PECT1, a rate-limiting enzyme in phosphatidylethanolamine biosynthesis, is involved in the regulation of stomatal movement in Arabidopsis.

An Arabidopsis mutant displaying impaired stomatal responses to CO2 , cdi4, was isolated by a leaf thermal imaging screening. The mutated gene PECT1 encodes CTP:phosphorylethanolamine cytidylyltransferase. The cdi4 exhibited a decrease in phosphatidylethanolamine levels and a defect in light-induced stomatal opening as well as low-CO2 -induced stomatal opening. We created RNAi lines in which PECT1 was specifically repressed in guard cells. These lines are impaired in their stomatal responses to low-CO2 concentrations or light. Fungal toxin fusicoccin (FC) promotes stomatal opening by activating plasma membrane H+ -ATPases in guard cells via phosphorylation. Arabidopsis H+ -ATPase1 (AHA1) has been reported to be highly expressed in guard cells, and its activation by FC induces stomatal opening. The cdi4 and PECT1 RNAi lines displayed a reduced stomatal opening response to FC. However, similar to in the wild-type, cdi4 maintained normal levels of phosphorylation and activation of the stomatal H+ -ATPases after FC treatment. Furthermore, the cdi4 displayed normal localization of GFP-AHA1 fusion protein and normal levels of AHA1 transcripts. Based on these results, we discuss how PECT1 could regulate CO2 - and light-induced stomatal movements in guard cells in a manner that is independent and downstream of the activation of H+ -ATPases. [Correction added on 15 May 2023, after first online publication: The third sentence is revised in this version.].

Corrigendum: Editorial: Proceedings of ASPL2019 - 8th Asian-Oceanian Symposium on Plant Lipids.

[This corrects the article DOI: 10.3389/fpls.2020.617094.].

Phosphatidylserine Is Required for the Normal Progression of Cell Plate Formation in Arabidopsis Root Meristems.

Phosphatidylserine (PS) is involved in various cellular processes in yeast and animals. However, PS functions in plants remain unclear. In Arabidopsis, PS is relatively enriched in flower and root tissues, and the genetic disturbance of PS biosynthesis in phosphatidylserine synthase1 (PSS1)/ pss1 heterozygotes induces sporophytic and gametophytic defects during pollen maturation. This study functionally characterized PS in Arabidopsis roots and observed that pss1 seedlings exhibited a short-root phenotype by reducing the meristem size and cell elongation capacity. Confocal microscopy imaging analyses of PS with GFP-LactC2 and the endocytic activity with FM 4-64 revealed that although GFP-LactC2 (or PS) was localized in the plasma membrane and endocytic membranes, the lack of PS in pss1 roots did not affect the constitutive endocytosis. Instead, a fluorescence imaging analysis of the cytokinetic phases in the dividing zone of pss1-2 roots revealed a significant delay in telophase progression, requiring active cargo vesicle trafficking for cell plate formation. Confocal microscopy imaging analysis of transgenic GFP-LactC2 root cells with developing cell plates indicated that GFP-LactC2 was localized at the cell plate. Moreover, confocal microscopy images of transgenic pss1-2 and PSS1 roots expressing the cell plate-specific syntaxin construct ProKNOLLE:eGFP-KNOLLE showed abnormal cell plate development in pss1-2ProKNOLLE:eGFP-KNOLLE roots. These results suggested that PS is required for root cytokinesis, possibly because it helps mediate the cargo vesicular trafficking required for cell plate formation.

Eukaryotic lipid metabolic pathway is essential for functional chloroplasts and CO2 and light responses in Arabidopsis guard cells.

Stomatal guard cells develop unique chloroplasts in land plant species. However, the developmental mechanisms and function of chloroplasts in guard cells remain unclear. In seed plants, chloroplast membrane lipids are synthesized via two pathways: the prokaryotic and eukaryotic pathways. Here we report the central contribution of endoplasmic reticulum (ER)-derived chloroplast lipids, which are synthesized through the eukaryotic lipid metabolic pathway, in the development of functional guard cell chloroplasts. We gained insight into this pathway by isolating and examining an Arabidopsis mutant, gles1 (green less stomata 1), which had achlorophyllous stomatal guard cells and impaired stomatal responses to CO2 and light. The GLES1 gene encodes a small glycine-rich protein, which is a putative regulatory component of the trigalactosyldiacylglycerol (TGD) protein complex that mediates ER-to-chloroplast lipid transport via the eukaryotic pathway. Lipidomic analysis revealed that in the wild type, the prokaryotic pathway is dysfunctional, specifically in guard cells, whereas in gles1 guard cells, the eukaryotic pathway is also abrogated. CO2-induced stomatal closing and activation of guard cell S-type anion channels that drive stomatal closure were disrupted in gles1 guard cells. In conclusion, the eukaryotic lipid pathway plays an essential role in the development of a sensing/signaling machinery for CO2 and light in guard cell chloroplasts.

