Machine learning model of the catalytic efficiency and substrate specificity of acyl-ACP thioesterase variants generated from natural and in vitro directed evolution.

Modulating the catalytic activity of acyl-ACP thioesterase (TE) is an important biotechnological target for effectively increasing flux and diversifying products of the fatty acid biosynthesis pathway. In this study, a directed evolution approach was developed to improve the fatty acid titer and fatty acid diversity produced by E. coli strains expressing variant acyl-ACP TEs. A single round of in vitro directed evolution, coupled with a high-throughput colorimetric screen, identified 26 novel acyl-ACP TE variants that convey up to a 10-fold increase in fatty acid titer, and generate altered fatty acid profiles when expressed in a bacterial host strain. These in vitro-generated variant acyl-ACP TEs, in combination with 31 previously characterized natural variants isolated from diverse phylogenetic origins, were analyzed with a random forest classifier machine learning tool. The resulting quantitative model identified 22 amino acid residues, which define important structural features that determine the catalytic efficiency and substrate specificity of acyl-ACP TE.

Dynamic relationships among pathways producing hydrocarbons and fatty acids of maize silk cuticular waxes.

The hydrophobic cuticle is the first line of defense between aerial portions of plants and the external environment. On maize (Zea mays L.) silks, the cuticular cutin matrix is infused with cuticular waxes, consisting of a homologous series of very long-chain fatty acids (VLCFAs), aldehydes, and hydrocarbons. Together with VLC fatty-acyl-CoAs (VLCFA-CoAs), these metabolites serve as precursors, intermediates and end-products of the cuticular wax biosynthetic pathway. To deconvolute the potentially confounding impacts of the change in silk microenvironment and silk development on this pathway, we profiled cuticular waxes on the silks of the inbreds B73 and Mo17, and their reciprocal hybrids. Multivariate interrogation of these metabolite abundance data demonstrates that VLCFA-CoAs and total free VLCFAs are positively correlated with the cuticular wax metabolome, and this metabolome is primarily affected by changes in the silk microenvironment and plant genotype. Moreover, the genotype effect on the pathway explains the increased accumulation of cuticular hydrocarbons with a concomitant reduction in cuticular VLCFA accumulation on B73 silks, suggesting that the conversion of VLCFA-CoAs to hydrocarbons is more effective in B73 than Mo17. Statistical modeling of the ratios between cuticular hydrocarbons and cuticular VLCFAs reveals a significant role of precursor chain length in determining this ratio. This study establishes the complexity of the product-precursor relationships within the silk cuticular wax-producing network by dissecting both the impact of genotype and the allocation of VLCFA-CoA precursors to different biological processes, and demonstrates that longer chain VLCFA-CoAs are preferentially utilized for hydrocarbon biosynthesis.

Roles of the MYB94/FUSED LEAVES1 (ZmFDL1) and GLOSSY2 (ZmGL2) genes in cuticle biosynthesis and potential impacts on Fusarium verticillioides growth on maize silks.

Maize silks, the stigmatic portions of the female flowers, have an important role in reproductive development. Silks also provide entry points for pathogens into host tissues since fungal hyphae move along the surface of the silks to reach the site of infection, i.e., the developing kernel. The outer extracellular surface of the silk is covered by a protective hydrophobic cuticle, comprised of a complex array of long-chain hydrocarbons and small amounts of very long chain fatty acids and fatty alcohols. This work illustrates that two previously characterized cuticle-related genes separately exert roles on maize silk cuticle deposition and function. ZmMYB94/FUSED LEAVES 1 (ZmFDL1) MYB transcription factor is a key regulator of cuticle deposition in maize seedlings. The ZmGLOSSY2 (ZmGL2) gene, a putative member of the BAHD superfamily of acyltransferases with close sequence similarity to the Arabidopsis AtCER2 gene, is involved in the elongation of the fatty acid chains that serve as precursors of the waxes on young leaves. In silks, lack of ZmFDL1 action generates a decrease in the accumulation of a wide number of compounds, including alkanes and alkenes of 20 carbons or greater and affects the expression of cuticle-related genes. These results suggest that ZmFDL1 retains a regulatory role in silks, which might be exerted across the entire wax biosynthesis pathway. Separately, a comparison between gl2-ref and wild-type silks reveals differences in the abundance of specific cuticular wax constituents, particularly those of longer unsaturated carbon chain lengths. The inferred role of ZmGL2 is to control the chain lengths of unsaturated hydrocarbons. The treatment of maize silks with Fusarium verticillioides conidia suspension results in altered transcript levels of ZmFDL1 and ZmGL2 genes. In addition, an increase in fungal growth was observed on gl2-ref mutant silks 72 hours after Fusarium infection. These findings suggest that the silk cuticle plays an active role in the response to F. verticillioides infection.

