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Trichomes are unicellular or multicellular hair-like appendages developed on the aerial plant epidermis of most plant species that act as a protective barrier against natural hazards. For this reason, evaluating the density of trichomes is a valuable approach for elucidating plant defence responses to a continuous challenging environment. However, previous methods for trichome counting, although reliable, require the use of specialised equipment, software or previous manipulation steps of the plant tissue, which poses a complicated hurdle for many laboratories. Here, we propose a new fast, accessible and user-friendly method to quantify trichomes that overcomes all these drawbacks and makes trichome quantification a reachable option for the scientific community. Particularly, this new method is based on the use of machine learning as a reliable tool for quantifying trichomes, following an Ilastik-Fiji tandem approach directly performed on 2D images. Our method shows high reliability and efficacy on trichome quantification in Arabidopsis thaliana by comparing manual and automated results in Arabidopsis accessions with diverse trichome densities. Due to the plasticity that machine learning provides, this method also showed adaptability to other plant species, demonstrating the ability of the method to spread its scope to a greater scientific community.

12 Pág. Centro de Biotecnología y Genómica de Plantas, During barley germination, cysteine proteases are essential in the mobilization of storage compounds providing peptides and amino acids to sustain embryo growth until photosynthesis is completely established. Knockdown barley plants, generated by artificial miRNA, for the cathepsins B- and F-like HvPap-19 and HvPap-1 genes, respectively, showed less cysteine protease activities and consequently lower protein degradation. The functional redundancy between proteases triggered an enzymatic compensation associated with an increase in serine protease activities in both knockdown lines, which was not sufficient to maintain germination rates and behaviour. Concomitantly, these transgenic lines showed alterations in the accumulation of protein and carbohydrates in the grain. While the total amount of protein increased in both transgenic lines, the starch content decreased in HvPap-1 knockdown lines and the sucrose concentration was reduced in silenced HvPap-19 grains. Consequently, phenotypes of HvPap-1 and HvPap-19 artificial miRNA lines showed a delay in the grain germination process. These data demonstrate the potential of exploring the properties of barley proteases for selective modification and use in brewing or in the livestock feeding industry., This research was supported by the Ministerio de Economia, Industria y Competitividad of Spain (projects BIO2014-53508-R, BIO17-83472-R, and RyC17MESFB). RyC grant (RyC2017-21814) from the MEIC and Plan Propio from the UPM-financed MES. We gratefully appreciate comments from Dr Ljudmilla Borisjuk and Dr Volodymyr Radchuk from the IPK-Gatersleben (Germany).

Plant-pest interactions involve multifaceted processes encompassing a complex crosstalk of pathways, molecules, and regulators aimed at overcoming defenses developed by each interacting organism. Among plant defensive compounds against phytophagous arthropods, cyanide-derived products are toxic molecules that directly target pest physiology. Here, we identified the Arabidopsis (Arabidopsis thaliana) gene encoding hydroxynitrile lyase (AtHNL, At5g10300) as one gene induced in response to spider mite (Tetranychus urticae) infestation. AtHNL catalyzes the reversible interconversion between cyanohydrins and derived carbonyl compounds with free cyanide. AtHNL loss- and gain-of-function Arabidopsis plants showed that specific activity of AtHNL using mandelonitrile as substrate was higher in the overexpressing lines than in wild-type (WT) and mutant lines. Concomitantly, mandelonitrile accumulated at higher levels in mutant lines than in WT plants and was significantly reduced in the AtHNL overexpressing lines. After mite infestation, mandelonitrile content increased in WT and overexpressing plants but not in mutant lines, while hydrogen cyanide (HCN) accumulated in the three infested Arabidopsis genotypes. Feeding bioassays demonstrated that the AtHNL gene participated in Arabidopsis defense against T. urticae. The reduced leaf damage detected in the AtHNL overexpressing lines reflected the mite's reduced ability to feed on leaves, which consequently restricted mite fecundity. In turn, mites upregulated TuCAS1 encoding β-cyanoalanine synthase to avoid the respiratory damage produced by HCN. This detoxification effect was functionally demonstrated by reduced mite fecundity observed when dsRNA-TuCAS-treated mites fed on WT plants and hnl1 mutant lines. These findings add more players in the Arabidopsis-T. urticae interplay to overcome mutual defenses., Grants BIO2017-83472-R, PDC2021-121055-I00, RED2018-102407-T, RED2018-102397-T and RyC17MESFB funded by MCIN/AEI/10.13039/501100011033, as appropriate, by “ERDF A way of making Europe” and by the “European Union” supported this work. The Convenio Plurianual between Comunidad de Madrid and UPM through the Apoyo-Jóvenes investigadores Program (grant SUR6Q9-22-YTFC3Z) supported this work. Grants LCF/PR18/51130007 from “La Caixa” Foundation, RyC2017-21814 and PRE2018-083375 from MCIN/AEI supported A.A., M.E.S., and I.R-D, respectively. The Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-2018.04538) supported S.D.

