Transcriptional changes of aquaporin genes in leaves of black medic induced by arbuscular mycorrhizal fungal inoculation under water deficit

One of the current research directions in plant-microbe interactions focuses on the mechanisms of plant adaptation to environmental stress through symbioses with various microorganisms. While the role of arbuscular mycorrhizal fungi in plant adaptation to drought is well-known, the underlying mechanisms of these processes remain poorly understood, particularly in leaf tissues. It is suggested that certain genes from the aquaporin family play a critical role both in adaptation to water deficit and in the development of an effective arbuscular mycorrhizal symbiosis. Thus, the important task in this study of plant-microbe symbioses is to assess the effect of arbuscular mycorrhizal fungal inoculation on the expression of aquaporin genes in leaves. This study utilizes the highly effective plant-microbe model system “Medicago lupulina + Rhizophagus irregularis” under drought stress conditions. A comparative assessment of gene transcription was carried out using the 2–∆∆CT method based on real-time quantitative PCR results: normalization was performed relative to the actin reference gene with non-inoculated plants serving as the control. The study was conducted both at the initial development stage (the 2nd leaf stage), and at the stage of active plant-microbe interaction (the flowering stage). The study revealed genes with significant differential expression under drought conditions when comparing mycorrhizal and non-mycorrhizal Medicago lupulina plants: NIP3;1, NIP4;2, specific NIP7;1, TIP5;1 at the 2nd leaf stage; genes NIP3;1, NIP5;1, NIP6;4, NIP7;1 (specific), PIP1;4, TIP2;3 and specific XIP1;1 at the flowering stage. Previously, in a similar experiment, under well-watering conditions, the same genes did not have differential expression between mycorrhizal and non-mycorrhizal plants. Thus, the listed genes likely participate in the adaptation of the studied plants to drought conditions. The obtained information can be used to develop highly productive plant-microbe systems involving arbuscular mycorrhizal fungi, aimed at transitioning to organic farming, minimizing negative environmental impact, and enhancing plant resistance to water deficit.

1. Asadollahi M., Iranbakhsh A., Ahmadvand R., Ebadi M., Mehregan I. Synergetic effect of water deficit and arbuscular mycorrhizal symbiosis on the expression of aquaporins in wheat (Triticum aestivum L.) roots: insights from NGS RNA-sequencing. Physiol Mol Biol Plants. 2023;29(2):195-208. doi 10.1007/s12298-023-01285-w

2. Bárzana G., Aroca R., Paz J.A., Chaumont F., Martinez-Ballesta M.C., Carvajal M., Ruiz-Lozano J.M. Arbuscular mycorrhizal symbiosis increases relative apoplastic water flow in roots of the host plant under both well-watered and drought stress conditions. Ann Bot. 2012;109(5):1009-1017. doi 10.1093/aob/mcs007

3. Bárzana G., Aroca R., Bienert G.P., Chaumont F., Ruiz-Lozano J.M. New insights into the regulation of aquaporins by the arbuscular mycorrhizal symbiosis in maize plants under drought stress and possible implications for plant performance. Mol Plant Microbe Interact. 2014;27(4):349-363. doi 10.1094/MPMI-09-13-0268-R

4. Daneliia G.V., Yemelyanov V.V., Shishova M.F. The role of watertransporting aquaporins of the PIP and TIP subfamilies in plant development and adaptation to stress factors. Ecological Genetics. 2024;22(4):343-368. doi 10.17816/ecogen637037

5. Dean R.M., Rivers R.L., Zeidel M.L., Roberts D.M. Purification and functional reconstitution of soybean nodulin 26. An aquaporin with water and glycerol transport properties. Biochemistry. 1999;38(1): 347-353. doi 10.1021/bi982110c

6. He F., Zhang H., Tang M. Aquaporin gene expression and physiological responses of Robinia pseudoacacia L. to the mycorrhizal fungus Rhizophagus irregularis and drought stress. Mycorrhiza. 2016; 26(4):311-323. doi 10.1007/s00572-015-0670-3

