Maize ß-amylase7 encodes 2 proteins using alternative transcriptional start sites: Nuclear BAM7 and plastidic BAM2.

An unusual ß-amylase7 (BAM7) gene in some angiosperms, including grasses such as maize (Zea mays), appears to encode 2 functionally distinct proteins: a nuclear-localized transcription factor (BAM7) and a plastid-localized starch hydrolase (BAM2). In Arabidopsis (Arabidopsis thaliana), these 2 proteins are encoded by separate genes on different chromosomes but their physiological functions are not well established. Using the maize BAM7 gene as a model, we detected 2 populations of transcripts by 5'-RACE which encode the predicted proteins. The 2 transcripts are apparently synthesized independently using separate core promoters about 1 kb apart, the second of which is located in the first intron of the full-length gene. The N-terminus of the shorter protein, ZmBAM7-S, begins near the 3' end of the first intron of ZmBAM7-L and starts with a predicted chloroplast transit peptide. We previously showed that ZmBAM7-S is catalytically active with properties like those of AtBAM2. Here, we report that ZmBAM7-S targets green fluorescent protein to plastids. The transcript encoding the longer protein, ZmBAM7-L, encodes an additional DNA-binding domain containing a functional nuclear localization signal. This putative dual-function gene originated at least 400 Mya, prior to the emergence of ferns, and has persisted in some angiosperms that lack a separate BAM2 gene. It appears to have been duplicated and subfunctionalized in at least 4 lineages of land plants, resulting in 2 genes resembling Arabidopsis BAM2 and BAM7. Targeting of 2 products from a single gene to different subcellular locations is not uncommon in plants, but it is unusual when they are predicted to serve completely different functions in the 2 locations.

The BAM7 gene in Zea mays encodes a protein with similar structural and catalytic properties to Arabidopsis BAM2.

Starch accumulates in the plastids of green plant tissues during the day to provide carbon for metabolism at night. Starch hydrolysis is catalyzed by members of the ß-amylase (BAM) family, which in Arabidopsis thaliana (At) includes nine structurally and functionally diverse members. One of these enzymes, AtBAM2, is a plastid-localized enzyme that is unique among characterized ß-amylases since it is tetrameric and exhibits sigmoidal kinetics. Sequence alignments show that the BAM domains of AtBAM7, a catalytically inactive, nuclear-localized transcription factor with an N-terminal DNA-binding domain, and AtBAM2 are more closely related to each other than they are to any other AtBAM. Since the BAM2 gene is found in more ancient lineages, it was hypothesized that the BAM7 gene evolved from BAM2. However, analysis of the genomes of 48 flowering plants revealed 12 species that appear to possess a BAM7 gene but lack a BAM2 gene. Upon closer inspection, these BAM7 proteins have a greater percent identity to AtBAM2 than to AtBAM7, and they share all of the AtBAM2 functional residues that BAM7 proteins normally lack. It is hypothesized that these genes may encode BAM2-like proteins although they are currently annotated as BAM7-like genes. To test this hypothesis, a cDNA for the short form of corn BAM7 (ZmBAM7-S) was designed for expression in Escherichia coli. Small-angle X-ray scattering data indicate that ZmBAM7-S has a tetrameric solution structure that is more similar to that of AtBAM2 than to that of AtBAM1. In addition, partially purified ZmBAM7-S is catalytically active and exhibits sigmoidal kinetics. Together, these data suggest that some BAM7 genes may encode a functional BAM2. Exploring and understanding the ß-amylase gene structure could have an impact on the current annotation of genes.

Solution structure and assembly of ß-amylase 2 from Arabidopsis thaliana.

Starch is a key energy-storage molecule in plants that requires controlled synthesis and breakdown for effective plant growth. ß-Amylases (BAMs) hydrolyze starch into maltose to help to meet the metabolic needs of the plant. In the model plant Arabidopsis thaliana there are nine BAMs, which have apparently distinct functional and domain structures, although the functions of only a few of the BAMs are known and there are no 3D structures of BAMs from this organism. Recently, AtBAM2 was proposed to form a tetramer based on chromatography and activity assays of mutants; however, there was no direct observation of this tetramer. Here, small-angle X-ray scattering data were collected from AtBAM2 and its N-terminal truncations to describe the structure and assembly of the tetramer. Comparison of the scattering of the AtBAM2 tetramer with data collected from sweet potato (Ipomoea batatas) BAM5, which is also reported to form a tetramer, showed there were differences in the overall assembly. Analysis of the N-terminal truncations of AtBAM2 identified a loop sequence found only in BAM2 orthologs that appears to be critical for AtBAM2 tetramer assembly as well as for activity.

Involvement of five catalytically active Arabidopsis ß-amylases in leaf starch metabolism and plant growth.

