Fatty acid de novo biosynthesis in plastids: Key enzymes and their critical roles for male reproduction and other processes in plants.

Fatty acid de novo biosynthesis in plant plastids is initiated from acetyl-CoA and catalyzed by a series of enzymes, which is required for the vegetative growth, reproductive growth, seed development, stress response, chloroplast development and other biological processes. In this review, we systematically summarized the fatty acid de novo biosynthesis-related genes/enzymes and their critical roles in various plant developmental processes. Based on bioinformatic analysis, we identified fatty acid synthase encoding genes and predicted their potential functions in maize growth and development, especially in anther and pollen development. Finally, we highlighted the potential applications of these fatty acid synthases in male-sterility hybrid breeding, seed oil content improvement, herbicide and abiotic stress resistance, which provides new insights into future molecular crop breeding.

Regulators of early maize leaf development inferred from transcriptomes of laser capture microdissection (LCM)-isolated embryonic leaf cells.

The superior photosynthetic efficiency of C4 leaves over C3 leaves is owing to their unique Kranz anatomy, in which the vein is surrounded by one layer of bundle sheath (BS) cells and one layer of mesophyll (M) cells. Kranz anatomy development starts from three contiguous ground meristem (GM) cells, but its regulators and underlying molecular mechanism are largely unknown. To identify the regulators, we obtained the transcriptomes of 11 maize embryonic leaf cell types from five stages of pre-Kranz cells starting from median GM cells and six stages of pre-M cells starting from undifferentiated cells. Principal component and clustering analyses of transcriptomic data revealed rapid pre-Kranz cell differentiation in the first two stages but slow differentiation in the last three stages, suggesting early Kranz cell fate determination. In contrast, pre-M cells exhibit a more prolonged transcriptional differentiation process. Differential gene expression and coexpression analyses identified gene coexpression modules, one of which included 3 auxin transporter and 18 transcription factor (TF) genes, including known regulators of Kranz anatomy and/or vascular development. In situ hybridization of 11 TF genes validated their expression in early Kranz development. We determined the binding motifs of 15 TFs, predicted TF target gene relationships among the 18 TF and 3 auxin transporter genes, and validated 67 predictions by electrophoresis mobility shift assay. From these data, we constructed a gene regulatory network for Kranz development. Our study sheds light on the regulation of early maize leaf development and provides candidate leaf development regulators for future study.

Modeling reveals posttranscriptional regulation of GA metabolism enzymes in response to drought and cold.

The hormone gibberellin (GA) controls plant growth and regulates growth responses to environmental stress. In monocotyledonous leaves, GA controls growth by regulating division-zone size. We used a systems approach to investigate the establishment of the GA distribution in the maize leaf growth zone to understand how drought and cold alter leaf growth. By developing and parameterizing a multiscale computational model that includes cell movement, growth-induced dilution, and metabolic activities, we revealed that the GA distribution is predominantly determined by variations in GA metabolism. Considering wild-type and UBI::GA20-OX-1 leaves, the model predicted the peak in GA concentration, which has been shown to determine division-zone size. Drought and cold modified enzyme transcript levels, although the model revealed that this did not explain the observed GA distributions. Instead, the model predicted that GA distributions are also mediated by posttranscriptional modifications increasing the activity of GA 20-oxidase in drought and of GA 2-oxidase in cold, which we confirmed by enzyme activity measurements. This work provides a mechanistic understanding of the role of GA metabolism in plant growth regulation.

Rhizosphere enzyme activities and microorganisms drive the transformation of organic and inorganic carbon in saline-alkali soil region.

