EgMADS3 directly regulates EgLPAAT to mediate medium-chain fatty acids (MCFA) anabolism in the mesocarp of oil palm.

KEY MESSAGE: EgMADS3, a pivotal transcription factor, positively regulates MCFA accumulation via binding to the EgLPAAT promoter, advancing lipid content in mesocarp of oil palm. Lipids function as the structural components of cell membranes, which serve as permeable barriers to the external environment of cells. The medium-chain fatty acid in the stored lipids of plants is an important renewable energy. Most research on MCFA production in plant lipid synthesis is based on biochemical methods, and the importance of transcriptional regulation in MCFA synthesis and its incorporation into TAGs needs further research. Oil palm is the most productive oil crop in the world and has the highest productivity among the main oil crops. In this study, the MADS transcription factor (EgMADS3) in the mesocarp of oil palm was characterized. Through the VIGS-virus induced gene silencing, it was determined that the potential target gene of EgMADS3 was related to the biosynthesis of medium-chain fatty acid (MCFA). Transient transformation in protoplasts and qRT-PCR analysis showed that EgMADS3 positively regulated the expression of EgLPAAT. The results of the yeast one-hybrid assays and EMSA indicated the interaction between EgMADS3 and EgLPAAT promoter. Through genetic transformation and fatty acid analysis, it is concluded that EgMADS3 directly regulates the mid-chain fatty acid synthesis pathway of the potential target gene EgLPAAT, thus promotes the accumulation of MCFA and improves the total lipid content. This study is innovative in the functional analysis of the MADS family transcription factor in the metabolism of medium-chain fatty acids (MCFA) of oil palm, provides a certain research basis for improving the metabolic pathway of chain fatty acids in oil palm, and improves the synthesis of MCFA in plants. Our results will provide a reference direction for further research on improving the oil quality through biotechnology of oil palm.

Identification and Analysis of MADS-Box Genes Expressed in the Mesocarp of Oil Palm Fruit (Elaeis guineensis Jacq.).

Oil palm (Elaeis guineensis) is the most important tropical oil-bearing crop species worldwide. MADS-box proteins, which play crucial roles in plant growth and development and are involved in various physiological and biochemical processes, compose one of the largest families of plant transcription factors. In this study, 42 MADS-box genes were screened from the mesocarp transcriptome database of oil palm fruit, and their phylogenetic relationships with Arabidopsis thaliana MADS-box genes were analyzed. Based on the results, MADS-box genes from oil palm mesocarp were classified into four groups: MIKCc-type, MIKC*-type, Mα-type, and Mγ-type MADS-box genes. Members of the subfamilies were classified according to the presence of three specific protein motifs. To explore the differential expression of the MADS-box genes, the dynamic expression of all selected MADS-box genes in oil palm was measured by RNA-seq. The high expression of specific MADS-box genes in the mesocarp of oil palm during different developmental stages indicates that those genes may play important roles in the cell division of and metabolite accumulation in the fruit and could become important targets for fruit development and oil accumulation research in oil palm.

Characterization and functional analysis of the MADS-box EgAGL9 transcription factor from the mesocarp of oil palm (Elaeis guineensis Jacq.).

Oil palm (Elaeis guineensis Jacq.) is one of the most important oil crops in the world, and compared to all oil crops, it has the highest productive efficiency. In the present study, a MADS-box transcription factor of the AGAMOUS class, named EgAGL9, was identified by expression profile analysis in the different developmental stages of oil palm mesocarp. Real-time quantitative PCR results confirmed that the expression of EgAGL9 increased rapidly during the last stages of oil palm mesocarp development. Then, three downstream genes, including EgSAD (Stearoyl-ACP desaturase), EgTSA (Tryptophan synthase) and EgSDH (Succinate dehydrogenase), were screened by ChIP-Seq and data analysis. EMSA analysis verified that EgAGL9 interacted with the promoter regions of EgSAD, EgTSA and EgSDH. Moreover, the expression levels of EgSAD, EgTSA and EgSDH were downregulated in EgAGL9-overexpressing protoplasts and calli of oil palm. Compared to WT, the total lipid content and ratio of unsaturated fatty acids in transgenic calli (including oleic acid, linoleic acid and linolenic acid) were significantly decreased. Together, these results revealed that these three EgAGL9-regulated genes are involved in regulatory pathways in the oil palm mesocarp. Compared with previous studies, the present study provides a new research strategy for understanding of the molecular regulatory pathways of lipid metabolism in mesocarp of oil palm. The obtained results will bring a new perspective for a comprehensive understanding of the regulation of the metabolic accumulation in the oil palm mesocarp.