7. He J.-D., Dong T., Wu H.-H., Zou Y.-N., Wu Q.-S., Kuča K. Mycorrhizas induce diverse responses of root TIP aquaporin gene expression to drought stress in trifoliate orange. Sci Hortic. 2019;243:64-69. doi 10.1016/j.scienta.2018.08.010

8. Heymann J.B., Engel A. Aquaporins: phylogeny, structure, and physiology of water channels. News Physiol Sci. 1999;14:187-193. doi 10.1152/physiologyonline.1999.14.5.187

9. Hussain A., Tanveer R., Mustafa G., Farooq M., Amin I., Mansoor S. Comparative phylogenetic analysis of aquaporins provides insight into the gene family expansion and evolution in plants and their role in drought tolerant and susceptible chickpea cultivars. Genomics. 2020;112(1):263-275. doi 10.1016/j.ygeno.2019.02.005

10. Jia Y., Liu X. Polyploidization and pseudogenization in allotetraploid frog Xenopus laevis promote the evolution of aquaporin family in higher vertebrates. BMC Genomics. 2020;21(1):525. doi 10.1186/s12864-020-06942-y

11. Kryukov A.A., Gorbunova A.O., Kudriashova T.R., Ivanchenko O.B., Shishova M.F., Yurkov A.P. SWEET transporters of Medicago lupulina in the arbuscular-mycorrhizal system in the presence of medium level of available phosphorus. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov J Genet Breed. 2023;27(3):189-196. doi 10.18699/VJGB-23-25

12. Kryukov A.A., Kudriashova T.R., Belyaeva A.I., Gorenkova A.I., Yurkov A.P. Effect of Rhizophagus irregularis inoculation on aquaporin gene expression in the roots of Medicago lupulina in drought conditions. Ecological Genetics. 2025;23(3):263-275. doi 10.17816/ecogen643544 (in Russian)

13. Kruse E., Uehlein N., Kaldenhoff R. The aquaporins. Genome Biol. 2006;7(2):206. doi 10.1186/gb-2006-7-2-206

14. Kudoyarova G., Veselov D., Yemelyanov V., Shishova M. The role of aquaporins in plant growth under conditions of oxygen deficiency. Int J Mol Sci. 2022;23(17):10159. doi 10.3390/ijms231710159

15. Kudryashova T.R., Kryukov A.A., Gorbunova A.O., Ivanchenko O.B., YurkovA.P. SWEET genes of Medicago lupulina during the development of symbiosis with arbuscular mycorrhiza under conditions of low phosphorus levels. Butlerov Communications. 2024;78(5):119- 127. doi 10.37952/ROIjbc-01/24-78-5-119/ROI-jbc-C/24-7-2-8 (in Russian)

16. Kudriashova T.R., Kryukov A.A., Gorenkova A.I., Yurkov A.P. Aquaporins and their role in plant-microbial systems. Vavilov Journal of Genetics and Breeding = Vavilov J Genet Breed. 2025;29(2):238- 247. doi 10.18699/vjgb-25-27

17. Lopez D., Amira M.B., Brown D., Muries B., Brunel-Michac N., Bourgerie S., Porcheron B., … Fumanal B., Label P., Pujade-Renaud V., Auguin D., Venisse J.-S. The Hevea brasiliensis XIP aquaporin subfamily: genomic, structural and functional characterizations with relevance to intensive latex harvesting. Plant Mol Biol. 2016; 91(4-5):375-396. doi 10.1007/s11103-016-0462-y

18. Luo Y., Ma L., Du W., Yan S., Wang Z., Pang Y. Identification and characterization of salt- and drought-responsive AQP family genes in Medicago sativa L. Int J Mol Sci. 2022;23(6):3342. doi 10.3390/ijms23063342

19. Ma J.F., Tamai K., Yamaji N., Mitani N., Konishi S., Katsuhara M., Ishiguro M., Murata Y., Yano M. A silicon transporter in rice. Nature. 2006;440(7084):688-691. doi 10.1038/nature04590

20. MacRae E. Extraction of plant RNA. In: Hilario E., Mackay J. (Eds) Protocols for Nucleic Acid Analysis by Nonradioactive Probes. Methods in Molecular Biology. Vol. 353. Humana Press, 2007; 15-24. doi 10.1385/1-59745-229-7:15