Western Jilin Province is one of the world's three major saline-alkali land distribution areas, and is also an important area of global climate change and carbon cycle research. Rhizosphere soil microorganisms and enzymes are the most active components in soil, which are closely related to soil carbon cycle and can reflect soil organic carbon (SOC) dynamics sensitively. Soil inorganic carbon (SIC) is the main existing form of soil carbon pool in arid saline-alkali land, and its quantity distribution affects the pattern of soil carbon accumulation and storage. Previous studies mostly focus on SOC, and pay little attention to SIC. Illumina Miseq high-throughput sequencing technology was used to reveal the changes of community structure in three maize fields (M1, M2 and M3) and three rice fields (R1, R2 and R3), which were affected by different levels of salinization during soil development. It is a new research topic of soil carbon cycle in saline-alkali soil region to investigate the effects of soil microorganisms and soil enzymes on the transformation of SOC and SIC in the rhizosphere. The results showed that the root-soil-microorganism interaction was changed by saline-alkali stress. The activities of catalase, invertase, amylase and ß-glucosidase decreased with increasing salinity. At the phylum level, most bacterial abundance decreases with increasing salinity. However, the relative abundance of Proteobacteria and Firmicutes in maize field and Firmicutes, Proteobacteria and Nitrospirae in rice field increased sharply under saline-alkali stress. The results of redundancy analysis showed that the differences of rhizosphere soil between the three maize and three rice fields were mainly affected by ESP, pH and soil salt content. In saline-alkali soil region, ß-glucosidase activity and amylase were significantly positively correlated with SOC content in maize fields, while catalase and ß-glucosidase were significantly positively correlated with SOC content in rice fields. Actinobacteria, Bacteroidetes and Verrucomicrobia had significant positive effects on SOC content of maize and rice fields. Proteobacteria, Gemmatimonadetes and Nitrospirae were positively correlated with SIC content. These enzymes and microorganisms are beneficial to soil carbon sequestration in saline-alkali soils.

Genome-wide analysis of the NAAT, DMAS, TOM, and ENA gene families in maize suggests their roles in mediating iron homeostasis.

BACKGROUND: Nicotianamine (NA), 2'-deoxymugineic acid (DMA), and mugineic acid (MA) are chelators required for iron uptake and transport in plants. Nicotianamine aminotransferase (NAAT), 2'-deoxymugineic acid synthase (DMAS), transporter of MAs (TOM), and efflux transporter of NA (ENA) are involved in iron uptake and transport in rice (Oryza sativa), wheat (Triticum aestivum), and barley (Hordeum vulgare); however, these families have not been fully identified and comprehensively analyzed in maize (Zea mays L.). RESULTS: Here, we identified 5 ZmNAAT, 9 ZmDMAS, 11 ZmTOM, and 2 ZmENA genes by genome mining. RNA-sequencing and quantitative real-time PCR analysis revealed that these genes are expressed in various tissues and respond differently to high and low iron conditions. In particular, iron deficiency stimulated the expression of ZmDMAS1, ZmTOM1, ZmTOM3, and ZmENA1. Furthermore, we determined protein subcellular localization by transient expression of green fluorescent protein fusions in maize mesophyll protoplasts. ZmNAAT1, ZmNAAT-L4, ZmDMAS1, and ZmDMAS-L1 localized in the cytoplasm, whereas ZmTOMs and ZmENAs targeted to plasma and tonoplast membranes, endomembranes, and vesicles. CONCLUSIONS: Our results suggest that the different gene expression profiles and subcellular localizations of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA family members may enable specific regulation of phytosiderophore metabolism in different tissues and under different external conditions, shedding light on iron homeostasis in maize and providing candidate genes for breeding iron-rich maize varieties.

Functional characterization of three maize SIZ/PIAS-type SUMO E3 ligases.

SUMOylation is a critical post-translational modification that regulates the nature and activity of protein substrates. The reaction is usually enhanced by a SIZ/PIAS-type of SUMO E3 ligase, but the functions of its homologs in maize have not yet been reported. In this study, we functionally characterized three members of this family of SUMO ligases, ZmSIZ1a, ZmSIZ1b, and ZmSIZ1c, from Zea mays. These maize SIZ1 homologs harbor conserved domains and structures with AtSIZ1, suggesting that they are potential functional SUMO ligases, which is supported by further biochemical data. The expression of these maize SIZ1 genes was detectable ubiquitously in different maize tissues and was usually induced by abiotic stresses. Expression of ZmSIZ1 members complements the leaf developmental defects of the AtSIZ1 mutant, suggesting their conserved function in development regulation. Interestingly, overexpression of ZmSIZ1c, but not ZmSIZ1a or ZmSIZ1b, in the wild-type Arabidopsis resulted in early flowering, implying that these members differ in terms of flowering control. Besides, overexpression of these ZmSIZ1 genes also improved salt tolerance in Arabidopsis. Collectively, our functional characterization of the ZmSIZ1 members provides hints for further investigation on the functions of SUMOylation in the development and stress responses in maize.

Preparation and structural properties of starch phosphate modified by alkaline phosphatase.

