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SUMMARY: 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. [ABSTRACT FROM AUTHOR]

The absorption of soil water by roots allows plants to maintain their water status. At the endodermis, water transport can be affected by initial formation of a Casparian strip and further deposition of suberin lamellas and regulated by the function of aquaporins. Four Casparian strip membrane domain protein‐like (CASPL; CASPL1B1, CASPL1B2, CASPL1D1, and CASPL1D2) were previously shown to interact with PIP2;1. The present work shows that CASPL1B1, CASPL1B2, and CASPL1D2 are exclusively expressed in suberized endodermal cells, suggesting a cell‐specific role in suberization and/or water transport regulation. When compared with wild‐type plants, and by contrast to caspl1b1*caspl1b2 double loss of function, caspl1d1*caspl1d2 double mutants showed, in some control or NaCl stress experiments and not upon abscisic acid (ABA) treatment, a weak enlargement of the continuous suberization zone. None of the mutants showed root hydraulic conductivity (Lpr) phenotype, whether in control, NaCl, or ABA treatment conditions. The data suggest a slight negative role for CASPL1D1 and CASPL1D2 in suberization under control or salt stress conditions, with no major impact on whole root transport functions. At the molecular level, CASPL1B1 was able to physically interact with PIP2;1 and potentially could influence the regulation of aquaporins by acting on their phosphorylated form. At the endodermis, water transport can be affected by initial formation of a Casparian strip and further deposition of suberin lamellas and regulated by the function of aquaporins. Four Casparian strip membrane domain protein‐like (CASPL; CASPL1B1, CASPL1B2, CASPL1D1, and CASPL1D2) were recently shown to interact with PIP2;1. The present work shows an exclusive expression of CASPL1B1, CASPL1B2 and CASPL1D2 in suberized endodermal cells and describes a slight negative role for CASPL1D1 and CASPL1D2 in suberization under control or salt stress conditions, with no major impact on whole root transport functions. At the molecular level, CASPL1B1 was able to physically interact with PIP2;1 and potentially could influence the regulation of aquaporins by acting on their phosphorylated form. [ABSTRACT FROM AUTHOR]

The hydraulic conductivity of plant roots ( Lpr) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post-translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 ( PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lpr of knockout A rabidopsis plants for four Ca2+-dependent protein kinases. cpk7 plants showed a 30% increase in Lpr because of a higher aquaporin activity. A quantitative proteomic analysis of wild-type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lpr of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7-dependent stability of specific membrane proteins. [ABSTRACT FROM AUTHOR]

Osmotic stress can be detrimental to plants, whose survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic-mediated stress. In this study, we used the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry to compile a list of 719 ubiquitinated lysine (K-Ub) residues from 450 Arabidopsis root membrane proteins (58% of which are transmembrane proteins), thereby adding to the database of ubiquitinated substrates in plants. Although no ubiquitin (Ub) motifs could be identified, the presence of acidic residues close to K-Ub was revealed. Our ubiquitinome analysis pointed to a broad role of ubiquitination in the internalization and sorting of cargo proteins. Moreover, the simultaneous proteome and ubiquitinome quantification showed that ubiquitination is mostly not involved in membrane protein degradation in response to short osmotic treatment but that it is putatively involved in protein internalization, as described for the aquaporin PIP2;1. Our in silico analysis of ubiquitinated proteins shows that two E2 Ub-conjugating enzymes, UBC32 and UBC34, putatively target membrane proteins under osmotic stress. Finally, we revealed a positive role for UBC32 and UBC34 in primary root growth under osmotic stress. [ABSTRACT FROM AUTHOR]

Abstract: Background: Aquaporins are membrane channels that facilitate the transport of water and small neutral molecules across biological membranes of most living organisms. Scope of review: Here, we present comprehensive insights made on plant aquaporins in recent years, pointing to their molecular and physiological specificities with respect to animal or microbial counterparts. Major conclusions: In plants, aquaporins occur as multiple isoforms reflecting a high diversity of cellular localizations and various physiological substrates in addition to water. Of particular relevance for plants is the transport by aquaporins of dissolved gases such as carbon dioxide or metalloids such as boric or silicic acid. The mechanisms that determine the gating and subcellular localization of plant aquaporins are extensively studied. They allow aquaporin regulation in response to multiple environmental and hormonal stimuli. Thus, aquaporins play key roles in hydraulic regulation and nutrient transport in roots and leaves. They contribute to several plant growth and developmental processes such as seed germination or emergence of lateral roots. General significance: Plants with genetically altered aquaporin functions are now tested for their ability to improve plant resistance to stresses. This article is part of a Special Issue entitled Aquaporins. [Copyright &y& Elsevier]

Methylation of biologically active molecules is achieved by methyltransferases (MTases). MTases can act on proteins through N- or O-carboxylmethylation reactions. Methylation of lysine and glutamic acid residues was recently described on the N-terminal tail of AtPIP2;1, a plasma membrane aquaporin of plants. In this study, we combine a bioinformatic and a biochemical screen and identify two MTases of Arabidopsis thaliana, SDG7 (At2g44150) and OMTF3 (At3g61990), as acting on the N-terminal tail of AtPIP2;1, at Lys3 and Glu6, respectively. Confocal microscopy imaging showed the two enzymes to be associated with the endoplasmic reticulum. An in vitro assay using various AtPIP2;1 N-terminal peptides as a bait allowed characterization of the enzymatic properties of recombinant SDG7 and OMTF3. The two enzymes showed minimal apparent Km values for their substrates, S-adenosylmethionine and peptide, in the range of 5–8 and 2–9 μM, respectively. SDG7 was shown to almost exclusively mono- or di-methylate Lys3. In contrast, OMTF3 specifically methylated Glu6, this methylation being dependent on the methylation profile of the neighboring Lys3 residue. In conclusion, this study allows the characterization of the first MTases able to methylate plant transmembrane proteins and provides the first identification of a glutamate-MTase in eukaryotes. [ABSTRACT FROM AUTHOR]

Summary Mechanisms that regulate water channels in the plant plasma membrane (PM) were investigated in Arabidopsis suspension cells. Cell hydraulic conductivity was measured with a cell pressure probe and was reduced 4-fold as compared to control values when calcium was added in the pipette and in bathing solution. To assess the significance of these effects in vitro , PM vesicles were isolated by aqueous two-phase partitioning and their water transport properties were characterized by stopped-flow spectrophotometry. Membrane vesicles isolated in standard conditions exhibited reduced water permeability (P f ) together with a lack of active water channels. In contrast, when prepared in the presence of chelators of divalent cations, PM vesicles showed a 2.3-fold higher P f and active water channels. Furthermore, equilibration of purified PM vesicles with divalent cations reduced their P f and water channel activity down to the basal level of membranes isolated in standard conditions. Ca2+ was the most efficient with a half-inhibition of P f at 50–100 µm free Ca2+ . Water transport in purified PM vesicles was also reversibly blocked by H+ , with a half-inhibition of P f at pH 7.2–7.5. Thus, both Ca2+ and H+ contribute to a membrane-delimited switch from active to inactive water channels that may allow coupling of water transport to cell signalling and metabolism. [ABSTRACT FROM AUTHOR]