SlALKBH9B is involved in drought-induced flower drop by regulating ethylene production

Abstract

Drought induces tomato (Solanum lycopersicum) flowers and fruits drop, which causes serious yield and economic losses in agriculture. However, the mechanism of action remains unclear. N6-methyladenosine (m6A) methylation is a prevalent epigenetic change integral to the growth, development, and adaptation of plants to abiotic stress factors. However, whether it participates in drought-induced abscission remains to be further studied. Here, we report that tomato demethylase alpha-ketoglutarate-dependent dioxygenase B (AlkB) homolog 9B (SlALKBH9B) exerts a detrimental influence on the regulation of drought-induced flower drop by mediating ethylene production. We found that drought markedly reduced the expression of SlALKBH9B, and knockout of SlALKBH9B enhanced flower drop, while overexpression of SlALKBH9B delayed the flower drop. Under drought conditions, the ethylene production of Slalkbh9b exhibited a considerably greater yield than that of the wild type (WT), while SlALKBH9B overexpression plants had lower ethylene production. Application of ethylene could abolish the delayed abscission effect of overexpression of SlALKBH9B. Further studies showed that drought downregulated SlALKBH9B expression, which specifically enhanced the methylation level of the 3′ untranslated region (UTR) of tomato ethylene excess producer 1 (SlETO1), leading to a decrease in the stability of SlETO1 mRNA and its protein translation efficiency. The loss of SlETO1 resulted in the accumulation of tomato 1-aminocyclopropane-1-carboxylic acid synthase 3 (SlACS3) and SlACS8 in the abscission zone (AZ) and then boosted ethylene production to accelerate abscission. Our results show that SlALKBH9B is an important inhibitor for drought-induced abscission and reveal a new mechanism through which drought-enhanced ethylene production leads to flower drop.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grants 32330097, 32372687, and 32302645), Liaoning Province Key Research and Development Project (2024JH2/102400031), and Agricultural Project of Liaoning Province (2023JH1/10200010). We are grateful to all of our team’s graduate students for their assistance in collecting the phenotypic data.

Author contributions

T.X. conceived the original research plans. T.X. and T.L. supervised the experiments. T.X., T.L., and C.-Z.J. designed the research. Y.C., X.L., L.C., R.L., Y.L.,S.G., S.W., J.L., C.T., S.M., and M.Q. performed the experiments. Y.C., analyzed the data and produced the plots. T.X., and Y.C., wrote and modified the manuscript. T.X. agrees to serve as the author responsible for contact and communication. All authors have read and approved the contents of this paper.

Data availability

The National Center for Biotechnology Information has received the m6A-seq data gathered from this investigation. The paper and the Supplementary Information files provide all the data that backs up the study’s findings. Moreover, this manuscript encompasses source data.

Conflict of interest statement

The authors declare no conflicts of interest.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Taylor JE, Whitelaw CA. Signals in abscission. New Phytol. 2001; 151:323-40

[2] Addicott FT. Plant hormones IN the control of abscission. Biol Rev. 1970; 45:485-524

[3] Florkiewicz AB, Ku ćko A, Kapusta M. et al. Drought disrupts auxin localization in abscission zone and modifies cell wall structure leading to flower separation in yellow lupine. Int J Mol Sci. 2020; 21:6848

[4] Botton A, Ruperti B. The yes and no of the ethylene involvement in abscission. Plants (Basel). 2019; 8:8

[5] Sundaresan S, Philosoph-Hadas S, Ma C. et al. The tomato hybrid proline-rich protein regulates the abscission zone competence to respond to ethylene signals. Hortic Res. 2018; 5:28

[6] Olson DC, Oetiker JH, Yang SF. Analysis of LE-ACS3, a 1-Aminocyclopropane-1-carboxylic acid synthase gene expressed during flooding in the roots of tomato plants ∗. JBiolChem. 1995; 270:14056-61

[7] Pattyn J, Vaughan-Hirsch J, Van de Poel B. The regulation of ethylene biosynthesis: a complex multilevel control circuitry. New Phytol. 2021; 229:770-82

[8] Booker MA, DeLong A. Producing the ethylene signal: regulation and diversification of ethylene biosynthetic enzymes. Plant Phys-iol. 2015; 169:42-50

[9] Yoon GM. New insights into the protein turnover regulation in ethylene biosynthesis. Mol Cells. 2015; 38:597-603

[10] Tatsuki M, Mori H. Phosphorylation of tomato 1-aminocyclopropane-1-carboxylic acid synthase, LE-ACS2, at the C-terminal region∗. JBiolChem. 2001; 276:28051-7

