Showing papers by "Ray A. Bressan published in 2016"

The miR165/166 Mediated Regulatory Module Plays Critical Roles in ABA Homeostasis and Response in Arabidopsis thaliana

Abstract: The function of miR165/166 in plant growth and development has been extensively studied, however, its roles in abiotic stress responses remain largely unknown. Here, we report that reduction in the expression of miR165/166 conferred a drought and cold resistance phenotype and hypersensitivity to ABA during seed germination and post-germination seedling development. We further show that the ABA hypersensitive phenotype is associated with a changed transcript abundance of ABA-responsive genes and a higher expression level of ABI4, which can be directly regulated by a miR165/166 target. Additionally, we found that reduction in miR165/166 expression leads to elevated ABA levels, which occurs at least partially through the increased expression of BG1, a gene that is directly regulated by a miR165/166 target. Taken together, our results uncover a novel role for miR165/166 in the regulation of ABA and abiotic stress responses and control of ABA homeostasis.

A Single Amino-Acid Substitution in the Sodium Transporter HKT1 Associated with Plant Salt Tolerance

Abstract: A crucial prerequisite for plant growth and survival is the maintenance of potassium uptake, especially when high sodium surrounds the root zone. The Arabidopsis HIGH-AFFINITY K+ TRANSPORTER1 (HKT1), and its homologs in other salt-sensitive dicots, contributes to salinity tolerance by removing Na+ from the transpiration stream. However, TsHKT1;2, one of three HKT1 copies in Thellungiella salsuginea, a halophytic Arabidopsis relative, acts as a K+ transporter in the presence of Na+ in yeast (Saccharomyces cerevisiae). Amino-acid sequence comparisons indicated differences between TsHKT1;2 and most other published HKT1 sequences with respect to an Asp residue (D207) in the second pore-loop domain. Two additional T. salsuginea and most other HKT1 sequences contain Asn (n) in this position. Wild-type TsHKT1;2 and altered AtHKT1 (AtHKT1N-D) complemented K+-uptake deficiency of yeast cells. Mutant hkt1-1 plants complemented with both AtHKT1N-D and TsHKT1;2 showed higher tolerance to salt stress than lines complemented by the wild-type AtHKT1. Electrophysiological analysis in Xenopus laevis oocytes confirmed the functional properties of these transporters and the differential selectivity for Na+ and K+ based on the n/d variance in the pore region. This change also dictated inward-rectification for Na+ transport. Thus, the introduction of Asp, replacing Asn, in HKT1-type transporters established altered cation selectivity and uptake dynamics. We describe one way, based on a single change in a crucial protein that enabled some crucifer species to acquire improved salt tolerance, which over evolutionary time may have resulted in further changes that ultimately facilitated colonization of saline habitats.

Pyl9 and uses thereof

Abstract: The present invention provides for transgenic plants and methods of producing such transgenic plants, wherein the transgenic plants express an increased amounts of PYL9 to interact with abscisic acid (ABA) thereby activating enhanced drought resistance and leaf senescence relative to control or wild-type plants.

A computational analysis of Salt Overly Sensitive 1 homologs in halophytes and glycophytes

