Amanda R. Storm

Societal Impact Statement: Cotton is a global crop cultivated worldwide, yet a significant portion of the cotton genome remains functionally unannotated. Attempts have been made to automate this annotation but have been unable to completely bridge the gap. Human curation is still needed for accurate interpretations and predictions to be made. We present a solution leveraging a course‐based approach to annotating protein function in a format tailored for undergraduate students. This format produces significant insight into individual proteins while introducing students to research in a low‐ to no‐cost footprint. In this way, this work provides a case study to bridge the gap between the next generation of scientists to deliver cutting‐edge science. Summary: Cotton suffers from a modest fraction of functionally unannotated proteome. This problem is unsolvable by our current automated annotation tools. We set out to develop a solution that brings undergraduates into the research pipeline. We systematically identified all unannotated proteins and their corresponding genes and compiled a set of thousands of proteins suitable for manual downstream analysis. We devised a mutually beneficial solution, which provides manual annotation of unknown function genes through an analysis pipeline that can be used by undergraduate students as an authentic research experience. This approach ultimately provides valuable annotations disseminated in the form of a microPublication that would otherwise remain inaccessible. This analysis pipeline solution is modular and adaptable as technology improves and has successfully produced multiple microPublications, each of which provided novel predictions of form and function for an uncharacterized protein in the cotton genome. These microPublication reports are then shared directly with the cotton community by linking them to the gene in the community database, which ensures the results are broadly findable to provide valuable functional information for downstream research purposes.

A gene of unknown function, Gohir.A02G161000.1, identified in Gossypium hirsutum was studied using computational sequence and structure bioinformatics tools. The associated protein GhRUS4-A0A1U8JPV7 (UniProt A0A1U8JPV7) is predicted to be a plastid-localized, transmembrane root UVB-sensitive 4 (RUS4) protein with a newly identified potential dimerization surface. Evidence from homology and sequence conservation suggest involvement in auxin transport and pollen maturation.

A protein of unknown function encoded by gene Gohir.A02G039501.1 in Gossypium hirsutum , was studied using sequence and structure bioinformatic tools leading to its proposed function as a nuclear, DNA-binding ALOG protein involved in gene regulation during organ boundary specification and maintenance. The encoded protein contains a predicted nuclear localization sequence, an ALOG domain with conserved residues in the modeled DNA-binding regions and nearly identical sequence identity to Arabidopsis homologs involved in development of organ boundaries at the shoot apical meristem. The protein was modeled by AlphaFold2 to have a four-helix bundle that is structurally analogous to DNA-binding domains of XerC/D-like recombinases.

A gene of unknown function identified in Gossypium hirsutum , Gohir.A03G0737001.1, was studied using sequence and bioinformatic tools. The encoded protein (referred to here as GhCPP1-A0A1U8HKT6) was predicted to function as a Chaperone-like protein of protochlorophyllide oxidoreductase (CPP1), which is involved with initiation of photochemical reactions of chlorophyll biosynthesis. Sequence analysis indicates it is embedded in the chloroplast envelope membrane through four transmembrane regions and contains a J-like domain that is structurally similar to the J domain of DnaJ/Hsp40 "holdase" chaperone proteins.

β-amylase3 (BAM3) is an enzyme essential for starch degradation in plant leaves and is also transcriptionally-induced under cold stress. However, we recently reported that BAM3’s enzymatic activity decreased in cold-stressed Arabidopsis leaves, although activity of BAM1, a homologous leaf β-amylase was largely unaffected. This decrease in BAM3 activity may relate to starch accumulation reported in cold-stressed plants. The aim of this study was to explore the disparity between BAM3 transcript and activity levels under cold stress and we present evidence suggesting BAM3 is being inhibited by post-translational modification (PTM). A mechanism of enzyme inhibition was suggested by observing that BAM3 protein levels remained unchanged under cold stress. Cold stress induces nitric oxide (NO) signaling with one result being alteration of protein activity by nitrosylation or glutathionylation through agents such as S-nitrosoglutathione (GSNO). To test whether NO-induction correlates with inhibition of BAM3 in vivo, plants were treated with sodium nitroprusside, which releases NO, and a decline in BAM3 but not BAM1 activity was again observed. Treatment of recombinant BAM3 and BAM1 with GSNO caused significant, dose-dependent inhibition of BAM3 activity while BAM1 was largely unaffected. Site-directed mutagenesis, anti-glutathione Western blots, and mass spectrometry were then used to determine that in vitro BAM3 inhibition was caused by glutathionylation at cysteine-433. Further, we generated a BAM1 mutant resembling BAM3 that was sensitive to GSNO inhibition. These findings demonstrate a differential response of two BAM paralogs to the Cys-modifying reagent GSNO and provide a possible molecular basis for reduced BAM3 activity in cold-stressed plants.

The Arabidopsis (Arabidopsis thaliana) genome contains nine β-amylase (BAM) genes, some of which play important roles in starch hydrolysis. However, little is known about BAM2, a plastid-localized enzyme reported to have extremely low catalytic activity. Using conservation of intron positions, we determined that the nine Arabidopsis BAM genes fall into two distinct subfamilies. A similar pattern was found in each major lineage of land plants, suggesting that these subfamilies diverged prior to the origin of land plants. Moreover, phylogenetic analysis indicated that BAM2 is the ancestral member of one of these subfamilies. This finding, along with the conservation of amino acids in the active site of BAM2, suggested that it might be catalytically active. We then identified KCl as necessary for BAM2 activity. Unlike BAM1, BAM3, and BAM5, three Arabidopsis BAMs that all exhibited hyperbolic kinetics, BAM2 exhibited sigmoidal kinetics with a Hill coefficient of over 3. Using multi-angle light scattering, we determined that BAM2 was a tetramer, whereas BAM5 was a monomer. Conserved residues from a diverse set of BAM2 orthologs were mapped onto a homology model of the protein, revealing a large, conserved surface away from the active site that we hypothesize is a secondary carbohydrate-binding site. Introduction of bulky methionine for glycine at two points on this surface reduced catalytic activity significantly without disrupting the tetrameric structure. Expression analysis indicated that BAM2 is more closely coexpressed with other starch degradation enzymes than any other BAM, suggesting that BAM2 may play an important role in starch degradation in plants.

A gene of unknown function, Gohir.A02G044702.1, identified in Gossypium hirsutum was studied using sequence and structure bioinformatic tools. The encoded protein (UniProt A0A1U8MGX4) was predicted to localize to the nucleus, was found to retain the B3 transcription factor domain with conserved DNA-binding residues and to most closely cluster with REM subfamily members of B3-domain containing proteins.