Seed-Specific Overexpression of the Pyruvate Transporter BASS2 Increases Oil Content in Arabidopsis Seeds.

Seed oil is important not only for human and animal nutrition, but also for various industrial applications. Numerous genetic engineering strategies have been attempted to increase the oil content per seed, but few of these strategies have involved manipulating the transporters. Pyruvate is a major source of carbon for de novo fatty acid biosynthesis in plastids, and the embryo's demand for pyruvate is reported to increase during active oil accumulation. In this study, we tested our hypothesis that oil biosynthesis could be boosted by increasing pyruvate flux into plastids. We expressed the known plastid-localized pyruvate transporter BILE ACID:SODIUM SYMPORTER FAMILY PROTEIN 2 (BASS2) under the control of a seed-specific soybean (Glycine max) glycinin-1 promoter in Arabidopsis thaliana. The resultant transgenic Arabidopsis plants (OEs), which expressed high levels of BASS2, produced seeds that were larger and heavier and contained 10-37% more oil than those of the wild type (WT), but were comparable to the WT seeds in terms of protein and carbohydrate contents. The total seed number did not differ significantly between the WT and OEs. Therefore, oil yield per plant was increased by 24-43% in the OE lines compared to WT. Taken together, our results demonstrate that seed-specific overexpression of the pyruvate transporter BASS2 promotes oil production in Arabidopsis seeds. Thus, manipulating the level of specific transporters is a feasible approach for increasing the seed oil content.

Identification of a Chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content.

Despite a strong interest in microalgal oil production, our understanding of the biosynthetic pathways that produce algal lipids and the genes involved in the biosynthetic processes remains incomplete. Here, we report that Chlamydomonas reinhardtii Cre09.g398289 encodes a plastid-targeted 2-lysophosphatidic acid acyltransferase (CrLPAAT1) that acylates the sn-2 position of a 2-lysophosphatidic acid to form phosphatidic acid, the first common precursor of membrane and storage lipids. In vitro enzyme assays showed that CrLPAAT1 prefers 16:0-CoA to 18:1-CoA as an acyl donor. Fluorescent protein-tagged CrLPAAT1 was localized to the plastid membrane in C. reinhardtii cells. Furthermore, expression of CrLPAAT1 in plastids led to a > 20% increase in oil content under nitrogen-deficient conditions. Taken together, these results demonstrate that CrLPAAT1 is an authentic plastid-targeted LPAAT in C. reinhardtii, and that it may be used as a molecular tool to genetically increase oil content in microalgae.

The small molecule fenpropimorph rapidly converts chloroplast membrane lipids to triacylglycerols in Chlamydomonas reinhardtii.

Concern about global warming has prompted an intense interest in developing economical methods of producing biofuels. Microalgae provide a promising platform for biofuel production, because they accumulate high levels of lipids, and do not compete with food or feed sources. However, current methods of producing algal oil involve subjecting the microalgae to stress conditions, such as nitrogen deprivation, and are prohibitively expensive. Here, we report that the fungicide fenpropimorph rapidly causes high levels of neutral lipids to accumulate in Chlamydomonas reinhardtii cells. When treated with fenpropimorph (10 µg mL(-1)) for 1 h, Chlamydomonas cells accumulated at least fourfold the amount of triacylglycerols (TAGs) present in the untreated control cells. Furthermore, the quantity of TAGs present after 1 h of fenpropimorph treatment was over twofold higher than that formed after 9 days of nitrogen starvation in medium with no acetate supplement. Biochemical analysis of lipids revealed that the accumulated TAGs were derived mainly from chloroplast polar membrane lipids. Such a conversion of chloroplast polar lipids to TAGs is desirable for biodiesel production, because polar lipids are usually removed during the biodiesel production process. Thus, our data exemplified that a cost and time effective method of producing TAGs is possible using fenpropimorph or similar drugs.

Photoassimilation, assimilate translocation and plasmodesmal biogenesis in the source leaves of Arabidopsis thaliana grown under an increased atmospheric CO2 concentration.

