Comparative transcriptome and functional analyses provide insights into the key factors regulating shoot regeneration in highbush blueberry

Abstract

Establishing an efficient plant regeneration system is a crucial prerequisite for genetic engineering technology in plants. However, the regeneration rate exhibits considerable variability among genotypes, and the key factors underlying shoot regeneration capacity remain largely elusive. Blueberry leaf explants cultured on a medium rich in cytokinins exhibit direct shoot organogenesis without prominent callus formation, which holds promise for expediting genetic transformation while minimizing somatic mutations during culture. The objective of this study is to unravel the molecular and genetic determinants that govern cultivar-specific shoot regeneration potential in highbush blueberry ( Vaccinium corymbosum L.). We conducted comparative transcriptome analysis using two highbush blueberry genotypes: ‘Blue Muffin’ (‘BM’) displaying a high regeneration rate ( > 80%) and ‘O’Neal’ (‘ON’) exhibiting a low regeneration rate ( < 10%). The findings revealed differential expression of numerous auxin-related genes; notably, ‘BM’ exhibited higher expression of auxin signaling genes compared to ‘ON’. Among blueberry orthologs of transcription factors involved in meristem formation in Arabidopsis, expression of VcENHANCER OF SHOOT REGENERATION ( VcESR ), VcWUSCHEL ( VcWUS ), and VcCUP-SHAPED COTYLEDON 2.1 were significantly higher in ‘BM’ relative to ‘ON’. Exogenous application of auxin promoted regeneration, as well as VcESR and VcWUS expression, whereas inhibition of auxin biosynthesis yielded the opposite effects. Overexpression of VcESR in ‘BM’ promoted shoot regeneration under phytohormone-free conditions by activating the expression of cytokinin- and auxin-related genes. These findings provide new insights into the molecular mechanisms underlying blueberry regeneration and have practical implications for enhancing plant regeneration and transformation techniques.

Acknowledgements

This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (No. 19KK0156) to H.Y. and R.T. and Grant-in-Aid for JSPS Fellows for (No. 21J22977) to M.O.

Author Contributions

H.Y. and M.O. designed the project and M.O. performed the experiments and wrote the manuscript. H.Y. and R.T. supervised all the work and revised the manuscript.

Supplementary Data

Supplementary data is available at Horticulture Research Journal online.

Conflict of interest statement

The authors declare no conflicts of interest.

Data availability

The obtained sequencing reads by RNA-seq study are available from the NCBI/DDBJ Sequence Read Archive under the following accession number, DRA017168.

References

[1] Reinert J, Backs D. Control of totipotency in plant cells growing in vitro. Nature. 1968; 220 :1340-1

[2] Thorpe TA. History of plant tissue culture. Mol Biotechnol. 2007; 37 :169-80

[3] Su YH, Tang LP, Zhao XY. et al. Plant cell totipotency: insights into cellular reprogramming. J Integr Plant Biol. 2020; 63 :228-43

[4] Altpeter F, Springer NM, Bartley LE. et al. Advancing crop transformation in the era of genome editing. Plant Cell. 2016; 28 :1510-20

[5] Lee K, Seo PJ. Dynamic epigenetic changes during plant regeneration. Trends Plant Sci. 2018; 23 :235-47

[6] Mathew MM, Prasad K. Model systems for regeneration: Arabidopsis. Development. 2021; 148 :dev195347

[7] Hicks GS. Patterns of organ development in plant tissue culture and the problem of organ determination. Bot Rev. 1980; 46 :1-23

[8] Wang L, Ji Y, Hu Y. et al. The architecture of intra-organism mutation rate variation in plants. PLoS Biol. 2019; 17 :e3000191

[9] Skała E, Grąbkowska R, Sitarek P. et al. Rhaponticum carthamoides regeneration through direct and indirect organogenesis, molecular profiles and secondary metabolite production. Plant Cell Tissue Organ Cult. 2015; 123 :83-98

[10] Pérez-Alonso N, Martin R, Capote A. et al. Efficient direct shoot organogenesis, genetic stability and secondary metabolite production of micropropagated Digitalis purpurea L. Ind Crop Prod. 2018; 116 :259-66

[11] Bhusare BP, John CK, Bhatt VP. et al. In vitro propagation of Digitalis lanata Ehrh. Through direct shoot regeneration-a source of cardiotonic glycosides. Ind Crop Prod. 2018; 121 :313-9

