The genetic architecture of the maize progenitor, teosinte, and how it was altered during maize domestication.

The genetics of domestication has been extensively studied ever since the rediscovery of Mendel's law of inheritance and much has been learned about the genetic control of trait differences between crops and their ancestors. Here, we ask how domestication has altered genetic architecture by comparing the genetic architecture of 18 domestication traits in maize and its ancestor teosinte using matched populations. We observed a strongly reduced number of QTL for domestication traits in maize relative to teosinte, which is consistent with the previously reported depletion of additive variance by selection during domestication. We also observed more dominance in maize than teosinte, likely a consequence of selective removal of additive variants. We observed that large effect QTL have low minor allele frequency (MAF) in both maize and teosinte. Regions of the genome that are strongly differentiated between teosinte and maize (high FST) explain less quantitative variation in maize than teosinte, suggesting that, in these regions, allelic variants were brought to (or near) fixation during domestication. We also observed that genomic regions of high recombination explain a disproportionately large proportion of heritable variance both before and after domestication. Finally, we observed that about 75% of the additive variance in both teosinte and maize is "missing" in the sense that it cannot be ascribed to detectable QTL and only 25% of variance maps to specific QTL. This latter result suggests that morphological evolution during domestication is largely attributable to very large numbers of QTL of very small effect.

A cost-effective maize ear phenotyping platform enables rapid categorization and quantification of kernels.

High-throughput phenotyping systems are powerful, dramatically changing our ability to document, measure, and detect biological phenomena. Here, we describe a cost-effective combination of a custom-built imaging platform and deep-learning-based computer vision pipeline. A minimal version of the maize (Zea mays) ear scanner was built with low-cost and readily available parts. The scanner rotates a maize ear while a digital camera captures a video of the surface of the ear, which is then digitally flattened into a two-dimensional projection. Segregating GFP and anthocyanin kernel phenotypes are clearly distinguishable in ear projections and can be manually annotated and analyzed using image analysis software. Increased throughput was attained by designing and implementing an automated kernel counting system using transfer learning and a deep learning object detection model. The computer vision model was able to rapidly assess over 390 000 kernels, identifying male-specific transmission defects across a wide range of GFP-marked mutant alleles. This includes a previously undescribed defect putatively associated with mutation of Zm00001d002824, a gene predicted to encode a vacuolar processing enzyme. Thus, by using this system, the quantification of transmission data and other ear and kernel phenotypes can be accelerated and scaled to generate large datasets for robust analyses.

Population-tailored mock genome enables genomic studies in species without a reference genome.

Based on molecular markers, genomic prediction enables us to speed up breeding schemes and increase the response to selection. There are several high-throughput genotyping platforms able to deliver thousands of molecular markers for genomic study purposes. However, even though its widely applied in plant breeding, species without a reference genome cannot fully benefit from genomic tools and modern breeding schemes. We used a method to assemble a population-tailored mock genome to call single-nucleotide polymorphism (SNP) markers without an available reference genome, and for the first time, we compared the results with standard genotyping platforms (array and genotyping-by-sequencing (GBS) using a reference genome) for performance in genomic prediction models. Our results indicate that using a population-tailored mock genome to call SNP delivers reliable estimates for the genomic relationship between genotypes. Furthermore, genomic prediction estimates were comparable to standard approaches, especially when considering only additive effects. However, mock genomes were slightly worse than arrays at predicting traits influenced by dominance effects, but still performed as well as standard GBS methods that use a reference genome. Nevertheless, the array-based SNP markers methods achieved the best predictive ability and reliability to estimate variance components. Overall, the mock genomes can be a worthy alternative for genomic selection studies, especially for those species where the reference genome is not available.

Evolutionary Metabolomics Identifies Substantial Metabolic Divergence between Maize and Its Wild Ancestor, Teosinte.

