Department of Biological Sciences

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Conserved Plasmodium Protein (PF3D7_0406000) of Unknown Function: In-silico Analysis and Cellular Localization
(Elsiever, 2021-08) Das, Ashis Kumar
In spite of a decrease in malaria cases, the threat of malaria due to Plasmodium falciparum still prevails. The sequencing of Plasmodium falciparum reveals that approximately 60% of the Plasmodium genes code for hypothetical/putative proteins. Here we report an in silico characterization and localization of one such protein. This was encoded by one of the hub genes, in a weighted gene co-expression based systems network, from in-vivo samples of patients suffering from uncomplicated malaria or complicated malaria disease like jaundice and jaundice with renal failure. Interestingly, the protein PF3D7_0406000 (PFD0300w) is classified as a conserved protein of unknown function and shows no identity with any protein from the human host. The transcriptomic data shows up-regulation of transcripts in cases of malaria induced disease complications. PFD0300w peptide antibody based immunolocalization studies using a, gametocyte producing P. falciparum strain RKL-9, shows presence of the protein in the cytoplasm of both asexual and sexual stage parasites.
Reduction of Sphingosine Kinase 1 Phosphorylation and Activity in Plasmodium-Infected Erythrocytes
(Frontiers, 2020-03-03) Das, Ashis Kumar
Sphingosine-1-phosphate (S1P), a bioactive lipid mediator is involved in an array of biological processes and linked to pathological manifestations. Erythrocyte is known as the major reservoir for S1P as they lack S1P-degrading enzymes (S1P lyase and S1P phosphohydrolase) and harbor sphingosine kinase-1 (SphK-1) essential for sphingosine conversion to S1P. Reduced S1P concentration in serum was correlated with disease severity in patients with Plasmodium falciparum and Plasmodium vivax infections. Herein, we aimed to identify the underlying mechanism and contribution of host erythrocytes toward depleted S1P levels in Plasmodium-infected patients vs. healthy individuals. The level and activity of SphK-1 were measured in vitro in both uninfected and cultured P. falciparum-infected erythrocytes. Infected erythrocytes demonstrated a significant decrease in SphK-1 level in a time-dependent manner. We found that 10–42 h post invasion (hpi), SphK1 level was predominantly reduced to ∼50% in rings, trophozoites, and schizonts compared to uninfected erythrocytes. We next analyzed the phosphorylation status of SphK-1, a modification responsible for its activity and S1P production, in both uninfected control and Plasmodium-infected erythrocytes. Almost ∼50% decrease in phosphorylation of SphK-1 was observed that could be corroborated with significant reduction in the production and release of S1P in infected erythrocytes. Serum S1P levels were studied in parallel in P. falciparum (N = 15), P. vivax (N = 36)-infected patients, and healthy controls (N = 6). The findings revealed that S1P concentration was significantly depleted in uncomplicated malaria cases and was found to be lowest in complicated malaria and thrombocytopenia in both P. falciparum and P. vivax-infected groups (∗∗p < 0.01). The lower serum S1P level could be correlated with the reduced platelet count defining the role of S1P level in platelet formation. In conclusion, erythrocyte SphK-1 and S1P levels were studied in Plasmodium-infected individuals and erythrocytes that helped in characterizing the complications associated with malaria and thrombocytopenia, providing insights into the contribution of host erythrocyte biology in malaria pathogenesis. Finally, this study proposes the use of S1P and its analog as a novel adjunct therapy for malaria complications.
