Sari A. Ruuska

Contrapuntal Networks of Gene Expression during Arabidopsis Seed Filling

We have used cDNA microarrays to examine changes in gene expression during Arabidopsis seed development and to compare wild-type and mutant wrinkled1 (wri1) seeds that have an 80% reduction in oil. Between 5 and 13 days after flowering, a period preceding and including the major accumulation of storage oils and proteins, ∼35% of the genes represented on the array changed at least twofold, but a larger fraction (65%) showed little or no change in expression. Genes whose expression changed most tended to be expressed more in seeds than in other tissues. Genes related to the biosynthesis of storage components showed several distinct temporal expression patterns. For example, a number of genes encoding core fatty acid synthesis enzymes displayed a bell-shaped pattern of expression between 5 and 13 days after flowering. By contrast, the expression of storage proteins, oleosins, and other known abscisic acid–regulated genes increased later and remained high. Genes for photosynthetic proteins followed a pattern very similar to that of fatty acid synthesis proteins, implicating a role in CO2 refixation and the supply of cofactors for oil synthesis. Expression profiles of key carbon transporters and glycolytic enzymes reflected shifts in flux from cytosolic to plastid metabolism. Despite major changes in metabolism between wri1 and wild-type seeds, <1% of genes differed by more than twofold, and most of these were involved in central lipid and carbohydrate metabolism. Thus, these data define in part the downstream responses to disruption of the WRI1 gene.

Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database.

The genome of Arabidopsis has been searched for sequences of genes involved in acyl lipid metabolism. Over 600 encoded proteins have been identified, cataloged, and classified according to predicted function, subcellular location, and alternative splicing. At least one-third of these proteins were previously annotated as “unknown function” or with functions unrelated to acyl lipid metabolism; therefore, this study has improved the annotation of over 200 genes. In particular, annotation of the lipolytic enzyme group (at least 110 members total) has been improved by the critical examination of the biochemical literature and the sequences of the numerous proteins annotated as “lipases.” In addition, expressed sequence tag (EST) data have been surveyed, and more than 3,700 ESTs associated with the genes were cataloged. Statistical analysis of the number of ESTs associated with specific cDNA libraries has allowed calculation of probabilities of differential expression between different organs. More than 130 genes have been identified with a statistical probability > 0.95 of preferential expression in seed, leaf, root, or flower. All the data are available as a Web-based database, the Arabidopsis Lipid Gene database (http://www.plantbiology.msu.edu/lipids/genesurvey/index.htm). The combination of the data of the Lipid Gene Catalog and the EST analysis can be used to gain insights into differential expression of gene family members and sets of pathway-specific genes, which in turn will guide studies to understand specific functions of individual genes.

The Capacity of Green Oilseeds to Utilize Photosynthesis to Drive Biosynthetic Processes

Seeds of many plant species are green during embryogenesis. To directly assess the influence of light on the physiological status of green oilseeds in planta, Brassica napus and soybean (Glycine max) seeds were rapidly dissected from plants growing in the light or dark. The activation state of malate dehydrogenase, which reflects reduced thioredoxin and NADP/NADPH ratios, was found to be as high in seeds exposed to light as in leaves and to decrease in the dark. Rubisco was highly activated (carbamylated) in both light and dark, most likely reflecting high seed CO2 concentrations. Activities of Rubisco and phosphoribulokinase were sufficient to account for significant refixation of CO2 produced during B. napus oil biosynthesis. To determine the influence of light on oil synthesis in planta, siliques on intact plants in full sunlight or detached siliques fed 3H2O were partly covered with aluminum foil. Seeds from light and dark sections were analyzed, and fatty acid accumulation was found to be higher in seeds exposed to light than seeds from dark sections. The spectrum of light filtering through silique walls and the pigment composition of developing B. napus embryos were determined. In addition to a low chlorophyll a/b ratio, the carotenoid pigments of seeds can provide additional capture of the green light that filters through siliques. Together, these results demonstrate that even the low level of light reaching seeds plays a substantial role in activating light-regulated enzymes, increasing fatty acid synthesis, and potentially powering refixation of CO2.

Xanthophyll cycle, light energy dissipation and electron transport in transgenic tobacco with reduced carbon assimilation capacity.

