Holger Fahnenstich

Systems Analysis of a Maize Leaf Developmental Gradient Redefines the Current C4 Model and Provides Candidates for Regulation

This work reports on a comprehensive systems biology analysis of maize leaves, concluding that C4 photosynthesis is established from sink tissue without an intermediate phase of C3 or C2 photosynthesis. We systematically analyzed a developmental gradient of the third maize (Zea mays) leaf from the point of emergence into the light to the tip in 10 continuous leaf slices to study organ development and physiological and biochemical functions. Transcriptome analysis, oxygen sensitivity of photosynthesis, and photosynthetic rate measurements showed that the maize leaf undergoes a sink-to-source transition without an intermediate phase of C3 photosynthesis or operation of a photorespiratory carbon pump. Metabolome and transcriptome analysis, chlorophyll and protein measurements, as well as dry weight determination, showed continuous gradients for all analyzed items. The absence of binary on–off switches and regulons pointed to a morphogradient along the leaf as the determining factor of developmental stage. Analysis of transcription factors for differential expression along the leaf gradient defined a list of putative regulators orchestrating the sink-to-source transition and establishment of C4 photosynthesis. Finally, transcriptome and metabolome analysis, as well as enzyme activity measurements, and absolute quantification of selected metabolites revised the current model of maize C4 photosynthesis. All data sets are included within the publication to serve as a resource for maize leaf systems biology.

Generation of Hydrogen Peroxide in Chloroplasts of Arabidopsis Overexpressing Glycolate Oxidase as an Inducible System to Study Oxidative Stress

Arabidopsis (Arabidopsis thaliana) overexpressing glycolate oxidase (GO) in chloroplasts accumulates both hydrogen peroxide (H2O2) and glyoxylate. GO-overexpressing lines (GO plants) grown at 75 μmol quanta m−2 s−1 show retarded development, yellowish rosettes, and impaired photosynthetic performance, while at 30 μmol quanta m−2 s−1, this phenotype virtually disappears. The GO plants develop oxidative stress lesions under photorespiratory conditions but grow like wild-type plants under nonphotorespiratory conditions. GO plants coexpressing enzymes that further metabolize glyoxylate but still accumulate H2O2 show all features of the GO phenotype, indicating that H2O2 is responsible for the GO phenotype. The GO plants can complete their life cycle, showing that they are able to adapt to the stress conditions imposed by the accumulation of H2O2 during the light period. Moreover, the data demonstrate that a response to oxidative stress is installed, with increased expression and/or activity of known oxidative stress-responsive components. Hence, the GO plants are an ideal noninvasive model system in which to study the effects of H2O2 directly in the chloroplasts, because H2O2 accumulation is inducible and sustained perturbations can reproducibly be provoked by exposing the plants to different ambient conditions.

Alteration of Organic Acid Metabolism in Arabidopsis Overexpressing the Maize C4 NADP-Malic Enzyme Causes Accelerated Senescence during Extended Darkness

The full-length cDNA encoding the maize (Zea mays) C4 NADP-malic enzyme was expressed in Arabidopsis (Arabidopsis thaliana) under the control of the cauliflower mosaic virus 35S promoter. Homozygous transgenic plants (MEm) were isolated with activities ranging from 6- to 33-fold of those found in the wild type. The transformants did not show any differences in morphology and development when grown in long days; however, dark-induced senescence progressed more rapidly in MEm plants compared to the wild type. Interestingly, senescence could be retarded in the transgenic lines by exogenously supplying glucose, sucrose, or malate, suggesting that the lack of a readily mobilized carbon source is likely to be the initial factor leading to the premature induction of senescence in MEm plants. A comprehensive metabolic profiling on whole rosettes allowed determination of approximately 80 metabolites during a diurnal cycle as well as following dark-induced senescence and during metabolic complementation assays. MEm plants showed no differences in the accumulation and degradation of carbohydrates with respect to the wild type in all conditions tested, but accumulated lower levels of intermediates used as respiratory substrates, prominently malate and fumarate. The data indicated that extremely low levels of malate and fumarate are responsible for the accelerated dark-induced senescence encountered in MEm plants. Thus, in prolonged darkness these metabolites are consumed faster than in the wild type and, as a consequence, MEm plants enter irreversible senescence more rapidly. In addition, the data revealed that both malate and fumarate are important forms of fixed carbon that can be rapidly metabolized under stress conditions in Arabidopsis.