Natural antisense transcripts in Plasmodium falciparum isolates from patients with complicated malaria
(Elsiever, 2014-06) Das, Ashis Kumar; Saxena, Vishal; Garg, Shilpi
Mechanisms regulating gene expression in malaria parasites are not well understood. Little is known about how the parasite regulates its gene expression during transition from one developmental stage to another and in response to various environmental conditions. Parasites in a diseased host face environments which differ from the static, well adapted in vitro conditions. Parasites thus need to adapt quickly and effectively to these conditions by establishing transcriptional states which are best suited for better survival. With the discovery of natural antisense transcripts (NATs) in this parasite and considering the various proposed mechanisms by which NATs might regulate gene expression, it has been speculated that these might be playing a critical role in gene regulation. We report here the diversity of NATs in this parasite, using isolates taken directly from patients with differing clinical symptoms caused by malaria infection. Using a custom designed strand specific whole genome microarray, a total of 797 NATs targeted against annotated loci have been detected. Out of these, 545 NATs are unique to this study. The majority of NATs were positively correlated with the expression pattern of the sense transcript. However, 96 genes showed a change in sense/antisense ratio on comparison between uncomplicated and complicated disease conditions. The antisense transcripts map to a broad range of biochemical/metabolic pathways, especially pathways pertaining to the central carbon metabolism and stress related pathways. Our data strongly suggests that a large group of NATs detected here are unannotated transcription units antisense to annotated gene models. The results reveal a previously unknown set of NATs that prevails in this parasite, their differential regulation in disease conditions and mapping to functionally well annotated genes. The results detailed here call for studies to deduce the possible mechanism of action of NATs, which would further help in understanding the in vivo pathological adaptations of these parasites.
Revealing natural antisense transcripts from Plasmodium vivax isolates: Evidence of genome regulation in complicated malaria
(Elsiever, 2013-12) Das, Ashis Kumar; Garg, Shilpi; Saxena, Vishal
Plasmodium vivax is the most geographically widespread human malaria parasite causing approximately 130–435 million infections annually. It is an economic burden in many parts of the world and poses a public health challenge along with the other Plasmodium sp. The biology of this parasite is less studied and poorly understood, in spite of these facts. Emerging evidence of severe complications due to infections by this parasite provides an impetus to focus research on the same. Investigating the parasite directly from infected patients is the best way to study its biology and pathogenic mechanisms. Gene expression studies of this parasite directly obtained from the patients has provided evidence of gene regulation resulting in varying amount of transcript levels in the different blood stages. The mechanisms regulating gene expression in malaria parasites are not well understood. Discovery of Natural Antisense Transcripts (NATs) in Plasmodium falciparum has suggested that these might play an important role in regulating gene expression. We report here the genome-wide occurrence of NATs in P. vivax parasites from patients with differing clinical symptoms. A total of 1348 NATs against annotated gene loci have been detected using a custom designed microarray with strand
A glimpse into the clinical proteome of human malaria parasites Plasmodium falciparum and Plasmodium vivax
(Wiley, 2009-11-09) Das, Ashis Kumar; Garg, Shilpi
Malaria causes a worldwide annual mortality of about a million people. Rapidly evolving drug-resistant species of the parasite have created a pressing need for the identification of new drug targets and vaccine candidates. By developing fractionation protocols to enrich parasites from low-parasitemia patient samples, we have carried out the first ever proteomics analysis of clinical isolates of early stages of Plasmodium falciparum (Pf) and P. vivax. Patient-derived malarial parasites were directly processed and analyzed using shotgun proteomics approach using high-sensitivity MS for protein identification. Our study revealed about 100 parasite-coded gene products that included many known drug targets such as Pf hypoxanthine guanine phosphoribosyl transferase, Pf L-lactate dehydrogenase, and Plasmepsins. In addition, our study reports the expression of several parasite proteins in clinical ring stages that have never been reported in the ring stages of the laboratory-cultivated parasite strain. This proof-of-principle study represents a noteworthy step forward in our understanding of pathways elaborated by the parasite within the malaria patient and will pave the way towards identification of new drug and vaccine targets that can aid malaria therapy.
Plasmodium vivax Malaria
(Emerging Infectious Diseases, 2005-01) Das, Ashis Kumar
We report 11 cases of severe Plasmodium vivax malaria in Bikaner (western India). Patients exhibited cerebral malaria, renal failure, circulatory collapse, severe anemia, hemoglobinurea, abnormal bleeding, acute respiratory distress syndrome, and jaundice. Peripheral blood microscopy, parasite antigen–based assays, and parasite 18s rRNA gene–based polymerase chain reaction showed the presence of P. vivax and absence of P. falciparum.