In this study, a method for the synthesis of starch phosphate using the transferase properties of alkaline phosphatase was explored. Maize starch was treated with a pyrophosphate solution containing alkaline phosphatase and catalytic ions under pH 8 at 37 °C. The synthesis of starch phosphate was evaluated and compared with untreated and treated starch controls. The phosphorus content of the samples increased up to 8500% with the catalytic ion concentration, whereas the peak viscosity by up to 41.4% decreased. The crystallinity and enthalpy of the phosphorylated samples were reduced by up to 26.8% and 23.3%, respectively; however, no significant was observed by Fourier-transform infrared spectrometer. The roughness of the starch surface and the distribution of elemental phosphorus were observed by scanning electron microscopy and energy dispersive Spectrometry. X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry results further indicated the grafting of the phosphate radical.

Two calcium-dependent protein kinases enhance maize drought tolerance by activating anion channel ZmSLAC1 in guard cells.

Stomatal closure is an important process to prevent water loss in plants response to drought stress, which is finely modulated by ion channels together with their regulators in guard cells, especially the S-type anion channel AtSLAC1 in Arabidopsis. However, the functional characterization and regulation analyses of anion channels in gramineous crops, such as in maize guard cells are still limited. In this study, we identified an S-type anion channel ZmSLAC1 that was preferentially expressed in maize guard cells and involved in stomatal closure under drought stress. We found that two Ca2+ -dependent protein kinases ZmCPK35 and ZmCPK37 were expressed in maize guard cells and localized on the plasma membrane. Lesion of ZmCPK37 resulted in drought-sensitive phenotypes. Mutation of ZmSLAC1 and ZmCPK37 impaired ABA-activated S-type anion currents in maize guard cells, while the S-type anion currents were increased in the guard cells of ZmCPK35- and ZmCPK37-overexpression lines. Electrophysiological characterization in maize guard cells and Xenopus oocytes indicated that ZmCPK35 and ZmCPK37 could activate ZmSLAC1-mediated Cl- and NO3- currents. The maize inbred and hybrid lines overexpressing ZmCPK35 and ZmCPK37 exhibited enhanced tolerance and increased yield under drought conditions. In conclusion, our results demonstrate that ZmSLAC1 plays crucial roles in stomatal closure in maize, whose activity is regulated by ZmCPK35 and ZmCPK37. Elevation of ZmCPK35 and ZmCPK37 expression levels is a feasible way to improve maize drought tolerance as well as reduce yield loss under drought stress.

Loss-of-function alleles of ZmPLD3 cause haploid induction in maize.

Doubled haploid technology has been widely applied to multiple plant species and is recognized as one of the most important technologies for improving crop breeding efficiency. Although mutations in MATRILINEAL/Zea mays PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/ZmPLA1/NLD) and Zea mays DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) have been shown to generate haploids in maize, knowledge of the genetic basis of haploid induction (HI) remains incomplete. Therefore, cloning of new genes underlying HI is important for further elucidating its genetic architecture. Here, we found that loss-of-function mutations of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), one of the members from the phospholipase D subfamily, could trigger maternal HI in maize. ZmPLD3 was identified through a reverse genetic strategy based on analysis of pollen-specifically expressed phospholipases, followed by validation through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) system. Mutations of ZmPLD3 resulted in a haploid induction rate (HIR) similar to that of mtl/zmpla1/nld and showed synergistic effects rather than functional redundancy on tripling the HIR (from 1.19% to 4.13%) in the presence of mtl/zmpla1/nld. RNA-seq profiling of mature pollen indicated that a large number of pollen-specific differentially expressed genes were enriched in processes related to gametogenesis development, such as pollen tube development and cell communication, during the double-fertilization process. In addition, ZmPLD3 is highly conserved among cereals, highlighting the potential application of these in vivo haploid-inducer lines for other important crop plant species. Collectively, our discovery identifies a novel gene underlying in vivo maternal HI and provides possibility of breeding haploid inducers with further improved HIR.

Comparative transcriptome and metabolome analysis reveal glutathione metabolic network and functional genes underlying blue and red-light mediation in maize seedling leaf.

