Phytoglobins in the nuclei, cytoplasm and chloroplasts modulate nitric oxide signaling and interact with abscisic acid.

Symbiotic hemoglobins provide O2 to N2 -fixing bacteria within legume nodules, but the functions of non-symbiotic hemoglobins or phytoglobins (Glbs) are much less defined. Immunolabeling combined with confocal microscopy of the Glbs tagged at the C-terminus with green fluorescent protein was used to determine their subcellular localizations in Arabidopsis and Lotus japonicus. Recombinant proteins were used to examine nitric oxide (NO) scavenging in vitro and transgenic plants to show S-nitrosylation and other in vivo interactions with NO and abscisic acid (ABA) responses. We found that Glbs occur in the nuclei, chloroplasts and amyloplasts of both model plants, and also in the cytoplasm of Arabidopsis cells. The proteins show similar NO dioxygenase activities in vitro, are nitrosylated in Cys residues in vivo, and scavenge NO in the stomatal cells. The Cys/Ser mutation does not affect NO dioxygenase activity, and S-nitrosylation does not significantly consume NO. We demonstrate an interaction between Glbs and ABA on several grounds: Glb1 and Glb2 scavenge NO produced in stomatal guard cells following ABA supply; plants overexpressing Glb1 show higher constitutive expression of the ABA responsive genes Responsive to ABA (RAB18), Responsive to Dehydration (RD29A) and Highly ABA-Induced 2 (HAI2), and are more tolerant to dehydration; and ABA strongly upregulates class 1 Glbs. We conclude that Glbs modulate NO and interact with ABA in crucial physiological processes such as the plant's response to dessication.

DNA-dependent homodimerization, sub-cellular partitioning, and protein destabilization control WUSCHEL levels and spatial patterning.

The homeodomain transcription factor WUSCHEL (WUS) promotes stem cell maintenance in inflorescence meristems of Arabidopsis thaliana WUS, which is synthesized in the rib meristem, migrates and accumulates at lower levels in adjacent cells. Maintenance of WUS protein levels and spatial patterning distribution is not well-understood. Here, we show that the last 63-aa stretch of WUS is necessary for maintaining different levels of WUS protein in the rib meristem and adjacent cells. The 63-aa region contains the following transcriptional regulatory domains: the acidic region, the WUS-box, which is conserved in WUS-related HOMEOBOX family members, and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR-like) domain. Our analysis reveals that the opposing functions of WUS-box, which is required for nuclear retention, and EAR-like domain, which participates in nuclear export, are necessary to maintain higher nuclear levels of WUS in cells of the rib meristem and lower nuclear levels in adjacent cells. We also show that the N-terminal DNA binding domain, which is required for both DNA binding and homodimerization, along with the homodimerization sequence located in the central part of the protein, restricts WUS from spreading excessively and show that the homodimerization is critical for WUS function. Our analysis also reveals that a higher level of WUS outside the rib meristem leads to protein destabilization, suggesting a new tier of regulation in WUS protein regulation. Taken together our data show that processes that influence WUS protein levels and spatial distribution are highly coupled to its transcriptional activity.

Threshold-dependent transcriptional discrimination underlies stem cell homeostasis.

Transcriptional mechanisms that underlie the dose-dependent regulation of gene expression in animal development have been studied extensively. However, the mechanisms of dose-dependent transcriptional regulation in plant development have not been understood. In Arabidopsis shoot apical meristems, WUSCHEL (WUS), a stem cell-promoting transcription factor, accumulates at a higher level in the rib meristem and at a lower level in the central zone where it activates its own negative regulator, CLAVATA3 (CLV3). How WUS regulates CLV3 levels has not been understood. Here we show that WUS binds a group of cis-elements, cis- regulatory module, in the CLV3-regulatory region, with different affinities and conformations, consisting of monomers at lower concentration and as dimers at a higher level. By deleting cis elements, manipulating the WUS-binding affinity and the homodimerization threshold of cis elements, and manipulating WUS levels, we show that the same cis elements mediate both the activation and repression of CLV3 at lower and higher WUS levels, respectively. The concentration-dependent transcriptional discrimination provides a mechanistic framework to explain the regulation of CLV3 levels that is critical for stem cell homeostasis.