Predicting moisture content during maize nixtamalization using machine learning with NIR spectroscopy.

KEY MESSAGE: Moisture content during nixtamalization can be accurately predicted from NIR spectroscopy when coupled with a support vector machine (SVM) model, is strongly modulated by the environment, and has a complex genetic architecture. Lack of high-throughput phenotyping systems for determining moisture content during the maize nixtamalization cooking process has led to difficulty in breeding for this trait. This study provides a high-throughput, quantitative measure of kernel moisture content during nixtamalization based on NIR scanning of uncooked maize kernels. Machine learning was utilized to develop models based on the combination of NIR spectra and moisture content determined from a scaled-down benchtop cook method. A linear support vector machine (SVM) model with a Spearman's rank correlation coefficient of 0.852 between wet laboratory and predicted values was developed from 100 diverse temperate genotypes grown in replicate across two environments. This model was applied to NIR spectra data from 501 diverse temperate genotypes grown in replicate in five environments. Analysis of variance revealed environment explained the highest percent of the variation (51.5%), followed by genotype (15.6%) and genotype-by-environment interaction (11.2%). A genome-wide association study identified 26 significant loci across five environments that explained between 5.04% and 16.01% (average = 10.41%). However, genome-wide markers explained 10.54% to 45.99% (average = 31.68%) of the variation, indicating the genetic architecture of this trait is likely complex and controlled by many loci of small effect. This study provides a high-throughput method to evaluate moisture content during nixtamalization that is feasible at the scale of a breeding program and provides important information about the factors contributing to variation of this trait for breeders and food companies to make future strategies to improve this important processing trait.

Genetic control of kernel compositional variation in a maize diversity panel.

Maize (Zea mays L.) is a multi-purpose row crop grown worldwide, which, over time, has often been bred for increased yield at the detriment of lower composition grain quality. Some knowledge of the genetic factors that affect quality traits has been discovered through the study of classical maize mutants; however, much of the underlying genetic control of these traits and the interaction between these traits remains unknown. To better understand variation that exists for grain compositional traits in maize, we evaluated 501 diverse temperate maize inbred lines in five unique environments and predicted 16 compositional traits (e.g., carbohydrates, protein, and starch) based on the output of near-infrared (NIR) spectroscopy. Phenotypic analysis found substantial variation for compositional traits and the majority of variation was explained by genetic and environmental factors. Correlations and trade-offs among traits in different maize types (e.g., dent, sweetcorn, and popcorn) were explored, and significant differences and meaningful correlations were detected. In total, 22.9-71.0% of the phenotypic variation across these traits could be explained using 2,386,666 single nucleotide polymorphism (SNP) markers generated from whole-genome resequencing data. A genome-wide association study (GWAS) was conducted using these same markers and found 72 statistically significant SNPs for 11 compositional traits. This study provides valuable insights in the phenotypic variation and genetic control underlying compositional traits that can be used in breeding programs for improving maize grain quality.

A multigenotype maize silk expression atlas reveals how exposure-related stresses are mitigated following emergence from husk leaves.

