The Ubiquitin Receptor DA1 Regulates Seed and Organ Size by Modulating the Stability of the Ubiquitin-Specific Protease UBP15/SOD2 in Arabidopsis

Although the control of organ size is a fundamental question in developmental biology, little is known about the genetic and molecular mechanisms that determine the final size of seeds in plants. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligases DA2 and ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to restrict seed growth in Arabidopsis thaliana. Here, we describe UBIQUITIN-SPECIFIC PROTEASE15 (UBP15), encoded by SUPPRESSOR2 OF DA1 (SOD2), which acts maternally to regulate seed size by promoting cell proliferation in the integuments of ovules and developing seeds. The sod2/ubp15 mutants form small seeds, while overexpression of UBP15 increases seed size of wild-type plants. Genetic analyses indicate that UBP15 functions antagonistically in a common pathway with DA1 to influence seed size, but does so independently of DA2 and EOD1. Further results reveal that DA1 physically associates with UBP15 in vitro and in vivo and modulates the stability of UBP15. Therefore, our findings establish a genetic and molecular framework for the regulation of seed size by four ubiquitin-related proteins DA1, DA2, EOD1, and UBP15 and suggest that they are promising targets for increasing seed size in crops.     

Reduced expression of FatA thioesterases in Arabidopsis affects the oil content and fatty acid composition of the seeds

Acyl–acyl carrier protein (ACP) thioesterases are enzymes that control the termination of intraplastidial fatty acid synthesis by hydrolyzing the acyl–ACP complexes. Among the different thioesterase gene families found in plants, the FatA-type fulfills a fundamental role in the export of the C18 fatty acid moieties that will be used to synthesize most plant glycerolipids. A reverse genomic approach has been used to study the FatA thioesterase in seed oil accumulation by screening different mutant collections of Arabidopsis thaliana for FatA knockouts. Two mutants were identified with T-DNA insertions in the promoter region of each of the two copies of FatA present in the Arabidopsis genome, from which a double FatA Arabidopsis mutant was made. The expression of both forms of FatA thioesterases was reduced in this double mutant (fata1 fata2), as was FatA activity. This decrease did not cause any evident morphological changes in the mutant plants, although the partial reduction of this activity affected the oil content and fatty acid composition of the Arabidopsis seeds. Thus, dry mutant seeds had less triacylglycerol content, while other neutral lipids like diacylglycerols were not affected. Furthermore, the metabolic flow of the different glycerolipid species into seed oil in the developing seeds was reduced at different stages of seed formation in the fata1 fata2 line. This diminished metabolic flow induced increases in the proportion of linolenic and erucic fatty acids in the seed oil, in a similar way as previously reported for the wri1 Arabidopsis mutant that accumulates oil poorly. The similarities between these two mutants and the origin of their phenotype are discussed in function of the results.     

AtEXP2 Is Involved in Seed Germination and Abiotic Stress Response in Arabidopsis

Expansins are cell wall proteins that promote cell wall loosening by inducing pH-dependent cell wall extension and stress relaxation. Expansins are required in a series of physiological developmental processes in higher plants such as seed germination. Here we identified an Arabidopsis expansin gene AtEXPA2 that is exclusively expressed in germinating seeds and the mutant shows delayed germination, suggesting that AtEXP2 is involved in controlling seed germination. Exogenous GA application increased the expression level of AtEXP2 during seed germination, while ABA application had no effect on AtEXP2 expression. Furthermore, the analysis of DELLA mutants show that RGL1, RGL2, RGA, GAI are all involved in repressing AtEXP2 expression, and RGL1 plays the most dominant role in controlling AtEXP2 expression. In stress response, exp2 mutant shows higher sensitivity than wild type in seed germination, while overexpression lines of AtEXP2 are less sensitive to salt stress and osmotic stress, exhibiting enhanced tolerance to stress treatment. Collectively, our results suggest that AtEXP2 is involved in the GA-mediated seed germination and confers salt stress and osmotic stress tolerance in Arabidopsis.     

Influence of systemic insecticides on lipid biosynthesis in seeds of Indian mustard (Brassica juncea L.)

