Genome-wide identification of HD-Zip transcription factors in blueberry and functional characterization of their drought-responsive mechanisms

SONG Jingrong, ZHANG Lingyun, CAO Yibo, LIU Jiali, ZHAO Wenyu, ZHANG Mi, ZHAI Rongxuan

Journal of Nanjing Forestry University (Natural Sciences Edition） ›› 0

PDF(1260 KB)
南京林业大学学报（自然科学版）
PDF(1260 KB)
Journal of Nanjing Forestry University (Natural Sciences Edition） ›› 0 DOI: 10.12302/j.issn.1000-2006.202512006

Genome-wide identification of HD-Zip transcription factors in blueberry and functional characterization of their drought-responsive mechanisms

  • SONG Jingrong, ZHANG Lingyun*, CAO Yibo*, LIU Jiali, ZHAO Wenyu, ZHANG Mi, ZHAI Rongxuan
Author information +
History +

Abstract

【Objective】Blueberry (Vaccinium spp.) is a perennial shrub belonging to the Ericaceae family, genus Vaccinium. Its fruits are rich in anthocyanins, organic acids, and various vitamins, possessing high nutritional and medicinal value and significant economic benefits. However, blueberries have shallow roots, lack root hairs, exhibit weak water absorption capacity, and are highly sensitive to drought stress. Drought has become a key environmental factor limiting the development of the blueberry industry. The HD-Zip transcription factor family is a plant-specific and important regulatory factor family that plays a critical role in abiotic stress responses, with the HD-Zip I subfamily being widely involved in drought and other stress responses. This study aims to conduct a genome-wide identification and bioinformatics analysis of the blueberry HD-Zip gene family, clarify their sequence characteristics, evolutionary relationships, and cis-regulatory element composition, and reveal the expression patterns of the HD-Zip I subfamily under drought stress, thereby identifying key genes involved in blueberry 'Bluecrop'（Vaccinium corymbosum 'Bluecrop'）drought response, and providing genetic resources and theoretical basis for dissecting the molecular mechanism of drought tolerance and drought-resistant genetic breeding in blueberry.【Method】Using the highbush blueberry cultivar‘Legacy’(Vaccinium corymbosum ‘Legacy’) as the experimental material, and based on the reference genome data of blueberry‘Draper’(Vaccinium corymbosum 'Draper'), a genome-wide identification of the HD-Zip gene family was performed using bioinformatics methods. Physicochemical properties, phylogeny, gene structure, conserved motifs, cis-acting elements, scaffold localization, and interspecific collinearity of the family members were analyzed. Based on our previously published blueberry drought transcriptome data, differentially expressed genes of the HD-Zip I subfamily were screened, and their tissue-specific expression patterns under moderate drought were analyzed. Blueberry seedlings were treated with 20% PEG-6000 to simulate drought stress. Six key differentially expressed genes were selected, and their expression levels at different time points were detected using RT-qPCR to verify the reliability of the transcriptome results.【Result】A total of 54 HD-Zip genes were identified in the blueberry genome, named VcHDZ1-VcHDZ54. They were classified into two subfamilies: HD-Zip I (32 members) and HD-Zip II (22 members); no members of subfamilies III or IV were found. The encoded proteins ranged from 191 to 584 aa in length, with molecular weights from 22.0 to 65.6 kDa; all were unstable hydrophilic proteins localized in the nucleus. Gene structure analysis showed that the exon-intron arrangement was highly conserved within the same subfamily but differed significantly between subfamilies. Conserved motif analysis identified 10 motifs, among which Motif 1, 2, and 3 were shared by all members, corresponding to the HD and LZ domains; Motif 4 was unique to the HD-Zip II subfamily. Promoter cis-acting element analysis revealed abundant elements related to light response, hormone response, and abiotic stress responses including drought and low temperature, among which were the drought-induced MYB binding site (MBS) and the ABA response element (ABRE). Scaffold localization showed that the 54 genes were distributed on 35 scaffolds. Drought expression analysis indicated that 10 differentially expressed genes of the HD-Zip I subfamily exhibited tissue-specific patterns: VcHDZ1, VcHDZ2, and VcHDZ3 were down-regulated in leaves but up-regulated in roots; VcHDZ4, VcHDZ8, VcHDZ21, and VcHDZ24 were down-regulated in both leaves and roots; VcHDZ16, VcHDZ17, and VcHDZ18 were up-regulated in both leaves and roots. The RT-qPCR validation results were generally consistent with the trends obtained from transcriptome analysis.【Conclusion】The blueberry HD-Zip gene family is structurally conserved and functionally diverse. The promoters contain abundant stress- and hormone-responsive elements, indicating their broad involvement in stress regulation. Members of the HD-Zip I subfamily participate in drought stress responses in a tissue-specific manner. VcHDZ16, VcHDZ2, and other genes can serve as important candidate genes for drought-resistant molecular breeding in blueberry. This study systematically reveals the basic characteristics and drought response patterns of the blueberry HD-Zip gene family, providing genetic resources and theoretical support for further elucidating the molecular mechanisms of drought tolerance and breeding new drought-resistant blueberry varieties.

