Identification and Characterization of a Thermotolerant TILLING Allele of Heat Shock Binding Protein 1 in Tomato.

The identification of heat stress (HS)-resilient germplasm is important to ensure food security under less favorable environmental conditions. For that, germplasm with an altered activity of factors regulating the HS response is an important genetic tool for crop improvement. Heat shock binding protein (HSBP) is one of the main negative regulators of HS response, acting as a repressor of the activity of HS transcription factors. We identified a TILLING allele of Solanum lycopersicum (tomato) HSBP1. We examined the effects of the mutation on the functionality of the protein in tomato protoplasts, and compared the thermotolerance capacity of lines carrying the wild-type and mutant alleles of HSBP1. The methionine-to-isoleucine mutation in the central heptad repeats of HSBP1 leads to a partial loss of protein function, thereby reducing the inhibitory effect on Hsf activity. Mutant seedlings show enhanced basal thermotolerance, while mature plants exhibit increased resilience in repeated HS treatments, as shown by several physiological parameters. Importantly, plants that are homozygous for the wild-type or mutant HSBP1 alleles showed no significant differences under non-stressed conditions. Altogether, these results indicate that the identified mutant HSBP1 allele can be used as a genetic tool in breeding, aiming to improve the thermotolerance of tomato varieties.

Recombinant Arabidopsis HSP70 Sustains Cell Survival and Metastatic Potential of Breast Cancer Cells.

The chaperone HSP70 protein is widely present in many different tumors and its expression correlates with an increased cell survival, low differentiation, and poor therapeutic outcome in human breast cancer. The intracellular protein has prevalently a cytoprotective function, while the extracellular HSP70 mediates immunologic responses. Evolutionarily, HSPs are well conserved from prokaryotes to eukaryotes, and human HSP70 shows a strong similarity to that of plant origin. In the current article, we have tested the potential effect of recombinant HSP70, from Arabidopsis thaliana, on cell survival and metastatic properties of breast cancer cells. Our data show that HSP70 sustains cell viability in MCF-7 and MDA-MB-231 breast tumoral cells and increases Cyclin D1 and Survivin expression. The extracellular HSP70 triggers cell migration and the activation of MMPs particularly in MDA-MB-231 cells. Furthermore, under UV-induced stress condition, the low levels of phospho-AKT were increased by exogenous HSP70, together with the upregulation of Cyclin D1, particularly in the tumoral cell phenotype. On the other hand, UV increased TP53 expression, and the coincubation of HSP70 lowers the TP53 levels similar to the control. These findings correlate with the cytoprotective and antiapoptotic role of HSPs, as reported in different cellular contexts. This is the first study on mammary cells that highlights how the heterologous HSP70 from Arabidopsis thaliana sustains cell survival prevalently in breast cancer cell types, thus maintaining their metastatic potential. Therefore, targeting HSP70 would be of clinical importance since HSP70 blocking selectively targets tumor cells, in which it supports cell growth and survival. Mol Cancer Ther; 15(5); 1063-73. ©2016 AACR.

Heat stress regimes for the investigation of pollen thermotolerance in crop plants.

KEY MESSAGE: Pollen thermotolerance. Global warming is predicted to increase the frequency and severity of extreme weather phenomena such as heat waves thereby posing a major threat for crop productivity and food security. The yield in case of most crop species is dependent on the success of reproductive development. Pollen development has been shown to be highly sensitive to elevated temperatures while the development of the female gametophyte as well as sporophytic tissues might also be disturbed under mild or severe heat stress conditions. Therefore, assessing pollen thermotolerance is currently of high interest for geneticists, plant biologists and breeders. A key aspect in pollen thermotolerance studies is the selection of the appropriate heat stress regime, the developmental stage that the stress is applied to, as well as the method of application. Literature search reveals a rather high variability in heat stress treatments mainly due to the lack of standardized protocols for different plant species. In this review, we summarize and discuss experimental approaches that have been used in various crops, with special focus on tomato, rice and wheat, as the best studied crops regarding pollen thermotolerance. The overview of stress treatments and the major outcomes of each study aim to provide guidelines for similar research in other crops.

Identification of novel small ncRNAs in pollen of tomato.