BACKGROUND: Light quality severely affects biosynthesis and metabolism-associated process of glutathione. However, the role of specific light is still unclear on the glutathione metabolism. In this article, comparatively transcriptome and metabolome methods are used to fully understand the blue and red-light conditions working on the glutathione metabolism in maize seedling leaf. RESULTS: There are 20 differently expressed genes and 4 differently expressed metabolites in KEGG pathway of glutathione metabolism. Among them, 12 genes belong to the glutathione S-transferase family, 3 genes belong to the ascorbate peroxidase gene family and 2 genes belong to the ribonucleoside-diphosphate reductase gene family. Three genes, G6PD, SPDS1, and GPX1 belong to the gene family of glucose 6-phosphate dehydrogenase, spermidine synthase, and glutathione peroxidase, respectively. Four differently expressed metabolites are identified. Three of them, Glutathione disulfide, Glutathione, and l-γ-Glutamyl-L-amino acid are decreased while L-Glutamate is increased. In addition, Through PPI analysis, two annotated genes gst16 and DAAT, and 3 unidentified genes 100381533, pco105094 and umc2770, identified as RPP13-like3, BCAT-like1and GMPS, were obtained. By the analysis of protein sequence and PPI network, we predict that pco105094 and umc2770 were involved in the GSSG-GSH and AsA-GSH cycle in the network of glutathione metabolism. CONCLUSIONS: Compared to red light, blue light remarkably changed the transcription signal transduction and metabolism of glutathione metabolism. Differently expressed genes and metabolic mapped to the glutathione metabolism signaling pathways. In total, we obtained three unidentified genes, and two of them were predicted in current glutathione metabolism network. This result will contribute to the research of glutathione metabolism of maize.

Phenotypic effects from the expression of a deregulated AtGAD1 transgene and GABA pathway suppression mutants in maize.

Glutamate decarboxylase (GAD; EC 4.1.1.15) catalyzes the irreversible decarboxylation of glutamate to produce γ-aminobutyric acid (GABA); a ubiquitous non-protein amino acid involved in the regulation of several aspects of plant metabolism and physiology. To study the function of GAD and GABA in maize, we have; 1) introduced native and deregulated forms of AtGAD1 into maize with the intent of increasing the synthesis of GABA and 2) introduced constructs into maize designed to suppress the activity of several GABA shunt, GABA transport and GABA pathway genes. Maize plants expressing the deregulated AtGAD1 exhibit a severe chlorosis and retarded growth phenotype and have high levels of GABA, and Ca++/CaM-independent GAD activity. Plants expressing the suppression constructs for GABA biosynthetic and transport pathway genes had no observable phenotype whereas a knockout of GABA catabolic pathway genes led to growth and developmental defects under standard growth conditions. The implications of this study to our understanding of the action and function of GABA and GAD in crops are discussed.

ZmXTH, a xyloglucan endotransglucosylase/hydrolase gene of maize, conferred aluminum tolerance in Arabidopsis.

Aluminum (Al) toxicity is one of the primary factors limiting crop production in acid soils worldwide. The cell wall is the major target of Al toxicity owing to the presence of many Al binding sites. Previous studies have found that XTH, encoding xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET), could participate in cell wall extension and affect the binding ability of the cell wall to Al by impeding the activities of these two enzymes. In this study, we found that ZmXTH, an XTH gene in maize, was involved in Al detoxification. The Al-induced up-regulation of ZmXTH occurred in the roots, prominently in the root tips. Additionally, the expression of ZmXTH was specifically induced by Al3+ but no other divalent or trivalent cations. Compared with the wild-type Arabidopsis, ZmXTH overexpressing plants grew more healthy and had decreased Al content in their root and root cell wall after Al stress. Overall, the results suggest that ZmXTH could confer the Al tolerance of transgenic Arabidopsis plants by reducing the Al accumulation in their roots and cell walls.

Impacts of environmental conditions, and allelic variation of cytosolic glutamine synthetase on maize hybrid kernel production.

Cytosolic glutamine synthetase (GS1) is the enzyme mainly responsible of ammonium assimilation and reassimilation in maize leaves. The agronomic potential of GS1 in maize kernel production was investigated by examining the impact of an overexpression of the enzyme in the leaf cells. Transgenic hybrids exhibiting a three-fold increase in leaf GS activity were produced and characterized using plants grown in the field. Several independent hybrids overexpressing Gln1-3, a gene encoding cytosolic (GS1), in the leaf and bundle sheath mesophyll cells were grown over five years in different locations. On average, a 3.8% increase in kernel yield was obtained in the transgenic hybrids compared to controls. However, we observed that such an increase was simultaneously dependent upon both the environmental conditions and the transgenic event for a given field trial. Although variable from one environment to another, significant associations were also found between two GS1 genes (Gln1-3 and Gln1-4) polymorphic regions and kernel yield in different locations. We propose that the GS1 enzyme is a potential lead for producing high yielding maize hybrids using either genetic engineering or marker-assisted selection. However, for these hybrids, yield increases will be largely dependent upon the environmental conditions used to grow the plants.

Ameliorative effect of melatonin improves drought tolerance by regulating growth, photosynthetic traits and leaf ultrastructure of maize seedlings.