EgMYB108 regulates very long-chain fatty acid (VLCFA) anabolism in the mesocarp of oil palm.

KEY MESSAGE: EgMYB108 regulates VLCFA anabolism in oil palm. Very long-chain fatty acids (VLCFAs), which are fatty acids with more than 18 C, can not only be used as a form of triglyceride (TAG) but also provide precursors for the biosynthesis of cuticle wax, and they exist in plant epidermal cells in the form of wax in higher plants. However, which and how transcriptional factors (TFs) regulate this process is largely unknown in oil palm. In this study, a MYB transcription factor (EgMYB108) with high expression in the mesocarp of oil palm fruit was characterized. Overexpression of EgMYB108 promoted not only total lipid content but also VLCFA accumulation in oil palm embryoids. Subsequently, transient transformation in protoplasts and qRT-PCR analysis indicated that the EgKCS5 and EgLACS4 genes were significantly increased with the overexpression of EgMYB108. Furthermore, yeast one­hybrid assays, dual-luciferase assays and EMSAs demonstrated that EgMYB108 binds to the promoters of EgKCS5 and EgLACS4 and regulates their transcription. Finally, EgMYB108 interacts with the promoters of EgLACS and EgKCS simultaneously and finally improves the VLCFA and total lipid contents; a pathway summarizing this interaction was depicted.. The results provide new insight into the mechanism by which EgMYB108 regulates lipid and VLCFA accumulation in oil palm.

EgmiR5179 Regulates Lipid Metabolism by Targeting EgMADS16 in the Mesocarp of Oil Palm (Elaeis guineensis).

EgMADS16, one of the MADS-box transcription factors in oil palm, has a high expression level in the late fruit development of the oil palm fruit mesocarp. At the same time, it is also predicted to be the target gene of EgmiR5179, which has been identified in previous research. In this paper, we focused on the function and regulatory mechanism of the EgMADS16 gene in oil palm lipid metabolism. The results indicated that the transcription level of EgMADS16 was highest in the fourth stage, and a dual-luciferase reporter assay proved that the EgMADS16 expression level was downregulated by EgmiR5179. In both the OXEgMADS16 Arabidopsis seeds and oil palm embryonic calli, the total lipid contents were significantly decreased, but the contents of C18:0 and C18:3 in OXEgMADS16 lines were significantly increased. As expected, EgmiR5179 weakened the inhibitory effect of EgMADS16 on the oil contents in transgenic Arabidopsis plants that coexpressed EgmiR5179 and EgMADS16 (OXEgmiR5179-EgMADS16). Moreover, yeast two-hybrid and BiFC analyses suggested that there was an interaction between the EgMADS16 protein and EgGLO1 protein, which had been proven to be capable of regulating fatty acid synthesis in our previous research work. In summary, a model of the molecular mechanism by which miRNA5179 targets EgMADS16 to regulate oil biosynthesis was hypothesized, and the research results provide new insight into lipid accumulation and molecular regulation in oil palm.

CnMADS1, a MADS transcription factor, positively modulates cell proliferation and lipid metabolism in the endosperm of coconut (Cocos nucifera L.).

MAIN CONCLUSION: The function of the first MADS-box transcription factor from endosperm of coconut, CnMADS1, was characterized via seed-specific overexpression in Arabidopsis seeds and further confirmed in protoplasts of coconut. Coconut (Cocos nucifera L.), which belongs to the palm family (Arecaceae), is one of the world's most useful economical tropical crops. However, few genes related to coconut endosperm development have been studied. In previous research, an AGAMOUS-like (AGL) MADS-box transcription factor, named CnMADS1, was identified in the endosperm of coconut through the SSH cDNA library. In this paper, functional characterization of the CnMADS1 gene was carried out by seed-specific overexpression in A. thaliana seeds and protoplasts of coconut. The results indicated that in the twelve independent T2 transgenic Arabidopsis lines with high overexpression of CnMADS1, the size of the mature seeds of transgenic plants was increased significantly (19.64% increase in the long axis and 8.6% increase in the short axis) compared to that of the wild-type seeds. Moreover, the total lipid content also increased significantly in mature seeds of transgenic plants. After comparing the expression of related genes in wild-type and transgenic plants and confirmation by EMSA, AtOSR1, a regulatory gene related to seed size, was proven to be significantly up-regulated by CnMADS1 in transgenic plants. Moreover, the transient transformation of protoplasts of coconut also proved that CnLECRK3 (the homologous gene of AtOSR1 in coconut) is up-regulated by the CnMADS1 gene in the same way. All these results indicated that a similar regulation mode existed in Arabidopsis and the endosperm of coconut and ultimately affected the yield and quality of coconut copra.