Abstract: Soil salinity is one of the most serious impediments to global agricultural productivity. Although most terrestrial plants are glycophytes which cannot tolerate high salt concentrations, a small fraction of species are halophytes. Exactly what allows these extremophile plants to survive in saline conditions is not yet well understood. Several studies have established the Salt Overly Sensitive (SOS) pathway as the canonical model for the mechanism responsible for salt tolerance. The SOS pathway involves interplay among Na-H antiporters for transporting sodium, and the activation of the kinase that phosphorylates the transporter. Among them, SOS1, a plasma membrane Na-H antiporter, has been shown to be a critical component for maintaining salt homeostasis by pumping sodium out of cells upon activation. Therefore, it is of great interest to evaluate any differences of SOS1 in halophytes as compared to glycophytes. Here we report a computational analysis of the primary and secondary structures of eight halophytes and seven glycophytes. ClustalW alignment of the protein sequences as a whole reveals no regions conserved specifically in only halophytes or in only glycophytes. In addition, the key regulatory residues at the C-terminus of SOS1, S1136 and S1138, which were shown to be the phosphorylation sites by the kinase SOS2, were completely conserved in all 15 halophytes and glycophytes. The four amino acids, G136, R365, G777, and G784, in which alterations affect the function of SOS1, are mostly conserved in the 15 species. The 14-3-3 binding site in the C-terminus which is important in the phosphorylation step of SOS1 in the SOS signal transduction cascade is also well conserved. Furthermore, the number of transmembrane helices for each species is between 9 and 12 and there is no significant difference between halophytes and glycophytes. If halophytes present any special feature of SOS1, it likely involves the presence (halophytes) or absence (glycophytes) of a SOS1-interacting component. PeerJ PrePrints | https://doi.org/10.7287/peerj.preprints.1668v1 | CC-BY 4.0 Open Access | rec: 21 Jan 2016, publ: 21 Jan 2016 Introduction Salt stress is a major abiotic stress factor that significantly impacts agricultural output. Approximately 20% of the world's cultivated land and nearly half of all irrigated lands are affected by salinity (Zhu, 2001). High salinity can adversely affect plants by inducing physiological responses at the cellular level such as ion imbalance and hyperosmotic stress as well as by altering outward physical characteristics through wilting, drying, and death (Zhu, 2001). However, plants differ in their ability to tolerate high salt concentrations. Halophytes, which make up around 1% of the world's flora, are an extremophile group of plants that are able to survive in highly saline soils (Flowers and Colmer, 2008; Flowers and Colmer, 2015). In contrast, the majority of plants, including most commercial crops, are glycophytes, which are not necessarily salt-intolerant but cannot survive in environments where the salt concentration is too high (Garg et al., 2013; Ji et al., 2013). Even so, the line separating halophytes from glycophytes is not discrete. There is actually a continuum of salt tolerance degrees with very salt-sensitive glycophytes at the low end and highly salt-tolerant halophytes at the high end (Rozema and Schat, 2012). Furthermore, within halophytes, many dicotyledonous halophytes grow optimally in 50-250 mM NaCl while monocotyledonous halophytes prefer less than 50 mM NaCl (Flowers and Colmer, 2008). We limit our species to dicotyledonous halophytes only in order to minimize any conflicting effects due to phylogenetics on salt tolerance ability. The factors that make halophytes different from glycophytes are not yet well understood. However, the mechanism(s) by which plants respond to increased salt concentrations has been extensively studied. The protein kinase cascade involving Salt Overly Sensitive (SOS) 1,2, and 3 has emerged as a predominant model for salt stress responses, as shown in Fig. 1. The SOS signal transduction pathway is activated when SOS3, a calcium-binding protein, binds with Ca, which is well-known as a "warning" signal for Na toxicity (Ji et al., 2013; Zhu, 2001). SOS3 then interacts with and activates SOS2, a serine/threonine kinase, and SOS2 phosphorylates SOS1, a plasma membrane Na-H antiporter, resulting in Na+ being transported out from the cell. SOS1 in particular has been identified as playing a key role in the reactions of plants to salinity stress. Mutants of SOS1 display stunted growth and significant leaf discoloration in environments with high Na, whereas transgenic sos1-1 mutants containing the wild-type SOS1 gene controlled by the cauliflower mosaic virus 35s promoter show no or little difference in PeerJ PrePrints | https://doi.org/10.7287/peerj.preprints.1668v1 | CC-BY 4.0 Open Access | rec: 21 Jan 2016, publ: 21 Jan 2016 growth in salt