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Industrial hemp (Cannabis sativa L.) is receiving increasing attention for its multiple end-uses; therefore, an improvement in its production is needed to meet the increased demand. In the present study, the effect of seed inoculation with two Trichoderma afroharzianum strains, T-AA and T-22, on plant growth and root morphology of hemp plants at sixth-leaf (S6) and tenth-leaf (S10) stages was assessed for two consecutive years (2020 and 2021). In addition, the ability of the two strains to enhance the accumulation of phenolic compounds in hemp leaves was also evaluated. The results obtained revealed the ability of T-22 to improve the growth and root morphology of hemp plants both in 2020 and 2021, although with different impact, probably ascribable to the different weather conditions in the two years. In 2020, the positive effects of T-22 were detected at S10 stage with significant increases in the shoot and root length (38% and 17%, respectively) and dried biomass (35% and 30%, respectively) compared to untreated plants. The total root surface area and the number of tips, forks, and crossings also increased significantly (24–36%) at this stage. In 2021, significant increases in the shoot length and dried biomass (40% and 30%, respectively) were observed at S6 stage, whereas root length and dried biomass increased significantly at S6 (55% and 47%, respectively) and S10 stage (121% and 40%, respectively). Significant increases in the total surface area and volume, as well as in the number of tips, forks, and crossings were also observed at both S6 and S10 stage (50–63% and 105–187%, respectively). Interestingly, in both years and at both stages, the two strains induced significant increases in the leaf accumulation of phenolic compounds and the antioxidant activity, which were greater in T-22- compared to T-AA-treated plants (18–102% and 13–34%, respectively). The results are discussed in light of the potential practical applications of T-22 as a biostimulant of hemp plant growth under favorable and unfavorable environmental conditions, and of both strains as promising tools for the improvement of the leaves' economic value as a source of health-promoting compounds. [ABSTRACT FROM AUTHOR]

In the present study, the phytochemical content and the antioxidant activity in the inflorescences of the monoecious hemp cultivar Codimono grown in southern Italy were assessed, and their elicitation was induced by foliar spray application of 50 mg/L and 250 mg/L of chitosan (CHT) at three different molecular weights (low, CHT L; medium, CHT M; high CHT H). The analysis of the phytochemical profile confirmed that cannabinoids were the most abundant class (54.2%), followed by flavonoids (40.3%), tocopherols (2.2%), phenolic acids (1.9%), and carotenoids (1.4%). Cannabinoids were represented almost exclusively by cannabidiol, whereas cannabigerol and Δ9-tetrahydrocannabinol were detected at very low levels (the latter was below the legal limit of 0.3%). The most abundant flavonoids were orientin and vitexin, whereas tocopherols were mainly represented by α-tocopherol. The antioxidant activity was found to be positively correlated with flavonoids and tocopherols. Statistical analysis revealed that the CHT treatments significantly affected the phytochemical content and the antioxidant activity of hemp inflorescences. Notably, a significant increase in the total phenolic content (from +36% to +69%), the α-tocopherol (from +45% to +75%) and β+γ-tocopherol (from +35% to +82%) contents, and the ABTS radical scavenging activity (from +12% to +28%) was induced by all the CHT treatments. In addition, treatments with CHT 50 solutions induced an increase in the total flavonoid content (from +12% to +27%), as well as in the vitexin (from +17% to +20%) and orientin (from +20% to +30%) contents. Treatment with CHT 50 L almost always resulted in the greatest increases. Overall, our findings indicated that CHT could be used as a low-cost and environmentally safe elicitor to improve the health benefits and the economic value of hemp inflorescences, thus promoting their employment in the food, pharmaceutical, nutraceutical, and cosmetic supply chains. [ABSTRACT FROM AUTHOR]

