Plasma Membrane Intrinsic Proteins Research Articles

Aquaporins: Multifunctional Players in Plant Growth, Development and Stress Responses

Aquaporins are small membrane proteins belonging to the group of Major Intrinsic proteins (MIPs) that facilitate the transport of water and small molecules across biological membranes. They were initially identified as water channels and later it has become clear that they also play important roles in several other physiological processes in plants. Plants have different types of aquaporins in terms of their location, gating properties, and solute specificity and can mainly divide into four major subfamilies. They are tonoplast intrinsic proteins (TIPs), nodulin26-like intrinsic proteins (NIPs), plasma membrane intrinsic proteins (PIPs), and the small basic intrinsic proteins (SIPs). All these aquaporins play a variety of physiological activities at all stages of plant growth and development. Also, they are believed to play a significant part in the plant’s defense mechanisms against biotic and abiotic stresses. This review comprehensively addresses the roles of diverse aquaporin homologs in growth and development and their response to different environmental factors.

Improved Waterlogging Tolerance in Roots of Cucumber Plants after Inoculation with Arbuscular Mycorrhizal Fungi

Mycorrhizal symbiosis enhances host plant resistance to various unfavorable environmental stresses, but whether and how it also enhances waterlogging tolerance in cucumber plants is not known. The objective of this study was to analyze the effect of Paraglomus occultum inoculation on biomass production, osmolyte levels, and the expression of 12 heat shock protein 70 (Hsp70) genes and 14 plasma membrane intrinsic protein (PIP) genes in the roots of cucumber plants under a short-term waterlogging stress (WS) (5 days) condition. Although the short-term WS treatment significantly inhibited the arbuscular mycorrhizal fungal colonization of roots, the inoculation with arbuscular mycorrhizal fungi (AMFs) significantly increased leaf, stem, and root biomass under WS. AMF inoculation also significantly increased root glucose, sucrose, betaine, and proline contents, along with decreased fructose levels, compared with the uninoculated control. More CsHsp70 and CsPIP genes were up-regulated in AMF-inoculated plants than in AMF-uninoculated plants in response to WS. AMF inoculation showed no significant effect on the expression of any of the examined CsHsp70 genes under no-waterlogging stress, but it did raise the expression of 11 of 12 CsHsp70 genes under WS. AMF colonization also down-regulated or had no effect on CsPIP expression under no-waterlogging stress, whereas it up-regulated the expression of 12 of the 14 CsPIP genes under WS. It is concluded that AMF inoculation enhances waterlogging tolerance in cucumber plants by increasing osmolyte levels and stress-responsive gene (CsPIP and CsHsp70) expression.

Plasma membrane intrinsic proteins SlPIP2;5 gene regulates tolerance to high VPD in tomato

Plasma membrane intrinsic proteins (PIPs) play important roles in plant growth, development, and abiotic stress responses. However, the functions and regulatory mechanisms of PIPs under high vapor pressure deficit (VPD) stress are not yet well understood. In this study, we used tobacco rattle virus (TRV) - mediated virus-induced gene silencing (VIGS) technology to discover that SlPIP2;5 is involved in the acclimation process of tomato plants to high VPD. Under high VPD, the TRV-SlPIP2;5 strain exhibited improved growth compared to the TRV strain, and silencing SlPIP2;5 increased the enzyme activities of the antioxidant system of tomato plants and reduced the sensitivity of plants to atmospheric drought stress by eliminating reactive oxygen species produced by the plants. Additionally, leaf structure was improved, with decreased thickness of the epidermis and spongy tissue, leading to enhanced water transport efficiency. Furthermore, the TRV-SlPIP2;5 strain had increased stomatal length and individual stomatal area, which changed net assimilation rate and stomatal conductance to air drought. In addition, SlPIP2;5 was localized on the plasma membrane, and its promoter region contained numerous core elements and V-myb avian myeloblastosis viral oncogene homolog (MYB) binding sites. Yeast One-Hybrid Assay (Y1H) and Dual Luciferase (LUC) assays showed that the transcription factor SlMYB110 can bind to SlPIP2;5 and regulate its expression. qRT-PCR analysis revealed that high VPD inhibited the expression of SlMYB110, consistent with the expression pattern of SlPIP2;5. Therefore, we speculate that under high VPD, SlMYB110 negatively regulates the expression of SlPIP2;5, thereby modulating the tolerance of tomato plants to atmospheric drought. These findings provide a theoretical basis for improving tomato response to high VPD, and SlPIP2;5 was added to the high VPD-responsive gene pool as a valuable gene that could be used to improve high VPD tolerance in tomato through genetic engineering.