An unexpected cause of fever and seizures
(Elsiever, 2008-09) Das, Ashis Kumar
Fungi hijack a plant apoplastic endoglucanase to release a ROS scavenging β-glucan decasaccharide to subvert immune responses
(2021-05-10) Chandrasekar, Balakumaran
Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner, insoluble cell wall (CW) layer and an outer, soluble extracellular polysaccharide (EPS) matrix. Here we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, implicating different biological functions. The barley (Hordeum vulgare) β-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved β-1,3;1,6-glucan decasaccharide (β-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight β-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by β-1,3-endoglucanases due to the presence of three β-1,6-linked glucose branches and can scavenge reactive oxygen species. Additionally, exogenous application of β-GD leads to enhanced fungal colonization in barley. Our data highlights the hitherto undescribed capacity of this often overseen fungal EPS layer to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant-microbe interface.
Design, construction and validation of a Plasmodium vivax microarray for the transcriptome profiling of clinical isolates
(Elsiever, 2016-12) Das, Ashis Kumar
High density oligonucleotide microarrays have been used on Plasmodium vivax field isolates to estimate whole genome expression. However, no microarray platform has been experimentally optimized for studying the transcriptome of field isolates. In the present study, we adopted both bioinformatics and experimental testing approaches to select best optimized probes suitable for detecting parasite transcripts from field samples and included them in designing a custom 15K P. vivax microarray. This microarray has long oligonucleotide probes (60 mer) that were in-situ synthesized onto glass slides using Agilent SurePrint technology and has been developed into an 8X15K format (8 identical arrays on a single slide). Probes in this array were experimentally validated and represents 4180 P. vivax genes in sense orientation, of which 1219 genes have also probes in antisense orientation. Validation of the 15K array by using field samples (n = 14) has shown 99% of parasite transcript detection from any of the samples. Correlation analysis between duplicate probes (n = 85) present in the arrays showed perfect correlation (r2 = 0.98) indicating the reproducibility. Multiple probes representing the same gene exhibited similar kind of expression pattern across the samples (positive correlation, r ≥ 0.6). Comparison of hybridization data with the previous studies and quantitative real-time PCR experiments were performed to highlight the microarray validation procedure. This array is unique in its design, and results indicate that the array is sensitive and reproducible. Hence, this microarray could be a valuable functional genomics tool to generate reliable expression data from P. vivax field isolates.
Disease specific modules and hub genes for intervention strategies: A co-expression network based approach for Plasmodium falciparum clinical isolates
(Elsiever, 2015-10) Das, Ashis Kumar
Systems biology approaches that are based on gene expression and bioinformatics analysis have been successful in predicting the functions of many genes in Plasmodium falciparum, a protozoan parasite responsible for most of the deaths due to malaria. However, approaches that can provide information about the biological processes that are active in this parasite in vivo during complicated malaria conditions have been scarcely deployed. Here we report the analysis of a weighted gene co-expression based network for P. falciparum, from non-cerebral clinical complications. Gene expression profiles of 20 P. falciparum clinical isolates were utilized to construct the same. A total of 20 highly interacting modules were identified post network creation. In 12 of these modules, at least 10% of the member genes, were found to be differentially regulated in parasites from patient isolates showing complications, when compared with those from patients with uncomplicated disease. Enrichment analysis helped identify biological processes like oxidation–reduction, electron transport chain, protein synthesis, ubiquitin dependent catabolic processes, RNA binding and purine nucleotide metabolic processes as associated with these modules. Additionally, for each module, highly connected hub genes were identified. Detailed functional analysis of many of these, which have known annotated functions underline their importance in parasite development and survival. This suggests, that other hub genes with unknown functions may also be playing crucial roles in parasite biology, and, are potential candidates for intervention strategies.
