Neil P. Schultes

High Glycolate Oxidase Activity Is Required for Survival of Maize in Normal Air

A mutant in the maize (Zea mays) Glycolate Oxidase1 (GO1) gene was characterized to investigate the role of photorespiration in C4 photosynthesis. An Activator-induced allele of GO1 conditioned a seedling lethal phenotype when homozygous and had 5% to 10% of wild-type GO activity. Growth of seedlings in high CO2 (1%–5%) was sufficient to rescue the mutant phenotype. Upon transfer to normal air, the go1 mutant became necrotic within 7 d and plants died within 15 d. Providing [1-14C]glycolate to leaf tissue of go1 mutants in darkness confirmed that the substrate is inefficiently converted to 14CO2, but both wild-type and GO-deficient mutant seedlings metabolized [1-14C]glycine similarly to produce [14C]serine and 14CO2 in a 1:1 ratio, suggesting that the photorespiratory pathway is otherwise normal in the mutant. The net CO2 assimilation rate in wild-type leaves was only slightly inhibited in 50% O2 in high light but decreased rapidly and linearly with time in leaves with low GO. When go1 mutants were shifted from high CO2 to air in light, they accumulated glycolate linearly for 6 h to levels 7-fold higher than wild type and 11-fold higher after 25 h. These studies show that C4 photosynthesis in maize is dependent on photorespiration throughout seedling development and support the view that the carbon oxidation pathway evolved to prevent accumulation of toxic glycolate.

An aerobiological framework for assessing cross-pollination in maize

Abstract Maize (Zea mays L.) is one of the world’s most important crops. Until recent times, improvement in maize resulted from the manipulation and exchange of genetic information within the genus Zea. With the advent of genetic engineering, genetic information from other species is routinely incorporated into the maize genome. Since maize is a wind-pollinated outcrossing species, questions arise concerning the flow of genetic information between genetically modified (GM) and non-GM maize. Because of the rapidly accelerating introduction of GM corn into agricultural production, improved aerobiological models to predict how far and to what extent maize pollen can be transported in the atmosphere are needed today. Models should provide quantitative understanding of both short- and long-range pollen dispersal, thereby offering a means to evaluate deposition of viable maize pollen in seed production fields as well as on neighboring farms. Central to pollen dispersal is the rich interplay between physics and biology and between space and time. We present an aerobiological framework for assessing corn pollen movement in the atmosphere that could also be applied to other crops and to uncultivated species. An important aim of this paper is to spark interest in the scientific community to generate a more complete framework of corn pollen dispersal. As such this manuscript presents a multidisciplinary albeit incomplete perspective to an emerging applied problem.

Functional Characterization of a Maize Purine Transporter by Expression in Aspergillus nidulans

We have characterized the function of Leaf Permease1 (LPE1), a protein that is necessary for proper chloroplast development in maize, by functional expression in the filamentous fungus Aspergillus nidulans. The choice of this ascomycete was dictated by the similarity of its endogenous purine transporters to LPE1 and by particular genetic and physiological features of purine transport and metabolism in A. nidulans. When Lpe1 was expressed in a purine transport–deficient A. nidulans strain, the capacity for uric acid and xanthine transport was acquired. This capacity was directly dependent on Lpe1 copy number and expression level. Interestingly, overexpression of LPE1 from >10 gene copies resulted in transformants with pleiotropically reduced growth rates on various nitrogen sources and the absolute inability to transport purines. Kinetic analysis established that LPE1 is a high-affinity (Km = 30 ± 2.5 μM), high-capacity transporter specific for the oxidized purines xanthine and uric acid. Competition studies showed that high concentrations of ascorbic acid (>30 mM) competitively inhibit LPE1-mediated purine transport. This work defines the biochemical function of LPE1, a plant representative of a large and ubiquitous transporter family. In addition, A. nidulans is introduced as a novel model system for the cloning and/or functional characterization of transporter genes.

Leaf permease1 gene of maize is required for chloroplast development.