Key words

blueberry (Vaccinium spp.) / HD-Zip gene family / drought stress / identification and analysis

Cite this article

EndNote

Ris (Procite)

Bibtex

Download Citations
SONG Jingrong, ZHANG Lingyun, CAO Yibo, LIU Jiali, ZHAO Wenyu, ZHANG Mi, ZHAI Rongxuan. Genome-wide identification of HD-Zip transcription factors in blueberry and functional characterization of their drought-responsive mechanisms[J]. Journal of Nanjing Forestry University (Natural Sciences Edition）. 0 https://doi.org/10.12302/j.issn.1000-2006.202512006

References

[1] LIU X,TAN X L,SU Y D,et al.Anthocyanin attenuates pulmonary arterial hypertension and associated heart failure via improving mitochondrial function through Nrf2-dependent mechanism[J].Clinical and Experimental Hypertension, 2025, 47(1):2503805. DOI:10.1080/10641963.2025.2503805.
[2] 王昌涛, 许小征. 蓝莓综合加工利用技术[M]. 北京: 中国农业科学技术出版社, 2013.Wang C T, Xu X Z. Comprehensive processing and application of blueberry[M]. Beijing: China Agricultural Science and Technology Press, 2013.
[3] WANG A B, WANG L, LIU K, et al.Comparative transcriptome profiling reveals the defense pathways and mechanisms in the leaves and roots of blueberry to drought stress[J]. Fruit Research, 2022, 2(18):18. DOI:10.48130/FruRes-2022-0018.
[4] FENG X H, BAI S N, ZHOU L X, et al.Integrated analysis of transcriptome and metabolome provides insights into flavonoid biosynthesis of blueberry leaves in response to drought stress[J]. International Journal of Molecular Sciences, 2024, 25(20):11135. DOI:10.3390/ijms252011135.
[5] MOLNAR S, CLAPA D, MITRE V.Response of the five highbush blueberry cultivars to in vitro induced drought stress by polyethylene glycol[J]. Agronomy, 2022, 12(3):732. DOI:10.3390/agronomy12030732.
[6] LI L Y, MA W W, WANG Y, et al.Foliar spraying of melatonin as a pretreatment enhances drought resistance in half-highbush blueberries: optimal concentrations for varied drought stress conditions[J]. New Zealand Journal of Crop and Horticultural Science, 2025, 53(5):2924-2941. DOI:10.1080/01140671.2025.2517407.
[7] 田琴,刘奎,吴翔纬,等.转录因子VcMYB17调控蓝莓抗旱性的功能研究[J].生物技术通报,2025,41(4):198-210.Tian Q,Liu K,Wu X W,et al.Functional study of transcription factor VcMYB17 in regulating drought tolerance in blueberry[J].Biotechnology Bulletin,2025,41(4):198-210. DOI:10.13560/j.cnki.biotech.bull.1985.2024-1031.
[8] 陈文荣,潘霞,邵俊怡,等.蓝莓VcLon1基因的克隆、表达及抗旱性分析[J].林业科学,2018,54(6):73-84.Chen W R,Pan X,Shao J Y,et al.Cloning,expression and analysis of drought resistance of VcLon1 gene from blueberry[J].Scientia Silvae Sinicae,2018,54(6):73-84.DOI:10.11707/j.1001-7488.20180609.
[9] WANG F B, ZHU H, CHEN D H, et al.A grape bHLH transcription factor gene, VvbHLH1, increases the accumulation of flavonoids and enhances salt and drought tolerance in transgenic Arabidopsis thaliana[J]. Plant Cell, Tissue and Organ Culture, 2016, 125(2):387-398. DOI:10.1007/s11240-016-0953-1.
[10] LI X Y, XU Y X, LIU K, et al.VcTT2 enhances drought tolerance in blueberry by regulating ROS scavenging[J]. Fruit Research, 2025, 5: e025. DOI:10.48130/frures-0025-0016.
[11] ARIEL F D, MANAVELLA P A, DEZAR C A, et al.The true story of the HD-Zip family[J]. Trends in Plant Science, 2007, 12(9):419-426. DOI:10.1016/j.tplants.2007.08.003.
[12] ARIEL F, DIET A, VERDENAUD M, et al.Environmental regulation of lateral root emergence in Medicago truncatula requires the HD-Zip I transcription factor HB1[J]. The Plant Cell, 2010, 22(7):2171-2183. DOI:10.1105/tpc.110.074823.
