Functional characterization of two type-1 diacylglycerol acyltransferase (DGAT1) genes from rice (Oryza sativa) embryo restoring the triacylglycerol accumulation in yeast.

KEY MESSAGE: Two OsDGAT1 genes showed the ability to restore TAG and LB synthesis in yeast H1246. Alterations in the N-terminal region of OsDGAT1-1 gene revealed its regulatory role in gene function. Accumulation of triacylglycerol (TAG) or oil in vegetative tissues has emerged as a promising approach to meet the global needs of food, feed, and fuel. Rice (Oryza sativa) has been recognized as an important cereal crop containing nutritional rice bran oil with high economic value for renewable energy production. To identify the key component involved in storage lipid biosynthesis, two type-1 diacylglycerol acyltransferases (DGAT1) from rice were characterized for its in vivo function in the H1246 (dga1, lro1, are1 and are2) yeast quadruple mutant. The ectopic expression of rice DGAT1 (designated as OsDGAT1-1 and OsDGAT1-2) genes restored the capability of TAG synthesis and lipid body (LB) formation in H1246. OsDGAT1-1 showed nearly equal substrate preferences to C16:0-CoA and 18:1-CoA whereas OsDGAT1-2 displayed substrate selectivity for C16:0-CoA over 18:1-CoA, indicating that these enzymes have contrasting substrate specificities. In parallel, we have identified the intrinsically disordered region (IDR) at the N-terminal domains of OsDGAT1 proteins. The regulatory role of the N-terminal domain was dissected. Single point mutations at the phosphorylation sites and truncations of the N-terminal region highlighted reduced lipid accumulation capabilities among different OsDGAT1-1 variants.

Characterization of oleosin genes from forage sorghum in Arabidopsis and yeast reveals their role in storage lipid stability.

MAIN CONCLUSION: Overexpression of forage sorghum oleosin genes in Arabidopsis oleosin-deficient mutant and yeast showed increased germination rate, triacylglycerol content, and protection against lipase-mediated TAG degradation. Plant lipids are an important source of ration for cattle or other livestock animals to fulfil their energy needs. Poor energy containing green forages are still one of the major sources of food for livestock animals, leaving the animals undernourished. This lowers the milk and meat production efficiency, thereby affecting human consumption. Oleosin, an essential oil body surface protein, is capable of enhancing and stabilizing the lipid content in plants. We identified and functionally characterized three forage sorghum oleosin genes (SbOle1, SbOle2, and SbOle3) in Arabidopsis and yeast. Phylogenetic analysis of SbOle proteins showed a close relationship with rice and maize oleosins. Expression analysis of SbOle genes determined a higher expression pattern in embryo followed by endosperm, while its expression in the non-seed tissues remained negligible. Overexpression of SbOle genes in Arabidopsis ole1-deficient mutants showed restoration of normal germination whereas control mutant seeds showed lower germination rates. Heterologous overexpression of SbOle in yeast cells resulted in increased TAG accumulation. Additionally, the TAG turnover assay showed the effectiveness of SbOle genes in reducing the yeast endogenous and rumen bacterial lipase-mediated TAG degradation. Taken together, our findings not only provide insights into forage sorghum oleosin for increasing the energy content in non-seed organs but also opened up the direction towards implication of oleosin in rumen protection of fodders.

Identification and molecular characterization of rice bran-specific lipases.

KEY MESSAGE: Among the 113 lipases present in rice genome, bran and endosperm-specific lipases were identified and lipase activity for one of the selected lipase gene is demonstrated in yeast. Rice bran is nutritionally superior than endosperm as it has major reservoirs of various minerals, vitamins, essential mineral oils and other bioactive compounds, however it is often under-utilized as a food product due to bran instability after milling. Various hydrolytic enzymes, such as lipases, present in bran causes degradation of the lipids present and are responsible for the bran instability. Here, in this study, we have systematically analyzed the 113 lipase genes present in rice genome, and identified 21 seed-specific lipases. By analyzing the expression of these genes in different seed tissues during seed development, we have identified three bran-specific and three endosperm-specific lipases, and one lipase which expresses in both bran and endosperm tissues. Further analysis of these genes during seed maturation and seed germination revealed that their expression increases during seed maturation and decreases during seed germination. Finally, we have shown the lipase activity for one of the selected genes, LOC_Os05g30900, in heterologous system yeast. The bran-specific lipases identified in this study would be very valuable for engineering designer rice varieties having increased bran stability in post-milling.

Can wheat survive in heat? Assembling tools towards successful development of heat stress tolerance in Triticum aestivum L.

Wheat is an important cereal crop that fulfils the calorie demands of the global humanity. Rapidly expanding populations are exposed to a fast approaching acute shortage in the adequate supply of food and fibre from agricultural resources. One of the significant threats to food security lies in the constantly increasing global temperatures which inflict serious injuries to the plants in terms of various physiological, biochemical and molecular processes. Wheat being a cool season crop is majorly impacted by the heat stress which adversely affects crop productivity and yield. These challenges would be potentially defeated with the implementation of genetic engineering strategies coupled with the new genome editing approaches. Development of transgenic plants for various crops has proved very effective for the incorporation of improved varietal traits in context of heat stress. With a similar approach, we need to target for the generation of heat stress tolerant wheat varieties which are capable of survival in such adverse conditions and yet produce well. In this review, we enumerate the current status of research on the heat stress responsive genes/factors and their potential role in mitigating heat stress in plants particularly in wheat with an aim to help the researchers get a holistic view of this topic. Also, we discuss on the prospective signalling pathway that is triggered in plants in general under heat stress.

