**3. Evolutionary relationship between coconut palm and oil palm**

Coconut and oil palm are important oil trees grown in tropical region and closely related species with approximately 50% lauric acid (C12:0) in their endosperm. There are 806 and 840 lipid-related genes annotated for coconut and oil palm, respectively [13]. The majority of lipid-related genes between coconut and oil palm were homologous genes, while 72.8% (438/601) of genes in coconut palm were located in homologous segments with oil palm. The two species have a close evolutionary relationship between predominant gene isoforms and high conservation of gene expression bias in the lipid metabolism pathways.

Since coconut and oil palm have high lauric acid (C12:0) in their endosperm, key genes responsible for MCFA also shared high homology in gene copy and expression pattern. Both coconut and oil palm have five FATB genes, but only three *EgFatB* genes highly expressed in oil palm mesocarp or endosperm and four *CnFatB* genes were highly expressed in endosperm or embryo. Homologous gene pair—*CnFatB3* and *EgFatB3—*were both highly expressed in their endosperms, which were validated as key genes for MCFA biosynthesis [3, 6]. Another key enzyme—LPAAT, three AtLPAAT1, or AtLPAAT2 orthologs were found in each of coconut and oil palm [5]. The LPAAT1 genes were clustered into class I and class II, and the class I genes of both species had higher expression levels in endosperm tissue. Moreover, the LPAAT2 genes were also clustered into two classes, and genes in class II had low or no expression.

For the key transcription factor associated with lipid synthesis—*WRI1*, Xiao et al. [5] identified three *WRI1* genes in coconut and six in oil palm and classified

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*Genes Involved in Lipid Metabolism in Coconut DOI: http://dx.doi.org/10.5772/intechopen.90998*

*Arabidopsis*, rice, yeast or *Escherichia coli (E. coli)*.

**4.1 Testing enzyme activity in vitro**

(LPAAT) had been modified.

**4.2 Testing enzyme activity in vivo**

deposition at the sn-2 position [11].

substrates [10].

the genes into three groups based on conserved amino acid sequences. The coconut and oil palm *WRI1* genes in the same group indicated the same expression pattern: group I was highly expressed both in the coconut endosperm and the oil palm

Coconut palm has a long life cycle and takes 5–10 years to start reproductive stage. Since that, using gene overexpression or knockout to analyze gene function in its own plant system will take years to observe the traits related to fruits. At present, no stable transformation system has been constructed in coconut palm. The convenient ways to validate gene function in coconut are testing biochemical feature of proteins *in vitro* or transforming gene into a heterologous system, such as

Lipid metabolism is composed of more than 120 enzymatic reactions. Validation of gene function related to lipid metabolism could be done by testing enzyme activity in vitro. Davies et al. have isolated CnLPAAT protein from immature coconut seeds and tested the LPAAT activity by adding Acyl-CoA and LPA as

Laurate is found enriched in sn-2, which indicates that a laurate-CoApreferring LPAAT is active during endosperm maturation. Davies et al. were able to detect such an enzyme from this tissue, which allowed Knutzon et al. [11] to perform protein purification and cloning of a cDNA encoding the 299-amino acid CLP protein from coconut. When expressed in *E. coli*, and using 12:0-LPA as an acceptor, this enzyme preferred medium-chain CoAs over 18:1-CoA as acyl donors. This is a direct evidence that in coconut endosperm, not only had the common fatty acid biosynthesis pathway been modified to produce almost entirely saturated medium chains but at least one enzyme of lipid biosynthesis

Gene function validation has been conducted through gene overexpression in heterologous plant systems which have stable gene transformation system, such as *Arabidopsis*, rice, and tobacco. Functional characterization of *CnWRI1* was done by gene overexpression in *Arabidopsis* and rice [15]. Overexpression of CnWRI1 in *Arabidopsis* seeds caused fatty acid composition changes but not for oil content, while overexpression of the gene in rice endosperm increased the starch content and decreased the protein contents [15]. For gene function validation of CnLPAAT (CCG001603.1), this gene was overexpressed in a transgenic oilseed (*Brassica napus*) plant, which expressed a 12:0-ACP thioesterase from California bay laurel (*Umbellularia californica*). The transgenic lines that coexpressed a 12:0-ACP thioesterase and CnLPAAT had increase laurate content from 50 mol% to total laurate levels, which suggested that CnLPAAT facilitates efficient laurate

Transient transgenic expression system of tobacco is also widely used for gene function analysis. Genes belonging to lipid metabolism were also validated by this system, investigating the possibility of oil production in non-sees biomass [18].

endosperm/mesocarp; group II or III has very low or no expression.

