Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds

The seed oil content in oilseed crops is a major selection trait to breeders. In Arabidopsis (Arabidopsis thaliana), LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) are key regulators of fatty acid biosynthesis. Overexpression of AtLEC1 and its orthologs in canola (Brassica napus), BnLEC1 and BnL1L, causes an increased fatty acid level in transgenic Arabidopsis plants, which, however, also show severe developmental abnormalities. Here, we use truncated napin A promoters, which retain the seed-specific expression pattern but with a reduced expression level, to drive the expression of BnLEC1 and BnL1L in transgenic canola. Conditional expression of BnLEC1 and BnL1L increases the seed oil content by 2% to 20% and has no detrimental effects on major agronomic traits. In the transgenic canola, expression of a subset of genes involved in fatty acid biosynthesis and glycolysis is up-regulated in developing seeds. Moreover, the BnLEC1 transgene enhances the expression of several genes involved in Suc synthesis and transport in developing seeds and the silique wall. Consistently, the accumulation of Suc and Fru is increased in developing seeds of the transgenic rapeseed, suggesting the increased carbon flux to fatty acid biosynthesis. These results demonstrate that BnLEC1 and BnL1L are reliable targets for genetic improvement of rapeseed in seed oil production.     

A heteromeric plastidic pyruvate kinase complex involved in seed oil biosynthesis in Arabidopsis

Glycolysis is a ubiquitous pathway thought to be essential for the production of oil in developing seeds of Arabidopsis thaliana and oil crops. Compartmentation of primary metabolism in developing embryos poses a significant challenge for testing this hypothesis and for the engineering of seed biomass production. It also raises the question whether there is a preferred route of carbon from imported photosynthate to seed oil in the embryo. Plastidic pyruvate kinase catalyzes a highly regulated, ATP-producing reaction of glycolysis. The Arabidopsis genome encodes 14 putative isoforms of pyruvate kinases. Three genes encode subunits alpha, beta(1), and beta(2) of plastidic pyruvate kinase. The plastid enzyme prevalent in developing seeds likely has a subunit composition of 4alpha4beta(1), is most active at pH 8.0, and is inhibited by Glu. Disruption of the gene encoding the beta(1) subunit causes a reduction in plastidic pyruvate kinase activity and 60% reduction in seed oil content. The seed oil phenotype is fully restored by expression of the beta(1) subunit-encoding cDNA and partially by the beta(2) subunit-encoding cDNA. Therefore, the identified pyruvate kinase catalyzes a crucial step in the conversion of photosynthate into oil, suggesting a preferred plastid route from its substrate phosphoenolpyruvate to fatty acids.     

Oxygen control of ethylene biosynthesis during seed development in Arabidopsis thaliana (L.) Heynh

An unforeseen side-effect on plant growth in reduced oxygen is the loss of seed production at concentrations around 25% atmospheric (50 mmol mol-1 O2). In this study, the model plant Arabidopsis thaliana (L.) Heynh. cv. 'Columbia' was used to investigate the effect of low oxygen on ethylene biosynthesis during seed development. Plants were grown in a range of oxygen concentrations (210 [equal to ambient], 160, 100, 50 and 25 mmol mol-1) with 0.35 mmol mol-1 CO2 in N2. Ethylene in full-sized siliques was sampled using gas chromatography, and viable seed production was determined at maturity. Molecular analysis of ethylene biosynthesis was accomplished using cDNAs encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase in ribonuclease protection assays and in situ hybridizations. No ethylene was detected in siliques from plants grown at 50 and 25 mmol mol-1 O2. At the same time, silique ACC oxidase mRNA increased three-fold comparing plants grown under the lowest oxygen with ambient controls, whereas ACC synthase mRNA was unaffected. As O2 decreased, tissue-specific patterning of ACC oxidase and ACC synthase gene expression shifted from the embryo to the silique wall. These data demonstrate how low O2 modulates the activity and expression of the ethylene biosynthetic pathway during seed development in Arabidopsis.     

Biochemical and physiological studies of Arabidopsis thaliana transgenic lines with repressed expression of the mitochondrial pyruvate dehydrogenase kinase

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. Previously, the cloning of a PDHK cDNA from Arabidopsis thaliana and the effects of constitutively down-regulating its expression on plant growth and development has been reported. The first detailed analyses of the biochemical and physiological effects of partial silencing of the mtPDHK in A. thaliana using antisense constructs driven by both constitutive and seed-specific promoters are reported here. The studies revealed an increased level of respiration in leaves of the constitutive antisense PDHK transgenics; an increase in respiration was also found in developing seeds of the seed-specific antisense transgenics. Both constitutive and seed-specific partial silencing of the mtPDHK resulted in increased seed oil content and seed weight at maturity. Feeding 3-(14)C pyruvate to bolted stems containing siliques (constitutive transgenics), or to isolated siliques or immature seeds (seed-specific transgenics) confirmed a higher rate of incorporation of radiolabel into all seed lipid species, particularly triacylglycerols. Neither constitutive nor seed-specific partial silencing of PDHK negatively affected overall silique and seed development. Instead, oil and seed yield, and overall plant productivity were improved. These findings suggest that a partial reduction of the repression of the mtPDC by antisense PDHK expression can alter carbon flux and, in particular, the contribution of carbon moieties from pyruvate to fatty acid biosynthesis and storage lipid accumulation in developing seeds, implicating a role for mtPDC in fatty acid biosynthesis in seeds.     

