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, ‫ف‬ 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 CO 2 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, Ͻ 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.

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 Ͼ 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.Acyl lipids can be defined as fatty acids and their naturally occurring ester, ether, or amide derivatives. In plants, these include acylglycerols such as triacylglycerols (TAGs), phospholipids, galactolipids, and sulfolipids, plus sphingolipids, acylated steryl glycosides, oxylipins, cutins, suberins, estolides and wax, and sterol esters. The list may be extended if we consider molecules immediately derived from acyl groups, such as the epicuticular wax components (hydrocarbons, alcohols, ketones, and so on) or natural products such as anacardic acids that impart protection to predation. Polar lipids are amphipathic and as such self-associate in water to produce a variety of structures. Therefore, they provide the building blocks for biological membranes. There is substantial evidence indicating that the composition of acyl lipids in membranes influences the targeting, distribution, and functional properties of both integral and membrane-associated proteins (Sprong et al., 2001;Wallis and Browse, 2002). Furthermore, many polar lipids and the intermediates in their synthesis and degradation serve as signaling molecules. In summary, acyl lipids function in a wide range of biological processes, such as carbon and free energy storage, cell signaling, modulation of enzyme activity and protein localization, vesicle budding and fusion, waterproofing, and surface protection (Browse and Somerville, 1994).Some acyl lipids such as TAGs, the major constituent of vegetable oils, are ...

Linear electron transport in chloroplasts produces a number of reduced components associated with photosystem I (PS I) that may subsequently participate in reactions that reduce O 2 . The two primary reactions that have been extensively studied are: ¢rst, the direct reduction of O 2 to superoxide by reduced donors associated with PS I (the Mehler reaction), and second, the rubisco oxygenase (ribulose 1,5-bisphosphate carboxylase oxygenase EC 4.1.1.39) reaction and associated peroxisomal and mitochondrial reactions of the photorespiratory pathway. This paper reviews a number of recent and past studies with higher plants, algae and cyanobacteria that have attempted to quantify O 2 £uxes under various conditions and their contributions to a number of roles, including photon energy dissipation. In C 3 and Crassulacean acid metabolism (CAM) plants, a Mehler O 2 uptake reaction is unlikely to support a signi¢cant £ow of electron transport (probably less than 10%). In addition, if it were present it would appear to scale with photosynthetic carbon oxidation cycle (PCO) and photosynthetic carbon reduction cycle (PCR) activity. This is supported by studies with antisense tobacco plants with reduced rubisco at low and high temperatures and high light, as well as studies with potatoes, grapes and madrone during water stress. The lack of signi¢cant Mehler in these plants directly argues for a strong control of Mehler reaction in the absence of ATP consumption by the PCR and PCO cycles. The di¡erence between C 3 and C 4 plants is primarily that the level of light-dependent O 2 uptake is generally much lower in C 4 plants and is relatively insensitive to the external CO 2 concentration. Such a major di¡erence is readily attributed to the operation of the C 4 CO 2 concentrating mechanism. Algae show a range of lightdependent O 2 uptake rates, similar to C 4 plants. As in C 4 plants, the O 2 uptake appears to be largely insensitive to CO 2 , even in species that lack a CO 2 concentrating mechanism and under conditions that are clearly limiting with respect to inorganic carbon supply. A part explanation for this could be that many algal rubsicos have considerably di¡erent oxygenase kinetic properties and exhibit far less oxygenase activity in air. This would lead to the conclusion that perhaps a greater proportion of the observed O 2 uptake may be due to a Mehler reaction and less to rubisco, compared with C 3 plants. In contrast to algae and higher plants, cyanobacteria appear to have a high capacity for Mehler O 2 uptake, which appears to be not well coupled or limited by ATP consumption. It is likely that in all higher plants and algae, which have a well-developed non-photochemical quenching mechanism, non-radiative energy dissipation is the major mechanism for dissipating excess photons absorbed by the light-harvesting complexes under stressful conditions. However, for cyanobacteria, with a lack of signi¢cant nonphotochemical quenching, the situation may well be di¡erent.

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 CO 2 concentrations. Activities of Rubisco and phosphoribulokinase were sufficient to account for significant refixation of CO 2 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 3 H 2 O 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 CO 2 .Oilseeds provide a major source of calories for human consumption and are an increasingly significant source of renewable industrial materials. Interest in engineering enhanced seed oil quantity and quality has prompted efforts to better understand the biosynthesis of oil and other storage products of seeds. Several of the major oilseed crops (e.g. soybean [Glycine max], rapeseed, cotton, and linseed) produce seeds that are green during their development, and this fact has prompted questions regarding the contributions of seed photosynthesis to oilseed metabolism.In leaf chloroplasts, fatty acid synthesis (FAS) is light dependent and utilizes ATP and reducing power generated by photosynthesis (Browse et al., 1986;Roughan and Ohlrogge, 1996). By contrast, plastids isolated from heterotrophic tissues, such as pea roots and cauliflower floral buds, require externally supplied ATP and/or reducing power for FAS (Mö hlmann et al., 1994;Xue et al., 1997). Similarly, plastids isolated from developing chlorophylless seeds of safflower and castor bean are heterotrophic in nature and require added ATP or reducing equivalents (Browse and Slack, 1985;Smith et al., 1992). The situation is more complicated in photosynthetic oilseeds that are green during development. Although these seeds are predominantly sink tissues, they contain chloroplasts with the thylakoid structures and enzymes of typical photosynthetic machinery. However, rather than producing carbon for ex...

