Triploid loquat maintains photosynthetic stability under freezing stress through excessive accumulation of unsaturated lipids

Abstract

Loquat (Eriobotrya japonica Lindl.), a subtropical evergreen species of the Rosaceae family, faces industry constraints in industrial development due to its sensitivity to freezing temperatures and low photosynthetic efficiency. Polyploid loquats, particularly triploids, exhibit enhanced stress resistance, vigorous growth, and seedless fruit production. In this study, triploid F1 progeny (B431 × GZ23) was obtained through hybridization between diploid (GZ23) and tetraploid (B431) parents. Under − 3^◦ C stress, the triploid lines exhibited significantly improved freezing tolerance and photosynthetic performance, as evidenced by chlorophyll fluorescence parameters and ultrastructural integrity. Lipidomics profiling across all B431 × GZ23 lines revealed that phosphatidylcholine (PC), particularly the abundant unsaturated PC species 18:2/18:2, played a key role in these adaptive advantages. Compared to the parental lines, EjFAD2 expression was specifically upregulated in triploid loquats under freezing stress. Consistent lipidomic and gene expression patterns across three B431 × GZ23 lines ruled out line-specific mutations. Heterologous expression of EjFAD2 in Arabidopsis increased freezing tolerance. Co-expression analysis identified EjMYBS3 as a regulator that binds to the EjFAD2 promoter, and its overexpression in transgenic Arabidopsis enhanced freezing tolerance. Transient expression of EjFAD2 and EjMYBS3 increased the content of PC 18:2/18:2 in loquat, which contributed to the maintenance of photosystem activity under freezing stress, thereby enhancing the freezing tolerance of loquat. Collectively, these findings provide preliminary insights into the molecular mechanisms underlying cold resistance in polyploid loquats and highlight the regulatory role of EjFAD2 and EjMYBS3 in freezing stress response.

References

[1] Hamann E, Denney D, Day S, et al. Review: plant eco-evolutionary responses to climate change: emerging directions. Plant Sci. 2021; 304: 110737

[2] Zhu JK . Abiotic stress signaling and responses in plants. Cell. 2016; 167: 313-24

[3] Román-Figueroa C, Bravo L, Paneque M, et al. Chemical products for crop protection against freezing stress: a review. J Agron Crop Sci. 2021; 207: 391-403

[4] Zhou P, Li X, Liu X, et al. Transcriptome profiling of Malus sieversii under freezing stress after being cold-acclimated. BMC Genomics. 2021; 22: 628

[5] Ambroise V, Legay S, Guerriero G, et al. The roots of plant frost hardiness and tolerance. Plant Cell Physiol. 2020; 61: 3-20

[6] Alix K, Gérard PR, Schwarzacher T, et al. Polyploidy and interspecific hybridization: partners for adaptation, speciation and evolution in plants. Ann Bot. 2017; 120: 183-94

[7] Folk RA, Siniscalchi CM, Soltis DE . Angiosperms at the edge: extremity, diversity, and phylogeny. Plant Cell Environ. 2020; 43: 2871-93

[8] Van de Peer Y, Ashman TL, Soltis PS, et al. Polyploidy: an evolutionary and ecological force in stressful times. Plant Cell. 2021; 33: 2899

[9] Renny-Byfield S, Wendel JF . Doubling down on genomes: polyploidy and crop plants. Am J Bot. 2014; 101: 1711-25

[10] Doyle JJ, Coate JE . Polyploidy, the nucleotype, and novelty: the impact of genome doubling on the biology of the cell. Int J Plant Sci. 2019; 180: 1-52

[11] Oustric J, Morillon R, Luro F, et al. Tetraploid Carrizo citrange rootstock (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.) enhances natural chilling stress tolerance of common clementine (Citrus clementina Hort. ex Tan). J Plant Physiol. 2017; 214: 108-15

