Pistil-derived lipids influence pollen tube growth and male fertility in Arabidopsis thaliana.

Pollen germination and pollen tube elongation require rapid phospholipid production and remodeling in membrane systems that involve both de novo synthesis and turnover. Phosphatidic acid phosphohydrolase (PAH) and lysophosphatidylcholine acyltransferase (LPCAT) are two key enzymes in membrane lipid maintenance. PAH generates diacylglycerol (DAG), a necessary precursor for the de novo synthesis of phosphatidylcholine (PC), while LPCAT reacylates lysophosphatidylcholine (LPC) to PC and plays an essential role in the remodeling of membrane lipids. In this study, we investigated the synthetic defects of pah and lpcat mutations in sexual reproduction of Arabidopsis (Arabidopsis thaliana) and explored the prospect of pistil lipid provision to pollen tube growth. The combined deficiencies of lpcat and pah led to decreased pollen tube growth in the pistil and reduced male transmission. Interestingly, pistils of the lipid mutant dgat1 ameliorated the male transmission deficiencies of pah lpcat pollen. In contrast, pollination with a non-specific phospholipase C (NPC) mutant exacerbated the fertilization impairment of the pah lpcat pollen. Given the importance of DAG in lipid metabolism and its contrasting changes in the dgat1 and npc mutants, we further investigated whether DAG supplement in synthetic media could influence pollen performance. DAG was incorporated into phospholipids of germinating pollen and stimulated pollen tube growth. Our study provides evidence that pistil derived lipids contribute to membrane lipid synthesis in pollen tube growth, a hitherto unknown role in synergistic pollen-pistil interactions.

Applying a non-GMO breeding approach with an identified natural variation to reduce food allergen Len c3 in Lens culinaris seeds.

Lentils (Lens culinaris) are produced in diverse agroecological regions and are consumed as one of the most important food legumes worldwide. Lentils possess a nutritional profile from a human health perspective that is not only nutrient dense but also offers a better balance between protein and carbohydrates. However, lentil causes food allergy, which has been a significant concern due to increased consumption in parts of the world. Len c3, a non-specific lipid transfer protein (LTP), was identified as one of the allergens in lentil seeds. In this study, we identified an LTP gene Lcu.2RBY.4g013600 that encodes the lentil allergen Len c3. We then focused on gene screening from a collection of natural accessions to search for natural mutations of the Len c3 allergen-encoding gene. A natural lentil line M11 was identified with mutations at LcLTP3b and low accumulation of vicilin through genomic-assisted approaches. Furthermore, we generated a pool of lentil germplasms with LcLTP3b mutation background through crossing the identified lentil plant M11 with two lentil cultivars, CDC Redmoon and CDC Gold. These generated lentil hybrids can be used as a breeding resource targeting at reducing allergen risk in lentil consumption.

Heterologous Overexpression of Apple MdKING1 Promotes Fruit Ripening in Tomato.

Fruit ripening is governed by a complex regulatory network, and ethylene plays an important role in this process. MdKING1 is a γ subunit of SNF1-related protein kinases (SnRKs), but the function was unclear. Here, we characterized the role of MdKING1 during fruit ripening, which can promote fruit ripening through the ethylene pathway. Our findings reveal that MdKING1 has higher expression in early-ripening cultivars than late-ripening during the early stage of apple fruit development, and its transcription level significantly increased during apple fruit ripening. Overexpression of MdKING1 (MdKING1 OE) in tomatoes could promote early ripening of fruits, with the increase in ethylene content and the loss of fruit firmness. Ethylene inhibitor treatment could delay the fruit ripening of both MdKING1 OE and WT fruits. However, MdKING1 OE fruits turned fruit ripe faster, with an increase in carotenoid content compared with WT. In addition, the expression of genes involved in ethylene biosynthesis (SlACO1, SlACS2, and SlACS4), carotenoid biosynthesis (SlPSY1 and SlGgpps2a), and fruit firmness regulation (SlPG2a, SlPL, and SlCEL2) was also increased in the fruits of MdKING1 OE plants. In conclusion, our results suggest that MdKING1 plays a key role in promoting tomato fruit ripening, thus providing a theoretical basis for apple fruit quality improvement by genetic engineering in the future.

