Engineering saline-alkali-tolerant apple rootstock by knocking down MdGH3 genes in M9-T337

Abstract

Soil salinization and alkalization have become an increasingly severe global issues, significantly limiting both the yield and quality of apples (Malus ×  domestica). M9-T337 is a widely used apple dwarfing rootstock; however, it is sensitive to saline-alkali stress. Therefore, developing saline-alkali tolerant apple rootstocks is essential. In this study, we utilized RNAi (RNA interference) technology to knock down GH3 genes in the M9-T337 background, aiming to engineer a dwarfing and stress-tolerant apple rootstock. We found that MdGH3 RNAi plants exhibited superior morphology compared to M9-T337 under saline-alkali stress conditions, characterized by more robust root systems, increased plant height, a lower Na⁺/K⁺ ratio, and enhanced photosynthetic and antioxidant capacities. Moreover, when MdGH3 RNAi plants were used as rootstocks, the GL-3/MdGH3 RNAi plants also displayed greater plant height, root vitality, photosynthetic ability, and antioxidant capacity compared to GL-3 grafted onto M9-T337 rootstock. Taken together, our study constructed a saline-alkali-tolerant apple rootstock by knocking down MdGH3 genes.

Keywords

Apple / Saline-alkali stress / M9-T337 / MdGH3 RNAi / Rootstock

References

[1] Casanova-SáezR, Mateo-BonmatíE, ŠimuraJ, PěnčíkA, NovákO, StaswickP, LjungK. Inactivation of the entire Arabidopsis group II GH3s confers tolerance to salinity and water deficit. New Phytol, 2022, 235: 263-275

[2] ChangS, PuryearJ, CairneyJ. A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Report, 1993, 11(2): 113-116

[3] Chen C, He G, Li J, Perez-Hormaeche J, Becker T, Luo M, Wallrad L, Gao J, Li J, Pardo JM, Kudla J, and Guo Y (2023) A salt stress-activated GSO1-SOS2-SOS1 module protects the Arabidopsis root stem cell niche by enhancing sodium ion extrusion. EMBO J 42(13):e113004. https://doi.org/10.15252/embj.2022113004

[4] Cimen B, Yesiloglu T (2016) Rootstock breeding for abiotic stress tolerance in citrus. In: Abiotic and biotic stress in plants - recent advances and future perspectives. IntechOpen. https://doi.org/10.5772/62047

[5] CuarteroJ, BolarínMC, AsínsMJ, MorenoV. Increasing salt tolerance in the tomato. J Exp Bot, 2006, 57: 1045-1058

[6] DingC, ZhangW, LiD, DongY, LiuJ, HuangQ, SuX. Effect of overexpression of JERFs on intracellular K+/Na+ balance in transgenic poplar (Populus alba × P. berolinensis) under salt stress. Front Plant Sci, 2020, 11: 1192

[7] DuH, WuN, FuJ, WangS, LiX, XiaoJ, XiongL. A GH3 family member, OsGH3-2, modulates auxin and abscisic acid levels and differentially affects drought and cold tolerance in rice. J Exp Bot, 2012, 63: 6467-6480

[8] Fan S, Yang S, Shi K, Yang L, An M, Wang F, Qi Y, Feng M, Wang M, Geng P et al (2024) Genome-wide identification of the LRX gene family in Cucurbitaceae and expression analysis under salt and drought stress in cucumber. Veg Res 4. https://doi.org/10.48130/vegres-0024-0025

[9] Fang S, Hou X, Liang X (2021) Response mechanisms of plants under saline-alkali stress. Front Plant Sci 12. https://doi.org/10.3389/fpls.2021.667458

[10] Goldschmidt EE (2014) Plant grafting: new mechanisms, evolutionary implications. Front Plant Sci 5. https://doi.org/10.3389/fpls.2014.00727

[11] GuoX, PengW, XuX, XieK, YangX. The potential of endophytes in improving salt-alkali tolerance and salinity resistance in plants. Int J Mol Sci, 2023, 24: 16917

[12] Guo Y, Wang Z, Deng CH, Dong J, Shu S, Wang Y, Han Z, Li W, Guo Y, Wang Z et al (2024) Improving the root system of apple rootstocks based on Agrobacterium rhizogenes-mediated transformation system. Fruit Res 4. https://doi.org/10.48130/frures-0024-0027

[13] GuptaBK, SahooKK, AnwarK, NongpiurRC, DeshmukhR, PareekA, Singla-PareekSL. Silicon nutrition stimulates Salt-Overly Sensitive (SOS) pathway to enhance salinity stress tolerance and yield in rice. Plant Physiol Biochem, 2021, 166: 593-604

[14] HabibiF, LiuT, FoltaK, SarkhoshA. Physiological, biochemical, and molecular aspects of grafting in fruit trees. Hortic Res, 2022, 9: uhac032