BACKGROUND: Melatonin is considered a potential plant growth regulator to enhance the growth of plants and increase tolerance to various abiotic stresses. Nevertheless, melatonin's role in mediating stress response in different plant species and growth cycles still needs to be explored. This study was conducted to understand the impact of different melatonin concentrations (0, 50, 100, and 150 µM) applied as a soil drench to maize seedling under drought stress conditions. A decreased irrigation approach based on watering was exposed to maize seedling after drought stress was applied at 40-45% of field capacity. RESULTS: The results showed that drought stress negatively affected the growth behavior of maize seedlings, such as reduced biomass accumulation, decreased photosynthetic pigments, and enhanced the malondialdehyde and reactive oxygen species (ROS). However, melatonin application enhanced plant growth; alleviated ROS-induced oxidative damages by increasing the photosynthetic pigments, antioxidant enzyme activities, relative water content, and osmo-protectants of maize seedlings. CONCLUSIONS: Melatonin treatment also enhanced the stomatal traits, such as stomatal length, width, area, and the number of pores under drought stress conditions. Our data suggested that 100 µM melatonin application as soil drenching could provide a valuable foundation for improving plant tolerance to drought stress conditions.

Lipid anchoring and electrostatic interactions target NOT-LIKE-DAD to pollen endo-plasma membrane.

Phospholipases cleave phospholipids, major membrane constituents. They are thus essential for many developmental processes, including male gamete development. In flowering plants, mutation of phospholipase NOT-LIKE-DAD (NLD, also known as MTL or ZmPLA1) leads to peculiar defects in sexual reproduction, notably the induction of maternal haploid embryos. Contrary to previous reports, NLD does not localize to cytosol and plasma membrane of sperm cells but to the pollen endo-plasma membrane (endo-PM), a specific membrane derived from the PM of the pollen vegetative cell that encircles the two sperm cells. After pollen tube burst, NLD localizes at the apical region of the egg apparatus. Pharmacological approaches coupled with targeted mutagenesis revealed that lipid anchoring together with electrostatic interactions are involved in the attachment of NLD to this atypical endo-PM. Membrane surface-charge and lipid biosensors indicated that phosphatidylinositol-4,5-bisphosphate is enriched in the endo-PM, uncovering a unique example of how membrane electrostatic properties can define a specific polar domain (i.e., endo-PM), which is critical for plant reproduction and gamete formation.

Creation of aromatic maize by CRISPR/Cas.

Aroma is an important quality parameter for breeding in rice (Oryza sativa). For example, the aromatic rice varieties basmati and jasmine rice, with a popcorn-like scent, are popular worldwide and routinely command a price premium. 2-acetyl-1-pyrroline (2AP) is a key flavor compound among over 200 volatiles identified in fragrant rice. A naturally fragrant germplasm exists in multiple plant species besides rice, which all exhibit lower activity of BETAINE ALDEHYDE DEHYDROGENASE 2 (BADH2). However, no equivalent aromatic germplasm has been described in maize (Zea mays). Here, we characterized the two maize BADH2 homologs, ZmBADH2a and ZmBADH2b. We generated zmbadh2a and zmbadh2b single mutants and the zmbadh2a-zmbadh2b double mutant by CRISPR/Cas in four inbred lines. A popcorn-like scent was only noticeable in seeds from the double mutant, but not from either single mutant or in wild type. In agreement, we only detected 2AP in fresh kernels and dried mature seeds from the double mutant, which accumulated between 0.028 and 0.723 mg/kg 2AP. These results suggest that ZmBADH2a and ZmBADH2b redundantly participate in 2AP biosynthesis in maize, and represent the creation of the world's first aromatic maize by simultaneous genome editing of the two BADH2 genes.

Safety assessment of phytase transgenic maize 11TPY001 by 90-day feeding study in rats.

11TPY001 is a transgenic maize that expresses the Aspergillus niger phyA2 gene which could significantly improve phosphorus bioavailability in monogastric animals. The present study was conducted to investigate the potential health effects of phytase transgenic maize 11TPY001 through a 90-day subchronic rodent feeding study. Maize grains from 11TPY001 or its parental counterpart maize OSL963 were incorporated into rodent diets at 12.5%, 25% and 50% concentrations by mass and administered to Sprague-Dawley rats (n = 10/sex/group) for 90 days. An additional control group of rats (n = 10/sex/group) were fed with common maize Zhengdan958 diets at 50% by mass. All formulated diets were nutritionally balanced. Body weights, food intake, hematology, serum chemistry, absolute and relative organ weights were measured, and gross as well as microscopic pathology were examined. Compared with rats fed OSL963 maize and the common maize diet groups, no adverse diet-related differences were observed in rats fed 11TPY001 maize diets with respect to clinical signs of toxicity, body weight/gain, food consumption/efficiency, hematology, clinical chemistry, organ weights, and gross and microscopic pathology. Under the conditions of this study, the results indicated that 11TPY001 did not cause any treatment related adverse effects in rats compared with its non-transgenic parental maize OSL963.