The extraordinarily long stigmatic silks of corn (Zea mays L.) are critical for grain production but the biology of their growth and emergence from husk leaves has remained underexplored. Accordingly, gene expression was assayed for inbreds 'B73' and 'Mo17' across five contiguous silk sections. Half of the maize genes (∼20,000) are expressed in silks, mostly in spatiotemporally dynamic patterns. In particular, emergence triggers strong differential expression of ∼1,500 genes collectively enriched for gene ontology terms associated with abiotic and biotic stress responses, hormone signaling, cell-cell communication, and defense metabolism. Further, a meta-analysis of published maize transcriptomic studies on seedling stress showed that silk emergence elicits an upregulated transcriptomic response that overlaps strongly with both abiotic and biotic stress responses. Although the two inbreds revealed similar silk transcriptomic programs overall, genotypic expression differences were observed for 5,643 B73-Mo17 syntenic gene pairs and collectively account for >50% of genome-wide expression variance. Coexpression clusters, including many based on genotypic divergence, were identified and interrogated via ontology-term enrichment analyses to generate biological hypotheses for future research. Ultimately, dissecting how gene expression changes along the length of silks and between husk-encased and emerged states offers testable models for silk development and plant response to environmental stresses.

Maize Glossy2 and Glossy2-like Genes Have Overlapping and Distinct Functions in Cuticular Lipid Deposition.

Plant epidermal cells express unique molecular machinery that juxtapose the assembly of intracellular lipid components and the unique extracellular cuticular lipids that are unidirectionally secreted to plant surfaces. In maize (Zea mays), mutations at the glossy2 (gl2) locus affect the deposition of extracellular cuticular lipids. Sequence-based genome scanning identified a new Gl2 homolog in the maize genome, namely Gl2-like Both the Gl2-like and Gl2 genes are members of the BAHD superfamily of acyltransferases, with close sequence similarity to the Arabidopsis (Arabidopsis thaliana) CER2 gene. Transgenic experiments demonstrated that Gl2-like and Gl2 functionally complement the Arabidopsis cer2 mutation, with differential influences on the cuticular lipids and the lipidome of the plant, particularly affecting the longer alkyl chain acyl lipids, especially at the 32-carbon chain length. Site-directed mutagenesis of the putative BAHD catalytic HXXXDX-motif indicated that Gl2-like requires this catalytic capability to fully complement the cer2 function, but Gl2 can accomplish complementation without the need for this catalytic motif. These findings demonstrate that Gl2 and Gl2-like overlap in their cuticular lipid function, but have evolutionarily diverged to acquire nonoverlapping functions.

Genetic and environmental variation impact the cuticular hydrocarbon metabolome on the stigmatic surfaces of maize.

BACKGROUND: Simple non-isoprenoid hydrocarbons accumulate in discrete regions of the biosphere, including within bacteria and algae as a carbon and/or energy store, and the cuticles of plants and insects, where they may protect against environmental stresses. The extracellular cuticular surfaces of the stigmatic silks of maize are rich in linear hydrocarbons and therefore provide a convenient system to study the biological origins and functions of these unique metabolites. RESULTS: To test the hypotheses that genetics and environment influence the accumulation of surface hydrocarbons on silks and to examine the breadth of metabolome compositions across diverse germplasm, cuticular hydrocarbons were analyzed on husk-encased silks and silks that emerged from the husk leaves from 32 genetically diverse maize inbred lines, most of which are commonly utilized in genetics experiments. Total hydrocarbon accumulation varied ~ 10-fold among inbred lines, and up to 5-fold between emerged and husk-encased silks. Alkenes accounted for 5-60% of the total hydrocarbon metabolome, and the majority of alkenes were monoenes with a double bond at either the 7th or 9th carbon atom of the alkyl chain. Total hydrocarbon accumulation was impacted to similar degrees by genotype and husk encasement status, whereas genotype predominantly impacted alkene composition. Only minor differences in the metabolome were observed on silks that were emerged into the external environment for 3- versus 6-days. The environmental influence on the metabolome was further investigated by growing inbred lines in 2 years, one of which was warmer and wetter. Inbred lines grown in the drier year accumulated up to 2-fold more hydrocarbons and up to a 22% higher relative abundance of alkenes. In summary, the surface hydrocarbon metabolome of silks is primarily governed by genotype and husk encasement status, with smaller impacts of environment and genotype-by-environment interactions. CONCLUSIONS: This study reveals that the composition of the cuticular hydrocarbon metabolome on silks is affected significantly by genetic factors, and is therefore amenable to dissection using quantitative genetic approaches. Such studies will clarify the genetic mechanisms responsible for the accumulation of these metabolites, enabling detailed functional investigations of the diverse and complex protective roles of silk surface lipids against environmental stresses.