Abstract Background The yellow colour of pasta products is one of the main criteria used by consumers to assess pasta quality. This character is due to the presence of carotenoid pigments in semolina. During pasta processing, oxidative degradation of carotenoid pigments occurs mainly due to lipoxygenase (LOX). In durum wheat (Triticum durum Desf.), two Lpx-1 genes have been identified on chromosome 4B, Lpx-B1.1 and Lpx-B1.2, and evidences have been reported that the deletion of Lpx-B1.1 is associated with a strong reduction in LOX activity in semolina. In the present study, we characterised the Lpx-B1 gene family identified in a durum wheat germplasm collection and related the distribution and expression of the Lpx-B1 genes and alleles to variations in LOX activity in the mature grains. Results In addition to the already known Lpx-B1.1 and Lpx-B1.2 genes, a new gene was identified, Lpx-B1.3, along with three different Lpx-B1.1 alleles, Lpx-B1.1a, Lpx-B1.1b and the partially deleted Lpx-B1.1c. Screening of the germplasm collection showed that all of the genotypes have one of the three Lpx-B1.1 alleles, associated with either Lpx-B1.2 or Lpx-B1.3, thus showing that in this collection the two genes are alternatives. Therefore, based on Lpx-B1 distribution, three different haplotypes were distinguished: haplotype I, carrying Lpx-B1.3 and the Lpx-B1.1b allele; haplotype II carrying Lpx-B1.2 and the Lpx-B1.1a allele; and haplotype III carrying Lpx-B1.2 and the Lpx-B1.1c allele. Determination of Lpx-B1 transcript abundance and total LOX activity in mature grains revealed differences among these three haplotypes: haplotypes I, II and III showed high, intermediate and low levels, respectively, of functional Lpx-B1 transcripts and enzymatic activity. Conclusions In this germplasm collection, the Lpx-B1 gene family accounts for most of the total LOX activity in the mature grains. Information on these Lpx-B1 haplotypes provides significant improvement for prediction of LOX-1 activity levels in mature grains, and will therefore help in breeding programmes aimed at selection of new durum wheat genotypes with higher carotenoid contents in their end products.

We previously isolated four cDNAs in durum wheat, TdsPLA 2 I , TdsPLA 2 II , TdsPLA 2 III and TdsPLA 2 IV , that encode proteins with homology to plant secretory phospholipases A 2 (sPLA 2 s) (Verlotta et al., Int. J. Mol. Sci. , 14, 2013, 5146–5169). In this study, we have further characterized TdsPLA 2 II and TdsPLA 2 III sequences that, on the basis of our previous findings, might encode sPLA 2 isoforms with different features. Functional analysis revealed that, similarly to other known sPLA 2 s, TdsPLA 2 II and TdsPLA 2 III have an optimum at pH 9.0, require Ca 2+ , are heat stable, and are inhibited by the disulfide-bond-reducing agent dithiothreitol. However, differences emerged between these TdsPLA 2 isoforms. Transcript analysis revealed that the TdsPLA 2 III gene is highly up-regulated under different environmental stresses; conversely, the TdsPLA 2 II gene is expressed at constant levels under almost all of the stress conditions examined. Moreover, TdsPLA 2 II is saturated at micromolar substrate and Ca 2+ concentrations, whereas TdsPLA 2 III requires millimolar concentrations to reach maximal activity. This suggests that TdsPLA 2 II normally functions under optimal conditions in vivo , whereas TdsPLA 2 III is only partially activated, depending on the specific phospholipid and Ca 2+ levels. Altogether these data lead to the hypothesis that in vivo TdsPLA 2 II and TdsPLA 2 III are differently regulated at both molecular and biochemical level and that TdsPLA 2 III plays a major role in durum wheat response to adverse environmental conditions. [ABSTRACT FROM AUTHOR]