AtPIP1;4 and AtPIP2;4 Cooperatively Mediate H2O2 Transport to Regulate Plant Growth and Disease Resistance.

The rapid production of hydrogen peroxide (H2O2) is a hallmark of plants' successful recognition of pathogen infection and plays a crucial role in innate immune signaling. Aquaporins (AQPs) are membrane channels that facilitate the transport of small molecular compounds across cell membranes. In plants, AQPs from the plasma membrane intrinsic protein (PIP) family are utilized for the transport of H2O2, thereby regulating various biological processes. Plants contain two PIP families, PIP1s and PIP2s. However, the specific functions and relationships between these subfamilies in plant growth and immunity remain largely unknown. In this study, we explore the synergistic role of AtPIP1;4 and AtPIP2;4 in regulating plant growth and disease resistance in Arabidopsis. We found that in plant cells treated with H2O2, AtPIP1;4 and AtPIP2;4 act as facilitators of H2O2 across membranes and the translocation of externally applied H2O2 from the apoplast to the cytoplasm. Moreover, AtPIP1;4 and AtPIP2;4 collaborate to transport bacterial pathogens and flg22-induced apoplastic H2O2 into the cytoplasm, leading to increased callose deposition and enhanced defense gene expression to strengthen immunity. These findings suggest that AtPIP1;4 and AtPIP2;4 cooperatively mediate H2O2 transport to regulate plant growth and immunity.

Comparative differences in maintaining membrane fluidity and remodeling cell wall between Glycine soja and Glycine max leaves under drought

Water shortage is one of the most important environmental factors limiting crop yield. In this study, we used wild soybean (Glycine soja Sieb. et Zucc.) and soybean (Glycinemax (L.) Merr.) seedlings as experimental materials, simulated drought stress using soil gravimetry, measured growth and physiological parameters, and analyzed differentially expressed genes and metabolites in the leaves of seedling by integrated transcriptomics and metabolomics techniques. The results indicate that under water deficit, Glycine soja maintained stable photosynthate by accumulating Mg2+, Fe3+, Mn2+, Zn2+ and B3+, and improved water absorption by increasing root growth. Notably, Glycine soja enhanced linoleic acid metabolism and plasma membrane intrinsic protein (PIP1) gene expression to maintain membrane fluidity, and increased pentose, glucuronate and galactose metabolism and thaumatin protein genes expression to remodel the cell wall, thereby increasing water-absorption to better tolerate to drought stress. In addition, it was found that secondary phenolic metabolism, such as phenylpropane biosynthesis, flavonoid biosynthesis and ascobate and aldarate metabolism were weakened, resulting in the collapse of the antioxidant system, which was the main reason for the sensitivity of Glycine max to drought stress. These results provide new insights into plant adaptation to water deficit and offer a theoretical basis for breeding soybean varieties with drought tolerance.

CmoPIP1-4 confers drought tolerance in pumpkin by altering hydrogen sulfide signaling

Drought is a major limiting factor for the growth and development of pumpkins. Plasma membrane intrinsic proteins (PIPs) are major water channels that play a crucial role in the regulation of cellular water status and solute trafficking during drought conditions. CmoPIP1-4 is a plasma membrane-localized protein that is significantly upregulated in roots and leaves under drought-stress conditions. In this study, the overexpression of CmoPIP1-4 enhances drought resistance in yeast. In contrast, CRISPR-mediated CmoPIP1-4 knockout in pumpkin roots increased drought sensitivity. This increased drought sensitivity of CmoPIP1-4 knockout plants is associated with a decline in the levels of hydrogen sulfide (H2S) and abscisic acid (ABA), accompanied by an increase in water loss caused by greater levels of transpiration and stomatal conductance. In addition, the sensitivity of CmoPIP1-4 CRISPR plants is further aggravated by reduced antioxidative enzyme activity, decreased proline and sugar contents, and extensive root damage. Furthermore, expression profiles of genes such as CmoHSP70s, CmoNCED3, CmoNCED4, and others involved in metabolic activities were markedly reduced in CmoPIP1-4 CRISPR plants. Moreover, we also discovered an interaction between the drought-responsive gene CmoDCD and CmoPIP1-4, indicating their potential role in activating H2S-mediated signaling in pumpkin, which could confer drought tolerance. The findings of our study collectively demonstrate CmoPIP1-4 plays a crucial role in the regulation of H2S-mediated signaling, influencing stomatal density and aperture in pumpkin plants, and thereby enhancing their drought tolerance.

