Identification and Analysis of Genes Involved in Double Fertilization in Rice.

Double fertilization is a key determinant of grain yield, and the failure of fertilization during hybridization is one important reason for reproductive isolation. Therefore, fertilization has a very important role in the production of high-yield and well-quality hybrid of rice. Here, we used RNA sequencing technology to study the change of the transcriptome during double fertilization with the help of the mutant fertilization barrier (feb) that failed to finish fertilization process and led to seed abortion. The results showed that 1669 genes were related to the guided growth of pollen tubes, 332 genes were involved in the recognition and fusion of the male-female gametes, and 430 genes were associated with zygote formation and early free endosperm nuclear division. Among them, the genes related to carbohydrate metabolism; signal transduction pathways were enriched in the guided growth of pollen tubes, the genes involved in the photosynthesis; fatty acid synthesis pathways were activated by the recognition and fusion of the male-female gametes; and the cell cycle-related genes might play an essential role in zygote formation and early endosperm nuclear division. Furthermore, among the 1669 pollen tube-related genes, it was found that 7 arabinogalactan proteins (AGPs), 1 cysteine-rich peptide (CRP), and 15 receptor-like kinases (RLKs) were specifically expressed in anther, while 2 AGPs, 7 CRPs, and 5 RLKs in pistil, showing obvious unequal distribution which implied they might play different roles in anther and pistil during fertilization. These studies laid a solid foundation for revealing double fertilization mechanism of rice and for the follow-up investigation.

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.

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.

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.

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.