Interaction between long noncoding RNA (lnc663) and microRNA (miR1128) regulates PDAT-like gene activity in bread wheat (Triticum aestivum L.).

Amylose, a starch subcomponent, can bind lipids within its helical groove and form an amylose-lipid complex, known as resistant starch type 5 (RS-5). RS contributes to lower glycaemic index of grain with health benefits. Unfortunately, genes involved in lipid biosynthesis in wheat grain remain elusive. Our study aims to characterize the lipid biosynthesis gene and its post-transcriptional regulation using the parent bread wheat variety 'C 306' and its EMS-induced mutant line 'TAC 75' varying in amylose content. Quantitative analyses of starch-bound lipids showed that 'TAC 75' has significantly higher lipid content in grains than 'C 306' variety. Furthermore, expression analyses revealed the higher expression of wheat phospholipid: diacylglycerol acyltransferase-like (PDAT-like) in the 'TAC 75' compared to the 'C 306'. Overexpression and ectopic expression of TaPDAT in yeast and tobacco leaf confirmed its ability to accumulate lipids in vivo. Enzyme activity assay showed that TaPDAT catalyzes the triacylglycerol synthesis by acylating 1,2-diacylglycerol. Interestingly, the long non-coding RNA, lnc663, was upregulated with the TaPDAT gene, while the miRNA, miR1128, downregulated in the 'TAC 75', indicating a regulatory relationship. The GFP reporter assay confirmed that the lnc663 acts as a positive regulator, and the miR1128 as a negative regulator of the TaPDAT gene, which controls lipid accumulation in wheat grain. Our findings outline TaPDAT-mediated biosynthesis of lipid accumulation and reveal the molecular mechanism of the lnc663 and miR1128 mediated regulation of the TaPDAT gene in wheat grain.

Identification and functional characterization of two acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) genes from forage sorghum (Sorghum bicolor) embryo.

Elevating the lipid content in high-biomass forage crops has emerged as a new research platform for increasing energy density and improving livestock production efficiency associated with improved human health beneficial meat and milk quality. To gain insights of triacylglycerol (TAG) biosynthesis in forage sorghum, two type-1 diacylglycerol acyltransferase (designated as SbDGAT1-1 and SbDGAT1-2) were characterized for its in vivo function. SbDGAT1-2 is more abundantly expressed in embryo and bran during the early stage of the grain development in comparison to SbDGAT1-1. Heterologous expression of SbDGAT1 genes in TAG deficient H1246 strain restored the TAG accumulation capability with high substrate predilection towards 16:0, 16:1 and 18:1 fatty acids (FA). In parallel, we have identified N-terminal intrinsically disordered region (IDR) in SbDGAT1 proteins. To test the efficacy of the N-terminal region, truncated variants of SbDGAT1-1 (designated as SbDGAT1-1(39-515) and SbDGAT1-1(89-515)) were generated and expressed in yeast H1246 strain. Deletion in the N-terminal region resulted in decreased accumulation of TAG and FA (16:0 and 18:0) when compared to the SbDGAT1-1 variant expressed in yeast H1246 strain. The present study provides significant insight in forage sorghum DGAT1 gene function, useful for enhancing the green-forage TAG content through metabolic engineering.

Mobilization of storage lipid reserve and expression analysis of lipase and lipoxygenase genes in rice (Oryza sativa var. Pusa Basmati 1) bran during germination.

Storage lipid mobilization by lipases and lipoxygenases (LOXs) in response to developmental cues take place during seed germination. After rice grain milling, the endogenous lipases and LOXs present in the bran fraction come in contact with the storage lipid reserve or triacylglycerol (TAG). Lipases catalyze the hydrolysis of TAGs to non-esterified fatty acids (NEFAs) and glycerol. The NEFAs, especially linoleic acid (18:2) produced, are further subjected to oxidative rancidity via peroxidation reaction catalyzed by LOXs. This results in the production of conjugated hydroperoxides of 18:2 that influence the off-flavors in rice bran lipids. The aim of this study is to understand how lipid mobilization and expression of lipase and LOX genes occur in the bran of germinating rice grains (Oryza sativavar. Pusa Basmati 1). Our results show that the primary source of storage lipids in bran is TAG, and its mobilization starts at 4 days after imbibition (4 DAI). Using publically available RNA-seq data and phylogeny analyses, we selected a total of 18 lipase and 16 LOX genes in rice for their expression profiles during onset of lipid mobilization. Gene expression analyses revealed OsLip1, OsLip9, and OsLip13; and OsLOX3 and OsLOX14 as the predominantly expressed genes in bran of germinating rice grains. This study explores two important events in the germinating rice grains, namely, mobilization of storage lipids and expression pattern of lipase and LOX genes. The information generated in this study can be used to efficiently manipulate the genes to enhance the shelf-stability of bran lipid reserve.