Insights into the molecular mechanisms underlying responses of apple trees to abiotic stresses.

Apple (Malus[Formula: see text]domestica) is a popular temperate fruit crop worldwide. However, its growth, productivity, and quality are often adversely affected by abiotic stresses such as drought, extreme temperature, and high salinity. Due to the long juvenile phase and highly heterozygous genome, the conventional breeding approaches for stress-tolerant cultivars are time-consuming and resource-intensive. These issues may be resolved by feasible molecular breeding techniques for apples, such as gene editing and marker-assisted selection. Therefore, it is necessary to acquire a more comprehensive comprehension of the molecular mechanisms underpinning apples' response to abiotic stress. In this review, we summarize the latest research progress in the molecular response of apples to abiotic stressors, including the gene expression regulation, protein modifications, and epigenetic modifications. We also provide updates on new approaches for improving apple abiotic stress tolerance, while discussing current challenges and future perspectives for apple molecular breeding.

A chloroplast diacylglycerol lipase modulates glycerolipid pathway balance in Arabidopsis.

Two parallel pathways compartmentalized in the chloroplast and the endoplasmic reticulum contribute to thylakoid lipid synthesis in plants, but how these two pathways are coordinated during thylakoid biogenesis and remodeling remains unknown. We report here the molecular characterization of a homologous ADIPOSE TRIGLYCERIDE LIPASE-LIKE gene, previously referred to as ATGLL. The ATGLL gene is ubiquitously expressed throughout development and rapidly upregulated in response to a wide range of environmental cues. We show that ATGLL is a chloroplast non-regioselective lipase with a hydrolytic activity preferentially towards 16:0 of diacylglycerol (DAG). Comprehensive lipid profiling and radiotracer labeling studies revealed a negative correlation of ATGLL expression and the relative contribution of the chloroplast lipid pathway to thylakoid lipid biosynthesis. Additionally, we show that genetic manipulation of ATGLL expression resulted in changes in triacylglycerol levels in leaves. We propose that ATGLL, through affecting the level of prokaryotic DAG in the chloroplast, plays important roles in balancing the two glycerolipid pathways and in maintaining lipid homeostasis in plants.

Application of γ-aminobutyric acid (GABA) improves fruit quality and rootstock drought tolerance in apple.

GABA (γ-aminobutyric acid) plays a multifaceted role in plant growth, fruit quality, and tolerance to abiotic stresses. However, its physiological roles and mechanisms in the fruit quality and response to long-term drought stress in apple remain unelucidated. To investigate the effect of GABA on apple fruit quality and drought tolerance, we sprayed exogenous GABA on apple cultivar "Cripps Pink" and irrigated rootstock M.9-T337 with GABA, respectively. Results showed that exogenous GABA could effectively improve the fruit quality of "Cripps Pink", including increased sugar-to-acid ratio, flesh firmness, pericarp malleability, and GABA content, as well as reduced fruit acidity. In addition, pretreatment of M.9-T337 plants with GABA improved their tolerance to both long- and short-term drought stress. Specifically, 1 mM exogenous GABA increased the net photosynthetic rate, relative leaf water content, root-to-shoot ratio, and water use efficiency under long-term drought stress, and delayed the increased of the relative electrolyte leakage under short-term drought stress. RNA-seq analysis identified 1271 differentially expressed genes (DEGs) between nontreated and GABA-pretreated plants under short-term drought stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these DEGs revealed that GABA may enhance plant drought resistance by upregulating the expression of genes related to "Biosynthesis of secondary metabolites", "MAPK signaling pathway", "Glutathione metabolism", and "Carbon fixation in photosynthetic organisms". In conclusion, these results revealed that exogenous GABA can improve fruit quality and enhance drought tolerance in apple.