Adjacent bundle sheath and mesophyll cells cooperate for carbon fixation in the leaves of C4 plants. Mutants with compromised plastid development should reveal the degree to which this cooperation is obligatory, because one can assay whether mesophyll cells with defective bundle sheath neighbors retain C4 characteristics or revert to C3 photosynthesis. The leaf permease1-mutable1 (lpe1-m1) mutant of maize exhibits disrupted chloroplast ultrastructure, preferentially affecting bundle sheath choroplasts under lower light. Despite the disrupted ultrastructure, the metabolic cooperation of bundle sheath and mesophyll cells for C4 photosynthesis remains intact. To investigate this novel mutation, the Activator transposon-tagged allele and cDNAs corresponding to the Lpe1 mRNA from wild-type plants were cloned. The Lpe1 gene encodes a polypeptide with significant similarity to microbial pyrimidine and purine transport proteins. An analysis of revertant sectors generated by Activator excision suggests that the Lpe1 gene product is cell autonomous and can be absent up to the last cell divisions in the leaf primordium without blocking bundle sheath chloroplast development.

AtAzg1 and AtAzg2 Comprise a Novel Family of Purine Transporters in Arabidopsis

In plants, nucleobase biochemistry is highly compartmented relying upon a well‐regulated and selective membrane transport system. In Arabidopsis two proteins, AtAzg1 and AtAzg2, show substantial amino acid sequence similarity to the adenine–guanine–hypoxanthine transporter AzgA of Aspergillus nidulans. Analysis of single and double mutant lines harboring T‐DNA insertion alleles AtAzg1‐1 and AtAzg2‐1 reveal a marked resistance to growth in the presence of 8‐azaadenine and 8‐azaguanine but not to other toxic nucleobase analogues. Conversely, yeast strains expressing AtAzg1 and AtAzg2 gain heightened sensitivity to growth on 8‐azaadenine and 8‐azaguanine. Radio‐labeled purine uptake experiments in yeast and in planta confirm the function of AtAzg1 and AtAzg2 as plant adenine–guanine transporters.

Genetic and molecular characterization reveals a unique nucleobase cation symporter 1 in Arabidopsis

Locus At5g03555 encodes a nucleobase cation symporter 1 (AtNCS1) in the Arabidopsis genome. Arabidopsis insertion mutants, AtNcs1‐1 and AtNcs1‐3, were used for in planta toxic nucleobase analog growth studies and radio‐labeled nucleobase uptake assays to characterize solute transport specificities. These results correlate with similar growth and uptake studies of AtNCS1 expressed in Saccharomyces cerevisiae. Both in planta and heterologous expression studies in yeast revealed a unique solute transport profile for AtNCS1 in moving adenine, guanine and uracil. This is in stark contrast to the canonical transport profiles determined for the well‐characterized S. cerevisiae NCS1 proteins FUR4 (uracil transport) or FCY2 (adenine, guanine, and cytosine transport).

The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility

The solute specificity profiles (transport and binding) for the nucleobase cation symporter 1 (NCS1) proteins, from the closely related C4 grasses Zea mays and Setaria viridis, differ from that of Arabidopsis thaliana and Chlamydomonas reinhardtii NCS1. Solute specificity profiles for NCS1 from Z. mays (ZmNCS1) and S. viridis (SvNCS1) were determined through heterologous complementation studies in NCS1-deficient Saccharomyces cerevisiae strains. The four Viridiplantae NCS1 proteins transport the purines adenine and guanine, but unlike the dicot and algal NCS1, grass NCS1 proteins fail to transport the pyrimidine uracil. Despite the high level of amino acid sequence similarity, ZmNCS1 and SvNCS1 display distinct solute transport and recognition profiles. SvNCS1 transports adenine, guanine, hypoxanthine, cytosine, and allantoin and competitively binds xanthine and uric acid. ZmNCS1 transports adenine, guanine, and cytosine and competitively binds, 5-fluorocytosine, hypoxanthine, xanthine, and uric acid. The differences in grass NCS1 profiles are due to a limited number of amino acid alterations. These amino acid residues do not correspond to amino acids essential for overall solute and cation binding or solute transport, as previously identified in bacterial and fungal NCS1, but rather may represent residues involved in subtle solute discrimination. The data presented here reveal that within Viridiplantae, NCS1 proteins transport a broad range of nucleobase compounds and that the solute specificity profile varies with species.

