Soybean (Glycine max L.) triacylglycerol lipase GmSDP1 regulates the quality and quantity of seed oil.

Seeds of soybean (Glycine max L.) are a major source of plant-derived oils. In the past, improvements have been made in the quantity and quality of seed oil. Triacylglycerols (TAGs) are the principal components of soybean seed oil, and understanding the metabolic regulation of TAGs in soybean seeds is essential. Here, we identified four soybean genes encoding TAG lipases, designated as SUGAR DEPENDENT1-1 (GmSDP1-1), GmSDP1-2, GmSDP1-3 and GmSDP1-4; these are homologous to Arabidopsis thaliana SDP1 (AtSDP1). To characterize the function of these genes during grain filling, transgenic lines of soybean were generated via RNA interference to knockdown the expression of all four GmSDP1 genes. The seed oil content of the transgenic soybean lines was significantly increased compared with the wild type (WT). Additionally, fatty acid profiles of the WT and transgenic soybean lines were altered; the content of linoleic acid, a major fatty acid in soybean seeds, was significantly reduced, whereas that of oleic acid was increased in transgenic soybean seeds compared with the WT. Substrate specificity experiments showed that TAG lipase preferentially cleaved oleic acid than linoleic acid in the oil body membrane in WT soybean. This study demonstrates that the GmSDP1 proteins regulate both the TAG content and fatty acid composition of soybean seeds during grain filling. These results provide a novel strategy for improving both the quantity and quality of soybean seed oil.

Extension of oil biosynthesis during the mid-phase of seed development enhances oil content in Arabidopsis seeds.

Regulation of oil biosynthesis in plant seeds has been extensively studied, and biotechnological approaches have been designed to increase seed oil content. Oil and protein synthesis is negatively correlated in seeds, but the mechanisms controlling interactions between these two pathways are unknown. Here, we identify the molecular mechanism controlling oil and protein content in seeds. We utilized transgenic Arabidopsis thaliana plants overexpressing WRINKLED1 (WRI1), a master transcription factor regulating seed oil biosynthesis, and knockout mutants of major seed storage proteins. Oil and protein biosynthesis in wild-type plants was sequentially activated during early and late seed development, respectively. The negative correlation between oil and protein contents in seeds arises from competition between the pathways. Extension of WRI1 expression during mid-phase of seed development significantly enhanced seed oil content. This study demonstrates that temporal activation of genes involved in oil or storage protein biosynthesis determines the oil/protein ratio in Arabidopsis seeds. These results provide novel insights into potential breeding strategies to generate crops with high oil contents in seeds.

The plastidic DEAD-box RNA helicase 22, HS3, is essential for plastid functions both in seed development and in seedling growth.

Plants accumulate large amounts of storage products in seeds to provide an energy reserve and to supply nutrients for germination and post-germinative growth. Arabidopsis thaliana belongs to the Brassica family, and oil is the main storage product in Arabidopsis seeds. To elucidate the regulatory mechanisms of oil biosynthesis in seeds, we screened for high density seeds (heavy seed) that have a low oil content. HS3 (heavy seed 3) encodes the DEAD-box RNA helicase 22 that is localized to plastids. The triacylglycerol (TAG) content of hs3-1 seeds was 10% lower than that of wild-type (WT) seeds, while the protein content was unchanged. The hs3-1 plants displayed a pale-green phenotype in developing seeds and seedlings, but not in adult leaves. The HS3 expression level was high in developing seeds and seedlings, but was low in stems, rosette leaves and flowers. The plastid gene expression profile of WT developing seeds and seedlings differed from that of hs3-1 developing seeds and seedlings. The expression of several genes was reduced in developing hs3-1 seeds, including accD, a gene that encodes the ß subunit of carboxyltransferase, which is one component of acetyl-CoA carboxylase in plastids. In contrast, no differences were observed between the expression profiles of WT and hs3-1 rosette leaves. These results show that HS3 is essential for proper mRNA accumulation of plastid genes during seed development and seedling growth, and suggest that HS3 ensures seed oil biosynthesis by maintaining plastid mRNA levels.