Plant immunity depends on fast and specific transcriptional reprogramming triggered by the perception of biotic stresses. Numerous studies have been conducted to better understand the response of plants to the generalist herbivore two-spotted spider mite (Tetranychus urticae). However, how plants perceive mites and how this perception is translated into changes in gene expression are largely unknown. In this work, we identified a gene induced in Arabidopsis (Arabidopsis thaliana) upon spider mite attack that encodes a two-domain protein containing predicted lectin and Toll/Interleukin-1 receptor domains. The gene, previously named PP2-A5, belongs to the Phloem Protein2 family. Biotic assays showed that PP2-A5 confers tolerance to T. urticae. Overexpression or knockout of PP2-A5 leads to transcriptional reprogramming that alters the balance of hormone accumulation and corresponding signaling pathways. The nucleocytoplasmic location of this protein supports a direct interaction with regulators of gene transcription, suggesting that the combination of two putative signaling domains in a single protein may provide a novel mechanism for regulating gene expression. Together, our results suggest that PP2-A5 improves the ability to defend against T. urticae by participating in the tight regulation of hormonal cross talk upon mite feeding. Further research is needed to determine the mechanism by which this two-domain protein functions and to clarify its molecular role in signaling following a spider mite attack.

Departamento de Biotecnología (INIA), The success in the response of a plant to a pest depends on the regulatory networks that connect plant perception and plant response. Meta-analyses of transcriptomic responses are valuable tools to discover novel mechanisms in the plant/herbivore interplay. Considering the quantity and quality of available transcriptomic analyses, Arabidopsis thaliana was selected to test the ability of comprehensive meta-analyses to disentangle plant responses. The analysis of the transcriptomic data showed a general induction of biological processes commonly associated with the response to herbivory, like jasmonate signaling or glucosinolate biosynthesis. However, an uneven induction of many genes belonging to these biological categories was found, which was likely associated with the particularities of each specific Arabidopsis-herbivore interaction. A thorough analysis of the responses to the lepidopteran Pieris rapae and the spider mite Tetranychus urticae highlighted specificities in the perception and signaling pathways associated with the expression of receptors and transcription factors. This information was translated to a variable alteration of secondary metabolic pathways. In conclusion, transcriptomic meta-analysis has been revealed as a potent way to sort out relevant physiological processes in the plant response to herbivores. Translation of these transcriptomic-based analyses to crop species will permit a more appropriate design of biotechnological programs., Research was supported by the Ministerio de Economía, Industria y Competitividad (MEIC, Grants BIO2017-83472-R, RED2018-102407-T, and RyC17MESFB) and by the Convenio Plurianual between Comunidad de Madrid (CM) and Universidad Politécnica de Madrid (UPM) through the Programa de Apoyo a la Realización de Proyectos de I+D para Jóvenes Investigadores (APOYO-jovenes-SUR6Q9-22-Y). RyC grant (RyC2017-21814) from the MEIC and Plan Propio from the UPM financed MES. Programa Propio UPM financed AG., 15 Pág.

HOPs (HSP70–HSP90 organizing proteins) are a highly conserved family of HSP70 and HSP90 co-chaperones whose role in assisting the folding of various hormonal receptors has been extensively studied in mammals. In plants, HOPs are mainly associated with stress response, but their potential involvement in hormonal networks remains completely unexplored. In this article we describe that a member of the HOP family, HOP3, is involved in the jasmonic acid (JA) pathway and is linked to plant defense responses not only to pathogens, but also to a generalist herbivore. The JA pathway regulates responses to Botrytis cinerea infection and to Tetranychus urticae feeding; our data demonstrate that the Arabidopsis (Arabidopsis thaliana) hop3-1 mutant shows an increased susceptibility to both. The hop3-1 mutant exhibits reduced sensitivity to JA derivatives in root growth assays and downregulation of different JA-responsive genes in response to methyl jasmonate, further revealing the relevance of HOP3 in the JA pathway. Interestingly, yeast two-hybrid assays and in planta co-immunoprecipitation assays found that HOP3 interacts with COI1, suggesting that COI1 is a target of HOP3. Consistent with this observation, COI1 activity is reduced in the hop3-1 mutant. All these data strongly suggest that, specifically among HOPs, HOP3 plays a relevant role in the JA pathway by regulating COI1 activity in response to JA and, consequently, participating in defense signaling to biotic stresses., One-sentence summary: The co-chaperone protein HOP3 (HSP70-HSP90 ORGANIZING PROTEIN 3) regulates the activity of jasmonic acid co-receptor CORONATINE INSENSITIVE 1 and functions in plant defense.