Plasma membrane aquaporins function in moisture regulation during seed germination and leaf hydration in eelgrass

Eelgrass (Zostera marina) is a marine angiosperm distributed in shallow seas that has a unique mechanism for regulating water content. Water transfer across the plasma membrane is facilitated by aquaporins, which are membrane proteins. In this study, we investigated the physiological functions of plasma membrane intrinsic proteins (PIPs) in eelgrass. ZoPIP1;1 and ZoPIP2;1 in eelgrass leaves were amplified by RT-PCR. Both ZoPIPs localized to the plasma membrane, and only ZoPIP2;1 showed water permeability. ZoPIP1;1 and ZoPIP2;1 were expressed in all tissues except male flowers. ZoPIPs were expressed during seed and fruit development, and their expression levels were low in mature seeds and elevated in germinating seeds. Moisture content decreased in the later phases of seed development and increased during seed germination. These findings imply that ZoPIPs participate in water uptake during seed germination. In leaves, ZoPIP expression was upregulated by dehydration but downregulated by rehydration. Water reabsorption of leaves was inhibited by HgCl2, an inhibitor of aquaporins. These findings indicate that ZoPIPs are upregulated by dehydration during extremely low tides and promote water uptake when the tide turns. Therefore, eelgrass PIPs function in moisture regulation during growth, seed germination, and the flood-ebb tidal cycle in seawater.

Plasma membrane intrinsic protein OsPIP2;6 is involved in root-to-shoot arsenic translocation in rice (Oryza sativa L.).

This study demonstrates the crucial role of OsPIP2;6 for translocation of arsenic from roots to shoots, which can decrease arsenic accumulation in rice for improved food safety. Arsenic (As) contamination in food and water, primarily through rice consumption, poses a significant health risk due to its natural tendency to accumulate inorganic arsenic (iAs). Understanding As transport mechanisms is vital for producing As-free rice. This study investigates the role of rice plasma membrane intrinsic protein, OsPIP2;6, for AsIII tolerance and accumulation. RNAi-mediated suppression of OsPIP2;6 expression resulted in a substantial (35-65%) reduction in As accumulation in rice shoots, while root arsenic levels remained largely unaffected. Conversely, OsPIP2;6 overexpression led to 15-76% higher arsenic accumulation in shoots, with no significant change in root As content. In mature plants, RNAi suppression caused (19-26%) decrease in shoot As, with flag leaves and grains showing a 16% reduction. OsPIP2;6 expression was detected in both roots and shoots, with higher transcript levels in shoots. Localization studies revealed its presence in vascular tissues of both roots and shoots. Overall, our findings highlight OsPIP2;6's role in root-to-shoot As translocation, attributed to its specific localization in the vascular tissue of roots and leaves. This knowledge can facilitate the development of breeding programs to mitigate As accumulation in rice and other food crops for improved food safety and increasing productivity on As-contaminated soils.

Arabidopsis SNARE SYP132 impacts on PIP2;1 trafficking and function in salinity stress.

In plants so-called plasma membrane intrinsic proteins (PIPs) are major water channels governing plant water status. Membrane trafficking contributes to functional regulation of major PIPs and is crucial for abiotic stress resilience. Arabidopsis PIP2;1 is rapidly internalised from the plasma membrane in response to high salinity to regulate osmotic water transport, but knowledge of the underlying mechanisms is fragmentary. Here we show that PIP2;1 occurs in complex with SYNTAXIN OF PLANTS 132 (SYP132) together with the plasma membrane H+-ATPase AHA1 as evidenced through in vivo and in vitro analysis. SYP132 is a multifaceted vesicle trafficking protein, known to interact with AHA1 and promote endocytosis to impact growth and pathogen defence. Tracking native proteins in immunoblot analysis, we found that salinity stress enhances SYP132 interactions with PIP2;1 and PIP2;2 isoforms to promote redistribution of the water channels away from the plasma membrane. Concurrently, AHA1 binding within the SYP132-complex was significantly reduced under salinity stress and increased the density of AHA1 proteins at the plasma membrane in leaf tissue. Manipulating SYP132 function in Arabidopsis thaliana enhanced resilience to salinity stress and analysis in heterologous systems suggested that the SNARE influences PIP2;1 osmotic water permeability. We propose therefore that SYP132 coordinates AHA1 and PIP2;1 abundance at the plasma membrane and influences leaf hydraulics to regulate plant responses to abiotic stress signals.

