Functional Study of the VaMYB4a-VaPAL Module in Response to Cold Stress in Vitis amurensis

doi:10.16420/j.issn.0513-353x.2025-0877

Acta Horticulturae Sinica ›› 2026, Vol. 53 ›› Issue (9): 2531-2544.doi: 10.16420/j.issn.0513-353x.2025-0877

• Genetic & Breeding · Germplasm Resources · Molecular Biology • Next Articles

Functional Study of the VaMYB4a-VaPAL Module in Response to Cold Stress in Vitis amurensis

LI Jiaxin¹, ZHANG Zhen¹^,²^,³, YU Qinhan⁵, WU Wenkang¹, ZHANG Ningbo¹^,²^,³, XU Weirong¹^,²^,³^,⁴^,⁵^,^*()

¹ School of Enology & Horticulture， Ningxia University， Yinchuan 750021， China
² Engineering Research Center of Grape and Wine， Ministry of Education，Ningxia University， Yinchuan 750021， China
³ Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia， Yinchuan 750021， China
⁴ State Key Laboratory of Efficient Production of Forest Resources， Yinchuan 750021， China
⁵ School of Life Science， Ningxia University， Yinchuan 750021， China

Received:2026-03-17 Revised:2026-05-21 Online:2026-09-25 Published:2026-09-20
Contact: XU Weirong

Abstract

Abstract:

Low-temperature stress is a major abiotic factor limiting the growth and productivity of grapevine（Vitis spp.），yet the integrated mechanisms underlying the integration of cold signaling and metabolic regulation remain poorly understood. Here，a wild grapevine（Vitis amurensis）MYB transcriptionfactor（VaMYB4a）were characterized. Through the generation of VaMYB4a overexpression and CRISPR/Cas9 knockout lines，combined with physiological analyses，we found that VaMYB4a significantly enhanced the stability of photosystem Ⅱ，reduced membrane lipid peroxidation and reactive oxygen species accumulation，and thereby improved plant cold tolerance. qRT-PCR analysis showed that VaMYB4a activated the CBF-COR pathway and significantly induced the expression of CBF1-CBF4 and the downstream gene RD29A. Yeast one-hybrid，dual-luciferase reporter，and electrophoretic mobility shift assays demonstrated that VaMYB4a directly binds to the CAACCA cis-element in the VaPAL promoter and enhances its transcriptional activity，revealing a potential molecular mechanism by which VaMYB4a coordinately regulates the phenylpropanoid metabolic pathway. Overexpression of VaPAL markedly alleviated oxidative damage under low-temperature stress and enhanced cold tolerance in callus tissues. Promoter activity analysis further indicated that VaPAL is a key low-temperature-responsive gene with the highest sensitivity within the phenylpropanoid pathway. Co-expression analysis revealed that VaPAL can feedback-regulate the response intensity of the CBF pathway，thereby acting as a bridge between signal transduction and metabolic regulation. Collectively，the VaMYB4a-VaPAL module plays an important role in the coordinated“signal-metabolism”regulation of grapevine responses to low-temperature stress，providing valuable gene resources and a theoretical basis for the genetic improvement of cold tolerance in fruit crops.

Key words: grapevine, low-temperature stress, VaMYB4a, PAL, cold tolerance

CLC Number:

S663.1

LI Jiaxin, ZHANG Zhen, YU Qinhan, WU Wenkang, ZHANG Ningbo, XU Weirong. Functional Study of the VaMYB4a-VaPAL Module in Response to Cold Stress in Vitis amurensis[J]. Acta Horticulturae Sinica, 2026, 53(9): 2531-2544.

