Phosphorylation influences water and ion channel function of AtPIP2;1

The phosphorylation state of two serine residues within the C‐terminal domain of AtPIP2;1 (S280, S283) regulates its plasma membrane localization in response to salt and osmotic stress. Here, we investigated whether the phosphorylation state of S280 and S283 also influence AtPIP2;1 facilitated water and cation transport. A series of single and double S280 and S283 phosphomimic and phosphonull AtPIP2;1 mutants were tested in heterologous systems. In Xenopus laevis oocytes, phosphomimic mutants AtPIP2;1 S280D, S283D, and S280D/S283D had significantly greater ion conductance for Na+ and K+, whereas the S280A single phosphonull mutant had greater water permeability. We observed a phosphorylation‐dependent inverse relationship between AtPIP2;1 water and ion transport with a 10‐fold change in both. The results revealed that phosphorylation of S280 and S283 influences the preferential facilitation of ion or water transport by AtPIP2;1. The results also hint that other regulatory sites play roles that are yet to be elucidated. Expression of the AtPIP2;1 phosphorylation mutants in Saccharomyces cerevisiae confirmed that phosphorylation influences plasma membrane localization, and revealed higher Na+ accumulation for S280A and S283D mutants. Collectively, the results show that phosphorylation in the C‐terminal domain of AtPIP2;1 influences its subcellular localization and cation transport capacity.This research was supported by the Australian Research Council (ARC) in the form of DP190102725, and Future Fellowship for CB (FT180100476); J. Q. and S. T. were supported through the ARC Centre of Excellence in Plant Energy Biology (CE140100008). M. G. was funded by the ARC Centre of Excellence for Translational Photosynthesis (CE1401000015)

A Survey of Barley PIP Aquaporin Ionic Conductance Reveals Ca2+-Sensitive HvPIP2;8 Na+ and K+ Conductance

Some plasma membrane intrinsic protein (PIP) aquaporins can facilitate ion transport. Here we report that one of the 12 barley PIPs (PIP1 and PIP2) tested, HvPIP2;8, facilitated cation transport when expressed in Xenopus laevis oocytes. HvPIP2;8-associated ion currents were detected with Na+ and K+, but not Cs+, Rb+, or Li+, and was inhibited by Ba2+, Ca2+, and Cd2+ and to a lesser extent Mg2+, which also interacted with Ca2+. Currents were reduced in the presence of K+, Cs+, Rb+, or Li+ relative to Na+ alone. Five HvPIP1 isoforms co-expressed with HvPIP2;8 inhibited the ion conductance relative to HvPIP2;8 alone but HvPIP1;3 and HvPIP1;4 with HvPIP2;8 maintained the ion conductance at a lower level. HvPIP2;8 water permeability was similar to that of a C-terminal phosphorylation mimic mutant HvPIP2;8 S285D, but HvPIP2;8 S285D showed a negative linear correlation between water permeability and ion conductance that was modified by a kinase inhibitor treatment. HvPIP2;8 transcript abundance increased in barley shoot tissues following salt treatments in a salt-tolerant cultivar Haruna-Nijo, but not in salt-sensitive I743. There is potential for HvPIP2;8 to be involved in barley salt-stress responses, and HvPIP2;8 could facilitate both water and Na+/K+ transport activity, depending on the phosphorylation status

A single nucleotide substitution in TaHKT1;5-D controls shoot Na+ accumulation in bread wheat

Improving salinity tolerance in the most widely cultivated cereal, bread wheat (Triticum aestivum L.), is essential to increase grain yields on saline agricultural lands. A Portuguese landrace, Mocho de Espiga Branca accumulates up to sixfold greater leaf and sheath sodium (Na+) than two Australian cultivars, Gladius and Scout, under salt stress in hydroponics. Despite high leaf and sheath Na+ concentrations, Mocho de Espiga Branca maintained similar salinity tolerance compared to Gladius and Scout. A naturally occurring single nucleotide substitution was identified in the gene encoding a major Na+ transporter TaHKT1;5-D in Mocho de Espiga Branca, which resulted in a L190P amino acid residue variation. This variant prevents Mocho de Espiga Branca from retrieving Na+ from the root xylem leading to a high shoot Na+ concentration. The identification of the tissue-tolerant Mocho de Espiga Branca will accelerate the development of more elite salt-tolerant bread wheat cultivars

Barley sodium content is regulated by natural variants of the Na+ transporter HvHKT1;5