[12] Verma SK, Yücesan BB, Cingöz G. et al. Direct shoot regeneration from leaf explants of Digitalis lamarckii, an endemic medicinal species. Turk J Bot. 2011; 35 :689-95

[13] Galán-Ávila A, García-Fortea E, Prohens J. et al. Development of a direct in vitro plant regeneration protocol from Cannabis sativa L. seedling explants: developmental morphology of shoot regeneration and ploidy level of regenerated plants. Front Plant Sci. 2020; 11 :645

[14] Morinaka H, Mamiya A, Tamaki H. et al. Transcriptome dynamics of epidermal reprogramming during direct shoot regeneration in Torenia fournieri. Plant Cell Physiol. 2021; 62 :1335-54

[15] Qiu D, Wei X, Fan S. et al. Regeneration of blueberry cultivars through indirect shoot organogenesis. HortScience. 2018; 53 :1045-9

[16] Omori M, Yamane H, Osakabe K. et al. Targeted mutagenesis of CENTRORADIALIS using CRISPR/Cas9 system through the improvement of genetic transformation efficiency of tetraploid highbush blueberry. J Hortic Sci Biotechnol. 2021; 96 :153-61

[17] Cappelletti R, Sabbadini S, Mezzetti B. The use of TDZ for the efficient in vitro regeneration and organogenesis of strawberry and blueberry cultivars. Sci Hortic. 2016; 207 :117-24

[18] Schuchovski C, Sant’Anna-Santos BF, Marra RC. et al. Morphological and anatomical insights into de novo shoot organogenesis of in vitro ‘Delite’ rabbiteye blueberries. Heliyon. 2020; 6 :e05468

[19] Georgieva M. Regenerative capacity of leaves and stem segments of six genotypes of Vaccinium corymbosum L. Scientif Bull Series F Biotechnol. 2020; 24 :11-6

[20] LoSchiavo F, Pitto L, Giuliano G. et al. DNA methylation of embryogenic carrot cell cultures and its variations as caused by mutation, differentiation, hormones and hypomethylating drugs. Theor Appl Genet. 1989; 77 :325-31

[21] Neelakandan AK, Wang K. Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications. Plant Cell Rep. 2012; 31 :597-620

[22] Tetsumura T, Matsumoto Y, Sato M. et al. Evaluation of basal media for micropropagation of four highbush blueberry cultivars. Sci Hortic. 2008; 119 :72-4

[23] Colle M, Leisner CP, Wai CM. et al. Haplotype-phased genome and evolution of phytonutrient pathways of tetraploid blueberry. GigaScience. 2019; 8 :giz012

[24] Nagasaka K, Yamane H, Nishiyama S. et al. Insights into the physiological and molecular mechanisms underlying highbush blueberry fruit growth affected by the pollen source. Hort J. 2022; 91 :140-51

[25] Zhao Y, Christensen SK, Fankhauser C. et al. A role for flavin monooxygenase-like enzymes in auxin biosynthesis. Science. 2001; 291 :306-9

[26] Ikeuchi M, Favero DS, Sakamoto Y. et al. Molecular mechanisms of plant regeneration. Annu Rev Plant Biol. 2019; 70 :377-406

[27] Richard C, Lescot M, Inzé D. et al. Effect of auxin, cytokinin, and sucrose on cell cycle gene expression in Arabidopsis thaliana cell suspension cultures. Plant Cell Tissue Organ Cult. 2002; 69 :167-76

[28] Vázquez-Ramos JM, Lara-Nuñez A. Cyclins D, phytoregulators and cell cycle onset in germinating maize. Plant Signal Behav. 2008; 3 :578-9

[29] Kakei Y, Yamazaki C, Suzuki M. et al. Small-molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function. Plant J. 2015; 84 :827-37

[30] Nomura Y, Matsuo N, Banno H. A domain containing the ESR motif in enhancer of shoot regeneration 1 functions as a transactivation domain. Plant Biotechnol (Tokyo). 2009; 26 :395-401

[31] Matsuo N, Makino M, Banno H. Arabidopsis enhancer of shoot regeneration ( ESR ) 1 and ESR2 regulate in vitro shoot regeneration and their expressions are differentially regulated. Plant Sci. 2011; 181 :39-46