Figures/Tables 8

References 42

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用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
双荧光素酶报告实验 Dual-luciferase reporter assay	0800-PAL(P1)-F	ACTCACTATAGGGCGAATTGGGTACCGCGATTCCAATTTTTTCAAAACT
双荧光素酶报告实验 Dual-luciferase reporter assay	0800-PAL(P1)-R	ATATCAAGCTTATCGATACCGTCGACCTCCAAGTGTTTGATCAAATGCC
酵母单杂交 Yeast one-hybrid	pHIS2-PAL(P1)-F	gactcactatagggcgaattcGCGATTCCAATTTTTTCAAAACT
	pHIS2-PAL(P1)-R	gattcgcgaacgcgtgagctcCTCCAAGTGTTTGATCAAATGCC
	pHIS2-PAL(P2)-F	gactcactatagggcgaattcGCAACACTCCACGTCGAAGC
	pHIS2-PAL(P2)-R	gattcgcgaacgcgtgagctcCGGAAACCTAGTTTCAAAAATTAATATG
电泳迁移率实验 Electrophoretic mobility shift assay	marker(PAL-P1)-F	atagaagcctctttaaacaaccaaaaacttataaaaaaaa
	marker(PAL-P1)-R	TTTTTTTTATAAGTTTTTGGTTGTTTAAAGAGGCTTCTAT
	competitor(PAL-P1)-F	atagaagcctctttaaacaaccaaaaacttataaaaaaaa
	competitor(PAL-P1)-R	TTTTTTTTATAAGTTTTTGGTTGTTTAAAGAGGCTTCTAT
	mutation(PAL-P1)-F	atagaagcctctttaaaccccccaaaacttataaaaaaaa
	mutation(PAL-P1)-R	TTTTTTTTATAAGTTTTGGGGGGTTTAAAGAGGCTTCTAT
实时荧光定量PCR qRT-PCR	4CL-RT-F	GATCCTCCTCCAGAACTGCG
	4CL-RT-R	AAGTGTAGAAGGGGTTGGCG
	C4H-RT-F	GAACCACCTGAACCTCTCCG
	C4H-RT-R	ATCCGAACTCCACTCCCTGA
	PAL-RT-F	GACTGGTGAAACGGTGAGGT
	PAL-RT-R	ACCGTTCCAAGCACTGAGAC
	VvCBF1-RT-F	ACGAGGAGGCAATGTTCAATA
	VvCBF1-RT-R	TCATCCCAGCTGAATCCTTTAC
	VvCBF2-RT-F	GAGAAAGCCGGAGATGGTTT
	VvCBF2-RT-R	CACACATAGCAGGTGGAGTAAG
	VvCBF3-RT-F	ATGGATGAGGAGGCATTGTTTA
	VvCBF3-RT-R	AAGTCCGTGTAGGAAGTTGTATC
	VvCBF4-RT-F	ACCCTCACCCGCTCGTATG
	VvCBF4-RT-R	CCGCGTCTCCCGAAACTT
	VvRD29A-RT-F	GGTGATGGTGGTGAAGAAGTGGTA
	VvRD29A-RT-R	AACGGTTGTTCTGGCTTATCCTGTA
内参基因 Reference gene	VvActin1-F	CTATCCTTCGTCTTGACCTTGCTG
内参基因 Reference gene	VvActin1-R	AGTGGTGAACATGTAACCCCTCTC
RUBY报告基因分析启动子活性 RUBY reporter gene analysis of promoter activity	PAL-RUBY-F	attaccgcctttgagtgagctctagaGAGCTAAGGCAACAAAGGTAAAAAT
	PAL-RUBY-R	gcgagggtcgcatgatccatggtaccATCTTAGAATCCCAACAAAGAAACAA
	4CL-RUBY-F	attaccgcctttgagtgagctctagaGAACCCAACCTGTCCAACTTATACT
	4CL-RUBY-R	gcgagggtcgcatgatccatggtaccGGTGGCACAGAGAGAGAGAGAGA
	C4H-RUBY-F	attaccgcctttgagtgagctctagaGAGTGAGTCAGAGTTAGATTATTGTCCG
	C4H-RUBY-R	gcgagggtcgcatgatccatggtaccGGTTGTGGCGGTGGATGG