21. Mammadov J., Buyyarapu R., Guttikonda S., Parliament K., Abdurakhmonov I.Y., Kumpatla S.P. Wild relatives of maize, rice, cotton, and soybean: treasure troves for tolerance to biotic and abiotic stresses. Front Plant Sci. 2018;9:886. doi 10.3389/fpls.2018.00886

22. Maurel C., Verdoucq L., Luu D.T., Santoni V. Plant aquaporins: membrane channels with multiple integrated functions. Annu Rev Plant Biol. 2008;59(1):595-624. doi 10.1146/annurev.arplant.59.032607.092734

23. Maurel C., Boursiac Y., Luu D.T., Santoni V., Shahzad Z., Verdoucq L. Aquaporins in plants. Physiol Rev. 2015;95(4):1321-1358. doi 10.1152/physrev.00008.2015

24. Min X., Wu H., Zhang Z., Wei X., Jin X., Ndayambaza B., Wang Y., Liu W. Genome-wide identification and characterization of the aquaporin gene family in Medicago truncatula. J Plant Biochem Biotechnol. 2019;28(3):320-335. doi 10.1007/s13562-018-0484-4

25. Mizutani M., Watanabe S., Nakagawa T., Maeshima M. Aquaporin NIP2;1 is mainly localized to the ER membrane and shows rootspecific accumulation in Arabidopsis thaliana. Plant Cell Physiol. 2006;47(10):1420-1426. doi 10.1093/pcp/pcl004

26. Murata K., Mitsuoka K., Hirai T., Walz T., Agre P., Heymann J.B., Engel A., Fujiyoshi Y. Structural determinants of water permeation through aquaporin-1. Nature. 2000;407(6804):599-605. doi 10.1038/35036519

27. Noronha H., Araújo D., Conde C., Martins A.P., Soveral G., Chaumont F., Delrot S., Gerós H. The grapevine uncharacterized intrinsic protein 1 (VvXIP1) is regulated by drought stress and transports glycerol, hydrogen peroxide, heavy metals but not water. PLoS One. 2016;11(8):e0160976. doi 10.1371/journal.pone.0160976

28. Phillips J.M., Hayman D.S. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br Mycol Soc. 1970;55(1):158- 161. doi 10.1016/S0007-1536(70)80110-3

29. Porcel R., Gómez M., Kaldenhoff R., Ruiz-Lozano J.M. Impairment of NtAQP1 gene expression in tobacco plants does not affect root colonization pattern by arbuscular mycorrhizal fungi but decreases their symbiotic efficiency under drought. Mycorrhiza. 2005;15:417-423. doi 10.1007/s00572-005-0346-5

30. Porcel R., Aroca R., Azcon R., Ruiz-Lozano J.M. PIP aquaporin gene expression in arbuscular mycorrhizal Glycine max and Lactuca sativa plants in relation to drought stress tolerance. Plant Mol Biol. 2006;60:389-404. doi 10.1007/s11103-005-4210-y

31. Quiroga G., Erice G., Aroca R., Chaumont F., Ruiz-Lozano J.M. Contribution of the arbuscular mycorrhizal symbiosis to the regulation of radial root water transport in maize plants under water deficit. Environ Exp Bot. 2019;167:103821. doi 10.1016/j.envexpbot.2019.103821

32. Remy W., Taylor T.N., Hass H., Kerp H. Four hundred-million-year-old vesicular arbuscular mycorrhizae. Proc Natl Acad Sci USA. 1994; 91(25):11841-11843. doi 10.1073/pnas.91.25.11841

33. Ruiz-Lozano J.M., Aroca R. Host response to osmotic stresses: stomatal behaviour and water use efficiency of arbuscular mycorrhizal plants. In: Koltai H., Kapulnik Y. (Eds) Arbuscular Mycorrhizas: Physiology and Function. Dordrecht: Springer, 2010;239-256. doi 10.1007/978-90-481-9489-6_11

34. Sandal N.N., Marcker K.A. Soybean nodulin 26 is homologous to the major intrinsic protein of the bovine lens fiber membrane. Nucleic Acids Res. 1988;16:9347-9348. doi 10.1093/nar/16.19.9347