[12] Lourkisti R, Froelicher Y, Herbette S, et al. Triploid citrus genotypes have a better tolerance to natural chilling conditions of photosynthetic capacities and specific leaf volatile organic compounds. Front Plant Sci. 2020; 11: 330

[13] Viswanathan C, Zhu JK . Molecular genetic analysis of cold-regulated gene transcription. Philos Trans R Soc B Biol Sci. 2002; 357: 877-86

[14] Mazur R, Gieczewska K, Kowalewska Ł, et al. Specific composition of lipid phases allows retaining an optimal thylakoid membrane fluidity in plant response to low-temperature treatment. Front Plant Sci. 2020; 11: 723

[15] Takahashi D, Uemura M, Kawamura Y . Freezing tolerance of plant cells: from the aspect of plasma membrane and microdomain. Adv Exp Med Biol. 2018; 1081: 61-79

[16] Kargiotidou A, Deli D, Galanopoulou D, et al. Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum). J Exp Bot. 2008; 59: 2043-56

[17] Upchurch RG . Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress. Biotechnol Lett. 2008; 30: 967-77

[18] Miura K, Furumoto T . Cold signaling and cold response in plants. Int J Mol Sci. 2013; 14: 5312-37

[19] Vijayan P, Browse J . Photoinhibition in mutants of Arabidopsis deficient in thylakoid unsaturation. Plant Physiol. 2002; 129: 876-85

[20] Lou Y, Schwender J, Shanklin J . FAD2 and FAD3 desaturases form heterodimers that facilitate metabolic channeling in vivo. J Biol Chem. 2014; 289: 17996-8007

[21] Bhunia RK, Kaur R, Maiti MK . Metabolic engineering of fatty acid biosynthetic pathway in sesame (Sesamum indicum L.): assembling tools to develop nutritionally desirable sesame seed oil. Phytochem Rev. 2016; 15: 799-811

[22] Tang S, Guan R, Zhang H, et al. Cloning and expression analysis of three cDNAs encoding omega-3 fatty acid desaturases from Descurainia sophia. Biotechnol Lett. 2007; 29: 1417-24

[23] Teixeira MC, Coelho N, Olsson ME, et al. Molecular cloning and expression analysis of three omega-6 desaturase genes from purslane (Portulaca oleracea L.). Biotechnol Lett. 2009; 31: 1089-101

[24] Yu C, Wang HS, Yang S, et al. Overexpression of endoplasmic reticulum omega-3 fatty acid desaturase gene improves chilling tolerance in tomato. Plant Physiol Biochem. 2009; 47: 1102-12

[25] Routaboul JM, Fischer SF, Browse J . Trienoic fatty acids are required to maintain chloroplast function at low temperatures. Plant Physiol. 2000; 124: 1697-705

[26] Zhou Z, Wang MJ, Zhao ST, et al. Changes in freezing tolerance in hybrid poplar caused by up- and down-regulation of PtFAD2 gene expression. Transgenic Res. 2010; 19: 647-54

[27] Schuler I, Milon A, Nakatani Y, et al. Differential effects of plant sterols on water permeability and on acyl chain ordering of soybean phosphatidylcholine bilayers. Proc Natl Acad Sci USA. 1991; 88: 6926-30

[28] Thomashow MF . Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Biol. 1999; 50: 571-99

[29] Lee JS, Adams KL . Global insights into duplicated gene expression and alternative splicing in polyploid Brassica napus under heat, cold, and drought stress. Plant Genome. 2020; 13: e20057

[30] Lu H, Xue L, Cheng J, et al. Polyploidization-driven differentiation of freezing tolerance in Solidago canadensis. Plant Cell Environ. 2020; 43: 1394-403

[31] Ji W, Li GR, Luo YX, et al. In vitro embryo rescue culture of F1 progenies from crosses between different ploidy grapes. Genet Mol Res. 2015; 14: 18616-22