Arabidopsis NAD-Malic Enzyme Functions As a Homodimer and Heterodimer and Has a Major Impact on Nocturnal Metabolism

Although the nonphotosynthetic NAD-malic enzyme (NAD-ME) was assumed to play a central role in the metabolite flux through the tricarboxylic acid cycle, the knowledge on this enzyme is still limited. Here, we report on the identification and characterization of two genes encoding mitochondrial NAD-MEs from Arabidopsis (Arabidopsis thaliana), AtNAD-ME1 and AtNAD-ME2. The encoded proteins can be grouped into the two clades found in the plant NAD-ME phylogenetic tree. AtNAD-ME1 belongs to the clade that includes known α-subunits with molecular masses of approximately 65 kD, while AtNAD-ME2 clusters with the known β-subunits with molecular masses of approximately 58 kD. The separated recombinant proteins showed NAD-ME activity, presented comparable kinetic properties, and are dimers in their active conformation. Native electrophoresis coupled to denaturing electrophoresis revealed that in vivo AtNAD-ME forms a dimer of nonidentical subunits in Arabidopsis. Further support for this conclusion was obtained by reconstitution of the active heterodimer in vitro. The characterization of loss-of-function mutants for both AtNAD-MEs indicated that both proteins also exhibit enzymatic activity in vivo. Neither the single nor the double mutants showed a growth or developmental phenotype, suggesting that NAD-ME activity is not essential for normal autotrophic development. Nevertheless, metabolic profiling of plants completely lacking NAD-ME activity revealed differential patterns of modifications in light and dark periods and indicates a major role for NAD-MEs during nocturnal metabolism.

Maize Source Leaf Adaptation to Nitrogen Deficiency Affects Not Only Nitrogen and Carbon Metabolism But Also Control of Phosphate Homeostasis

Crop plant development is strongly dependent on the availability of nitrogen (N) in the soil and the efficiency of N utilization for biomass production and yield. However, knowledge about molecular responses to N deprivation derives mainly from the study of model species. In this article, the metabolic adaptation of source leaves to low N was analyzed in maize (Zea mays) seedlings by parallel measurements of transcriptome and metabolome profiling. Inbred lines A188 and B73 were cultivated under sufficient (15 mm) or limiting (0.15 mm) nitrate supply for up to 30 d. Limited availability of N caused strong shifts in the metabolite profile of leaves. The transcriptome was less affected by the N stress but showed strong genotype- and age-dependent patterns. N starvation initiated the selective down-regulation of processes involved in nitrate reduction and amino acid assimilation; ammonium assimilation-related transcripts, on the other hand, were not influenced. Carbon assimilation-related transcripts were characterized by high transcriptional coordination and general down-regulation under low-N conditions. N deprivation caused a slight accumulation of starch but also directed increased amounts of carbohydrates into the cell wall and secondary metabolites. The decrease in N availability also resulted in accumulation of phosphate and strong down-regulation of genes usually involved in phosphate starvation response, underlining the great importance of phosphate homeostasis control under stress conditions.

Transgenic Introduction of a Glycolate Oxidative Cycle into A. thaliana Chloroplasts Leads to Growth Improvement.

The photorespiratory pathway helps illuminated C3-plants under conditions of limited CO2 availability by effectively exporting reducing equivalents in form of glycolate out of the chloroplast and regenerating glycerate-3-P as substrate for RubisCO. On the other hand, this pathway is considered as probably futile because previously assimilated CO2 is released in mitochondria. Consequently, a lot of effort has been made to reduce this CO2 loss either by reducing fluxes via engineering RubisCO or circumventing mitochondrial CO2 release by the introduction of new enzyme activities. Here we present an approach following the latter route, introducing a complete glycolate catabolic cycle in chloroplasts of Arabidopsis thaliana comprising glycolate oxidase (GO), malate synthase (MS), and catalase (CAT). Results from plants bearing both GO and MS activities have already been reported (Fahnenstich et al., 2008). This previous work showed that the H2O2 produced by GO had strongly negative effects. These effects can be prevented by introducing a plastidial catalase activity, as reported here. Transgenic lines bearing all three transgenic enzyme activities were identified and some with higher CAT activity showed higher dry weight, higher photosynthetic rates, and changes in glycine/serine ratio compared to the wild type. This indicates that the fine-tuning of transgenic enzyme activities in the chloroplasts seems crucial and strongly suggests that the approach is valid and that it is possible to improve the growth of A. thaliana by introducing a synthetic glycolate oxidative cycle into chloroplasts.

Analysis of Arabidopsis with highly reduced levels of malate and fumarate sheds light on the role of these organic acids as storage carbon molecules.

While malate and fumarate participate in a multiplicity of pathways in plant metabolism, the function of these organic acids as carbon stores in C3 plants has not been deeply addressed. Here, Arabidopsis (Arabidopsis thaliana) plants overexpressing a maize (Zea mays) plastidic NADP-malic enzyme (MEm plants) were used to analyze the consequences of sustained low malate and fumarate levels on the physiology of this C3 plant. When grown in short days (sd), MEm plants developed a pale-green phenotype with decreased biomass and increased specific leaf area, with thin leaves having lower photosynthetic performance. These features were absent in plants growing in long days. The analysis of metabolite levels of rosettes from transgenic plants indicated similar disturbances in both sd and long days, with very low levels of malate and fumarate. Determinations of the respiratory quotient by the end of the night indicated a shift from carbohydrates to organic acids as the main substrates for respiration in the wild type, while MEm plants use more reduced compounds, like fatty acids and proteins, to fuel respiration. It is concluded that the alterations observed in sd MEm plants are a consequence of impairment in the supply of carbon skeletons during a long dark period. This carbon starvation phenotype observed at the end of the night demonstrates a physiological role of the C4 acids, which may be a constitutive function in plants.

