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The excessive accumulation of chloride (Cl−) in leaves due to salinity is frequently related to decreased yield in citrus. Two salt tolerance experiments to detect quantitative trait loci (QTLs) for leaf concentrations of Cl−, Na+, and other traits using the same reference progeny derived from the salt-tolerant Cleopatra mandarin (Citrus reshni) and the disease-resistant donor Poncirus trifoliata were performed with the aim to identify repeatable QTLs that regulate leaf Cl− (and/or Na+) exclusion across independent experiments in citrus, as well as potential candidate genes involved. A repeatable QTL controlling leaf Cl− was detected in chromosome 6 (LCl-6), where 23 potential candidate genes coding for transporters were identified using the C. clementina genome as reference. Transcriptomic analysis revealed two important candidate genes coding for a member of the nitrate transporter 1/peptide transporter family (NPF5.9) and a major facilitator superfamily (MFS) protein. Cell wall biosynthesis- and secondary metabolism-related processes appeared to play a significant role in differential gene expression in LCl-6. Six likely gene candidates were mapped in LCl-6, showing conserved synteny in C. reshni. In conclusion, markers to select beneficial Cleopatra mandarin alleles of likely candidate genes in LCl-6 to improve salt tolerance in citrus rootstock breeding programs are provided.

Salt tolerance is a target trait in plant science and tomato breeding programs. Wild tomato accessions have been often explored for this purpose. Since shoot Na/K is a key component of salt tolerance, RNAi-mediated knockdown isogenic lines obtained for Solanum galapagense alleles encoding both class I Na transporters HKT1;1 and HKT1;2 were used to investigate the silencing effects on the Na and K contents of the xylem sap, and source and sink organs of the scion, and their contribution to salt tolerance in all 16 rootstock/scion combinations of non-silenced and silenced lines, under two salinity treatments. The results show that SgHKT1;1 is operating differently from SgHKT1;2 regarding Na circulation in the tomato vascular system under salinity. A model was built to show that using silenced SgHKT1;1 line as rootstock would improve salt tolerance and fruit quality of varieties carrying the wild type SgHKT1;2 allele. Moreover, this increasing effect on both yield and fruit soluble solids content of silencing SgHKT1;1 could explain that a low expressing HKT1;1 variant was fixed in S. lycopersicum during domestication, and the paradox of increasing agronomic salt tolerance through silencing the HKT1;1 allele from S. galapagense, a salt adapted species.

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.

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.

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.

Confirming eradication success can be notoriously difficult and costly, especially when the species is still present but remains undetected, due to very low population densities and imperfect detection methods. There has been a lack of research on appropriate guidelines and estimation procedures for declaring eradication success for programs aimed at eradicating alien reptiles. Here we develop quantitative rules for confirmation monitoring in eradication campaigns of the red-eared slider turtle (Trachemys scripta elegans). We used a database of slider trapping data from control and eradication campaigns conducted in localities across the Iberian Peninsula and southern France to construct models for inferring appropriate trapping efforts for confirming slider turtle eradication. Basking traps were slightly more efficient than net traps in capturing sliders, although trapping was an inefficient monitoring method given the low capture probabilities estimated. The results of our spatially-explicit eradication scenarios revealed the importance of habitat configuration in declaring eradication success. Declaration of eradication success is contingent on the thresholds set to minimise false positives (i.e., falsely declaring eradication successful), but in any scenario large trapping efforts were required to confirm eradication. Given the low estimated capture probabilities, alternative methods such as eDNA and visual surveys should be considered for monitoring sliders. We suggest that if the costs associated with the impact of alien sliders can be adequately estimated, then eradication can be confirmed by rules minimising both false positive and negative error rates. Otherwise., rules minimising false positive errors would be more appropriate.

The olive tree is a crop of high socio-economical importance in the Mediterranean area. Sexual reproduction in this plant is an essential process, which determines the yield. Successful fertilization is mainly favored and sometimes needed of the presence of pollen grains from a different cultivar as the olive seizes a self-incompatibility system allegedly determined of the sporophytic type. The purpose of the present study was to identify key gene products involved in the function of olive pollen and pistil, in order to help elucidate the events and signaling processes, which happen during the courtship, pollen grain germination, and fertilization in olive. The use of subtractive SSH libraries constructed using, on the one hand one specific stage of the pistil development with germinating pollen grains, and on the other hand mature pollen grains may help to reveal the specific transcripts involved in the cited events. Such libraries have also been created by subtracting vegetative mRNAs (from leaves), in order to identify reproductive sequences only. A variety of transcripts have been identified in the mature pollen grains and in the pistil at the receptive stage. Among them, those related to defense, transport and oxidative metabolism are highlighted mainly in the pistil libraries where transcripts related to stress, and response to biotic and abiotic stimulus have a prominent position. Extensive lists containing information as regard to the specific transcripts determined for each stage and tissue are provided, as well as functional classifications of these gene products. Such lists were faced up to two recent datasets obtained in olive after transcriptomic and genomic approaches. The sequences and the differential expression level of the SSH-transcripts identified here, highly matched the transcriptomic information. Moreover, the unique presence of a representative number of these transcripts has been validated by means of qPCR approaches. The construction of SSH