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy-2 and Icy-4 cystatin genes. Artificial microRNA lines independently silencing the two drought-induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay-green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy-2 and Icy-4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap-1, HvPap-12, and HvPap-16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.

21 Pàg. Centro de Biotecnología y Genómica de Plantas, The indole-3-pyruvic acid pathway is the main route for auxin biosynthesis in higher plants. Tryptophan aminotransferases (TAA1/TAR) and members of the YUCCA family of flavin-containing monooxygenases catalyze the conversion of l-tryptophan via indole-3-pyruvic acid to indole-3-acetic acid (IAA). It has been described that jasmonic acid (JA) locally produced in response to mechanical wounding triggers the de novo formation of IAA through the induction of two YUCCA genes, YUC8 and YUC9. Here, we report the direct involvement of a small number of basic helix-loop-helix transcription factors of the MYC family in this process. We show that the JA-mediated regulation of the expression of the YUC8 and YUC9 genes depends on the abundance of MYC2, MYC3, and MYC4. In support of this observation, seedlings of myc knockout mutants displayed a strongly reduced response to JA-mediated IAA formation. Furthermore, transactivation assays provided experimental evidence for the binding of MYC transcription factors to a particular tandem G-box motif abundant in the promoter regions of YUC8 and YUC9, but not in the promoters of the other YUCCA isogenes. Moreover, we demonstrate that plants that constitutively overexpress YUC8 and YUC9 show less damage after spider mite infestation, thereby underlining the role of auxin in plant responses to biotic stress signals., This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO), grant number BFU2017-82826-R

Background The model species Tetranychus urticae produces important plant injury and economic losses in the field. The current accepted method for the quantification of the spider mite damage in Arabidopsis whole rosettes is time consuming and entails a bottleneck for large-scale studies such as mutant screening or quantitative genetic analyses. Here, we describe an improved version of the existing method by designing an automatic protocol. The accuracy, precision, reproducibility and concordance of the new enhanced approach are validated in two Arabidopsis accessions with opposite damage phenotypes. Results are compared to the currently available manual method. Results Image acquisition experiments revealed that the automatic settings plus 10 values of brightness and the black background are the optimal conditions for a specific recognition of spider mite damage by software programs. Among the different tested methods, the Ilastik-Fiji tandem based on machine learning was the best procedure able to quantify the damage maintaining the differential range of damage between accessions. In addition, the Ilastik-Fiji tandem method showed the lowest variability within a set of conditions and the highest stability under different lighting or background surroundings. Bland-Altman concordance results pointed out a negative value for Ilastik-Fiji, which implies a minor estimation of the damage when compared to the manual standard method. Conclusions The novel approach using Ilastik and Fiji programs entails a great improvement for the quantification of the specific spider mite damage in Arabidopsis whole rosettes. The automation of the proposed method based on interactive machine learning eliminates the subjectivity and inter-rater-variability of the previous manual protocol. Besides, this method offers a robust tool for time saving and to avoid the damage overestimation observed with other methods.

Departamento de Biotecnología (INIA), Plants experience different abiotic/biotic stresses, which trigger their molecular machinery to cope with them. Besides general mechanisms prompted by many stresses, specific mechanisms have been introduced to optimize the response to individual threats. However, these key mechanisms are difficult to identify. Here, we introduce an in-depth species-specific transcriptomic analysis and conduct an extensive meta-analysis of the responses to related species to gain more knowledge about plant responses. The spider mite Tetranychus urticae was used as the individual species, several arthropod herbivores as the related species for meta-analysis, and Arabidopsis thaliana plants as the common host. The analysis of the transcriptomic data showed typical common responses to herbivory, such as jasmonate signaling or glucosinolate biosynthesis. Also, a specific set of genes likely involved in the particularities of the Arabidopsis-spider mite interaction was discovered. The new findings have determined a prominent role in this interaction of the jasmonate-induced pathways leading to the biosynthesis of anthocyanins and tocopherols. Therefore, tandem individual/general transcriptomic profiling has been revealed as an effective method to identify novel relevant processes and specificities in the plant response to environmental stresses., Research was supported by the Ministerio de Economía, Industria y Competitividad (MEIC, grants BIO2017‐83472‐R, RED2018‐102407‐T, and RyC17MESFB) and by the Convenio Plurianual between Comunidad de Madrid (CM) and Universidad Politécnica de Madrid (UPM) through the Programa de Apoyo a la Realización de Proyectos de I+D para Jóvenes Investigadores (APOYO‐JOVENES‐SUR6Q9‐22‐Y). RyC grant (RyC2017‐21814) from the MEIC and Plan Propio from the UPM financed MES. Studentships from: MEIC to I.R‐D (PRE2018‐083375), Comunidad de Madrid to G.R‐H (PEJD‐2019‐PRE/BIO‐15882), Programa Propio UPM to A.G., 15 Pág.

