Multifaceted role and regulation of aquaporins for efficient stomatal movements.

Stomata are micropores on the leaf epidermis that allow carbon dioxide (CO2) uptake for photosynthesis at the expense of water loss through transpiration. Stomata coordinate the plant gas exchange of carbon and water with the atmosphere through their opening and closing dynamics. In the context of global climate change, it is essential to better understand the mechanism of stomatal movements under different environmental stimuli. Aquaporins (AQPs) are considered important regulators of stomatal movements by contributing to membrane diffusion of water, CO2 and hydrogen peroxide. This review compiles the most recent findings and discusses future directions to update our knowledge of the role of AQPs in stomatal movements. After highlighting the role of subsidiary cells (SCs), which contribute to the high water use efficiency of grass stomata, we explore the expression of AQP genes in guard cells and SCs. We then focus on the cellular regulation of AQP activity at the protein level in stomata. After introducing their post-translational modifications, we detail their trafficking as well as their physical interaction with various partners that regulate AQP subcellular dynamics towards and within specific regions of the cell membranes, such as microdomains and membrane contact sites.

Plasma membrane aquaporins interact with the endoplasmic reticulum resident VAP27 proteins at ER-PM contact sites and endocytic structures.

Plasma membrane (PM) intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water and small solutes. The functional importance of the PM organisation of PIPs in the interaction with other cellular structures is not completely understood. We performed a pull-down assay using maize (Zea mays) suspension cells expressing YFP-ZmPIP2;5 and validated the protein interactions by yeast split-ubiquitin and bimolecular fluorescence complementation assays. We expressed interacting proteins tagged with fluorescent proteins in Nicotiana benthamiana leaves and performed water transport assays in oocytes. Finally, a phylogenetic analysis was conducted. The PM-located ZmPIP2;5 physically interacts with the endoplasmic reticulum (ER) resident ZmVAP27-1. This interaction requires the ZmVAP27-1 cytoplasmic major sperm domain. ZmPIP2;5 and ZmVAP27-1 localise in close vicinity in ER-PM contact sites (EPCSs) and endocytic structures upon exposure to salt stress conditions. This interaction enhances PM water permeability in oocytes. Similarly, the Arabidopsis ZmVAP27-1 paralogue, AtVAP27-1, interacts with the AtPIP2;7 aquaporin. Together, these data indicate that the PIP2-VAP27 interaction in EPCSs is evolutionarily conserved, and suggest that VAP27 might stabilise the aquaporins and guide their endocytosis in response to salt stress.

Major cereal crops benefit from biological nitrogen fixation when inoculated with the nitrogen-fixing bacterium Pseudomonas protegens Pf-5 X940.

A main goal of biological nitrogen fixation research has been to expand the nitrogen-fixing ability to major cereal crops. In this work, we demonstrate the use of the efficient nitrogen-fixing rhizobacterium Pseudomonas protegens Pf-5 X940 as a chassis to engineer the transfer of nitrogen fixed by BNF to maize and wheat under non-gnotobiotic conditions. Inoculation of maize and wheat with Pf-5 X940 largely improved nitrogen content and biomass accumulation in both vegetative and reproductive tissues, and this beneficial effect was positively associated with high nitrogen fixation rates in roots. 15 N isotope dilution analysis showed that maize and wheat plants obtained substantial amounts of fixed nitrogen from the atmosphere. Pf-5 X940-GFP-tagged cells were always reisolated from the maize and wheat root surface but never from the inner root tissues. Confocal laser scanning microscopy confirmed root surface colonization of Pf-5 X940-GFP in wheat plants, and microcolonies were mostly visualized at the junctions between epidermal root cells. Genetic analysis using biofilm formation-related Pseudomonas mutants confirmed the relevance of bacterial root adhesion in the increase in nitrogen content, biomass accumulation and nitrogen fixation rates in wheat roots. To our knowledge, this is the first report of robust BNF in major cereal crops.

Exploring the Ancestral Mechanisms of Regulation of Horizontally Acquired Nitrogenases.

The vast majority of Pseudomonas species are unable to fix atmospheric nitrogen. Although several studies have demonstrated that some strains belonging to the genus Pseudomonas sensu stricto do have the ability to fix nitrogen by the expression of horizontally acquired nitrogenase, little is known about the mechanisms of nitrogenase adaptation to the new bacterial host. Recently, we transferred the nitrogen fixation island from Pseudomonas stutzeri A1501 to the non-nitrogen-fixing bacterium Pseudomonas protegens Pf-5, and interestingly, the resulting recombinant strain Pf-5 X940 showed an uncommon phenotype of constitutive nitrogenase activity. Here, we integrated evolutionary and functional approaches to elucidate this unusual phenotype. Phylogenetic analysis showed that polyhydroxybutyrate (PHB) biosynthesis genes from natural nitrogen-fixing Pseudomonas strains have been acquired by horizontal transfer. Contrary to Pf-5 X940, its derived PHB-producing strain Pf-5 X940-PHB exhibited the inhibition of nitrogenase activity under nitrogen-excess conditions, and displayed the typical switch-on phenotype observed in natural nitrogen-fixing strains after nitrogen deficiency. This indicates a competition between PHB production and nitrogen fixation. Therefore, we propose that horizontal transfer of PHB biosynthesis genes could be an ancestral mechanism of regulation of horizontally acquired nitrogenases in the genus Pseudomonas.