Altering the Substrate Specificity of Acetyl-CoA Synthetase by Rational Mutagenesis of the Carboxylate Binding Pocket.

Acetyl-CoA synthetase (ACS) is a member of a large superfamily of enzymes that display diverse substrate specificities, with a common mechanism of catalyzing the formation of a thioester bond between Coenzyme A and a carboxylic acid, while hydrolyzing ATP to AMP and pyrophosphate. As an activated form of acetate, acetyl-CoA is a key metabolic intermediate that links many metabolic processes, including the TCA cycle, amino acid metabolism, fatty acid metabolism and biosynthetic processes that generate many polyketides and some terpenes. We explored the structural basis of the specificity of ACS for only activating acetate, whereas other members of this superfamily utilize a broad range of other carboxylate substrates. By computationally modeling the structure of the Arabidopsis ACS and the Pseudomonas chlororaphis isobutyryl-CoA synthetase using the experimentally determined tertiary structures of homologous ACS enzymes as templates, we identified residues that potentially comprise the carboxylate binding pocket. These predictions were systematically tested by mutagenesis of four specific residues. The resulting rationally redesigned carboxylate binding pocket modified the size and chemo-physical properties of the carboxylate binding pocket. This redesign successfully switched a highly specific enzyme from using only acetate, to be equally specific for using longer linear (up to hexanoate) or branched chain (methylvalerate) carboxylate substrates. The significance of this achievement is that it sets a precedent for understanding the structure-function relationship of an enzyme without the need for an experimentally determined tertiary structure of that target enzyme, and rationally generates new biocatalysts for metabolic engineering of a broad range of metabolic processes.

Development and application of a quantitative bioassay to evaluate maize silk resistance to corn earworm herbivory among progenies derived from Peruvian landrace Piura.

Corn earworm (CEW), Helicoverpa zea (Boddie), (Lepidoptera: Noctuidae), is a major insect pest of corn (Zea mays spp. mays L.). CEW larvae feed on silks, kernels and cobs, causing substantial yield and quality losses both through herbivory and by vectoring pathogens. The long-term goal of this work is to elucidate the genetic and biochemical basis of a potentially novel CEW resistance source discovered in silk tissue of Piura 208, a Peruvian landrace of maize (PI 503849). We developed a quantitative CEW bioassay and tested it on four populations that contrast alleles from Piura 208 with those from GT119, a CEW-susceptible maize inbred line. In replicated analyses of two populations of F1:2 families, corn genotype accounts for 84% and 68% of the variance in CEW larval weights, and up to 60% of the variance in CEW pupation percentage, demonstrating both the success of the quantitative bioassay and the strength of the Piura 208 resistance mechanism. Analyses of two corresponding populations of BC1:2 families revealed substantially diminished effects of corn genotype on CEW weight gain and pupation. This loss of Piura 208-derived CEW resistance during backcrossing suggests complex (multi-genic) inheritance of a threshold-dependent mechanism. Technical factors in bioassay performance were also assessed, often by analyzing the 1,641 CEW larvae that were raised on control diet (meridic with no corn silks added). Minor, but statistically significant impacts on CEW weight gain, pupation, and mortality were attributable to multiple technical factors in the preparation, incubation and evaluation phases of the bioassay, demonstrating the importance of randomization, stratification, replication, and variable-tracking across the many steps of this quantitative CEW bioassay. Overall, these findings indicate that this scaled-up, quantitative CEW bioassay is fundamentally sound and that Piura 208-derived resistance alleles are experimentally tractable for genetic and mechanistic research using this approach.

Identification of active site residues implies a two-step catalytic mechanism for acyl-ACP thioesterase.