The systemic insecticides namely phorate (Thimet 10 G) oxydemeton methyl (metasystox 25 EC) and dimethoate (Rogor 30 EC) decreased oil content in the developing seeds of Indian mustard (Brassica juncea L.) but showed an increase in the mature seeds. The inhibitory effect in the developing seed was accompanied by an increase in soluble sugars and a corresponding decrease in malate dehydrogenase and G6P dehydrogenase activity. The relative proportion of triacylglycerols and glycolipids decreased significantly while that of phospholipids and free fatty acids increased in the developing seeds. In the mature seeds, the proportion of triacylglycerols did not change appreciably from that in control. The erucic acid synthesis which was less at 10 and 20 days after fertilization (DAF) increased at 30 DAF with oxydemeton methyl and dimethoate; phorate was ineffective. In mature seeds, the proportion of erucic acid increased at the cost of linoleic and linolenic acids. All the insecticides appreciably decreased the rate of [1-¹⁴C]acetate incorporation into lipids both in vivo and in vitro experiments. In the in vivo experiment, the synthesis of polar lipids was enhanced at 10 and 20 DAF, and the higher doses of oxydemeton methyl and dimethoate at 30 DAF. On the other hand, the ¹⁴C-incorporation into triacylglycerols showed an opposite trend to that of polar lipids. In the in vitro experiment, oxydemeton methyl and dimethoate enhanced the synthesis of polar lipids at 10 and 20 DAF while these inhibited it at 30 DAF. The synthesis of triacylglycerols was inhibited by the use of these insecticides.     

Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> T-DNA insertion in the rice <Emphasis Type="Italic">DWARF SHOOT AND DEFECTIVE PANICLE1</Emphasis> (<Emphasis Type="Italic">DSDP1</Emphasis>) gene causes a severe dwarf phenotype,reduces plant vigour,and affects seed germination

We report a new T-DNA-tagged rice plant chi7, which displays a severe dwarf phenotype, reduced plant vigour, and impaired panicle development in the homozygous state. By chromosome walking, T-DNA integration was mapped in chromosome 2, 1054-bp upstream of the translation start site of a gene (Os02g0820400), which we designate as DWARF SHOOT AND DEFECTIVE PANICLE1 (DSDP1). DSDP1 expression was unexpectedly higher in the homozygous mutant leaves than in the hemizygous mutant and control plant leaves. Mutant dsdp1 seeds, stored for 24 weeks, failed to germinate in soil. The growth vigour of the dsdp1 mutant reduced with increasing seed storage period. The dsdp1 mutant plants, grown in vitro on MS medium, formed short, stout, and ageotropic roots with lesser number of root hairs. The findings suggest that DSDP1 may function as a negative regulator of many developmental processes in rice.     

Natural Variation in Seed Very Long Chain Fatty Acid Content Is Controlled by a New Isoform of KCS18 in Arabidopsis thaliana

Oil from oleaginous seeds is mainly composed of triacylglycerols. Very long chain fatty acids (VLCFAs) are major constituents of triacylglycerols in many seed oils and represent valuable feedstock for industrial purposes. To identify genetic factors governing natural variability in VLCFA biosynthesis, a quantitative trait loci (QTL) analysis using a recombinant inbred line population derived from a cross between accessions Bay-0 and Shahdara was performed in Arabidopsis thaliana. Two fatty acid chain length ratio (CLR) QTL were identified, with one major locus, CLR.2, accounting for 77% of the observed phenotypic variation. A fine mapping and candidate gene approach showed that a key enzyme of the fatty acid elongation pathway, the β-ketoacyl-CoA synthase 18 (KCS18), was responsible for the CLR.2 QTL detected between Bay-0 and Shahdara. Association genetics and heterologous expression in yeast cells identified a single point mutation associated with an alteration of KCS18 activity, uncovering the molecular bases for the modulation of VLCFA content in these two natural populations of Arabidopsis. Identification of this kcs18 mutant with altered activity opens new perspectives for the modulation of oil composition in crop plants.     

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, approximately 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, <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 Auxin-Deficient <Emphasis Type="Italic">Defective Kernel18 (dek18)</Emphasis> Mutation Alters the Expression of Seed-Specific Biosynthetic Genes in Maize

The dek18 mutant of maize was previously classified as a collapsed kernel mutant named cp*-931A, which has a decreased auxin content in kernels. Molecular and functional characterization of this mutant line offers the possibility to better understand auxin biology during maize seed development. Seeds of the dek18 mutants are smaller compared to wild-type seeds and the vegetative development of dek18 is delayed. Here we analyzed the expression of several auxin-related genes in dek18 homozygous seeds and normal-sized seeds (Dek18/-) segregating on the same ear. Three genes related to auxin biosynthesis ZmAlliinase/Tar3, ZmTar1, and ZmYuc1 were highly downregulated in the mutant compared to the wild type. Sequence analysis of these genes revealed that no nucleotide difference is present in dek18 homozygous seeds compared to Dek18/-, except for ZmYuc1. Two different ZmYuc1 cDNAs sequences are produced: a normal-sized sequence of 1197 bp and a shorter coding sequence lacking the third exon. Ectopic expression of ZmYuc1 cDNAs in Arabidopsis indicates that (i) the ZmYuc1 gene is functional in Arabidopsis and (ii) the third exon is required for the enzymatic activity of the YUCCA1 protein. Because ZmYuc1, ZmTar1, and ZmAlliinase are barely expressed in dek18 homozygous seeds, it is proposed that the mutation responsible for the dek18 phenotype alters the upstream regulation of the auxin biosynthetic pathway.     

