FUNCTIONAL DIVERSIFICATION OF MEDICAGO TRUNCATULA PIP AQUAPORIN SUBFAMILY

VITALI, VICTORIA; JOZEFKOWICZ, CINTIA; CANESSA FORTUNA, AGUSTINA; SCOCHERA, FLORENCIA; FRARE, ROMINA; BIENERT GERD PATRICK; AYUB, NICOLÁS; SOTO, GABRIELA; ALLEVA, KARINA

Congreso; LVI Congreso de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2020

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

Water movement across plant plasma membrane (PM) depends on the activity of PIP (plasma membrane intrinsic protein)aquaporins. About 10 to 30 PIP genes are encoded by seed plant species divided in two groups of paralogs (PIP1 and PIP2).Regulatory mechanisms such as heterotetramerization between paralogous, interaction with endoplasmic reticulum proteins,phosphorylation and pH/calcium triggered gating, have been well studied in this subfamily. However, it has not yet beenresolved whether the high number of PIPs per plant is due to functional overlapping or functional divergence. In order toaddress this question, we performed a comprehensive analysis of the PIP subfamily in Medicago truncatula by combiningcoding sequences analysis, survey of tissue expression patterns, characterization of the biological activity and structural studies. Medicago truncatula is a legume plant model with 10 PIP coded in its genome whose full MtPIP functionalcharacterization has not yet been addressed. By analysing different developmental stages and plant tissues (publicly availablemicroarrays), we found that M. truncatula PIP channels are ubiquitously expressed. However, the transcriptional expressionprofile of MtPIP2;2 and MtPIP2;3 stood out for their diverse response and high identity (> 85%). Interestingly, RNAseq resultsshow that these two PIP genes are present in a variety of tissues, in particular in mature nodules, where two PIP1 (MtPIP1;1and MtPIP1;4) are also expresse d. These four PIPs show a highly correlated response when M. truncatula grows under differentnitrogen sources or drought conditions, suggesting that the individual isoforms or after heterotetramerization they may becrucial for their role in the plants? response reaction to those physiological drivers. Functional studies carried out inheterologous systems show that both MtPIP2: i- localize in the PM, while the two MtPIP1 only locate in PM upon coexpression with any of the MtPIP2, ii- transport H2O and H2O2 but not ammonia, urea or boric acid, iii- are inhibited by cytosolic acidification showing the same pH0,5 but different cooperative response, iv- do not present the typical increase in water transport when coexpressed with MtPIP1 (previously reported in other plant species). Through structural studies, we found thatMtPIP2;2 and MtPIP2;3 differ in the extracellular electrostatic topologies and the structural-communication signatures, withthe main primary sequence differences concentrated in the N-termini, and the extracellular loops A, C and E. These findingssuggest a functional divergence of two MtPIP2 focused in the cooperative response and the formation of hetero-oligomerswhere minimal changes in the extracellular loops should be involved. Future research will encompass a wider spectrum offunctions in all PIP family members to unravel the spectrum of their biological roles and evolutionary process that give accountfor the conservation of multiple isoforms.