Phytophagous mites and thrips are global pests affecting a wide range of agricultural crops (Mouden et al., 2017; Agut et al., 2018). Among the arthropods, they are phylogenetically distant, but both classes harbor species ranging from highly specialized to extremely polyphagous (Rioja et al., 2017; Wu et al., 2018). Through convergent evolution, mites and thrips evolved stylets to facilitate feeding from mesophyll or epidermal cells (Bensoussan et al., 2016; Rioja et al., 2017; Wu et al., 2018). Despite large crops losses (Agut et al., 2018; Steenbergen et al., 2018) that are expected to become more severe with global warming (Ximenez-Embun et al., 2017; Urbaneja-Bernat et al., 2019), the interactions between mites/thrips and their host plants have been understudied. Hence, understanding how plants defend themselves against these pests is essential for developing crop protection strategies. This Research Topic provides an update on recent advances in the plant molecular and physiological mechanisms associated with phytophagous mite/thrips-plant interactions, and provides an overview of different approaches for improving crop resistance sustainably, either through repellence, feeding disruption or prevention of feeding damage. Here, we highlight some of the major points arising from these reports.

To survive under water deficiency, plants alter gene expression patterns, make structural and physiological adjustments, and optimize the use of water. Rapid degradation and turnover of proteins is required for effective nutrient recycling. Here, we examined the transcriptional responses of the C1A cysteine protease family to drought in barley and found that four genes were up-regulated in stressed plants. Knock-down lines for the protease-encoding genes HvPap-1 and HvPap-19 showed unexpected changes in leaf cuticle thickness and stomatal pore area. The efficiency of photosystem II and the total amount of proteins were almost unaltered in stressed transgenic plants while both parameters decreased in stressed wild-type plants. Although the patterns of proteolytic activities in the knock-down lines did not change, the amino acid accumulation increased in response to drought, concomitant with a higher ABA content. Whilst jasmonic acid (JA) and JA-Ile concentrations increased in stressed leaves of the wild-type and the HvPap-1 knock-down lines, their levels were lower in the HvPap-19 knock-down lines, suggesting the involvement of a specific hormone interaction in the process. Our data indicate that the changes in leaf cuticle thickness and stomatal pore area had advantageous effects on leaf defense against fungal infection and mite feeding mediated by Magnaporthe oryzae and Tetranychus urticae, respectively.

Cadmium treatment induces transient peroxisome proliferation in Arabidopsis leaves. To determine whether this process is regulated by pexophagy and to identify the mechanisms involved, we analysed time course-dependent changes in ATG8, an autophagy marker, and the accumulation of peroxisomal marker PEX14a. After 3 hr of Cd exposure, the transcript levels of ATG8h, ATG8c, a, and i were slightly up-regulated and then returned to normal. ATG8 protein levels also increased after 3 hr of Cd treatment, although an opposite pattern was observed in PEX14. Arabidopsis lines expressing GFP-ATG8a and CFP-SKL enabled us to demonstrate the presence of pexophagic processes in leaves. The Cd-dependent induction of pexophagy was demonstrated by the accumulation of peroxisomes in autophagy gene (ATG)-related Arabidopsis knockout mutants atg5 and atg7. We show that ATG8a colocalizes with catalase and NBR1 in the electron-dense peroxisomal core, thus suggesting that NBR1 may be an autophagic receptor for peroxisomes, with catalase being possibly involved in targeting pexophagy. Protein carbonylation and peroxisomal redox state suggest that protein oxidation may trigger pexophagy. Cathepsine B, legumain, and caspase 6 may also be involved in the regulation of pexophagy. Our results suggest that pexophagy could be an important step in rapid cell responses to cadmium., Ministerio de Ciencia e Innovación, Grant/Award Numbers: BIO2015‐67657‐P and BIO2015‐68957‐REDT, BIO2015‐68957‐REDT, BIO2016‐76633‐P and PGC2018‐ 098372‐B‐I00; TRANSAUTOPHAGY EU Framework Program Horizon, Grant/Award Number: OC‐2015‐1‐19840; ERDF, Grant/Award Numbers: PGC2018‐098372‐B‐I00, BIO2016‐76633‐P, BIO2015‐68957‐REDT and BIO2015‐67657‐P

