Genetic and Biochemical Investigation of Seed Fatty Acid Accumulation in Arabidopsis

Abstract

As a vegetable oil, consisting principally of triacylglycerols, is the major storage form of photosynthetically-fixed carbon in oilseeds which are of significant agricultural and industrial value. Photosynthesis in chlorophyll-containing green seeds, along with photosynthesis in leaves and other green organs, generates ATP and reductant (NADPH and NADH) needed for seed fatty acid production. However, contribution of seed photosynthesis to fatty acid accumulation in seeds have not been well-defined. Here, we report the contribution of seed-photosynthesis to fatty acid production by probing segregating green (photosynthetically-competent) and non-green or yellow (photosynthetically-non-competent) seeds in siliques of an Arabidopsis chlorophyll synthase mutant. Using this mutant, we found that yellow seeds lacking photosynthetic capacity reached 80% of amounts of oil in green seeds at maturity. Combining this with studies using shaded siliques, we determined that seed-photosynthesis accounts for 20% and silique and leaf/stem photosynthesis each account for ~40% of the ATP and reductant for seed oil production. Transmission electron microscopy (TEM) and pyridine nucleotides and ATP analyses revealed that seed photosynthesis provides ATP and reductant for oil production mostly during early development, as evidenced by delayed oil accumulation in non-green seeds. Transcriptomic analyses suggests that the oxidative pentose phosphate pathway could be the source of carbon, energy and reductants required for fatty acid synthesis beyond the early stages of seed development.

Keywords: Arabidopsis; NADPH; chlorophyll synthase; fatty acid biosynthesis; oil bodies; oxidative pentose phosphate pathway; photosynthesis; seed development.

Figure 1

Analyses of seed biomass, oil, and protein contents. (A) Comparison of seed weight in photosynthetic, green seeds (red) and non-photosynthetic, yellow seeds (non-red) from two (CHLSYN7 and CHLSYN8) independent Atchlsyn1-1/CHLSYN lines. (B) FAMEs analysis of seed oil content of red/green (photosynthetic) seeds and, non-red/yellow (non-photosynthetic) seeds from two independent mutant lines CHLSYN7 and CHLSYN8, respectively. (C) Comparison of red/green (photosynthetic) seeds and, non-red/yellow (non-photosynthetic) seeds protein content in two independent complemented lines, CHLSYN7 and CHLSYN8. The two independent lines are represented by orange and green bars, respectively. (D) Fatty acid composition of seed wildtype and mutant seeds. Orange and cyan bar indicates green (photosynthetic) and yellow (non-photosynthetic) seeds of CHLSYN7, yellow and light-green bar indicates green (photosynthetic) and yellow (non-photosynthetic) seeds from CHLSYN8, respectively. Values are means ± SD (n = 3 biological replicates). Statistical significance was analyzed using the two-sided Student’s t-test. The asterisk indicates a significant difference (^*p < 0.05).

Figure 2

Analysis of seed oil and chlorophyll content in WT and Atchlsyn1-1 mutant during development. (A) Biochemical estimation of total FAs and chlorophyll content in WT. Oil content is on the left or the primary Y-axis and chlorophyll content is on the right or secondary Y-axis. (B) The seed oil content of seeds from photosynthetic, green seeds (red), and non-photosynthetic, yellow (non-red) seeds. Blue lines indicate WT seed chlorophyll content. The Red line indicates WT seed oil content. The green line indicates photosynthetic, green/(red) seed oil content. The orange line indicates non-photosynthetic, yellow/(non-red) seed oil content. Values are means ±SD (n = 3 biological replicates). Statistical significance was analyzed using the two-sided Student’s t-test. The asterisk indicates a significant difference (^*p < 0.05).