Engineering drought-tolerant apple by knocking down six GH3 genes and potential application of transgenic apple as a rootstock.

Drought poses a major threat to apple fruit production and quality. Because of the apple's long juvenile phase, developing varieties with improved drought tolerance using biotechnology approaches is needed. Here, we used the RNAi approach to knock down six GH3 genes in the apple. Under prolonged drought stress, the MdGH3 RNAi plants performed better than wild-type plants and had stronger root systems, higher root-to-shoot ratio, greater hydraulic conductivity, increased photosynthetic capacity, and increased water use efficiency. Moreover, MdGH3 RNAi plants promoted the drought tolerance of the scion when they were used as rootstock, compared with wild-type and M9-T337 rootstocks. Scions grafted onto MdGH3 RNAi plants showed increased plant height, stem diameter, photosynthetic capacity, specific leaf weight, and water use efficiency. The use of MdGH3 RNAi plants as rootstocks can also increase the C/N ratio of the scion and achieve the same effect as the M9-T337 rootstock in promoting the flowering and fruiting of the scion. Notably, using MdGH3 RNAi plants as rootstocks did not reduce fruit weight and scion quality compared with using M9-T337 rootstock. Our research provides candidate genes and demonstrates a general approach that could be used to improve the drought tolerance of fruit trees without sacrificing the yield and quality of scion fruits.

Zinc-finger protein MdBBX7/MdCOL9, a target of MdMIEL1 E3 ligase, confers drought tolerance in apple.

Water deficit is one of the main challenges for apple (Malus × domestica) growth and productivity. Breeding drought-tolerant cultivars depends on a thorough understanding of the drought responses of apple trees. Here, we identified the zinc-finger protein B-BOX 7/CONSTANS-LIKE 9 (MdBBX7/MdCOL9), which plays a positive role in apple drought tolerance. The overexpression of MdBBX7 enhanced drought tolerance, whereas knocking down MdBBX7 expression reduced it. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis identified one cis-element of MdBBX7, CCTTG, as well as its known binding motif, the T/G box. ChIP-seq and RNA-seq identified 1,197 direct targets of MdBBX7, including ETHYLENE RESPONSE FACTOR (ERF1), EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15), and GOLDEN2-LIKE 1 (GLK1) and these were further verified by ChIP-qPCR and electronic mobility shift assays. Yeast two-hybrid screen identified an interacting protein of MdBBX7, RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (MIEL1). Further examination revealed that MdMIEL1 could mediate the ubiquitination and degradation of MdBBX7 by the 26S proteasome pathway. Genetic interaction analysis suggested that MdMIEL1 acts as an upstream factor of MdBBX7. In addition, MdMIEL1 was a negative regulator of the apple drought stress response. Taken together, our results illustrate the molecular mechanisms by which the MdMIEL1-MdBBX7 module influences the response of apple to drought stress.

MdGH3.6 is targeted by MdMYB94 and plays a negative role in apple water-deficit stress tolerance.

Drought significantly limits apple fruit production and quality. Decoding the key genes involved in drought stress tolerance is important for breeding varieties with improved drought resistance. Here, we identified GRETCHEN HAGEN3.6 (GH3.6), an indole-3-acetic acid (IAA) conjugating enzyme, to be a negative regulator of water-deficit stress tolerance in apple. Overexpressing MdGH3.6 reduced IAA content, adventitious root number, root length and water-deficit stress tolerance, whereas knocking down MdGH3.6 and its close paralogs increased IAA content, adventitious root number, root length and water-deficit stress tolerance. Moreover, MdGH3.6 negatively regulated the expression of wax biosynthetic genes under water-deficit stress and thus negatively regulated cuticular wax content. Additionally, MdGH3.6 negatively regulated reactive oxygen species scavengers, including antioxidant enzymes and metabolites involved in the phenylpropanoid and flavonoid pathway in response to water-deficit stress. Further study revealed that the homolog of transcription factor AtMYB94, rather than AtMYB96, could bind to the MdGH3.6 promoter and negatively regulated its expression under water-deficit stress conditions in apple. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees.

