Alterations in the Expression of Seed Development Genes Affect Seed Size and Storage Compound Allocation in <em>Arabidopsis thaliana</em>

Seeds are major components of the human diet and seed oil represents a source of renewable and environmentally friendly replacements for fossil-based raw materials. During maturation seeds accumulate storage reserves to support the growth of the seedling before it gains its photosynthetic activity. The main storage reserves are oil in the form of triacylglycerols, proteins, and carbohydrates in the form of starch. Increasing the seed size and therefore storage reserve content is of major economical and agricultural importance. In this study we investigated the effects of alteration in seed development gene expression on storage allocation in seeds of Arabidopsis thaliana. New alleles of the seed development genes FIE1, PHE1, MINI3, and IKU2 were isolated and the homozygous mutant lines of the candidate genes were characterized. While the amount of storage proteins and sucrose was similar to the WT controls, the oil content was strongly reduced in all mutants to varying extents. This finding indicates that the carbon flow from carbohydrates to fatty acids biosynthesis is affected. To investigate the effects of the overexpression of FIE, PHE1, MINI3, and IKU2 on seed size and storage reserve accumulation, cDNAs encoding the candidate genes were overexpressed in Arabidopsis thaliana ecotype Col-0 using the embryo-specific glycinin promoter from soybean. Thus, the transgenes under control of the glycinin promoter, and the endogenous genes are differentially expressed, spatially and temporally. While the overexpression of FIE, PHE1, and MINI3 did not have any effects on seed size and storage reserve accumulation, IKU2-OE lines displayed a strong increase in seed size and oil content, but no change in protein and sucrose content. Moreover, non-transgenic segregant seeds of a heterozygous IKU2-OE line also showed an increase in seed size and oil content indicating a maternal effect of IKU2-OE overexpression in the seed coat or silique of the heterozygous plants. The increase in seed size and oil content was only observed in seeds of heterozygous IKU2-OE lines but not in homozygous seeds. RT-PCR analysis showed that IKU2 is highly expressed in seeds of heterozygous IKU2-OE lines, but is co-suppressed in homozygous IKU2-OE lines, which show similar low expression level as the control Col-0-EV. The increase in seed size of heterozygous IKU2-OE seeds was associated with a reduction in total seed yield per plant compared to the empty vector controls, while the siliques size and number of seeds per silique were not affected. It has been previously shown that the seed specific overexpression of seed development genes using strong promoters induces an increase in seed size associated with developmental abnormalities in the transgenic plants. However, the overexpression of the same genes under control of truncated or mutated promoters induces an increase in seed size without detrimental effects on plant development. Therefore, it might be possible to overcome the negative correlation between seed size and seed yield per plant by overexpression IKU2 using a modified glycinin promoter. The future goals of this study are the overexpression of IKU2 in oilseed crops such as soybean and rapeseed with the aim to increase the seed size and oil content in the transgenic lines

Metabolic reconstructions identify plant 3‐methylglutaconyl‐CoA hydratase that is crucial for branched‐chain amino acid catabolism in mitochondria

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144600/1/tpj13955_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144600/2/tpj13955.pd

The Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in \u3ci\u3eArabidopsis\u3c/i\u3e

It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. Here, we demonstrate that Arabidopsis thaliana uses for that purpose two separate biosynthetic branches stemming from phenylalanine and tyrosine. Gene network modeling and characterization of T-DNA mutants indicated that acyl-activating enzyme encoded by At4g19010 contributes to the biosynthesis of ubiquinone specifically from phenylalanine. CoA ligase assays verified that At4g19010 prefers para-coumarate, ferulate, and caffeate as substrates. Feeding experiments demonstrated that the at4g19010 knockout cannot use para-coumarate for ubiquinone biosynthesis and that the supply of 4-hydroxybenzoate, the side-chain shortened version of para-coumarate, can bypass this blockage. Furthermore, a trans-cinnamate 4-hydroxylase mutant, which is impaired in the conversion of trans-cinnamate into para-coumarate, displayed similar defects in ubiquinone biosynthesis to that of the at4g19010 knockout. Green fluorescent protein fusion experiments demonstrated that At4g19010 occurs in peroxisomes, resulting in an elaborate biosynthetic architecture where phenylpropanoid intermediates have to be transported from the cytosol to peroxisomes and then to mitochondria where ubiquinone is assembled. Collectively, these results demonstrate that At4g19010 activates the propyl side chain of para-coumarate for its subsequent β-oxidative shortening. Evidence is shown that the peroxisomal ABCD transporter (PXA1) plays a critical role in this branch. Includes supplementary files

Seed-specific suppression of ADP-glucose pyrophosphorylase in Camelina sativa increases seed size and weight

Abstract Background Camelina (Camelina sativa L.) is a promising oilseed crop that may provide sustainable feedstock for biofuel production. One of the major drawbacks of Camelina is its smaller seeds compared to other major oil crops such as canola, which limit oil yield and may also pose challenges in successful seedling establishment, especially in dryland cultivation. Previous studies indicate that seed development may be under metabolic control. In oilseeds, starch only accumulates temporarily during seed development but is almost absent in mature seeds. In this study, we explored the effect of altering seed carbohydrate metabolism on Camelina seed size through down-regulating ADP-glucose pyrophosphorylase (AGPase), a major enzyme in starch biosynthesis. Results An RNAi construct comprising sequences of the Camelina small subunit of an AGPase (CsAPS) was expressed in Camelina cultivar Suneson under a seed-specific promoter. The RNAi suppression reduced AGPase activities which concurred with moderately decreased starch accumulation during seed development. Transcripts of genes examined that are involved in storage products were not affected, but contents of sugars and water were increased in developing seeds. The transgenic seeds were larger than wild-type plants due to increased cell sizes in seed coat and embryos, and mature seeds contained similar oil but more protein contents. The larger seeds showed advantages on seedling emergence from deep soils. Conclusions Changing starch and sugar metabolism during seed development may increase the size and mass of seeds without affecting their final oil content in Camelina. Increased seed size may improve seedling establishment in the field and increase seed yield