Reactive oxygen species (ROS) are molecules that play a prominent role in plant response to numerous stresses, including plant interactions with herbivores. Previous findings indicate that Arabidopsis plants showed an increase in H2O2 accumulation after Tetranychus urticae infestation. Despite its importance, no information has been reported on the relationships between ROS-metabolizing systems and the spider mite-triggered plant-induced responses. In this work, four ROS-related genes that were differentially expressed between the resistant Bla-2 and the susceptible Kon Arabidopsis accessions were selected for the analysis. These genes encode proteins putatively involved in the generation (BBE22) and degradation (GPX7 and GSTU4) of H2O2, and in the degradation of ascorbate (AO). Overexpressing BBE22 and silencing GPX7, GSTU4 and AO resulted in higher leaf damage and better mite performance relative to the wild-type plants. Minor effects on H2O2 accumulation obscure major effects on the expression of genes related to ROS-metabolism and JA and SA signaling pathways, and on ROS-related enzymatic activities. In conclusion, the integration of ROS and ROS-related compounds and enzymes in the response of Arabidopsis to the spider mite T. urticae was confirmed. However, the complex network involved in ROS signaling makes difficult to predict the impact of a specific genetic manipulation.

Plant–pest relationships involve complex processes encompassing a network of molecules, signals, and regulators for overcoming defenses they develop against each other. Phytophagous arthropods identify plants mainly as a source of food. In turn, plants develop a variety of strategies to avoid damage and survive. The success of plant defenses depends on rapid and specific recognition of the phytophagous threat. Subsequently, plants trigger a cascade of short-term responses that eventually result in the production of a wide range of compounds with defense properties. This review deals with the main features involved in the interaction between plants and phytophagous insects and acari, focusing on early responses from the plant side. A general landscape of the diverse strategies employed by plants within the first hours after pest perception to block the capability of phytophagous insects to develop mechanisms of resistance is presented, with the potential of providing alternatives for pest control.

BACKGROUND Sulfoxaflor, a new insecticide from the sulfoximine chemical family, and imidacloprid, a widely used neonicotinoid insecticide, were tested to assess the susceptibility and feeding behaviour of two populations of Myzus persicae: Mp61, which exhibited target-site R81T resistance to neonicotinoids, and Mp1989, a laboratory clone maintained since 1989 as a susceptible reference. RESULTS The imidacloprid LC50 value for Mp61 was 16 times higher than for Mp1989, showing a moderate level of resistance. Sulfoxaflor LC50 values for Mp61 and Mp1989 were much closer. The probing behaviour, as assessed by electrical penetration graphs (EPGs), of both populations was clearly altered by sulfoxaflor, which reduced the ability of aphids to find and feed from the phloem. The feeding behaviour of the susceptible Mp1989 population was much more severely affected than the moderately resistant Mp61 population on imidacloprid-treated plants. PCR assays of both aphid populations followed by DNA sequencing identified differences between populations in the point mutation in the β-subunit of the nicotinic acetylcholine receptor linked to the resistant gene against the neonicotinoid insecticide. CONCLUSIONS Sulfoxaflor provoked feeding cessation more rapidly than imidacloprid in both aphid populations. Sharp differences in feeding behaviour were detected between the susceptible and the moderately resistant neonicotinoid-resistant aphid populations. The EPG technique can be used as a useful tool to give new insights into the functional effects of new chemical compounds and for early detection of low to moderate levels of resistance of sap-feeding insects to insecticides. The potential of this technique was validated by molecular analysis of the R81T mutation target site. © 2015 Society of Chemical Industry

16 p.-8 fig.-1 tab.-1 tab. supl.- 1 mat. supl., Microspores are reprogrammed towards embryogenesis by stress. Many microspores die after this stress, limiting the efficiency of microspore embryogenesis. Autophagy is a degradation pathway that plays critical roles in stress response and cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; less is known in plants. Plant cathepsins are papain-like C1A cysteine proteases involved in many physiological processes, including programmed cell death. We have analysed the involvement of autophagy in cell death, in relation to cathepsin activation, during stress-induced microspore embryogenesis in Hordeum vulgare. After stress, reactive oxygen species (ROS) and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8, and autophagosomes. Concomitantly, cathepsin L/F-, B-, and H-like activities were induced, cathepsin-like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6, and HvPap-19 proteins increased and localized in the cytoplasm, resembling autophagy structures. Inhibitors of autophagy and cysteine proteases reduced cell death and promoted embryogenesis. The findings reveal a role for autophagy in stress-induced cell death during microspore embryogenesis, and the participation of cathepsins. Similar patterns of activation, expression, and localization suggest a possible connection between cathepsins and autophagy. The results open up new possibilities to enhance microspore embryogenesis efficiency with autophagy and/or cysteine protease modulators., This work is supported by projects AGL2014-52028-R and AGL2017-82447-R funded by the Spanish Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (ERDF/FEDER). YPP is the recipient of a grant (PEJ15/BIO/AI-01S8) funded by Comunidad de Madrid and European Commission through ERDF/FEDER.

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy-2 and Icy-4 cystatin genes. Artificial microRNA lines independently silencing the two drought-induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay-green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy-2 and Icy-4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap-1, HvPap-12, and HvPap-16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.

