Spatiotemporal profiles of gene activity in stamen delineate nucleo-cytoplasmic interaction in a male-sterile somatic cybrid citrus

Abstract

Cytoplasmic male sterility (CMS) has long been used to produce seedless fruits in perennial woody crops like citrus. A male-sterile somatic cybrid citrus (G1 + HBP) was generated by protoplast fusion between a CMS callus parent ‘Guoqing No. 1’ Satsuma mandarin (Citrus unshiu, G1) and a fertile mesophyll parent Hirado Buntan pummelo (Citrus grandis, HBP). To uncover the male-sterile mechanism of G1 + HBP, we compared the transcriptome profiles of stamen organ and cell types at five stages between G1 + HBP and HBP, including the initial stamen primordia, enlarged stamen primordia, pollen mother cells, tetrads, and microspores captured by laser microdissection. The stamen organ and cell types showed distinct gene expression profiles. A majority of genes involved in stamen development were differentially expressed, especially CgAP3.2, which was downregulated in enlarged stamen primordia and upregulated in tetrads of G1 + HBP compared with HBP. Jasmonic acid- and auxin-related biological processes were enriched among the differentially expressed genes of stamen primordia, and the content of jasmonic acid biosynthesis metabolites was higher in flower buds and anthers of G1 + HBP. In contrast, the content of auxin biosynthesis metabolites was lower in G1 + HBP. The mitochondrial tricarboxylic acid cycle and oxidative phosphorylation processes were enriched among the differentially expressed genes in stamen primordia, meiocytes, and microspores, indicating the dysfunction of mitochondria in stamen organ and cell types of G1 + HBP. Taken together, the results indicate that malfunction of mitochondria-nuclear interaction might cause disorder in stamen development, and thus lead to male sterility in the citrus cybrid.

Acknowledgements

This research was financially supported by the Ministry of Science and Technology of China (2022YFF1003101), the National Natural Science Foundation of China (31530065, 31820103011, 32202451), and the Foundation of Hubei Hongshan Laboratory (2021hszd009).

Author contributions

X.M.W. and W.W.G. conceived and supervised the research; N.J. performed the experiments and analyzed the data with contributions from M.Q.F., L.C.C., L.H.K., C.C.L., Z.P.Y., R.W., and K.D.X; N.J. and X.M.W. wrote the manuscript with contributions from all co-authors.

Data availability

The raw data that support the findings of this study are openly available in NCBI database under the BioProject accession number PRJNA938902.

Conflict of interest

The authors declared that they have no conflicts of interest.

References

[1] Chen L, Liu Y-G. Male sterility and fertility restoration in crops. Annu Rev Plant Biol. 2014; 65: 579-606.

[2] Kim Y-J, Zhang D . Molecular control of male fertility for crop hybrid breeding. Trends Plant Sci. 2018; 23: 53-65.

[3] Xu F, Yang X, Zhao N et al. Exploiting sterility and fertility variation in cytoplasmic male sterile vegetable crops. Hortic Res. 2022; 9: uhab039.

[4] Wang K, Gao F, Ji Y et al. ORFH79 impairs mitochondrial function via interaction with a subunit of electron transport chain complex III in Honglian cytoplasmic male sterile rice. New Phytol. 2013; 198: 408-18.

[5] Touzet PH, Meyer E . Cytoplasmic male sterility and mitochondrial metabolism in plants. Mitochondrion. 2014; 19: 166-71.

[6] Luo D, Xu H, Liu Z et al. A detrimental mitochondrial-nuclear interaction causes cytoplasmic male sterility in rice. Nat Genet. 2013; 45: 573-7.

[7] Jiang H, Lu Q, Qiu S et al. Fujian cytoplasmic male sterility and the fertility restorer gene OsRf19 provide a promising breeding system for hybrid rice . Proc Natl Acad Sci USA. 2022; 119: e2208759119.

[8] L’Homme Y, Stahl RJ, Li X-Q et al. Brassica nap cytoplasmic male sterility is associated with expression of a mtDNA region containing a chimeric gene similar to the pol CMS-associated orf224 gene . Curr Genet. 1997; 31: 325-35.

[9] Kuwabara K, Harada I, Matsuzawa Y et al. Organelle genome assembly uncovers the dynamic genome reorganization and cytoplasmic male sterility associated genes in tomato. Hortic Res. 2021; 8: 1-11.

[10] Kuwabara K, Arimura S-I, Shirasawa K et al. orf137 triggers cytoplasmic male sterility in tomato. Plant Physiol. 2022; 189: 465-8.