Plasmodium falciparum complicated malaria: Modulation and connectivity between exportome and variant surface antigen gene families
(Elsiever, 2015-05) Das, Ashis Kumar
In temperate and sub-tropical regions of Asia and Latin America, complicated malaria manifested as hepatic dysfunction or renal dysfunction is seen in all age groups. There has been a concerted focus on understanding the patho-physiological and molecular basis of complicated malaria in children, much less is known about it in adults. We report here, the analysis of data from a custom, cross strain microarray (Agilent Platform) using material from adult patient samples, showing hepatic dysfunction or renal failure. These are the most common manifestations seen in adults along with cerebral malaria. The data has been analyzed with reference to variant surface antigens, encoded by the var, rifin and stevor gene families. The differential regulation profiles of key genes (comparison between Plasmodium falciparum complicated and uncomplicated isolates) have been observed. The exportome has been analyzed using similar parameters. Gene ontology term based functional enrichment of differentially regulated genes identified, up-regulated genes statistically enriched (P < 0.05) to critical biological processes like generation of precursor metabolite and energy, chromosome organization and electron transport chain. Systems network based functional enrichment of overall differentially regulated genes yielded a similar result. We are reporting here, up-regulation of var group B and C genes whose proteins are predicted to interact with CD36 receptor in the host, the up-regulation of domain cassette 13 (DC13) containing var group A, as also the up-regulation of group A rifins and many of the stevors. This is contrary to most other reports from pediatric patients, with cerebral malaria where the up-regulation of mostly var A group genes have been seen. A protein–protein interaction based network has been created and analysis performed. This co-expression and text mining based network has shown overall connectivity between the variant surface antigens (VSA) and the exportome. The up-regulation of var group B and C genes encoding PfEMP1 with different domain architecture would be important for deciding strategies for disease prevention.
The synthesis of xyloglucan, an abundant plant cell wall polysaccharide, requires CSLC function
(PNAS, 2020-08-18) Chandrasekar, Balakumaran
Xyloglucan (XyG) is an abundant component of the primary cell walls of most plants. While the structure of XyG has been well studied, much remains to be learned about its biosynthesis. Here we employed reverse genetics to investigate the role of Arabidopsis cellulose synthase like-C (CSLC) proteins in XyG biosynthesis. We found that single mutants containing a T-DNA in each of the five Arabidopsis CSLC genes had normal levels of XyG. However, higher-order cslc mutants had significantly reduced XyG levels, and a mutant with disruptions in all five CSLC genes had no detectable XyG. The higher-order mutants grew with mild tissue-specific phenotypes. Despite the apparent lack of XyG, the cslc quintuple mutant did not display significant alteration of gene expression at the whole-genome level, excluding transcriptional compensation. The quintuple mutant could be complemented by each of the five CSLC genes, supporting the conclusion that each of them encodes a XyG glucan synthase. Phylogenetic analyses indicated that the CSLC genes are widespread in the plant kingdom and evolved from an ancient family. These results establish the role of the CSLC genes in XyG biosynthesis, and the mutants described here provide valuable tools with which to study both the molecular details of XyG biosynthesis and the role of XyG in plant cell wall structure and function.
Glycosidase and glycan polymorphism control hydrolytic release of immunogenic flagellin peptides
(Science AAAS, 2019-04) Chandrasekar, Balakumaran
Plants and animals recognize conserved flagellin fragments as a signature of bacterial invasion. These immunogenic elicitor peptides are embedded in the flagellin polymer and require hydrolytic release before they can activate cell surface receptors. Although much of flagellin signaling is understood, little is known about the release of immunogenic fragments. We discovered that plant-secreted β-galactosidase 1 (BGAL1) of Nicotiana benthamiana promotes hydrolytic elicitor release and acts in immunity against pathogenic Pseudomonas syringae strains only when they carry a terminal modified viosamine (mVio) in the flagellin O-glycan. In counter defense, P. syringae pathovars evade host immunity by using BGAL1-resistant O-glycans or by producing a BGAL1 inhibitor. Polymorphic glycans on flagella are common to plant and animal pathogenic bacteria and represent an important determinant of host immunity to bacterial pathogens.