A MADS-box gene, EgMADS21, negatively regulates EgDGAT2 expression and decreases polyunsaturated fatty acid accumulation in oil palm (Elaeis guineensis Jacq.).

KEY MESSAGE: EgMADS21 regulates PUFA accumulation in oil palm. Oil palm (Elaeis guineensis Jacq.) is the most productive world oil crop, accounting for 36% of world plant oil production. However, the molecular mechanism of the transcriptional regulation of fatty acid accumulation and lipid synthesis in the mesocarp of oil palm by up- or downregulating the expression of genes involved in related pathways remains largely unknown. Here, an oil palm MADS-box gene, EgMADS21, was screened in a yeast one-hybrid assay using the EgDGAT2 promoter sequence as bait. EgMADS21 is preferentially expressed in early mesocarp developmental stages in oil palm fruit and presents a negative correlation with EgDGAT2 expression. The direct binding of EgMADS21 to the EgDGAT2 promoter was confirmed by electrophoretic mobility shift assay. Subsequently, transient expression of EgMADS21 in oil palm protoplasts revealed that EgMADS21 not only binds to the EgDGAT2 promoter but also negatively regulates the expression of EgDGAT2. Furthermore, EgMADS21 was stably overexpressed in transgenic oil palm embryoids by Agrobacterium-mediated transformation. In three independent transgenic lines, EgDGAT2 expression was significantly suppressed by the expression of EgMADS21. The content of linoleic acid (C18:2) in the three transgenic embryoids was significantly decreased, while that of oleic acid (C18:1) was significantly increased. Combined with the substrate preference of EgDGAT2 identified in previous research, the results demonstrate the molecular mechanism by which EgMADS21 regulates EgDGAT2 expression and ultimately affects fatty acid accumulation in the mesocarp of oil palm.

Multigene engineering of medium-chain fatty acid biosynthesis in transgenic Arabidopsis thaliana by a Cre/LoxP multigene expression system.

Medium-chain fatty acids (MCFAs) are an ideal feedstock for biodiesel and a range of oleochemical products. In this study, different combinations of CnFATB3, CnLPAAT-B and CnKASI from coconut (Cocos nucifera L.) were coexpressed in transgenic Arabidopsis thaliana by a Cre/LoxP multigene expression system. Transgenic lines expressing different combinations of these genes were designated FL (FatB3 + LPAAT-B), FK (FatB3 + KASI) and FLK (FatB3 + LPAAT-B + KASI). The homozygous seeds of transgenic Arabidopsis thaliana expressing high levels of these genes were screened, and their fatty acid composition and lipid contents were determined. Compared with its content in wild-type A. thaliana, the lauric acid (C12:0) content was significantly increased by at least 395%, 134% and 124% in FLK, FL and FK seeds, respectively. Meanwhile, the myristic acid (C14:0) content was significantly increased by at least 383%, 106% and 102% in FL, FLK and FK seeds, respectively, compared to its level in wild-type seeds. Therefore, the FLK plants exhibited the best effects to increase the level of C12:0, and FL expressed the optimal combination of genes to increase the level of 14:0 MCFA.

Isolation and characterization of the EgWRI1 promoter from oil palm (Elaeis guineensis Jacq.) and its response to environmental stress and ethylene.

