Natalia Palacios-Rojas

Genome-Wide Reprogramming of Primary and Secondary Metabolism, Protein Synthesis, Cellular Growth Processes, and the Regulatory Infrastructure of Arabidopsis in Response to Nitrogen

Transcriptome analysis, using Affymetrix ATH1 arrays and a real-time reverse transcription-PCR platform for >1,400 transcription factors, was performed to identify processes affected by long-term nitrogen-deprivation or short-term nitrate nutrition in Arabidopsis. Two days of nitrogen deprivation led to coordinate repression of the majority of the genes assigned to photosynthesis, chlorophyll synthesis, plastid protein synthesis, induction of many genes for secondary metabolism, and reprogramming of mitochondrial electron transport. Nitrate readdition led to rapid, widespread, and coordinated changes. Multiple genes for the uptake and reduction of nitrate, the generation of reducing equivalents, and organic acid skeletons were induced within 30 min, before primary metabolites changed significantly. By 3 h, most genes assigned to amino acid and nucleotide biosynthesis and scavenging were induced, while most genes assigned to amino acid and nucleotide breakdown were repressed. There was coordinate induction of many genes assigned to RNA synthesis and processing and most of the genes assigned to amino acid activation and protein synthesis. Although amino acids involved in central metabolism increased, minor amino acids decreased, providing independent evidence for the activation of protein synthesis. Specific genes encoding expansin and tonoplast intrinsic proteins were induced, indicating activation of cell expansion and growth in response to nitrate nutrition. There were rapid responses in the expression of many genes potentially involved in regulation, including genes for trehalose metabolism and hormone metabolism, protein kinases and phosphatases, receptor kinases, and transcription factors.

Extension of the visualization tool mapman to allow statistical analysis of arrays, display of coresponding genes, and comparison with known responses

MapMan is a user-driven tool that displays large genomics datasets onto diagrams of metabolic pathways or other processes. Here, we present new developments, including improvements of the gene assignments and the user interface, a strategy to visualize multilayered datasets, the incorporation of statistics packages, and extensions of the software to incorporate more biological information including visualization of coresponding genes and horizontal searches for similar global responses across large numbers of arrays.

Metabolic and Phenotypic Responses of Greenhouse-Grown Maize Hybrids to Experimentally Controlled Drought Stress

Adaptation to abiotic stresses like drought is an important acquirement of agriculturally relevant crops like maize. Development of enhanced drought tolerance in crops grown in climatic zones where drought is a very dominant stress factor therefore plays an essential role in plant breeding. Previous studies demonstrated that corn yield potential and enhanced stress tolerance are associated traits. In this study, we analyzed six different maize hybrids for their ability to deal with drought stress in a greenhouse experiment. We were able to combine data from morphophysiological parameters measured under well-watered conditions and under water restriction with metabolic data from different organs. These different organs possessed distinct metabolite compositions, with the leaf blade displaying the most considerable metabolome changes following water deficiency. Whilst we could show a general increase in metabolite levels under drought stress, including changes in amino acids, sugars, sugar alcohols, and intermediates of the TCA cycle, these changes were not differential between maize hybrids that had previously been designated based on field trial data as either drought-tolerant or susceptible. The fact that data described here resulted from a greenhouse experiment with rather different growth conditions compared to natural ones in the field may explain why tolerance groups could not be confirmed in this study. We were, however, able to highlight several metabolites that displayed conserved responses to drought as well as metabolites whose levels correlated well with certain physiological traits.

Deficiency of a Plastidial Adenylate Kinase in Arabidopsis Results in Elevated Photosynthetic Amino Acid Biosynthesis and Enhanced Growth

An Arabidopsis (Arabidopsis thaliana) L. Heynh mutant deficient in an isoform of adenylate kinase (ADK; At2g37250) was isolated by reverse genetics. It contains a T-DNA insertion 377 bp downstream of the start point of transcription. The mutant lacks At2g37250 transcripts and has a mild reduction in total cellular ADK activity. Green fluorescent protein-fusion based cellular localization experiments, carried out with the full-length At2g37250, suggested a plastidial localization for this isoform. In keeping with this observation, organelle isolation experiments revealed that the loss in ADK activity was confined to the inner plastid. This plastid stroma ADK gene was found to be expressed tissue constitutively but at much higher levels in illuminated leaves. Phenotypic and biochemical analyses of the mutant revealed that it exhibited higher amino acid biosynthetic activity in the light and was characterized by an enhanced root growth. When the mutant was subjected to either continuous light or continuous dark, growth phenotypes were also observed in the shoots. While the levels of adenylates were not much altered in the leaves, the pattern of change observed in the roots was consistent with the inhibition of an ATP-consuming reaction. Taken together, these data suggest a role for the plastid stromal ADK in the coordination of metabolism and growth, but imply that the exact importance of this isoform is tissue dependent.

