Overexpression of the soybean transcription factor GmDof4 significantly enhances the lipid content of Chlorella ellipsoidea.

BACKGROUND: The lipid content of microalgae is regarded as an important indicator for biodiesel. Many attempts have been made to increase the lipid content of microalgae through biochemical and genetic engineering. Significant lipid accumulation in microalgae has been achieved using biochemical engineering, such as nitrogen starvation, but the cell growth was severely limited. However, enrichment of lipid content in microalgae by genetic engineering is anticipated. In this study, GmDof4 from soybean (Glycine max), a transcription factor affecting the lipid content in Arabidopsis, was transferred into Chlorella ellipsoidea. We then investigated the molecular mechanism underlying the enhancement of the lipid content of transformed C. ellipsoidea. RESULTS: We constructed a plant expression vector, pGmDof4, and transformed GmDof4 into C. ellipsoidea by electroporation. The resulting expression of GmDof4 significantly enhanced the lipid content by 46.4 to 52.9%, but did not affect the growth rate of the host cells under mixotrophic culture conditions. Transcriptome profiles indicated that 1,076 transcripts were differentially regulated: of these, 754 genes were significantly upregulated and 322 genes were significantly downregulated in the transgenic strains under mixotrophic culture conditions. There are 22 significantly regulated genes (|log2 ratio| >1) involved in lipid and fatty acid metabolism. Quantitative real-time PCR and an enzyme activity assay revealed that GmDof4 significantly up-regulated the gene expression and enzyme activity of acetyl-coenzyme A carboxylase, a key enzyme for fatty acid synthesis, in transgenic C. ellipsoidea cells. CONCLUSIONS: The hetero-expression of a transcription factor GmDof4 gene from soybean can significantly increase the lipid content but not affect the growth rate of C. ellipsoidea under mixotrophic culture conditions. The increase in lipid content could be attributed to the large number of genes with regulated expression. In particular, the acetyl-coenzyme A carboxylase gene expression and enzyme activity were significantly upregulated in the transgenic cells. Our research provides a new way to increase the lipid content of microalgae by introducing a specific transcription factor to microalgae strains that can be used for the biofuel and food industries.

Identification and characterization of high-molecular-weight glutenin subunits from Agropyron intermedium.

High-molecular-weight glutenin subunit (HMW-GS) is a primary determinant of processing quality of wheat. Considerable progress has been made in understanding the structure, function and genetic regulation of HMW-GS in wheat and some of its related species, but less is known about their orthologs in Agropyron intermedium, a useful related species for wheat improvement. Here seven HMW-GSs in Ag. intermedium were identified using SDS-PAGE and Western blotting experiments. Subsequently, the seven genes (Glu-1Aix1 ∼ 4 and Glu-1Aiy1 ∼ 3) encoding the seven HMW-GSs were isolated using PCR technique with degenerate primers, and confirmed by bacterial expression and Western blotting. Sequence analysis indicated that the seven Ag. intermedium HMW-GSs shared high similarity in primary structure to those of wheat, but four of the seven subunits were unusually small compared to the representatives of HMW-GS from wheat and two of them possessed extra cysteine residues. The alignment and clustering analysis of deduced amino acid sequences revealed that 1Aix1 and 1Aiy1 subunits had special molecular structure, belonging to the hybrid type compounding between typical x- and y-type subunit. The xy-type subunit 1Aix1 is composed of the N-terminal of x-type and C-terminal of y-type, whereas yx-type subunit 1Aiy1 comprises the N-terminal of y-type and C-terminal of x-type. This result strongly supported the hypothesis of unequal crossover mechanism that might generate the novel coding sequence for the hybrid type of HMW-GSs. In addition to the aforementioned, the other novel characteristics of the seven subunits were also discussed. Finally, phylogenetic analysis based on HMW-GS genes was carried out and provided new insights into the evolutionary biology of Ag. intermedium.

Identification of the substrate recognition region in the Δ6-fatty acid and Δ8-sphingolipid desaturase by fusion mutagenesis.

