Cloning and Expression Activity Analysis of the WOX4a Promoter of Populus×xiaohei

doi:10.7525/j.issn.1673-5102.2026.03.006

Abstract

Abstract:

The stem， as one of the main storage organs of trees， plays a crucial role in wood production； enhancing its storage capacity（sink strength） is of great significance for increasing wood yield. WOX4 belongs to the WUSCHEL-related homeobox（WOX） transcription factor family and functions in maintaining vascular cambium stem cell activity and promoting cell proliferation， thereby regulating secondary growth in stems and roots. It is highly expressed during xylem development in stems and roots. In this study， RT-qPCR was used to analyze the relative expression levels of the PxWOX4a gene in different tissues. In addition， transgenic plants harboring the pPxWOX4a：：GUS construct were subjected to GUS staining to further analyze the expression pattern of the PxWOX4a promoter. The results showed that WOX4a was highly expressed in the stems of Populus×xiaohei. The upstream 1 742 bp promoter region was cloned， and cis-acting element analysis revealed that it contained multiple light-responsive elements， including G-box， I-box， and Box4. A reporter vector（pBI121-pPxWOX4a：：GUS） was generated and transformed into P. alba×P. glandulosa ‘84K’. Molecular analysis confirmed stable integration of the transgene into the genome. RT-qPCR and GUS histochemical staining demonstrated that the PxWOX4a promoter drove strong expression of the GUS gene in stems. These results indicated that the PxWOX4a promoter cloned from Populus × xiaohei exhibited stem-specific and high activity. It therefore provides a useful tool for stem-targeted genetic engineering to regulate wood formation and improve biomass yield in poplar and other forest trees.

Key words: poplar, stem, PxWOX4a promoter, promoter activity, GUS staining

CLC Number:

S714.7

Xu ZHANG, Yeling QIN, Ruoxin SHI, Nan LIU, Zhiru XU, Guanjun LIU. Cloning and Expression Activity Analysis of the WOX4a Promoter of Populus×xiaohei[J]. Bulletin of Botanical Research, 2026, 46(3): 449-457.