[11] Berkowitz O, De Clercq I, Van Breusegem F et al. Interaction between hormonal and mitochondrial signalling during growth, development and in plant defence responses. Plant Cell Environ. 2016; 39: 1127- 39.

[12] Welchen E, Canal MV, Gras DE et al. Cross-talk between mitochondrial function, growth, and stress signalling pathways in plants. J Exp Bot. 2021; 72: 4102-18.

[13] Yang J, Zhang M, Yu J . Mitochondrial retrograde regulation tuning fork in nuclear genes expressions of higher plants. J Genet Genomics. 2008; 35: 65-71.

[14] Wang B, Farooq Z, Chu L et al. High-generation near-isogenic lines combined with multi-omics to study the mechanism of polima cytoplasmic male sterility. BMC Plant Biol. 2021; 21: 130.

[15] Dong X, Kim WK, Lim Y-P et al. Ogura-CMS in Chinese cabbage (Brassica rapa ssp. pekinensis) causes delayed expression of many nuclear genes . Plant Sci. 2013; 199-200: 7-17.

[16] Li P, Zhang D, Su T et al. Genome-wide analysis of mRNA and lncRNA expression and mitochondrial genome sequencing provide insights into the mechanisms underlying a novel cytoplasmic male sterility system, BVRC-CMS96, in Brassica rapa. Theor Appl Genet. 2020; 133: 2157-70.

[17] Ng S, De Clercq I, Van Aken O et al. Anterograde and retrograde regulation of nuclear genes encoding mitochondrial proteins during growth, development, and stress. Mol Plant. 2014; 7: 1075-93.

[18] Gaborieau L, Brown GG, Mireau H . The propensity of pentatricopeptide repeat genes to evolve into restorers of cytoplasmic male sterility. Front Plant Sci. 2016; 7: 1816.

[19] Hu J, Wang K, Huang W et al. The rice pentatricopeptide repeat protein RF5 restores fertility in Hong-Lian cytoplasmic male-sterile lines via a complex with the glycine-rich protein GRP162. Plant Cell. 2012; 24: 109-22.

[20] Liu Z, Yang Z, Wang X et al. A mitochondria-targeted PPR protein restores pol cytoplasmic male sterility by reducing orf224 transcript levels in oilseed rape . Mol Plant. 2016; 9: 1082-4.

[21] Wang C, Lezhneva L, Arnal N et al. The radish Ogura fertility restorer impedes translation elongation along its cognate CMS-causing mRNA. Proc Natl Acad Sci USA. 2021; 118: e2105274118.

[22] Sun X, Liu Y, Wang L et al. Molecular characterization of the Rs-Rf₁ gene and molecular marker-assisted development of elite radish (Raphanus sativus L.) CMS lines with a functional marker for fertility restoration . Mol Breed. 2012; 30: 1727-36.

[23] Wei B, Bosland PW, Zhang Z et al. A predicted NEDD8 conjugating enzyme gene identified as a Capsicum candidate Rf gene using bulk segregant RNA sequencing . Hortic Res. 2020; 7: 210.

[24] Carlsson J, Leino M, Sohlberg J et al. Mitochondrial regulation of flower development. Mitochondrion. 2008; 8: 74-86.

[25] Guo W-W, Grosser JW . Somatic hybrid vigor in Citrus: direct evidence from protoplast fusion of an embryogenic callus line with a transgenic mesophyll parent expressing the GFP gene . Plant Sci. 2005; 168: 1541-5.

[26] Guo W-W, Xiao S-X, Deng X-X . Somatic cybrid production via protoplast fusion for citrus improvement. Sci Hortic. 2013; 163: 20-6.

[27] Omar AA, Murata M, Yu Q et al. Production of three new grapefruit cybrids with potential for improved citrus canker resistance. In Vitro Cell Dev Biol - Plant. 2017; 53: 256-69.

[28] Yamamoto M, Matsumoto R, Okudai N et al. Aborted anthers of Citrus result from gene-cytoplasmic male sterility . Sci Hortic. 1997; 70: 9-14.

[29] Nakano M, Nesumi H, Yoshioka T et al. Segregation of plants with undeveloped anthers among hybrids derived from the seed parent, ‘kiyomi’ (Citrus unshiu × C sinensis). J Jpn Soc Hortic Sci. 2001; 70: 539-45.