[11] Meng Y, Ma N, Zhang Q. et al. Precise spatio-temporal modula-tion of ACC synthase by MPK6 cascade mediates the response of rose flowers to rehydration. Plant J. 2014; 79:941-50

[12] Li G, Meng X, Wang R. et al. Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis. PLoS Genet. 2012; 8:e1002767

[13] Skottke KR, Yoon GM, Kieber JJ. et al. Protein phosphatase 2A controls ethylene biosynthesis by differentially regulating the turnover of ACC synthase isoforms. PLoS Genet. 2011; 7:e1001370

[14] Ludwików A, Cie´sla A, Kasprowicz-Malu´ski A. et al. Arabidop-sis protein phosphatase 2C ABI1 interacts with type I ACC synthases and is involved in the regulation of ozone-induced ethylene biosynthesis. Mol Plant. 2014; 7:960-76

[15] Wang KLC, Yoshida H, Lurin C. et al. Regulation of ethylene gas biosynthesis by the Arabidopsis ETO1 protein. Nature. 2004; 428: 945-50

[16] Tan ST, Xue HW. Casein kinase 1 regulates ethylene synthesis by phosphorylating and promoting the turnover of ACS5. Cell Rep. 2014; 9:1692-702

[17] Ma X, Yuan Y, Li C. et al. Brassinosteroids suppress ethylene-induced fruitlet abscission through LcBZR1/2-mediated transcriptional repression of LcACS1/4 and LcACO2/3 in litchi. Hortic Res. 2021; 8:105

[18] Zhong H, Tang HF, Kai Y. N6-methyladenine RNA modification (m(6)a): an emerging regulator of metabolic diseases. Curr Drug Targets. 2020; 21:1056-67

[19] Shao Y, Wong CE, Shen L. et al. N(6)-methyladenosine modifica-tion underlies messenger RNA metabolism and plant develop-ment. Curr Opin Plant Biol. 2021; 63:102047

[20] Shen L, Ma J, Li P. et al. Recent advances in the plant epitran-scriptome. Genome Biol. 2023; 24:43

[21] Jia G, Fu Y, Zhao X. et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol. 2011; 7:885-7

[22] Fu Y, Dominissini D, Rechavi G. et al. Gene expression regulation mediated through reversible m6A RNA methylation. Nat Rev Genet. 2014; 15:293-306

[23] Arribas-Hernández L, Bressendorff S, Hansen MH. et al. An m(6)A-YTH module controls developmental timing and mor-phogenesis in Arabidopsis. Plant Cell. 2018; 30:952-67

[24] Zhong S, Li H, Bodi Z. et al. MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor. Plant Cell. 2008; 20:1278-88

[25] Martínez-Pérez M, Aparicio F, López-Gresa MP. et al. Arabidopsis m(6)A demethylase activity modulates viral infection of a plant virus and the m(6)A abundance in its genomic RNAs. Proc Natl Acad Sci USA. 2017; 114:10755-60

[26] Chen Y, Zhao Y, Chen J. et al. ALKBH5 suppresses malignancy of hepatocellular carcinoma via m(6)A-guided epigenetic inhi-bition of LYPD1. Mol Cancer. 2020; 19:123

[27] Yanlin S, Chui Eng W, Lisha S. et al. N6-methyladenosine modi-fication underlies messenger RNA metabolism and plant devel-opment. Curr Opin Plant Biol. 2021; 63:102047

[28] Zhou L, Tian S, Qin G. RNA methylomes reveal the m(6)A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening. Genome Biol. 2019; 20:156

[29] Zhou L, Tang R, Li X. et al. N6-methyladenosine RNA modification regulates strawberry fruit ripening in an ABA-dependent man-ner. Genome Biol. 2021; 22:168

[30] Guo T, Liu C, Meng F. et al. The m(6) A reader MhYTP2 regulates MdMLO19 mRNA stability and antioxidant genes translation efficiency conferring powdery mildew resistance in apple. Plant Biotechnol J. 2022; 20:511-25

[31] Luo J-H, Wang M, Jia G-F. et al. Transcriptome-wide analysis of epitranscriptome and translational efficiency associated with heterosis in maize. JExp Bot. 2021; 72:2933-46

[32] Zhao X, Yang Y, Sun BF. et al. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res. 2014; 24:1403-19

[33] Tang C, Klukovich R, Peng H. et al. ALKBH5-dependent m6A demethylation controls splicing and stability of long 3´-UTR mRNAs in male germ cells. Proc Natl Acad Sci USA. 2018;115:E325-e333