[13] AOYAMA T, DONG C H, WU Y, et al.Ectopic expression of the Arabidopsis transcriptional activator Athb-1 alters leaf cell fate in tobacco[J]. The Plant Cell, 1995, 7(11):1773-1785. DOI:10.1105/tpc.7.11.1773.
[14] GONG S H, DING Y F, HU S S, et al.The role of HD-Zip class I transcription factors in plant response to abiotic stresses[J]. Physiologia Plantarum, 2019, 167(4):516-525. DOI:10.1111/ppl.12965.
[15] LIU K, HAN X L, LIANG Z L, et al.Genome-wide identification, classification, and expression analysis of the HD-Zip transcription factor family in apple (Malus domestica)[J]. International Journal of Molecular Sciences, 2022, 23(5):2632. DOI:10.3390/ijms23052632.
[16] ELHITI M, STASOLLA C.Structure and function of homodomain-leucine zipper (HD-Zip) proteins[J]. Plant Signaling and Behavior, 2009, 4(2):86-88. DOI:10.4161/psb.4.2.7692.
[17] VALDÉS A E, ÖVERNÄS E, JOHANSSON H, et al. The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities[J]. Plant Molecular Biology, 2012, 80(4):405-418. DOI:10.1007/s11103-012-9956-4.
[18] RÉ D A, CAPELLA M, BONAVENTURE G, et al.Arabidopsis AtHB7 and AtHB12 evolved divergently to fine tune processes associated with growth and responses to water stress[J]. BMC Plant Biology, 2014, 14: 150. DOI:10.1186/1471-2229-14-150.
[19] ZHAO Y, MA Q, JIN X L, et al.A novel maize homeodomain-leucine zipper (HD-Zip) I gene, Zmhdz10, positively regulates drought and salt tolerance in both rice and Arabidopsis[J]. Plant and Cell Physiology, 2014, 55(6):1142-1156. DOI:10.1093/pcp/pcu054.
[20] GAO Y M, LIU H L, ZHANG K M, et al.A moso bamboo transcription factor, Phehdz1, positively regulates the drought stress response of transgenic rice[J]. Plant Cell Reports, 2021, 40(1):187-204. DOI:10.1007/s00299-020-02625-w.
[21] FANG Y, WANG L, LIU K, et al.Genome-wide investigation of HD-ZIP gene family and functional characterization of BnaHDZ149 and BnaHDZ22 in salt and drought response in Brassica napus[J]. Plant Science, 2024, 346: 112130. DOI:10.1016/j.plantsci.2024.112130.
[22] ZHANG X, LIU J, LIU Y, et al.Genome-wide analysis of HD-Zip genes in walnut identifies JrHDZ28 as a key regulator of drought and salt stress tolerance[J]. Industrial Crops and Products, 2025, 236: 121933. DOI:10.1016/j.indcrop.2025.121933.
[23] Colle M, Leisner C P, Wai C M, et al. Colle M, Leisner C P, Wai C M, et al. Haplotype-phased genome and evolution of phytonutrient pathways of tetraploid blueberry[J]. Giga Science, 2019, 8(3):giz012.DOI:10.1093/gigascience/giz012.
[24] WANG D, GONG Y, LI Y, et al.Genome-wide analysis of the homeodomain-leucine zipper family in Lotus japonicus and the overexpression of LjHDZ7 in Arabidopsis for salt tolerance[J]. Frontiers in Plant Science, 2022, 13: 955199. DOI:10.3389/fpls.2022.955199.
[25] 黄丹, 彭兵阳, 张盼盼, 等.油茶HD-Zip基因家族鉴定及其在非生物胁迫下的表达分析[J].生物技术通报, 2025,41(6):191-207.Huang D, Peng B Y, Zhang P P, et al. Identification of HD-Zip gene family in camellia oleifera and analysis of its expression under abiotic stress[J]. Biotechnology Bulletin, 2025, 41(6):191-207. DOI:10.13560/j.cnki.biotech.bull.1985.2024-1146.
[26] YE K Y, LI J W, WANG F M, et al.Genome-wide analysis and expression profiling of the HD-Zip gene family in kiwifruit[J]. BMC Genomics, 2024, 25(1):354. DOI:10.1186/s12864-024-10025-7.
[27] KUMAR S, STECHER G, LI M, et al.MEGA X: molecular evolutionary genetics analysis across computing platforms[J]. Molecular Biology and Evolution, 2018, 35(6):1547-1549. DOI:10.1093/molbev/msy096.
[28] BAILEY T L, MIKAEL B, BUSKE F A, et al.MEME SUITE: tools for motif discovery and searching[J]. Nucleic Acids Research, 2009, 37: W202-W208. DOI:10.1093/nar/gkp335.
