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The plant hormones salicylic acid (SA) and jasmonic acid (JA) regulate defense mechanisms capable of overcoming different plant stress conditions and constitute distinct but interconnected signaling pathways. Interestingly, several other molecules are reported to trigger stress-specific defense responses to biotic and abiotic stresses. In this study, we investigated the effect of 14 elicitors against diverse but pivotal types of abiotic (drought) and biotic (the chewing insect Ascia monuste , the hemibiotrophic bacterium Pseudomonas syringae DC 3000 and the necrotrophic fungus Alternaria alternata ) stresses on broccoli and Arabidopsis . Among the main findings, broccoli pre-treated with SA and chitosan showed the highest drought stress recovery in a dose-dependent manner. Several molecules led to increased drought tolerance over a period of three weeks. The enhanced drought tolerance after triggering the SA pathway was associated with stomata control. Moreover, methyl jasmonate (MeJA) reduced A. monuste insect development and plant damage, but unexpectedly, other elicitors increased both parameters. GUS reporter assays indicated expression of the SA-dependent PR1 gene in plants treated with nine elicitors, whereas the JA-dependent LOX2 gene was only expressed upon MeJA treatment. Overall, elicitors capable of tackling drought and biotrophic pathogens mainly triggered the SA pathway, but adversely also induced systemic susceptibility to chewing insects. These findings provide directions for potential future in-depth characterization and utilization of elicitors and induced resistance in plant protection.

BackgroundPathological calcification ((PC), i.e. the deposition of calcium-containing crystals) in tendons is a hallmark of calcific tendinopathy (CT), a disease associated with pain, tendon rupture, and disability. Currently, only symptomatic treatments exist for CT and none of them target specifically calcification. Intriguingly, the gasotransmitter H2S has recently emerged as a potential anti-calcifying molecule [1].ObjectivesTo establish in CT the potential anti-calcifying effect of hydrogen sulfide (H2S) and of the H2S-producing enzyme cystathionine gamma lyase (CSE), and the underlying mechanisms involved.MethodsWild type (WT) and CSE knock-out (KO) tenocytes were isolated from adult mice Achilles’ tendons. Tenocyte were cultured in αMEM + 10% FBS + 50μg/ml ascorbic acid (Control medium) in presence of 10% calciprotein particles (calcification medium, CM). Spontaneous Achilles’ tendon CT in old mice (35 weeks old) and surgery-induced Achilles’ tendon CT in8-12 weeks old mice were quantified by micro computed tomography. Dynamic Young’s modulus was measured in 35 weeks old mice as previously described [2]. Immunohistochemistry was performed on human and murine tendon sections with anti-CSE and anti-LOX and anti-LOXL2rabbit polyclonal antibodies. LOX expression was analyzed by qPCR and by Western-blot. LOX(L) activity was quantified in cells supernatants and lysates.ResultsIn vitro, tenocyte calcification (in CM) was inhibited by exogenous H2S-donors, while it was exacerbated in CSE KO tenocytes, producing as expected less H2S than WT. Reduced calcification in tenocytes exposed to H2S was accompanied by decreased expression of genes coding for BMP2, BMP4 and decreased activation of the BMP signaling pathway (pSMAD1/5/8). Accordingly, BMPs expression and BMPs-pathway activation were exacerbated in CSE KO tenocytes compared to WT tenocytes.The protective role of CSE-H2S was confirmed in vivo. Indeed, in aged mice, micro computed tomography revealed exacerbated Achilles’ tendon calcification in CSE KO mice compared to WT. Interestingly, CSE deficiency led to reduced biomechanical strength, as the dynamic Young’s modulus was significantly decreased upon increased tendon displacement. Furthermore, using a tenotomy model of CT, we found an inverse correlation between CSE expression and calcification in tendons. This was confirmed in human tendons from CT patients, which exhibited decreased CSE expression where calcification was present.In parallel experiments, we found that calcification in tenocytes was significantly reduced by addition of BAPN, a pan-inhibitor of lysyl oxidases (LOX(L)) enzymes. LOX(L) family includes 5 enzymes LOX and LOX1-4, catalysing elastin and collagen cross-links. We next investigated if the anti-calcfiying effect of H2S in CT could be mediated by inhibition of LOX(L). Indeed, in CM-stimulated tenocytes we found that H2S impacts both LOX/LOX2 expression (as CSE deficiency increased significantly LOX/LOX2 gene expression) and LOX(L) activity (as CSE deficiency increased LOX(L) activity and conversely H2S dose-dependently inhibited LOX(L) activity in WT tenocytes. Finally, in vivo we discovered that, CSE was inversely correlated with calcification and LOX and LOXL2 expression in mouse and human tendons.ConclusionAltogether, our results suggest that increasing H2S levels in tenocytes could represent a future strategy to prevent or decrease calcification in CT, likely via down-modulation of LOX(L) expression and activity.References[1]Castelblanco, M., et al., The role of the gasotransmitter hydrogen sulfide in pathological calcification. Br J Pharmacol, 2020. 177(4): p. 778-792.[2]Kronenberg, D., et al., Increased Collagen Turnover Impairs Tendon Microstructure and Stability in Integrin alpha2beta1-Deficient Mice. Int J Mol Sci, 2020. 21(8).Acknowledgements:NIL.Disclosure of InterestsNone Declared.