Two aquaporins, PIP1;1 and PIP2;1, mediate the uptake of neonicotinoid pesticides in plants

Neonicotinoids (NEOs), a large class of organic compounds, are a type of commonly used pesticide for crop protection. Their uptake and accumulation in plants are prerequisites for their intra- and intercellular movements, transformation, and function. Understanding the molecular mechanisms that underpin NEO uptake by plants is crucial for effective application, which remains elusive. Here, we demonstrate that NEOs enter plant cells primarily through the transmembrane symplastic pathway and accumulate mainly in the cytosol. Two plasma membrane intrinsic proteins discovered in Brassica rapa, BraPIP1;1 and BraPIP2;1, were found to encode aquaporins (AQPs) that are highly permeable to NEOs in different plant species and facilitate NEO subcellular diffusion and accumulation. Their conserved transport function was further demonstrated in Xenopus laevis oocyte and yeast assays. BraPIP1;1 and BraPIP2;1 gene knockouts and interaction assays suggested that their proteins can form functional heterotetramers. Assessment of the potential of mean force indicated a negative correlation between NEO uptake and the energy barrier of BraPIP1;1 channels. This study shows that AQPs transport organic compounds with greater osmolarity than previously thought, providing new insight into the molecular mechanisms of organic compound uptake and facilitating innovations in systemic pesticides.

Primary, seminal and lateral roots of maize show type-specific growth and hydraulic responses to water deficit.

The water uptake capacity of a root system is determined by its architecture and hydraulic properties, which together shape the root hydraulic architecture. Here, we investigated root responses to water deficit (WD) in seedlings of a maize (Zea mays) hybrid line (B73H) grown in hydroponic conditions, taking into account the primary root (PR), the seminal roots (SR), and their respective lateral roots. WD was induced by various polyethylene glycol concentrations and resulted in dose-dependent inhibitions of axial and lateral root growth, lateral root formation, and hydraulic conductivity (Lpr), with slightly distinct sensitivities to WD between PR and SR. Inhibition of Lpr by WD showed a half-time of 5 to 6 min and was fully (SR) or partially (PR) reversible within 40 min. In the two root types, WD resulted in reduced aquaporin expression and activity, as monitored by mRNA abundance of 13 plasma membrane intrinsic protein (ZmPIP) isoforms and inhibition of Lpr by sodium azide, respectively. An enhanced suberization/lignification of the epi- and exodermis was observed under WD in axial roots and in lateral roots of the PR but not in those of SR. Inverse modeling revealed a steep increase in axial conductance in root tips of PR and SR grown under WD that may be due to the decreased growth rate of axial roots in these conditions. Overall, our work reveals that these root types show quantitative differences in their anatomical, architectural, and hydraulic responses to WD, in terms of sensitivity, amplitude and reversibility. This distinct functionalization may contribute to integrative acclimation responses of whole root systems to soil WD.

Populus euphratica CPK21 interacts with heavy metal stress-associated proteins to mediate Cd tolerance in Arabidopsis

