A vegetative storage protein improves drought tolerance in maize.

Vegetative storage proteins (VSPs) are known to serve as nitrogen reserves in many dicot plants but remain undiscovered in grasses, most widely grown group of crops globally. We identified and characterized a VSP in maize and demonstrated that its overexpression improved drought tolerance. Nitrogen supplementation selectively induced a mesophyll lipoxygenase (ZmLOX6), which was targeted to chloroplasts by a novel N-terminal transit peptide of 62 amino acids. When ectopically expressed under the control of various tissue-specific promoters, it accumulated to a fivefold higher level upon expression in the mesophyll cells than the wild-type plants. Constitutive expression or targeted expression specifically to the bundle sheath cells increased its accumulation by less than twofold. The overexpressed ZmLOX6 was remobilized from the leaves like other major proteins during grain development. Evaluated in the field over locations and years, transgenic hybrids overexpressing ZmLOX6 in the mesophyll cells significantly outyielded nontransgenic sibs under managed drought stress imposed at flowering. Additional storage of nitrogen as a VSP in maize leaves ameliorated the effect of drought on grain yield.

Chitinase-like1 Plays a Role in Stalk Tensile Strength in Maize.

Stalk lodging in maize (Zea mays) causes significant yield losses due to breaking of stalk tissue below the ear node before harvest. Here, we identified the maize brittle stalk4 (bk4) mutant in a Mutator F2 population. This mutant was characterized by highly brittle aerial parts that broke easily from mechanical disturbance or in high-wind conditions. The bk4 plants displayed a reduction in average stalk diameter and mechanical strength, dwarf stature, senescence at leaf tips, and semisterility of pollen. Histological studies demonstrated a reduction in lignin staining of cells in the bk4 mutant leaves and stalk, and deformation of vascular bundles in the stalk resulting in the loss of xylem and phloem tissues. Biochemical characterization showed a significant reduction in p-coumaric acid, Glc, Man, and cellulose contents. The candidate gene responsible for bk4 phenotype is Chitinase-like1 protein (Ctl1), which is expressed at its highest levels in elongated internodes. Expression levels of secondary cell wall cellulose synthase genes (CesA) in the bk4 single mutant, and phenotypic observations in double mutants combining bk4 with bk2 or null alleles for two CesA genes, confirmed interaction of ZmCtl1 with CesA genes. Overexpression of ZmCtl1 enhanced mechanical stalk strength without affecting plant stature, senescence, or fertility. Biochemical characterization of ZmCtl1 overexpressing lines supported a role for ZmCtl1 in tensile strength enhancement. Conserved identity of CTL1 peptides across plant species and analysis of Arabidopsis (Arabidopsis thaliana) ctl1-1 ctl2-1 double mutants indicated that Ctl1 might have a conserved role in plants.

Identification and characterization of the maize arogenate dehydrogenase gene family.

In plants, the amino acids tyrosine and phenylalanine are synthesized from arogenate by arogenate dehydrogenase and arogenate dehydratase, respectively, with the relative flux to each being tightly controlled. Here the characterization of a maize opaque endosperm mutant (mto140), which also shows retarded vegetative growth, is described The opaque phenotype co-segregates with a Mutator transposon insertion in an arogenate dehydrogenase gene (zmAroDH-1) and this led to the characterization of the four-member family of maize arogenate dehydrogenase genes (zmAroDH-1-zmAroDH-4) which share highly similar sequences. A Mutator insertion at an equivalent position in AroDH-3, the most closely related family member to AroDH-1, is also associated with opaque endosperm and stunted vegetative growth phenotypes. Overlapping but differential expression patterns as well as subtle mutant effects on the accumulation of tyrosine and phenylalanine in endosperm, embryo, and leaf tissues suggest that the functional redundancy of this gene family provides metabolic plasticity for the synthesis of these important amino acids. mto140/arodh-1 seeds shows a general reduction in zein storage protein accumulation and an elevated lysine phenotype typical of other opaque endosperm mutants, but it is distinct because it does not result from quantitative or qualitative defects in the accumulation of specific zeins but rather from a disruption in amino acid biosynthesis.

Superior field performance of waxy corn engineered using CRISPR-Cas9.

We created waxy corn hybrids by CRISPR-Cas9 editing of a waxy allele in 12 elite inbred maize lines, a process that was more than a year faster than conventional trait introgression using backcrossing and marker-assisted selection. Field trials at 25 locations showed that CRISPR-waxy hybrids were agronomically superior to introgressed hybrids, producing on average 5.5 bushels per acre higher yield.

Isotopic ratio outlier analysis improves metabolomics prediction of nitrogen treatment in maize.