In plants and bacteria that use a Type II fatty acid synthase, isozymes of acyl-acyl carrier protein (ACP) thioesterase (TE) hydrolyze the thioester bond of acyl-ACPs, terminating the process of fatty acid biosynthesis. These TEs are therefore critical in determining the fatty acid profiles produced by these organisms. Past characterizations of a limited number of plant-sourced acyl-ACP TEs have suggested a thiol-based, papain-like catalytic mechanism, involving a triad of Cys, His, and Asn residues. In the present study, the sequence alignment of 1019 plant and bacterial acyl-ACP TEs revealed that the previously proposed Cys catalytic residue is not universally conserved and therefore may not be a catalytic residue. Systematic mutagenesis of this residue to either Ser or Ala in three plant acyl-ACP TEs, CvFatB1 and CvFatB2 from Cuphea viscosissima and CnFatB2 from Cocos nucifera, resulted in enzymatically active variants, demonstrating that this Cys residue (Cys348 in CvFatB2) is not catalytic. In contrast, the multiple sequence alignment, together with the structure modeling of CvFatB2, suggests that the highly conserved Asp309 and Glu347, in addition to previously proposed Asn311 and His313, may be involved in catalysis. The substantial loss of catalytic competence associated with site-directed mutants at these positions confirmed the involvement of these residues in catalysis. By comparing the structures of acyl-ACP TE and the Pseudomonas 4-hydroxybenzoyl-CoA TE, both of which fold in the same hotdog tertiary structure and catalyze the hydrolysis reaction of thioester bond, we have proposed a two-step catalytic mechanism for acyl-ACP TE that involves an enzyme-bound anhydride intermediate.

Two distinct domains contribute to the substrate acyl chain length selectivity of plant acyl-ACP thioesterase.

The substrate specificity of acyl-ACP thioesterase (TE) plays an essential role in controlling the fatty acid profile produced by type II fatty acid synthases. Here we identify two groups of residues that synergistically determine different substrate specificities of two acyl-ACP TEs from Cuphea viscosissima (CvFatB1 and CvFatB2). One group (V194, V217, N223, R226, R227, and I268 in CvFatB2) is critical in determining the structure and depth of a hydrophobic cavity in the N-terminal hotdog domain that binds the substrate's acyl moiety. The other group (255-RKLSKI-260 and 285-RKLPKL-289 in CvFatB2) defines positively charged surface patches that may facilitate binding of the ACP moiety. Mutagenesis of residues within these two groups results in distinct synthetic acyl-ACP TEs that efficiently hydrolyze substrates with even shorter chains (C4- to C8-ACPs). These insights into structural determinants of acyl-ACP TE substrate specificity are useful in modifying this enzyme for tailored fatty acid production in engineered organisms.

A robust and efficient method for the extraction of plant extracellular surface lipids as applied to the analysis of silks and seedling leaves of maize.

Aerial plant organs possess a diverse array of extracellular surface lipids, including both non-polar and amphipathic constituents that collectively provide a primary line of defense against environmental stressors. Extracellular surface lipids on the stigmatic silks of maize are composed primarily of saturated and unsaturated linear hydrocarbons, as well as fatty acids, and aldehydes. To efficiently extract lipids of differing polarities from maize silks, five solvent systems (hexanes; hexanes:diethyl ether (95:5); hexanes:diethyl ether (90:10); chloroform:hexanes (1:1) and chloroform) were tested by immersing fresh silks in solvent for different extraction times. Surface lipid recovery and the relative composition of individual constituents were impacted to varying degrees depending on solvent choice and duration of extraction. Analyses were performed using both silks and leaves to demonstrate the utility of the solvent- and time-optimized protocol in comparison to extraction with the commonly used chloroform solvent. Overall, the preferred solvent system was identified as hexanes:diethyl ether (90:10), based on its effectiveness in extracting surface hydrocarbons and fatty acids as well as its reduced propensity to extract presumed internal fatty acids. Metabolite profiling of wildtype and glossy1 seedlings, which are impaired in surface lipid biosynthesis, demonstrated the ability of the preferred solvent to extract extracellular surface lipids rich in amphipathic compounds (aldehydes and alcohols). In addition to the expected deficiencies in dotriacontanal and dotriacontan-1-ol for gl1 seedlings, an unexpected increase in fatty acid recovery was observed in gl1 seedlings extracted in chloroform, suggesting that chloroform extracts lipids from internal tissues of gl1 seedlings. This highlights the importance of extraction method when evaluating mutants that have altered cuticular lipid compositions. Finally, metabolite profiling of silks from maize inbreds B73 and Mo17, exposed to different environments and harvested at different ages, revealed differences in hydrocarbon and fatty acid composition, demonstrating the dynamic nature of surface lipid accumulation on silks.

