Identification of Sex-Associated Genetic Markers in Pistacia lentiscus var. chia for Early Male Detection.

Pistacia lentiscus var. chia is a valuable crop for its high-added-value mastic, a resin with proven pharmaceutical and cosmeceutical properties harvested from the male tree trunk. To achieve the maximum economic benefits from the cultivation of male mastic trees, it is important to develop early sex diagnosis molecular tools for distinguishing the sex type. Thus far, the work on sex identification has focused on Pistacia vera with promising results; however, the low transferability rates of these markers in P. lentiscus necessitates the development of species-specific sex-linked markers for P. lentiscus var. chia. To our knowledge, this is the first report regarding: (i) the development of species-specific novel transcriptome-based markers for P. lentiscus var. chia and their assessment on male, female and monoecious individuals using PCR-HRM analysis, thus, introducing a cost-effective method for sex identification with high accuracy that can be applied with minimum infrastructure, (ii) the effective sex identification in mastic tree using a combination of different sex-linked ISSR and SCAR markers with 100% accuracy, and (iii) the impact evaluation of sex type on the genetic diversity of different P. lentiscus var. chia cultivars. The results of this study are expected to provide species-specific markers for accurate sex identification that could contribute to the selection process of male mastic trees at an early stage for mass propagation systems and to facilitate future breeding efforts related to sex-linked productivity and quality of mastic resin.

Seasonal Shifts in Soil Microbiome Structure Are Associated with the Cultivation of the Local Runner Bean Variety around the Lake Mikri Prespa.

Leguminous crops play a key role in food production and agroecosystem sustainability. However, climate change and agricultural intensification have a significant impact on the available arable land, soil microbiome functions, and ultimately, crop productivity. The "Prespa bean" (Phaseolous coccineous L.) is an important leguminous crop for the agricultural economy of the rural areas surrounding the lake, Mikri Prespa, which is of significant ecological importance. The seasonal effects on soil microbiome structure, diversity and functions associated with the runner bean cultivation were investigated using 16S rRNA amplicon sequencing. The results indicated that the presence of the runner bean differentially shaped the soil microbial community structure. The runner bean was implicated in the recruitment of specific bacteria, by favouring or excluding specific classes or even phyla. Soil functions involved in nutrient availability and carbon metabolism, among other pathways, were associated with microbiome-plant interactions. The temporal relative abundance shifts could be explained by the impact of soil organic matter, the fertilization regime, and the equilibrium in carbon metabolic processes. This research has shown the effect of runner bean cultivation on the soil microbiome which, in future, may potentially contribute to research into sustainable agricultural productivity and the protection of soil ecosystem services.

Diagnosis of Induced Resistance State in Tomato Using Artificial Neural Network Models Based on Supervised Self-Organizing Maps and Fluorescence Kinetics.

The aim of this study was to develop three supervised self-organizing map (SOM) models for the automatic recognition of a systemic resistance state in plants after application of a resistance inducer. The pathosystem Fusarium oxysporum f. sp. radicis-lycopersici (FORL) + tomato was used. The inorganic, defense inducer, Acibenzolar-S-methyl (benzo-[1,2,3]-thiadiazole-7-carbothioic acid-S-methyl ester, ASM), reported to induce expression of defense genes in tomato, was applied to activate the defense mechanisms in the plant. A handheld fluorometer, FluorPen FP 100-MAX-LM by SCI, was used to assess the fluorescence kinetics response of the induced resistance in tomato plants. To achieve recognition of resistance induction, three models of supervised SOMs, namely SKN, XY-F, and CPANN, were used to classify fluorescence kinetics data, in order to determine the induced resistance condition in tomato plants. To achieve this, a parameterization of fluorescence kinetics curves was developed corresponding to fluorometer variables of the Kautsky Curves. SKN was the best supervised SOM, achieving 97.22% to 100% accuracy. Gene expression data were used to confirm the accuracy of the supervised SOMs.

Biosolid-Amended Soil Enhances Defense Responses in Tomato Based on Metagenomic Profile and Expression of Pathogenesis-Related Genes.