WRI1 is a plant-specific transcription factor that enhances the accumulation of oils through the upregulation of the expression of genes involved in glycolysis and fatty acid biosynthesis. In this study, the EgWRI1 promoter from oil palm was isolated and characterized in transgenic Arabidopsis. The sequence analysis results revealed that various putative plant regulatory elements are present in the EgWRI1 promoter region. The EgWRI1 promoter and beta-glucuronidase (GUS) reporter gene were transcriptionally fused and transformed into Arabidopsis thaliana. Histochemical analysis revealed that GUS staining was very strong in whole seedlings, especially the stems, leaves, and siliques. Moreover, GUS staining was strong in the silique coats but weak in the seeds. Furthermore, to detect whether EgWRI1 was induced by environmental stress, we detected the expression efficiency of the EgWRI1 promoter in transgenic Arabidopsis treated with low temperature, darkness, and exogenous ethylene. The results showed that the activity of the EgWRI1 promoter was induced by darkness but suppressed significantly when exposed to exogenous ethylene. When treated with low temperature, the activity of the EgWRI1 promoter was first reduced after 24 hours but recovered after 48 hours. Taken together, these results reveal the features of the EgWRI1 promoter from oil palm, which will be helpful for improving oil accumulation in oil palm via reasonable cultivation methods.

Identification and function analysis of a type 2 diacylglycerol acyltransferase (DGAT2) from the endosperm of coconut (Cocos nucifera L.).

Coconut (Cocos nucifera L.) is one of the most characteristic plants of tropical areas. Coconut oil and its derivatives have been widely used in various industries. In this paper, a type 2 diacylglycerol acyltransferase (DGAT2), which is one of the key enzymes involved in triacylglycerol (TAG) biosynthesis, was first characterized in coconut pulp (endosperm). The results indicated that CoDGAT2 was highly expressed in coconut pulp approximately 7 months after pollination. The heterologous expression of CoDGAT2 in the mutant yeast H1246 restored TAG biosynthesis in the yeast, which exhibited substrate preference for two unsaturated fatty acids (UFAs), palmitoleic acid (C16:1) and oleic acid (C18:1). Moreover, the seed-specific overexpression of CoDGAT2 in Arabidopsis thaliana led to a significant increase in the linoleic acid (C18:2) content (approximately 6%) compared with that in the wild type. In contrast, the proportions of eicosadienoic acid (C20:1) and arachidic acid (C20:0) were decreased. These results offer new insights on the function of CoDGAT2 in coconut and provide a novel molecular target for lipid genetic modification to change the fatty acid (FA) composition of oils.

Genome-wide association analysis of the lipid and fatty acid metabolism regulatory network in the mesocarp of oil palm (Elaeis guineensis Jacq.) based on small noncoding RNA sequencing.

Oil palm (Elaeis guineensis Jacq.) is the highest oil-yielding crop in the plant kingdom and accumulates 90% of palm oil in the mesocarp. However, the regulatory mechanisms of lipid and fatty acid (FA) metabolism in oil palm are just beginning to be understood, and more studies are needed, especially in the understanding of small noncoding RNA (ncRNA) and mRNA. Based on the deep sequencing of small noncoding RNAs and the degradome in five developmental mesocarp stages, 452 microRNAs (miRNAs), including 170 conserved known-miRNAs (kn-miRNAs) and 282 novel-miRNA (nov-miRNAs), were identified. After predicting the targets of those miRNAs to 37 FA synthesis-related genes, we found that 22 kn-miRNAs and 14 nov-miRNAs might be involved in FA metabolism pathways. Among them, eg-miR156c, eg-miR397, eg-miR444b and nov-miR129 regulated FA synthesis in plastids and the transport of FA-ACP from plastids to the endoplasmic reticulum by targeting acetyl-CoA carboxylase 1 (ACC1), long-chain acyl-CoA synthetase 9 (LACS9), LACS4 and enoyl-ACP reductase (ENR), respectively. Nov-miR138 and nov-miR59 targeted glycerol-3-phosphate acyltransferase (GPAT), and nov-miR274 targeted phosphatidate phosphatase 1 (PAP1). Both target genes are involved in triacylglycerol synthesis in the endoplasmic reticulum. Eg-miR156e and eg-miR156j played pivotal roles by targeting ß-ketoacyl-CoA synthase 12 (KCS12), and nov-miR201 targets very-long-chain enoyl-CoA reductase (ECR). Several miRNAs were also predicted to indirectly regulate FA synthesis and lipid metabolism through the squamosa promoter-binding protein-like gene (SPL), NAC and MYB transcription factors. As a whole, indications of a complex and extensive miRNA-mRNA regulatory network associated with FA metabolism in the mesocarp of the oil palm is presented. The results help to broaden the knowledge of potential mechanisms that might be regulated by miRNAs through modulation of the expression of FA-related target gene metabolism in the oil palm.