Using 18-day-old Arabidopsis thaliana seedlings grown under increased (780 p.p.m., experimental plants) or ambient (390 p.p.m., control plants) CO2 conditions, we evaluated (14)CO2 photoassimilation in and translocation from representative source leaves. The total (14)CO2 photoassimilation amounts increased in the third leaves of the experimental plants in comparison with that found for the third leaves of the control plants, but the rates were comparable for the first leaves of the two groups. In contrast, translocation of labeled assimilates doubled in the first leaves of the experimental group, whereas translocation was, at best, passively enhanced even though photoassimilation increased in their third leaves. The transcript levels of the companion cell-specific sucrose:H(+) symporter gene SUC2 were not significantly affected in the two groups of plants, whereas those of the sucrose effluxer gene SWEET12 and the sieve element-targeted sucrose:H(+) symporter gene SUT4 were up-regulated in the experimental plants, suggesting up-regulation of SUT4-dependent apoplastic phloem loading. Compared with SUC2, SUT4 is a minor component that is expressed in companion cells but functions in sieve elements after transfer through plasmodesmata. The number of aniline blue-stained spots for plasmodesma-associated callose in the midrib wall increased in the first leaf of the experimental plants but was comparable in the third leaf between the experimental and control plants. These results suggest that A. thaliana responds to greater than normal concentrations of CO2 differentially in the first and third leaves in regards to photoassimilation, assimilate translocation and plasmodesmal biogenesis.

Rapid induction of lipid droplets in Chlamydomonas reinhardtii and Chlorella vulgaris by Brefeldin A.

Algal lipids are the focus of intensive research because they are potential sources of biodiesel. However, most algae produce neutral lipids only under stress conditions. Here, we report that treatment with Brefeldin A (BFA), a chemical inducer of ER stress, rapidly triggers lipid droplet (LD) formation in two different microalgal species, Chlamydomonas reinhardtii and Chlorella vulgaris. LD staining using Nile red revealed that BFA-treated algal cells exhibited many more fluorescent bodies than control cells. Lipid analyses based on thin layer chromatography and gas chromatography revealed that the additional lipids formed upon BFA treatment were mainly triacylglycerols (TAGs). The increase in TAG accumulation was accompanied by a decrease in the betaine lipid diacylglyceryl N,N,N-trimethylhomoserine (DGTS), a major component of the extraplastidic membrane lipids in Chlamydomonas, suggesting that at least some of the TAGs were assembled from the degradation products of membrane lipids. Interestingly, BFA induced TAG accumulation in the Chlamydomonas cells regardless of the presence or absence of an acetate or nitrogen source in the medium. This effect of BFA in Chlamydomonas cells seems to be due to BFA-induced ER stress, as supported by the induction of three homologs of ER stress marker genes by the drug. Together, these results suggest that ER stress rapidly triggers TAG accumulation in two green microalgae, C. reinhardtii and C. vulgaris. A further investigation of the link between ER stress and TAG synthesis may yield an efficient means of producing biofuel from algae.

Mitochondrial phosphatidylethanolamine level modulates Cyt c oxidase activity to maintain respiration capacity in Arabidopsis thaliana rosette leaves.

Phosphatidylethanolamine is the predominant phospholipid of the mitochondrial inner membrane. In Arabidopsis, pect1-4 mutants exhibit reduced cellular phosphatidylethanolamine levels owing to reduced CTP:phosphorylethanolamine cytidylyltransferase (PECT; EC 2.7.7.14) activity. Consequently, pect1-4 mutants may have decreased mitochondrial phosphatidylethanolamine levels, thereby affecting respiration capacity. Wild-type and pect1-4 plants grew similarly under a short-day condition until 5 weeks, when pect1-4 leaves had slightly less Chl. Total respiration was comparable between wild-type and pect1-4 leaves at 3 weeks and then increased 2-fold in the wild-type but only 1.1-fold in pect1-4 leaves. Compared with the wild type, the Cyt oxidase pathway capacity was reduced by 36% in pect1-4 leaves at 5 weeks and by 43% in pect1-4 mitochondria in 5-week-old rosette leaves. Maximal Cyt c oxidase (COX) activity was 20% lower in pect1-4 mitochondria than in wild-type mitochondria at 5 weeks despite comparable COX II protein levels in mitochondria at that time. Furthermore, COX II protein levels doubled in both wild-type and pect1-4 mitochondria between 3 and 5 weeks. Phosphatidylethanolamine levels were similar between mitochondria from these plants at 3 weeks and then increased by 6.4% in wild-type mitochondria and decreased by 6.5% in pect1-4 mitochondria by 5 weeks. Phosphatidylcholine levels compensated for the decreases in phosphatidylethanolamine levels. COX activity was lower in pect1-4 mitochondria at 5 weeks, most probably due to reduced phosphatidylethanolamine levels and/or an altered phosphatidylethanolamine:phosphatidylcholine ratio. Thus, PECT1 regulates mitochondrial phosphatidylethanolamine levels, which are important for maintaining respiration capacity in Arabidopsis leaves during prolonged growth under short-day conditions.

Acyl-lipid metabolism.

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.