[29] LESCOT M.PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J]. Nucleic Acids Research, 2002, 30(1):325-327. DOI:10.1093/nar/30.1.325.
[30] LI Y, XIONG H Y, GUO D J, et al.Genome-wide characterization and expression profiling of the relation of the HD-Zip gene family to abiotic stress in barley (Hordeum vulgare)[J]. Plant Physiology and Biochemistry, 2019, 141: 250-258. DOI:10.1016/j.plaphy.2019.05.026.
[31] WANG A, LIANG K, YANG S, et al.Genome-wide analysis of MYB transcription factors of Vaccinium corymbosum and their positive responses to drought stress[J]. BMC Genomics, 2021, 22(1):565. DOI:10.1186/s12864-021-07850-5.
[32] VASHISTH T, JOHNSON L K, MALLADI A.An efficient RNA isolation procedure and identification of reference genes for normalization of gene expression in blueberry[J]. Plant Cell Reports, 2011, 30(12):2167-2176. DOI:10.1007/s00299-011-1121-z.
[33] GE X X, LIU Z, WU X M, CHAI L J, GUO W W.Genome-wide identification, classification and analysis of HD-ZIP gene family in citrus, and its potential roles in somatic embryogenesis regulation[J]. Gene, 2015, 574(1):61-68. DOI:10.1016/j.gene.2015.07.079.
[34] 刘梦梦,刘晓,尤倩,等.半夏HD-Zip基因家族全基因组鉴定及表达分析[J].农业生物技术学报,2024,32(11):2540-2551.Liu M M, Liu X, You Q, et al. Genome wide identification and expression analysis of the HD-Zip gene family in Pinellia ternata[J]. Journal of Agricultural Biotechnology, 2024, 32(11):2540-2551. DOI:10.3969/j.issn.1674-7968.2024.11.008.
[35] ZHANG J, CHEN C, YANG Q, et al.Evolution of HD-Zip transcription factors and their function in cabbage leafy head formation[J]. Frontiers in Plant Science, 2025, 16: 1583110. DOI:10.3389/fpls.2025.1289476.
[36] SHARIF R, RAZA A, CHEN P, et al.HD-ZIP gene family: potential roles in improving plant growth and regulating stress-responsive mechanisms in plants[J]. Genes, 2021, 12(8):1256. DOI:10.3390/genes12081256.
[37] ZYLA N, BABULA-SKOWRONSKA D.Evolutionary consequences of functional and regulatory divergence of HD-Zip I transcription factors as a source of diversity in protein interaction networks in plants[J]. Journal of Molecular Evolution, 2023, 91(5):581-597. DOI:10.1007/s00239-023-10121-4.
[38] 罗少,唐翠明,戴凡炜,等.植物转录因子HD-Zip基因家族参与逆境胁迫的研究进展[J].南方农业,2021,15(35):173-175.Luo S, Tang C M, Dai F W, et al. Research progress on the involvement of plant transcription factor HD-Zip gene family in abiotic stress responses[J]. South China Agriculture, 2021, 15(35):173-175. DOI:10.19415/j.cnki.1673-890x.2021.35.058.
[39] XU Y Q, LIU C L.Genome-wide identification and analysis of the HD-Zip transcription factor family in oats[J]. Frontiers in Molecular Biosciences, 2024, 11: 1475276. DOI:10.3389/fmolb.2024.1475276.
[40] CARABELLI M, TURCHI L, RUZZA V, et al.Homeodomain-Leucine Zipper II family of transcription factors to the limelight: central regulators of plant development[J]. Plant Signaling & Behavior, 2013, 8(9):e25447. DOI:10.4161/psb.25447.
[41] FRANKLIN K A, PRAEKELT U, STODDART W M, et al.Phytochromes B, D, and E act redundantly to control multiple physiological responses in Arabidopsis[J]. Plant Physiology, 2003, 131(3):1340-1346. DOI:10.1104/pp.102.015487.
[42] SAMPA, DAS, SENJUTI, et al. Role of HD-Zip transcription factors in plant development and stress responses[J]. International Journal of Agriculture, Environment and Biotechnology, 2016, 9(5):711-718. DOI:10.1007/s40003-016-0235-x.
[43] ROODBARKELARI F, GROOT E P.Regulatory function of homeodomain-leucine zipper (HD-ZIP) family proteins during embryogenesis[J]. New Phytologist, 2016, 213(1):147-160. DOI:10.1111/nph.14132.