Insights into the effect of human civilization on Malus evolution and domestication.

The evolutionary history of the Malus genus has not been well studied. In the current study, we presented genetic evidence on the origin of the Malus genus based on genome sequencing of 297 Malus accessions, revealing the genetic relationship between wild species and cultivated apples. Our results demonstrated that North American and East Asian wild species are closer to the outgroup (pear) than Central Asian species, and hybrid species including natural (separated before the Pleistocene, about 2.5 Mya) and artificial hybrids (including ornamental trees and rootstocks) are between East and Central Asian wild species. Introgressions from M. sylvestris in cultivated apples appeared to be more extensive than those from M. sieversii, whose genetic background flowed westward across Eurasia and eastward to wild species including M. prunifolia, M. × asiatica, M. × micromalus, and M. × robust. Our results suggested that the loss of ancestral gene flow from M. sieversii in cultivated apples accompanied the movement of European traders around the world since the Age of Discovery. Natural SNP variations showed that cultivated apples had higher nucleotide diversity than wild species and more unique SNPs than other apple groups. An apple ERECTA-like gene that underwent selection during domestication on 15th chromosome was identified as a likely major determinant of fruit length and diameter, and an NB-ARC domain-containing gene was found to strongly affect anthocyanin accumulation using a genome-wide association approach. Our results provide new insights into the origin and domestication of apples and will be useful in new breeding programmes and efforts to increase fruit crop productivity.

Elucidating the biochemical basis of trans-16:1 fatty acid change in leaves during cold acclimation in wheat.

In plant cells, phosphatidylglycerol (PG) in the chloroplast has a characteristic trans-∆3-hexadecenoic acid (t16:1) at the sn-2 position. The t16:1 content in wheat leaf tissues decreases during cold treatment, but the significance of this fatty acid compositional change and the underlying biochemical mechanism remains poorly understood. Using a large collection of wheat cultivars displaying a varying capacity of freezing tolerance, we show for the first time under field conditions that this low temperature induced t16:1 change is associated with winter hardiness. To explore the metabolic mechanism responsible for the reduction of t16:1, we performed detailed lipid analysis and comparative transcriptome study with four selected wheat lines under cold acclimation. Our results show that wheat leaf tissues experience a gradual decrease in chloroplast lipid pathway activity during cold acclimation and that the decline in chloroplast lipid synthesis manifests itself in the decrease of t16:1 in PG. Comparative RNA-seq analyses with leaf tissues further reveal concerted transcriptome shifts indicating a rebalancing of chloroplast and cytosolic lipid synthesis during cold acclimation. Our study, thus, provides mechanistic understanding on chloroplast lipid adjustments as a "molecular ideotype" and the t16:1 change as a specific metabolite marker for screening freezing tolerance in wheat.

On the Role of DGAT1 in Seed Glycerolipid Metabolic Network and Critical Stages of Plant Development in Arabidopsis.

Studies on the model plant Arabidopsis thaliana have uncovered the identities of most enzymatic components involved in seed storage lipid biosynthesis. However, much remains to be learned on how pathway interactions operate in the seed metabolic network. In this study, we dissected seed glycerolipid molecular compositional changes in the Arabidopsis mutant deficient in diacylglycerol acyltransferase 1 (DGAT1). Our results indicate that metabolic adjustments occurred in both phosphatidylcholine synthesis and deacylation in developing seeds. Ultrastructural changes of perturbed oil and protein bodies were also evident in cotyledon parenchyma cells. To unmask the physiological and developmental role associated with DGAT1-mediated neutral lipid biosynthesis, we attempted to combine dgat1 mutation with lpcat2 that harbors a defect in lysophosphatidylcholine acyltransferase 2 (LPCAT2). Disruption in both DGAT1 and LPCAT2 led to an apparent defect in pollen development that manifested as pollen sterility. Collectively, our results highlight a role of DGAT1 in both storage lipid synthesis and plant development.