During plant growth, sodium (Na+) in the soil is transported via the xylem from the root to the shoot. While excess Na+ is toxic to most plants, non-toxic concentrations have been shown to improve crop yields under certain conditions, such as when soil K+ is low. We quantified grain Na+ across a barley genome-wide association study panel grown under non-saline conditions and identified variants of a Class 1 HIGH-AFFINITY-POTASSIUM-TRANSPORTER (HvHKT1;5)-encoding gene responsible for Na+ content variation under these conditions. A leucine to proline substitution at position 189 (L189P) in HvHKT1;5 disturbs its characteristic plasma membrane localisation and disrupts Na+ transport. Under low and moderate soil Na+, genotypes containing HvHKT1:5P189 accumulate high concentrations of Na+ but exhibit no evidence of toxicity. As the frequency of HvHKT1:5P189 increases significantly in cultivated European germplasm, we cautiously speculate that this non-functional variant may enhance yield potential in non-saline environments, possibly by offsetting limitations of low available K+.R.W., M.S., and M.M. acknowledge support from ERC project 669182 ‘SHUFFLE’ to R.W. K.H., P.S., M.B., J.R., and R.W. acknowledge the Rural & Environment Science & Analytical Services Division of the Scottish Government. C.B., J.Q., and Y.Q. acknowledge support from Rutherford Fund Strategic Partner Grants 2018 - Award Reference: RF-2018-30 to C.H. and R.W. S.R. is grateful for financial assistance from The Australian Research Council Industrial Transformation Research Hub for Wheat in a Hot and Dry Climate (IH130200027), the Grains Research and Development Corporation (ACP00009) and The Waite Research Institute, University of Adelaide. C.B. acknowledges support from the Grains Research and Development Corporation (GRDC) and the Australian Research Council (FT180100476). We are thankful to the Australian Research Council for funding through CE140100008 to M.G. (J.Q. and Y.Q.), FT180100476 to C.S.B. and DE160100804 to S.W. (A.S.). D.E.S. acknowledges support from BBSRC Grant BB/L000113/1. MH acknowledges financial support from the Huaiyin Normal University, Chin

A chloroplast retrograde signal, 3'-phosphoadenosine 5'-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination

Organelle-nuclear retrograde signaling regulates gene expression, but its roles in specialized cells and integration with hormonal signaling remain enigmatic. Here we show that the SAL1-PAP (3′-phosphoadenosine 5′- phosphate) retrograde pathway interacts with abscisic acid (ABA) signaling to regulate stomatal closure and seed germination in Arabidopsis. Genetically or exogenously manipulating PAP bypasses the canonical signaling components ABA Insensitive 1 (ABI1) and Open Stomata 1 (OST1); priming an alternative pathway that restores ABA-responsive gene expression, ROS bursts, ion channel function, stomatal closure and drought tolerance in ost1-2. PAP also inhibits wild type and abi1-1 seed germination by enhancing ABA sensitivity. PAP-XRN signaling interacts with ABA, ROS and Ca2+; up-regulating multiple ABA signaling components, including lowly-expressed Calcium Dependent Protein Kinases (CDPKs) capable of activating the anion channel SLAC1. Thus, PAP exhibits many secondary messenger attributes and exemplifies how retrograde signals can have broader roles in hormone signaling, allowing chloroplasts to fine-tune physiological responsesCE140100008; DE14010114

