BITS Faculty Publications
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Item Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination(OUP, 2015-06) Chandrasekar, BalakumaranCysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteinsItem Beta galactosidases in Arabidopsis and tomato–a mini review(Portland Press, 2016-02) Chandrasekar, BalakumaranBeta 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.Item The synthesis of xyloglucan, an abundant plant cell wall polysaccharide, requires CSLC function(PNAS, 2020-08-18) Chandrasekar, BalakumaranXyloglucan (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.