18 p.-8 fig., The defense response of the plants against herbivores relies on a complex network of interconnected signaling pathways. In this work, we characterized a new key player in the response of Arabidopsis against the two-spotted spider mite Tetranychus urticae, the MATI (Mite Attack Triggered Immunity) gene. This gene was differentially induced in resistant Bla-2 strain relative to susceptible Kon Arabidopsis accessions after mite attack, suggesting a potential role in the control of spider mites. To study the MATI gene function, it has been performed a deep molecular characterization of the gene combined with feeding bioassays using modified Arabidopsis lines and phytophagous arthropods. The MATI gene belongs to a new gene family that had not been previously characterized. Biotic assays showed that it confers a high tolerance not only to T. urticae, but also to the chewing lepidopteran Spodoptera exigua. Biochemical analyses suggest that MATI encodes a protein involved in the accumulation of reducing agents upon herbivore attack to control plant redox homeostasis avoiding oxidative damage and cell death. Besides, molecular analyses demonstrated that MATI is involved in the modulation of different hormonal signaling pathways, affecting the expression of genes involved in biosynthesis and signaling of the jasmonic acid and salicylic acid hormones. The fact that MATI is also involved in defense through the modulation of the levels of photosynthetic pigments highlights the potential of MATI proteins to be exploited as biotechnological tools for pest control., This work was supported by projects from Ministerio de Economía y Competitividad of Spain (projects BIO2014-53508- R and 618105-FACCE-Era Net Plus) and the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI–046), the Ontario Research Fund (RE08-067) and the Natural Sciences and Engineering Research Council of Canada.

Most molecular-genetic studies of plant defense responses to arthropod herbivores have focused on insects. However, plant-feeding mites are also pests of diverse plants, and mites induce different patterns of damage to plant tissues than do well-studied insects (e.g. lepidopteran larvae or aphids). The two-spotted spider mite (Tetranychus urticae) is among the most significant mite pests in agriculture, feeding on a staggering number of plant hosts. To understand the interactions between spider mite and a plant at the molecular level, we examined reciprocal genome-wide responses of mites and its host Arabidopsis (Arabidopsis thaliana). Despite differences in feeding guilds, we found that transcriptional responses of Arabidopsis to mite herbivory resembled those observed for lepidopteran herbivores. Mutant analysis of induced plant defense pathways showed functionally that only a subset of induced programs, including jasmonic acid signaling and biosynthesis of indole glucosinolates, are central to Arabidopsis’s defense to mite herbivory. On the herbivore side, indole glucosinolates dramatically increased mite mortality and development times. We identified an indole glucosinolate dose-dependent increase in the number of differentially expressed mite genes belonging to pathways associated with detoxification of xenobiotics. This demonstrates that spider mite is sensitive to Arabidopsis defenses that have also been associated with the deterrence of insect herbivores that are very distantly related to chelicerates. Our findings provide molecular insights into the nature of, and response to, herbivory for a representative of a major class of arthropod herbivores.

Plant senescence is a complex physiological process consequence either of the natural lifespan or externally induced by abiotic and biotic factors. It comprises a coordinated sequence of molecular and biochemical events, phenotypically illustrated by changes in plant colour. Senescence is associated with alterations in chlorophyll and pigment content, reduction of photosynthesis, hydrolysis of macromolecules to produce more simple compounds and dismantling of cell organelles, to finally produce cell death. At the end, relocation of nutrients from the senescent tissues towards sink organs or growing tissues takes place to complete a recycling process. Consequently, the major part of the nitrogen is released as ammonium after being re-assimilated into amino acids to be exported via the phloem to the developing grains, fruits and tubers. During senescence, the reprograming of thousands of genes is triggered in response to specific senescence-promoting factors under a restricted regulatory control. The actual high-throughput omics technologies have led to the generation of integrative information, which has been used to understand the physiological changes during the onset and progression of senescence. This chapter covers an overview on plant senescence, particularly focussed on the senescence of the leaf, including the most recent findings about features, signalling, regulation and pathways involved in this natural or induced process.

