Elevated CO2 Modulates Plant Hydraulic Conductance Through Regulation of PIPs Under Progressive Soil Drying in Tomato Plants

Increasing atmospheric CO2 concentrations accompanied by abiotic stresses challenge food production worldwide. Elevated CO2 (e[CO2]) affects plant water relations via multiple mechanisms involving abscisic acid (ABA). Here, two tomato (Solanum lycopersicum) genotypes, Ailsa Craig (AC) and its ABA-deficient mutant (flacca), were used to investigate the responses of plant hydraulic conductance to e[CO2] and drought stress. Results showed that e[CO2] decreased transpiration rate (E) increased plant water use efficiency only in AC, whereas it increased daily plant water consumption and osmotic adjustment in both genotypes. Compared to growth at ambient [CO2], AC leaf and root hydraulic conductance (Kleaf and Kroot) decreased at e[CO2], which coincided with the transcriptional regulations of genes of plasma membrane intrinsic proteins (PIPs) and OPEN STOMATA 1 (OST1), and these effects were attenuated in flacca during soil drying. Severe drought stress could override the effects of e[CO2] on plant water relation characteristics. In both genotypes, drought stress resulted in decreased E, Kleaf, and Kroot accompanied by transcriptional responses of PIPs and OST1. However, under conditions combining e[CO2] and drought, some PIPs were not responsive to drought in AC, indicating that e[CO2] might disturb ABA-mediated drought responses. These results provide some new insights into mechanisms of plant hydraulic response to drought stress in a future CO2-enriched environment.

Permeability profiling of all 13 Arabidopsis PIP aquaporins using a high throughput yeast approach

Plant aquaporins have many more functions than just transporting water. Within the diversity of plant aquaporins are isoforms capable of transporting signaling molecules, nutrients, metalloids and gases. It is established that aquaporin substrate discrimination depends on combinations of factors such as solute size, pore size and polarity, and post-translational protein modifications. But our understanding of the relationships between variation in aquaporin structures and the implications for permeability is limited. High-throughput yeast-based assays were developed to assess diverse substrate permeabilities to water, H2O2, boric acid, urea and Na+. All 13 plasma membrane intrinsic proteins (PIPs) from Arabidopsis (AtPIPs) were permeable to both water and H2O2, although their effectiveness varied, and none were permeable to urea. AtPIP2 isoforms were more permeable to water than AtPIP1s, while AtPIP1s were more efficient at transporting H2O2 with AtPIP1;3 and AtPIP1;4 being the most permeable. Among the AtPIP2s, AtPIP2;2 and AtPIP2;7 were also permeable to boric acid and Na+. Linking AtPIP substrate profiles with phylogenetics and gene expression data enabled us to align substrate preferences with known biological roles of AtPIPs and importantly guide towards unidentified roles hidden by functional redundancy at key developmental stages and within tissue types. This analysis positions us to more strategically test in planta physiological roles of AtPIPs in order to unravel their complex contributions to the transport of important substrates, and secondly, to resolve links between aquaporin protein structure, substrate discrimination, and transport efficiency.

The pip1s Quintuple Mutants Demonstrate the Essential Roles of PIP1s in the Plant Growth and Development of Arabidopsis

Plasma membrane intrinsic proteins (PIPs) transport water, CO2 and small neutral solutes across the plasma membranes. In this study, we used the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 system (CRISPR/Cas9) to mutate PIP1;4 and PIP1;5 in a pip1;1,2,3 triple mutant to generate a pip1;1,2,3,4,5 (pip1s−) quintuple mutant. Compared to the wild-type (WT) plant, the pip1s− mutants had smaller sized rosette leaves and flowers, less rosette leaf number, more undeveloped siliques, shorter silique and less seeds. The pollen germination rate of the pip1s− mutant was significantly lower than that of the WT and the outer wall of the pip1s− mutant’s pollen was deformed. The transcriptomic analysis showed significant alterations in the expression of many key genes and transcription factors (TFs) in the pip1s− mutant which involved in the development of leaf, flower and pollen, suggesting that the mutant of PIP1s not only directly affects hydraulics and carbon fixation, but also regulates the expression of related genes to affect plant growth and development.

Interaction Between the SNARE SYP121 and the Plasma Membrane Aquaporin PIP2;7 Involves Different Protein Domains

Plasma membrane intrinsic proteins (PIPs) are channels facilitating the passive diffusion of water and small solutes. Arabidopsis PIP2;7 trafficking occurs through physical interaction with SNARE proteins including the syntaxin SYP121, a plasma membrane Qa-SNARE involved in membrane fusion. To better understand the interaction mechanism, we aimed at identifying the interaction motifs in SYP121 and PIP2;7 using ratiometric bimolecular fluorescence complementation assays in Nicotiana benthamiana. SYP121 consists of four regions, N, H, Q, and C, and sequential deletions revealed that the C region, containing the transmembrane domain, as well as the H and Q regions, containing the Habc and Qa-SNARE functional domains, interact with PIP2;7. Neither the linker between the Habc and the Qa-SNARE domains nor the H or Q regions alone could fully restore the interaction with PIP2;7, suggesting that the interacting motif depends on the conformation taken by the HQ region. When investigating the interacting motif(s) in PIP2;7, we observed that deletion of the cytosolic N- and/or C- terminus led to a significant decrease in the interaction with SYP121. Shorter deletions revealed that at the N-terminal amino acid residues 18–26 were involved in the interaction. Domain swapping experiments between PIP2;7 and PIP2;6, a PIP isoform that does not interact with SYP121, showed that PIP2;7 N-terminal part up to the loop C was required to restore the full interaction signal, suggesting that, as it is the case for SYP121, the interaction motif(s) in PIP2;7 depend on the protein conformation. Finally, we also showed that PIP2;7 physically interacted with other Arabidopsis SYP1s and SYP121 orthologs.

