María E. Santamaría

C1A cysteine‐proteases and their inhibitors in plants

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 plants 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.

Gene Pyramiding of Peptidase Inhibitors Enhances Plant Resistance to the Spider Mite Tetranychus urticae

The two-spotted spider mite Tetranychus urticae is a damaging pest worldwide with a wide range of host plants and an extreme record of pesticide resistance. Recently, the complete T. urticae genome has been published and showed a proliferation of gene families associated with digestion and detoxification of plant secondary compounds which supports its polyphagous behaviour. To overcome spider mite adaptability a gene pyramiding approach has been developed by co-expressing two barley proteases inhibitors, the cystatin Icy6 and the trypsin inhibitor Itr1 genes in Arabidopsis plants by Agrobacterium-mediated transformation. The presence and expression of both transgenes was studied by conventional and quantitative real time RT-PCR assays and by indirect ELISA assays. The inhibitory activity of cystatin and trypsin inhibitor was in vitro analysed using specific substrates. Single and double transformants were used to assess the effects of spider mite infestation. Double transformed lines showed the lowest damaged leaf area in comparison to single transformants and non-transformed controls and different accumulation of H2O2 as defence response in the leaf feeding site, detected by diaminobenzidine staining. Additionally, an impact on endogenous mite cathepsin B- and L-like activities was observed after feeding on Arabidopsis lines, which correlates with a significant increase in the mortality of mites fed on transformed plants. These effects were analysed in view of the expression levels of the target mite protease genes, C1A cysteine peptidase and S1 serine peptidase, identified in the four developmental mite stages (embryo, larvae, nymphs and adults) performed using the RNA-seq information available at the BOGAS T. urticae database. The potential of pyramiding different classes of plant protease inhibitors to prevent plant damage caused by mites as a new tool to prevent pest resistance and to improve pest control is discussed.

Cysteine peptidases and their inhibitors in Tetranychus urticae: a comparative genomic approach

BackgroundCysteine peptidases in the two-spotted spider mite Tetranychus urticae are involved in essential physiological processes, including proteolytic digestion. Cystatins and thyropins are inhibitors of cysteine peptidases that modulate their activity, although their function in this species has yet to be investigated. Comparative genomic analyses are powerful tools to obtain advanced knowledge into the presence and evolution of both, peptidases and their inhibitors, and could aid to elucidate issues concerning the function of these proteins.ResultsWe have performed a genomic comparative analysis of cysteine peptidases and their inhibitors in T. urticae and representative species of different arthropod taxonomic groups. The results indicate: i) clade-specific proliferations are common to C1A papain-like peptidases and for the I25B cystatin family of inhibitors, whereas the C1A inhibitors thyropins are evolutionarily more conserved among arthropod clades; ii) an unprecedented extensive expansion for C13 legumain-like peptidases is found in T. urticae; iii) a sequence-structure analysis of the spider mite cystatins suggests that diversification may be related to an expansion of their inhibitory range; and iv) an in silico transcriptomic analysis shows that most cathepsin B and L cysteine peptidases, legumains and several members of the cystatin family are expressed at a higher rate in T. urticae feeding stages than in embryos.ConclusionComparative genomics has provided valuable insights on the spider mite cysteine peptidases and their inhibitors. Mite-specific proliferations of C1A and C13 peptidase and I25 cystatin families and their over-expression in feeding stages of mites fit with a putative role in mite’s feeding and could have a key role in its broad host feeding range.

Plant protein peptidase inhibitors: an evolutionary overview based on comparative genomics

BackgroundPeptidases are key proteins involved in essential plant physiological processes. Although protein peptidase inhibitors are essential molecules that modulate peptidase activity, their global presence in different plant species remains still unknown. Comparative genomic analyses are powerful tools to get advanced knowledge into the presence and evolution of both, peptidases and their inhibitors across the Viridiplantae kingdom.ResultsA genomic comparative analysis of peptidase inhibitors and several groups of peptidases in representative species of different plant taxonomic groups has been performed. The results point out: i) clade-specific presence is common to many families of peptidase inhibitors, being some families present in most land plants; ii) variability is a widespread feature for peptidase inhibitory families, with abundant species-specific (or clade-specific) gene family proliferations; iii) peptidases are more conserved in different plant clades, being C1A papain and S8 subtilisin families present in all species analyzed; and iv) a moderate correlation among peptidases and their inhibitors suggests that inhibitors proliferated to control both endogenous and exogenous peptidases.ConclusionsComparative genomics has provided valuable insights on plant peptidase inhibitor families and could explain the evolutionary reasons that lead to the current variable repertoire of peptidase inhibitors in specific plant clades.

Phytocystatins: Defense Proteins against Phytophagous Insects and Acari

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.