Temperature and relative humidity (RH) influence post-harvest ripening, a crucial stage for quality promotion in some oil plants or fruits. Torreya grandis cv. Merrillii nuts, which are rich in unsaturated fatty acids (UFA), are easily affected by temperature and humidity, and they oxidize quickly during the post-harvest ripening stage, leading to the deterioration of nut quality. In this study, the main nutraceutical components, fatty acid composition, and related metabolic parameters of lipid rancidity under four treatments (20°C and 70% RH, T20-LH; 30°C and 70% RH, T30-LH; 20°C and 90% RH, T20-HH; 30°C and 90% RH, T30-HH) were measured. The post-harvest ripening process was advanced under HH treatments (T20-HH and T30-HH) compared to LH treatments (T20-LH and T30-LH) and was associated with a shorter time for the seed coat to turn dark black and a faster reduction in starch content. The amount of unsaturated fatty acids significantly increased under the T20-HH treatment, but significantly decreased under the T30-HH treatment from 12 to 16 d of ripening time. The acid value (AV) and lipase activity under the T30-HH treatment remained virtually constant from 12 to 16 d of ripening time, and this was accompanied by a dramatic increase in peroxide value (POV), lipoxygenase (LOX) activity, and relative expression of the LOX2 gene. Meanwhile, a significant positive correlation between LOX activity and POV, malondialdehyde (MDA) content, and O2⋅– content was observed. The results imply that the lower amount of oxidative rancidity induced by the T20-HH treatment is related to the LOX activity induced by down-regulation of the LOX2 gene during the late after-ripening stage. Therefore, the T20-HH treatment not only promoted the post-harvest process of T. grandis ‘Merrillii’ nuts but also delayed lipid oxidation, which was ultimately associated with better oil quality at the late after-ripening stage.

Little is known about molecular responses in plants to phloem feeding by insects. The induction of genes associated with wound and pathogen response pathways was investigated following green peach aphid (Myzus persicae) feeding on Arabidopsis. Aphid feeding on rosette leaves induced transcription of two genes associated with salicylic acid (SA)-dependent responses to pathogens (PR-1 and BGL2) 10- and 23-fold, respectively. Induction of PR-1 and BGL2 mRNA was reduced in npr1 mutant plants, which are deficient in SA signaling. Application of the SA analog benzothiadiazole led to decreases in aphid reproduction on leaves of both wild-type plants and mutant plants deficient in responsiveness to SA, suggesting that wild-type SA-dependent responses do not influence resistance to aphids. Two-fold increases occurred in mRNA levels of PDF1.2, which encodes defensin, a peptide involved in the jasmonate (JA)-/ethylene-dependent response pathway. Transcripts encoding JA-inducible lipoxygenase (LOX2) and SA/JA-inducible Phe-ammonia lyase increased 1.5- to 2-fold. PDF1.2 and LOX2 induction by aphids did not occur in infested leaves of the JA-resistant coi1-1 mutant. Aphid feeding induced 10-fold increases in mRNA levels of a stress-related monosaccharide symporter gene, STP4. Phloem feeding on Arabidopsis leads to stimulation of response pathways associated with both pathogen infection and wounding.