Maize (Zea mays subsp mays) was domesticated from its wild ancestor, teosinte (Zea mays subsp parviglumis). Maize's distinct morphology and adaptation to diverse environments required coordinated changes in various metabolic pathways. However, how the metabolome was reshaped since domestication remains poorly understood. Here, we report a comprehensive assessment of divergence in the seedling metabolome between maize and teosinte. In total, 461 metabolites exhibited significant divergence due to selection. Interestingly, teosinte and tropical and temperate maize, representing major stages of maize evolution, targeted distinct sets of metabolites. Alkaloids, terpenoids, and lipids were specifically targeted in the divergence between teosinte and tropical maize, while benzoxazinoids were specifically targeted in the divergence between tropical and temperate maize. To identify genetic factors controlling metabolic divergence, we assayed the seedling metabolome of a large maize-by-teosinte cross population. We show that the recent metabolic divergence between tropical and temperate maize tended to have simpler genetic architecture than the divergence between teosinte and tropical maize. Through integrating transcriptome data, we identified candidate genes contributing to metabolic divergence, many of which were under selection at the nucleotide and transcript levels. Through overexpression or mutant analysis, we verified the roles of Flavanone 3-hydroxylase1, Purple aleurone1, and maize terpene synthase1 in the divergence of their related biosynthesis pathways. Our findings not only provide important insights into domestication-associated changes in the metabolism but also highlight the power of combining omics data for trait dissection.

Identification and Fine Mapping of the Recessive Gene BK-5, Which Affects Cell Wall Biosynthesis and Plant Brittleness in Maize.

The cellulose of the plant cell wall indirectly affects the cell shape and straw stiffness of the plant. Here, the novel brittleness mutant brittle stalk-5 (bk-5) of the maize inbred line RP125 was characterized. We found that the mutant displayed brittleness of the stalk and even the whole plant, and that the brittleness phenotype existed during the whole growth period from germination to senescence. The compressive strength was reduced, the cell wall was thinner, and the cellulose content was decreased compared to that of the wild type. Genetic analysis and map-based cloning indicated that bk-5 was controlled by a single recessive nuclear gene and that it was located in a 90.2-Kb region on chromosome 3 that covers three open reading frames (ORFs). Sequence analysis revealed a single non-synonymous missense mutation, T-to-A, in the last exon of Zm00001d043477 (B73: version 4, named BK-5) that caused the 951th amino acid to go from leucine to histidine. BK-5 encodes a cellulose synthase catalytic subunit (CesA), which is involved with cellulose synthesis. We found that BK-5 was constitutively expressed in all tissues of the germinating stage and silking stage, and highly expressed in the leaf, auricula, and root of the silking stage and the 2-cm root and bud of the germinating stage. We found that BK-5 mainly localized to the Golgi apparatus, suggesting that the protein might move to the plasma membrane with the aid of Golgi in maize. According to RNA-seq data, bk-5 had more downregulated genes than upregulated genes, and many of the downregulated genes were enzymes and transcription factors related to cellulose, hemicellulose, and lignin biosynthesis of the secondary cell wall. The other differentially expressed genes were related to metabolic and cellular processes, and were significantly enriched in hormone signal transduction, starch and sucrose metabolism, and the plant-pathogen interaction pathway. Taken together, we propose that the mutation of gene BK-5 causes the brittle stalk phenotype and provides important insights into the regulatory mechanism of cellulose biosynthesis and cell wall development in maize.

Genetic diversity and population structure of early and extra-early maturing maize germplasm adapted to sub-Saharan Africa.

BACKGROUND: Assessment and effective utilization of genetic diversity in breeding programs is crucial for sustainable genetic improvement and rapid adaptation to changing breeding objectives. During the past two decades, the commercialization of the early and extra-early maturing cultivars has contributed to rapid expansion of maize into different agro-ecologies of sub-Saharan Africa (SSA) where maize has become an important component of the agricultural economy and played a vital role in food and nutritional security. The present study aimed at understanding the population structure and genetic variability among 439 early and extra-early maize inbred lines developed from three narrow-based and twenty-seven broad-based populations by the International Iinstitute of Tropical Agriculture Maize Improvement Program (IITA-MIP). These inbreds were genotyped using 9642 DArTseq-based single nucleotide polymorphism (SNP) markers distributed uniformly throughout the maize genome. RESULTS: About 40.8% SNP markers were found highly informative and exhibited polymorphic information content (PIC) greater than 0.25. The minor allele frequency and PIC ranged from 0.015 to 0.500 and 0.029 to 0.375, respectively. The STRUCTURE, neighbour-joining phylogenetic tree and principal coordinate analysis (PCoA) grouped the inbred lines into four major classes generally consistent with the selection history, ancestry and kernel colour of the inbreds but indicated a complex pattern of the genetic structure. The pattern of grouping of the lines based on the STRUCTURE analysis was in concordance with the results of the PCoA and suggested greater number of sub-populations (K = 10). Generally, the classification of the inbred lines into heterotic groups based on SNP markers was reasonably reliable and in agreement with defined heterotic groups of previously identified testers based on combining ability studies. CONCLUSIONS: Complete understanding of potential heterotic groups would be difficult to portray by depending solely on molecular markers. Therefore, planned crosses involving representative testers from opposing heterotic groups would be required to refine the existing heterotic groups. It is anticipated that the present set of inbreds could contribute new beneficial alleles for population improvement, development of hybrids and lines with potential to strengthen future breeding programs. Results of this study would help breeders in formulating breeding strategies for genetic enhancement and sustainable maize production in SSA.