Biosolid application is an effective strategy, alternative to synthetic chemicals, for enhancing plant growth and performance and improving soil properties. In previous research, biosolid application has shown promising results with respect to tomato resistance against Fusarium oxysporum f. sp. radicis-lycopersici (Forl). Herein, we aimed at elucidating the effect of biosolid application on the plant-microbiome response mechanisms for tomato resistance against Forl at a molecular level. More specifically, plant-microbiome interactions in the presence of biosolid application and the biocontrol mechanism against Forl in tomato were investigated. We examined whether biosolids application in vitro could act as an inhibitor of growth and sporulation of Forl. The effect of biosolid application on the biocontrol of Forl was investigated based on the enhanced plant resistance, measured as expression of pathogen-response genes, and pathogen suppression in the context of soil microbiome diversity, abundance, and predicted functions. The expression of the pathogen-response genes was variably induced in tomato plants in different time points between 12 and 72 h post inoculation in the biosolid-enriched treatments, in the presence or absence of pathogens, indicating activation of defense responses in the plant. This further suggests that biosolid application resulted in a successful priming of tomato plants inducing resistance mechanisms against Forl. Our results have also demonstrated that biosolid application alters microbial diversity and the predicted soil functioning, along with the relative abundance of specific phyla and classes, as a proxy for disease suppression. Overall, the use of biosolid as a sustainable soil amendment had positive effects not only on plant health and protection, but also on growth of non-pathogenic antagonistic microorganisms against Forl in the tomato rhizosphere and thus, on plant-soil microbiome interactions, toward biocontrol of Forl.

Characterization of the Genetic Diversity Present in a Diverse Sesame Landrace Collection Based on Phenotypic Traits and EST-SSR Markers Coupled With an HRM Analysis.

A selection of sesame (Sesamum indicum L.) landraces of different eco-geographical origin and breeding history have been characterized using 28 qualitative morpho-physiological descriptors and seven expressed sequence tag-simple sequence repeat (EST-SSR) markers coupled with a high-resolution melting (HRM) analysis. The most variable qualitative traits that could efficiently discriminate landraces, as revealed by the correlation analyses, were the plant growth type and position of the branches, leaf blade width, stem pubescence, flowering initiation, capsule traits and seed coat texture. The agglomerative hierarchical clustering analysis based on a dissimilarity matrix highlighted three main groups among the sesame landraces. An EST-SSR marker analysis revealed an average polymorphism information content (PIC) value of 0.82, which indicated that the selected markers were highly polymorphic. A principal coordinate analysis and dendrogram reconstruction based on the molecular data classified the sesame genotypes into four major clades. Both the morpho-physiological and molecular analyses showed that landraces from the same geographical origin were not always grouped in the same cluster, forming heterotic groups; however, clustering patterns were observed for the Greek landraces. The selective breeding of such traits could be employed to unlock the bottleneck of local phenotypic diversity and create new cultivars with desirable traits.

Overexpression of A Biotic Stress-Inducible Pvgstu Gene Activates Early Protective Responses in Tobacco under Combined Heat and Drought.

Drought and heat stresses are major factors limiting crop growth and productivity, and their effect is more devastating when occurring concurrently. Plant glutathione transferases (GSTs) are differentially expressed in response to different stimuli, conferring tolerance to a wide range of abiotic stresses. GSTs from drought-tolerant Phaseolus vulgaris var. "Plake Megalosperma Prespon" is expected to play an important role in the response mechanisms to combined and single heat and drought stresses. Herein, we examined wild-type N. tabacum plants (cv. Basmas Xanthi) and T1 transgenic lines overexpressing the stress-induced Pvgstu3-3 and Pvgstu2-2 genes. The overexpression of Pvgstu3-3 contributed to potential thermotolerance and greater plant performance under combined stress. Significant alterations in the primary metabolism were observed in the transgenic plants between combined stress and stress-free conditions. Stress-responsive differentially expressed genes (DEGs) and transcription factors (TFs) related to photosynthesis, signal transduction, starch and sucrose metabolism, osmotic adjustment and thermotolerance, were identified under combined stress. In contrast, induction of certain DEGs and TF families under stress-free conditions indicated that transgenic plants were in a primed state. The overexpression of the Pvgstu3-3 is playing a leading role in the production of signaling molecules, induction of specific metabolites and activation of the protective mechanisms for enhanced protection against combined abiotic stresses in tobacco.

The Effects of Moderate and Severe Salinity on Composition and Physiology in the Biomass Crop Miscanthus × giganteus.

Saline land represents a growing resource that could be utilised for growing biomass crops, such as Miscanthus × giganteus (Greef et Deu.), for eliminating competition with staple food crops. However, the response mechanisms to different salinity regimes, in relation to the impact on quality of the harvested biomass and the combustion properties are largely unknown. Herein, the focus was on the salt-induced compositional changes of ion flux and compartmentalization in the rhizome, stems, and leaves in relation to their impact on salinity tolerance and the combustion quality through investigating the photophysiological, morphophysiological, and biochemical responses of M. × giganteus to moderate and a severe salinity. Severe salinity induced an immediate and sustained adverse response with a reduction in biomass yield, photoinhibition, and metabolic limitations in photosynthesis. Moderate salinity resulted in a slower cumulative response with low biomass losses. Biomass composition, variations in ion compartmentalisation and induction of proline were dependent on the severity and duration of salinity. Ash behaviour indices, including the base percentage and base-to-acid ratio, indicated lower corrosion potential and lower risk of slagging under salinity. Understanding the impact of salinity on the potential for growth on saline land may identify new targets for breeding salinity-tolerant bioenergy crops.