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.

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.

In the life cycle of a flowering plant, the male gametophyte (pollen grain) produced in the anther reaches the stigmatic surface and initiates the pollen–pistil interaction, an important step in plant reproduction, which ultimately leads to the delivery of two sperm cells to the female gametophyte (embryo sac) inside the ovule. The pollen tube undergoes a strictly apical expansion characterized by a high growth rate, whose targeting should be tightly regulated. A continuous exchange of signals therefore takes place between the haploid pollen and diploid tissue of the pistil until fertilization. In compatible interactions, theses processes result in double fertilization to form a zygote (2n) and the triploid endosperm. Among the large number of signaling mechanisms involved, the redox network appears to be particularly important. Respiratory burst oxidase homologs (Rbohs) are superoxide-producing enzymes involved in a broad range of processes in plant physiology. In this study, we review the latest findings on understanding Rboh activity in sexual plant reproduction, with a particular focus on the male gametophyte from the anther development stages to the crowning point of fertilization. Rboh isoforms have been identified in both the male and female gametophyte and have proven to be tightly regulated. Their role at crucial points such as proper growth of pollen tube, self-incompatibility response and eventual fertilization is discussed. [EN]

Enzyme activities regulating ROS metabolism in the olive seeds are relatively unknown, in spite of the economical importance of this tree and the increasing interest of these seeds for multiple purposes. The aim of this work was to provide a comprehensive analysis of the genes encoding major antioxidant enzymes in mature seed. For this purpose, we have used transcriptomic sequence information available through our own experiments and in specific databases (Olea EST). Numerous sequences have been retrieved and analysed by using basic descriptive tools. Expression of transcripts encoding several of these enzymes has been carried out. These include catalases, superoxide dismutase, dehydroascorbate reductase, GSNO reductase, NADPH oxidase, lipoxygenase and thioredoxins. Cellular localization of several of these enzymes and histochemical detection of enzyme activities have been also carried out. We discuss how these enzyme activities may be involved in the olive seed metabolism through germination, and how they may contribute to the antioxidant capacity, stability and organoleptic properties of olive oils.

Olive transcriptome has been recently built as the result of library screening through the use of different NGS strategies. Such transcriptome mainly relies on vegetative reads. However, the peculiarity of the reproductive tissues, and the widely reported presence of numerous tissue-specific transcripts in both stigma and pollen grains made us to attempt a similar approach in the later tissues. mRNA was isolated from olive stigmas and pollen grains at different developmental stages and during in vitro pollen germination, respectively. cDNA libraries were built from the isolated mRNA, and subjected to 454/Roche Titanium+ sequencing. Moreover, NGS readings were combined with additional sequences obtained from subtractive libraries, in order to produce a de novo assembly of the olive reproductive transcriptome. Several public software and other tools developed by authors were used for this purpose. Sequences were assembled together into 58 098 (stigma) and 26 422 (pollen) contigs, respectively. Functional annotation is currently underway, however numerous gene products have been previously analysed, particularly those corresponding to major enzymes regulating ROS metabolism. Some of them, including catalases, superoxide dismutase, dehydroascorbate reductase, GSNO reductase, NADPH oxidase, lipoxygenase and thioredoxins, have been further analysed using basic descriptive tools.

Transcriptome analysis of the olive stigma and pollen grain was carried out at different developmental stages and during in vitro pollen germination, respectively. Isolated mRNA was used to build cDNA libraries, which were subjected to 454+ sequencing. Readings were combined with sequences obtained from subtractive libraries, in order to produce a de novo assembly of the olive reproductive transcriptome. The presence of transcripts corresponding to major enzymes regulating ROS metabolism was assessed. Sequences corresponding to catalases, superoxide dismutase, dehydroascorbate reductase, GSNO reductase, NADPH oxidase, lipoxygenase and thioredoxins were retrieved and analysed using basic descriptive tools. Expression of transcripts in pollen and pistil was analysed by semiquantitative RT-PCR. Cellular localization of several enzymes was also carried out by using immunocytochemistry. We discuss how these enzyme activities may be involved in the olive pollen metabolism through germination, in the olive stigma over the periods before, after and during receptivity, and how they may contribute to the interaction pollen-stigma.