Molecular characterization, expression and functional analysis of acyl-CoA-binding protein gene family in maize (Zea mays).

BACKGROUND: Acyl-CoA-binding proteins (ACBPs) possess a conserved acyl-CoA-binding (ACB) domain that facilitates binding to acyl-CoA esters and trafficking in eukaryotic cells. Although the various functions of ACBP have been characterized in several plant species, their structure, molecular evolution, expression profile, and function have not been fully elucidated in Zea mays L. RESULTS: Genome-wide analysis identified nine ZmACBP genes in Z. mays, which could be divided into four distinct classes (class I, class II, class III, and class IV) via construction of a phylogenetic tree that included 48 ACBP genes from six different plant species. Transient expression of a ZmACBP-GFP fusion protein in tobacco (Nicotiana tabacum) epidermal cells revealed that ZmACBPs localized to multiple different locations. Analyses of expression profiles revealed that ZmACBPs exhibited temporal and spatial expression changes during abiotic and biotic stresses. Eight of the nine ZmACBP genes were also found to have significant association with agronomic traits in a panel of 500 maize inbred lines. The heterologous constitutive expression of ZmACBP1 and ZmACBP3 in Arabidopsis enhanced the resistance of these plants to salinity and drought stress, possibly through alterations in the level of lipid metabolic and stress-responsive genes. CONCLUSION: The ACBP gene family was highly conserved across different plant species. ZmACBP genes had clear tissue and organ expression specificity and were responsive to both biotic and abiotic stresses, suggesting their roles in plant growth and stress resistance.

A universal chromosome identification system for maize and wild Zea species.

Maize was one of the first eukaryotic species in which individual chromosomes can be identified cytologically, which made maize one of the oldest models for genetics and cytogenetics research. Nevertheless, consistent identification of all 10 chromosomes from different maize lines as well as from wild Zea species remains a challenge. We developed a new technique for maize chromosome identification based on fluorescence in situ hybridization (FISH). We developed two oligonucleotide-based probes that hybridize to 24 chromosomal regions. Individual maize chromosomes show distinct FISH signal patterns, which allow universal identification of all chromosomes from different Zea species. We developed karyotypes from three Zea mays subspecies and two additional wild Zea species based on individually identified chromosomes. A paracentric inversion was discovered on the long arm of chromosome 4 in Z. nicaraguensis and Z. luxurians based on modifications of the FISH signal patterns. Chromosomes from these two species also showed distinct distribution patterns of terminal knobs compared with other Zea species. These results support that Z. nicaraguensis and Z. luxurians are closely related species.

Parallel altitudinal clines reveal trends in adaptive evolution of genome size in Zea mays.

While the vast majority of genome size variation in plants is due to differences in repetitive sequence, we know little about how selection acts on repeat content in natural populations. Here we investigate parallel changes in intraspecific genome size and repeat content of domesticated maize (Zea mays) landraces and their wild relative teosinte across altitudinal gradients in Mesoamerica and South America. We combine genotyping, low coverage whole-genome sequence data, and flow cytometry to test for evidence of selection on genome size and individual repeat abundance. We find that population structure alone cannot explain the observed variation, implying that clinal patterns of genome size are maintained by natural selection. Our modeling additionally provides evidence of selection on individual heterochromatic knob repeats, likely due to their large individual contribution to genome size. To better understand the phenotypes driving selection on genome size, we conducted a growth chamber experiment using a population of highland teosinte exhibiting extensive variation in genome size. We find weak support for a positive correlation between genome size and cell size, but stronger support for a negative correlation between genome size and the rate of cell production. Reanalyzing published data of cell counts in maize shoot apical meristems, we then identify a negative correlation between cell production rate and flowering time. Together, our data suggest a model in which variation in genome size is driven by natural selection on flowering time across altitudinal clines, connecting intraspecific variation in repetitive sequence to important differences in adaptive phenotypes.