In this project, we studied the gene regulation of 15-lipoxygenase 2 (15-LOX2), the most abundant arachidonate-metabolizing LOX in adult human prostate and a negative cell-cycle regulator in normal human prostate (NHP) epithelial cells. Through detailed in silico promoter examination and promoter deletion and activity analysis, we found that several Sp1 sites (i.e., three GC boxes and one CACCC box) in the proximal promoter region play a critical role in regulating 15-LOX2 expression in NHP cells. Several pieces of evidence further suggest that the Sp1 and Sp3 proteins play a physiologically important role in positively and negatively regulating the 15-LOX2 gene expression, respectively. First, mutations in the GC boxes affected the 15-LOX2 promoter activity. Second, both Sp1 and Sp3 proteins were detected in the protein complexes that bound the GC boxes revealed by electrophoretic mobility shift assay. Third, importantly, inhibition of Sp1 activity or overexpression of Sp3 both inhibited the endogenous 15-LOX2 mRNA expression. Since 15-LOX2 is normally expressed in the prostate luminal epithelial cells, we subsequently explored whether androgen/androgen receptor may directly regulate its gene expression. The results indicate that androgen does not directly regulate 15-LOX2 gene expression. Together, these observations provide insight on how 15-LOX2 gene expression may be regulated in NHP cells.

Fusarium stalk rot caused by Fusarium verticillioides is one of the most devastating diseases of maize that causes significant yield losses and poses potential security concerns for foods worldwide. The underlying mechanisms of maize plants regulating defence against the disease remain poorly understood. Here, integrative proteomic and transcriptomic analyses were employed to identify pathogenesis‐related protein genes by comparing differentially expressed proteins (DEPs) and differentially expressed genes (DEGs) in maize stalks after inoculation with F. verticillioides. Functional enrichment analysis showed that DEGs and DEPs were mainly enriched in glutathione metabolism, starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, linoleic acid metabolism, and phenylpropanoid biosynthesis. Fourteen DEGs and DEGs that were highly elevated after inoculation with F. verticillioides were confirmed with parallel reaction monitoring and reverse transcription‐quantitative PCR, demonstrating the accountability and reliability of proteomic and transcriptomic data. We also assessed the potential roles of defence‐related genes ZmCTA1, ZmWIP1, and ZmLOX2, identified from the multi‐omics analysis, during the process of F. verticillioides infection through virus‐induced gene silencing. The elevation of stalk rot symptomatic characteristics in the silenced plants revealed their contribution to resistance. We further functionally characterized the roles of ZmLOX2 expression in the defence response of maize plants conditioning fungal invasion via the salicylic acid‐dependent pathway. Collectively, this study provides a comprehensive analysis of transcriptome and proteome of maize stalks following F. verticillioides inoculation, and defence‐related genes that could inform selection of new genes as targets in breeding strategies. This study analysed the transcriptome and proteome of maize stalks following Fusarium verticillioides infection and characterizes the roles of ZmLOX2 in the maize defence response via the salicylic acid‐dependent pathway.

High-salinity and blast disease are two major stresses that cause dramatic yield loss in rice production. GF14 ( 14-3-3 ) genes have been reported to play important role in biotic and abiotic stresses in plants. However, the roles of OsGF14C remain unknown. To understand the functions and regulatory mechanisms of OsGF14C in regulating salinity tolerance and blast resistance in rice, we have conducted OsGF14C -overexpressing transgenic experiments in the present study. Our results showed that overexpression of OsGF14C enhanced salinity tolerance but reduced blast resistance in rice. The enhanced salinity tolerance is related to the reduction of methylglyoxal and Na + uptake instead of exclusion or compartmentation and the negative role of OsGF14C in blast resistance is associated with the suppression of OsGF14E , OsGF14F and PR genes. Our results together with the results from the previous studies suggest that the lipoxygenase gene LOX2 which is regulated by OsGF14C may play roles in coordinating salinity tolerance and blast resistance in rice. The current study for the first time revealed the possible roles of OsGF14C in regulating salinity tolerance and blast resistance in rice, and laid down a foundation for further functional study and crosstalk regulation between salinity and blast resistance in rice.