Adaptation of maize source leaf metabolism to stress related disturbances in carbon, nitrogen and phosphorus balance

BackgroundAbiotic stress causes disturbances in the cellular homeostasis. Re-adjustment of balance in carbon, nitrogen and phosphorus metabolism therefore plays a central role in stress adaptation. However, it is currently unknown which parts of the primary cell metabolism follow common patterns under different stress conditions and which represent specific responses.ResultsTo address these questions, changes in transcriptome, metabolome and ionome were analyzed in maize source leaves from plants suffering low temperature, low nitrogen (N) and low phosphorus (P) stress. The selection of maize as study object provided data directly from an important crop species and the so far underexplored C4 metabolism. Growth retardation was comparable under all tested stress conditions. The only primary metabolic pathway responding similar to all stresses was nitrate assimilation, which was down-regulated. The largest group of commonly regulated transcripts followed the expression pattern: down under low temperature and low N, but up under low P. Several members of this transcript cluster could be connected to P metabolism and correlated negatively to different phosphate concentration in the leaf tissue. Accumulation of starch under low temperature and low N stress, but decrease in starch levels under low P conditions indicated that only low P treated leaves suffered carbon starvation.ConclusionsMaize employs very different strategies to manage N and P metabolism under stress. While nitrate assimilation was regulated depending on demand by growth processes, phosphate concentrations changed depending on availability, thus building up reserves under excess conditions. Carbon and energy metabolism of the C4 maize leaves were particularly sensitive to P starvation.

OPTIMAS-DW: A comprehensive transcriptomics, metabolomics, ionomics, proteomics and phenomics data resource for maize

BackgroundMaize is a major crop plant, grown for human and animal nutrition, as well as a renewable resource for bioenergy. When looking at the problems of limited fossil fuels, the growth of the world’s population or the world’s climate change, it is important to find ways to increase the yield and biomass of maize and to study how it reacts to specific abiotic and biotic stress situations. Within the OPTIMAS systems biology project maize plants were grown under a large set of controlled stress conditions, phenotypically characterised and plant material was harvested to analyse the effect of specific environmental conditions or developmental stages. Transcriptomic, metabolomic, ionomic and proteomic parameters were measured from the same plant material allowing the comparison of results across different omics domains. A data warehouse was developed to store experimental data as well as analysis results of the performed experiments.DescriptionThe OPTIMAS Data Warehouse (OPTIMAS-DW) is a comprehensive data collection for maize and integrates data from different data domains such as transcriptomics, metabolomics, ionomics, proteomics and phenomics. Within the OPTIMAS project, a 44K oligo chip was designed and annotated to describe the functions of the selected unigenes. Several treatment- and plant growth stage experiments were performed and measured data were filled into data templates and imported into the data warehouse by a Java based import tool. A web interface allows users to browse through all stored experiment data in OPTIMAS-DW including all data domains. Furthermore, the user can filter the data to extract information of particular interest. All data can be exported into different file formats for further data analysis and visualisation. The data analysis integrates data from different data domains and enables the user to find answers to different systems biology questions. Finally, maize specific pathway information is provided.ConclusionsWith OPTIMAS-DW a data warehouse for maize was established, which is able to handle different data domains, comprises several analysis results that will support researchers within their work and supports systems biological research in particular. The system is available at http://www.optimas-bioenergy.org/optimas_dw.

An integrated functional approach to dissect systemic responses in maize to arbuscular mycorrhizal symbiosis

Most terrestrial plants benefit from the symbiosis with arbuscular mycorrhizal fungi (AMF) mainly under nutrient-limited conditions. Here the crop plant Zea mays was grown with and without AMF in a bi-compartmented system separating plant and phosphate (Pi) source by a hyphae-permeable membrane. Thus, Pi was preferentially taken up via the mycorrhizal Pi uptake pathway while other nutrients were ubiquitously available. To study systemic effects of mycorrhizal Pi uptake on leaf status, leaves of these plants that display an increased biomass in the presence of AMF were subjected to simultaneous ionomic, transcriptomic and metabolomic analyses. We observed robust changes of the leaf elemental composition, that is, increase of P, S and Zn and decrease of Mn, Co and Li concentration in mycorrhizal plants. Although changes in anthocyanin and lipid metabolism point to an improved P status, a global increase in C versus N metabolism highlights the redistribution of metabolic pools including carbohydrates and amino acids. Strikingly, an induction of systemic defence gene expression and concomitant accumulation of secondary metabolites such as the terpenoids alpha- and beta-amyrin suggest priming of mycorrhizal maize leaves as a mycorrhiza-specific response. This work emphasizes the importance of AM symbiosis for the physiological status of plant leaves and could lead to strategies for optimized breeding of crop species with high growth potential.