Deciphering the contribution of maize aquaporins regulated by arbuscular mycorrhizae to the transport in planta of water and/or solutes of physiological importance under drought

Resumen

Drought stress is one of the major abiotic factors affecting normal growth and development of plants from both natural and agroecosystems. Climate change is expected to intensify the periods of drought as well as to involve areas that were not threatened by this phenomenon in the past, which will consequently affect crop production and food security. The effect of drought in Mediterranean regions, which largely depend on agriculture, will have important social and economic consequences. The natural-occurring symbiosis between arbuscular mycorrhizal fungi and roots of approximately 80% of land plants, including numerous crops, is able to enhance the uptake of water and nutrients in the soil, thanks to an extended hyphal network that allows the uptake of nutrients out of the root depletion zone. The benefits of the AM symbiosis also include the protection of the plants against a range of abiotic and biotic stresses. In fact, the association is well known for conferring drought stress tolerance in different plant species, including maize. The fungi colonize root cortex cells, forming the arbuscules, which are the exchange structures between the two partners. During this process, the plasma membranes of these cells suffer extensive morphological alterations to surround the arbuscules. Among these modifications, changes in location or abundance of membrane proteins are commonly produced. Aquaporins (AQPs) are integral membrane proteins belonging to the major intrinsic protein (MIP) superfamily. These channels, present in all living organisms, facilitate the passive flux of water and a range of small solutes across cell membranes. AQPs have been mainly studied in relation to the hydraulic properties of plants. Nevertheless, the capacity of transporting different solutes has opened the possibility of their involvement in other physiological processes. In fact, AQPs participate in the symbiotic exchange at the plant-fungus interface, and several genes were modulated by the arbuscular mycorrhizal symbiosis under drought stress conditions. In maize, AM symbiosis has been shown to regulate mRNA abundance of a high number of aquaporins, including members of the different subfamilies. Additionally it was demonstrated that they can transport water as well as other solutes of physiological importance (such as glycerol, ammonia, urea, boron, silicon or hydrogen peroxide) under normal and drought stress conditions. Previous to this work, it was also shown that the AM symbiosis can modulate the switch between water transport pathways in the root of the host plant. This is understood as a way to provide higher flexibility in the response of AM plants to water deficit, according to the demands of the aerial part. The present PhD thesis is mainly focused on the identification of AM-regulated maize aquaporin isoforms key for drought tolerance, and the identification of their specific functions in planta. Moreover, it is a goal of this study to understand if these aquaporins have a key influence on root water transport capacity of the host plant and if they contribute to the higher flexibility of AM roots for switching between cell-to-cell and apoplastic water transport pathways. With this aim, the combination between Zea mays L. and Rhizophagus irregularis was used as a model in all the experiments carried out in this PhD thesis. As a first approach to understand the differential regulation of maize aquaporins by the AM symbiosis, two maize cultivars with contrasting drought sensitivity were compared under normal and drought stress conditions: PR34G13 (drought-tolerant) and PR34B39 (drought-sensitive). Results showed that the AM symbiosis improved physiological parameters to a higher extent in the drought-sensitive cultivar. This effect was reflected in the higher membrane stability, efficiency of photosystem II, accumulation of soluble sugars and plant biomass production. The benefits of the AM inoculation were also related to a higher and broader regulation of root aquaporins in the drought-sensitive cultivar. From this initial study, eight maize aquaporins were selected for being regulated by the AM symbiosis or for being putative transporters of solutes with relevance in drought stress tolerance. These aquaporins were analyzed in the subsequent experiments. This study is presented in the first chapter of this PhD thesis. Subsequently, the second chapter had the objective of elucidating if the key effect of the regulation of maize aquaporins by the AM symbiosis was the enhancement of root cell water transport capacity. With this aim, pressure probe and protoplast swelling assays were performed using intact cortical cells and root cell protoplasts, respectively from AM and non-AM plants subjected or not to drought stress. The obtained results showed that cells from droughted-AM roots maintained cell hydraulic conductivity (Lpc) and water permeability coefficient (Pf) values of non-stressed plants, whereas in non-AM plants these values declined drastically as a consequence of water deficit. Under these conditions, phosphorylation status of plant PIP2 aquaporins was increased by the symbiosis, which may be related to a higher activity of their water channels. Additionally, AM symbiosis also enhanced photosynthetic capacity thanks to an increased PEPc activity and CO2-saturated photosynthetic rate. In summary, this chapter demonstrated a better performance of AM root cells in water transport under water deficit, which is connected to a better performance of the shoot in terms of photosynthetic capacity. The third chapter of this PhD thesis intended to elucidate the possible involvement