Δ8-sphingolipid desaturase and Δ6-fatty acid desaturase share high protein sequence identity. Thus, it has been hypothesized that Δ6-fatty acid desaturase is derived from Δ8-sphingolipid desaturase; however, there is no direct proof. The substrate recognition regions of Δ6-fatty acid desaturase and Δ8-sphingolipid desaturase, which aid in understanding the evolution of these two enzymes, have not been reported. A blackcurrant Δ6-fatty acid desaturase and a Δ8-sphingolipid desaturase gene, RnD6C and RnD8A, respectively, share more than 80 % identity in their coding protein sequences. In this study, a set of fusion genes of RnD6C and RnD8A were constructed and expressed in yeast. The Δ6- and Δ8-desaturase activities of the fusion proteins were characterized. Our results indicated that (1) the exchange of the C-terminal 172 amino acid residues can lead to a significant decrease in both desaturase activities; (2) amino acid residues 114-174, 206-257, and 258-276 played important roles in Δ6-substrate recognition, and the last two regions were crucial for Δ8-substrate recognition; and (3) amino acid residues 114-276 of Δ6-fatty acid desaturase contained the substrate recognition site(s) responsible for discrimination between ceramide (a substrate of Δ8-sphingolipid desaturase) and acyl-PC (a substrate of Δ6-fatty acid desaturase). Substituting the amino acid residues 114-276 of RnD8A with those of RnD6C resulted in a gain of Δ6-desaturase activity in the fusion protein but a loss in Δ8-sphingolipid desaturase activity. In conclusion, several regions important for the substrate recognition of Δ8-sphingolipid desaturase and Δ6-fatty acid desaturase were identified, which provide clues in understanding the relationship between the structure and function in desaturases.

New insights into the organization, recombination, expression and functional mechanism of low molecular weight glutenin subunit genes in bread wheat.

The bread-making quality of wheat is strongly influenced by multiple low molecular weight glutenin subunit (LMW-GS) proteins expressed in the seeds. However, the organization, recombination and expression of LMW-GS genes and their functional mechanism in bread-making are not well understood. Here we report a systematic molecular analysis of LMW-GS genes located at the orthologous Glu-3 loci (Glu-A3, B3 and D3) of bread wheat using complementary approaches (genome wide characterization of gene members, expression profiling, proteomic analysis). Fourteen unique LMW-GS genes were identified for Xiaoyan 54 (with superior bread-making quality). Molecular mapping and recombination analyses revealed that the three Glu-3 loci of Xiaoyan 54 harbored dissimilar numbers of LMW-GS genes and covered different genetic distances. The number of expressed LMW-GS in the seeds was higher in Xiaoyan 54 than in Jing 411 (with relatively poor bread-making quality). This correlated with the finding of higher numbers of active LMW-GS genes at the A3 and D3 loci in Xiaoyan 54. Association analysis using recombinant inbred lines suggested that positive interactions, conferred by genetic combinations of the Glu-3 locus alleles with more numerous active LMW-GS genes, were generally important for the recombinant progenies to attain high Zeleny sedimentation value (ZSV), an important indicator of bread-making quality. A higher number of active LMW-GS genes tended to lead to a more elevated ZSV, although this tendency was influenced by genetic background. This work provides substantial new insights into the genomic organization and expression of LMW-GS genes, and molecular genetic evidence suggesting that these genes contribute quantitatively to bread-making quality in hexaploid wheat. Our analysis also indicates that selection for high numbers of active LMW-GS genes can be used for improvement of bread-making quality in wheat breeding.

Identification and functional analysis of the genes encoding Delta6-desaturase from Ribes nigrum.