Identification and characterization of a plastidial ω-3 fatty acid desaturase EgFAD8 from oil palm (Elaeis guineensis Jacq.) and its promoter response to light and low temperature.

In higher plants, ω-3 fatty acid desaturases are the key enzymes in the biosynthesis of alpha-linolenic acid (18:3), which plays key roles in plant metabolism as a structural component of both storage and membrane lipids. Here, the first ω-3 fatty acid desaturase gene was identified and characterized from oil palm. The bioinformatic analysis indicated it encodes a temperature-sensitive chloroplast ω-3 fatty acid desaturase, designated as EgFAD8. The expression analysis revealed that EgFAD8 is highly expressed in the oil palm leaves, when compared with the expression in the mesocarp. The heterologous expression of EgFAD8 in yeast resulted in the production of a novel fatty acid 18:3 (about 0.27%), when fed with 18:2 in the induction culture. Furthermore, to detect whether EgFAD8 could be induced by the environment stress, we detected the expression efficiency of the EgFAD8 promoter in transgenic Arabidopsis treated with low temperature and darkness, respectively. The results indicated that the promoter of EgFAD8 gene could be significantly induced by low temperature and slightly induced by darkness. These results reveal the function of EgFAD8 and the feature of its promoter from oil palm fruits, which will be useful for understanding the fuction and regulation of plastidial ω-3 fatty acid desaturases in higher plants.

Characterization and Functional Analysis of a Type 2 Diacylglycerol Acyltransferase (DGAT2) Gene from Oil Palm (Elaeis guineensis Jacq.) Mesocarp in Saccharomyces cerevisiae and Transgenic Arabidopsis thaliana.

Oil palm (Elaeis guineensis Jacq.) is the highest oil-yielding plant in the world, storing 90 and 60% (dry weight) oil in its mesocarp and kernel, respectively. To gain insights into the oil accumulation mechanism, one of the key enzymes involved in triacylglycerol (TAG) biosynthesis, a Type 2 diacylglycerol acyltransferase (DGAT2) from oil palm, was characterized for its in vivo activity. EgDGAT2 is highly expressed in mesocarp during the last two developmental stages while large amounts of oil are accumulated at the highest rate during ripening. Heterologous expression of EgDGAT2 in mutant yeast H1246 restored TAG biosynthesis with substrate preference toward unsaturated fatty acids (FAs) (16:1 and 18:1). Furthermore, seed-specific overexpression of EgDGAT2 in Arabidopsis thaliana enhanced the content of polyunsaturated FAs 18:2 and 18:3 (each by 6 mol%) in seed TAGs, when compared to that from wild-type Arabidopsis. In turn, the proportion of 18:0 and 20:0 FAs in seed TAGs from EgDGAT2 transgenic lines decreased accordingly. These results provide new insights into understanding the in vivo activity of EgDGAT2 from oil palm mesocarp, which will be of importance for metabolic enhancement of unsaturated FAs production.

Characterization and Ectopic Expression of CoWRI1, an AP2/EREBP Domain-Containing Transcription Factor from Coconut (Cocos nucifera L.) Endosperm, Changes the Seeds Oil Content in Transgenic Arabidopsis thaliana and Rice (Oryza sativa L.).