[15] HanS, JiaM, YangJ, JiangJ. The integration of ACS2-generated ACC with GH3-mediated IAA homeostasis in NaCl-stressed primary root elongation of Arabidopsis seedlings. Plant Growth Regul, 2019, 88: 151-158

[16] HuangD, WangQ, ZhangZ, JingG, MaM, MaF, LiC. Silencing MdGH3-2/12 in apple reduces drought resistance by regulating AM colonization. Hortic Res, 2021, 8: 1-14

[17] JelicMD, MandicAD, MaricicSM, SrdjenovicBU. Oxidative stress and its role in cancer. J Cancer Res Ther, 2021, 17: 22

[18] JezJM. Connecting primary and specialized metabolism: Amino acid conjugation of phytohormones by GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetases. Curr Opin Plant Biol, 2022, 66: 102194

[19] Jia B, Sun M, Sun X, Li R, Wang Z, Wu J, Wei Z, DuanMu H, Xiao J, and Zhu Y (2016) Overexpression of GsGSTU13 and SCMRP in Medicago sativa confers increased salt-alkaline tolerance and methionine content. Physiol Plant 156(2):176–189. https://doi.org/10.1111/ppl.12350.

[20] JiangL, ShenW, LiuC, TahirMM, LiX, ZhouS, MaF, GuanQ. Engineering drought-tolerant apple by knocking down six GH3 genes and potential application of transgenic apple as a rootstock. Hortic Res, 2022, 9: uhac122

[21] JiangL, ZhangD, LiuC, ShenW, HeJ, YueQ, NiuC, YangF, LiX, ShenX, et al.. MdGH3.6 is targeted by MdMYB94 and plays a negative role in apple water-deficit stress tolerance. Plant J, 2022, 109: 1271-1289

[22] KeD, XieY, LiH, HuL, HeY, GuoC, ZhaiY, GuoJ, LiK, ChuZ, et al.. Anchorene, a carotenoid-derived growth regulator, modulates auxin homeostasis by suppressing GH3-mediated auxin conjugation. J Integr Plant Biol, 2024, 66: 2490-2504

[23] KirunguJN, MagwangaRO, LuP, CaiX, ZhouZ, WangX, PengR, WangK, LiuF. Functional characterization of Gh_A08G1120 (GH3.5) gene reveal their significant role in enhancing drought and salt stress tolerance in cotton. BMC Genet, 2019, 20: 62

[24] KoochakH, Ludwig-MüllerJ. Physcomitrium patens mutants in auxin conjugating GH3 proteins show salt stress tolerance but auxin homeostasis is not involved in regulation of oxidative stress factors. Plants, 2021, 10: 1398

[25] LiR, ShiF, FukudaK. Interactive effects of various salt and alkali stresses on growth, organic solutes, and cation accumulation in a halophyte Spartina alterniflora (Poaceae). Environ Exp Bot, 2010, 68: 66-74

[26] LiX, LiM, ZhouB, YangY, ZhouJ, WeiQ, ZhangJ. Na+ efflux from apple dwarfing rootstocks is associated with high-salt resistance of their scions. J Plant Growth Regul, 2021, 40: 2139-2147

[27] LiX, ZhouS, LiuZ, LuL, DangH, LiH, ChuB, ChenP, MaZ, ZhaoS, et al.. Fine-tuning of SUMOylation modulates drought tolerance of apple. Plant Biotechnol J, 2022, 20: 903-919

[28] LiX, MaZ, SongY, ShenW, YueQ, KhanA, TahirMM, WangX, MalnoyM, MaF, et al.. Insights into the molecular mechanisms underlying responses of apple trees to abiotic stresses. Hortic Res, 2023, 10: uhad144

[29] LiuZ, BianN, GuoJ, ZhaoS, KhanA, ChuB, MaZ, NiuC, MaF, MaM, et al.. Interfering small ubiquitin modifiers (SUMO) improves the thermotolerance of apple by facilitating the activity of MdDREB2A. Stress Biol, 2023, 3: 10

[30] LiuZ, WuC, LiW, HuL, FuX, HuB, LiaoY, XiangZ, JiangH, HuangW, YangX, DuA, PuZ, TuS, MaX, LiH. OsCSLD4 confers salt–alkali tolerance by regulating gene expressions in photosynthesis and carbohydrate biosynthesis pathways, cell wall hemicellulose accumulation and physio-biochemical adaptability in rice. Plant Stress, 2024, 14: 100604

[31] LouL, YuF, TianM, LiuG, WuY, WuY, XiaR, PardoJM, GuoY, XieQ. ESCRT-I component VPS23A sustains salt tolerance by strengthening the SOS module in Arabidopsis. Mol Plant, 2020, 13: 1134-1148