Insights on the Proteases Involved in Barley and Wheat Grain Germination.

Seed storage proteins must be hydrolyzed by proteases to deliver the amino acids essential for embryo growth and development. Several groups of proteases involved in this process have been identified in both the monocot and the dicot species. This review focuses on the implication of proteases during germination in two cereal species, barley and wheat, where proteolytic control during the germination process has considerable economic importance. Formerly, the participation of proteases during grain germination was inferred from reports of proteolytic activities, the expression of individual genes, or the presence of individual proteins and showed a prominent role for papain-like and legumain-like cysteine proteases and for serine carboxypeptidases. Nowadays, the development of new technologies and the release of the genomic sequences of wheat and barley have permitted the application of genome-scale approaches, such as those used in functional genomics and proteomics. Using these approaches, the repertoire of proteases known to be involved in germination has increased and includes members of distinct protease families. The development of novel techniques based on shotgun proteomics, activity-based protein profiling, and comparative and structural genomics will help to achieve a general view of the proteolytic process during germination.

Differential response of silencing HvIcy2 barley plants against Magnaporthe oryzae infection and light deprivation.

BACKGROUND: Phytocystatins (PhyCys) act as endogenous regulators of cysteine proteases (CysProt) involved in various physiological processes. Besides, PhyCys are involved in plant reactions to abiotic stresses like drought or darkness and have been used as effective molecules against different pests and pathogens. The barley PhyCys-CysProt system is considered a model of protease-inhibitor regulation of protein turnover. Thirteen barley cystatins (HvCPI-1 to HvCPI-13) have been previously identified and characterized. Among them HvCPI-2 has been shown to have a relevant role in plant responses to pathogens and pests, as well as in the plant response to drought. RESULTS: The present work explores the multiple role of this barley PhyCys in response to both, biotic and abiotic stresses, focusing on the impact of silencing this gene. HvIcy-2 silencing lines behave differentially against the phytopathogenic fungus Magnaporthe oryzae and a light deprivation treatment. The induced expression of HvIcy-2 by the fungal stress correlated to a higher susceptibility of silencing HvIcy-2 plants. In contrast, a reduction in the expression of HvIcy-2 and in the cathepsin-L and -B like activities in the silencing HvIcy-2 plants was not accompanied by apparent phenotypical differences with control plants in response to light deprivation. CONCLUSION: These results highlight the specificity of PhyCys in the responses to diverse external prompts as well as the complexity of the regulatory events leading to the response to a particular stress. The mechanism of regulation of these stress responses seems to be focused in maintaining the balance of CysProt and PhyCys levels, which is crucial for the modulation of physiological processes induced by biotic or abiotic stresses.

Vacuolar processing enzyme 4 contributes to maternal control of grain size in barley by executing programmed cell death in the pericarp.

The angiosperm embryo and endosperm are limited in space because they grow inside maternal seed tissues. The elimination of cell layers of the maternal seed coat by programmed cell death (PCD) could provide space and nutrition to the filial organs. Using the barley (Hordeum vulgare L.) seed as a model, we elucidated the role of vacuolar processing enzyme 4 (VPE4) in cereals by using an RNAi approach and targeting the enzymatic properties of the recombinant protein. A comparative characterization of transgenic versus wild-type plants included transcriptional and metabolic profiling, flow cytometry, histology and nuclear magnetic imaging of grains. The recombinant VPE4 protein exhibited legumain and caspase-1 properties in vitro. Pericarp disintegration was delayed in the transgenic grains. Although the VPE4 gene and enzymatic activity was decreased in the early developing pericarp, storage capacity and the size of the endosperm and embryo were reduced in the mature VPE4-repressed grains. The persistence of the pericarp in the VPE4-affected grains constrains endosperm and embryo growth and leads to transcriptional reprogramming, perturbations in signalling and adjustments in metabolism. We conclude that VPE4 expression executes PCD in the pericarp, which is required for later endosperm filling, and argue for a role of PCD in maternal control of seed size in cereals.

Silencing barley cystatins HvCPI-2 and HvCPI-4 specifically modifies leaf responses to drought stress.

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.

Overexpression of HvIcy6 in Barley Enhances Resistance against Tetranychus urticae and Entails Partial Transcriptomic Reprogramming.