Alfalfa snakin-1 prevents fungal colonization and probably coevolved with rhizobia.

BACKGROUND: The production of antimicrobial peptides is a common defense strategy of living cells against a wide range of pathogens. Plant snakin peptides inhibit bacterial and fungal growth at extremely low concentrations. However, little is known of their molecular and ecological characteristics, including origin, evolutionary equivalence, specific functions and activity against beneficial microbes. The aim of this study was to identify and characterize snakin-1 from alfalfa (MsSN1). RESULTS: Phylogenetic analysis showed complete congruence between snakin-1 and plant trees. The antimicrobial activity of MsSN1 against bacterial and fungal pathogens of alfalfa was demonstrated in vitro and in vivo. Transgenic alfalfa overexpressing MsSN1 showed increased antimicrobial activity against virulent fungal strains. However, MsSN1 did not affect nitrogen-fixing bacterial strains only when these had an alfalfa origin. CONCLUSIONS: The results reported here suggest that snakin peptides have important and ancestral roles in land plant innate immunity. Our data indicate a coevolutionary process, in which alfalfa exerts a selection pressure for resistance to MsSN1 on rhizobial bacteria. The increased antimicrobial activity against virulent fungal strains without altering the nitrogen-fixing symbiosis observed in MsSN1-overexpressing alfalfa transgenic plants opens the way to the production of effective legume transgenic cultivars for biotic stress resistance.

Exploring the intrinsic limits of nitrogenase transfer from bacteria to eukaryotes.

Biological nitrogen fixation is widespread among the Eubacteria and Archae domains but completely absent in eukaryotes. The lack of lateral transfer of nitrogen-fixation genes from prokaryotes to eukaryotes has been partially attributed to the physiological requirements necessary for the function of the nitrogenase complex. However, symbiotic bacterial nitrogenase activity is protected by the nodule, a plant structure whose organogenesis can be trigged in the absence of bacteria. To explore the intrinsic potentiality of this plant organ, we generated rhizobium-independent nodules in alfalfa by overexpressing the MsDMI3 kinase lacking the autoinhibitory domain. These transgenic nodules showed similar levels of leghemoglobin, free oxygen, ATP, and NADPH to those of efficient Sinorhizobium meliloti B399-infected nodules, suggesting that the rhizobium-independent nodules can provide an optimal microenvironment for nitrogenase activity. Finally, we discuss the intrinsic evolutionary constraints on transfer of nitrogen-fixation genes between bacteria and eukaryotes.

AQPX-cluster aquaporins and aquaglyceroporins are asymmetrically distributed in trypanosomes.

Major Intrinsic Proteins (MIPs) are membrane channels that permeate water and other small solutes. Some trypanosomatid MIPs mediate the uptake of antiparasitic compounds, placing them as potential drug targets. However, a thorough study of the diversity of these channels is still missing. Here we place trypanosomatid channels in the sequence-function space of the large MIP superfamily through a sequence similarity network. This analysis exposes that trypanosomatid aquaporins integrate a distant cluster from the currently defined MIP families, here named aquaporin X (AQPX). Our phylogenetic analyses reveal that trypanosomatid MIPs distribute exclusively between aquaglyceroporin (GLP) and AQPX, being the AQPX family expanded in the Metakinetoplastina common ancestor before the origin of the parasitic order Trypanosomatida. Synteny analysis shows how African trypanosomes specifically lost AQPXs, whereas American trypanosomes specifically lost GLPs. AQPXs diverge from already described MIPs on crucial residues. Together, our results expose the diversity of trypanosomatid MIPs and will aid further functional, structural, and physiological research needed to face the potentiality of the AQPXs as gateways for trypanocidal drugs.

Toward understanding of the high number of plant aquaporin isoforms and multiple regulation mechanisms.