This review deals with phytocystatins, focussing on their potential role as defence proteins against phytophagous arthropods. Information about the evolutionary, molecular and biochemical features and inhibitory properties of phytocystatins are presented. Cystatin ability to inhibit heterologous cysteine protease activities is commented on as well as some approaches of tailoring cystatin specificity to enhance their defence function towards pests. A general landscape on the digestive proteases of phytophagous insects and acari and the remarkable plasticity of their digestive physiology after feeding on cystatins are highlighted. Biotechnological approaches to produce recombinant cystatins to be added to artificial diets or to be sprayed as insecticide–acaricide compounds and the of use cystatins as transgenes are discussed. Multiple examples and applications are included to end with some conclusions and future perspectives., This work was supported by projects from Ministerio de Economia y Competitividad (projects BIO2014-53508-R and 618105-FACCE-Era Net Plus). Maria Estrella Santamaria was recipient of post-doctoral grant from the subprogram Juan de la Cierva 2012 from the Spanish Ministerio de Economia y Competitividad. We thank Pablo Bielza (Universidad Politecnica de Cartagena, Spain) for one of the photos included in Figure 2. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

Senescence-associated proteolysis in plants is a complex and controlled process, essential for mobilization of nutrients from old or stressed tissues, mainly leaves, to growing or sink organs. Protein breakdown in senescing leaves involves many plastidial and nuclear proteases, regulators, different subcellular locations and dynamic protein traffic to ensure the complete transformation of proteins of high molecular weight into transportable and useful hydrolysed products. Protease activities are strictly regulated by specific inhibitors and through the activation of zymogens to develop their proteolytic activity at the right place and at the proper time. All these events associated with senescence have deep effects on the relocation of nutrients and as a consequence, on grain quality and crop yield. Thus, it can be considered that nutrient recycling is the common destiny of two processes, plant senescence and, proteolysis. This review article covers the most recent findings about leaf senescence features mediated by abiotic and biotic stresses as well as the participants and steps required in this physiological process, paying special attention to C1A cysteine proteases, their specific inhibitors, known as cystatins, and their potential targets, particularly the chloroplastic proteins as source for nitrogen recycling.

Protein breakdown and mobilization from old or stressed tissues to growing and sink organs are some of the metabolic features associated with abiotic/biotic stresses, essential for nutrient recycling. The massive degradation of proteins implies numerous proteolytic events in which cysteine-proteases are the most abundant key players. Analysing the role of barley C1A proteases in response to abiotic stresses is crucial due to their impact on plant growth and grain yield and quality. In this study, dark and nitrogen starvation treatments were selected to induce stress in barley. Results show that C1A proteases participate in the proteolytic processes triggered in leaves by both abiotic treatments, which strongly induce the expression of the HvPap-1 gene encoding a cathepsin F-like protease. Differences in biochemical parameters and C1A gene expression were found when comparing transgenic barley plants overexpressing or silencing the HvPap-1 gene and wild-type dark-treated leaves. These findings associated with morphological changes evidence a lifespan-delayed phenotype of HvPap-1 silenced lines. All these data elucidate on the role of this protease family in response to abiotic stresses and the potential of their biotechnological manipulation to control the timing of plant growth.

Use of meta-topolin (mT) [6-(3-hydroxybenzylamino) purine] to scale up the plantain clone ‘CEMSA ¾’ in temporary immersion systems (TIS), when compared with conventional micropropagation protocols, has been shown to improve production and quality of microplants. To evaluate the impact of those innovations on plant field performance, in vitro plants of ‘CEMSA ¾’ were subjected to either an additional multiplication cycle using TIS or semi-solid medium (SS) in the presence of 4.44 μM mT or N 6 -benzyladenine (BA). Growth and yield components in the microplants grown in field – plant crop – and in the developed plants of the following field generation – first ratoon – were evaluated. Also (epi)genomic traits were studied by flow cytometry, fluorimetry-based AFLP and HPLC analyses of global genomic DNA methylation. Neither in vitro microprogation systems – TIS and SS – nor cytokinins – mT and BA – had any factorial effect on yield productivity of the plant crop. In the first ratoon, significant differences in yield appeared, with TIS-BA plants being more productive than those generated from TIS-mT. For most of the measured variables, the plant crop had higher values than the first ratoon, but true-to-type and each off-type phenotype of the plant crop was always inherited by their corresponding offspring of the first ratoon. The proportion of true-to-type plants was higher for SS-BA and TIS-mT culture conditions. Irrespective of micropropagation protocol and phenotype, in the plant crop, average genomic DNA content was maintained and global genomic methylation levels were similar. Nevertheless, dwarf individuals showed more AFLP polymorphisms with respect to mother plants than the rest of the micropropagated plants, and a dwarfism-related AFLP marker is proposed. On the basis of the results obtained and previous reports, the innovative TIS-mT protocol was deemed to be the most suitable plantain multiplication technique among those tested.

Among the C1A cysteine proteases, the plant cathepsin F-like group has been poorly studied. This paper describes the molecular and functional characterization of the HvPap-1 cathepsin F-like protein from barley. This peptidase is N-glycosylated and has to be processed to become active by its own propeptide being an important modulator of the peptidase activity. The expression pattern of its mRNA and protein suggest that it is involved in different proteolytic processes in the barley plant. HvPap-1 peptidase has been purified in Escherichia coli and the recombinant protein is able to degrade different substrates, including barley grain proteins (hordeins, albumins, and globulins) stored in the barley endosperm. It has been localized in protein bodies and vesicles of the embryo and it is induced in aleurones by gibberellin treatment. These three features support the implication of HvPap-1 in storage protein mobilization during grain germination. In addition, a complex regulation exerted by the barley cystatins, which are cysteine protease inhibitors, and by its own propeptide, is also described.