Cystatins have been largely used for pest control against phytophagous species. However, cystatins have not been commonly overexpressed in its cognate plant species to test their pesticide capacity. Since the inhibitory role of barley HvCPI-6 cystatin against the phytophagous mite Tetranychus urticae has been previously demonstrated, the purpose of our study was to determine if barley transgenic lines overexpressing its own HvIcy6 gene were more resistant against this phytophagous infestation. Besides, a transcriptomic analysis was done to find differential expressed genes among wild-type and transformed barley plants. Barley plants overexpressing HvIcy6 cystatin gene remained less susceptible to T. urticae attack when compared to wild-type plants, with a significant lesser foliar damaged area and a lower presence of the mite. Transcriptomic analysis revealed a certain reprogramming of cellular metabolism and a lower expression of several genes related to photosynthetic activity. Therefore, although caution should be taken to discard potential deleterious pleiotropic effects, cystatins may be used as transgenes with impact on agricultural crops by conferring enhanced levels of resistance to phytophagous pests.

HvPap-1 C1A Protease and HvCPI-2 Cystatin Contribute to Barley Grain Filling and Germination.

Proteolysis is an essential process throughout the mobilization of storage proteins in barley (Hordeum vulgare) grains during germination. It involves numerous types of enzymes, with C1A Cys proteases the most abundant key players. Manipulation of the proteolytic machinery is a potential way to enhance grain yield and quality, and it could influence the mobilization of storage compounds along germination. Transgenic barley plants silencing or over-expressing the cathepsin F-like HvPap-1 Cys protease show differential accumulation of storage molecules such as starch, proteins, and free amino acids in the grain. It is particularly striking that the HvPap-1 artificial microRNA lines phenotype show a drastic delay in the grain germination process. Alterations to the proteolytic activities in the over-expressing and knock-down grains associated with changes in the level of expression of several C1A peptidases were also detected. Similarly, down-regulating cystatin Icy-2, one of the proteinaceous inhibitors of the cathepsin F-like protease, also has important effects on grain filling. However, the ultimate physiological influence of manipulating a peptidase or an inhibitor cannot be always predicted, since the plant tries to compensate the modified proteolytic effects by modulating the expression of some other peptidases or their inhibitors.

HvPap-1 C1A protease actively participates in barley proteolysis mediated by abiotic stresses.

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.

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.

Plant senescence and proteolysis: two processes with one destiny.

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.

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

BACKGROUND: Peptidases 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. RESULTS: A 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. CONCLUSIONS: Comparative 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.

C1A cysteine protease-cystatin interactions in leaf senescence.

Senescence-associated proteolysis in plants is a crucial process to relocalize nutrients from leaves to growing or storage tissues. The massive net degradation of proteins involves broad metabolic networks, different subcellular compartments, and several types of proteases and regulators. C1A cysteine proteases, grouped as cathepsin L-, B-, H-, and F-like according to their gene structures and phylogenetic relationships, are the most abundant enzymes responsible for the proteolytic activity during leaf senescence. Besides, cystatins as specific modulators of C1A peptidase activities exert a complex regulatory role in this physiological process. This overview article covers the most recent information on C1A proteases in leaf senescence in different plant species. Particularly, it is focussed on barley, as the unique species where the whole gene family members of C1A cysteine proteases and cystatins have been analysed.

Potato CONSTANS is involved in photoperiodic tuberization in a graft-transmissible manner.

CONSTANS (CO) is involved in the photoperiodic control of plant developmental processes, including flowering in several species and seasonal growth cessation and bud set in trees. It has been proposed that CO could also affect the day-length regulation of tuber induction in Solanum tuberosum (potato), a plant of great agricultural relevance. To address this question, we examined the role of CO in potato. A potato CO-like gene, StCO, was identified and found to be highly similar to a previously reported potato gene of unknown function. Potato plants overexpressing StCO tuberized later than wild-type plants under a weakly inductive photoperiod. StCO silencing promoted tuberization under both repressive and weakly inductive photoperiods, but did not have any effect under strongly inductive short days, demonstrating that StCO represses tuberization in a photoperiod-dependent manner. The effect of StCO on tuber induction was transmitted through grafts. In addition, StCO affected the mRNA levels of StBEL5 - a tuberization promoter, the mRNA of which moves long distances in potato plants - and StFT/StSP6A, a protein highly similar to FLOWERING LOCUS T (FT), which is a key component of systemic flowering signals in other species. We also found that StFT/StSP6A transcript levels correlate with the induction of tuber formation in wild-type plants. These results show that StCO plays an important role in photoperiodic tuberization and, together with the recent demonstration that StFT/StSP6A promotes tuberization, indicate that the CO/FT module participates in controlling this process. Moreover, they support the notion that StCO is involved in the expression of long-distance regulatory signals in potato, as CO does in other species.