Genome-Wide Identification and Characterization of Polygalacturonase Gene Family in Maize (Zea mays L.).

Polygalacturonase (PG, EC 3.2.1.15) is a crucial enzyme for pectin degradation and is involved in various developmental processes such as fruit ripening, pollen development, cell expansion, and organ abscission. However, information on the PG gene family in the maize (Zea mays L.) genome and the specific members involved in maize anther development are still lacking. In this study, we identified 55 PG family genes from the maize genome and further characterized their evolutionary relationship and expression patterns. Phylogenetic analysis revealed that ZmPGs are grouped into six Clades, and gene structures of the same Clade are highly conserved, suggesting their functional conservation. The ZmPGs are randomly distributed across maize chromosomes, and collinearity analysis showed that many ZmPGs might be derived from tandem duplications and segmental duplications, and these genes are under purifying selection. Furthermore, gene expression analysis provided insights into possible functional divergence among ZmPGs. Based on the RNA-seq data analysis, we found that many ZmPGs are expressed in various tissues while 18 ZmPGs are highly expressed in maize anther, and their detailed expression profiles in different anther developmental stages were further investigated by using RT-qPCR analysis. These results provide valuable information for further functional characterization and application of the ZmPGs in maize.

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.

Identification of the variety of maize seeds based on hyperspectral images coupled with convolutional neural networks and subregional voting.

BACKGROUND: Maize is one of the most important food crops in the world. Many different varieties of maize seeds are similar in size and appearance, so distinguishing the varieties of maize seed is a significant research topic. This study used hyperspectral image processing coupled with convolutional neural network (CNN) and a subregional voting method to recognize different varieties of maize seed. RESULTS: First, visible and near-infrared (NIR-visible) hyperspectral images were obtained. Savitzky-Golay (SG) smoothing and first derivative (FD) were used to pretreat the raw spectra and highlight the spectral differences of samples of different varieties. Second, the region of interest (ROI) of each sample was divided into several subregions according to the shape and the number of pixels. Then, a method was proposed for reshaping the images of pixel spectra for the CNN and the training model was established. Finally, using subregional voting, one prediction result was generated from the prediction results of several original subregions in one sample. The results showed that, for six varieties of normal maize seeds, the tests identified embryoid and non-embryoid forms with 93.33% and 95.56% accuracy, respectively. For six varieties of sweet maize seeds, the test accuracy in embryoid and non-embryoid forms was 97.78% and 98.15%, respectively. CONCLUSION: The maize seed was identified accurately. The present study demonstrated that the CNN model for spectral image coupled with subregional voting represents a new approach for the identification of varieties of maize seed. © 2021 Society of Chemical Industry.

Identification of the volatile profiles of 22 traditional and newly bred maize varieties and their porridges by PTR-QiTOF-MS and HS-SPME GC-MS.

BACKGROUND: Low adoption of maize varieties bred to address the nutritional needs of the growing African population limits their impact. Aroma is essential in consumer preference, but has hitherto hardly been studied. We analysed the volatile organic compounds of flours and porridges of 22 maize varieties belonging to four nutritionally distinct groups, namely provitamin A maize, quality protein maize, yellow and white maize. RESULTS: Proton-transfer-reaction quadrupole ion time-of-flight mass spectrometry (PTR-QiTOF-MS) analysis generated 524 mass peaks ranging from 16.007 to 448.089 m/z. Principal component analysis separated the varieties belonging to the four groups. With headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME GC-MS), 48 volatile compounds were identified in maize flour and 21 in maize porridge, including hexane, nonane, pentanoic acid, 1-octen-3-ol, 1-hexanol, hexanal, nonanal, 2-pentylfuran and 2-heptanone. Volatile compounds such as 1,2,4-trimethyl benzene, associated with thermal degradation of carotenoids, increased in the porridge of yellow and provitamin A maize. CONCLUSION: The results indicate that PTR-QiTOF-MS and HS-SPME GC-MS combined with multivariate analysis are instrumental to study the volatile aroma compounds of different maize varieties.

Using multiple reference genomes to identify and resolve annotation inconsistencies.