Investigation of chloride transport mechanisms in Arabidopsis thaliana root

Salinity tolerance is correlated with shoot chloride (Cl⁻) exclusion in many horticultural and crop species (e.g. grapevine, soybean). It is hypothesized that the key regulatory step in root-to-shoot transfer of Cl⁻ is conferred by plasma membrane-localised anion transporters associated within the root vasculature. Reducing long-distance Cl⁻ transport by manipulating the regulation of anion transporters in the root vasculature is therefore a strategy that promises to increase plant tolerance to saline environments. However, the information of which candidate genes are responsible for this process is limited. To gain a greater knowledge of the long distance Cl⁻ movement from a molecular aspect, a number of candidate anion transporters from Arabidopsis thaliana were identified from a preliminary microarray study. Quantitative PCR was used to indicate transcriptional levels of candidate anion transporters that decreased upon NaCl and ABA treatment. Based on this analysis, AtSLAH1, AtSLAH3 and AtNRT1.5 were selected as genes of interest (GOI) that were likely to be involved in the Cl⁻ movement between the root stele symplast and the xylem vessels. To functionally characterize the transport properties of all GOIs at a protein level, various heterologous systems were used to investigate the anion (Cl⁻ and NO₃⁻) transport capacity. Two-electrode voltage clamp electrophysiology was used to measure the currents that were generated by the target anions crossing oocyte membranes. A yeast expression system was also used to further study the anion transport properties in vitro. AtSLAH1 cRNA injected oocytes were not able to produce significant anion currents. Also, no evident anion currents were generated from a site-directed mutant of AtSLAH1 in a putative phosphorylation site injected into oocytes. Although there was evidence that anion currents were elicited from AtSLAH1 and AtSnRk2.3 co-injected oocytes, due to difficulties in the ability to reproduce these results, it is uncertain whether AtSLAH1 can function as an anion transporter in the conditions tested. Both wild type and site-mutated AtSLAH1 was also separately transformed into yeast for further examination without an observable phenotype. In order to examine the effect of altered AtSLAH1 expression on shoot anion accumulation, AtSLAH1 amiRNA knockdown and constitutive over expression of AtSLAH1 mutant plants were generated. AtSLAH1 knockdown lines (T2) exhibited strong repression in transcript abundance in low salt environments and resulted in a significant reduction in shoot Cl⁻ when compared to nulls. Constitutive over expression of AtSLAH1 showed increased shoot Cl⁻ contents under high salt stress. These results indicated the potential role of AtSLAH1 in Cl⁻ transport in plants. Electrophysiological characterization of AtSLAH3 in oocytes showed that AtSLAH3 was able to produce significant NO₃⁻ but not Cl⁻ currents suggesting a role in the efflux of NO₃⁻ out of cells in most of circumstances. Similar results were gained in AtSLAH3- transformed yeast. However, AtSLAH3 over-expression lines showed a decreased shoot Cl⁻ without an effect on shoot NO₃⁻ under high salt stress compared to null plants. The potential reasons for this are discussed and further experiments are proposed to test these hypotheses. Although AtNRT1.5 has been reported to transport NO₃⁻, electrophysiological characterization of AtNRT1.5 in X. Laevis oocytes was not able to detect any anion currents induced by the gene. Interestingly, AtNRT1.5 transformed yeast showed a significant inhibited phenotype (grow less well than empty vector control) when challenged with high concentration of Cl⁻ and NO₃⁻ within the growth media, indicating a role the transport of both anions. Constitutive over-expression lines showed a potent shoot Cl⁻ reduction under high salt stress compared to nulls. Interestingly, no significant NO₃⁻ accumulation in shoot was identified. These results might suggest that AtNRT1.5 was able to regulate both Cl⁻ and NO₃⁻ transport from root to shoot; however, the mechanism by which this occurs is unclear. Previous findings indicated the possibilities that Cl⁻ and NO₃⁻ can be transported through the same anion channel/transporter. To further study the regulation of Cl⁻ and NO₃⁻ uptake, an anion blocker (DIDS) was used to test the anion shoot accumulation under different salt conditions. Under high salt stress, DIDS was able to reduce the Cl⁻ accumulation and increase the NO₃⁻ contents in shoots. Further experiments are required at both a physiological and molecular level to further understand how plants recognize and respond to this blocker, as the molecular targets of this blocker are a potential way to improve the plant salt tolerance and nitrogen use efficiency under high salt stress. In summary, new information was revealed on several candidates that affect root-to-shoot loading of chloride and new research avenues have been proposed based on the findings of this study.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2015

Inoculation of Prickly Pear Litter with Microbial Agents Promotes the Efficiency in Aerobic Composting

Prickly pear (Rosa roxburghii Tratt), a shrub mainly distributed in South China, is an economically essential plant for helping the local people out of poverty. To efficiently provide sufficient nutrients to the plant in the soil for the ecological cultivation of prickly pear, we studied the aerobic composting of a prickly pear litter with three agents, including AC (Bacillus natto, Bacillus sp., Actinomycetes sp., Saccharomyces sp., Trichoderma sp., Azotobacter sp., and Lactobacillus sp.), BC (Bacillus subtilis, Lactobacillaceae sp., Bacillus licheniformis, Saccharomyces sp., and Enterococcus faecalis), and CC (Bacillus sp., Actinomycetes sp., Lactobacillaceae sp., Saccharomyces sp., and Trichoderma sp.) and a control without microbial agents. The results show that the physicochemical and microbial traits of three resultant prickly pear composts were different after the inoculation with AC, BC, or CC. The pH values of three composts ranged from 8.0 to 8.5, and their conductivity values were between 1.6 and 1.9 mS/cm. The seed germination index of all three composts exceeded 70%. The contents of volatile solids and organic matter of the three composts both decreased significantly. The BC maximally increased the total N (18%) of the compost, whereas the CC maximally increased the total P (48%) and total K (38%) contents. Contents of available P and available K of the three composts increased significantly, and the available N content in compost after BC inoculation increased by 16%. The physicochemical features showed that three composts were non-hazardous to plants, and the microbial agents improved nutrient availability. The richness, Chao1, and Shannon index in the bacterial communities of three composts increased significantly. At the phylum level, Proteobacteria, Bacteroidetes, and Firmicutes bacterium became dominant in the three composts, whereas at the family level, Microscillaceae and A4b (phylum Chloroflexi) became the dominant groups. Abundant cellulose-degrading bacteria existed at the dominant phylum level, which promoted fiber degradation in composts. Organic matter and the available N content regulated the composting bacterium. The inoculants enhanced the efficiency of composting: agents B and C were more suitable exogenous inoculants for the composting of a prickly pear litter