[32] Shin J, Bae S, Seo PJ. De novo shoot organogenesis during plant regeneration. J Exp Bot. 2020; 71 :63-72

[33] Liu C, Callow P, Rowland L. et al. Adventitious shoot regeneration from leaf explants of southern highbush blueberry cultivars. Plant Cell Tissue Organ Cult. 2010; 103 :137-44

[34] Jiang Z, Li J, Qu LJ. et al. Auxins in Hormone Metabolism and Signaling in Plants. Cambridge, MA: Academic Press; 2017:39-76

[35] Kato H, Kouno M, Takeda M. et al. The roles of the sole activator-type auxin response factor in pattern formation of Marchantia polymorpha. Plant Cell Physiol. 2017; 58 :1642-51

[36] Suzuki H, Kato H, Iwano M. et al. Auxin signaling is essential for organogenesis but not for cell survival in the liverwort Marchantia polymorpha. Plant Cell. 2023; 35 :1058-75

[37] Raspor M, Motyka V, Kaleri AR. et al. Integrating the roles for cytokinin and auxin in de novo shoot organogenesis: from hormone uptake to signaling outputs. Int J Mol Sci. 2021; 22 :8554

[38] Banno H, Ikeda Y, Niu QW. et al. Overexpression of Arabidopsis ESR1 induces initiation of shoot regeneration. Plant Cell. 2001; 13 :2609-18

[39] Ikeda Y, Banno H, Niu Q. et al. The enhancer of shoot regeneration 2 gene in Arabidopsis regulates cup-shaped cotyledon 1 at the transcriptional level and controls cotyledon development. Plant Cell Physiol. 2006; 47 :1443-56

[40] Yordanov YS, Ma C, Strauss SH. et al. Early bud-break 1 ( EBB1 ) is a regulator of release from seasonal dormancy in poplar trees. PNAS. 2014; 111 :10001-6

[41] Zhao X, Han X, Wang Q. et al. EARLY BUD BREAK 1 triggers BUD break in peach trees by regulating hormone metabolism, the cell cycle, and cell wall modifications. J Exp Bot. 2020; 71 :3512-23

[42] Kusnandar AS, Itoh J, Sato Y. et al. NARROW and DWARF LEAF 1, the ortholog of Arabidopsis enhancer of shoot regeneration1/dornröschen, mediates leaf development and maintenance of the shoot apical meristem in Oryza sativa L. Plant Cell Physiol. 2022; 63 :265-78

[43] Ishida S, Suzuki H, Iwaki A. et al. Diminished auxin signaling triggers cellular reprogramming by inducing a regeneration factor in the liverwort Marchantia polymorpha. Plant Cell Physiol. 2022; 63 :384-400

[44] Maren NA, Duan H, Da K. et al. Genotype-independent plant transformation. Hortic Res. 2022; 9 :uhac047

[45] Iwase A, Harashima H, Ikeuchi M. et al. WIND1 promotes shoot regeneration through transcriptional activation of enhancer of shoot regeneration1 in Arabidopsis. Plant Cell. 2017; 29 :54-69

[46] Murashige T, Skoog F. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant. 1962; 15 :473-97

[47] McCown BH, Lloyd G. Woody plant medium (WPM)-a mineral nutrient formulation for microculture of woody plant-species. HortScience. 1981; 16 :453

[48] Kawamoto T. Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants. Arch Histol Cytol. 2003; 66 :123-43

[49] Chen S, Zhou Y, Chen Y. et al. Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018; 34 :i884-90

[50] Langmead B, Salzberg SL. Fast gapped-read alignment with bowtie 2. Nat Methods. 2012; 9 :357-9

[51] Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011; 12 :323

[52] Ge SX, Son EW, Yao R. iDEP: an integrated web application for differential expression and pathway analysis of RNA-Seq data. BMC Bioinformatics. 2018; 19 :1-24

[53] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2. Genome Biol. 2014; 15 :550

[54] Young MD, Wakefield MJ, Smyth GK. et al. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010; 11 :R14-2

[55] Omori M, Yamane H, Osakabe K. et al. The evaluation of CRISPR-Cas9-mediated editing efficiency using endogenous promoters in tetraploid blueberry. Acta Hort. 2023; 1362 :49-56