Coconut (Cocos nucifera L.) is a key tropical crop and a member of the monocotyledonous family Arecaceae (Palmaceae). Few genes and related metabolic processes involved in coconut endosperm development have been investigated. In this study, a new member of the WRI1 gene family was isolated from coconut endosperm and was named CoWRI1. Its transcriptional activities and interactions with the acetyl-CoA carboxylase (BCCP2) promoter of CoWRI1 were confirmed by the yeast two-hybrid and yeast one-hybrid approaches, respectively. Functional characterization was carried out through seed-specific expression in Arabidopsis and endosperm-specific expression in rice. In transgenic Arabidopsis, high over-expressions of CoWRI1 in seven independent T2 lines were detected by quantitative real-time PCR. The relative mRNA accumulation of genes encoding enzymes involved in either fatty acid biosynthesis or triacylglycerols assembly (BCCP2, KASI, MAT, ENR, FATA, and GPDH) were also assayed in mature seeds. Furthermore, lipid and fatty acids C16:0 and C18:0 significantly increased. In two homozygous T2 transgenic rice lines (G5 and G2), different CoWRI1 expression levels were detected, but no CoWRI1 transcripts were detected in the wild type. Analyses of the seed oil content, starch content, and total protein content indicated that the two T2 transgenic lines showed a significant increase (P < 0.05) in seed oil content. The transgenic lines also showed a significant increase in starch content, whereas total protein content decreased significantly. Further analysis of the fatty acid composition revealed that palmitic acid (C16:0) and linolenic acid (C18:3) increased significantly in the seeds of the transgenic rice lines, but oleic acid (C18:1) levels significantly declined.

Identification of a Δ12 fatty acid desaturase from oil palm (Elaeis guineensis Jacq.) involved in the biosynthesis of linoleic acid by heterologous expression in Saccharomyces cerevisiae.

Oil palm (Elaeis guineensis Jacq.) is one of the highest oil-yield crops in the world. A Δ12-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from oil palm and their functions identified. The open reading frames (ORFs) of egFAD2 (GenBank accession: KT023602) consisted of 1176bp and code for 391 amino acids. Their deduced polypeptides showed 75-93% identity to microsomal Δ12-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. RT-PCR experiment indicated that the egFAD2 gene exhibited the highest accumulation in the mesocarp of fruits at 120-140 DAP (i.e. the fourth period of fruit development) and, despite having different expression levels, the other four stages were at significantly lower levels compared with the fourth stage. Plasmid pYES2-egFAD2 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Yeast cells transformed with plasmid constructs containing egFAD12 produced an appreciable amount of linoleic acids (18:2(Δ9,)(12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ12-desaturase enzymes.

Identification and computational annotation of genes differentially expressed in pulp development of Cocos nucifera L. by suppression subtractive hybridization.

BACKGROUND: Coconut (Cocos nucifera L.) is one of the world's most versatile, economically important tropical crops. Little is known about the physiological and molecular basis of coconut pulp (endosperm) development and only a few coconut genes and gene product sequences are available in public databases. This study identified genes that were differentially expressed during development of coconut pulp and functionally annotated these identified genes using bioinformatics analysis. RESULTS: Pulp from three different coconut developmental stages was collected. Four suppression subtractive hybridization (SSH) libraries were constructed (forward and reverse libraries A and B between stages 1 and 2, and C and D between stages 2 and 3), and identified sequences were computationally annotated using Blast2GO software. A total of 1272 clones were obtained for analysis from four SSH libraries with 63% showing similarity to known proteins. Pairwise comparing of stage-specific gene ontology ids from libraries B-D, A-C, B-C and A-D showed that 32 genes were continuously upregulated and seven downregulated; 28 were transiently upregulated and 23 downregulated. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT), phospholipase D, acetyl-CoA carboxylase carboxyltransferase beta subunit, 3-hydroxyisobutyryl-CoA hydrolase-like and pyruvate dehydrogenase E1 ß subunit were associated with fatty acid biosynthesis or metabolism. Triose phosphate isomerase, cellulose synthase and glucan 1,3-ß-glucosidase were related to carbohydrate metabolism, and phosphoenolpyruvate carboxylase was related to both fatty acid and carbohydrate metabolism. Of 737 unigenes, 103 encoded enzymes were involved in fatty acid and carbohydrate biosynthesis and metabolism, and a number of transcription factors and other interesting genes with stage-specific expression were confirmed by real-time PCR, with validation of the SSH results as high as 66.6%. Based on determination of coconut endosperm fatty acids content by gas chromatography-mass spectrometry, a number of candidate genes in fatty acid anabolism were selected for further study. CONCLUSION: Functional annotation of genes differentially expressed in coconut pulp development helped determine the molecular basis of coconut endosperm development. The SSH method identified genes related to fatty acids, carbohydrate and secondary metabolites. The results will be important for understanding gene functions and regulatory networks in coconut fruit.

Cloning and functional expression of a cDNA encoding stearoyl-ACP Δ9-desaturase from the endosperm of coconut (Cocos nucifera L.).

Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176 bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC-MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future.