Starch degradation in chloroplasts requires ß-amylase (BAM) activity, but in Arabidopsis, there are nine BAM proteins, five of which are thought to be catalytic. Although single-gene knockouts revealed the necessity of BAM3 for starch degradation, contributions of other BAMs are poorly understood. Moreover, it is not possible to detect the contribution of individual BAMs in plants containing multiple active BAMs. Therefore, we constructed a set of five quadruple mutants each expressing only one catalytically active BAM, and a quintuple mutant missing all of these BAMs (B-Null). Using these mutants, we assessed the influence of each individual BAM on plant growth and on leaf starch degradation. Both BAM1 and BAM3 alone support wild-type (WT) levels of growth. BAM3 alone is sufficient to degrade leaf starch completely whereas BAM1 alone can only partially degrade leaf starch. In contrast, BAM2, BAM5, and BAM6 have no detectable effect on starch degradation or plant growth, being comparable with the B-Null plants. B-Null plant extracts contained no measurable amylase activity, whereas BAM3 and BAM1 contributed about 70% and 14% of the WT activity, respectively. BAM2 activity was low but detectable and BAM6 contributed no measurable activity. Interestingly, activity of BAM1 and BAM3 in the mutants varied little developmentally or diurnally, and did not increase appreciably in response to osmotic or cold stress. With these genetic lines, we now have new opportunities to investigate members of this diverse gene family.

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.

ß-amylase-like proteins function as transcription factors in Arabidopsis, controlling shoot growth and development.

Plants contain ß-amylase-like proteins (BAMs; enzymes usually associated with starch breakdown) present in the nucleus rather than targeted to the chloroplast. They possess BRASSINAZOLE RESISTANT1 (BZR1)-type DNA binding domains--also found in transcription factors mediating brassinosteroid (BR) responses. The two Arabidopsis thaliana BZR1-BAM proteins (BAM7 and BAM8) bind a cis-regulatory element that both contains a G box and resembles a BR-responsive element. In protoplast transactivation assays, these BZR1-BAMs activate gene expression. Structural modeling suggests that the BAM domain's glucan binding cleft is intact, but the recombinant proteins are at least 1000 times less active than chloroplastic ß-amylases. Deregulation of BZR1-BAMs (the bam7bam8 double mutant and BAM8-overexpressing plants) causes altered leaf growth and development. Of the genes upregulated in plants overexpressing BAM8 and downregulated in bam7bam8 plants, many carry the cis-regulatory element in their promoters. Many genes that respond to BRs are inversely regulated by BZR1-BAMs. We propose a role for BZR1-BAMs in controlling plant growth and development through crosstalk with BR signaling. Furthermore, we speculate that BZR1-BAMs may transmit metabolic signals by binding a ligand in their BAM domain, although diurnal changes in the concentration of maltose, a candidate ligand produced by chloroplastic ß-amylases, do not influence their transcription factor function.

Genomics and bioinformatics in undergraduate curricula: Contexts for hybrid laboratory/lecture courses for entering and advanced science students.

Emerging interest in genomics in the scientific community prompted biologists at James Madison University to create two courses at different levels to modernize the biology curriculum. The courses are hybrids of classroom and laboratory experiences. An upper level class uses raw sequence of a genome (plasmid or virus) as the subject on which to base the experience of genomic analysis. Students also learn bioinformatics and software programs needed to support a project linking structure and function in proteins and showing evolutionary relatedness of similar genes. An optional entry-level course taken in addition to the required first-year curriculum and sponsored in part by the Howard Hughes Medical Institute, engages first year students in a primary research project. In the first semester, they isolate and characterize novel bacteriophages that infect soil bacteria. In the second semester, these young scientists annotate the genes on one or more of the unique viruses they discovered. These courses are demanding but exciting for both faculty and students and should be accessible to any interested faculty member.

An alpha-amylase (At4g25000) in Arabidopsis leaves is secreted and induced by biotic and abiotic stress.

Leaves are reported to contain a secreted alpha-amylase that accumulates during senescence or after biotic or abiotic stress; however, a gene encoding this enzyme has not been described. Because a secreted amylase is isolated from plastidic starch, the function of this enzyme is difficult to predict, but circumstantial evidence suggests that it may degrade starch after cell death. The Arabidopsis thaliana genome contains three alpha-amylase genes, one of which, AMY1 (At4g25000), has a putative signal sequence suggesting that the protein may be secreted. Two independent T-DNA insertion mutants in AMY1 lacked an amylase band on starch zymograms, which was previously named 'A1'. Washed leaf protoplasts contained reduced A1 activity suggesting that the enzyme is secreted. Native AMY1, fused to a weakly fluorescent form of GFP, was sensitive to proteinase K infiltrated into leaf apoplastic spaces, while a cytosolic form of GFP was unaffected until cell breakage, confirming that the AMY1 protein is secreted. Amylase A1 was transcriptionally induced in senescing leaves and in leaves exposed to heat stress, treated with abscisic acid or infected with Pseudomonas syringae pv. tomato expressing avrRpm1. The A1 amylase was also extremely heat resistant and its expression was up-regulated in cpr5-2, an activated defence response mutant.