To provide a broad analysis of gene expression in developing Arabidopsis seeds, microarrays have been produced that display approximately 2,600 seed-expressed genes. DNA for genes spotted on the arrays were selected from Ͼ10,000 clones partially sequenced from a cDNA library of developing seeds. Based on a series of controls, sensitivity of the arrays was estimated at one to two copies of mRNA per cell and cross hybridization was estimated to occur if closely related genes have Ͼ70% to 80% sequence identity. These arrays have been hybridized in a series of experiments with probes derived from seeds, leaves, and roots of Arabidopsis. 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 25% of the 2,600 genes were expressed at ratios Ն 2-fold higher in seeds than leaves or roots and 10% at ratios Ն 10. Included in this list are a large number of proteins of unknown function, and potential regulatory factors such as protein kinases, phosphatases, and transcription factors. The Arabidopsis arrays were also found to be useful for transcriptional profiling of mRNA isolated from developing oilseed rape (Brassica napus) seeds and expression patterns correlated well between the two species.

The water-soluble carbohydrate (WSC) that accumulates in the stems of wheat during growth can be an important contributor to grain filling, particularly under conditions when assimilation is limited, such as during end-of-season drought. WSC concentration was measured at anthesis across a diverse set of wheat genotypes over multiple environments. Environmental differences in WSC concentration were large (means for the set ranging between 108 and 203 mg g–1 dry weight), and there were significant and repeatable differences in WSC accumulation among genotypes (means ranging from 112 to 213 mg g–1 dry weight averaged across environments), associated with large broad-sense heritability (H = 0.90 ± 0.12). These results suggest that breeding for high WSC should be possible in wheat. The composition of the WSC, examined in selected genotypes, indicated that the variation in total WSC was attributed mainly to variation in the fructan component, with the other major soluble carbohydrates, sucrose and hexose, varying less. The degree of polymerisation (DP) of fructo-oligosaccharides was up to ~13 in samples where higher levels of WSC were accumulated, owing either to genotype or environment, but the higher DP components (DP > 6) were decreased in samples of lower total WSC. The results are consistent with fructan biosynthesis occurring via a sequential mechanism that is dependent on the availability of sucrose, and differences in WSC contents of genotypes are unlikely to be due to major mechanistic differences.

Several environmental factors including drought and disease can reduce leaf area and photosynthesis during grain-filling to decrease grain yield and kernel weight of cereal crops. Water-soluble carbohydrates (WSC) accumulated around anthesis can be mobilised to assist in filling of developing grains when post-anthesis assimilation is low. Cultivar differences support opportunities to select for high WSC but little is known of the extent or nature of genetic control for this trait in wheat. Three wheat mapping populations (Cranbrook/Halberd, Sunco/Tasman, and CD87/Katepwa) were phenotyped for WSC and other agronomic traits across multiple environments. The range for WSC concentration (WSC-C) was large among progeny contributing to moderate-to-high narrow-sense heritabilities within environments (h2 = 0.51–0.77). Modest genotype × environment interaction reduced the correlation of genotype means across environments (rp = 0.37–0.78, P < 0.01) to reduce heritability on a line-mean (h2 = 0.55–0.87) basis. Transgressive segregation was large and genetic control complex, with 7–16 QTLs being identified for WSC-C in each population. Heritability was smaller (h2 = 0.32–0.54) for WSC mass per unit area (WSC-A), reflecting large genotype × environment interaction and residual variance with estimating anthesis biomass. Fewer significant QTLs (4–8) were identified for this trait in each population, while sizes of individual genetic effects varied between populations but were repeatable across environments. Several genomic regions were common across populations including those associated with plant height (e.g. Rht-B1) and/or anthesis date (e.g. Ppd1). Genotypes with high WSC-C were commonly shorter, flowered earlier, and produced significantly (P < 0.01) fewer tillers than those of low WSC-C. This resulted in similar yields, lower final biomass, and fewer grains per m2, but greater dry weight partitioning to grain, kernel weight, and less grain screenings in high compared with low WSC-C genotypes. By contrast, lines high for WSC-A produced more fertile tillers associated with similar or greater anthesis and maturity biomass, grain number, and yield, yet similar kernel weight or size compared with genotypes with low WSC-A. The data support an important role for WSC-A in assuring stable yield and grain size. However, the small effects of many independent WSC QTLs may limit their direct use for marker-aided selection in breeding programs. We suggest using molecular markers to enrich populations for favourable height and anthesis date alleles before the more costly phenotypic selection among partially inbred families for greater WSC-A.

A gas-exchange system with a rapid response time was used to study the interplay between rate-limiting processes of C3 photosynthesis in wild-type tobacco (Nicotiana tabacum L. cv. W38) and transgenic tobaccos with antisense DNAs directed against the Rubisco small subunit (anti-SSu plants) or the chloroplast glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH plants). High ribulosebisphos-phate (RuBP) pools were generated in leaves by exposing them briefly to very low CO2, after which they were transferred to varying CO2 concentrations, and transient CO2 assimilation rates were measured within the first 2–3 s. Comparison of the transient (RuBP-saturated) and steady-state rates confirmed that the CO2 assimilation rate in anti-SSu plants was RuBP-saturated (i.e. Rubisco limited) at all intercellular CO2 partial pressures (Ci), and that, in anti-GAPDH plants, the transition from RuBP-saturation to RuBP-limitation occurred at lower assimilation rates and lower Ci as GAPDH activity was decreased. In addition, we investigated whether the integrated post-illumination CO2 uptake could be used as a non-destructive means of estimating RuBP pools in leaves. In wild-type plants there was generally a good agreement between RuBP pools extracted from leaves after rapid freeze-clamping and estimates made from post-illumination CO2 uptake. However, in the anti-SSu plants, the post- illumination CO2 uptake underestimated the actual RuBP content and the discrepancy became larger as the Rubisco content decreased. Possible explanations for this are discussed.