Detection and quantification of cashew in commercial tea products using High Resolution Melting (HRM) analysis.

Tea, a popular aromatic infusion and food supplement, prepared from Camellia sinensis (L.) Kuntze leaves, is often subjected to adulteration with various undeclared inorganic and plant-derived materials. Cashew (Anacardium occidentale L.) nut husk is one of the most common plant tea adulterants. To date, there are limited DNA-based technologies for tea authentication and quantitative detection of adulterants. Herein, we used a universal plant DNA barcoding marker coupled with High Resolution Melting (Bar-HRM) analysis to authenticate tea products from cashew ground nut. Additionally, cashew-specific markers coupled with HRM technology were used to detect and quantify adulteration of tea with cashew DNA. This methodology can reliably detect admixtures as low as 1% v/v cashew in commercial tea products. Overall, our results demonstrate that the HRM technology is a strong molecular approach in tea authentication, capable of detecting very low adulterations in DNA admixtures. PRACTICAL APPLICATION: In this study, we established the use of high-resolution DNA-based technologies for the detection of cashew adulteration in tea, even in very low quantities. The technology could be applied to a greater range of plant-based tea adulterants. This work is expected to facilitate the traceability and authenticity of tea products and form the basis for the development of strategies against fraudulent practices.

DNA Fingerprinting and Species Identification Uncovers the Genetic Diversity of Katsouni Pea in the Greek Islands Amorgos and Schinoussa.

Pea (P. sativum L.), one of the most important legume crops worldwide, has been traditionally cultivated in Lesser Cyclades since ancient times. The commonly known traditional pea cultivar, 'Katsouni', is endemic to the islands of Amorgos and Schinoussa and is of great local economic importance. Despite the widespread cultivation of 'Katsouni' in both islands, it is still unknown whether the current Schinoussa and Amorgos pea populations are distinct landraces, and if they have common evolutionary origin. To assist conservation and breeding of the pea crop, the genetic diversity and phylogenetic relationships of 39 pea samples from Amorgos and 86 from Schinoussa were studied using DNA barcoding and ISSR marker analyses. The results indicate that both populations are different landraces with distinct geographical distribution and are more closely related to P. sativum subsp. elatius than the P. abyssinicum and P. fulvum species. Further characterization of the 'Katsouni' landraces for functional polymorphisms regarding pathogen resistance, revealed susceptibility to the powdery mildew (Erysiphe pisi DC.). This work represents the first investigation on the genetic diversity and population structure of the 'Katsouni' cultivar. Exploiting the local genetic diversity of traditional landraces is fundamental for conservation practices and crop improvement through breeding strategies.

Novel Miscanthus genotypes selected for different drought tolerance phenotypes show enhanced tolerance across combinations of salinity and drought treatments.

BACKGROUND AND AIMS: Water deficit and salinity stresses are often experienced by plants concurrently; however, knowledge is limited about the effects of combined salinity and water deficit stress in plants, and especially in C4 bioenergy crops. Here we aim to understand how diverse drought tolerance traits may deliver tolerance to combinations of drought and salinity in C4 crops, and identify key traits that influence the productivity and biomass composition of novel Miscanthus genotypes under such conditions. METHODS: Novel genotypes used included M. sinensis and M. floridulus species, pre-screened for different drought responses, plus the commercial accession Miscanthus × giganteus (M×g.). Plants were grown under control treatments, single stress or combinations of water deficit and moderate salinity stress. Morphophysiological responses, including growth, yield, gas exchange and leaf water relations and contents of proline, soluble sugars, ash and lignin were tested for significant genotypic and treatment effects. KEY RESULTS: The results indicated that plants subjected to combined stresses showed more severe responses compared with single stresses. All novel drought-tolerant genotypes and M×g. were tolerant to moderate salinity stress. Biomass production in M. sinensis genotypes was more resilient to co-occurring stresses than that in M×g. and M. floridulus, which, despite the yield penalty produced more biomass overall. A stay-green M. sinensis genotype adopted a conservative growth strategy with few significant treatment effects. Proline biosynthesis was species-specific and was triggered by salinity and co-occurring stress treatments, mainly in M. floridulus. The ash content was compartmentalized differently in leaves and stems in the novel genotypes, indicating different mechanisms of ion accumulation. CONCLUSIONS: This study highlights the potential to select novel drought-tolerant Miscanthus genotypes that are resilient to combinations of stress and is expected to contribute to a deeper fundamental knowledge of different mechanistic responses identified for further exploitation in developing resilient Miscanthus crops.