[30] Guo WW, Cheng YJ, Serrano P et al. Targeted cybridization in citrus: transfer of Satsuma cytoplasm to seedy cultivars for potential seedlessness. Plant Cell Rep. 2004; 22: 752-8.

[31] Wang L, Pan Z-Y, Guo W-W . Proteomic analysis of leaves from a diploid cybrid produced by protoplast fusion between Satsuma mandarin and pummelo. Plant Cell Tissue Organ Cult. 2010; 103: 165-74.

[32] Zhang S, Yin Z-P, Wu X-M et al. Assembly of Satsuma mandarin mitochondrial genome and identification of cytoplasmic male sterility-specific ORFs in a somatic cybrid of pummelo. Tree Genet Genomes. 2020; 16: 84.

[33] Zheng B-B, Wu X-M, Ge X-X et al. Comparative transcript profiling of a male sterile cybrid pummelo and its fertile type revealed altered gene expression related to flower development. PLoS One. 2012; 7: e43758.

[34] Zheng B-B, Fang Y-N, Pan Z-Y et al. iTRAQ-based quantitative proteomics analysis revealed alterations of carbohydrate metabolism pathways and mitochondrial proteins in a male sterile cybrid pummelo. J Proteome Res. 2014; 13: 2998-3015.

[35] Fang Y-N, Zheng B-B, Wang L et al. High-throughput sequencing and degradome analysis reveal altered expression of miRNAs and their targets in a male-sterile cybrid pummelo (Citrus grandis). BMC Genomics. 2016; 17: 591.

[36] Fang Y-N, Yang X-M, Jiang N et al. Genome-wide identification and expression profiles of phased siRNAs in a male-sterile somatic cybrid of pummelo (Citrus grandis). Tree Genet Genomes. 2020; 16: 1-13.

[37] Wang R, Fang Y-N, Wu X-M et al. The miR399- CsUBC24 module regulates reproductive development and male fertility in citrus . Plant Physiol. 2020; 183: 1681-95.

[38] Wang N, Li C, Kuang L et al. Pan-mitogenomics reveals the genetic basis of cytonuclear conflicts in citrus hybridization, domestication, and diversification. Proc Natl Acad Sci USA. 2022; 119: e2206076119.

[39] Nakatsuka T, Saito M, Nishihara M . Functional characterization of duplicated B-class MADS-box genes in Japanese gentian. Plant Cell Rep. 2016; 35: 895-904.

[40] Galimba KD, Martínez-Gómez J, Di Stilio VS . Gene duplication and transference of function in the paleo AP3 lineage of floral organ identity genes . Front Plant Sci. 2018; 9: 334.

[41] Yuan Z, Zhang D . Roles of jasmonate signalling in plant inflorescence and flower development. Curr Opin Plant Biol. 2015; 27: 44-51.

[42] Scott RJ, Spielman M, Dickinson HG . Stamen structure and function. Plant Cell. 2004; 16: S46-60.

[43] Zhang S, Yao J, Wang L et al. Role of grapevine SEPALLATA-related MADS-box gene VvMADS39 in flower and ovule development . Plant J. 2022; 111: 1565-79.

[44] Irish V. The ABC model of floral development. Curr Biol. 2017; 27: R887-90.

[45] Zhang H, Han W, Linghu T et al. Overexpression of a pear B-class MADS-box gene in tomato causes male sterility. Fruit Res. 2023; 3: 1-11.

[46] Tan F-C, Swain SM . Functional characterization of AP3, SOC1 and WUS homologues from citrus (Citrus sinensis). Physiol Plant. 2007; 131: 481-95.

[47] Cecchetti V, Altamura MM, Brunetti P et al. Auxin controls Arabidopsis anther dehiscence by regulating endothecium lignification and jasmonic acid biosynthesis . Plant J. 2013; 74: 411-22.

[48] Acosta IF, Przybyl M . Jasmonate signaling during Arabidopsis stamen maturation . Plant Cell Physiol. 2019; 60: 2648-59.

[49] Zhou P, Fatima M, Ma X et al. Auxin regulation involved in gynoecium morphogenesis of papaya flowers. Hortic Res. 2019; 6: 119.

[50] Pak H, Guo Y, Chen M et al. The effect of exogenous methyl jasmonate on the flowering time, floral organ morphology, and transcript levels of a group of genes implicated in the development of oilseed rape flowers (Brassica napus L.). Planta. 2009; 231: 79-91.

[51] Tsai W-C, Lee P-F, Chen H-I et al. PeMADS6, a GLOBOSA/PISTILLATA-like gene in Phalaenopsis equestris involved in petaloid formation, and correlated with flower longevity and ovary development . Plant Cell Physiol. 2005; 46: 1125-39.