A Dedicated Type II NADPH Dehydrogenase Performs the Penultimate Step in the Biosynthesis of Vitamin K\u3csub\u3e1\u3c/sub\u3e in \u3ci\u3eSynechocystis\u3c/i\u3e and Arabidopsis

Mutation of Arabidopsis thaliana NAD(P)H DEHYDROGENASE C1 (NDC1; At5g08740) results in the accumulation of demethylphylloquinone, a late biosynthetic intermediate of vitamin K1. Gene coexpression and phylogenomics analyses showed that conserved functional associations occur between vitamin K biosynthesis and NDC1 homologs throughout the prokaryotic and eukaryotic lineages. Deletion of Synechocystis ndbB, which encodes for one such homolog, resulted in the same defects as those observed in the cyanobacterial demethylnaphthoquinone methyltransferase knockout. Chemical modeling and assay of purified demethylnaphthoquinone methyltransferase demonstrated that, by virtue of the strong electrophilic nature of S-adenosyl-Lmethionine, the transmethylation of the demethylated precursor of vitamin K is strictly dependent on the reduced form of its naphthoquinone ring. NDC1 was shown to catalyze such a prerequisite reduction by using NADPH and demethylphylloquinone as substrates and flavine adenine dinucleotide as a cofactor. NDC1 displayed Michaelis-Menten kinetics and was markedly inhibited by dicumarol, a competitive inhibitor of naphthoquinone oxidoreductases. These data demonstrate that the reduction of the demethylnaphthoquinone ring represents an authentic step in the biosynthetic pathway of vitamin K, that this reaction is enzymatically driven, and that a selection pressure is operating to retain type II NAD(P)H dehydrogenases in this process

Deciphering and modifying LAFL transcriptional regulatory network in seed for improving yield and quality of storage compounds

International audienc

Genetically Programmed Changes in Photosynthetic Cofactor Metabolism in Copper-deficient Chlamydomonas *

Genetic and genomic studies indicate that copper deficiency triggers changes in the expression of genes encoding key enzymes in various chloroplast-localized lipid/pigment biosynthetic pathways. Among these are CGL78 involved in chlorophyll biosynthesis and HPPD1, encoding 4-hydroxyphenylpyruvate dioxygenase catalyzing the committed step of plastoquinone and tocopherol biosyntheses. Copper deficiency in wild-type cells does not change the chlorophyll content, but a survey of chlorophyll protein accumulation in this situation revealed increased accumulation of LHCSR3, which is blocked at the level of mRNA accumulation when either CGL78 expression is reduced or in the crd1 mutant, which has a copper-nutrition conditional defect at the same step in chlorophyll biosynthesis. Again, like copper-deficient crd1 strains, cgl78 knock-down lines also have reduced chlorophyll content concomitant with loss of PSI-LHCI super-complexes and reduced abundance of a chlorophyll binding subunit of PSI, PSAK, which connects LHCI to PSI. For HPPD1, increased mRNA results in increased abundance of the corresponding protein in copper-deficient cells concomitant with CRR1-dependent increased accumulation of γ-tocopherols, but not plastoquinone-9 nor total tocopherols. In crr1 mutants, where increased HPPD1 expression is blocked, plastochromanol-8, derived from plastoquinone-9 and purported to also have an antioxidant function, is found instead. Although not previously found in algae, this metabolite may occur only in stress conditions

Acyl Editing and Headgroup Exchange Are the Major Mechanisms That Direct Polyunsaturated Fatty Acid Flux into Triacylglycerols

Triacylglycerols (TAG) in seeds of Arabidopsis (Arabidopsis thaliana) and many plant species contain large amounts of polyunsaturated fatty acids (PUFA). These PUFA are synthesized on the membrane lipid phosphatidylcholine (PC). However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the key enzymes/genes that control these fluxes known. By reverse genetics and metabolic labeling experiments, we demonstrate that two genes encoding the lysophosphatidylcholine acyltransferases LPCAT1 and LPCAT2 in Arabidopsis control the previously identified “acyl-editing” process, the main entry of fatty acids into PC. The lpcat1/lpcat2 mutant showed increased contents of very-long-chain fatty acids and decreased PUFA in TAG and the accumulation of small amounts of lysophosphatidylcholine in developing seeds revealed by [(14)C]acetate-labeling experiments. We also showed that mutations in LPCATs and the PC diacylglycerol cholinephosphotransferase in the reduced oleate desaturation1 (rod1)/lpcat1/lpcat2 mutant resulted in a drastic reduction of PUFA content in seed TAG, accumulating only one-third of the wild-type level. These results indicate that PC acyl editing and phosphocholine headgroup exchange between PC and diacylglycerols control the majority of acyl fluxes through PC to provide PUFA for TAG synthesis