High cadmium (Cd, 50 µM) caused transcriptional upregulation of Ca2+-dependent protein kinase 21 (CPK21) in roots and leaves of Populus euphratica. The promoter region (1803 bp) of PeCPK21 was engineered to fuse PeCPK21-pro::GUS and transformed in Arabidopsis. The activity of GUS significantly increased in roots, cotyledons, and hypocotyls under 100 μM CdCl2 treatment. The 1599 bp coding sequence of PeCPK21 was transferred into Arabidopsis, and overexpression of PeCPK21 increased Cd tolerance as evidenced by less reduction in root length and plant growth under CdCl2 stress conditions (100 µM, 7 d). PeCPK21-interacting proteins were enriched by HaloTag pull-down and corresponding expression profiles after Cd exposure indicate that PeCPK21 interacts with heavy metal stress-associated proteins (HMAPs) to mediate Cd tolerance in transgenic Arabidopsis. PeCPK21 interacted with the Cd-regulated cation/heavy metal transport proteins annexin, OPT3, COPT5, and PDF2.2 to limit Cd uptake and accumulation in transgenic plants. PeCPK21 may also interact with the V-type proton ATPase subunits, VHA-B1, VHA-C, and AVA-P2, which were transcriptionally upregulated by Cd to accelerate vacuolar Cd compartmentalization. Cd-triggered H2O2 production was limited in plants overexpressing PeCPK21, which was due to the activated antioxidant enzymes. The interaction of PeCPK21 with the antioxidant enzymes TAPX, APX1, APX2, TRXM4, GPX3, CDSP32, and PRXQ, which showed transcriptional upregulation by Cd, could enhance antioxidant defense in transgenic Arabidopsis. It is also possible that PeCPK21 interacts with plasma membrane intrinsic proteins, PIP1–1, PIP2A, and PIP2–7, which had an Cd-enhanced transcript, to improve water status in transgenic Arabidopsis. In summary, we hypothesize that the calcium sensor PeCPK21 can attenuate Cd toxicity by interacting with HMAPs to effectively limit Cd accumulation, increase ROS scavenging, and improve water status in transgenic Arabidopsis.

Combined Soil Microorganism Amendments and Foliar Micronutrient Nanofertilization Increased the Production of Allium cepa L. through Aquaporin Gene Regulation.

The aim of this study was to investigate the impact of changes in aquaporin expression on the growth of onion (Allium cepa L.) plants when subjected to dual applications of microorganism-based soil amendments and foliar nanoencapsulated mineral nutrients. Multiple physiological parameters related to water, gas exchange, and nutrient content in leaf, root, and bulb tissues were determined. Additionally, the gene expression of aquaporins, specifically PIP1, PIP2 (aquaporin subfamily plasma membrane intrinsic protein), and TIP2 (aquaporin subfamily tonoplast intrinsic protein), was analyzed. The findings revealed that the foliar application of nutrients in a nanoencapsulated form significantly enhanced nutrient penetration, mobilization, and overall plant growth to a greater extent than free-form fertilizers. Amendments with microorganisms alone did not promote growth but influenced the production of secondary metabolites in the bulbs. The combination of microorganisms and nanoencapsulated mineral nutrients demonstrated synergistic effects, increasing dry matter, mineral content, and aquaporin gene expression. This suggests that aquaporins play a pivotal role in the transport of nutrients from leaves to storage organs, resulting in the overexpression of PIP2 aquaporins in bulbs, improved water uptake, and enhanced cell growth. Therefore, the combined treatment with microorganisms and nanoencapsulated mineral nutrients may be an optimal approach for enhancing onion productivity by regulating aquaporins under field conditions.

Global characterization of OsPIP aquaporins reveals that the H2O2 transporter OsPIP2;6 increases resistance to rice blast

Plasma membrane intrinsic proteins (PIPs) are conserved plant aquaporins that transport small molecules across the plasma membrane to trigger instant stress responses and maintain cellular homeostasis under biotic and abiotic stress. To elucidate their roles in plant immunity to pathogen attack, we characterized the expression patterns, subcellular localizations, and H2O2-transport ability of 11 OsPIPs in rice (Oryza sativa), and identified OsPIP2;6 as necessary for rice disease resistance. OsPIP2;6 resides on the plasma membrane and facilitates cytoplasmic import of the immune signaling molecule H2O2. Knockout of OsPIP2;6 increases rice susceptibility to Magnaporthe oryzae, indicating a positive function in plant immunity. OsPIP2;6 interacts with OsPIP2;2, which has been reported to increase rice resistance to pathogens via H2O2 transport. Our findings suggest that OsPIP2;6 cooperates with OsPIP2;2 as a defense signal transporter complex during plant–pathogen interaction.

Sugarcane mosaic virus employs 6K2 protein to impair ScPIP2;4 transport of H2O2 to facilitate virus infection.