The Parasporal Body of Bacillus thuringiensis subsp. israelensis: A Unique Phage Capsid-Associated Prokaryotic Insecticidal Organelle.

The three most important commercial bacterial insecticides are all derived from subspecies of Bacillus thuringiensis (Bt). Specifically, Bt subsp. kurstaki (Btk) and Bt subsp. aizawai (Bta) are used to control larval lepidopteran pests. The third, Bt subsp. israelensis (Bti), is primarily used to control mosquito and blackfly larvae. All three subspecies produce a parasporal body (PB) during sporulation. The PB is composed of insecticidal proteins that damage the midgut epithelium, initiating a complex process that results in the death of the insect. Among these three subspecies of Bt, Bti is unique as it produces the most complex PB consisting of three compartments. Each compartment is bound by a multilaminar fibrous matrix (MFM). Two compartments contain one protein each, Cry11Aa1 and Cyt1Aa1, while the third contains two, Cry4Aa1/Cry4Ba1. Each compartment is packaged independently before coalescing into the mature spherical PB held together by additional layers of the MFM. This distinctive packaging process is unparalleled among known bacterial organelles, although the underlying molecular biology is yet to be determined. Here, we present structural and molecular evidence that the MFM has a hexagonal pattern to which Bti proteins Bt152 and Bt075 bind. Bt152 binds to a defined spot on the MFM during the development of each compartment, yet its function remains unknown. Bt075 appears to be derived from a bacteriophage major capsid protein (MCP), and though its sequence has markedly diverged, it shares striking 3-D structural similarity to the Escherichia coli phage HK97 Head 1 capsid protein. Both proteins are encoded on Bti's pBtoxis plasmid. Additionally, we have also identified a six-amino acid motif that appears to be part of a novel molecular process responsible for targeting the Cry and Cyt proteins to their cytoplasmic compartments. This paper describes several previously unknown features of the Bti organelle, representing a first step to understanding the biology of a unique process of sorting and packaging of proteins into PBs. The insights from this research suggest a potential for future applications in nanotechnology.

A 54-kilodalton protein encoded by pBtoxis is required for parasporal body structural integrity in Bacillus thuringiensis subsp. israelensis.

Strains of Bacillus thuringiensis such as B. thuringiensis subsp. israelensis (ONR-60A) and B. thuringiensis subsp. morrisoni (PG-14) pathogenic for mosquito larvae produce a complex parasporal body consisting of several protein endotoxins synthesized during sporulation that form an aggregate of crystalline inclusions bound together by a multilamellar fibrous matrix. Most studies of these strains focus on the molecular biology of the endotoxins, and although it is known that parasporal body structural integrity is important to achieving high toxicity, virtually nothing is known about the matrix that binds the toxin inclusions together. In the present study, we undertook a proteomic analysis of this matrix to identify proteins that potentially mediate assembly and stability of the parasporal body. In addition to fragments of their known major toxins, namely, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, we identified peptides with 100% identity to regions of Bt152, a protein coded for by pBtoxis of B. thuringiensis subsp. israelensis, the plasmid that encodes all endotoxins of this subspecies. As it is known that the Bt152 gene is expressed in B. thuringiensis subsp. israelensis, we disrupted its function and showed that inactivation destabilized the parasporal body matrix and, concomitantly, inclusion aggregation. Using fluorescence microscopy, we further demonstrate that Bt152 localizes to the parasporal body in both strains, is absent in other structural or soluble components of the cell, including the endospore and cytoplasm, and in ligand blots binds to purified multilamellar fibrous matrix. Together, the data show that Bt152 is essential for stability of the parasporal body of these strains.

Graft-transmissible induction of potato tuberization by the microRNA miR172.