The maize ZmMIEL1 E3 ligase and ZmMYB83 transcription factor proteins interact and regulate the hypersensitive defence response.

The plant hypersensitive response (HR), a rapid cell death at the point of pathogenesis, is mediated by nucleotide-binding site, leucine-rich repeat (NLR) resistance proteins (R-proteins) that recognize the presence of specific pathogen-derived proteins. Rp1-D21 is an autoactive maize NLR R-protein that triggers HR spontaneously. We previously mapped loci associated with variation in the strength of HR induced by Rp1-D21. Here we identify the E3 ligase ZmMIEL1 as the causal gene at a chromosome 10 modifier locus. Transient ZmMIEL1 expression in Nicotiana benthamiana reduced HR induced by Rp1-D21, while suppression of ZmMIEL1 expression in maize carrying Rp1-D21 increased HR. ZmMIEL1 also suppressed HR induced by another autoactive NLR, the Arabidopsis R-protein RPM1D505V, in N. benthamiana. We demonstrated that ZmMIEL1 is a functional E3 ligase and that the effect of ZmMIEL1 was dependent on the proteasome but also that levels of Rp1-D21 and RPM1D505V were not reduced when coexpressed with ZmMIEL1 in the N. benthamiana system. By comparison to a similar system in Arabidopsis, we identify ZmMYB83 as a potential target of ZmMIEL1. Suppression of ZmMYB83 expression in maize lines carrying Rp1-D21 suppressed HR. Suppression of ZmMIEL1 expression caused an increase in ZmMYB83 transcript and protein levels in N. benthamiana and maize. Using coimmunoprecipitation and bimolecular fluorescence complementation assays, we demonstrated that ZmMIEL1 and ZmMYB83 physically interacted. Additionally, ZmMYB83 and ZmMIEL1 regulated the expression of a set of maize very long chain fatty acid (VLCFA) biosynthetic genes that may be involved in regulating HR.

Functional Characterization of an Anthocyanin Dimalonyltransferase in Maize.

Anthocyanins are pigments with appealing hues that are currently being used as sources of natural colorants. The interaction of acylation on the stability of anthocyanin molecules has long been known. Maize is an abundant source of malonylglucoside and dimalonylglucoside anthocyanins. The enzyme Aat1 is an anthocyanin acyltransferase known to synthesize the majority of acylated anthocyanins in maize. In this paper, we characterize the substrate specificity and reaction kinetics of Aat1. It was found that Aat1 has anthocyanin 3-O-glucoside dimalonyltransferase activity and is only the second enzyme of this type characterized to this date. Our results indicate that Aat1 can utilize malonyl-CoA; succinyl-CoA and every anthocyanin 3-O-glucoside tested. Results of this study provide insight into the structure-function relations of dimalonyltransferases and give a unique insight into the activity of monocot anthocyanin acyltransferases.

Genetic Variation of the Serine Acetyltransferase Gene Family for Sulfur Assimilation in Maize.

Improving sulfur assimilation in maize kernels is essential due to humans and animals' inability to synthesize methionine. Serine acetyltransferase (SAT) is a critical enzyme that controls cystine biosynthesis in plants. In this study, all SAT gene members were genome-wide characterized by using a sequence homology search. The RNA-seq quantification indicates that they are highly expressed in leaves, other than root and seeds, consistent with their biological functions in sulfur assimilation. With the recently released 25 genomes of nested association mapping (NAM) founders representing the diverse maize stock, we had the opportunity to investigate the SAT genetic variation comprehensively. The abundant transposon insertions into SAT genes indicate their driving power in terms of gene structure and genome evolution. We found that the transposon insertion into exons could change SAT gene transcription, whereas there was no significant correlation between transposable element (TE) insertion into introns and their gene expression, indicating that other regulatory elements such as promoters could also be involved. Understanding the SAT gene structure, gene expression and genetic variation involved in natural selection and species adaption could precisely guide genetic engineering to manipulate sulfur assimilation in maize and to improve nutritional quality.