An integrated metabolomic and gene expression analysis identifies heat and calcium metabolic networks underlying postharvest sweet cherry fruit senescence.

MAIN CONCLUSION: Ηeat and calcium treatments reprogram sweet cherry fruit metabolism during postharvest senescence as evidenced by changes in respiration, amino acid metabolism, sugars, and secondary metabolites shift. Heat and calcium treatments are used to improve postharvest fruit longevity; however, the exact mechanism remains poorly understood. To characterize the impact of these treatments on sweet cherries metabolism, 'Lapins' fruits were treated with heat or CaCl2 solutions and their combination and subsequently were exposed at room temperature, for up to 4 days, defined as senescence period. Single and combined heat and calcium treatments partially delayed fruit senescence, as evidenced by changes in fruit colour darkening, skin penetration force, and respiration activity. Calcium content was noticeably increased by heat in Ca-treated fruit. Several primary metabolites, including amino acids, organic acids, and alcohols, were decreased in response to both treatments, while many soluble sugars and secondary metabolites were increased within 1 day post-treatment. Changes of several metabolites in heat-treated fruits, especially esculetin, peonidin 3-O-glucoside and peonidin 3-O-galactoside, ribose, pyroglutamate, and isorhamnetin-3-O-rutinoside, were detected. The metabolome of fruit exposed to calcium also displayed substantial modulations, particularly in the levels of galactose, glycerate, aspartate, tryptophan, phospharate rutin, and peonidin 3-O-glucoside. The expression of several genes involved in TCA cycle (MDH1, IDH1, OGDH, SUCLA2, and SDH1-1), pectin degradation (ADPG1) as well as secondary (SK1, 4CL1, HCT, and BAN), amino acids (ALDH18A1, ALDH4A1, GS, GAD, GOT2, OPLAH, HSDH, and SDS), and sugar (PDHA1 and DLAT) metabolism were affected by both treatments. Pathway-specific analysis further revealed the regulation of fruit metabolic programming by heat and calcium. This work provides a comprehensive understanding of metabolic regulation in response to heat and calcium during fruit senescence.

Tolerance of Transplastomic Tobacco Plants Overexpressing a Theta Class Glutathione Transferase to Abiotic and Oxidative Stresses.

Chloroplasts are organelles subjected to extreme oxidative stress conditions. Biomolecules produced in the chloroplasts act as signals guiding plant metabolism toward stress tolerance and play a major role in regulating gene expression in the nucleus. Herein, we used transplastomic plants as an alternative approach to expression of transgenes in the nucleus for conferring stress tolerance to abiotic stresses and herbicides. To investigate the morphophysiological and molecular mechanisms and the role of plastid expressed GSTs in tobacco stress detoxification and stress tolerance, we used transplastomic tobacco lines overexpressing a theta class glutathione transferase (GST) in chloroplasts. The transplastomic plants were tested under drought (0, 100, and 200 mM mannitol) and salinity (0, 150, and 300 mM NaCl) in vitro, and under herbicide stress (Diquat). Our results suggest that pt AtGSTT lines were tolerant to herbicide-induced oxidative and salinity stresses and showed enhanced response tolerance to mannitol-induced osmotic stress compared to WT plants. Overexpression of the Arabidopsis thaliana AtGSTT in the chloroplasts resulted in enhanced photo-tolerance and turgor maintenance under stress. Whole-genome transcriptome analysis revealed that genes related to stress tolerance, were upregulated in pt AtGSTT2a line under both control and high mannitol stress conditions. Transplastomic plants overexpressing the pt AtGSTT2a in the chloroplast showed a state of acclimation to stress, as only limited number of genes were upregulated in the pt AtGSTT2a transplastomic line compared to WT under stress conditions while at the same time genes related to stress tolerance were upregulated in pt AtGSTT2a plants compared to WT in stress-free conditions. In parallel, the metabolic profile indicated limited perturbations of the metabolic homeostasis in the transplastomic lines and greater accumulation of mannitol, and soluble sugars under high mannitol stress. Therefore, transplastomic lines seem to be in a state of acclimation to stress under stress-free conditions, which was maintained even under high mannitol stress. The results help to elucidate the role of GSTs in plant abiotic stress tolerance and the underlying mechanisms of the GSTs expressed in the chloroplast, toward environmental resilience of cultivated crops.