Heterologous complementation studies reveal the solute transport profiles of a two-member nucleobase cation symporter 1 (NCS1) family in Physcomitrella patens.

As part of an evolution-function analysis, two nucleobase cation symporter 1 (NCS1) from the moss Physcomitrella patens (PpNCS1A and PpNCS1B) are examined--the first such analysis of nucleobase transporters from early land plants. The solute specificity profiles for the moss NCS1 were determined through heterologous expression, growth and radiolabeled uptake experiments in NCS1-deficient Saccharomyces cerevisiae. Both PpNCS1A and 1B, share the same profiles as high affinity transporters of adenine and transport uracil, guanine, 8-azaguanine, 8-azaadenine, cytosine, 5-fluorocytosine, hypoxanthine, and xanthine. Despite sharing the same solute specificity profile, PpNCS1A and PpNCS1B move nucleobase compounds with different efficiencies. The broad nucleobase transport profile of PpNCS1A and 1B differs from the recently-characterized Viridiplantae NCS1 in breadth, revealing a flexibility in solute interactions with NCS1 across plant evolution.

Arabidopsis thaliana locus At5g62890, a nucleobase-ascorbate transporter family member, is preferentially expressed in carpel transmitting tract and tapetal cells

The expression pattern for Arabidopsis locus At5g62890 (MQB2.190) is investigated at the molecular and whole plant level. MQB2.190 belongs to the nucleobase-ascorbate transporter (NAT) family which includes transporters of purines, pyrimidines and ascorbate. A full-length cDNA clone was sequenced verifying that MQB2.190 is actively expressed and encodes a NAT protein. Northern blot and RT-PCR analysis reveal that MQB2.190 is expressed at a low level in a variety of tissues, but is preferentially expressed in flowers. Whole plant gene expression was investigated by generating transgneic Arabidopsis with the MQB2.190 promoter driving GUS gene. Visible GUS staining was detected in the central tissue of carpels and siliques and faintly in anthers, but was absent in young seedlings, roots, leaves, stems or other floral tissues. MQB2.190 transcripts were precisely localized to the central transmitting tract of the carpel and in the tapetal cell layer in anthers using RNA in situ hybridization.

Light-harvesting complex B7 shifts the irradiance response of photosynthetic light-harvesting regulation in leaves of Arabidopsis thaliana.

The nuclear LHCB7 gene is common in higher plants, encodes a transcript that is well expressed in a subset of leaf mesophyll cells, and is associated with a protein product that is homologous to pigment-binding components of the photosystem (PS) II peripheral antenna complex. We compared the physiological properties of wild type and LHCB7-deficient leaves [DNA insertion, Arabidopsis thaliana (At) ecotype Columbia] in terms of pigment content, CO2 exchange, in vivo transmittance at 810 nm, and chlorophyll fluorescence. The latter two techniques are functional indicators for PSI and PSII, respectively. Key features of the mutant phenotype were confirmed using antisense technology and a hemizygote of two independent AtLHCB7 DNA insertion lines. Growth, leaf pigment composition, white light absorptance, and levels of AtLHCB1-6 were not significantly different in the mutant compared to wild type. Likewise, neither intrinsic PSII light capture efficiency nor partitioning of absorbed radiation to PSII was affected by the mutation. The absence of AtLHCB7 is associated with lower rates of light-saturated photosynthesis and a diminished irradiance threshold for induction of photoprotective non-photochemical quenching. Overall, the pattern of change in light utilization parameters and plastoquinol level indicated that loss of AtLHCB7 expression led to slower Rubisco turnover characterized by pH-dependent balancing of electron transport to reduced carbon assimilation capacity (photosynthetic control). No effect of AtLHCB7 genotype on xanthophyll de-epoxidation state was detected suggesting that factors in addition to lumenal pH influence zeaxanthin accumulation.