The effects of reduced CO2 assimilation capacity on the leaf pigment composition and the dissipation of light energy were studied using transgenic tobacco (Nicotiana tabacum L. cv. W38). Two plant types were used: anti-SSu plants with reduced amounts of Rubisco and anti-GAPDH plants with reduced activity of chloroplast glycer-aldehyde 3-phosphate dehydrogenase. A moderate reduction in the photosynthetic capacity increased the de-epoxidation state of the xanthophyll-cycle pigments. In contrast, there was no large effect on the leaf pigment composition and the ratio of the xanthophyll cycle pigments to chlorophyll, and total carotenoids increased only in the most severe transgenic plants. The light induction of photosynthesis, fluorescence quenching and de-epoxida ion of the xanthophyll cycle pigments were also followed in wild-type and anti-SSu plants. Anti-SSu plants maintained high nonphotochemical quenching and increased xanthophyll de-epoxidation in the light but the reduction state of QA remained high. For both wild-type and anti-SSu plants, the electron transport rate estimated from chlorophyll a fluorescence appeared to be much higher than that required to support the observed rate of CO2 assimilation and photorespiration during the early phase of photosynthetic induction. However, the two estimates converged with the onset of steady-state photosynthesis.

Functional Genomics from a Plant Biochemist's Perspective

The lipids derived from plant seeds provide approximately 20% of dietary calories in developed nations. The fatty acid composition of these dietary oils can influence several components of long-term health. Almost all the genes involved in producing dietary oils are now isolated, allowing rational modifications in saturated and polyunsaturated fatty acid content in foods. Several of these genes have been identified using genomic approaches. However, many questions about the regulation of plant lipid metabolism seem difficult to solve using conventional biochemical or molecular approaches. For example, we have little understanding of why or how some seeds produce >50% oil whereas other seeds store largely carbohydrate or protein. Major control over complex plant biochemical pathways may only become possible by understanding regulatory networks which provide ‘global’ control over these pathways. To begin to discover such networks and provide a broad analysis of gene expression in developing oilseeds, we have taken two genomic approaches: 1) We have sequenced 10,500 expressed sequence tags (EST) from developing Arabidopsis seeds and analyzed these using different bioinformatics tools. Approximately 30% of the ESTs have no match in dbEST, suggesting many represent mRNAs derived from genes that are specifically expressed in seeds. ‘Electronic northern’ analysis of EST abundance data has provided insights into the import of photosynthate into developing embryos, its conversion into seed oil and the regulation of this pathway. 2) We have produced microarrays which display ca. 4000 seed-expressed Arabidopsis genes. Sensitivity of the arrays is estimated at 1–2 copies mRNA/cell. The arrays have been hybridized with probes derived from seeds, leaves, and roots and analysis of expression ratios between the different tissues has allowed the tissue-specific expression patterns of many hundreds of genes to be described for the first time. Approximately 10% of the genes were expressed at ratios ≥10-fold higher in seeds than leaves or roots. We also identified several transcription factors, as well as kinases and phosphatases, whose expression changed during the seed development, and which may be involved in the regulation of the storage compound synthesis. Moreover, cluster analysis of the expression profiles established distinct groups of co-regulated genes.

Rubisco Activation is Impaired in Transgenic Tobacco Plants with Reduced Electron Transport Capacity

Rubisco can be catalytically competent only after a specific lysyl residue within the active site has been carbamylated. Before carbamylation can occur, any inhibitory ligands bound at the site must be released, and this process is facilitated by another enzyme, Rubisco activase. It has been shown in vitro that Rubisco activase needs to hydrolyse ATP to function and is inhibited by ADP, and so presumably is sensitive to the chloroplast ATP/ADP ratio (1). However, there are indications that activase is also regulated by transthylakoid pH gradient (∆pH) and electron transport through PSI (2).

Regulation of CO 2 Assimilation Rate by the Chloroplast Cytochrome BF Complex

The photosynthetic electron transport pathway is responsible for the provision of ATP and NADPH for photosynthetic carbon reduction (PCR) and as such it has the potential to exert strong regulation over photosynthesis and growth. Transduction of light energy by the photosynthetic apparatus of chloroplasts depends on the co-ordinated capture of light energy by the two photosystems, PSI and PSII, along with electron transfer between these two centers. Generation of a trans-thylakoid proton gradient is required for synthesis of ATP via the ATP synthase complex, and NADPH production is dependent on linear electron flow from PSII to PSI and through ferredoxin to ferredoxin:NADP+ oxidoreductase. Functionally the cytochrome bf complex is located in a central position in the electron transport scheme between P680 and P700 because of its ability to operate as both a plastoquinol and plastocyanin oxidoreductase (1,2). The cytochrome bf complex can act in both linear electron transport (production of ATP and NADPH) and cyclic electron transport flow (ATP generation only). Price and coworkers have engineered transgenic tobacco plants where the nuclear-encoded Rieske FeS subunit of the cytochrome bf complex was specifically suppressed through antisense RNA technology (3). They produced plants with cytochrome bf content as low as 5% of wild type values. In this paper we have used these Rieske FeS antisense plants to examine the link between electron transport rate and PCR cycle activity. We conclude that the reduction in electron tranport rate reduces CO2 assimilation rate both by reducing the RuBP concentration and by reducing Rubisco carbamylation.