National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada.
BMC Plant Biol. 2012 Jan 10;12:4. doi: 10.1186/1471-2229-12-4.
The Arabidopsis thaliana dgat1 mutant, AS11, has an oil content which is decreased by 30%, and a strongly increased ratio of 18:3/20:1, compared to wild type. Despite lacking a functional DGAT1, AS11 still manages to make 70% of WT seed oil levels. Recently, it was demonstrated that in the absence of DGAT1, PDAT1 was essential for normal seed development, and is a dominant determinant in Arabidopsis TAG biosynthesis.
Biochemical, metabolic and gene expression studies combined with genetic crossing of selected Arabidopsis mutants have been carried out to demonstrate the contribution of Arabidopsis PDAT1 and LPCAT2 in the absence of DGAT1 activity.
Through microarray and RT-PCR gene expression analyses of AS11 vs. WT mid-developing siliques, we observed consistent trends between the two methods. FAD2 and FAD3 were up-regulated and FAE1 down-regulated, consistent with the AS11 acyl phenotype. PDAT1 expression was up-regulated by ca 65% while PDAT2 expression was up-regulated only 15%, reinforcing the dominant role of PDAT1 in AS11 TAG biosynthesis. The expression of LPCAT2 was up-regulated by 50-75%, while LPCAT1 expression was not significantly affected. In vitro LPCAT activity was enhanced by 75-125% in microsomal protein preparations from mid-developing AS11 seed vs WT. Co-incident homozygous knockout lines of dgat1/lpcat2 exhibited severe penalties on TAG biosynthesis, delayed plant development and seed set, even with a functional PDAT1; the double mutant dgat1/lpcat1 showed only marginally lower oil content than AS11.
Collectively, the data strongly support that in AS11 it is LPCAT2 up-regulation which is primarily responsible for assisting in PDAT1-catalyzed TAG biosynthesis, maintaining a supply of PC as co-substrate to transfer sn-2 moieties to the sn-3 position of the enlarged AS11 DAG pool.
拟南芥 dgat1 突变体 AS11 的油含量比野生型低 30%，18:3/20:1 的比例明显增加。尽管缺乏功能性的 DGAT1，AS11 仍能产生 70%WT 种子油水平。最近，研究表明，在缺乏 DGAT1 的情况下，PDAT1 对正常种子发育至关重要，是拟南芥 TAG 生物合成的主要决定因素。
通过生化、代谢和基因表达研究以及选择的拟南芥突变体的遗传杂交，证明了拟南芥 PDAT1 和 LPCAT2 在缺乏 DGAT1 活性时的作用。
通过 AS11 与 WT 中发育中期的蒴果进行微阵列和 RT-PCR 基因表达分析，我们观察到两种方法之间存在一致的趋势。FAD2 和 FAD3 上调，FAE1 下调，与 AS11 的酰基表型一致。PDAT1 的表达上调了约 65%，而 PDAT2 的表达仅上调了 15%，这强化了 PDAT1 在 AS11 TAG 生物合成中的主导作用。LPCAT2 的表达上调了 50-75%，而 LPCAT1 的表达没有明显受到影响。与 WT 相比，从中发育的 AS11 种子的微粒体蛋白制剂中，LPCAT 的活性提高了 75-125%。在 PDAT1 功能正常的情况下，dgat1/lpcat2 的纯合敲除系表现出严重的 TAG 生物合成缺陷、植物发育和种子结实延迟，即使是 dgat1/lpcat2 的双突变体，其油含量也仅略低于 AS11。
总的来说，这些数据有力地支持了这样一种观点，即在 AS11 中，LPCAT2 的上调主要负责协助 PDAT1 催化的 TAG 生物合成，维持 PC 作为共底物的供应，将 sn-2 位转移到扩大的 AS11 DAG 池的 sn-3 位。