[44] KAGALE S, ROZWADOWSKI L K.Genome-wide analysis of ethylene-responsive element binding factor-associated amphiphilic repression motif-containing transcriptional regulators in Arabidopsis[J]. Plant Physiology, 2010, 152(3):1109-1134. DOI:10.1104/pp.109.151704.
[45] MUKHERJEE K, BROCCHIERI L, BÜRGLIN T R. A comprehensive classification and evolutionary analysis of plant homeobox genes[J]. Molecular Biology and Evolution, 2009, 26(12):2775-2794.DOI:10.1093/molbev/msp201.
[46] HARRIS J C, HRMOVA M, LOPATO S, et al.Modulation of plant growth by HD-Zip class I and II transcription factors in response to environmental stimuli[J]. New Phytologist, 2011, 190(4):823-837. DOI:10.1111/j.1469-8137.2011.03733.x.
[47] ZHONG Y, YAN X C, WANG N, et al.ZmHB53, a Maize homeodomain-leucine zipper I transcription factor family gene, contributes to abscisic acid sensitivity and confers seedling drought tolerance by promoting the activity of ZmPYL4[J]. Plant Cell and Environment, 2025, 48(6):3829-3843. DOI:org/10.1111/pce.15394.
[48] ZHANG S X, HAIDER I, KOHLEN W, et al.Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice[J]. Plant Molecular Biology, 2012, 80(6):571-585.DOI:10.1007/s11103-012-9967-1.
[49] SÖDERMAN E, MATTSSON J, ENGSTRÖM P. The Arabidopsis homeobox gene ATHB-7 is induced by water deficit and by abscisic acid[J]. The Plant Journal, 1996, 10(2):375-381. DOI:10.1046/j.1365-313X.1996.10020375.x.
[50] LEE Y H, OH H S, CHEON C I, et al.Structure and expression of the Arabidopsis thaliana homeobox gene Athb-12[J]. Biochemical and Biophysical Research Communications, 2001, 284(1):133-141. DOI:10.1006/bbrc.2001.4904.
[51] JOHANNESSON H, WANG Y, HANSON J, et al.The Arabidopsis thaliana homeobox gene ATHB5 is a potential regulator of abscisic acid responsiveness in developing seedlings[J]. Plant Molecular Biology, 2003, 51(5):719-729. DOI:10.1023/A:1022567625228.
[52] ZHANG Z R, ZHU R R, JI X H, et al.Genome-wide characterization and expression analysis of the HD-Zip gene family in response to salt stress in pepper[J]. International Journal of Genomics, 2021, 2021: 8105124.DOI:10.1155/2021/8105124.
[53] TANG Y, WANG X, WANG Y, et al.Heterologous expression of physic nut JcHDZ25 confers tolerance to drought stress in transgenic rice[J]. BMC Genomics, 2025, 26(1):366. DOI:10.1186/s12864-025-11566-1.
[54] 郝陆洋, 张晓, 高晨曦,等.玉米HD-Zip转录因子基因Zmhdz6的克隆与功能分析[J].植物遗传资源学报,2022,23(3):823-831. Hao L Y, Zhang X J, Gao C X, et al. Cloning and functional analysis of HD-Zip transcription factor gene Zmhdz6 in maize[J]. Journal of Plant Genetic Resources, 2022, 23(3):823-831. DOI:10.13430/j.cnki.jpgr.20211124001.
[55] SÖDERMAN E, HJELLSTRÖM M, FAHLESON J, et al. The HD-Zip gene ATHB6 in Arabidopsis is expressed in developing leaves, roots and carpels and up-regulated by water deficit conditions[J]. Plant Molecular Biology, 1999, 40(6):1073-1083. DOI:10.1023/A:1006267013170.
PDF(1260 KB)

Accesses

Citation

Detail
Sections
Recommended
The full text is translated into English by AI, aiming to facilitate reading and comprehension. The core content is subject to the explanation in Chinese.

Author

Reader

Reviewer

www.nldxb.njfu.edu.cn

Edited ＆ Published by Editorial Department of Nanjing Forestry University(Natural Sciences Edition)
Address： No.159 Longpan Road,Nanjing 210037 Jiangsu,P.R.China
E-mail ：xuebao@njfu.edu.cn , xuebao@njfu.com.cn
Telephone +86-25-85428247,85427076
Distributed by China International Book Trading Corporation P.O. Box 399, Beijing ,P.R. China

/

〈 〉