Molecular identification and functional characterization of a cyanogenic glucosyltransferase from flax (Linum unsitatissimum).

Flax seed has become consumers' choice for not only polyunsaturated alpha-linolenic fatty acid but also nutraceuticals such as lignans and soluble fiber. There is, however, a major drawback of flax as a source of functional food since the seeds contain significant level of cyanogenic glucosides. The final step of cyanogenic glucoside biosynthesis is mediated by UDP-glucose dependent glucosyltransferase. To date, no flax cyanogenic glucosyl transferase genes have been reported with verified biochemical functionality. Here we present a study on the identification and enzymatic characterization of a first flax cyanogenic glucosyltransferase, LuCGT1. We show that LuCGT1 was highly active towards both aliphatic and aromatic substrates. The LuCGT1 gene is expressed in leaf tissues as well as in developing seeds, and its expression level was drastically reduced in flax mutant lines low in cyanogenic glucosides. Identification of LuCGT1 provides a molecular handle for developing gene specific markers for targeted breeding of low cyanogenic glucosides in flax.

CIPK9 is involved in seed oil regulation in Brassica napus L. and Arabidopsis thaliana (L.) Heynh.

BACKGROUND: Accumulation of storage compounds during seed development plays an important role in the life cycle of oilseed plants; these compounds provide carbon and energy resources to support the establishment of seedlings. RESULTS: In this study, we show that BnCIPK9 has a broad expression pattern in Brassica napus L. tissues and that wounding stress strongly induces its expression. The overexpression of BnCIPK9 during seed development reduced oil synthesis in transgenic B. napus compared to that observed in wild-type (WT) plants. Functional analysis revealed that seed oil content (OC) of complementation lines was similar to that of WT plants, whereas OC in Arabidopsis thaliana (L.) Heynh. Atcipk9 knockout mutants (cipk9) was higher than that of WT plants. Seedling of cipk9 mutants failed to establish roots on a sugar-free medium, but root establishment could be rescued by supplementation of sucrose or glucose. The phenotype of complementation transgenic lines was similar to that of WT plants when grown on sugar-free medium. Mutants, cipk9, cbl2, and cbl3 presented similar phenotypes, suggesting that CIPK9, CBL2, and CBL3 might work together and play similar roles in root establishment under sugar-free condition. CONCLUSION: This study showed that BnCIPK9 and AtCIPK9 encode a protein kinase that is involved in sugar-related response and plays important roles in the regulation of energy reserves. Our results suggest that AtCIPK9 negatively regulates lipid accumulation and has a significant effect on early seedling establishment in A. thaliana. The functional characterization of CIPK9 provides insights into the regulation of OC, and might be used for improving OC in B. napus. We believe that our study makes a significant contribution to the literature because it provides information on how CIPKs coordinate stress regulation and energy signaling.

The D-lactate dehydrogenase MoDLD1 is essential for growth and infection-related development in Magnaporthe oryzae.

Rice blast disease caused by Magnaporthe oryzae is initiated by the attachment of conidia to plant surfaces. Germ tubes emerging from conidia develop melanized appressoria to physically penetrate the host surface. Previous studies revealed that appressorium development requires the breakdown of storage lipids and glycogen that occur in peroxisomes and the cytosol respectively, culminating in production of pyruvate. However, the downstream product(s) entering the mitochondria for further oxidation is unclear. In this study, we aimed to investigate the molecular basis underlying the metabolic flux towards the mitochondria associated with the infectious-related development in M. oryzae. We showed that D-lactate is a key intermediate metabolite of the mobilization of lipids and glycogen, and its oxidative conversion to pyruvate is catalysed by a mitochondrial D-lactate dehydrogenase MoDLD1. Deletion of MoDLD1 caused defects in conidiogenesis and appressorium formation, and subsequently the loss of fungal pathogenicity. Further analyses demonstrated that MoDLD1 activity is involved in the maintenance of redox homeostasis during conidial germination. Thus, MoDLD1 is a critical modulator that channels metabolite flow to the mitochondrion coupling cellular redox state, and contributes to development and virulence of M. oryzae.