Sugarcane mosaic virus (SCMV), one of the main pathogens causing sugarcane mosaic disease, is widespread in sugarcane (Saccharum spp. hybrid) planting areas and causes heavy yield losses. RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) NADPH oxidases and plasma membrane intrinsic proteins (PIPs) have been associated with the response to SCMV infection. However, the underlying mechanism is barely known. In the present study, we demonstrated that SCMV infection upregulates the expression of ScRBOHs and the accumulation of hydrogen peroxide (H2O2), which inhibits SCMV replication. All eight sugarcane PIPs (ScPIPs) interacted with SCMV-encoded protein 6K2, whereby two PIP2s (ScPIP2;1 and ScPIP2;4) were verified as capable of H2O2 transport. Furthermore, we revealed that SCMV-6K2 interacts with ScPIP2;4 via transmembrane domain 5 to interfere with the oligomerization of ScPIP2;4, subsequently impairing ScPIP2;4 transport of H2O2. This study highlights a mechanism adopted by SCMV to employ 6K2 to counteract the host resistance mediated by H2O2 to facilitate virus infection and provides potential molecular targets for engineering sugarcane resistance against SCMV.

Aquaporins-mediated water availability in substrates for cannabis cultivation in relation to CBD yield

Abstract Aims The objective of this study was to identify the most suitable substrate for Cannabis sativa L. cultivation based on its effects on water relations and Cannabidiol (CBD) production. Methods Biomass, physiological parameters, minerals, changes in the expression levels of plasma membrane intrinsic Proteins (PIP) and CBD concentration was measured in C. sativa (var. Tiborszallasi) plants cultivated on 5 substrates with different physical–chemical characteristics. Results The substrates available water (AW) was the main factor affecting growth and production. The efficiency of the water use was governed fundamentally by transpiration. Experimental substrates(S) 1 and 3 (S1 and S3) were those in which the plants grew optimally and allows plants to invest energy in secondary metabolites production acquiring high levels of CBD. The plants grown in S2 and S5, composed by coconut fiber and perlite, showed the lowest growth in agreement with low transpiration rates which reduce the water uptake. S5 substrate, with some available water (AW) still present, is forcing plants to invest energy in improving water and nutrient transport, as observed by the high levels of nutrients in planta and PIPs expression levels. S4 plants presented the highest inflorescence production and CBD content, which can be attributed to plant stress due to the low levels of AW and high pH and electrical conductivity (EC). Conclusion The absorption of water and minerals by plants has been affected by PIP-mediated water transport, playing key roles for the optimal utilization of the water present in the substrates, with specific isoforms involved in these responses.

Genome-wide identification and characterisation of Aquaporins in Rosa chinensis

ABSTRACT Aquaporins (AQPs) are small integral membrane proteins that facilitate the efficient transport of small solutes like water, metalloids, urea, and CO2 across the membrane. Plant aquaporins are known to be involved in vital physiological processes like seed germination, stomatal movement, cell elongation, and reproductive growth. In the present study, 36 AQPs were identified through genome-wide computational analysis performed in the rose genome. Subsequent phylogenetic analysis classified the AQPs into five subfamilies, including 10 Plasma membrane intrinsic proteins (PIPs), 8 Tonoplast intrinsic proteins (TIPs), 9 Nodulin intrinsic proteins (NIPs), 3 Small intrinsic proteins (SIPs), and 6 uncharacterised intrinsic proteins (XIPs). Based on the prediction of silicon solute specificity, one of the NIPs is predicted to be a silicon transporter. Structural analysis revealed the characteristic six trans-membrane domains and two-half alpha helix harbouring two conserved NPA motifs in the majority of the predicted AQPs. The protein tertiary structures predicted with homology-based modelling were used to study the pore lining remains and to predict the solute transport activity of the AQPs. Expression profiling of the rose AQPs showed tissue-specific expression of many members. The data provided here will help in understanding the AQP-based solute transport system in roses and other related species.

Protein kinase SnRK2.4 is a key regulator of aquaporins and root hydraulics in Arabidopsis.