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
双荧光素酶报告实验 Dual-luciferase reporter assay	0800-PAL(P1)-F	ACTCACTATAGGGCGAATTGGGTACCGCGATTCCAATTTTTTCAAAACT
双荧光素酶报告实验 Dual-luciferase reporter assay	0800-PAL(P1)-R	ATATCAAGCTTATCGATACCGTCGACCTCCAAGTGTTTGATCAAATGCC
酵母单杂交 Yeast one-hybrid	pHIS2-PAL(P1)-F	gactcactatagggcgaattcGCGATTCCAATTTTTTCAAAACT
	pHIS2-PAL(P1)-R	gattcgcgaacgcgtgagctcCTCCAAGTGTTTGATCAAATGCC
	pHIS2-PAL(P2)-F	gactcactatagggcgaattcGCAACACTCCACGTCGAAGC
	pHIS2-PAL(P2)-R	gattcgcgaacgcgtgagctcCGGAAACCTAGTTTCAAAAATTAATATG
电泳迁移率实验 Electrophoretic mobility shift assay	marker(PAL-P1)-F	atagaagcctctttaaacaaccaaaaacttataaaaaaaa
	marker(PAL-P1)-R	TTTTTTTTATAAGTTTTTGGTTGTTTAAAGAGGCTTCTAT
	competitor(PAL-P1)-F	atagaagcctctttaaacaaccaaaaacttataaaaaaaa
	competitor(PAL-P1)-R	TTTTTTTTATAAGTTTTTGGTTGTTTAAAGAGGCTTCTAT
	mutation(PAL-P1)-F	atagaagcctctttaaaccccccaaaacttataaaaaaaa
	mutation(PAL-P1)-R	TTTTTTTTATAAGTTTTGGGGGGTTTAAAGAGGCTTCTAT
实时荧光定量PCR qRT-PCR	4CL-RT-F	GATCCTCCTCCAGAACTGCG
	4CL-RT-R	AAGTGTAGAAGGGGTTGGCG
	C4H-RT-F	GAACCACCTGAACCTCTCCG
	C4H-RT-R	ATCCGAACTCCACTCCCTGA
	PAL-RT-F	GACTGGTGAAACGGTGAGGT
	PAL-RT-R	ACCGTTCCAAGCACTGAGAC
	VvCBF1-RT-F	ACGAGGAGGCAATGTTCAATA
	VvCBF1-RT-R	TCATCCCAGCTGAATCCTTTAC
	VvCBF2-RT-F	GAGAAAGCCGGAGATGGTTT
	VvCBF2-RT-R	CACACATAGCAGGTGGAGTAAG
	VvCBF3-RT-F	ATGGATGAGGAGGCATTGTTTA
	VvCBF3-RT-R	AAGTCCGTGTAGGAAGTTGTATC
	VvCBF4-RT-F	ACCCTCACCCGCTCGTATG
	VvCBF4-RT-R	CCGCGTCTCCCGAAACTT
	VvRD29A-RT-F	GGTGATGGTGGTGAAGAAGTGGTA
	VvRD29A-RT-R	AACGGTTGTTCTGGCTTATCCTGTA
内参基因 Reference gene	VvActin1-F	CTATCCTTCGTCTTGACCTTGCTG
内参基因 Reference gene	VvActin1-R	AGTGGTGAACATGTAACCCCTCTC
RUBY报告基因分析启动子活性 RUBY reporter gene analysis of promoter activity	PAL-RUBY-F	attaccgcctttgagtgagctctagaGAGCTAAGGCAACAAAGGTAAAAAT
	PAL-RUBY-R	gcgagggtcgcatgatccatggtaccATCTTAGAATCCCAACAAAGAAACAA
	4CL-RUBY-F	attaccgcctttgagtgagctctagaGAACCCAACCTGTCCAACTTATACT
	4CL-RUBY-R	gcgagggtcgcatgatccatggtaccGGTGGCACAGAGAGAGAGAGAGA
	C4H-RUBY-F	attaccgcctttgagtgagctctagaGAGTGAGTCAGAGTTAGATTATTGTCCG
	C4H-RUBY-R	gcgagggtcgcatgatccatggtaccGGTTGTGGCGGTGGATGG

Functional Study of the VaMYB4a-VaPAL Module in Response to Cold Stress in Vitis amurensis

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