The amino acid permease AAP8 is important for early seed development in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The development of seeds depends on the import of carbohydrates and amino acids supplied by the maternal tissue via the phloem. Several amino acid transporters have been reported to be expressed during seed and silique development in Arabidopsis thaliana (L.) Heynh. Here we show that mutants lacking the high affinity amino acid permease 8 (At1g10010) display a severe seed phenotype. The overall number of seeds and the number of normally developed seed is reduced by ∼50% in siliques of the Ataap8 T-DNA insertion mutant. This result could be reproduced in plants where expression of AtAAP8 is targeted with an RNAi approach. The seed phenotype is correlated with a specifically altered amino acid composition of young siliques. Aspartic acid and glutamic acid are significantly reduced in young siliques of the mutants. In correlation with the fact that AAP8 is a high affinity transporter for acidic amino acids, translocation of ¹⁴C-labelled aspartate fed via the root system to seeds of the mutants is reduced. AAP8 plays a crucial role for the uptake of amino acids into the endosperm and supplying the developing embryo with amino acids during early embryogenesis.     

Analysis of the Arabidopsis cytosolic proteome highlights subcellular partitioning of central plant metabolism

The plant cell cytosol is a dynamic and complex intracellular matrix that, by definition, contains no compartmentalization. Nonetheless, it maintains a wide variety of biochemical networks and often links metabolic pathways across multiple organelles. There have been numerous detailed proteomic studies of organelles in the model plant Arabidopsis thaliana, although no such analysis has been undertaken on the cytosol. The cytosolic protein fraction from cell suspensions of Arabidopsis thaliana was isolated and analyzed using offline strong cation exchange liquid chromatography and LC-MS/MS. This generated a robust set of 1071 cytosolic proteins. Functional annotation of this set revealed major activities in protein synthesis and degradation, RNA metabolism and basic sugar metabolism. This included an array of important cytosol-related functions, specifically the ribosome, the set of tRNA catabolic enzymes, the ubiquitin-proteasome pathway, glycolysis and associated sugar metabolism pathways, phenylpropanoid biosynthesis, vitamin metabolism, nucleotide metabolism, an array of signaling and stress-responsive molecules, and NDP-sugar biosynthesis. This set of cytosolic proteins provides for the first time an extensive analysis of enzymes responsible for the myriad of reactions in the Arabidopsis cytosol and defines an experimental set of plant protein sequences that are not targeted to subcellular locations following translation and folding in the cytosol.     

Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca²⁺ handling relationship in plant cells

The existence of a transport protein that imports cytosolic NAD(+) into peroxisomes has been controversially discussed for decades. Nevertheless, the biosynthesis of NAD(+) in the cytosol necessitates the import of NAD(+) into peroxisomes for numerous reduction/oxidation (redox) reactions. However, a gene encoding such a transport system has not yet been identified in any eukaryotic organism. Here, we describe the peroxisomal NAD(+) carrier in Arabidopsis. Our candidate gene At2g39970 encodes for a member of the mitochondrial carrier family. We confirmed its peroxisomal localization using fluorescence microscopy. For a long time At2g39970 was assumed to represent the peroxisomal ATP transporter. In this study, we could show that the recombinant protein mediated the transport of NAD(+) . Hence, At2g39970 was named PXN for peroxisomal NAD(+) carrier. The loss of PXN in Arabidopsis causes defects in NAD(+) -dependent β-oxidation during seedling establishment. The breakdown of fatty acid released from storage oil was delayed, which led to the retention of oil bodies in pxn mutant seedlings. Based on our results, we propose that PXN delivers NAD(+) for optimal fatty acid degradation during storage oil mobilization.     

Keep an eye on PPi: the vacuolar-type H+-pyrophosphatase regulates postgerminative development in Arabidopsis

Postgerminative growth of seed plants requires specialized metabolism, such as gluconeogenesis, to support heterotrophic growth of seedlings until the functional photosynthetic apparatus is established. Here, we show that the Arabidopsis thaliana fugu5 mutant, which we show to be defective in AVP1 (vacuolar H(+)-pyrophosphatase), failed to support heterotrophic growth after germination. We found that exogenous supplementation of Suc or the specific removal of the cytosolic pyrophosphate (PPi) by the heterologous expression of the cytosolic inorganic pyrophosphatase1 (IPP1) gene from budding yeast (Saccharomyces cerevisiae) rescued fugu5 phenotypes. Furthermore, compared with the wild-type and AVP1(Pro):IPP1 transgenic lines, hypocotyl elongation in the fugu5 mutant was severely compromised in the dark but recovered upon exogenous supply of Suc to the growth media. Measurements revealed that the peroxisomal β-oxidation activity, dry seed contents of storage lipids, and their mobilization were unaffected in fugu5. By contrast, fugu5 mutants contained ~2.5-fold higher PPi and ~50% less Suc than the wild type. Together, these results provide clear evidence that gluconeogenesis is inhibited due to the elevated levels of cytosolic PPi. This study demonstrates that the hydrolysis of cytosolic PPi, rather than vacuolar acidification, is the major function of AVP1/FUGU5 in planta. Plant cells optimize their metabolic function by eliminating PPi in the cytosol for efficient postembryonic heterotrophic growth.     

Arabidopsis ADC genes involved in polyamine biosynthesis are essential for seed development

Arginine decarboxylase (ADC) is a rate-limiting enzyme that catalyzes the first step of polyamine (PA) biosynthesis in Arabidopsis thaliana. We generated a double mutant deficient in Arabidopsis two ADC genes (ADC1-/- ADC2-/-) and examined their roles in seed development. None of the F2 seedlings from crosses of adc1-1 and adc2-2 had the ADC1-/- ADC2-/- genotype. In addition, some abnormal seeds were observed among the ADC1+/- ADC2-/- and ADC1-/- ADC2+/- siliques. Viable offspring with the ADC1-/- ADC2-/- genotype could not be obtained from the ADC1+/- ADC2-/- and ADC1-/- ADC2+/- plants. These results indicate that AtADC genes are required for production of polyamines that are essential for normal seed development in Arabidopsis.