[34] Tang J, Yang J, Duan H. et al. ALKBH10B, an mRNA m(6)A demethylase, modulates ABA response during seed germination in Arabidopsis. Front Plant Sci. 2021; 12:712713

[35] Huong TT, Yang Z, Ngoc LNT. et al. ALKBH8B, a putative RNA demethylase, plays a role in the response of Arabidopsis to salt stress and Abscisic acid. J Plant Biol. 2022; 65:319-30

[36] Gu X, Smaill SJ, Wang B. et al. Reducing plant-derived ethylene concentrations increases the resistance of temperate grassland to drought. Sci Total Environ. 2022; 846:157408

[37] Miao Z, Zhang T, Qi Y. et al. Evolution of the RNA N (6)-methyladenosine methylome mediated by genomic duplication. Plant Physiol. 2020; 182:345-60

[38] Zhang G, Lv Z, Diao S. et al. Unique features of the m(6)A methylome and its response to drought stress in sea buckthorn (Hippophae rhamnoides Linn.). RNA Biol. 2021; 18: 794-803

[39] Hu J, Manduzio S, Kang H. Epitranscriptomic RNA methylation in plant development and abiotic stress responses. Front Plant Sci. 2019; 10:500

[40] Huong TT, Ngoc LNT, Kang H. Functional characterization of a putative RNA demethylase ALKBH6 in Arabidopsis growth and abiotic stress responses. Int J Mol Sci. 2020; 21:6707

[41] Yu Q, Liu S, Yu L. et al. RNA demethylation increases the yield and biomass of rice and potato plants in field trials. Nat Biotechnol. 2021; 39:1581-8

[42] Llop-Tous I, Barry CS, Grierson D. Regulation of ethylene biosyn-thesis in response to pollination in tomato flowers. Plant Physiol. 2000; 123:971-8

[43] Qi L, Yue H, Xichun L. et al. Rhein inhibits AlkB repair enzymes and sensitizes cells to methylated DNA damage∗. JBiolChem. 2016; 291:11083-93

[44] Arraes FB, Beneventi MA, Lisei de Sa ME. et al. Implications of ethylene biosynthesis and signaling in soybean drought stress tolerance. BMC Plant Biol. 2015; 15:213

[45] Hopper DW, Ghan R, Schlauch KA. et al. Transcriptomic net-work analyses of leaf dehydration responses identify highly connected ABA and ethylene signaling hubs in three grapevine species differing in drought tolerance. BMC Plant Biol. 2016; 16:118

[46] Ren MY, Feng RJ, Shi HR. et al. Expression patterns of members of the ethylene signaling-related gene families in response to dehydration stresses in cassava. PLoS One. 2017; 12:e0177621

[47] Schaller GE, Binder BM. Inhibitors of ethylene biosynthesis and signaling. Methods Mol Biol. 2017; 1573:223-35

[48] Luo GZ, MacQueen A, Zheng G. et al. Unique features of the m6A methylome in Arabidopsis thaliana. Nat Commun. 2014; 5:5630

[49] Wan Y, Tang K, Zhang D. et al. Transcriptome-wide high-throughput deep m(6)A-seq reveals unique differential m(6)A methylation patterns between three organs in Arabidopsis thaliana. Genome Biol. 2015; 16:272

[50] Heinz S, Benner C, Spann N. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell. 2010; 38:576-89

[51] Wei LH, Song P, Wang Y. et al. The m(6)a reader ECT2 controls trichome morphology by affecting mRNA stability in Arabidopsis. Plant Cell. 2018; 30:968-85

[52] Gingerich DJ, Gagne JM, Salter DW. et al. Cullins 3a and 3b assemble with members of the broad complex/tramtrack/bric-a-brac (BTB) protein family to form essential ubiquitin-protein ligases (E3s) in Arabidopsis. JBiolChem. 2005; 280:18810-21

[53] Du H, Wu N, Cui F. et al. A homolog of ethylene overproducer, OsETOL1, differentially modulates drought and submergence tolerance in rice. Plant J. 2014; 78:834-49

[54] Li J, Jia H, Wang J. cGMP and ethylene are involved in maintaining ion homeostasis under salt stress in Arabidopsis roots. Plant Cell Rep. 2014; 33:447-59

[55] Zhou L, Tang R, Li X. et al. N(6)-methyladenosine RNA modifi-cation regulates strawberry fruit ripening in an ABA-dependent manner. Genome Biol. 2021; 22:168