[32] Jiang S, An H, Xu F, et al. Proteome analysis provides new insight into major proteins involved in gibberellin-induced fruit setting in triploid loquat (Eriobotrya japonica). Genes Genom. 2020; 42: 383-92

[33] Chen ZJ . Genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids. Annu Rev Plant Biol. 2007; 58: 377-406

[34] Grant-Downton RT, Dickinson HG . Epigenetics and its implications for plant biology. The epigenetic network in plants. Ann Bot. 2005; 96: 1143-64

[35] Madlung A, Masuelli RW, Watson B, et al. Remodeling of DNA methylation and phenotypic and transcriptional changes in synthetic Arabidopsis allotetraploids. Plant Physiol. 2002; 129: 733-46

[36] Levy AA, Feldman M . Genetic and epigenetic reprogramming of the wheat genome upon allopolyploidization. Biol J Linn Soc. 2004; 82: 607-13

[37] Liu B, Wendel JF . Epigenetic phenomena and the evolution of plant allopolyploids. Mol Phylogenet Evol. 2003; 29: 365-79

[38] Allen DJ, Ort DR . Impacts of chilling temperatures on photosynthesis in warm-climate plants. Trends Plant Sci. 2001; 6: 36-42

[39] Li HM, Yu CW . Chloroplast galactolipids: the link between photosynthesis, chloroplast shape, jasmonates, phosphate starvation and freezing tolerance. Plant Cell Physiol. 2018; 59: 1128-34

[40] Kratsch HA, Wise RR . The ultrastructure of chilling stress. Plant Cell Environ. 2000; 23: 337-50

[41] Miquel M, James D, Dooner H, et al. Arabidopsis requires polyunsaturated lipids for low-temperature survival. Proc Natl Acad Sci USA. 1993; 90: 6208-12

[42] Song C, Yang Y, Yang T, et al. MaMYB4 recruits histone deacetylase MaHDA2 and modulates the expression of ω-3 fatty acid desaturase genes during cold stress response in banana fruit. Plant Cell Physiol. 2019; 60: 2410-22

[43] Hofmann NR . A global view of hybrid vigor: DNA methylation, small RNAs, and gene expression. Plant Cell. 2012; 24: 841

[44] Miller M, Song Q, Shi X, et al. Natural variation in timing of stress-responsive gene expression predicts heterosis in intraspecific hybrids of Arabidopsis. Nat Commun. 2015; 6: 7453

[45] Chen L, Bian J, Shi S, et al. Genetic analysis for the grain number heterosis of a super-hybrid rice WFYT025 combination using RNA-Seq. Rice. 2018; 11: 37

[46] Jing D, Liu X, He Q, et al. Genome assembly of wild loquat (Eriobotrya japonica) and resequencing provide new insights into the genomic evolution and fruit domestication in loquat. Hortic Res. 2022; 10: 2

[47] Jiang S, An H, Xu F, et al. Chromosome-level genome assembly and annotation of the loquat (Eriobotrya japonica) genome. GigaScience. 2020; 9: giaa015

[48] Su K, Bremer DJ, Jeannotte R, et al. Membrane lipid composition and heat tolerance in cool-season turfgrasses, including a hybrid bluegrass. J Am Soc Hortic Sci. 2009; 134: 511-20

[49] Jiang S, Luo J, Xu F, et al. Transcriptome analysis reveals candidate genes involved in gibberellin-induced fruit setting in triploid loquat (Eriobotrya japonica). Front Plant Sci. 2016; 7

[50] Strasser RJ, Tsimilli-Michael M, Srivastava A . Analysis of the chlorophyll a fluorescence transient. In: Chlorophyll a Fluorescence: A Signature of Photosynthesis.Springer Netherlands: Dordrecht, 2004

[51] Hernández JA, Almansa MS . Short-term effects of salt stress on antioxidant systems and leaf water relations of pea leaves. Physiol Plant. 2002; 115: 251-7