Three independent durum wheat mutant lines that show delayed leaf senescence or stay-green (SG) phenotype, SG196, SG310 and SG504, were compared to the parental genotype, cv. Trinakria, with respect to the photosynthetic parameters and the cellular redox state of the flag leaf in the period from flowering to senescence. The SG mutants maintained their chlorophyll content and net photosynthetic rate for longer than Trinakria, thus revealing a functional SG phenotype. They also showed a better redox state as demonstrated by: (1) a lower rate of superoxide anion production due to generally higher activity of the antioxidant enzymes superoxide dismutase and catalase in all of the SG mutants and also of the total peroxidase in SG196; (2) a higher thiol content that can be ascribed to a higher activity of the NADPH-providing enzyme glucose-6-phosphate dehydrogenase in all of the SG mutants and also of the NADP-dependent malic enzyme in SG196; (3) a lower pro-oxidant activity of lipoxygenase that characterises SG196 and SG504 mutants close to leaf senescence. Overall, these results show a general relationship in durum wheat between the SG phenotype and a better redox state. This relationship differs across the different SG mutants, probably as a consequence of the different set of altered genes underlying the SG trait in these independent mutant lines. [ABSTRACT FROM AUTHOR]

Phospholipases A2 (PLA2s) are known to mediate signaling cascades during plant growth and development, as well as biotic and abiotic stress responses. In this context, the present study provides extensive characterization of specific PLA2s in durum wheat, and assesses their involvement in durum wheat response to drought stress. In durum wheat leaves, four full-length expressed sequences encoding putative PLA2s were isolated and characterized as belonging to the class of secretory PLA2s (sPLA2s): TdsPLA2I, TdsPLA2II, TdsPLA2III and TdsPLA2IV. PLA2 activity was also detected, the characteristics of which resemble those of previously characterized plant sPLA2s: strong preference for phospholipids; requirement for millimolar Ca2+ concentrations; optimal activity at basic pH; heat stability; and inhibition by the reducing agent dithiothreitol. With drought stress imposed at both the vegetative and reproductive stages, accumulation of TdsPLA2I and TdsPLA2III transcripts, and to a lesser extent of TdsPLA2IV transcript, paralleled increased PLA2 activity; both transcript levels and enzymatic activity decreased as a consequence of stress recovery. Consistently, free fatty acid analysis of drought-stressed leaves revealed increased linoleate, linolenate and palmitate contents, which were reversed by plant re-watering. Overall, these findings strongly suggest that there are inducible sPLA2 isoforms in durum wheat that have roles in orchestrating the plant response to drought stress. [ABSTRACT FROM AUTHOR]

Abstract: The activity of mitochondrial phospholipase A2 (PLA2) was shown for the first time in plants. It was observed in etiolated seedlings from durum wheat, barley, tomato, spelt and green seedlings of maize, but not in potato and topinambur tubers and lentil etiolated seedlings. This result was achieved by a novel spectrophotometric assay based on the coupled PLA2/lipoxygenase reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine as substrate; the mitochondrial localisation was assessed by checking recovery of marker enzymes. Durum wheat mitochondrial PLA2 (DWM-PLA2) showed maximal activity at pH 9.0 and 1mM Ca2+, hyperbolic kinetics (K m =90±6μM, V max =29±1nmolmin−1 mg−1 of protein) and inhibition by methyl arachidonyl fluorophosphonate, 5-(4-benzyloxyphenyl)-4S-(7-phenylheptanoylamino)pentanoic acid and palmityl trifluoromethyl ketone. Reactive oxygen species had no effect on DWM-PLA2, that instead was activated by about 50% and 95%, respectively, under salt (0.21M NaCl) and osmotic (0.42M mannitol) stress imposed during germination. Contrarily, a secondary Ca2+-independent activity, having optimum at pH 7.0, was stress-insensitive. We propose that the activation of DWM-PLA2 is responsible for the strong increase of free fatty acids recently measured in mitochondria under the same stress conditions [Laus, et al., J. Exp. Bot. 62 (2011) 141–154] that, in turn, activate potassium channel and uncoupling protein, able to counteract hyperosmotic stress. [Copyright &y& Elsevier]