Spatial Mapping and Profiling of Metabolite Distributions during Germination.

Germination is a highly complex process by which seeds begin to develop and establish themselves as viable organisms. In this study, we utilize a combination of gas chromatography-mass spectrometry, liquid chromatography-fluorescence, and mass spectrometry imaging approaches to profile and visualize the metabolic distributions of germinating seeds from two different inbreds of maize (Zea mays) seeds, B73 and Mo17. Gas chromatography and liquid chromatography analyses demonstrate that the two inbreds are highly differentiated in their metabolite profiles throughout the course of germination, especially with regard to amino acids, sugar alcohols, and small organic acids. Crude dissection of the seed followed by gas chromatography-mass spectrometry analysis of polar metabolites also revealed that many compounds were highly sequestered among the various seed tissue types. To further localize compounds, matrix-assisted laser desorption/ionization mass spectrometry imaging was utilized to visualize compounds in fine detail in their native environments over the course of germination. Most notably, the fatty acyl chain-dependent differential localization of phospholipids and triacylglycerols was observed within the embryo and radicle, showing correlation with the heterogeneous distribution of fatty acids. Other interesting observations include unusual localization of ceramides on the endosperm/scutellum boundary and subcellular localization of ferulate in the aleurone.

High spatial resolution mass spectrometry imaging reveals the genetically programmed, developmental modification of the distribution of thylakoid membrane lipids among individual cells of maize leaf.

Metabolism in plants is compartmentalized among different tissues, cells and subcellular organelles. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption ionization (MALDI) has recently advanced to allow for the visualization of metabolites at single-cell resolution. Here we applied 5- and 10 µm high spatial resolution MALDI-MSI to the asymmetric Kranz anatomy of Zea mays (maize) leaves to study the differential localization of two major anionic lipids in thylakoid membranes, sulfoquinovosyldiacylglycerols (SQDG) and phosphatidylglycerols (PG). The quantification and localization of SQDG and PG molecular species, among mesophyll (M) and bundle sheath (BS) cells, are compared across the leaf developmental gradient from four maize genotypes (the inbreds B73 and Mo17, and the reciprocal hybrids B73 × Mo17 and Mo17 × B73). SQDG species are uniformly distributed in both photosynthetic cell types, regardless of leaf development or genotype; however, PG shows photosynthetic cell-specific differential localization depending on the genotype and the fatty acyl chain constituent. Overall, 16:1-containing PGs primarily contribute to the thylakoid membranes of M cells, whereas BS chloroplasts are mostly composed of 16:0-containing PGs. Furthermore, PG 32:0 shows genotype-specific differences in cellular distribution, with preferential localization in BS cells for B73, but more uniform distribution between BS and M cells in Mo17. Maternal inheritance is exhibited within the hybrids, such that the localization of PG 32:0 in B73 × Mo17 is similar to the distribution in the B73 parental inbred, whereas that of Mo17 × B73 resembles the Mo17 parent. This study demonstrates the power of MALDI-MSI to reveal unprecedented insights on metabolic outcomes in multicellular organisms at single-cell resolution.

Microbial production of bi-functional molecules by diversification of the fatty acid pathway.

Fatty acids that are chemically functionalized at their ω-ends are rare in nature yet offer unique chemical and physical properties with wide ranging industrial applications as feedstocks for bio-based polymers, lubricants and surfactants. Two enzymatic determinants control this ω-group functionality, the availability of an appropriate acyl-CoA substrate for initiating fatty acid biosynthesis, and a fatty acid synthase (FAS) variant that can accommodate that substrate in the initial condensation reaction of the process. In Type II FAS, 3-ketoacyl-ACP synthase III (KASIII) catalyses this initial condensation reaction. We characterized KASIIIs from diverse bacterial sources, and identified variants with novel substrate specificities towards atypical acyl-CoA substrates, including 3-hydroxybutyryl-CoA. Using Alicyclobacillus acidocaldarius KASIII, we demonstrate the in vivo diversion of FAS to produce novel ω-1 hydroxy-branched fatty acids from glucose in two bioengineered microbial hosts. This study unveils the biocatalytic potential of KASIII for synthesizing diverse ω-functionalized fatty acids.