Key messageTCX8 localizes to nucleus and has transcriptional repression activity. TCX8 binds to the promoter region of LOX2 encoding lipoxygenase, causing JA biosynthesis suppression, and thereby delays plant senescence.Conserved CXC domain-containing proteins are found in most eukaryotes. Eight TCX proteins, which are homologs of animal CXC–Hinge–CXC (CHC) proteins, were identified in Arabidopsis, and three of them, TSO1, TCX2/SOL2 and TCX3/SOL1, have been reported to affect cell-cycle control. TCX8, one of the TCX family proteins, was believed to be a TF but its precise function has not been reported. Yeast two-hybrid screening revealed TCP20, a TF that binds to the promoter of LOX2 encoding lipoxygenase, as a strong candidate for interaction with TCX8. We confirmed that TCX8 directly interacts with TCP20 using in vitro pull-down assay and in vivo BiFC and observed that TCX8, as a TF, localizes to nucleus. Using EMSA and by analyzing phenotypes of TCX8-overexpression lines, we demonstrated that TCX8 regulates the expression of LOX2 by binding to either cis-element of LOX2 promoter to which TCP20 or TCP4 binds, affecting JA biosynthesis, and thereby delaying plant senescence. Our study provides new information about the role of TCX8 in modulating plant senescence through regulating LOX2 expression.

Background: Phoronida is a small group of marine worm-like suspension feeders, which together with brachiopods and bryozoans form the clade Lophophorata. Although their development is well studied on the morphological level, data regarding gene expression during this process are scarce and restricted to the analysis of relatively few transcription factors. Here, we present a description of the expression patterns of Hox genes during the embryonic and larval development of the phoronid Phoronopsis harmeri. Results: We identified sequences of eight Hox genes in the transcriptome of Ph. harmeri and determined their expression pattern during embryonic and larval development using whole mount in situ hybridization. We found that none of the Hox genes is expressed during embryonic development. Instead their expression is initiated in the later developmental stages, when the larval body is already formed. In the investigated initial larval stages the Hox genes are expressed in the non-collinear manner in the posterior body of the larvae: in the telotroch and the structures that represent rudiments of the adult worm. Additionally, we found that certain head-specific transcription factors are expressed in the oral hood, apical organ, preoral coelom, digestive system and developing larval tentacles, anterior to the Hox-expressing territories. Conclusions: The lack of Hox gene expression during early development of Ph. harmeri indicates that the larval body develops without positional information from the Hox patterning system. Such phenomenon might be a consequence of the evolutionary intercalation of the larval form into an ancestral life cycle of phoronids. The observed Hox gene expression can also be a consequence of the actinotrocha representing a “head larva”, which is composed of the most anterior body region that is devoid of Hox gene expression. Such interpretation is further supported by the expression of head-specific transcription factors. This implies that the Hox patterning system is used for the positional information of the trunk rudiments and is, therefore, delayed to the later larval stages. We propose that a new body form was intercalated to the phoronid life cycle by precocious development of the anterior structures or by delayed development of the trunk rudiment in the ancestral phoronid larva.

Wounding is a constant threat to plant survival throughout their lifespan; therefore, understanding the biological responses to wounds at the cellular level is important. The protoplast system is versatile for molecular biology, however, no wounding studies on this system have been reported. We established a new approach for wounding research using mechanically damaged Arabidopsis mesophyll protoplasts. Wounded protoplasts showed typical wounding responses, such as increased MPK6 kinase activity and upregulated JAZ1 expression. We also assessed expression profiles and protein stability of the basic helix-loop-helix transcription factor MYC2 in wounded protoplasts. Promoter activity, gene expression, and protein stability of MYC2 were compromised, but recovered in the early stage of wounding. In the late stage, the promoter activity and expression of MYC2 were increased, but the protein stability was not changed. According to the results of the present study, this new cell-based approach will be of use in various molecular studies on plant wounding.