TEM studies indicate that storage lipids in jatropha developing endosperm are synthesized from 28 to 56 DAF. Consistent with this observation, almost all of the 68 genes identified in this study showed high or peak expression at either one or two or even three stages from 28 to 56 DAF. Genes with expression patterns of Categories I, II and IV may encode the core enzymes or proteins or their subunits that are required for storage lipid biosynthesis. Genes with expression patterns of Categories III and IV may be involved in biosynthesis of fatty acid and lipids or lipid signaling, which are essential for endosperm development. The analysis on differential expression of genes that encode enzymes or proteins with similar function in fatty acid and lipid biosynthesis may provide clues to identify these key genes that play pivotal roles in the limiting steps of storage lipid biosynthesis. The information on gene expression levels and patterns provides guideline on genetic breeding and genetic engineering of jatropha for increasing oil content or changing profiles of fatty acid and lipids in jatropha seeds.
KAS is involved in the formation of acetoacetyl ACP. All plants examined to date contain three KAS isoenzymes (I, II, and III) and each distinguishes by its substrate specificity [15
]. Our studies demonstrated that all three KAS genes showed an expression pattern of Category I (Figure A to C). The KAS I and KAS III genes showed peak expression at 28 DAF, whereas the KAS II gene was maximally expressed at 42 DAF (Figure A to C). The latter result was slightly different from a previous study, in which the KAS II gene showed a peak expression at 50 DAP (days after pollination), when the jatropha seeds were almost fully matured [12
KCS is a component of the elongation complex responsible for the synthesis of very long chains of monounsaturated fatty acids (VLCMFA) in the seeds of plants [16
]. The KCS gene showed an expression pattern of Category III, which was constitutively expressed throughout endosperm development (Figure H). Our result was similar to that of FAE1
gene in Brassica napus
]. The results indicate that KCS, which determines fatty acid profiles in storage lipids, is not regulated at the transcription level. Taylor et al. (2009) produced transgenic Arabidopsis and Brassica Carinata
plants that expressed Cardamine KCS gene. The seed-specific expression of the Cardamine KCS gene led to 55-fold and 15-fold increase in nervonic acid proportions in Arabidopsis and B. carinata
seed oil, respectively [16
FATA is a intraplastidial enzyme that terminates the synthesis of fatty acids in plants [18
]. It also facilitates the export of acyl moieties to endoplasmic reticulum where they can be used in the production of glycerolipids [18
]. The FATA gene showed gene expression of Category I in jatropha developing endosperm (Figure J). In Arabidopsis FATA mutant, palmitate (16:0) and stearate (18:0) contents were reduced to 56% and 30
% in seeds, suggesting that FATA plays a major role in determining the types of fatty acids. Analysis of individual glycerollipids revealed a 4-fold reduction of 16:0 and a 10-fold reduction of 18:0 in the FATA mutant [19
]. Further analysis showed that FATA is involved in biosynthesis of saturated fatty acids, which are essential for plant growth and development [19
The phospholipid biosynthetic enzyme, LPAT, catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, the major precursor of all glycerolipids [20
]. Four LPAT genes were identified in this study. The LPAT1, LPAT2 and LPAT5 genes showed a expression pattern of Category I, whereas the LPAT4 gene displayed an expression pattern of Category II, whose expression was constantly increased from 14 to 56 DAF (Figure H to J; Figure F). These results support the hypothesis that increasing the expression of glycerolipid acyltransferase in seeds leads to a greater flux of intermediates through the Kennedy pathway and enhanced triacylglycerol accumulation [21
]. Indeed, overexpression of two rapeseed LPAAT (LPAT) isozymes in Arabidopsis increased lipid content and seed mass in seeds [21
]. Considering the LPAT2 gene is the only LPAT gene that is highly expressed at both 42 and 56 DAF (Figure B), it may be used to be overexpressed in jatropha endosperm at late developmental stages to enhance storage lipid production.
DGAT catalyzes the final step of lipid synthesis in many plants. Its expression level is correlated with lipid accumulation. The DGAT1 gene in jatropha showed an expression pattern of Category I, which displayed high expressions at 28 and 42 DAF and a decreased expression at 56 DAF (Figure K). Previous studies have shown that a phenylalanine insertion in DGAT1-2 at position 469 (F469) is responsible for the increased oil and oleic-acid contents in maize [22
]. As one of the oil quantitative trait loci (QTLs), ectopic expression of the high-oil DGAT1-2
allele increases oil and oleic-acid contents up to 41
% and 107
%, respectively [22
]. The DGAT activity in developing seeds of transgenic lines was enhanced by 10
% to 70
]. In addition, overexpression of a diacylglycerol acyltransferase 2A from soil fungus Umbelopsis ramanniana
in soybean seed led to a 1.5
% increase in oil yield in the mature seed [23
]. Based on these reports, overexpression of the DGAT gene in transgenic jatropha plants may have high potential to increase the oil yield.
Desaturases play a pivotal role in fatty acid desaturation during fatty acid and lipid biosynthesis. Ten desaturase genes were identified to be expressed in developing jatropha endosperm. Most of the desaturase genes showed an expression pattern of Category I except that the SD gene displayed an expression pattern of Category II (Figure ). The expression pattern of the FAD6 gene in this study, which had a peak expression at 28 DAF, was different from a previous study, in which chloroplast-6 fatty acid desaturase (Chlo 6 or FAD6) gene showed the maximum expression at 50 DAP [12
]. The desaturase genes are good candidates for engineering oil plants to increase or decrease the production of polyunsaturated fatty acids. Recent study demonstrated that downregulation of JcFAD2-1
in jatropha by RNA interference technology caused a dramatic increase of oleic acid (> 78
%) and a corresponding reduction in polyunsaturated fatty acids (< 3
%) in its seed oil [24
]. The AAD, DALD, D12FAC and FAD2 genes were the major desaturase genes that were highly expressed at 42 DAF (Figure D). Likewise, both SD and FAD2 genes were the major desaturase genes that were highly expressed at 56 DAF (Figure D). These desaturase genes are potential candidates for genetic engineering to modify polyunsaturated fatty acids in jatropha seed oil.
In plants, storage lipds are generally stored in oil body that is enclosed with a single layer of phospholipid rich in oleosin proteins. Seeds with high oil content have more oleosins than those with low oil content [25
]. The exact role of oleosin in oil accumulation is unclear, although it may be involved in the biosynthesis and mobilization of plant oils. Previous study demonstrated that the relative net amounts of oleosin and oil accumulation during seed development are the major determinants of oil-body size in desiccation-tolerant seeds [26
]. Xu et al. (2011) found that Ole1
showed maximum expression at 50 DAF [12
]. Two oleosin genes, the Oleosin and Oleosin 3 genes, were identified in this study. The Oleosin gene showed an expression pattern of Category II (Figure C), whereas the Oleosin 3 genes displayed an expression pattern of Category I (Figure A). More importantly, the Oleosin gene was the major oleosin gene that was expressed at the late stages of endosperm development (Figure C). In this scenario, over-expression of the Oleosin gene in developing jatropha endosperm, especially at the late stage, may have potential to increase oil yield in jatropha seeds.