Dynamic Metabolic Profiles and Tissue-Specific Source Effects on the Metabolome of Developing Seeds of Brassica napus

Canola (Brassica napus) is one of several important oil-producing crops, and the physiological processes, enzymes, and genes involved in oil synthesis in canola seeds have been well characterized. However, relatively little is known about the dynamic metabolic changes that occur during oil accumulation in seeds, as well as the mechanistic origins of metabolic changes. To explore the metabolic changes that occur during oil accumulation, we isolated metabolites from both seed and silique wall and identified and characterized them by using gas chromatography coupled with mass spectrometry (GC-MS). The results showed that a total of 443 metabolites were identified from four developmental stages. Dozens of these metabolites were differentially expressed during seed ripening, including 20 known to be involved in seed development. To investigate the contribution of tissue-specific carbon sources to the biosynthesis of these metabolites, we examined the metabolic changes of silique walls and seeds under three treatments: leaf-detachment (Ld), phloem-peeling (Pe), and selective silique darkening (Sd). Our study demonstrated that the oil content was independent of leaf photosynthesis and phloem transport during oil accumulation, but required the metabolic influx from the silique wall. Notably, Sd treatment resulted in seed senescence, which eventually led to a severe reduction of the oil content. Sd treatment also caused a significant accumulation of fatty acids (FA), organic acids and amino acids. Furthermore, an unexpected accumulation of sugar derivatives and organic acid was observed in the Pe- and Sd-treated seeds. Consistent with this, the expression of a subset of genes involved in FA metabolism, sugar and oil storage was significantly altered in Pe and Sd treated seeds. Taken together, our studies suggest the metabolite profiles of canola seeds dynamically varied during the course of oil accumulation, which may provide a new insight into the mechanisms of the oil accumulation at the metabolite level.     

Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds

Oleosin is the most abundant protein in the oil bodies of plant seeds, playing an important role in regulating oil body formation and lipid accumulation. To investigate whether lipid accumulation in transgenic rice seeds depends on the expression level of oleosin, we introduced two soybean oleosin genes encoding 24 kDa proteins into rice under the control of an embryo-specific rice promoter REG-2. Overexpression of soybean oleosin in transgenic rice leads to an increase of seed lipid content up to 36.93 and 46.06 % higher than that of the non-transgenic control, respectively, while the overall fatty acid profiles of triacylglycerols remained unchanged. The overexpression of soybean oleosin in transgenic rice seeds resulted in more numerous and smaller oil bodies compared with wild type, suggesting that an inverse relationship exists between oil body size and the total oleosin level. The increase in lipid content is accompanied by a reduction in the accumulation of total seed protein. Our results suggest that it is possible to increase rice seed oil content for food use and for use as a low-cost feedstock for biodiesel by overexpressing oleosin in rice seeds.     

Control of Pratylenchus penetrans and Meloidogyne hapla and Yield Response of Alfalfa Due to Oxamyl Seed Treatments

Alfalfa (Medicago sativa L. cv. Saranac) seed were soaked for 20 minutes in water, acetone, or methanol containing 10 or 50 mg/ml of oxamyl (Vydate L) or coated with a 2% aqueous cellulose solution containing the same amounts of oxamyl. Seed were analyzed for oxamyl by HPLC immediately after treatment and after 9 and 26 months of storage. Oxamyl content of alfalfa seed did not decline after 26 months of storage. The effects of seed treatment on growth of alfalfa and nematode control were examined using soils infested with Pratylenchus penetrans and Meloidogyne hapla. Germination was not affected by any of the seed treatments. Twenty-one days after sowing, the total growth of alfalfa seedlings grown from seed treated with 50 mg/ml of oxamyl in P. penetrans-infested soils had increased by 62% over controls. Nodulation per pot increased by as much as 267%, and the densities of P. penetrans per gram of root were reduced by as much as 73% compared to control plants. In M. hapla-infested soils, increases in plant growth (32%) and nodulation (71%) also occurred with oxamyl-treated seeds. Root gall reduction (86%) was also substantial due to oxamyl seed treatment.