[32] LuS, WangP, NaiG, LiY, SuY, LiangG, ChenB, MaoJ. Insight into VvGH3 genes evolutional relationship from monocotyledons and dicotyledons reveals that VvGH3-9 negatively regulates the drought tolerance in transgenic Arabidopsis. Plant Physiol Biochem, 2022, 172: 70-86

[33] LuoP, LiT-T, ShiW-M, MaQ, DiD-W. The Roles of GRETCHEN HAGEN3 (GH3)-Dependent Auxin Conjugation in the Regulation of Plant Development and Stress Adaptation. Plants, 2023, 12(24): 4111

[34] LuoX, DaiY, ZhengC, YangY, ChenW, WangQ, ChandrasekaranU, DuJ, LiuW, ShuK. The ABI4-RbohD/VTC2 regulatory module promotes reactive oxygen species (ROS) accumulation to decrease seed germination under salinity stress. New Phytol, 2021, 229: 950-962

[35] Ma Q, Xia Z, Cai Z, Li L, Cheng Y, Liu J, Nian H (2019) GmWRKY16 enhances drought and salt tolerance through an ABA-mediated pathway in Arabidopsis thaliana. Front Plant Sci 9. https://doi.org/10.3389/fpls.2018.01979

[36] MitaniN, MatsumotoR, YoshiokaT, KunigaT. Citrus hybrid seedlings reduce initial time to flower when grafted onto shiikuwasha rootstock. Sci Hortic, 2008, 116: 452-455

[37] NiuC, JiangL, CaoF, LiuC, GuoJ, ZhangZ, YueQ, HouN, LiuZ, LiX, et al.. Methylation of a MITE insertion in the MdRFNR1-1 promoter is positively associated with its allelic expression in apple in response to drought stress. Plant Cell, 2022, 34: 3983-4006

[38] Padilla YG, Gisbert-Mullor R, López-Galarza S, Calatayud Á, Padilla YG, Gisbert-Mullor R, López-Galarza S, Calatayud Á (2024) Grafting in pepper to overcome drought, salinity, and high temperature. In: Abiotic stress in crop plants - ecophysiological responses and molecular approaches. IntechOpen. https://doi.org/10.5772/intechopen.114359

[39] QinH, WangJ, ChenX, WangF, PengP, ZhouY, MiaoY, ZhangY, GaoY, QiY, et al.. Rice OsDOF15 contributes to ethylene-inhibited primary root elongation under salt stress. New Phytol, 2019, 223: 798-813

[40] RigaP. Effect of rootstock on growth, fruit production and quality of tomato plants grown under low temperature and light conditions. Hortic Environ Biotechnol, 2015, 56: 626-638

[41] ShehataSA, OmarHS, ElfaidyAGS, EL-SayedSSF, AbuarabME, AbdeldaymEA. Grafting enhances drought tolerance by regulating stress-responsive gene expression and antioxidant enzyme activities in cucumbers. BMC Plant Biol, 2022, 22: 408

[42] Shi C, Yang F, Liu Z, Li Y, Di X, Wang J, Lin J (2021) Uniform water potential induced by salt, alkali, and drought stresses has different impacts on the seedling of Hordeum jubatum: from growth, photosynthesis, and chlorophyll fluorescence. Front Plant Sci 12. https://doi.org/10.3389/fpls.2021.733236

[43] StaswickPE, SerbanB, RoweM, TiryakiI, MaldonadoMT, MaldonadoMC, SuzaW. Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. Plant Cell, 2005, 17: 616-627

[44] SteinhorstL, HeG, MooreLK, SchültkeS, Schmitz-ThomI, CaoY, HashimotoK, AndrésZ, PiepenburgK, RagelP, et al.. A Ca2+-sensor switch for tolerance to elevated salt stress in Arabidopsis. Dev Cell, 2022, 57: 2081-2094.e7

[45] SunX, JiaX, HuoL, CheR, GongX, WangP, MaF. MdATG18a overexpression improves tolerance to nitrogen deficiency and regulates anthocyanin accumulation through increased autophagy in transgenic apple. Plant Cell Environ, 2018, 41: 469-480

[46] Sun Y, Liang W, Cheng H, Wang H, Lv D, Wang W, Liang M, Miao C (2021) NADPH Oxidase-derived ROS promote mitochondrial alkalization under salt stress in Arabidopsis root cells. Plant Signal Behav 16. https://doi.org/10.1080/15592324.2020.1856546

[47] Tabakov SG, Yordanov AI, Kaymakanov PV (2016) Comparative study of apple rootstocks ‘M9-T337’ and ‘Supporter 4 Pi 80’ with seven cultivars. Acta Hortic. https://doi.org/10.17660/actahortic.2016.1139.35

[48] TsaiYC, ChenKC, ChengTS, LeeC, LinSH, TungCW. Chlorophyll fluorescence analysis in diverse rice varieties reveals the positive correlation between the seedlings salt tolerance and photosynthetic efficiency. BMC Plant Biol, 2019, 19: 403