A single residue deletion in the barley HKT1;5 P189 variant restores plasma membrane localisation but not Na⁺ conductance

Available online 15 June 2021Leaf Na⁺ exclusion, mediated by plasma membrane-localised Class 1 High-affinity potassium (K⁺) Transporters (HKTs), is a key mechanism contributing to salinity tolerance of several major crop plants. We determined previously that the leucine to proline residue substitution at position 189 (L189P) in barley HvHKT1;5 disrupts its characteristic plasma membrane localisation and Na⁺ conductance. Here, we focus on a surprising observation that a single residue deletion of methionine at position 372 (M372del) within the conserved VMMYL motif in plant HKTs, restores plasma membrane localisation but not Na⁺ conductance in HvHKT1;5 P189. To clarify why the singular M372 deletion regains plasma membrane localisation, we built 3D models and defined α-helical assembly pathways of the P189 M372del mutant, and compared these findings to the wild-type protein, and the HvHKT1;5 L189 variant and its M372del mutant. We find that α-helical association and assembly pathways in HvHKT1;5 proteins fall in two contrasting categories. Inspections of structural flexibility through molecular dynamics simulations revealed that the conformational states of HvHKT1;5 P189 diverge from those of the L189 variant and M372del mutants. We propose that M372del in HvHKT1;5 P189 instigates structural rearrangements allowing routing to the plasma membrane, while the restoration of conductance would require further interventions. We integrate the microscopy, electrophysiology, and biocomputational data and discuss how a profound structural change in HvHKT1;5 P189 M372del impacts its α-helical protein association pathway and flexibility, and how these features underlie a delicate balance leading to restoring plasma membrane localisation but not Na⁺ conductance.Stefanie Wege, Jiaen Qiu, Caitlin Byrt, Kelly Houston, Robbie Waugh, Matthew Gilliham, Maria Hrmov

Divalent Cations Regulate the Ion Conductance Properties of Diverse Classes of Aquaporins

Aquaporins (AQPs) are known to facilitate water and solute fluxes across barrier membranes. An increasing number of AQPs are being found to serve as ion channels. Ion and water permeability of selected plant and animal AQPs (plant Arabidopsis thaliana AtPIP2;1, AtPIP2;2, AtPIP2;7, human Homo sapiens HsAQP1, rat Rattus norvegicus RnAQP4, RnAQP5, and fly Drosophila melanogaster DmBIB) were expressed in Xenopus oocytes and examined in chelator-buffered salines to evaluate the effects of divalent cations (Ca2+, Mg2+, Ba2+ and Cd2+) on ionic conductances. AtPIP2;1, AtPIP2;2, HsAQP1 and DmBIB expressing oocytes had ionic conductances, and showed differential sensitivity to block by external Ca2+. The order of potency of inhibition by Ca2+ was AtPIP2;2 > AtPIP2;1 > DmBIB > HsAQP1. Blockage of the AQP cation channels by Ba2+ and Cd2+ caused voltage-sensitive outward rectification. The channels with the highest sensitivity to Ca2+ (AtPIP2;1 and AtPIP2;2) showed a distinctive relief of the Ca2+ block by co-application of excess Ba2+, suggesting that divalent ions act at the same site. Recognizing the regulatory role of divalent cations may enable the discovery of other classes of AQP ion channels, and facilitate the development of tools for modulating AQP ion channels. Modulators of AQPs have potential value for diverse applications including improving salinity tolerance in plants, controlling vector-borne diseases, and intervening in serious clinical conditions involving AQPs, such as cancer metastasis, cardiovascular or renal dysfunction