BACKGROUND: The unprecedented role of sncRNAs in the regulation of pollen biogenesis on both transcriptional and epigenetic levels has been experimentally proven. However, little is known about their global regulation, especially under stress conditions. We used tomato pollen in order to identify pollen stage-specific sncRNAs and their target mRNAs. We further deployed elevated temperatures to discern stress responsive sncRNAs. For this purpose high throughput sncRNA-sequencing as well as Massive Analysis of cDNA Ends (MACE) were performed for three-replicated sncRNAs libraries derived from tomato tetrad, post-meiotic, and mature pollen under control and heat stress conditions. RESULTS: Using the omiRas analysis pipeline we identified known and predicted novel miRNAs as well as sncRNAs from other classes, responsive or not to heat. Differential expression analysis revealed that post-meiotic and mature pollen react most strongly by regulation of the expression of coding and non-coding genomic regions in response to heat. To gain insight to the function of these miRNAs, we predicted targets and annotated them to Gene Ontology terms. This approach revealed that most of them belong to protein binding, transcription, and Serine/Threonine kinase activity GO categories. Beside miRNAs, we observed differential expression of both tRNAs and snoRNAs in tetrad, post-meiotic, and mature pollen when comparing normal and heat stress conditions. CONCLUSIONS: Thus, we describe a global spectrum of sncRNAs expressed in pollen as well as unveiled those which are regulated at specific time-points during pollen biogenesis. We integrated the small RNAs into the regulatory network of tomato heat stress response in pollen.

Assessment of heat shock protein 70 induction by heat in alfalfa varieties and constitutive overexpression in transgenic plants.

Heat shock proteins (HSPs) are molecular chaperones involved in many cellular functions. It has been shown that mammalian cytosolic HSP70 binds antigenic peptides mediating the activation of the immune system, and that it plays a determining role in tumour immunogenicity. This suggests that HSP70 may be used for the production of conjugated vaccines. Human and plant HSPs share high sequence similarity and some important biological functions in vitro. In addition, plant HSPs have no endotoxic side effects. Extraction of HSP70 from plants for use as vaccine adjuvant requires enhancing its concentration in plant tissues. In this work, we explored the possibility to produce HSP70 in both transgenic and non-transgenic plants, using alfalfa as a model species. First, a transcriptional analysis of a constitutive and an inducible HSP70 genes was conducted in Arabidopsis thaliana. Then the coding sequence of the inducible form was cloned and introduced into alfalfa by Agrobacterium-mediated transformation, and the accumulation of the protein in leaf tissue of transgenic plants was demonstrated. We also tested diverse alfalfa varieties for heat-inducible expression of endogenous HSP70, revealing variety-specific responses to heat shock.

Cardiac and hepatic role of r-AtHSP70: basal effects and protection against ischemic and sepsis conditions.

Heat shock proteins (HSPs), highly conserved in all organisms, act as molecular chaperones activated by several stresses. The HSP70 class of stress-induced proteins is the most studied subtype in cardiovascular and inflammatory disease. Because of the high similarity between plant and mammalian HSP70, the aim of this work was to evaluate whether recombinant HSP70 of plant origin (r-AtHSP70) was able to protect rat cardiac and hepatic function under ischemic and sepsis conditions. We demonstrated for the first time that, in ex vivo isolated and perfused rat heart, exogenous r-AtHSP70 exerted direct negative inotropic and lusitropic effects via Akt/endothelial nitric oxide synthase pathway, induced post-conditioning cardioprotection via Reperfusion Injury Salvage Kinase and Survivor Activating Factor Enhancement pathways, and did not cause hepatic damage. In vivo administration of r-AtHSP70 protected both heart and liver against lipopolysaccharide-dependent sepsis, as revealed by the reduced plasma levels of interleukin-1ß, tumour necrosis factor alpha, aspartate aminotransferase and alanine aminotransferase. These results suggest exogenous r-AtHSP70 as a molecular modulator able to protect myocardial function and to prevent cardiac and liver dysfunctions during inflammatory conditions.

Engineered drought tolerance in tomato plants is reflected in chlorophyll fluorescence emission.