BACKGROUND: Advances in sequencing technologies have led to the release of reference genomes and annotations for multiple individuals within more well-studied systems. While each of these new genome assemblies shares significant portions of synteny between each other, the annotated structure of gene models within these regions can differ. Of particular concern are split-gene misannotations, in which a single gene is incorrectly annotated as two distinct genes or two genes are incorrectly annotated as a single gene. These misannotations can have major impacts on functional prediction, estimates of expression, and many downstream analyses. RESULTS: We developed a high-throughput method based on pairwise comparisons of annotations that detect potential split-gene misannotations and quantifies support for whether the genes should be merged into a single gene model. We demonstrated the utility of our method using gene annotations of three reference genomes from maize (B73, PH207, and W22), a difficult system from an annotation perspective due to the size and complexity of the genome. On average, we found several hundred of these potential split-gene misannotations in each pairwise comparison, corresponding to 3-5% of gene models across annotations. To determine which state (i.e. one gene or multiple genes) is biologically supported, we utilized RNAseq data from 10 tissues throughout development along with a novel metric and simulation framework. The methods we have developed require minimal human interaction and can be applied to future assemblies to aid in annotation efforts. CONCLUSIONS: Split-gene misannotations occur at appreciable frequency in maize annotations. We have developed a method to easily identify and correct these misannotations. Importantly, this method is generic in that it can utilize any type of short-read expression data. Failure to account for split-gene misannotations has serious consequences for biological inference, particularly for expression-based analyses.

Repetitive DNA content in the maize genome is uncoupled from population stratification at SNP loci.

BACKGROUND: Repetitive DNA is a major component of plant genomes and is thought to be a driver of evolutionary novelty. Describing variation in repeat content among individuals and between populations is key to elucidating the evolutionary significance of repetitive DNA. However, the cost of producing references genomes has limited large-scale intraspecific comparisons to a handful of model organisms where multiple reference genomes are available. RESULTS: We examine repeat content variation in the genomes of 94 elite inbred maize lines using graph-based repeat clustering, a reference-free and rapid assay of repeat content. We examine population structure using genome-wide repeat profiles, and demonstrate the stiff-stalk and non-stiff-stalk heterotic populations are homogenous with regard to global repeat content. In contrast, and similar to previously reported results, the same individuals show clear differentiation, and aggregate into two populations when examining population structure using genome-wide SNPs. Additionally, we develop a novel kmer based technique to examine the chromosomal distribution of repeat clusters in silico and show a cluster dependent association with gene density. CONCLUSION: Our results indicate global repeat content variation in the heterotic populations of maize has not diverged, and is uncoupled from population stratification at SNP loci. We show that repeat families exhibit divergent patterns with regard to chromosomal distribution, some repeat clusters accumulate in regions of high gene density, whereas others aggregate in regions of low gene density.

High-throughput retrotransposon-based genetic diversity of maize germplasm assessment and analysis.

Maize is one of the world's most important crops and a model for grass genome research. Long terminal repeat (LTR) retrotransposons comprise most of the maize genome; their ability to produce new copies makes them efficient high-throughput genetic markers. Inter-retrotransposon-amplified polymorphisms (IRAPs) were used to study the genetic diversity of maize germplasm. Five LTR retrotransposons (Huck, Tekay, Opie, Ji, and Grande) were chosen, based on their large number of copies in the maize genome, whereas polymerase chain reaction primers were designed based on consensus LTR sequences. The LTR primers showed high quality and reproducible DNA fingerprints, with a total of 677 bands including 392 polymorphic bands showing 58% polymorphism between maize hybrid lines. These markers were used to identify genetic similarities among all lines of maize. Analysis of genetic similarity was carried out based on polymorphic amplicon profiles and genetic similarity phylogeny analysis. This diversity was expected to display ecogeographical patterns of variation and local adaptation. The clustering method showed that the varieties were grouped into three clusters differing in ecogeographical origin. Each of these clusters comprised divergent hybrids with convergent characters. The clusters reflected the differences among maize hybrids and were in accordance with their pedigree. The IRAP technique is an efficient high-throughput genetic marker-generating method.

High-precision chronology for Central American maize diversification from El Gigante rockshelter, Honduras.