Figures/Tables 10

Table 1

Fig.1

Fig.2

Fig.3

Fig.4

Fig.5

Fig.6

Fig.7

Fig.8

Fig.9

References 25

[1]	韩茹，游乐，江鸣涛，等.台湾独蒜兰生长发育过程中源库关系转换研究［J］.热带作物学报，2023，44（11）：2330-2342.
	HAN R， YOU L， JIANG M T，et al.Source-sink exchange during the growth and development of Pleione formosana ［J］.Chinese Journal of Tropical Crops，2023，44（11）：2330-2342.
[2]	刘颖慧，贾海坤，高琼.植物同化物分配及其模型研究综述［J］.生态学报，2006，26（6）：1981-1992.
	LIU Y H， JIA H K， GAO Q.Review on researches of photoassimilates partitioning and its models［J］.Acta Ecologica Sinica，2006，26（6）：1981-1992.
[3]	WANG Y D， ZHANG H M， GU J W，et al.The sink-source relationship in cucumber（Cucumis sativus L.）is modulated by DNA methylation［J］.Plants，2024，13（1）：103.
[4]	LOU H C， LI S J， SHI Z H，et al.Engineering source-sink relations by prime editing confers heat-stress resilience in tomato and rice［J］.Cell，2025，188（2）：530-549.
[5]	宋想，王璐瑶，富博晓，等.高等植物启动子元件鉴定与合成研究进展［J］.植物学报，2024，59（5）：691-708.
	SONG X， WANG L Y， FU B X，et al.Advances in identification and synthesis of promoter elements in higher plants［J］.Chinese Bulletin of Botany，2024，59（5）：691-708.
[6]	曾丹琦，何春梅，俞振明，等.WOX转录因子在植物发育中的作用［J］.热带亚热带植物学报，2022，30（5）：742-752.
	ZENG D Q， HE C M， YU Z M，et al.Roles of WOX transcription factors in plant development［J］.Journal of Tropical and Subtropical Botany，2022，30（5）：742-752.
[7]	BREUNINGER H， RIKIRSCH E， HERMANN M，et al.Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo［J］.Developmental Cell，2008，14（6）：867-876.
[8]	COSTANZO E， TREHIN C， VANDENBUSSCHE M.The role of WOX genes in flower development［J］.Annals of Botany，2014，114（7）：1545-1553.
[9]	LU Y， LIU Z Y， LYU M L，et al.Characterization of JsWOX1 and JsWOX4 during callus and root induction in the shrub species Jasminum sambac ［J］.Plants，2019，8（4）：79.
[10]	ZHANG C L， WANG J F， WANG X，et al. UF，a WOX gene，regulates a novel phenotype of un-fused flower in tomato［J］.Plant Science，2020，297：110523.
[11]	SU Y H， ZHOU C， LI Y J，et al.Integration of pluripotency pathways regulates stem cell maintenance in the Arabidopsis shoot meristem［J］.Proceedings of the National Academy of Sciences of the United States of America，2020，117（36）：22561-22571.
[12]	JI J B， STRABLE J， SHIMIZU R，et al. WOX4 promotes procambial development［J］.Plant Physiology，2010，152（3）：1346-1356.
[13]	李建波，贾会霞，张进，等.毛白杨PtoWOX4a基因过表达对次生生长的影响［J］.林业科学，2018，54（2）：52-59.
	LI J B， JIA H X， ZHANG J，et al.Effect of overexpression of Populus tomentosa WUSCHEL-related homeobox 4（PtoWOX4a）on the secondary growth of poplar［J］.Scientia Silvae Sinicae，2018，54（2）：52-59.
[14]	叶青青，周明兵.木本植物形成层活动的分子调控机制［J］.浙江农林大学学报，2024，41（4）：879-886.
	YE Q Q， ZHOU M B.Molecular regulatory mechanisms of cambium activity in woody plants［J］.Journal of Zhejiang A&F University，2024，41（4）：879-886.
[15]	沈清越，康忠信，刘亚芹.杨树良种：小黑杨［J］.林业科技通讯，1979（7）：7-8.
	SHEN Q Y， KANG Z X， LIU Y Q.A superior poplar variety：Populus × xiaohei ［J］.Forestry Science and Technology Communication，1979（7）：7-8.
[16]	GU X Y， PENG J H， OU Q，et al. Dendrocalamus farinosus SWEET14 is involved in source-sink carbohydrate partitioning［J］.Journal of Experimental Botany，2025，76（10）：2727-2742.
[17]	刘冉冉.栾树维管形成层活动规律及其细胞分化发育的研究［D］.武汉：华中农业大学，2013.
	LIU R R.Studies on rhythm of vascular cambium activity and cell differentiation in Koelreuteria paniculata ［D］.Wuhan：Huazhong Agricultural University，2013.
[18]	KUCUKOGLU M， NILSSON J， ZHENG B，et al. WUSCHEL-RELATED HOMEOBOX4（WOX4）-like genes regulate cambial cell division activity and secondary growth in Populus trees［J］.New Phytologist，2017，215（2）：642-657.
[19]	KUZNETSOVA X A， DODUEVA I E， LUTOVA L A.The homeodomain of the Raphanus sativus WOX4 binds to the promoter of the LOG3 cytokinin biosynthesis gene［J］.Ecological genetics，2024，22（1）：33-46.
[20]	SUER S， AGUSTI J， SANCHEZ P，et al. WOX4 imparts auxin responsiveness to cambium cells in Arabidopsis ［J］.The Plant Cell，2011，23（9）：3247-3259.
[21]	TANG X F， WANG C P， CHAI G H，et al.Ubiquitinated DA1 negatively regulates vascular cambium activity through modulating the stability of WOX4 in Populus ［J］.The Plant Cell，2022，34（9）：3364-3382.
[22]	崔茂良，魏君，陈晓梅，等.小黑杨提纯复壮及优良无性系选育技术［J］.防护林科技，2003（2）：76.
	CUI M L， WEI J， CHEN X M，et al.Purification and rejuvenation techniques of Populus tomentosa and selection and breeding of superior clones［J］.Protection Forest Science and Technology，2003（2）：76.
[23]	ZHANG B， YUE D D， HAN B，et al. RAPID LEAF FALLING 1 facilitates chemical defoliation and mechanical harvesting in cotton［J］.Molecular Plant，2025，18（5）：765-782.
[24]	路星星. rolB/C基因对颠茄托品烷生物碱合成能力的影响［D］.贵阳：贵州大学，2022.
	LU X X.Effects of the rolB/C genes on biosynthesis ability of tropane alkaloid in Atropa belladonna ［D］.Guiyang：Guizhou University，2022.
[25]	俞婷，黄丹丹，朱炎辉，等.马铃薯Stpatatin 05基因转录调控因子筛选及互作验证［J］.生物技术通报，2025，41（3）：137-145.
	YU T， HUANG D D， ZHU Y H，et al.Screening and interaction verification of transcription factors Stpatatin 05 gene in potato［J］.Biotechnology Bulletin，2025，41（3）：137-145.

元件 Elements	序列 Sequence	功能 Function	数量 Amount
TGA-element	AACGAC	生长素响应元件Auxin-responsive element	1
ABRE	ACGTG/CACGTG	脱落酸响应元件Abscisic acid responsiveness	4
ARE	AAACCA	厌氧诱导元件Anaerobic induction	4
G-Box	CACGAC/TACGTG	光响应元件Light responsiveness	5
HD-Zip 1	CAAT（A/T）ATTG/TGATAATGT	参与栅栏组织生长元件Palisade mesophyll cells	1
MBS	CAACTG	MYB结合位点MYB binding site	1
Box 4	ATTAAT	光响应元件Light responsiveness	1
chs-CMA1a	TTACTTAA	光响应元件Light responsiveness	1
I-box	CCATATCCAAT/TGATAATGT	光响应元件Light responsiveness	3
TCCC-motif	TCTCCCT	光响应元件Light responsiveness	1
MYB binding site	CAACAG	MYB结合位点 MYB binding site	3