Drought stress is one of the most important factors that limit crop productivity worldwide. In order to obtain tomato plants with enhanced drought tolerance, we inserted the transcription factor gene ATHB-7 into the tomato genome. This gene was demonstrated earlier to be up-regulated during drought stress in Arabidopsis thaliana thus acting as a negative regulator of growth. We compared the performance of wild type and transgenic tomato line DTL-20, carrying ATHB-7 gene, under well-irrigated and water limited conditions. We found that transgenic plants had reduced stomatal density and stomatal pore size and exhibited an enhanced resistance to soil water deficit. We used the transgenic plants to investigate the potential of chlorophyll fluorescence to report drought tolerance in a simulated high-throughput screening procedure. Wild type and transgenic tomato plants were exposed to drought stress lasting 18 days. The stress was then terminated by rehydration after which recovery was studied for another 2 days. Plant growth, leaf water potential, and chlorophyll fluorescence were measured during the entire experimental period. We found that water potential in wild type and drought tolerant transgenic plants diverged around day 11 of induced drought stress. The chlorophyll fluorescence parameters: the non-photochemical quenching, effective quantum efficiency of PSII, and the maximum quantum yield of PSII photochemistry yielded a good contrast between wild type and transgenic plants from day 7, day 12, and day 14 of induced stress, respectively. We propose that chlorophyll fluorescence emission reports well on the level of water stress and, thus, can be used to identify elevated drought tolerance in high-throughput screens for selection of resistant genotypes.

9-lipoxygenase metabolism is involved in the almond/Aspergillus carbonarius interaction.

Phyto-oxylipins are a group of biologically active molecules that play an important role in plant defence. Their production begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases (LOX) to form 9- or 13-hydroperoxides that are substrates for several enzymes involved in the synthesis of final oxylipins, which can act as signal molecules and/or direct antimicrobials. In the present work, the response of the 9-LOX pathway in the almond/Aspergillus carbonarius (producer of ochratoxin A) interaction was studied. Both LOX gene expression and activity are up-regulated over the course of fungal infection in immature and mature almonds. The biochemical characterization of major LOX and hydroperoxide lyase (HPL) isoforms indicated that 9-LOX metabolism is specifically induced by A. carbonarius. Lipid peroxidation profiling showed that, in infected immature almonds, enzymatically produced 9-hydro(peroxy) fatty acids (HFAs) were the main HFAs and are further metabolized by HPL into C9-aldehydes. Both HPL gene expression and C9-aldehydes increased over the course of fungal infection. In mature almonds infected with A. carbonarius, levels of LOX expression and activity were lower than those found in immature seeds, and 9-HFA represented the minority of total HFA, which consisted of mostly 13- and non-enzymatically produced HFA. In these experimental conditions, no volatile aldehydes were recorded from these samples, even though HPL was up-regulated in infected mature almonds. The effects on the growth of A. carbonarius of the aldehydes produced by these enzymes were also tested in vitro. Results reported here led to the proposal that, in almond seed, the association of 9-LOX and HPL has an important role in seed defence mechanism against pathogen infection.

A homeodomain leucine zipper gene from Craterostigma plantagineum regulates abscisic acid responsive gene expression and physiological responses.

A subset of homeodomain leucine zipper proteins (HDZip) play a role in regulating adaptation responses including developmental adjustment to environmental cues in plants. Here we report the structural and functional characterisation of a dehydration responsive nuclear-targeted HDZip transcriptional regulator, CpHB-7. DNA-protein interaction studies suggest that CDeT6-19, a known ABA and dehydration responsive dehydrin gene, is a potential target gene of CpHB-7 in the desiccation-tolerant plant Craterostigma plantagineum. Transgenic plants that ectopically express CpHB-7 display reduced sensitivity towards ABA during seed germination and stomatal closure. Expression analysis reveals that genes with induced or repressed expression in CpHB-7 ectopic expression lines are either mostly repressed or induced by ABA, drought or salt treatment respectively, thus demonstrating that CpHB-7 modifies ABA-responsive gene expression as a negative regulator. CpHB-7 gene expression is also linked to early organ development, leading to the suggestion that CpHB-7 is functionally similar to the Arabidopsis transcription factor, ATHB-6.

Molecular cloning and characterization of an almond 9-hydroperoxide lyase, a new CYP74 targeted to lipid bodies.

Oxylipin metabolism represents one of many defence mechanisms employed by plants. It begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases to form fatty acid hydroperoxides that are substrates for several enzymes, including specialized cytochrome P450s known as CYP74s. The targeting of a new CYP74, a 9-hydroperoxide lyase (HPL) from almonds, to the endomembrane system and lipid bodies, both as enzyme activity in almond seeds and as GFP fusions transiently expressed in tobacco protoplasts, is described. Such association of a CYP74 with lipid bodies has not been reported previously. Also described are the properties of a 9-HPL gene, the developmental regulation of its expression, the production and characterization of recombinant 9-HPL in Escherichia coli, and the developmental correlation between gene expression, enzyme activity, and the appearance of volatile C9 aldehydes from HPL action.