compared with the wild type, demonstrating that the SOS1 locus is necessary, but not necessarily sufficient, for salt tolerance (Shi et al., 2000). In addition, SOS1 expression is upregulated in plants in response to NaCl stress (Shi et al., 2000). The C-terminal region, where phosphorylation sites and 14-3-3binding sites are located, is also proposed to be important for SOS1 function in plant salt tolerance (Shi et al., 2000). In this study we present the comparison of SOS1 protein sequences of eight halophytes and seven glycophytes. We looked for distinctly conserved regions as a whole, at key mutation positions, and near the C-terminus, and we determined the number of transmembrane helices in each species to analyze secondary structure. Materials & Methods SOS1 and SOS1 homologs were identified using the BLASTp search of the NCBI protein database, using previously identified SOS1 from Arabidopsis from The Arabidopsis Information Resource (TAIR) gene database (arabidopsis.org). The top BLASTp hit was used as the homolog for each species. Protein sequences were aligned using ClustalW alignment in the sequence alignment editor BioEdit. The number of transmembrane helices were determined using the TMHMM Server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), which predicts transmembrane helices in proteins. Results & Discussion To identify any regions conserved uniquely in either halophytes or glycophytes in SOS1, we first collected the SOS1 protein sequences of eight halophytes and seven glycophytes, using NCBI Blast with Arabidopsis thaliana SOS1 as a query. The halophyte species analyzed were Eutrema parvulum, Eutrema halophila, Salicornia dolichostachya, Aeluropus littoralis, Suaeda salsa, Salicornia brachiata, Mesembryanthemum crystallinum, and Distichlis spicata (Table 1). The glycophyte species analyzed were Arabidopsis thaliana, Solanum lycopersicum, Oryza sativa Indica group, Sorghum bicolor, Gossypium hirsutum, Selaginella moellendorffii, and Physcomitrella patens (Table 1). ClustalW alignment was used to compare the sequences at the primary level. Although there is variation among species, no differentiating regions between halophyte and glycophyte protein sequences were found at the primary level. That is, there were PeerJ PrePrints | https://doi.org/10.7287/peerj.preprints.1668v1 | CC-BY 4.0 Open Access | rec: 21 Jan 2016, publ: 21 Jan 2016 no regions where all of the halophyte sequences had the same (or similar) amino acid and all of the glycophyte sequences did not have that amino acid, or vice versa (Fig. 2). Several sos1 mutant alleles in Arabidopsis thaliana were initially isolated from a mutant screen looking for mutants having altered salt response. When treated with 100mM NaCl, sos1 mutants showed sensitive responses such as arrested growth, chlorosis in older leaves, and dark color in young leaves., The genetic mapping study revealed that four of these alleles (sos1-3, sos1-8, sos1-9, and sos1-12) had missense mutations in SOS1 which result in amino acid substitutions such as Gly 136 to Glu, Arg 365 to Cys, Gly 777 to Glu, and Gly 784 to Asp in sos1-3, sos1-8, sos1-9, and sos1-12, respectively. This indicates that the amino acids at these particular positions are crucial (Shi et al., 2000). Sequence alignment showed that amino acids are mostly conserved at these crucial missense mutation areas (Fig. 3A). At region A, only S. brachiata showed a deviation from the amino acids as its sequence had arginine instead of glycine at this position. Mutation regions B and D were completely conserved in all species and at region C, there was another substitution of arginine for glycine, this time with S. lycopersicum. It has been proposed that there are key regulatory residues at the C-terminus of SOS1. Previously, 1136 Serine and 1138 Serine in the C-terminus of Arabidopsis SOS1 were shown to be the phosphorylation sites by SOS2 (Quintero et al., 2010). These two amino acid positions were completely conserved in all 15 halophytes and glycophytes (Fig. 3B). Arabidopsis SOS2 phosphorylates these serine residues and facilitates binding by a 14-3-3 protein. An alignment of the SOS1 protein sequences of A. thaliana and P. patens shows strong conservation, suggesting a common mechanism of sodium tolerance shared by plants (Kleist et al., 2014). The sequences were also strongly conserved at this 14-3-3 binding site in the C-terminus (Fig. 3B). These data show that since both the sequences as a whole, key mutation areas, and important binding sites are all mostly totally conserved, halophyte-glycophyte differences are most likely not evident at the primary sequence level. The number of transmembrane helices was determined for each species. All species had between 9 and 12 transmembrane helices but no distinct pattern in either glycophytes or halophytes was found. In conclusion, there appears to be no major differences in SOS1 between halophytes and glycophytes at the primary and secondary levels. Considering