Multiplex Fluorescent, Activity-Based Protein Profiling Identifies Active α-Glycosidases and Other Hydrolases in Plants
(PMC, 2018-05) Chandrasekar, Balakumaran
With nearly 140 α-glycosidases in 14 different families, plants are well equipped with enzymes that can break the α-glucosidic bonds in a large diversity of molecules. Here, we introduce activity-based protein profiling (ABPP) of α-glycosidases in plants using α-configured cyclophellitol aziridine probes carrying various fluorophores or biotin. In Arabidopsis (Arabidopsis thaliana), these probes label members of the GH31 family of glycosyl hydrolases, including endoplasmic reticulum-resident α-glucosidase-II Radial Swelling3/Priority for Sweet Life5 (RSW3/PSL5) and Golgi-resident α-mannosidase-II Hybrid Glycosylation1 (HGL1), both of which trim N-glycans on glycoproteins. We detected the active state of extracellular α-glycosidases such as α-xylosidase XYL1, which acts on xyloglucans in the cell wall to promote cell expansion, and α-glucosidase AGLU1, which acts in starch hydrolysis and can suppress fungal invasion. Labeling of α-glycosidases generates pH-dependent signals that can be suppressed by α-glycosidase inhibitors in a broad range of plant species. To demonstrate its use on a nonmodel plant species, we applied ABPP on saffron crocus (Crocus sativus), a cash crop for the production of saffron spice. Using a combination of biotinylated glycosidase probes, we identified and quantified 67 active glycosidases in saffron crocus stigma, of which 10 are differentially active. We also uncovered massive changes in hydrolase activities in the corms upon infection with Fusarium oxysporum using multiplex fluorescence labeling in combination with probes for serine hydrolases and cysteine proteases. These experiments demonstrate the ease with which active α-glycosidases and other hydrolases can be analyzed through ABPP in model and nonmodel plants.
A Genotypic Comparison Reveals That the Improvement in Nitrogen Remobilization Efficiency in Oilseed Rape Leaves Is Related to Specific Patterns of Senescence-Associated Protease Activities and Phytohormones
(Frontiers Media S.A, 2019-02-04) Chandrasekar, Balakumaran
Oilseed rape (Brassica napus L.) is an oleoproteaginous crop characterized by low N use efficiency (NUE) that is mainly related to a weak Nitrogen Remobilization Efficiency (NRE) during the sequential leaf senescence of the vegetative stages. Based on the hypothesis that proteolysis efficiency is crucial for the improvement of leafNRE, our objective was to characterize key senescence-associated proteolytic mechanisms of two genotypes (Ténor and Samouraï) previously identified with contrasting NREs. To reach this goal, biochemical changes, protease activities and phytohormone patterns were studied in mature leaves undergoing senescence in two genotypes with contrasting NRE cultivated in a greenhouse under limiting or ample nitrate supply. The genotype with the higher NRE (Ténor) possessed enhanced senescence processes in response to nitrate limitation, and this led to greater degradation of soluble proteins compared to the other genotype (Samouraï). This efficient proteolysis is associated with (i) an increase in serine and cysteine protease (CP) activities and (ii) the appearance of new CP activities (RD21-like, SAG12-like, RD19-like, cathepsin-B, XBCP3-like and aleurain-like proteases) during senescence induced by N limitation. Compared to Samouraï, Ténor has a higher hormonal ratio ([salicylic acid] + [abscisic acid])/([cytokinins]) that promotes senescence, particularly under low N conditions, and this is correlated with the stronger protein degradation and serine/CP activities observed during senescence. Short statement: The improvement in N recycling during leaf senescence in a genotype of Brassica napus L. characterized by a high nitrogen remobilization efficiency is related to a high phytohormonal ratio ([salicylic acid] + [abscisic acid])/([cytokinins]) that promotes leaf senescence and is correlated with an increase or the induction of specific serine and cysteine protease activities.