We evaluated Isotope Ratio Outlier Analysis (IROA) as a metabolome-wide internal standard approach to improve the quality of LC/MS data collected from a large-scale greenhouse experiment designed to metric the ability of metabolomics to model quantitatively nitrogen treatments. We further looked at how IROA would be incorporated into a metabolomics workflow. For this we compared IROA processed data with that generated without the benefit of metabolome-wide internal standards using our current tool, Genedata Expressionist, from the same raw LC/MS data files. In our experiment, 367 maize plants were grown from kernel in a greenhouse under controlled conditions. Plants were treated from germination on with varying concentrations of nutrient nitrogen as one (treatment) variable. A second variable was the presence of one of two transgenes. Metabolomics analysis of leaves was performed by LC/MS positive and negative electrospray ionization modes, and raw data were processed with both our routine and IROA protocols. IROA data analysis detected 184 metabolites in each ionization mode. Analysis without IROA yielded 281 metabolites in positive ionization mode and 172 in negative ionization mode. Data from both protocols were normalized for sample dry weight, location in the greenhouse, extraction batch, sample run order, and internal standard. Normalized results were subjected to partial least squares (PLS) analysis to model the relationship between the metabolome and nitrogen treatment. Without IROA, regression coefficients of 0.819 and 0.849 for positive and negative modes, respectively were achieved. The IROA protocol improved on the values, yielding regression coefficients of 0.876 and 0.879 for positive and negative modes, respectively. In addition, IROA corrected for detector saturation for several high abundant peaks. Our experiment demonstrates that incorporating IROA into an LC/MS metabolomics experiment improves data quality and facilitates more precise modeling of a biological response.

Effect of Genetics, Environment, and Phenotype on the Metabolome of Maize Hybrids Using GC/MS and LC/MS.

We evaluated the variability of metabolites in various maize hybrids due to the effect of environment, genotype, phenotype as well as the interaction of the first two factors. We analyzed 480 forage and the same number of grain samples from 21 genetically diverse non-GM Pioneer brand maize hybrids, including some with drought tolerance and viral resistance phenotypes, grown at eight North American locations. As complementary platforms, both GC/MS and LC/MS were utilized to detect a wide diversity of metabolites. GC/MS revealed 166 and 137 metabolites in forage and grain samples, respectively, while LC/MS captured 1341 and 635 metabolites in forage and grain samples, respectively. Univariate and multivariate analyses were utilized to investigate the response of the maize metabolome to the environment, genotype, phenotype, and their interaction. Based on combined percentages from GC/MS and LC/MS datasets, the environment affected 36% to 84% of forage metabolites, while less than 7% were affected by genotype. The environment affected 12% to 90% of grain metabolites, whereas less than 27% were affected by genotype. Less than 10% and 11% of the metabolites were affected by phenotype in forage and grain, respectively. Unsupervised PCA and HCA analyses revealed similar trends, i.e., environmental effect was much stronger than genotype or phenotype effects. On the basis of comparisons of disease tolerant and disease susceptible hybrids, neither forage nor grain samples originating from different locations showed obvious phenotype effects. Our findings demonstrate that the combination of GC/MS and LC/MS based metabolite profiling followed by broad statistical analysis is an effective approach to identify the relative impact of environmental, genetic and phenotypic effects on the forage and grain composition of maize hybrids.

Effect of environment and genotype on commercial maize hybrids using LC/MS-based metabolomics.

We recently applied gas chromatography coupled to time-of-flight mass spectrometry (GC/TOF-MS) and multivariate statistical analysis to measure biological variation of many metabolites due to environment and genotype in forage and grain samples collected from 50 genetically diverse nongenetically modified (non-GM) DuPont Pioneer commercial maize hybrids grown at six North American locations. In the present study, the metabolome coverage was extended using a core subset of these grain and forage samples employing ultra high pressure liquid chromatography (uHPLC) mass spectrometry (LC/MS). A total of 286 and 857 metabolites were detected in grain and forage samples, respectively, using LC/MS. Multivariate statistical analysis was utilized to compare and correlate the metabolite profiles. Environment had a greater effect on the metabolome than genetic background. The results of this study support and extend previously published insights into the environmental and genetic associated perturbations to the metabolome that are not associated with transgenic modification.

Analytical method evaluation and discovery of variation within maize varieties in the context of food safety: transcript profiling and metabolomics.

Profiling techniques such as microarrays, proteomics, and metabolomics are used widely to assess the overall effects of genetic background, environmental stimuli, growth stage, or transgene expression in plants. To assess the potential regulatory use of these techniques in agricultural biotechnology, we carried out microarray and metabolomic studies of 3 different tissues from 11 conventional maize varieties. We measured technical variations for both microarrays and metabolomics, compared results from individual plants and corresponding pooled samples, and documented variations detected among different varieties with individual plants or pooled samples. Both microarray and metabolomic technologies are reproducible and can be used to detect plant-to-plant and variety-to-variety differences. A pooling strategy lowered sample variations for both microarray and metabolomics while capturing variety-to-variety variation. However, unknown genomic sequences differing between maize varieties might hinder the application of microarrays. High-throughput metabolomics could be useful as a tool for the characterization of transgenic crops. However, researchers will have to take into consideration the impact on the detection and quantitation of a wide range of metabolites on experimental design as well as validation and interpretation of results.