The first steps toward maize (Zea mays subspecies mays) domestication occurred in the Balsas region of Mexico by ∼9,000 calendar years B.P. (cal B.P.), but it remains unclear when maize was productive enough to be a staple grain in the Americas. Molecular and microbotanical data provide a partial picture of the timing and nature of morphological change, with genetic data indicating that alleles for some domestication traits were not yet fixed by 5,300 cal B.P. in the highlands of Mexico. Here, we report 88 radiocarbon dates on the botanical remains from El Gigante rockshelter (Honduras) to establish a Bayesian chronology over the past ∼11,000 y spanning the transition to maize-based food production. Botanical remains are remarkably well preserved and include over 10,000 maize macrofossils. We directly dated 37 maize cobs to establish the appearance and local change of maize at the site. Cobs are common in deposits dating between 4,340 and 4,020 cal B.P., and again between 2,350 and 980 cal B.P. The earliest cobs appear robustly domesticated, having 10-14 rows, suggesting strong selection for increased yield. The later cobs are comparable to these earliest ones, but show clear emergence of diverse traits, including increased cob width, rachis segment length, and cupule width. Our results indicate that domesticated landraces of maize productive enough to be a staple grain existed in Central America by 4,300 cal B.P.

Independent introductions and admixtures have contributed to adaptation of European maize and its American counterparts.

Through the local selection of landraces, humans have guided the adaptation of crops to a vast range of climatic and ecological conditions. This is particularly true of maize, which was domesticated in a restricted area of Mexico but now displays one of the broadest cultivated ranges worldwide. Here, we sequenced 67 genomes with an average sequencing depth of 18x to document routes of introduction, admixture and selective history of European maize and its American counterparts. To avoid the confounding effects of recent breeding, we targeted germplasm (lines) directly derived from landraces. Among our lines, we discovered 22,294,769 SNPs and between 0.9% to 4.1% residual heterozygosity. Using a segmentation method, we identified 6,978 segments of unexpectedly high rate of heterozygosity. These segments point to genes potentially involved in inbreeding depression, and to a lesser extent to the presence of structural variants. Genetic structuring and inferences of historical splits revealed 5 genetic groups and two independent European introductions, with modest bottleneck signatures. Our results further revealed admixtures between distinct sources that have contributed to the establishment of 3 groups at intermediate latitudes in North America and Europe. We combined differentiation- and diversity-based statistics to identify both genes and gene networks displaying strong signals of selection. These include genes/gene networks involved in flowering time, drought and cold tolerance, plant defense and starch properties. Overall, our results provide novel insights into the evolutionary history of European maize and highlight a major role of admixture in environmental adaptation, paralleling recent findings in humans.

Regulation of Sulfur Homeostasis in Mycorrhizal Maize Plants Grown in a Fe-Limited Environment.

Sulfur is an essential macronutrient for growth of higher plants. The entry of the sulfate anion into the plant, its importation into the plastids for assimilation, its long-distance transport through the vasculature, and its storage in the vacuoles require specific sulfate transporter proteins. In this study, mycorrhizal and non-mycorrhizal maize plants were grown for 60 days in an S-deprived substrate, whilst iron was provided to the plants in the sparingly soluble form of FePO4. On day 60, sulfate was provided to the plants. The gene expression patterns of a number of sulfate transporters as well as sulfate assimilation enzymes were studied in leaves and roots of maize plants, both before as well as after sulfate supply. Prolonged sulfur deprivation resulted in a more or less uniform response of the genes' expressions in the roots of non-mycorrhizal and mycorrhizal plants. This was not the case neither in the roots and leaves after the supply of sulfur, nor in the leaves of the plants during the S-deprived period of time. It is concluded that mycorrhizal symbiosis modified plant demands for reduced sulfur, regulating accordingly the uptake, distribution, and assimilation of the sulfate anion.

An integrated multi-layered analysis of the metabolic networks of different tissues uncovers key genetic components of primary metabolism in maize.

Primary metabolism plays a pivotal role in normal plant growth, development and reproduction. As maize is a major crop worldwide, the primary metabolites produced by maize plants are of immense importance from both calorific and nutritional perspectives. Here a genome-wide association study (GWAS) of 61 primary metabolites using a maize association panel containing 513 inbred lines identified 153 significant loci associated with the level of these metabolites in four independent tissues. The genome-wide expression level of 760 genes was also linked with metabolite levels within the same tissue. On average, the genetic variants at each locus or transcriptional variance of each gene identified here were estimated to have a minor effect (4.4-7.8%) on primary metabolic variation. Thirty-six loci or genes were prioritized as being worthy of future investigation, either with regard to functional characterization or for their utility for genetic improvement. This target list includes the well-known opaque 2 (O2) and lkr/sdh genes as well as many less well-characterized genes. During our investigation of these 36 loci, we analyzed the genetic components and variations underlying the trehalose, aspartate and aromatic amino acid pathways, thereby functionally characterizing four genes involved in primary metabolism in maize.