Plant cysteine-proteases (CysProt) represent a well-characterized type of proteolytic enzymes that fulfill tightly regulated physiological functions (senescence and seed germination among others) and defense roles. This article is focused on the group of papain-proteases C1A (family C1, clan CA) and their inhibitors, phytocystatins (PhyCys). In particular, the protease-inhibitor interaction and their mutual participation in specific pathways throughout the plant's life are reviewed. C1A CysProt and PhyCys have been molecularly characterized, and comparative sequence analyses have identified consensus functional motifs. A correlation can be established between the number of identified CysProt and PhyCys in angiosperms. Thus, evolutionary forces may have determined a control role of cystatins on both endogenous and pest-exogenous proteases in these species. Tagging the proteases and inhibitors with fluorescence proteins revealed common patterns of subcellular localization in the endoplasmic reticulum-Golgi network in transiently transformed onion epidermal cells. Further in vivo interactions were demonstrated by bimolecular fluorescent complementation, suggesting their participation in the same physiological processes.

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most polyphagous herbivores feeding on cell contents of over 1100 plant species including more than 150 crops. It is being established as a model for chelicerate herbivores with tools that enable tracking of reciprocal responses in plant-spider mite interactions. However, despite their important pest status and a growing understanding of the molecular basis of interactions with plant hosts, knowledge of the way mites interface with the plant while feeding and the plant damage directly inflicted by mites is lacking. Here, utilizing histology and microscopy methods, we uncovered several key features of T. urticae feeding. By following the stylet path within the plant tissue, we determined that the stylet penetrates the leaf either in between epidermal pavement cells or through a stomatal opening, without damaging the epidermal cellular layer. Our recordings of mite feeding established that duration of the feeding event ranges from several minutes to more than half an hour, during which time mites consume a single mesophyll cell in a pattern that is common to both bean and Arabidopsis plant hosts. In addition, this study determined that leaf chlorotic spots, a common symptom of mite herbivory, do not form as an immediate consequence of mite feeding. Our results establish a cellular context for the plant-spider mite interaction that will support our understanding of the molecular mechanisms and cell signaling associated with spider mite feeding. © 2016 Bensoussan, Santamaria, Zhurov, Diaz, Grbi and Grbi.

31 p.-3 fig.-3 tab.-2 fig. supl.-1 tab. supl., Digestive proteases of the phytophagous mite Tetranychus urticae have been characterised by comparing their activity in body and faecal extracts. Aspartyl, cathepsin B- and L-like and legumain activities were detected in both mite bodies and faeces, with a specific activity of aspartyl and cathepsin L-like proteases about 5- and 2-fold higher, respectively, in mite faeces than in bodies. In general, all these activities were maintained independently of the host plant where the mites were reared (bean, tomato or maize). Remarkably, this is the first report in a phytophagous mite of legumain-like activity, which was characterised for its ability to hydrolyse the specific substrate Z-VAN-AMC, its activation by DTT and inhibition by IAA but not by E-64. Gel free nanoLC–nanoESI-QTOF MS/MS proteomic analysis of mite faeces resulted in the identification of four cathepsins L and one aspartyl protease (from a total of the 29 cathepsins L, 27 cathepsins B, 19 legumains and two aspartyl protease genes identified the genome of this species). Gene expression analysis reveals that four cathepsins L and the aspartyl protease identified in the mite faeces, but also two cathepsins B and two legumains that were not detected in the faeces, were expressed at high levels in the spider mite feeding stages (larvae, nymphs and adults) relative to embryos. Taken together, these results indicate a digestive role for cysteine and aspartyl proteases in T. urticae. The expression of the cathepsins B and L, legumains and aspartyl protease genes analysed in our study increased in female adults after feeding on Arabidopsis plants over-expressing the HvCPI-6 cystatin, that specifically targets cathepsins B and L, or the CMe trypsin inhibitor that targets serine proteases. This unspecific response suggests that in addition to compensation for inhibitor-targeted enzymes, the increase in the expression of digestive proteases in T. urticae may act as a first barrier against ingested plant defensive proteins., This work was supported by a Grant from CSIC (Grant CSIC-201040E049 to F.O), Ministerio de Ciencia e Innovación (Grant AGL11-23650 to I.D.) and the Government of Canada through Genome Canada and the Ontario Genomics Institute (Grant OGI-046 to V.G.), and the Ontario Research Fund-Global Leadership in Genomics and Life Sciences (Grant GL2-01-035 to V.G.).