35. Sharma K., Gupta S., Thokchom S.D., Jangir P., Kapoor R. Arbuscular mycorrhiza-mediated regulation of polyamines and aquaporins during abiotic stress: deep insights on the recondite players. Front Plant Sci. 2021;12:642101. doi 10.3389/fpls.2021.642101

36. Singh S., Bhatt V., Kumar V., Kumawat S., Khatri P., Singla P., Shivaraj S.M., Nadaf A., Deshmukh R., Sharma T.R., Sonah H. Evolutionary understanding of aquaporin transport system in the basal eudicot model species Aquilegia coerulea. Plants. 2020;9(6):799. doi 10.3390/plants9060799

37. Smith S.E., Read D.J. (Eds) Mycorrhizal Symbiosis. San Diego, CA: Academic Press, 2008. doi 10.1016/B978-0-12-370526-6.X5001-6

38. Trouvelot A., Kough J.L., Gianinazzi S. Mesure du taux de mycorhization VA d’un système radiculaire. Recherche de méthodes ayant une signification fonctionnelle. In: Gianinazzi-Pearson V., Gianinazzi S. (Eds) Physiological and Genetical Aspects of Mycorrhizae. Paris: INRA, 1986;217-221

39. Uehlein N., Lovisolo C., Siefritz F., Kaldenhoff R. The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiological functions. Nature. 2003;425(6959):734-737. doi 10.1038/nature02027

40. Vorobyev N.I., Yurkov A.P., Provorov N.A. Certificate N2010612112 about the Registration of the Computer Program “Program for Calculating the Mycorrhization Indices of Plant Roots” (Dated December 2, 2016). Moscow: The Federal Service for Intellectual Property, 2016 (in Russian)

41. Wang D., Ni Y., Xie K., Li Y., Wu W., Shan H., Cheng B., Li X. Aquaporin ZmTIP2;3 promotes drought resistance of maize through symbiosis with arbuscular mycorrhizal fungi. Int J Mol Sci. 2024;25(8): 4205. doi 10.3390/ijms25084205

42. YurkovA.P., Jacobi L.M., Gapeeva N.E., Stepanova G.V., Shishova M.F. Development of arbuscular mycorrhiza in highly responsive and mycotrophic host plant – black medick (Medicago lupulina L.). Russ J Dev Biol. 2015;46(5):263-275. doi 10.1134/S1062360415050082

43. Yurkov A., Kryukov A., Gorbunova A., Sherbakov A., Dobryakova K., Mikhaylova Y., Afonin A., Shishova M. AM-induced alteration in the expression of genes, encoding phosphorus transporters and enzymes of carbohydrate metabolism in Medicago lupulina. Plants. 2020; 9(4):486. doi 10.3390/plants9040486

44. Yurkov A., Puzanskiy R., Avdeeva G., Jacobi L., Gorbunova A., Kryukov A., Kozhemyakov A., … Yemelyanov V., Shavarda A., Kirpichnikova A., Smolikova G., Shishova M. Mycorrhiza-induced alterations in metabolome of Medicago lupulina leaves during symbiosis development. Plants. 2021;10(11):2506. doi 10.3390/plants10112506

45. Yurkov A.P., Afonin A.M., Kryukov A.A., Gorbunova A.O., Kudryashova T.R., Kovalchuk A.I., Gorenkova A.I., … Zhukov V.A., Puzanskiy R.K., Mikhailova Y.V., Yemelyanov V.V., Shishova M.F. The effects of Rhizophagus irregularis inoculation on transcriptome of Medicago lupulina leaves at early vegetative and flowering stages of plant development. Plants. 2023;12:3580. doi 10.3390/plants12203580

46. Zhou X., Yi D., Ma L., Wang X. Genome-wide analysis and expression of the aquaporin gene family in Avena sativa L. Front Plant Sci. 2024;14:1305299. doi 10.3389/fpls.2023.1305299

47. Zou Y.N., Wu H.H., Giri B., Wu Q.S., Kuča K. Mycorrhizal symbiosis down-regulates or does not change root aquaporin expression in trifoliate orange under drought stress. Plant Physiol Biochem. 2019; 144:292-299. doi 10.1016/j.plaphy.2019