Subcellular-level resolution MALDI-MS imaging of maize leaf metabolites by MALDI-linear ion trap-Orbitrap mass spectrometer.

A significant limiting factor in achieving high spatial resolution for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) imaging is the size of the laser spot at the sample surface. Here, we present modifications to the beam-delivery optics of a commercial MALDI-linear ion trap-Orbitrap instrument, incorporating an external Nd:YAG laser, beam-shaping optics, and an aspheric focusing lens, to reduce the minimum laser spot size from ~50 µm for the commercial configuration down to ~9 µm for the modified configuration. This improved system was applied for MALDI-MS imaging of cross sections of juvenile maize leaves at 5-µm spatial resolution using an oversampling method. A variety of different metabolites including amino acids, glycerolipids, and defense-related compounds were imaged at a spatial resolution well below the size of a single cell. Such images provide unprecedented insights into the metabolism associated with the different tissue types of the maize leaf, which is known to asymmetrically distribute the reactions of C4 photosynthesis among the mesophyll and bundle sheath cell types. The metabolite ion images correlate with the optical images that reveal the structures of the different tissues, and previously known and newly revealed asymmetric metabolic features are observed.

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity.

BACKGROUND: Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. RESULTS: To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. CONCLUSION: These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

Starch-branching enzyme IIa is required for proper diurnal cycling of starch in leaves of maize.

Starch-branching enzyme (SBE), a glucosyl transferase, is required for the highly regular pattern of α-1,6 bonds in the amylopectin component of starch. In the absence of SBEIIa, as shown previously in the sbe2a mutant of maize (Zea mays), leaf starch has drastically reduced branching and the leaves exhibit a severe senescence-like phenotype. Detailed characterization of the maize sbe2a mutant revealed that SBEIIa is the primary active branching enzyme in the leaf and that in its absence plant growth is affected. Both seedling and mature sbe2a mutant leaves do not properly degrade starch during the night, resulting in hyperaccumulation. In mature sbe2a leaves, starch hyperaccumulation is greatest in visibly senescing regions but also observed in green tissue and is correlated to a drastic reduction in photosynthesis within the leaf. Starch granules from sbe2a leaves observed via scanning electron microscopy and transmission electron microscopy analyses are larger, irregular, and amorphous as compared with the highly regular, discoid starch granules observed in wild-type leaves. This appears to trigger premature senescence, as shown by an increased expression of genes encoding proteins known to be involved in senescence and programmed cell death processes. Together, these results indicate that SBEIIa is required for the proper diurnal cycling of transitory starch within the leaf and suggest that SBEIIa is necessary in producing an amylopectin structure amenable to degradation by starch metabolism enzymes.

Nearly identical paralogs: implications for maize (Zea mays L.) genome evolution.

As an ancient segmental tetraploid, the maize (Zea mays L.) genome contains large numbers of paralogs that are expected to have diverged by a minimum of 10% over time. Nearly identical paralogs (NIPs) are defined as paralogous genes that exhibit > or = 98% identity. Sequence analyses of the "gene space" of the maize inbred line B73 genome, coupled with wet lab validation, have revealed that, conservatively, at least approximately 1% of maize genes have a NIP, a rate substantially higher than that in Arabidopsis. In most instances, both members of maize NIP pairs are expressed and are therefore at least potentially functional. Of evolutionary significance, members of many NIP families also exhibit differential expression. The finding that some families of maize NIPs are closely linked genetically while others are genetically unlinked is consistent with multiple modes of origin. NIPs provide a mechanism for the maize genome to circumvent the inherent limitation that diploid genomes can carry at most two "alleles" per "locus." As such, NIPs may have played important roles during the evolution and domestication of maize and may contribute to the success of long-term selection experiments in this important crop species.