Effects of genetics and environment on the metabolome of commercial maize hybrids: a multisite study.

This study was designed to elucidate the biological variation in expression of many metabolites due to environment, genotype, or both, and to investigate the potential utility of metabolomics to supplement compositional analysis for substantial equivalence assessments of genetically modified (GM) crops. A total of 654 grain and 695 forage samples from 50 genetically diverse non-GM DuPont Pioneer maize hybrids grown at six locations in the U.S. and Canada were analyzed by coupled gas chromatography time-of-flight-mass spectrometry (GC/TOF-MS). A total of 156 and 185 metabolites were measured in grain and forage samples, respectively. Univariate and multivariate statistical analyses were employed extensively to compare and correlate the metabolite profiles. We show that the environment had far more impact on the forage metabolome compared to the grain metabolome, and the environment affected up to 50% of the metabolites compared to less than 2% by the genetic background. The findings from this study demonstrate that the combination of GC/TOF-MS metabolomics and comprehensive multivariate statistical analysis is a powerful approach to identify the sources of natural variation contributed by the environment and genotype.

The maize low-phytic acid 3 encodes a myo-inositol kinase that plays a role in phytic acid biosynthesis in developing seeds.

Phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate or Ins P6, is the most abundant myo-inositol phosphate in plant cells, but its biosynthesis is poorly understood. Also uncertain is the role of myo-inositol as a precursor of phytic acid biosynthesis. We identified a low-phytic acid mutant, lpa3, in maize. The Mu-insertion mutant has a phenotype of reduced phytic acid, increased myo-inositol and lacks significant amounts of myo-inositol phosphate intermediates in seeds. The gene responsible for the mutation encodes a myo-inositol kinase (MIK). Maize MIK protein contains conserved amino acid residues found in pfkB carbohydrate kinases. The maize lpa3 gene is expressed in developing embryos, where phytic acid is actively synthesized and accumulates to a large amount. Characterization of the lpa3 mutant provides direct evidence for the role of myo-inositol and MIK in phytic acid biosynthesis in developing seeds. Recombinant maize MIK phosphorylates myo-inositol to produce multiple myo-inositol monophosphates, Ins1/3P, Ins4/6P and possibly Ins5P. The characteristics of the lpa3 mutant and MIK suggest that MIK is not a salvage enzyme for myo-inositol recycling and that there are multiple phosphorylation routes to phytic acid in developing seeds. Analysis of the lpa2/lpa3 double mutant implies interactions between the phosphorylation routes.

The maize low-phytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene.

Reduced phytic acid content in seeds is a desired goal for genetic improvement in several crops. Low-phytic acid mutants have been used in genetic breeding, but it is not known what genes are responsible for the low-phytic acid phenotype. Using a reverse genetics approach, we found that the maize (Zea mays) low-phytic acid lpa2 mutant is caused by mutation in an inositol phosphate kinase gene. The maize inositol phosphate kinase (ZmIpk) gene was identified through sequence comparison with human and Arabidopsis Ins(1,3,4)P(3) 5/6-kinase genes. The purified recombinant ZmIpk protein has kinase activity on several inositol polyphosphates, including Ins(1,3,4)P(3), Ins(3,5,6)P(3), Ins(3,4,5,6)P(4), and Ins(1,2,5,6)P(4). The ZmIpk mRNA is expressed in the embryo, the organ where phytic acid accumulates in maize seeds. The ZmIpk Mutator insertion mutants were identified from a Mutator F(2) family. In the ZmIpk Mu insertion mutants, seed phytic acid content is reduced approximately 30%, and inorganic phosphate is increased about 3-fold. The mutants also accumulate myo-inositol and inositol phosphates as in the lpa2 mutant. Allelic tests showed that the ZmIpk Mu insertion mutants are allelic to the lpa2. Southern-blot analysis, cloning, and sequencing of the ZmIpk gene from lpa2 revealed that the lpa2-1 allele is caused by the genomic sequence rearrangement in the ZmIpk locus and the lpa2-2 allele has a nucleotide mutation that generated a stop codon in the N-terminal region of the ZmIpk open reading frame. These results provide evidence that ZmIpk is one of the kinases responsible for phytic acid biosynthesis in developing maize seeds.

Guar seed beta-mannan synthase is a member of the cellulose synthase super gene family.

Genes for the enzymes that make plant cell wall hemicellulosic polysaccharides remain to be identified. We report here the isolation of a complementary DNA (cDNA) clone encoding one such enzyme, mannan synthase (ManS), that makes the beta-1, 4-mannan backbone of galactomannan, a hemicellulosic storage polysaccharide in guar seed endosperm walls. The soybean somatic embryos expressing ManS cDNA contained high levels of ManS activities that localized to Golgi. Phylogenetically, ManS is closest to group A of the cellulose synthase-like (Csl) sequences from Arabidopsis and rice. Our results provide the biochemical proof for the involvement of the Csl genes in beta-glycan formation in plants.