Gamma-linolenic acid (gamma-linolenic acid, GLA; C18:3 Delta(6, 9, 12)) belongs to the omega-6 family and exists primarily in several plant oils, such as evening primrose oil, blackcurrant oil, and borage oil. Delta(6)-desaturase is a key enzyme involved in the synthesis of GLA. There have been no previous reports on the genes encoding Delta(6)-desaturase in blackcurrant (Ribes nigrum L.). In this research, five nearly identical copies of Delta(6)-desaturase gene-like sequences, named RnD8A, RnD8B, RnD6C, RnD6D, and RnD6E, were isolated from blackcurrant. Heterologous expression in Saccharomyces cerevisiae and/or Arabidopsis thaliana confirmed that RnD6C/D/E were Delta(6)-desaturases that could use both alpha-linolenic acids (ALA; C18:3 Delta(9,12,15)) and linoleic acid (LA; C18:2 Delta(9,12)) precursors in vivo, whereas RnD8A/B were Delta(8)-sphingolipid desaturases. Expression of GFP tagged with RnD6C/D/E showed that blackcurrant Delta(6)-desaturases were located in the mitochondrion (MIT) in yeast and the endoplasmic reticulum (ER) in tobacco. GC-MS results showed that blackcurrant accumulated GLA and octadecatetraenoic acids (OTA; C18:4 Delta(6,9,12,15)) mainly in seeds and a little in other organs and tissues. RT-PCR results showed that RnD6C and RnD6E were expressed in all the tissues at a low level, whereas RnD6D was expressed at a high level only in seeds, leading to the accumulation of GLA and OTA in seeds. This research provides new insights to our understanding of GLA synthesis and accumulation in plants and the evolutionary relationship of this class of desaturases, and new clues as to the amino acid determinants which define precise enzyme activity.

Proteomic response to iron deficiency in tomato root.

To know the root adjustment in response to iron deficiency, differentially displayed proteins in tomato roots of wild type and its iron uptake inefficient mutant T3238fer were analyzed by 2-DE and MALDI-TOF MS-based proteomic method under iron sufficiency and deficiency. Ninety-seven proteins were identified, 63 of them were classified in various metabolic pathways. About 40 proteins involved in starch degradation, TCA and ascorbate cycles were upregulated under iron deficiency and grouped in a network together with glycolysis, whereas proteins for fructose metabolism were decreased. Proteins involved in methionine synthesis, cell wall synthesis, mitochondria ATP synthesis, vacuole ATPase, HSP70/90, etc. also revealed enhanced expression under iron deficiency, while proteins about redox homeostasis, transcription factors, kinases, etc. showed diversified changes. The responses are closely associated with energy metabolism, organic acid formation, root morphological change, redox and sulfur homeostasis, and signal transduction, which enhance iron uptake, reutilization and other adaptive changes. Most of the proteins affected by iron deficiency and fer mutation showed similar effect on individual proteins or pathways, but the independent function of FER to iron deficiency were statistically indicated.

Characterization of the HMW glutenin subunits from Aegilops searsii and identification of a novel variant HMW glutenin subunit.

High molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe a more detailed characterization of the HMW glutenin subunits from Aegilops searsii, which is diploid and contains the S(s) genome related to the S genome of Aegilops speltoides and the A, B and D genomes of hexaploid wheat. SDS-PAGE experiments revealed two subunits (one x and one y) for each of the nine Ae. searsii accessions analyzed, indicating that the HMW glutenin subunit gene locus of Ae. searsii is similar to the Glu-1 locus found in wheat in containing both x and y genes. The primary structure of the four molecularly cloned subunits (from two Ae. searsii accessions) was highly similar to that of the previously reported x and y subunits. However, in one accession (IG49077), the last 159 residues of the x subunit (1S(s)x49077), which contained the sequence element GHCPTSPQQ, were identical to those of the y subunit (1S(s)y49077) from the same accession. Consequently, 1S(s)x49077 contains an extra cysteine residue located at the C-terminal part of its repetitive domain, which is novel compared to the x-type subunits reported so far. Based on this and previous studies, the structure and expression of the Glu-1 locus in Ae. searsii is discussed. A hypothesis on the genetic mechanism generating the coding sequence for the novel 1S(s)x49077 subunit is presented.