 2017, 33: 555-575.
[29]	Xie YR, Zhou Q, Zhao YP, et al. FHY3 and FAR1 integrate light signals with the miR156-SPL module-mediated aging pathway to regulate Arabidopsis flowering [J]. Mol Plant, 2020, 13(3): 483-498.