The photoreceptor phytochrome B (PHYB) and the homeodomain protein BEL5 are involved in the response of potato tuber induction to the photoperiod. However, whether they act in the same tuberization pathway is unknown. Here we show the effect of a microRNA, miR172, on this developmental event. miR172 levels are higher under tuber-inducing short days than under non-inductive long days and are upregulated in stolons at the onset of tuberization. Overexpression of this microRNA in potato promotes flowering, accelerates tuberization under moderately inductive photoperiods and triggers tuber formation under long days. In plants with a reduced abundance of PHYB, which tuberize under long days, both BEL5 mRNA and miR172 levels are reduced in leaves and increased in stolons. This, together with the presence of miR172 in vascular bundles and the graft transmissibility of its effect on tuberization, indicates that either miR172 might be mobile or it regulates long-distance signals to induce tuberization. Consistent with this, plants overexpressing miR172 show increased levels of BEL5 mRNA, which has been reported to be transmissible through grafts. Furthermore, we identify an APETALA2-like mRNA containing a miR172 binding site, which is downregulated in plants overexpressing miR172 and plants in which PHYB is silenced. Altogether, our results suggest that miR172 probably acts downstream of the tuberization repressor PHYB and upstream of the tuberization promoter BEL5 and allow us to propose a model for the control of tuberization by PHYB, miR172 and BEL5.

Characterization of the entire cystatin gene family in barley and their target cathepsin L-like cysteine-proteases, partners in the hordein mobilization during seed germination.

Plant cystatins are inhibitors of cysteine-proteases of the papain C1A and legumain C13 families. Cystatin data from multiple plant species have suggested that these inhibitors act as defense proteins against pests and pathogens and as regulators of protein turnover. In this study, we characterize the entire cystatin gene family from barley (Hordeum vulgare), which contain 13 nonredundant genes, and identify and characterize their target enzymes, the barley cathepsin L-like proteases. Cystatins and proteases were expressed and purified from Escherichia coli cultures. Each cystatin was found to have different inhibitory capability against barley cysteine-proteases in in vitro inhibitory assays using specific substrates. Real-time reverse transcription-polymerase chain reaction revealed that inhibitors and enzymes present a wide variation in their messenger RNA expression patterns. Their transcripts were mainly detected in developing and germinating seeds, and some of them were also expressed in leaves and roots. Subcellular localization of cystatins and cathepsin L-like proteases fused to green fluorescent protein demonstrated the presence of both protein families throughout the endoplasmic reticulum and the Golgi complex. Proteases and cystatins not only colocalized but also interacted in vivo in the plant cell, as revealed by bimolecular fluorescence complementation. The functional relationship between cystatins and cathepsin L-like proteases was inferred from their common implication as counterparts of mobilization of storage proteins upon barley seed germination. The opposite pattern of transcription expression in gibberellin-treated aleurones presented by inhibitors and enzymes allowed proteases to specifically degrade B, C, and D hordeins stored in the endosperm of barley seeds.

Plant Proteases: From Key Enzymes in Germination to Allies for Fighting Human Gluten-Related Disorders.

Plant proteases play a crucial role in many different biological processes along the plant life cycle. One of the most determinant stages in which proteases are key protagonists is the plant germination through the hydrolysis and mobilization of other proteins accumulated in seeds and cereal grains. The most represented proteases in charge of this are the cysteine proteases group, including the C1A family known as papain-like and the C13 family also called legumains. In cereal species such as wheat, oat or rye, gluten is a very complex mixture of grain storage proteins, which may affect the health of sensitive consumers like celiac patients. Since gluten proteins are suitable targets for plant proteases, the knowledge of the proteases involved in storage protein mobilization could be employed to manipulate the amount of gluten in the grain. Some proteases have been previously found to exhibit promising properties for their application in the degradation of known toxic peptides from gluten. To explore the variability in gluten-degrading capacities, we have now analyzed the degradation of gluten from different wheat cultivars using several cysteine proteases from barley. The wide variability showed highlights the possibility to select the protease with the highest potential to alter grain composition reducing the gluten content. Consequently, new avenues could be explored combining genetic manipulation of proteolytic processes with silencing techniques to be used as biotechnological tools against gluten-related disorders.