Characterization of interactions between the soybean plasma membrane- intrinsic proteins GmPIP1s and GmPIP2s responding to salt stress

Background: Salt tolerance is a key trait in soybean breeding and plant responses to salt stress include physiological and biochemical changes that affect the movement of water across the plasma membrane. In this study, we report the interactions of a set of aquaporins, soybean (Glycine max) plasma membrane-intrinsic proteins (GmPIPs), in response to salt stress. Results: GmPIP1;5 and GmPIP1;6 formed hetero-tetramers with GmPIP2;4, GmPIP2;6, GmPIP2;8, GmPIP2;9, GmPIP2;11, and GmPIP2;13. We detected interactions between GmPIP1;6 and GmPIP1;7, but not between GmPIP1;6 and GmPIP1;5. Furthermore, GmPIP2;9 formed homo-tetramers, and this interaction was strengthened under salt and osmotic stress. Expression analysis indicated complex and unique responses to salt stress depending on the duration of the stress. For example, GmPIP2;8, encoding one of the heteromer-forming PIP proteins, was highly up-regulated under early salt stress.Conclusions: Our study highlights the vital role of hetero- and homo-tetramers, in salt tolerance; and improves understanding of the mechanisms by which soybean aquaporin isoforms respond to abiotic stress.

Evolutionary Understanding of Aquaporin Transport System in the Basal Eudicot Model Species Aquilegia coerulea

Aquaporins (AQPs) play a pivotal role in the cellular transport of water and many other small solutes, influencing many physiological and developmental processes in plants. In the present study, extensive bioinformatics analysis of AQPs was performed in Aquilegia coerulea L., a model species belonging to basal eudicots, with a particular focus on understanding the AQPs role in the developing petal nectar spur. A total of 29 AQPs were identified in Aquilegia, and their phylogenetic analysis performed with previously reported AQPs from rice, poplar and Arabidopsis depicted five distinct subfamilies of AQPs. Interestingly, comparative analysis revealed the loss of an uncharacterized intrinsic protein II (XIP-II) group in Aquilegia. The absence of the entire XIP subfamily has been reported in several previous studies, however, the loss of a single clade within the XIP family has not been characterized. Furthermore, protein structure analysis of AQPs was performed to understand pore diversity, which is helpful for the prediction of solute specificity. Similarly, an AQP AqcNIP2-1 was identified in Aquilegia, predicted as a silicon influx transporter based on the presence of features such as the G-S-G-R aromatic arginine selectivity filter, the spacing between asparagine-proline-alanine (NPA) motifs and pore morphology. RNA-seq analysis showed a high expression of tonoplast intrinsic proteins (TIPs) and plasma membrane intrinsic proteins (PIPs) in the developing petal spur. The results presented here will be helpful in understanding the AQP evolution in Aquilegia and their expression regulation, particularly during floral development.

Plasma Membrane Aquaporin Members PIPs Act in Concert to Regulate Cold Acclimation and Freezing Tolerance Responses in Arabidopsis thaliana

Aquaporins play a major role in plant water uptake at both optimal and environmentally stressed conditions. However, the functional specificity of aquaporins under cold remains obscure. To get a better insight to the role of aquaporins in cold acclimation and freezing tolerance, we took an integrated approach of physiology, transcript profiling and cell biology in Arabidopsis thaliana. Cold acclimation resulted in specific upregulation of PIP1;4 and PIP2;5 aquaporin (plasma membrane intrinsic proteins) expression, and immunoblotting analysis confirmed the increase in amount of PIP2;5 protein and total amount of PIPs during cold acclimation, suggesting that PIP2;5 plays a major role in tackling the cold milieu. Although single mutants of pip1;4 and pip2;5 or their double mutant showed no phenotypic changes in freezing tolerance, they were more sensitive in root elongation and cell survival response under freezing stress conditions compared with the wild type. Consistently, a single mutation in either PIP1;4 or PIP2;5 altered the expression of a number of aquaporins both at the transcriptional and translational levels. Collectively, our results suggest that aquaporin members including PIP1;4 and PIP2;5 function in concert to regulate cold acclimation and freezing tolerance responses.

Plant Aquaporins

This mini-review briefly presents the main types of plant aquaporins, highlighting their importance for different plant species and for plant cellular functions. Aquaporins (AQPs), families of water channel proteins (WCPs) are transmembrane proteins that are present in prokaryotes, animals, plants, and humans. The plant aquaporins are part of the Major Intrinsic Proteins (MIPs) family which resides in the following plant organs: roots, stems, leaves, flowers, fruits, and seeds. According to the sub-cellular localization, to their sequence homologies and to their phylogenetic distribution, plant aquaporins have been divided in five subgroups: (a) plasma membrane intrinsic proteins (PIPs); (b) tonoplast intrinsic proteins (TIPs); (c) Nodulin26-like intrinsic membrane proteins (NIPs); (d) small basic intrinsic proteins (SIPs) and (e) uncharacterized intrinsic proteins (XIPs). Different subclasses of the plant aquaporins allow several types of transport using: water, glycerol, urea, hydrogen peroxide, organic acids, ethanol, methanol, arsenite, lactic acid, and gaseous compounds. Plant aquaporins have a significant role in cell response to cold stress, photosynthesis, plant growth, cell elongation, reproduction, and seed germination.