Search bioRxiv⌕ Search

Biology subjects

Mayes, H. B.

Publications and source records attributed to Mayes, H. B..

3 recordsLinked to original sources

Functional Relations of CSLD2, CSLD3, and CSLD5 Proteins during Cell Wall Synthesis in Arabidopsis

Plant cell expansion is a dynamic process that is physically constrained by the deposition of new cell wall components. During tip growth, new cell wall materials are delivered at a restricted plasma membrane domain which results in a highly polarized expansion within that specified domain. Previous studies demonstrated that this process requires the activities of members of the Cellulose Synthase-Like D (CSLD) subfamily of cell wall synthases. CSLD3 displays {beta}-1,4 glucan synthase activity, but whether other members of CSLD subfamily share this conserved biochemical activity, and whether CSLD proteins form higher-order complexes to perform {beta}-1,4 glucan synthase activities have not been determined. Here, we use genetic methods to demonstrate that CSLD2 and CSLD3 functions are interchangeable during root hair elongation and cell plate formation, while CSLD5 provides a unique and irreplaceable function in the formation of cell plates. Importantly, genetic analysis with inactivated versions of CSLD3 show that, unlike CESA proteins, CSLDs do not require the simultaneous presence of different isoforms to perform catalytic cell wall synthase activities. In vitro biochemical activity experiments confirmed that CSLD2, CSLD3, and CSLD5 proteins displayed {beta}-1,4 glucan synthases activities. Taken together, these results indicated that while all three vegetatively expressed CSLD proteins possess conserved {beta}-1,4 glucan synthase activities, that CSLD5 has a more complicated and specialized role during cell plate formation.

cell biology↗

Functional Characterization of fer-ts, a ts- FERONIA Mutant Allele That Alters Root Hair Growth

In plants, root hairs undergo a highly-polarized form of cell expansion called tip-growth, in which cell wall deposition is restricted to the root hair apex. In order to identify essential cellular components that might have been missed in earlier genetic screens we identified conditional temperature sensitive (ts) root hair mutants by EMS mutagenesis. Here we describe one of these mutants, fer-ts (feronia-temperature sensitive). Mutant fer-ts seedlings grew normally at normal temperatures (20{degrees}C), but failed to form root hairs at elevated temperatures (30{degrees}C). Map based-cloning and whole genome sequencing revealed that fer-ts resulted from a G41S substitution in the extracellular domain of FERONIA (FER). A functional fluorescent fusion of FER containing the fer-ts mutation localized to plasma membranes, but was subject to enhanced protein turnover at elevated temperatures. While tip-growth was rapidly inhibited by addition of RALF1 peptides in both wild-type and fer-ts mutants at normal temperatures, root elongation of fer-ts seedlings was resistant to added RALF1 peptide at elevated temperatures. Additionally, at elevated temperatures fer-ts seedlings displayed altered ROS accumulation upon auxin treatment and phenocopied constitutive fer mutant responses to a variety of plant hormone treatments. Molecular modeling and sequence comparison with other CrRLK1L receptor family members revealed that the mutated glycine in fer-ts is highly conserved, but is not located in the recently characterized RALF23 and LORELI-LIKE-GLYCOPROTEIN (LLG2) binding domains, perhaps suggesting that fer-ts phenotypes may not be directly due to loss of binding to RALF1 peptides.

plant biology↗

Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis

Engineering of carbohydrate-active enzymes like glycosynthases for chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable directed evolution based protein engineering methods. Currently there are no ultrahigh-throughput screening methods available for rapid and highly sensitive single cell-based screening of evolved glycosynthase enzymes employing azido sugars as substrates. Here, we report a fluorescence-based approach employing click-chemistry for the selective detection of glycosyl azides (versus free inorganic azides) that facilitated ultrahigh-throughput in-vivo single cell-based assay of glycosynthase activity. This discovery has led to the development of a directed evolution methodology for screening and sorting glycosynthase mutants for synthesis of desired fucosylated oligosaccharides. Our screening technique facilitated rapid fluorescence activated cell sorting of a large library of glycosynthase variants (>106 mutants) expressed in E. coli to identify several novel mutants with increased activity for {beta}-fucosyl-azide activated donor sugars towards desired acceptor sugars, demonstrating the broader applicability of this methodology.

biochemistry↗