Role of Na+ transporters HKT1;1 and HKT1;2 in tomato salt tolerance. I. Function loss of cheesmaniae alleles in roots and aerial parts.

We analyzed the physiological impact of function loss on cheesmaniae alleles at the HKT1;1 and HKT1;2 loci in the roots and aerial parts of tomato plants in order to determine the relative contributions of each locus in the different tissues to plant Na+/K+ homeostasis and subsequently to tomato salt tolerance. We generated different reciprocal rootstock/scion combinations with non-silenced, single RNAi-silenced lines for ScHKT1;1 and ScHKT1;2, as well as a silenced line at both loci from a near isogenic line (NIL14), homozygous for the Solanum cheesmaniae haplotype containing both HKT1 loci and subjected to salinity under natural greenhouse conditions. Our results show that salt treatment reduced vegetative growth and altered the Na+/K+ ratio in leaves and flowers; negatively affecting fruit production, particularly in graft combinations containing single silenced ScHKT1;2- and double silenced ScHKT1;1/ScHKT1;2 lines when used as scion. We concluded that the removal of Na+ from the xylem by ScHKT1;2 in the aerial part of the plant can have an even greater impact than that on Na+ homeostasis at the root level under saline conditions. Also, ScHKT1;1 function loss in rootstock greatly reduced the Na+/K+ ratio in leaf and flower tissues, minimized yield loss under salinity. Our results suggest that, in addition to xylem Na+ unloading, ScHKT1;2 could also be involved in Na+ uploading into the phloem, thus promoting Na+ recirculation from aerial parts to the roots. This recirculation of Na+ to the roots through the phloem could be further favoured by ScHKT1;1 silencing at these roots.

Na+ transporter HKT1;2 reduces flower Na+ content and considerably mitigates the decline in tomato fruit yields under saline conditions.

Genes encoding HKT1-like Na+ transporters play a key role in the salinity tolerance mechanism in Arabidopsis and other plant species by retrieving Na+ from the xylem of different organs and tissues. In this study, we investigated the role of two HKT1;2 allelic variants in tomato salt tolerance in relation to vegetative growth and fruit yield in plants subjected to salt treatment in a commercial greenhouse under real production conditions. We used two near-isogenic lines (NILs), homozygous for either the Solanum lycopersicum (NIL17) or S. cheesmaniae (NIL14) allele, at HKT1;2 loci and their respective RNAi-Sl/ScHKT1;2 lines. The results obtained show that both ScHKT1;2- and SlHKT1;2-silenced lines display hypersensitivity to salinity associated with an altered leaf Na+/K+ ratio, thus confirming that HKT1;2 plays an important role in Na+ homeostasis and salinity tolerance in tomato. Both silenced lines also showed Na+ over-accumulation and a slight, but significant, reduction in K+ content in the flower tissues of salt-treated plants and consequently a higher Na+/K+ ratio as compared to the respective unsilenced lines. This altered Na+/K+ ratio in flower tissues is associated with a sharp reduction in fruit yield, measured as total fresh weight and number of fruits, in both silenced lines under salinity conditions. Our findings demonstrate that Na+ transporter HKT1;2 protects the flower against Na+ toxicity and mitigates the reduction in tomato fruit yield under salinity conditions.

Generation of Superoxide by OeRbohH, a NADPH Oxidase Activity During Olive (Olea europaea L.) Pollen Development and Germination.

Reactive oxygen species (ROS) are produced in the olive reproductive organs as the result of intense metabolism. ROS production and pattern of distribution depend on the developmental stage, supposedly playing a broad panel of functions, which include defense and signaling between pollen and pistil. Among ROS-producing mechanisms, plasma membrane NADPH-oxidase activity is being highlighted in plant tissues, and two enzymes of this type have been characterized in Arabidopsis thaliana pollen (RbohH and RbohJ), playing important roles in pollen physiology. Besides, pollen from different species has shown distinct ROS production mechanism and patterns of distribution. In the olive reproductive tissues, a significant production of superoxide has been described. However, the enzymes responsible for such generation are unknown. Here, we have identified an Rboh-type gene (OeRbohH), mainly expressed in olive pollen. OeRbohH possesses a high degree of identity with RbohH and RbohJ from Arabidopsis, sharing most structural features and motifs. Immunohistochemistry experiments allowed us to localize OeRbohH throughout pollen ontogeny as well as during pollen tube elongation. Furthermore, the balanced activity of tip-localized OeRbohH during pollen tube growth has been shown to be important for normal pollen physiology. This was evidenced by the fact that overexpression caused abnormal phenotypes, whereas incubation with specific NADPH oxidase inhibitor or gene knockdown lead to impaired ROS production and subsequent inhibition of pollen germination and pollen tube growth.