A peroxisomal ABC transporter promotes seed germination by inducing pectin degradation under the control of ABI5.

Seed dormancy is essential for most plants to control the timing of germination. In Arabidopsis thaliana, PED3 is a single-copy gene encoding an ATP-binding cassette transporter that is required for peroxisomal fatty acid beta-oxidation. PED3 is involved in the import of several biologically important molecules into the peroxisome, including very-long-chain fatty acids associated with the breakdown of seed-reserve lipids, and precursors of auxin and jasmonic acid. The germination of ped3 mutants is significantly impaired, suggesting that PED3 regulates dormancy and germination. A transcriptome analysis revealed that many genes containing the core motif of the ABA responsive element (ABRE) in their promoter regions, and the ABA insensitive 5 (ABI5) transcription factor that binds to ABRE, are abnormally up-regulated in imbibed ped3 seeds. Expression of polygalacturonase inhibiting proteins (PGIPs) is also up-regulated specifically in ped3 after imbibition. By contrast, the ped3 abi5 double mutant does not show any of these expression patterns. The results indicate that the abi5 mutation normalizes PGIP expression and rescues the impaired germination phenotype of the ped3 mutant. PGIPs are known to act as inhibitors of polygalacturonases that degrade pectin. The amount of PGIP1 transcript regulates the timing of radicle protrusion. The impaired germination of ped3 could also be rescued by removal of pectin from the seed coat using exogenous polygalacturonase or acidic conditions. Overall, our results suggest that PED3, a peroxisomal ABC transporter, promotes seed germination by suppressing PGIPs under the control of ABI5.

Polyubiquitin promoter-based binary vectors for overexpression and gene silencing in Lotus japonicus.

In this study, we compared the transcriptional activities between Cauliflower mosaic virus (CaMV)35S promoter and polyubiquitin (Ljubq1) promoter from Lotus japonicus using beta-glucuronidase (gus) reporter gene in transgenic plants of L. japonicus. The promoter analysis demonstrated that the Ljubq1 promoter possessed higher activity than the CaMV35S promoter in leaves, stems, roots, nodules, and pollen. Finally, we created GATEWAY conversion technology-compatible binary vectors for over-expression and RNA interference under the Ljubq1 promoter. These materials could provide alternative choice for studies in L. japonicus.

Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots.

The roots of most higher plants form arbuscular mycorrhiza, an ancient, phosphate-acquiring symbiosis with fungi, whereas only four related plant orders are able to engage in the evolutionary younger nitrogen-fixing root-nodule symbiosis with bacteria. Plant symbioses with bacteria and fungi require a set of common signal transduction components that redirect root cell development. Here we present two highly homologous genes from Lotus japonicus, CASTOR and POLLUX, that are indispensable for microbial admission into plant cells and act upstream of intracellular calcium spiking, one of the earliest plant responses to symbiotic stimulation. Surprisingly, both twin proteins are localized in the plastids of root cells, indicating a previously unrecognized role of this ancient endosymbiont in controlling intracellular symbioses that evolved more recently.

Pollen development and tube growth are affected in the symbiotic mutant of Lotus japonicus, crinkle.

The symbiotic mutant of Lotus japonicus, crinkle (crk), exhibits abnormal nodulation and other alterations in the root hairs, trichomes, and seedpods. Defective nodulation in crk mutant is due to the arrested infection thread growth from the epidermis into the cortex. Here, we describe that crk is also affected in male fertility that causes the production of small pods with few seeds. Under in vitro conditions, pollen germination and tube growth were markedly reduced in the crk mutant. A swollen tip phenotype with disorganized filamentous actin (F-actin) was observed in the mutant pollen tubes after prolonged in vitro culture. During pollen development, the striking difference noted in the mutant was the small size of the microspores that remained spherical. Histological examination of ovule development, as well as outcrosses of the mutant as female to wild type as male, showed no evidence of abnormality in the female gametophyte development. Based on these findings, the Crk gene, aside from its role in the infection process during nodulation, is also involved in male gametophyte development and function. Therefore, this gene represents a connection between nodule symbiosis, polar tip growth, and other plant developmental processes.