Gossypium hirsutum gene of unknown function Gohir.A02G161000 encodes a potential transmembrane Root UVB Sensitive 4 Protein with a putative protein-protein interaction interface.

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.

Gossypium hirsutum gene of unknown function Gohir.A02G131900 encodes a potential plant-specific, dual-domain exo-1,3-ß-glucosidase.

A gene of unknown function, Gohir.A02G131900.1, identified in Gossypium hirsutum was studied using computational sequence and structure bioinformatic tools. The encoded protein GhGH5BG-A0A1U8NW40 (UniProt A0A1U8NW40) is predicted to be secreted and localized to the cell wall. Homology and conserved residues indicate it belongs to a plant-specific subgroup of the glycoside hydrolase family 5 and likely has exo-1,3-ß-glucosidase activity. This subgroup is unique in containing a fascin-like domain which may have evolved a unique glucan binding site of interest for further research.

Gossypium hirsutum gene of unknown function, Gohir.A02G039501.1, encodes a potential DNA-binding ALOG protein involved in gene regulation.

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.

Gossypium hirsutum gene of unknown function Gohir.A03G0737001 encodes a potential Chaperone-like Protein of protochlorophyllide oxidoreductase (CPP1).

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.

Gossypium hirsutum gene of unknown function Gohir.A03G007700.1 encodes a potential VAN3-binding protein with a phosphoinositide-binding site.

A gene of unknown function, Gohir.A03G007700.1 (gene ID: Gohir.A03G007700_UTX-TM1_v2.1; transcript ID: Gohir.A03G007700.1_UTX-TM1_v2.1), identified in Gossypium hirsutum was studied using bioinformatic analyses of the sequence and structure of its encoded protein. Results from domain prediction, conserved residues and structure comparison indicate the encoded plant-specific protein (UniProt A0A1U8N485) is part of the VAN3-binding protein family with a conserved phosphoinositide-binding site. Homology comparison suggests functional similarity with Arabidopsis FORKED-like FL5 and 6, which localize to the Golgi apparatus and are linked to vein development and leaf size phenotypes.

Gossypium hirsutum gene of unknown function, Gohir.A02G044702.1, encodes a potential B3 Transcription Factor of the REM subfamily.

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.

Review: The Arabidopsis ß-amylase (BAM) gene family: Diversity of form and function.

Multi-gene families present a rich research area to study how related proteins evolve to acquire new structures and functions. The ß-amylase (BAM) gene family is named for catalytic members' ability to hydrolyze starch into maltose units. However, the family also contains proteins that are catalytically inactive, have additional domains, or are not localized with a starch substrate. Here we review the current knowledge of each of the nine Arabidopsis BAMs, including information on their localization, structural features, expression patterns, regulation and potential functions. We also discuss unique characteristics of studying multi-gene families, such as the consideration of different kinetic parameters when performing assays on leaf extracts, and suggest approaches that may be fruitful in learning more about their unique functions.

Quaternary Structure, Salt Sensitivity, and Allosteric Regulation of ß-AMYLASE2 From Arabidopsis thaliana.

The ß-amylase family in Arabidopsis thaliana has nine members, four of which are both plastid-localized and, based on active-site sequence conservation, potentially capable of hydrolyzing starch to maltose. We recently reported that one of these enzymes, BAM2, is catalytically active in the presence of physiological levels of KCl, exhibits sigmoidal kinetics with a Hill coefficient of over 3, is tetrameric, has a putative secondary binding site (SBS) for starch, and is highly co-expressed with other starch metabolizing enzymes. Here we generated a tetrameric homology model of Arabidopsis BAM2 that is a dimer of dimers in which the putative SBSs of two subunits form a deep groove between the subunits. To validate this model and identify key residues, we generated a series of mutations and characterized the purified proteins. (1) Three point mutations in the putative subunit interfaces disrupted tetramerization; two that interfered with the formation of the starch-binding groove were largely inactive, whereas a third mutation prevented pairs of dimers from forming and was active. (2) The model revealed that a 30-residue N-terminal acidic region, not found in other BAMs, appears to form part of the putative starch-binding groove. A mutant lacking this acidic region was active and did not require KCl for activity. (3) A conserved tryptophan residue in the SBS is necessary for activation and may form π-bonds with sugars in starch. (4) Sequence alignments revealed a conserved serine residue next to one of the catalytic glutamic acid residues, that is a conserved glycine in all other active BAMs. The serine side chain points away from the active site and toward the putative starch-binding groove. Mutating the serine in BAM2 to a glycine resulted in an enzyme with a VMax similar to that of the wild type enzyme but with a 7.5-fold lower KM for soluble starch. Interestingly, the mutant no longer exhibited sigmoidal kinetics, suggesting that allosteric communication between the putative SBS and the active site was disrupted. These results confirm the unusual structure and function of this widespread enzyme, and suggest that our understanding of starch degradation in plants is incomplete.

Glutathionylation Inhibits the Catalytic Activity of Arabidopsis ß-Amylase3 but Not That of Paralog ß-Amylase1.

ß-Amylase3 (BAM3) is an enzyme that is 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 the activity of BAM1, a homologous leaf ß-amylase, was largely unaffected. This decrease in BAM3 activity may relate to the accumulation of starch 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. 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, 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. In addition, 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.

Arabidopsis ß-Amylase2 Is a K+-Requiring, Catalytic Tetramer with Sigmoidal Kinetics.

The Arabidopsis (Arabidopsis thaliana) genome contains nine ß-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.

ß-Amylase1 and ß-amylase3 are plastidic starch hydrolases in Arabidopsis That Seem to Be Adapted for Different Thermal, pH, and stress conditions.

Starch degradation in chloroplasts requires ß-amylase (BAM) activity, which is encoded by a multigene family. Of nine Arabidopsis (Arabidopsis thaliana) BAM genes, six encode plastidic enzymes, but only four of these are catalytically active. In vegetative plants, BAM1 acts during the day in guard cells, whereas BAM3 is the dominant activity in mesophyll cells at night. Plastidic BAMs have been difficult to assay in leaf extracts, in part because of a cytosolic activity encoded by BAM5. We generated a series of double mutants lacking BAM5 and each of the active plastidic enzymes (BAM1, BAM2, BAM3, and BAM6) and found that most of the plastidic activity in 5-week-old plants was encoded by BAM1 and BAM3. Both of these activities were relatively constant during the day and the night. Analysis of leaf extracts from double mutants and purified BAM1 and BAM3 proteins revealed that these proteins have distinct properties. Using soluble starch as the substrate, BAM1 and BAM3 had optimum activity at pH 6.0 to 6.5, but at high pH, BAM1 was more active than BAM3, consistent with its known daytime role in the guard cell stroma. The optimum temperature for BAM1, which is transcriptionally induced by heat stress, was about 10°C higher than that of BAM3, which is transcriptionally induced by cold stress. The amino acid composition of BAM1 and BAM3 orthologs reflected differences that are consistent with known adaptations of proteins from heat- and cold-adapted organisms, suggesting that these day- and night-active enzymes have undergone thermal adaptation.