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.].

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.

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.

AtABCA9 transporter supplies fatty acids for lipid synthesis to the endoplasmic reticulum.

Fatty acids, the building blocks of biological lipids, are synthesized in plastids and then transported to the endoplasmic reticulum (ER) for assimilation into specific lipid classes. The mechanism of fatty acid transport from plastids to the ER has not been identified. Here we report that AtABCA9, an ABC transporter in Arabidopsis thaliana, mediates this transport. AtABCA9 was localized to the ER, and atabca9 null mutations reduced seed triacylglycerol (TAG) content by 35% compared with WT. Developing atabca9 seeds incorporated 35% less (14)C-oleoyl-CoA into TAG compared with WT seeds. Furthermore, overexpression of AtABCA9 enhanced TAG deposition by up to 40%. These data strongly support a role for AtABCA9 as a supplier of fatty acid substrates for TAG biosynthesis at the ER during the seed-filling stage. AtABCA9 may be a powerful tool for increasing lipid production in oilseeds.

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.

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.

PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana.

Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.

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

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

Isozyme-specific modes of activation of CTP:phosphorylcholine cytidylyltransferase in Arabidopsis thaliana at low temperature.

Arabidopsis thaliana increases cellular phosphatidylcholine (PC) content during cold acclimation by up-regulating PC biosynthesis. The A. thaliana genes CCT1 and CCT2 encode CTP:phosphorylcholine cytidylyltransferases (CCTs; EC 2.7.7.15), which regulate PC biosynthesis via the CDP-choline pathway. We isolated the T-DNA-tagged knockout mutants cct1 and cct2 of A. thaliana (Wassilevskaja; WS). CCT activity in cct1 and cct2 plants accounted for 29 and 79% to the cellular CCT activity of WS plants, respectively. When plants were exposed to 2 degrees C for 7 d, CCT activity increased in both cct1 and cct2 plants, and immunoblot analyses revealed that cct1 contained an increased level of CCT2 protein whereas cct2 exhibited little increase in CCT1 level. For each mutant grown at 23 degrees C, CCT activity was mainly enriched in the particulate (15,000 x g pellet) and microsomal (150,000 x g pellet) fractions from rosette leaf homogenates. After exposure to cold, the particulate and microsomal fractions of cct1 plants had higher total CCT activity due to increased levels of CCT2; in contrast, the levels of CCT1 in cct2 plants remained unchanged in particulate and microsomal fractions despite a significant increase in the total CCT activity. We conclude that the CDP-choline pathway of A. thaliana is up-regulated at low temperature via isogene-specific modes: enhanced expression of CCT2 and post-translational activation/inactivation of CCT1 in membranes. PC levels were similarly maintained in both mutants and WS plants after 14 d at 2 degrees C, suggesting that either of the CCT genes is sufficient for PC biosynthesis at low temperature.

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.