[56] Luo JH, Wang Y, Wang M. et al. Natural variation in RNA m(6)a methylation and its relationship with translational status. Plant Physiol. 2020; 182:332-44

[57] Yoshida H, Nagata M, Saito K. et al. Arabidopsis ETO1 specif-ically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases. BMC Plant Biol. 2005; 5:14

[58] Trusov Y, Botella JR. Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.]. JExp Bot. 2006; 57:3953-60

[59] Muñoz-Robredo P, Rubio P, Infante R. et al. Ethylene biosynthesis in apricot: identification of a ripening-related 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene. Postharvest Biol Technol. 2012; 63:85-90

[60] Nakatsuka A, Shiomi S, Kubo Y. et al. Expression and internal feedback regulation of ACC synthase and ACC oxidase genes in ripening tomato fruit. Plant Cell Physiol. 1997; 38:1103-10

[61] Salman-Minkov A, Levi A, Wolf S. et al. ACC synthase genes are polymorphic in watermelon (Citrullus spp.) and differentially expressed in flowers and in response to auxin and gibberellin. Plant Cell Physiol. 2008; 49:740-50

[62] Dejian Z, Renxue X, Xiu C. Ethylene modulates root hair devel-opment in trifoliate orange through auxin-signaling pathway. Sci Hortic. 2016; 213:252-9

[63] Yu Y, Yang D, Zhou S. et al. The ethylene response factor OsERF109 negatively affects ethylene biosynthesis and drought tolerance in rice. Protoplasma. 2017; 254:401-8

[64] Schellingen K, Van Der Straeten D, Vandenbussche F. et al. Cadmium-induced ethylene production and responses in Ara-bidopsis thaliana rely on ACS2 and ACS 6 gene expression. BMC Plant Biol. 2014; 14:214

[65] Khan MA, Meng Y, Liu D. et al. Responses of rose RhACS1 and RhACS2 promoters to abiotic stresses in transgenic Arabidopsis thaliana. Plant Cell Rep. 2015; 34:795-804

[66] Khan S, Alvi AF, Saify S. et al. The ethylene biosynthetic enzymes, 1-Aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO): the less explored players in abiotic stress tolerance. Biomolecules. 2024; 14:90

[67] Guzmán P, Ecker JR. Exploiting the triple response of Arabidopsis to identify ethylene-related mutants. Plant Cell. 1990; 2:513-23

[68] Engel M, Eggert C, Kaplick PM. et al. The role of m(6)a/m-RNA methylation in stress response regulation. Neuron. 2018; 99:389-403.e9

[69] Zhou L, Gao G, Tang R. et al. M(6) A-mediated regulation of crop development and stress responses. Plant Biotechnol J. 2022; 20: 1447-55

[70] Reichardt S, Piepho HP, Stintzi A. et al. Peptide signaling for drought-induced tomato flower drop. Science. 2020; 367:1482-5

[71] Wang S, Ge S, Liu X. et al. A regulatory network involving calmod-ulin controls phytosulfokine peptide processing during drought-induced flower abscission. Plant Cell. 2024;37:koaf013.

[72] Sade N, Galkin E, Moshelion M. Measuring Arabidopsis, tomato and barley leaf relative water content (RWC). BIO-PROTOCOL. 2015; 5:5

[73] Wang R, Li R, Cheng L. et al. SlERF 52 regulates SlTIP1;1 expression to accelerate tomato pedicel abscission. Plant Physiol. 2021; 185: 1829-46

[74] Roberts JA, Schindler CB, Tucker GA. Ethylene-promoted tomato flower abscission and the possible involvement of an inhibitor. Planta. 1984; 160:159-63

[75] Cheng L, Li R, Wang X. et al. A SlCLV3-SlWUS module regulates auxin and ethylene homeostasis in low light-induced tomato flower abscission. Plant Cell. 2022; 34:4388-408

[76] Liu Y, Schiff M, Dinesh-Kumar SP. Virus-induced gene silencing in tomato. Plant J. 2002; 31:777-86

[77] Wang JL, Wang M, Zhang L. et al. WAV E3 ubiquitin ligases mediate degradation of IAA32/34 in the TMK1-mediated auxin signaling pathway during apical hook development. Proc Natl Acad Sci USA. 2024; 121:e2314353121

[78] Wang X, Lu Z, Gomez A. et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014; 505:117-20

[79] Merchante C, Brumos J, Yun J. et al. Gene-specific translation regulation mediated by the hormone-signaling molecule EIN2. Cell. 2015; 163:684-97

[80] Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008; 3:1101-8