Metabolite Profiles of Maize Leaves in Drought, Heat, and Combined Stress Field Trials Reveal the Relationship between Metabolism and Grain Yield

Foliar metabolite levels, including myoinositol, show correlation with grain yield in tropical maize field trials during drought, heat, and simultaneous drought/heat stresses. The development of abiotic stress-resistant cultivars is of premium importance for the agriculture of developing countries. Further progress in maize (Zea mays) performance under stresses is expected by combining marker-assisted breeding with metabolite markers. In order to dissect metabolic responses and to identify promising metabolite marker candidates, metabolite profiles of maize leaves were analyzed and compared with grain yield in field trials. Plants were grown under well-watered conditions (control) or exposed to drought, heat, and both stresses simultaneously. Trials were conducted in 2010 and 2011 using 10 tropical hybrids selected to exhibit diverse abiotic stress tolerance. Drought stress evoked the accumulation of many amino acids, including isoleucine, valine, threonine, and 4-aminobutanoate, which has been commonly reported in both field and greenhouse experiments in many plant species. Two photorespiratory amino acids, glycine and serine, and myoinositol also accumulated under drought. The combination of drought and heat evoked relatively few specific responses, and most of the metabolic changes were predictable from the sum of the responses to individual stresses. Statistical analysis revealed significant correlation between levels of glycine and myoinositol and grain yield under drought. Levels of myoinositol in control conditions were also related to grain yield under drought. Furthermore, multiple linear regression models very well explained the variation of grain yield via the combination of several metabolites. These results indicate the importance of photorespiration and raffinose family oligosaccharide metabolism in grain yield under drought and suggest single or multiple metabolites as potential metabolic markers for the breeding of abiotic stress-tolerant maize.

Reliable and inexpensive colorimetric method for determining protein-bound tryptophan in maize kernels.

Biofortification programs in maize have led to the development of quality protein maize (QPM) with increased contents of the essential amino acids lysine and tryptophan, and increased nutritional value for protein deficient populations where maize is a staple food. Because multiple genetic systems control and modify the protein quality of QPM, tryptophan or lysine monitoring is required to maximize genetic gain in breeding programs. The objective of this work was to develop an accurate, reliable, and inexpensive method for tryptophan analysis in whole-grain maize flour to support QPM research efforts around the world. Tryptophan reacts with glyoxylic acid in the presence of sulfuric acid and ferric chloride, producing a colored compound that absorbs at 560 nm. A series of experiments varying the reagent concentrations, hydrolysis time, and length of the colorimetric reaction resulted in an optimized protocol which uses 0.1 M glyoxylic acid in 7 N sulfuric acid and 1.8 mM ferric chloride, and 30 min reaction time. This method produced stable and reproducible results for tryptophan concentration in whole-grain maize flour and was validated by comparison with data obtained using an acetic acid-based colorimetric procedure (r(2) = 0.80) and high pressure liquid chromatography (HPLC) (r(2) = 0.71). We describe adaptations that permit high throughput application of this tryptophan analysis method using a microplate platform.

Advances in maize genomics and their value for enhancing genetic gains from breeding

Maize is an important crop for food, feed, forage, and fuel across tropical and temperate areas of the world. Diversity studies at genetic, molecular, and functional levels have revealed that, tropical maize germplasm, landraces, and wild relatives harbor a significantly wider range of genetic variation. Among all types of markers, SNP markers are increasingly the marker-of-choice for all genomics applications in maize breeding. Genetic mapping has been developed through conventional linkage mapping and more recently through linkage disequilibrium-based association analyses. Maize genome sequencing, initially focused on gene-rich regions, now aims for the availability of complete genome sequence. Conventional insertion mutation-based cloning has been complemented recently by EST- and map-based cloning. Transgenics and nutritional genomics are rapidly advancing fields targeting important agronomic traits including pest resistance and grain quality. Substantial advances have been made in methodologies for genomics-assisted breeding, enhancing progress in yield as well as abiotic and biotic stress resistances. Various genomic databases and informatics tools have been developed, among which MaizeGDB is the most developed and widely used by the maize research community. In the future, more emphasis should be given to the development of tools and strategic germplasm resources for more effective molecular breeding of tropical maize products.