Proteomic Investigations of Proteases Involved in Cotyledon Senescence: A Model to Explore the Genotypic Variability of Proteolysis Machinery Associated with Nitrogen Remobilization Efficiency during the Leaf Senescence of Oilseed Rape
(MDPI, 2017-12) Chandrasekar, Balakumaran
Oilseed rape is characterized by a low nitrogen remobilization efficiency during leaf senescence, mainly due to a lack of proteolysis. Because cotyledons are subjected to senescence, it was hypothesized that contrasting protease activities between genotypes may be distinguishable early in the senescence of cotyledons. To verify this assumption, our goals were to (i) characterize protease activities in cotyledons between two genotypes with contrasting nitrogen remobilization efficiency (Ténor and Samouraï) under limiting or ample nitrate supply; and (ii) test the role of salicylic acid (SA) and abscisic acid (ABA) in proteolysis regulation. Protease activities were measured and identified by a proteomics approach combining activity-based protein profiling with LC-MS/MS. As in senescing leaves, chlorophyll and protein contents decrease in senescing cotyledons and are correlated with an increase in serine and cysteine protease activities. Two RD21-like and SAG-12 proteases previously associated with an efficient proteolysis in senescing leaves of Ténor are also detected in senescing cotyledons. The infiltration of ABA and SA provokes the induction of senescence and several cysteine and serine protease activities. The study of protease activities during the senescence of cotyledons seems to be a promising experimental model to investigate the regulation and genotypic variability of proteolysis associated with efficient N remobilization
Low Protease Content in Medicago truncatula Cell Cultures Facilitates Recombinant Protein Production
(Wiley, 2018-03) Chandrasekar, Balakumaran
Medicago truncatula is an established model for studying legume biology. More recently, it has also been exploited as a Molecular Farming platform for the production of recombinant proteins, with the successful expression of fungal and human proteins in plants and cell suspension cultures of this species. One of the challenges that now must be overcome is the degradation of final products during production and downstream processing stages. In the M. truncatula genome, there are more than 400 putative protease-encoding genes, but to date, the proteolytic content of Medicago cell cultures has not been studied. In this report, the proteolytic activities that can potentially hamper the successful production of recombinant proteins in this system are evaluated. The potential proteases responsible for the degradation of target proteins are identified. Interestingly, the number of proteases found in Medicago spent medium is considerably lower than that of the well-established tobacco bright yellow 2 (BY-2) system. Papain-like cysteine proteases are found to be the major contributors to recombinant protein degradation in Medicago. This knowledge is used to engineer a cell line with reduced endogenous protease activity by expressing a selective protease inhibitor, further improving this expression platform.
Beta galactosidases in Arabidopsis and tomato - a mini review
(Portlandpress, 2016-02-09) Chandrasekar, Balakumaran
Beta galactosidases (BGALs) are glycosyl hydrolases that remove terminal β-D-galactosyl residues from β-D-galactosides. There are 17 predicted BGAL genes in the genomes of both Arabidopsis (BGAL1–17) and tomato (TBG1–17). All tested BGALs have BGAL activity but their distinct expression profiles and ancient phylogenetic separation indicates that these enzymes fulfil diverse, non-redundant roles in plant biology. The majority of these BGALs are predicted to have signal peptide and thought to act during cell wall-related biological processes. Interestingly, deletion of BGAL6 and BGAL10 in Arabidopsis causes reduced mucilage release during seed imbibition and shorter siliques respectively, whereas TBG4 depletion by RNAi decreases in fruit softening in tomato. The majority of plant BGALs remain to be characterized.
Inhibitor Discovery by Convolution ABPP
(Springer, 2017) Chandrasekar, Balakumaran
Activity-based protein profiling (ABPP) has emerged as a powerful proteomic approach to study the active proteins in their native environment by using chemical probes that label active site residues in proteins. Traditionally, ABPP is classified as either comparative or competitive ABPP. In this protocol, we describe a simple method called convolution ABPP, which takes benefit from both the competitive and comparative ABPP. Convolution ABPP allows one to detect if a reduced signal observed during comparative ABPP could be due to the presence of inhibitors. In convolution ABPP, the proteomes are analyzed by comparing labeling intensities in two mixed proteomes that were labeled either before or after mixing. A reduction of labeling in the mix-and-label sample when compared to the label-and-mix sample indicates the presence of an inhibitor excess in one of the proteomes. This method is broadly applicable to detect inhibitors in proteomes against any proteome containing protein activities of interest. As a proof of concept, we applied convolution ABPP to analyze secreted proteomes from Pseudomonas syringae-infected Nicotiana benthamiana leaves to display the presence of a beta-galactosidase inhibitor
Activity-based protein profiling of hydrolytic enzymes induced by gibberellic acid in isolated aleurone layers of malting barley
(Wiley, 2016-07) Chandrasekar, Balakumaran
During barley germination, the aleurone layer secretes most of the enzymes required to degrade the endosperm, many of which are yet to be characterized. We used activity-based protein profiling (ABPP) to detect a range of active enzymes extracted from aleurone layers isolated from grains of a commercial malting barley variety incubated with or without gibberellic acid (GA). Enzymes found to be induced by GA were putative aleurains, cathepsin-B-like proteases and serine hydrolases. By using an inhibitory sugar panel, a specific active retaining β-glycosidase in the barley aleurone was identified as a putative xylanase. Our results show that ABPP can be used rapidly to identify a variety of active enzyme isoforms in cereal aleurone without the need for enzyme purification.