Figure 3

TEM analysis of oil bodies of embryos cotyledons from the same silique of Arabidopsis chlorophyll synthase mutant line CHLSYN7. (A) Section of developing embryos from photosynthetic, green(red) seeds at 8 DAF. (B) Section of developing embryos from non-photosynthetic, yellow(non-red) seeds at 8 DAF. (C) Percentage area of oil bodies per cell of embryos from photosynthetic, green/(red) and non-photosynthetic yellow(non-red) seeds at 8 DAF. (D) Section of developing embryos from photosynthetic, green(red) at 12 DAF. (E) Section of developing embryos from non-photosynthetic, yellow(non-red) seeds at 12 DAF. (F) Percentage area of oil bodies per cell of embryos cotyledons from photosynthetic, green/(Red) and non-photosynthetic, yellow (Non-red) seeds at 12 DAF. (G) Section of embryos from dry seeds (Red). (H) Section of embryos from dry non-red seeds. (I) Percentage area of oil bodies per cell of embryos cotyledons from dry seeds (40 DAF) red and non-red. Red triangles indicate the oil body. Green and orange bars indicate red and non-red seeds of CHLSYN7 seeds. Scale bar = 2 μm. Values are means from 5 to 6 section obtained from three replicates. Statistical significance was analyzed using the two-sided Student t-test. The asterisk indicates a significant difference (^*p < 0.05).

Figure 4

Photosynthetic organ contribution to total oil production in seeds. (A) Analysis of the weight of dry shaded and control seeds (40 DAF). (B) The total oil content of dry shaded and control seeds. (C) The protein content of dry shaded and control seeds. (D) Fatty acid composition of shaded and control seeds. (E) The relative decrease in oil content per plant. (F) Contribution of different photosynthetic tissues to seed oil production (seed chlorophyll ± error = 1.53, silique chlorophyll ± error = 1.97). Values are means ± SD (n = 3 biological replicates). Green and orange bars indicate control and shaded. Statistical significance was analyzed using the two-sided Student’s t-test. The asterisk indicates a significant difference (^*p < 0.05).

Figure 5

Gene expression analysis of developing yellow and green seeds at 10 DAF. (A) Number of DEGs in the non-photosynthetic background. (B) Heat map showing expression profile of DEGs. (C) Showing the GO classification of DEGs in the non-photosynthetic background. (D) Differentially enrich pathways of DEGs in the non-photosynthetic background. (E) Real-time PCR analysis of TAG biosynthetic pathway genes. (F) Differentially expressed gene of the cytosolic and plastic branch of the oxidative pentose phosphate pathway. (G) Non-oxidative pentose phosphate pathway. (H) Sugar transport. Green and yellow bars indicate photosynthetic green seeds and non-photosynthetic seeds. Values are means ±SD (n = 3 biological replicates, i.e., GD, GE, GF are three replicated biological samples belonging to green seeds. While YB, YA, YC are three replicated biological samples of yellow seeds). Statistical significance was analyzed using the two-sided Student t-test. The asterisk indicates a significant difference (^*p < 0.05).

Figure 6

Measurement of pyridine nucleotides and ATP analyses of photosynthetic and non-photosynthetic seeds at 8, and 10 DAF. (A) ATP content. (B) ADP content. (C) ATP/ADP ratio. (D) NADH content. (E) NAD content. (F) NADH/NAD ratio. (G) NADPH content. (H) NADP content. (I) NADPH/NADP ratio. Green bar represents photosynthetic seeds, and the orange bar represents non-photosynthetic seeds. Values are means ± SD (n = 3 biological replicates). Statistical significance was analyzed using a two-sided Student t-test. The asterisk indicates a significant difference (^*p < 0.05).

Figure 7

Proposed model of reductant supply for FA biosynthesis seed. This model shows that green developing seeds have two pathways for generating NADPH for FA synthesis during seed development. i.e., (A) Photosynthesis (B) Oxidative pentose phosphate pathway (OPPP). Six to eleven days after flowering were chosen because seeds de-greening starts at 11 DAF.