Near-Infrared Reflectance Spectroscopy (NIRS) for Protein, Tryptophan, and Lysine Evaluation in Quality Protein Maize (QPM) Breeding Programs

Quality protein maize (QPM) has approximately twice the tryptophan (Trp) and lysine (Lys) concentrations in protein compared to normal maize. Because several genetic systems control the protein quality of QPM, it is essential to regularly monitor Trp and/or Lys in breeding programs. Our objective was to examine the potential of near-infrared reflectance spectroscopy (NIRS) to enhance the efficiency of QPM research efforts by partially replacing more expensive and time-consuming wet chemistry analysis. More than 276 maize samples were used to develop NIRS models for protein content (PC), Trp, and Lys. The standard error of prediction (SEP) for the calibration and the coefficient of determination for validation (R(2)(v)) were 0.26 and 0.96 for PC, 0.005 and 0.85 for Trp, and 0.02 and 0.75 for Lys. When the NIRS models were used to evaluate 266 S2 lines from five QPM breeding populations, the coefficients of determination between NIRS and the chemical data were 0.94, 0.76, and 0.80 for PC, Trp, and Lys, respectively. Therefore, the NIRS models can be used to support the QPM breeding efforts.

Near-Infrared Reflectance Spectroscopy (NIRS) Assessment of δ18O and Nitrogen and Ash Contents for Improved Yield Potential and Drought Adaptation in Maize

The oxygen isotope composition (δ(18)O), accumulation of minerals (ash content), and nitrogen (N) content in plant tissues have been recently proposed as useful integrative physiological criteria associated with yield potential and drought resistance in maize. This study tested the ability of near-infrared reflectance spectroscopy (NIRS) to predict δ(18)O and ash and N contents in leaves and mature kernels of maize. The δ(18)O and ash and N contents were determined in leaf and kernel samples from a set of 15 inbreds and 18 hybrids grown in Mexico under full irrigation and two levels of drought stress. Calibration models between NIRS spectra and the measured variables were developed using modified partial least-squares regressions. Global models (which included inbred lines and hybrids) accurately predicted ash and N contents, whereas prediction of δ(18)O showed lower results. Moreover, in hybrids, NIRS clearly reflected genotypic differences in leaf and kernel ash and N contents within each water treatment. It was concluded that NIRS can be used as a rapid, cost-effective, and accurate method for predicting ash and N contents and as a method for screening δ(18)O in maize with promising applications in crop management and maize breeding programs for improved water and nitrogen use efficiency and grain quality.

Maize genotype and food matrix affect the provitamin a carotenoid bioefficacy from staple and carrot-fortified feeds in Mongolian gerbils (Meriones unguiculatus)

Biofortification to increase provitamin A carotenoids is an agronomic approach to alleviate vitamin A deficiency. Two studies compared biofortified foods using in vitro and in vivo methods. Study 1 screened maize genotypes (n = 44) using in vitro analysis, which demonstrated decreasing micellarization with increasing provitamin A. Thereafter, seven 50% biofortified maize feeds that hypothesized a one-to-one equivalency between β-cryptoxanthin and β-carotene were fed to Mongolian gerbils. Total liver retinol differed among the maize groups (P = 0.0043). Study 2 assessed provitamin A bioefficacy from 0.5% high-carotene carrots added to 60% staple-food feeds, followed by in vitro screening. Liver retinol was highest in the potato and banana groups, maize group retinol did not differ from baseline, and all treatments differed from control (P < 0.0001). In conclusion, β-cryptoxanthin and β-carotene have similar bioefficacy; meal matrix effects influence provitamin A absorption from carrot; and in vitro micellarization does not predict bioefficacy.