S-nitroso- and nitro- proteomes in the olive (Olea europaea L.) pollen. Predictive versus experimental data by nano-LC-MS.

The data presented here are related to the research article entitled "Generation of nitric oxide by olive (Olea europaea L.) pollen during in vitro germination and assessment of the S-nitroso- and nitro-proteomes by computational predictive methods" doi:10.1016/j.niox.2017.06.005 (Jimenez-Quesada et al., 2017) [1]. Predicted cysteine S-nitrosylation and Tyr-nitration sites in proteins derived from a de novo assembled and annotated pollen transcriptome from olive tree (Olea europaea L.) were obtained after using well-established predictive tools in silico. Predictions were performed using both default and highly restrictive thresholds. Numerous gene products identified with these characteristics are listed here. An experimental validation of the data, consisting in nano-LC-MS (Liquid Chromatography-Mass Spectrometry) determination of olive pollen proteins after immunoprecipitation with antibodies to anti-S-nitrosoCys and anti-3-NT (NitroTyrosine) allowed identification of numerous proteins subjected to these two post-translational modifications, which are listed here together with information regarding their cross-presence among the predictions.

Generation of nitric oxide by olive (Olea europaea L.) pollen during in vitro germination and assessment of the S-nitroso- and nitro-proteomes by computational predictive methods.

Nitric oxide is recognized as a signaling molecule involved in a broad range of physiological processes in plants including sexual reproduction. NO has been detected in the pollen grain at high levels and regulates pollen tube growth. Previous studies demonstrated that NO as well as ROS are produced in the olive reproductive tissues in a stage- and tissue-specific manner. The aim of this study was to assess the production of NO throughout the germination of olive (Olea europaea L.) pollen in vitro. The NO fluorescent probe DAF-2DA was used to image NO production in situ, which was correlated to pollen viability. Moreover, by means of a fluorimetric assay we showed that growing pollen tubes release NO. GSNO -a mobile reservoir of NO, formed by the S-nitrosylation of NO with reduced glutathione (GSH) - was for the first time detected and quantified at different stages of pollen tube growth using a LC-ES/MS analysis. Exogenous NO donors inhibited both pollen germination and pollen tube growth and these effects were partially reverted by the specific NO-scavenger c-PTIO. However, little is known about how NO affects the germination process. With the aim of elucidating the putative relevance of protein S-nitrosylation and Tyr-nitration as important post-translational modifications in the development and physiology of the olive pollen, a de novo assembled and annotated reproductive transcriptome from olive was challenged in silico for the putative capability of transcripts to become potentially modified by S-nitrosylation/Tyr-nitration according to well-established criteria. Numerous gene products with these characteristics were identified, and a broad discussion as regards to their potential role in plant reproduction was built after their functional classification. Moreover, the importance of both S-nitrosylation/Tyr-nitrations was experimentally assessed and validated by using Western blotting, immunoprecipitation and proteomic approaches.

The conformational stability and biophysical properties of the eukaryotic thioredoxins of Pisum sativum are not family-conserved.

Thioredoxins (TRXs) are ubiquitous proteins involved in redox processes. About forty genes encode TRX or TRX-related proteins in plants, grouped in different families according to their subcellular localization. For instance, the h-type TRXs are located in cytoplasm or mitochondria, whereas f-type TRXs have a plastidial origin, although both types of proteins have an eukaryotic origin as opposed to other TRXs. Herein, we study the conformational and the biophysical features of TRXh1, TRXh2 and TRXf from Pisum sativum. The modelled structures of the three proteins show the well-known TRX fold. While sharing similar pH-denaturations features, the chemical and thermal stabilities are different, being PsTRXh1 (Pisum sativum thioredoxin h1) the most stable isoform; moreover, the three proteins follow a three-state denaturation model, during the chemical-denaturations. These differences in the thermal- and chemical-denaturations result from changes, in a broad sense, of the several ASAs (accessible surface areas) of the proteins. Thus, although a strong relationship can be found between the primary amino acid sequence and the structure among TRXs, that between the residue sequence and the conformational stability and biophysical properties is not. We discuss how these differences in the biophysical properties of TRXs determine their unique functions in pea, and we show how residues involved in the biophysical features described (pH-titrations, dimerizations and chemical-denaturations) belong to regions involved in interaction with other proteins. Our results suggest that the sequence demands of protein-protein function are relatively rigid, with different protein-binding pockets (some in common) for each of the three proteins, but the demands of structure and conformational stability per se (as long as there is a maintained core), are less so.