[49] TuranS, TripathyBC. Salt-stress induced modulation of chlorophyll biosynthesis during de-etiolation of rice seedlings. Physiol Plant, 2015, 153: 477-491

[50] WangX, GengS, MaY, ShiD, YangC, WangH. Growth, photosynthesis, solute accumulation, and ion balance of tomato plant under sodium- or potassium-salt stress and alkali stress. Agron J, 2015, 107: 651-661

[51] WangY, LiW, XuX, QiuC, WuT, WeiQ, MaF, HanZ. Progress of apple rootstock breeding and its use. Hortic Plant J, 2019, 5: 183-191

[52] WangG, ZengF, SongP, SunB, WangQ, WangJ. Effects of reduced chlorophyll content on photosystem functions and photosynthetic electron transport rate in rice leaves. J Plant Physiol, 2022, 272: 153669

[53] Wang J, Ding C, Cui C, Song J, Ji G, Sun N, Qi yue S, Li J, Xu ru Z, Zhang H (2024) Physiological and molecular responses of poplar to salt stress and salt tolerance functional analysis of Paggrxc9. https://doi.org/10.2139/ssrn.4875917

[54] WangL, ZhangC, WangY, WangY, YangC, LuM, WangC. Tamarix hispida aquaporin ThPIP2;5 confers salt and osmotic stress tolerance to transgenic Tamarix and Arabidopsis. Environ Exp Bot, 2018, 152: 158-166

[55] WangQ, WuC, XieB, LiuY, CuiJ, ChenG, ZhangY. Model analysing the antioxidant responses of leaves and roots of switchgrass to NaCl-salinity stress. Plant Physiol Biochem, 2012, 58: 288-296

[56] WebsterAD. Rootstock and interstock effects on deciduous fruit tree vigour, precocity, and yield productivity. N Z J Crop Hortic Sci, 1995, 23: 373-382

[57] XianX, ZhangZ, WangS, ChengJ, GaoY, MaN, LiC, WangY. Exogenous melatonin strengthens saline-alkali stress tolerance in apple rootstock M9–T337 seedlings by initiating a variety of physiological and biochemical pathways. Chem Biol Technol Agric, 2024, 11: 58

[58] XieY, ZhuY, WangN, LuoM, OtaT, GuoR, TakahashiI, YuZ, AizeziY, ZhangL, et al.. Chemical genetic screening identifies nalacin as an inhibitor of GH3 amido synthetase for auxin conjugation. Proc Natl Acad Sci, 2022, 119: e2209256119

[59] YaoP, ZhangC, QinT, LiuY, LiuZ, XieX, BaiJ, SunC, BiZ. Comprehensive analysis of GH3 gene family in potato and functional characterization of StGH3.3 under drought stress. Int J Mol Sci, 2023, 24: 15122

[60] ZhangZ, CaoB, GaoS, XuK. Grafting improves tomato drought tolerance through enhancing photosynthetic capacity and reducing ROS accumulation. Protoplasma, 2019, 256: 1013-1024

[61] ZhangD, HeJ, ChengP, ZhangY, KhanA, WangS, LiZ, ZhaoS, ZhanX, MaF, et al.. 4-methylumbelliferone (4-MU) enhances drought tolerance of apple by regulating rhizosphere microbial diversity and root architecture. Hortic Res, 2023, 10: uhad099

[62] ZhangH, YuF, XieP, SunS, QiaoX, TangS, ChenC, YangS, MeiC, YangD, et al.. A Gγ protein regulates alkaline sensitivity in crops. Science, 2023, 379: eade8416

[63] Zhao K, Cheng Z, Guo Q, Yao W, Liu H, Zhou B, Jiang T (2020) Characterization of the poplar R2R3-MYB gene family and over-expression of PsnMYB108 confers salt tolerance in transgenic tobacco. Front Plant Sci 11. https://doi.org/10.3389/fpls.2020.571881

[64] ZhouY, HuangL, LiuS, ZhaoM, LiuJ, LinL, LiuK. Physiological and transcriptomic analysis of IAA-induced antioxidant defense and cell wall metabolism in postharvest mango fruit. Food Res Int, 2023, 174: 113504

[65] ZhuY, FazioG, MazzolaM. Elucidating the molecular responses of apple rootstock resistant to ARD pathogens: challenges and opportunities for development of genomics-assisted breeding tools. Horticulture Research, 2014, 1: 14043

Funding

National Natural Science Foundation of China(32202459)

Chinese Universities Scientific Fund(2452023067)

the Key S&T Special Projects of Xinjiang, China(2023B02018-2)

Xinjiang Apple Research System of China(XJLGCYJSTX04)

RIGHTS & PERMISSIONS

The Author(s)