Soil water uptake by roots is a key component of plant water homeostasis contributing to plant growth and survival under ever-changing environmental conditions. The water transport capacity of roots (root hydraulic conductivity; Lpr ) is mostly contributed by finely regulated Plasma membrane Intrinsic Protein (PIP) aquaporins. In this study, we used natural variation of Arabidopsis for the identification of quantitative trait loci (QTLs) contributing to Lpr . Using recombinant lines from a biparental cross (Cvi-0 x Col-0), we show that the gene encoding class 2 Sucrose-Non-Fermenting Protein kinase 2.4 (SnRK2.4) in Col-0 contributes to >30% of Lpr by enhancing aquaporin-dependent water transport. At variance with the inactive and possibly unstable Cvi-0 SnRK2.4 form, the Col-0 form interacts with and phosphorylates the prototypal PIP2;1 aquaporin at Ser121 and stimulates its water transport activity upon coexpression in Xenopus oocytes and yeast cells. Activation of PIP2;1 by Col-0 SnRK2.4 in yeast also requires its protein kinase activity and can be counteracted by clade A Protein Phosphatases 2C. SnRK2.4 shows all hallmarks to be part of core abscisic acid (ABA) signaling modules. Yet, long-term (>3 h) inhibition of Lpr by ABA possibly involves a SnRK2.4-independent inhibition of PIP2;1. SnRK2.4 also promotes stomatal aperture and ABA-induced inhibition of primary root growth. The study identifies a key component of Lpr and sheds new light on the functional overlap and specificity of SnRK2.4 with respect to other ABA-dependent or independent SnRK2s.

Plant Aquaporin Gating Is Reversed by Phosphorylation on Intracellular Loop D-Evidence from Molecular Dynamics Simulations.

Aquaporins (AQPs) constitute a wide and ancient protein family of transmembrane channels dedicated to the regulation of water exchange across biological membranes. In plants, higher numbers of AQP homologues have been conserved compared to other kingdoms of life such as in animals or in bacteria. As an illustration of this plant-specific functional diversity, plasma membrane intrinsic proteins (PIPs, i.e., a subfamily of plant AQPs) possess a long intracellular loop D, which can gate the channel by changing conformation as a function of the cellular environment. However, even though the closure of the AQP by loop D conformational changes is well described, the opening of the channel, on the other hand, is still misunderstood. Several studies have pointed to phosphorylation events as the trigger for the transition from closed- to open-channel states. Nonetheless, no clear answer has been obtained yet. Hence, in order to gain a more complete grasp of plant AQP regulation through this intracellular loop D gating, we investigated the opening of the channel in silico through molecular dynamics simulations of the crystallographic structure of Spinacia oleracea PIP2;1 (SoPIP2;1). Through this technique, we addressed the mechanistic details of these conformational changes, which eventually allowed us to propose a molecular mechanism for PIP functional regulation by loop D phosphorylation. More precisely, our results highlight the phosphorylation of loop D serine 188 as a trigger of SoPIP2;1 water channel opening. Finally, we discuss the significance of this result for the study of plant AQP functional diversity.

Plasma membrane aquaporins regulate root hydraulic conductivity in the model plant Setaria viridis.

The high rate of productivity observed in panicoid crops is in part due to their extensive root system. Recently, green foxtail (Setaria viridis) has emerged as a genetic model system for panicoid grasses. Natural accessions of S. viridis originating from different parts of the world, with differential leaf physiological behavior, have been identified. This work focused on understanding the physiological and molecular mechanisms controlling root hydraulic conductivity and root-to-shoot gas exchange signaling in S. viridis. We identified 2 accessions, SHA and ZHA, with contrasting behavior at the leaf, root, and whole-plant levels. Our results indicated a role for root aquaporin (AQP) plasma membrane (PM) intrinsic proteins in the differential behavior of SHA and ZHA. Moreover, a different root hydraulic response to low levels of abscisic acid between SHA and ZHA was observed, which was associated with root AQPs. Using cell imaging, biochemical, and reverse genetic approaches, we identified PM intrinsic protein 1;6 (PIP1;6) as a possible PIP1 candidate that regulates radial root hydraulics and root-to-shoot signaling of gas exchange in S. viridis. In heterologous systems, PIP1;6 localized in the endoplasmic reticulum, and upon interaction with PIP2s, relocalization to the PM was observed. PIP1;6 was predominantly expressed at the root endodermis. Generation of knockout PIP1;6 plants (KO-PIP1;6) in S. viridis showed altered root hydraulic conductivity, altered gas exchange, and alteration of root transcriptional patterns. Our results indicate that PIPs are essential in regulating whole-plant water homeostasis in S. viridis. We conclude that root hydraulic conductivity and gas exchange are positively associated and are regulated by AQPs.