The success of sexual reproduction in plants involves (i) the proper formation of the plant gametophytes (pollen and embryo sac) containing the gametes, (ii) the accomplishment of specific interactions between pollen grains and the stigma, which subsequently lead to (iii) the fusion of the gametes and eventually to (iv) the seed setting. Owing to the lack of mobility, plants have developed specific regulatory mechanisms to control all developmental events underlying the sexual plant reproduction according to environmental challenges. Over the last decade, redox regulation and signaling have come into sight as crucial mechanisms able to manage critical stages during sexual plant reproduction. This regulation involves a complex redox network which includes reactive oxygen species (ROS), reactive nitrogen species (RNS), glutathione and other classic buffer molecules or antioxidant proteins, and some thiol/disulphide-containing proteins belonging to the thioredoxin superfamily, like glutaredoxins (GRXs) or thioredoxins (TRXs). These proteins participate as critical elements not only in the switch between the mitotic to the meiotic cycle but also at further developmental stages of microsporogenesis. They are also implicated in the regulation of pollen rejection as the result of self-incompatibility. In addition, they display precise space-temporal patterns of expression and are present in specific localizations like the stigmatic papillae or the mature pollen, although their functions and subcellular localizations are not clear yet. In this review we summarize insights and perspectives about the presence of thiol/disulphide-containing proteins in plant reproduction, taking into account the general context of the cell redox network. [EN]

Superoxide dismutases (SODs) are a class of antioxidant enzymes which catalyze the dismutation of superoxide into oxygen and hydrogen peroxide, therefore controlling cellular levels of Reactive Oxygen Species (ROS). In the mature pollen grains of the olive tree, the presence of several forms of CuZn-SOD and the cytosolic localization of the enzyme have been described. The present study was aimed to elucidate the adaptation of the oxidative metabolism to the changing conditions occurring during the course of olive pollen formation, hydration and pollen tube emergence and growth. We used a polyclonal antibody (raised against a KLH-linked synthetic peptide including a consensus sequence for CuZn-SODs in olive pollen) in immunocytochemical experiments carried out by Fluorescence (FM) and Transmission Electron Microcopy (TEM).

The presence of high levels of NO has been described in several pollen grains, where their presence has been shown to regulate pollen tube growth and orientation. Here we have assessed the production of NO during in vitro germination in the olive pollen by using exogenous NO donors like sodium nitroprusside and S-nitrosoglutathione. Both chemical induced a remarkable inhibition of pollen germination and tube growth, which were partially reverted by the addition of the specific NO-scavenger carboxy-PTIO (cPTIO). The presence and localization of NO was determined either by using a fluorimetric assay and by confocal microscopy using the fluorescent probe diaminoflurescein-2 diacetate (DAF-2DA). As olive pollen is also a highly allergenic source, the putative role of NO in eliciting the hypersensitive response in humans is discussed.

ROS and NO are low molecular weight molecules implicated in plant signalling. Their relevant implication in pollen tube growth and reorientation has been shown particularly in plant model species. In olive reproductive tissues, their presence has been described in a stage- and tissue- specific manner. Accurate detection and quantification of minor changes in ROS/NO and the chemical species resulting or interacting with their metabolism in reproductive tissues is a challenging task at present. The goal of the present study was to elucidate the implication of the NO metabolism in the process of olive pollen germination in vítro. For this purpose we used a recently published method based in the use of liquid chromatography-electrospray/mass spectrometry to detect different antioxidant metabolites in volved in NO metabolism. We have analysed the levels of key molecules like GSH (reduced glutathione), GSSH (oxidized glutathione), GSNO {Snitrosoglutathione) and ascorbic acid (vitamin e) in the mature pollen, and the pollen grain and pollen tube at different times of in vitro germination. These data correspond to both dormant and activated metabolism, respectively. The results revealed remarkalble changes in the levels of GSNO, which is a cellular NO reservoir, throughout the germination process. These data confirm the involvement of NO and antioxidant molecules in the pollen tube growth, as observed previously by using CLSM, and point to potential new specific roles for these molecules as signalling intermediates.