Basic Procedures for Epigenetic Analysis in Plant Cell and Tissue Culture

In vitro culture is one of the most studied techniques, and it is used to study many developmental processes, especially in forestry species, because of growth timing and easy manipulation. Epigenetics has been shown as an important influence on many research analyses such as cancer in mammals and developmental processes in plants such as flowering, but regarding in vitro culture, techniques to study DNA methylation or chromatin modifications were mainly limited to identify somaclonal variation of the micropropagated material. Because in vitro culture is not only a way to generate plant material but also a bunch of differentially induced developmental processes, an approach of techniques and some research carried out to study the different changes regarding DNA methylation and chromatin and translational modifications that take place during these processes is reviewed.

Dehydration Stress Contributes to the Enhancement of Plant Defense Response and Mite Performance on Barley

Under natural conditions, plants suffer different stresses simultaneously or in a sequential way. At present, the combined effect of biotic and abiotic stressors is one of the most important threats to crop production. Understanding how plants deal with the panoply of potential stresses affecting them is crucial to develop biotechnological tools to protect plants. As well as for drought stress, the economic importance of the spider mite on agriculture is expected to increase due to climate change. Barley is a host of the polyphagous spider mite Tetranychus urticae and drought produces important yield losses. To obtain insights on the combined effect of drought and mite stresses on the defensive response of this cereal, we have analyzed the transcriptomic responses of barley plants subjected to dehydration (water-deficit) treatment, spider mite attack, or to the combined dehydration-spider mite stress. The expression patterns of mite-induced responsive genes included many jasmonic acid responsive genes and were quickly induced. In contrast, genes related to dehydration tolerance were later up-regulated. Besides, a higher up-regulation of mite-induced defenses was showed by the combined dehydration and mite treatment than by the individual mite stress. On the other hand, the performance of the mite in dehydration stressed and well-watered plants was tested. Despite the stronger defensive response in plants that suffer dehydration and mite stresses, the spider mite demonstrates a better performance under dehydration condition than in well-watered plants. These results highlight the complexity of the regulatory events leading to the response to a combination of stresses and emphasize the difficulties to predict their consequences on crop production.

Epigenetics, the Role of DNA Methylation in Tree Development

During development of multicellular organisms, cells become differentiated by modulating different programs of gene expression. Cells have their own epigenetic signature which reflects genotype, developmental history, and environmental influences, and it is ultimately reflected in the phenotype of the cells and the organism. However, in normal development or disease situations, such as adaptation to climate change or during in vitro culture, some cells undergo major epigenetic reprogramming involving the removal of epigenetic marks in the nuclei followed by the establishment of a different new set of marks. Compared with animal cells, biotech-mediated achievements are reduced in plants despite the presence of cell polypotency. In forestry, any sustainable developments using biotech tools remain restricted to the lab, without progressing to the field for application. Such barriers in the translation between development and implementation need to be addressed by organizations that have the power to integrate these two fields. However, a lack of understanding of gene regulation is also to blame for this barrier. In recent years, great progress has been made in unraveling the control of gene expression. These advances are discussed in this chapter, including the possibility of applying this knowledge in forestry practice.

Senescence-Associated Genes in Response to Abiotic/Biotic Stresses

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.

Host plant use by two distinct lineages of the tomato red spider mite, Tetranychus evansi , differing in their distribution range

Abstract The tomato red spider mite, Tetranychus evansi, is an emerging pest of solanaceous crops. Two distinct genetic lineages (I and II) have been identified, lineage I having a much wider geographic distribution than lineage II. This has been attributed to differences in cold hardiness that make lineage I better adapted to colonize the coldest parts of the invaded area. However, other factors such as the ability to exploit different hosts may also be involved. In this work, we compared the performance of the Nice (lineage I) and Perpignan (lineage II) strains of T. evansi on two frequent host plants for this species: black nightshade, Solanum nigrum, and cultivated tomato, S. lycopersicum. In general, Nice strain mites performed better (higher fecundity, lower offspring mortality, bigger egg size and lower percentage of males) than Perpignan strain mites when both: (1) they were reared and tested on the same host plant (S. lycopersicum or S. nigrum); and (2) when shifted from S. nigrum to S. lycopersicum and vice versa. Digestive proteases showed also higher expression in Nice strain mites than in Perpignan strain mites, independently of their plant host, potentially reflecting a more efficient proteolytic digestion of plant proteins. However, no differences in detoxification enzyme (P450, esterases and glutathione S-transferases) activities were found when the two strains were compared. In conclusion, our results demonstrate that Nice strain mites exhibited life history traits leading to higher fitness on two different hosts, which may be related with the higher invasive potential and outbreak risks of mites from lineage I.