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Clubroot, caused by the obligate parasite Plasmodiophora brassicae , is one of the most devastating diseases of canola ( Brassica napus ) in Canada. The identification of novel genes that contribute to clubroot resistance is important for the sustainable management of clubroot, as these genes may be used in the development of resistant canola cultivars. Phospholipase As (PLAs) play important roles in plant defense signaling and stress tolerance, and thus are attractive targets for crop breeding. However, since canola is an allopolyploid and has multiple copies of each PLA gene, it is time-consuming to test the functions of PLAs directly in this crop. In contrast, the model plant Arabidopsis thaliana has a simpler genetic background and only one copy of each PLA . Therefore, it would be reasonable and faster to validate the potential utility of PLA genes in Arabidopsis first. In this study, we identified seven homozygous atpla knockout/knockdown mutants of Arabidopsis , and tested their performance following inoculation with P. brassicae . Four mutants ( pla 1 -ii α, pla 1 -i γ 3 , pla 1 -iii , ppla-iii β, ppla-iii δ) developed more severe clubroot than the wild-type, suggesting increased susceptibility to P. brassicae . The homologs of these Arabidopsis PLAs ( AtPLAs ) in B. napus ( BnPLAs ) were identified through Blast searches and phylogenic analysis. Expression of the BnPLAs was subsequently examined in transcriptomic datasets generated from canola infected by P. brassicae , and promising candidates for further characterization identified.

Key messageAIL7 over-expression modulates fatty acid biosynthesis and triacylglycerol accumulation in Arabidopsis developing seeds through the transcriptional regulation of associated genes.Seed fatty acids (FAs) and triacylglycerol (TAG) contribute to many functions in plants, and seed lipids have broad food, feed and industrial applications. As a result, an enormous amount of attention has been dedicated towards uncovering the regulatory cascade responsible for the fine-tuning of the lipid biosynthetic pathway in seeds, which is regulated in part through the action of LEAFY COTYLEDON1, ABSCISSIC ACID INSENSITIVE 3, FUSCA3 and LEC2 (LAFL) transcription factors. Although AINTEGUMENTA-LIKE 7 (AIL7) is involved in meristematic function and shoot phyllotaxy, its effect in the context of lipid biosynthesis has yet to be assessed. Here, we generated AIL7 seed-specific over-expression lines and found that they exhibited significant alterations in FA composition and decreased total lipid accumulation in seeds. Seeds and seedlings from transgenic lines also exhibited morphological deviations compared to wild type. Correspondingly, RNA-Seq analysis demonstrated that the expression of many genes related to FA biosynthesis and TAG breakdown were significantly altered in developing siliques from transgenic lines compared to wild-type plants. The seed-specific over-expression of AIL7 also altered the expression profiles of many genes related to starch metabolism, photosynthesis and stress response, suggesting further roles for AIL7 in plants. These findings not only advance our understanding of the lipid biosynthetic pathway in seeds, but also provide evidence for additional functions of AIL7, which could prove valuable in downstream breeding and/or metabolic engineering endeavors.

Rising emissions of anthropogenic greenhouse gases such as carbon dioxide (CO2), nitrous oxide (N2O) and methane (CH4) are a key driver of climate change, which is predicted to have myriad detrimental consequences in coming years if not kept in check. Given the potency of CH4 in terms of trapping heat in the atmosphere in the short term, as well as the fact that ruminant production currently contributes approximately 30% of anthropogenic emissions, there is an impetus to substantially decrease the generation of ruminant-derived CH4. While various strategies are being assessed in this context, a multi-faceted approach is likely required to achieve significant reductions. Feed supplementation is one strategy that has shown promise in this field by attenuating methanogenesis in rumen archaea; however, this can be costly and sometimes impractical. In this review, we examine and discuss the prospect of directly modulating forages and/or rumen archaea themselves in a manner that would reduce methanogenesis using CRISPR/Cas-mediated gene editing platforms. Such an approach could provide a valuable alternative to supplementation and has the potential to contribute to the sustainability of agriculture, as well as the mitigation of climate change, in the future.

Key message Seed-specific down-regulation of AtCESA1 and AtCESA9 , which encode cellulose synthase subunits, differentially affects seed storage compound accumulation in Arabidopsis. High amounts of cellulose can negatively affect crop seed quality, and, therefore, diverting carbon partitioning from cellulose to oil, protein and/or starch via molecular breeding may improve seed quality. To determine the effect of seed cellulose content reduction on levels of storage compounds, Arabidopsis thaliana CELLULOSE SYNTHASE1 ( AtCESA1 ) and AtCESA9 genes, which both encode cellulose synthase subunits, were individually down-regulated using seed-specific intron-spliced hairpin RNA (hpRNAi) constructs. The selected seed-specific AtCESA1 and AtCESA9 Arabidopsis RNAi lines displayed reduced cellulose contents in seeds, and exhibited no obvious visual phenotypic growth defects with the exception of a minor effect on early root development in AtCESA1 RNAi seedlings and early hypocotyl elongation in the dark in both types of RNAi line. The seed-specific down-regulation of AtCESA9 resulted in a reduction in seed weight compared to empty vector controls, which was not observed in AtCESA1 RNAi lines. In terms of effects on carbon partitioning, AtCESA1 and AtCESA9 RNAi lines exhibited distinct effects. The down-regulation of AtCESA1 led to a ~ 3% relative increase in seed protein content ( P  = 0.04) and a ~ 3% relative decrease in oil content ( P  = 0.02), but caused no alteration in soluble glucose levels. On the contrary, AtCESA9 RNAi lines did not display a significant reduction in seed oil, protein or soluble glucose content. Taken together, our results indicate that the seed-specific down-regulation of AtCESA1 causes alterations in seed storage compound accumulation, while the effect of AtCESA9 on carbon partitioning is absent or minor in Arabidopsis.

GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE (GPAT) genes encode enzymes involved in glycerolipid biosynthesis in plants. Ten GPAT homologues have been identified in Arabidopsis. GPATs 4–8 have been shown to be involved in the production of extracellular lipid barrier polyesters. Recently, GPAT9 was reported to be essential for triacylglycerol (TAG) biosynthesis in developing Arabidopsis seeds. The enzymatic properties and possible functions of GPAT9 in surface lipid, polar lipid and TAG biosynthesis in non-seed organs, however, have not been investigated. Here we show that Arabidopsis GPAT9 exhibits sn-1 acyltransferase activity with high specificity for acyl-coenzyme A, thus providing further evidence that this GPAT is involved in storage lipid biosynthesis. We also confirm a role for GPAT9 in seed oil biosynthesis and further demonstrate that GPAT9 contributes to the biosynthesis of both polar lipids and TAG in developing leaves, as well as lipid droplet production in developing pollen grains. Conversely, alteration of constitutive GPAT9 expression had no obvious effects on surface lipid biosynthesis. Taken together, these studies expand our understanding of GPAT9 function to include modulation of several different intracellular glycerolipid pools in plant cells.

Soilborne pathogens can be highly devastating, and clubroot, caused by Plasmodiophora brassicae, is particularly destructive to cruciferous plants. Although many AP2/ERF family transcription factors (TFs) have crucial physiological functions, very little is known regarding their functions in the context of soilborne diseases. Here we investigated the roles of AINTEGUMENTA-LIKE 7 (AIL7), an AIL sub-family TF in the AP2/ERF family, in plant immunity against clubroot. Unexpectedly, both AIL7 overexpression and mutant Arabidopsis lines exhibited increased tolerance to P. brassicae. Subsequent analysis revealed significant transcriptional alterations in genes linked to pathogen response, along with notable differences in genes associated with salicylic acid (SA) and jasmonic acid (JA) defense pathways, compared to wild-type plants. Interestingly, there was a tendency for up-regulation of SA- and JA-related genes in AIL7 overexpression and mutant lines in the absence, rather than presence, of P. brassicae. Subsequent phytohormone analyses confirmed these results. Taken together, AIL7 has an important role in maintaining constitutive systemic acquired resistance, involving phytohormone mediated defense, and this, rather than an accumulation of SA following P. brassicae challenge, primes the plants for improved clubroot resistance, which would shed light on exploring the functions of other AP2/ERF family TFs in plant immunity against soilborne pathogens.

Microalgal oils in the form of triacylglycerols (TAGs) are broadly used as nutritional supplements and biofuels. Diacylglycerol acyltransferase (DGAT) catalyzes the final step of acyl-CoA-dependent biosynthesis of TAG and is considered a key target for manipulating oil production. Although a growing number of DGAT1s have been identified and over-expressed in some algal species, the detailed structure-function relationship, as well as the improvement of DGAT1 performance via protein engineering, remain largely untapped. Here, we explored the structure-function features of the hydrophilic N-terminal domain of DGAT1 from the green microalga Chromochloris zofingiensis (CzDGAT1). The results indicated that the N-terminal domain of CzDGAT1 was less disordered than those of the higher eukaryotic enzymes and its partial truncation or complete removal could substantially decrease enzyme activity, suggesting its possible role in maintaining enzyme performance. Although the N-terminal domains of animal and plant DGAT1s were previously found to bind acyl-CoAs, replacement of CzDGAT1 N-terminus by an acyl-CoA binding protein (ACBP) could not restore enzyme activity. Interestingly, the fusion of ACBP to the N-terminus of the full-length CzDGAT1 could enhance the enzyme affinity for acyl-CoAs and augment protein accumulation levels, which ultimately drove oil accumulation in yeast cells and tobacco leaves to higher levels than the full-length CzDGAT1. Overall, our findings unravel the distinct features of the N-terminus of algal DGAT1 and provide a strategy to engineer enhanced performance in DGAT1 via protein fusion, which may open a vista in generating improved membrane-bound acyl-CoA-dependent enzymes and boosting oil biosynthesis in plants and oleaginous microorganisms.