**4. Methods used in validation gene function in coconut palm**

*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

point between storage and membrane lipid synthesis. Coconut palm has three orthologs of AT2G19450 (*AtDGAT1*) and two orthologs of AT3G51520 (*AtDGAT2*). Coconut *DGATs* genes had higher expression level in coconut endosperm than in the leaf and embryo, especially for *DGAT1* isoform CCG007098.3 and *DGAT2* isoform

Zheng et al. cloned a DGAT2 gene from coconut pulp and transferred the gene into the deficient yeast H1246 and *Arabidopsis* [13]. The DGAT2 gene that is expressed in the deficient yeast had DGAT catalysis activity and restored TAG synthesis in the yeast. Further lipid composition analysis showed that *CnDGAT2* has a substrate preference for two UFAs (C16:1 and C18:1) in yeast and linoleic acid (C18:2) in transgenic plants. These results provide knowledge on CnDGAT2 and

*WRINKLED1* (WRI1, AT3G54320) directly controls the transcriptional activation of the fatty acid biosynthetic pathway in *Arabidopsis* and belongs to the APETALA2-ethylene-responsive element-binding protein (AP2-EREBP) family [17]. The ortholog of *AtWRI1* in oil palm was validated as a key transcription factor associated with lipid synthesis [3]. In coconut, three AT3G54320 orthologs were found— CCG005292.1, CCG012597.1, and CCG019741.1. CCG005292.1 and CCG012597.1 were expressed in the endosperm but had low expression in leaf and endosperm, while CCG019741.1 has no expression in leaf, embryo, or endosperm [5]. The *CnWRI1* gene copy (CCG012597.1) validated its interaction with the promoter sequence of acetyl-CoA carboxylase by yeast one-hybrid system [15]. Overexpression of *CnWRI1* (CCG012597.1) specifically in *Arabidopsis* seed showed an increase of palmitic acid

(C16:0) and linolenic acid (C18:3) but a decrease in oleic acid content [15].

**3. Evolutionary relationship between coconut palm and oil palm**

gene expression bias in the lipid metabolism pathways.

related species with approximately 50% lauric acid (C12:0) in their endosperm. There are 806 and 840 lipid-related genes annotated for coconut and oil palm, respectively [13]. The majority of lipid-related genes between coconut and oil palm were homologous genes, while 72.8% (438/601) of genes in coconut palm were located in homologous segments with oil palm. The two species have a close evolutionary relationship between predominant gene isoforms and high conservation of

Coconut and oil palm are important oil trees grown in tropical region and closely

Since coconut and oil palm have high lauric acid (C12:0) in their endosperm, key genes responsible for MCFA also shared high homology in gene copy and expression pattern. Both coconut and oil palm have five FATB genes, but only three *EgFatB* genes highly expressed in oil palm mesocarp or endosperm and four *CnFatB* genes were highly expressed in endosperm or embryo. Homologous gene pair—*CnFatB3* and *EgFatB3—*were both highly expressed in their endosperms, which were validated as key genes for MCFA biosynthesis [3, 6]. Another key enzyme—LPAAT, three AtLPAAT1, or AtLPAAT2 orthologs were found in each of coconut and oil palm [5]. The LPAAT1 genes were clustered into class I and class II, and the class I genes of both species had higher expression levels in endosperm tissue. Moreover, the LPAAT2 genes were also clustered into two classes, and genes in class II had low or no expression. For the key transcription factor associated with lipid synthesis—*WRI1*, Xiao et al. [5] identified three *WRI1* genes in coconut and six in oil palm and classified

offer new insights into TAG assembly in coconut.

**2.2 Transcription factors regulating fatty acid biosynthesis**

CCG026159.1 [5].

**226**

the genes into three groups based on conserved amino acid sequences. The coconut and oil palm *WRI1* genes in the same group indicated the same expression pattern: group I was highly expressed both in the coconut endosperm and the oil palm endosperm/mesocarp; group II or III has very low or no expression.