of the AM-regulated aquaporins in the in planta transport of boron (B) under well-watered or drought stress conditions. With this objective, different B concentrations were applied in the nutrient solution to both non-AM and AM plants that were submitted or not to a water deficit treatment. It was shown that aquaporins and B efflux transports were generally down-regulated in AM plants, suggesting that other mechanisms contribute to B homeostasis in these plants, probably more related to the enhancement of water transport which would concomitantly increase the passive transport of this micronutrient. In this study, different aquaporins (ZmPIP2;2, ZmTIP2;3 and ZmNIP1;1) and B efflux transporters (RTE, RTE2 and RTE3) were transcriptionally regulated by B levels in planta, which confirms their previously proposed role in B transport. Results showed that the AM symbiosis improved plant physiology and performance in absence of N fertilization or under low urea or ammonium fertilization, regardless of the watering regime. In contrast, under high N supply, no effect of the AM symbiosis was observed. Similarly to the third chapter, in chapter four it was evaluated the possible involvement of the AM-regulated aquaporins in the in planta transport of ammonium or urea as a mechanism contributing to drought stress tolerance by the AM symbiosis. Thus, AM and non-AM maize plants were cultivated under different ammonium or urea levels in the growing substrate. The up-regulation of ZmTIP1;1 mRNA levels with both N forms suggested that this protein could be transporting both urea and ammonium in planta. Moreover, the differential regulation of ZmTIP4;1 and ZmPIP2;4 hinted at the possibility of a role of these two aquaporins in N mobilization in planta. These aquaporin genes were also differentially regulated by the AM symbiosis, suggesting a possible role in the AM-mediated plant N homeostasis that deserves future studies. In the fifth and sixth chapters is presented the research work corresponding to the elucidation of the fourth specific objective of this PhD thesis: Deciphering if the higher flexibility of AM plants to switch between water transport pathways is due to aquaporin regulation mediated by salicylic acid (SA) orindol-3-acetic-acid. In chapter five, exogenous SA was applied to non-AM and AM plants subjected or not to drought stress treatment. Additionally, an inhibitor of SA biosynthesis (2-aminoindan-2-phosphonic acid, AIP) was also applied to half of the plants. It was demonstrated that exogenous SA application altered root hydraulic parameters decreasing root hydraulic conductivity (Lpr) and osmotic root hydraulic conductivity (Lo) under drought stress conditions. This effect could be related to the regulation of root aquaporins (as ZmPIP2;4 and ZmTIP1;1), whose protein levels correlated with Lo under water deficit. Furthermore, SA differently modulated the percentage of water flowing by the apoplastic pathway, decreasing its contribution to total root water flow in AM plants and increasing it in non-AM plants. In chapter six, IAA was applied, following the same experimental approach than with SA. Here, it was revealed that IAA affected root hydraulic parameters (mainly Lo) during water stress conditions, similarly to SA, which was decreased in both non-AM and AM plants. The regulation of the internal cell component of root water conductivity (Lo) suggested that aquaporins are involved in the IAA-dependent inhibition of this internal cell pathway. Interestingly, similarly to SA application, IAA regulated differently apoplastic water flow in AM and non-AM plants under water deficit, which confirms the previous hypothesis. In both experiments, exogenous application of the hormone altered endogenous levels of other phytohormones (such as ABA, SA, JA or JA-Ile), revealing the complex network that regulates water transport in roots. The study described in chapter seven intended to understand if the AM symbiosis alters radial root water transport in the host plant and whether this modification is due to alteration of plant aquaporins activity or amounts and/or changes in apoplastic barriers. For that we measured osmotic (Lo) and hydrostatic (Lpr) root hydraulic conductivities and we used sodium azide (NaN3) as inhibitor of aquaporins activity and of cell-to-cell water transport. Additionally, the study constitutes a first approach to elucidate the role of the AM fungus on the modification of apoplastic barriers. Once more, it was confirmed that the AM fungus modifies water transport in roots, increasing all hydraulic parameters compared to non-AM plants. NaN3 inhibition of Lo was lower in AM plants than in non-AM plants. The former plants also had higher relative apoplastic water flow values, suggesting a compensatory mechanism for aquaporin activity inhibition in these plants and leading to higher Lpr values as compared to non-AM plants. The lower inhibition of Lo in AM plants seems to be related to the regulation of aquaporins activity through posttranslational mechanisms. Casparian bands increased with drought but also in AM plants, although this did not decrease water flow values in these plants. There is the possibility that apoplastic barriers of AM roots have a different composition, which could explain the different water transport of these roots. In summary, the study conducted in this PhD Thesis increases the general knowledge about the plant drought tolerance induced by the AM symbiosis. It is evidenced that the AM symbiosis has a role in the modulation of cell water conductivity in roots, which is probably related to aquaporins activity. Moreover, the higher flexibility of AM roots to modulate water transport is confirmed in independent experiments, which is translated into the better performance of these plants under water scarcity.