[HER2 protein testing in gastric cancer: a retrospective analysis of 1 471 cases during two different periods in a single medical center].

OBJECTIVE: To study the potential factors in influencing the performance of immunohistochemical testing for HER2 protein in gastric cancers. METHODS: The HER2 protein expression status of 1 471 surgically resected archival gastric cancer cases in Drum Tower Hospital collected during two different periods was retrospectively analyzed. The materials included 957 cases tested during the period from 2007 to 2009 (group 1) and 514 cases from 2012 to 2013 (group 2). The test procedures and results observed during these two periods were compared. RESULTS: The percentages of score 3 HER2 protein expression (14.4%, 74/514 versus 9.5%, 91/957) and score 2 or score 3 HER2 protein expression (27.2%, 140/514 versus 21.7%, 208/957) were both higher in group 2 than in group 1 (P < 0.05). In group 1, the cancer tissue was fixed in 10% formalin, stained manually with HER2 antibody A0485 (Dako) and assessed by different pathologists.In group 2, the tissue was fixed in 10% neutral buffered formalin (pH 7.2), stained using automated immunostaining system (Roche Benchmark XT) with HER2 antibody 4B5 (Ventana) and assessed by a specialized team of pathologists. CONCLUSION: The results of HER2 immunostaining in gastric cancer are influenced by a number of factors including type of fixative, clone number of primary antibody, staining methods and experience of pathologists.

Molecular cloning and characterisation of an acyl carrier protein thioesterase gene (CocoFatB1) expressed in the endosperm of coconut (Cocos nucifera) and its heterologous expression in Nicotiana tabacum to engineer the accumulation of different fatty acids.

Coconut (Cocos nucifera L.) contains large amounts of medium chain fatty acids, which mostly recognise acyl-acyl carrier protein (ACP) thioesterases that hydrolyse acyl-ACP into free fatty acids to terminate acyl chain elongation during fatty acid biosynthesis. A full-length cDNA of an acyl-ACP thioesterase, designated CocoFatB1, was isolated from cDNA libraries prepared from coconut endosperm during fruit development. The gene contained an open reading frame of 1254 bp, encoding a 417-amino acid protein. The amino acid sequence of the CocoFatB1 protein showed 100% and 95% sequence similarity to CnFatB1 and oil palm (Elaeis guineensis Jacq.) acyl-ACP thioesterases, respectively. Real-time fluorescent quantitative PCR analysis indicated that the CocoFatB1 transcript was most abundant in the endosperm from 8-month-old coconuts; the leaves and endosperm from 15-month-old coconuts had ~80% and ~10% of this level. The CocoFatB1 coding region was overexpressed in tobacco (Nicotiana tabacum L.) under the control of the seed-specific napin promoter following Agrobacterium tumefaciens-mediated transformation. CocoFatB1 transcript expression varied 20-fold between different transgenic plants, with 21 plants exhibiting detectable levels of CocoFatB1 expression. Analysis of the fatty acid composition of transgenic tobacco seeds showed that the levels of myristic acid (14 : 0), palmitic acid (16 : 0) and stearic acid (18 : 0) were increased by 25%, 34% and 17%, respectively, compared with untransformed plants. These results indicated that CocoFatB1 acts specifically on 14 : 0-ACP, 16 : 0-ACP and 18 : 0-ACP, and can increase medium chain saturated fatty acids. The gene may valuable for engineering fatty acid metabolism in crop improvement programmes.

Effect of harvest date on the nutritional quality and antioxidant capacity in 'Hass' avocado during storage.

The effect of harvest date on nutritional compounds and antioxidant activity (AOC) in avocado (Persea americana Mill. cv Hass) fruit during storage was determined. The fruits were harvested at seven different dates and ripened at 25 °C following 21 or 35 days of cold storage. The results indicated that the phenolic and glutathione contents were increased and the ascorbic acid content was not significantly different in early harvested fruit (January to March), and the phenolic, ascorbic acid and glutathione contents were increased slightly and then decreased on late harvested fruit (April to June). Similar trends were observed in the changes of AOC. Furthermore, AOC in early harvested fruit after storage for 35 days was much higher than that in late harvested fruit after storage for 21 days. Therefore, avocado can be harvested earlier for economic benefits according to the market and can keep high nutritional value for human health benefits.