Since the discovery of the first plant aquaporin (AQP) in 1993, our conception of the way plants control cell water homeostasis as well as their global water balance has been revisited. Plant AQPs constitute a large family of evolutionarily related channels that, in addition to water, can also facilitate the membrane diffusion of a number of small solutes, such as urea, CO2, H2O2, ammonia, metalloids, and even ions, indicating a wide range of cellular functions. At the cellular level, AQPs are subject to various regulation mechanisms leading to active/inactive channels in their target membranes. In this review, we discuss several specific questions that need to be addressed in future research. Why are so many different AQPs simultaneously expressed in specific cellular types? How is their selectivity to different solutes controlled (in particular in the case of multiple permeation properties)? What does the molecular interaction between AQPs and other molecules tell us about their regulation and their involvement in specific cellular and physiological processes? Resolving these questions will definitely help us better understand the physiological advantages that plants have to express and regulate so many AQP isoforms.

A proteome map of a quadruple photoreceptor mutant sustains its severe photosynthetic deficient phenotype.

Light is the environmental factor that most affects plant growth and development through its impact on photomorphogenesis and photosynthesis. A quadruple photoreceptor mutant lacking four of the most important photoreceptors in plants, phytochromes A and B (phyA, phyB) and cryptochromes 1 and 2 (cry1, cry2), is severely affected in terms of growth and development. Previous studies have suggested that in addition to a photomorphogenic disorder, the phyA phyB cry1 cry2 quadruple mutant might have severe alterations in photosynthetic ability. Here, we investigated the photosynthetic processes altered in the quadruple mutant and performed a proteomic profiling approach to identify some of the proteins involved. The phyA phyB cry1 cry2 quadruple mutant showed reduced leaf area and total chlorophyll content. Photosynthetic rates at high irradiances were reduced approximately 65% compared to the wild type (WT). Light-saturated photosynthesis and the response of net CO2 exchange to low and high internal CO2 concentrations suggest that the levels or activity of the components of the Calvin cycle and electron transport might be reduced in the quadruple mutant. Most of the under-expressed proteins in the phyA phyB cry1 cry2 quadruple mutant consistently showed a chloroplastic localization, whereas components of the Calvin cycle and light reaction centers were overrepresented. Additionally, Rubisco expression was reduced threefold in the phyA phyB cry1 cry2 quadruple mutant. Together, these results highlight the importance of the phytochrome and cryptochrome families in proper autotrophy establishment in plants. They also suggest that an overall limitation in the chlorophyll levels, expression of Rubisco, and enzymes of the Calvin Cycle and electron transport that affect ribulose-1,5-biphosphate (RuBP) regeneration reduced photosynthetic capacity in the phyA phyB cry1 cry2 quadruple mutant.

Understanding the function of bacterial and eukaryotic thiolases II by integrating evolutionary and functional approaches.

Acetoacetyl-CoA thiolase (EC 2.3.1.9), commonly named thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA and CoA. This enzyme acts in anabolic processes as the first step in the biosynthesis of isoprenoids and polyhydroxybutyrate in eukaryotes and bacteria, respectively. We have recently reported the evolutionary and functional equivalence of these enzymes, suggesting that thiolase II could be the rate limiting enzyme in these pathways and presented evidence indicating that this enzyme modulates the availability of reducing equivalents during abiotic stress adaptation in bacteria and plants. However, these results are not sufficient to clarify why thiolase II was evolutionary selected as a critical enzyme in the production of antioxidant compounds. Regarding this intriguing topic, we propose that thiolase II could sense changes in the acetyl-CoA/CoA ratio induced by the inhibition of the tricarboxylic acid cycle under abiotic stress. Thus, the high level of evolutionary and functional constraint of thiolase II may be due to the connection of this enzyme with an ancient and conserved metabolic route.

Engineering Pseudomonas protegens Pf-5 for nitrogen fixation and its application to improve plant growth under nitrogen-deficient conditions.

Nitrogen is the second most critical factor for crop production after water. In this study, the beneficial rhizobacterium Pseudomonas protegens Pf-5 was genetically modified to fix nitrogen using the genes encoding the nitrogenase of Pseudomonas stutzeri A1501 via the X940 cosmid. Pf-5 X940 was able to grow in L medium without nitrogen, displayed high nitrogenase activity and released significant quantities of ammonium to the medium. Pf-5 X940 also showed constitutive expression and enzymatic activity of nitrogenase in ammonium medium or in nitrogen-free medium, suggesting a constitutive nitrogen fixation. Similar to Pseudomonas protegens Pf-5, Pseudomonas putida, Pseudomonas veronii and Pseudomonas taetrolens but not Pseudomonas balearica and Pseudomonas stutzeri transformed with cosmid X940 showed constitutive nitrogenase activity and high ammonium production, suggesting that this phenotype depends on the genome context and that this technology to obtain nitrogen-fixing bacteria is not restricted to Pf-5. Interestingly, inoculation of Arabidopsis, alfalfa, tall fescue and maize with Pf-5 X940 increased the ammonium concentration in soil and plant productivity under nitrogen-deficient conditions. In conclusion, these results open the way to the production of effective recombinant inoculants for nitrogen fixation on a wide range of crops.