[52] Song S, Qi T, Huang H et al. The jasmonate-ZIM domain proteins interact with the R2R3-MYB transcription factors MYB21 and MYB24 to affect jasmonate-regulated stamen development in Arabidopsis. Plant Cell. 2011; 23: 1000-13.

[53] Ghelli R, Brunetti P, Napoli N et al. A newly identified flower-specific splice variant of AUXIN RESPONSE FACTOR8 regulates stamen elongation and endothecium lignification in Arabidopsis. Plant Cell. 2018; 30: 620-37.

[54] Liu Z, Miao L, Huo R et al. ARF2-ARF4 and ARF5 are essential for female and male gametophyte development in Arabidopsis. Plant Cell Physiol. 2018; 59: 179-89.

[55] Zhang D, Wilson ZA . Stamen specification and anther development in rice. Chin Sci Bull. 2009; 54: 2342-53.

[56] Wan X, Wu S, Li Z et al. Maize genic male-sterility genes and their applications in hybrid breeding: progress and perspectives. Mol Plant. 2019; 12: 321-42.

[57] Lei X, Liu B . Tapetum-dependent male meiosis progression in plants: increasing evidence emerges. Front Plant Sci. 2020; 10: 1667.

[58] Gu J-N, Zhu J, Yu Y et al. DYT1 directly regulates the expression of TDF1 for tapetum development and pollen wall formation in Arabidopsis. Plant J. 2014; 80: 1005-13.

[59] Lou Y, Zhou H-S, Han Y et al. Positive regulation of AMS by TDF1 and the formation of a TDF1-AMS complex are required for anther development in Arabidopsis thaliana. New Phytol. 2018; 217: 378-91.

[60] Siqueira JA, da Fonseca-Pereira P, Fernie AR et al. Recycling amino acids ensures meiosis and seed development. Trends Plant Sci. 2022; 27: 1084-6.

[61] Kianian PMA, Kianian SF . Mitochondrial dynamics and the cell cycle. Front Plant Sci. 2014; 5: 222.

[62] Nguyen TD, Moon S, Nguyen VNT et al. Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage. Plant Mol Biol. 2016; 92: 71-88.

[63] Zhang H, Yang H, Hu D et al. Single-cell RNA sequencing of meiocytes and microspores reveals the involvement of the Rf4 gene in redox homeostasis of CMS-C maize . Crop J. 2021; 9: 1237-47.

[64] Ivanova A, Law SR, Narsai R et al. A functional antagonistic relationship between auxin and mitochondrial retrograde signaling regulates alternative oxidase1a expression in Arabidopsis. Plant Physiol. 2014; 165: 1233-54.

[65] Li M, Yu G, Cao C et al. Metabolism, signaling, and transport of jasmonates. Plant Commun. 2021; 2: 100231.

[66] Serrano I, Audran C, Rivas S . Chloroplasts at work during plant innate immunity. J Exp Bot. 2016; 67: 3845-54.

[67] Adriano N-N, Araújo WL, Fernie AR . Targeting mitochondrial metabolism and machinery as a means to enhance photosynthesis. Plant Physiol. 2011; 155: 101-7.

[68] Van Aken O . Mitochondrial redox systems as central hubs in plant metabolism and signaling. Plant Physiol. 2021; 186: 36-52.

[69] Schwarzländer M, König A-C, Sweetlove LJ et al. The impact of impaired mitochondrial function on retrograde signalling: a meta-analysis of transcriptomic responses. J Exp Bot. 2012; 63: 1735-50.

[70] Pertea M, Kim D, Pertea GM et al. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat Protoc. 2016; 11: 1650-67.

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

[72] Kumar L, Futschik ME . Mfuzz: a software package for soft clustering of microarray data. Bioinformation. 2007; 2: 5-7.

[73] Langfelder P, Horvath S . WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008; 9: 559.

[74] Shannon P, Markiel A, Ozier O et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13: 2498-504.

[75] Cantalapiedra CP, Hernández-Plaza A, Letunic I et al. eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Mol Biol Evol. 2021; 38: 5825-9.

[76] Wu T, Hu E, Xu S et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation. 2021; 2: 100141.

[77] Feng M-Q, Lu M-D, Long J-M et al. miR156 regulates somatic embryogenesis by modulating starch accumulation in citrus. J Exp Bot. 2022; 73: 6170-85.