Effects of eIFiso4G1 mutation on seed oil biosynthesis.

Fatty acid biosynthesis is a primary metabolic pathway that occurs in plastids, whereas the formation of glycerolipid molecules for the majority of cellular membrane systems and the deposition of storage lipid in seeds takes place in the cytosolic compartment. In this report, we present a study of an Arabidopsis mutant, ar21, with a novel seed fatty acid phenotype showing higher contents of eicosanoic acid (20:1) and oleic acid (18:1) and a reduced level of α-linolenic acid (18:3). A combination of map-based cloning and whole-genome sequencing identified the genetic basis underlying the fatty acid phenotype as a lesion in the plant-specific eukaryotic translation initiation factor eIFiso4G1. Transcriptome analysis on developing seeds revealed a reduced level of plastid-encoded genes. Specifically, decreases in both transcript and protein levels of an enzyme involved in fatty acid biosynthesis, the ß-subunit of the plastidic heteromeric acetyl-CoA carboxylase (htACCase) encoded by accD, were evident in the mutant. Biochemical assays showed that the developing seeds of the mutant possessed a decreased htACCase activity in the plastid but an elevated activity of homomeric acetyl-CoA carboxylase (hmACCase). These results suggested that the increased 20:1 was attributable at least in part to the enhanced cytosolic hmACCase activity. We also detected a significant repression of FATTY ACID DESATURASE 3 (FAD3) during seed development, which correlated with a decreased 18:3 level in seed oil. Together, our study on a mutant of eIFiso4G1 uncovered multifaceted interactions between the cytosolic and plastidic compartments in seed lipid biosynthesis that impact major seed oil traits.

Adjustments of lipid pathways in plant adaptation to temperature stress.

Modulation of membrane lipid composition under varying environmental conditions is an important part of plant stress adaptation. Most notably, proportional changes of lipid composition in response to temperature changes are a major cellular response to requirements of membrane fluidity adjustment. In higher plants, synthesis of glycerolipids is accomplished by 2 major pathways, the prokaryotic and eukaryotic pathway, located in the chloroplast and the endoplasmic reticulum (ER), respectively. Recently, we systematically investigated the re-adjustments of glycerolipid pathways under temperature stress at the metabolite and transcript levels using 3 plant species with distinct lipid profiles. The relative contributions of 2 pathways and lipid channeling from the ER and chloroplast were both observed in plants under temperature stress. Potential factors controlling the lipid flux were identified through transcriptome analysis.

Understanding the biochemical basis of temperature-induced lipid pathway adjustments in plants.

Glycerolipid biosynthesis in plants proceeds through two major pathways compartmentalized in the chloroplast and the endoplasmic reticulum (ER). The involvement of glycerolipid pathway interactions in modulating membrane desaturation under temperature stress has been suggested but not fully explored. We profiled glycerolipid changes as well as transcript dynamics under suboptimal temperature conditions in three plant species that are distinctively different in the mode of lipid pathway interactions. In Arabidopsis thaliana, a 16:3 plant, the chloroplast pathway is upregulated in response to low temperature, whereas high temperature promotes the eukaryotic pathway. Operating under a similar mechanistic framework, Atriplex lentiformis at high temperature drastically increases the contribution of the eukaryotic pathway and correspondingly suppresses the prokaryotic pathway, resulting in the switch of lipid profile from 16:3 to 18:3. In wheat (Triticum aestivum), an 18:3 plant, low temperature also influences the channeling of glycerolipids from the ER to chloroplast. Evidence of differential trafficking of diacylglycerol moieties from the ER to chloroplast was uncovered in three plant species as another layer of metabolic adaptation under temperature stress. We propose a model that highlights the predominance and prevalence of lipid pathway interactions in temperature-induced lipid compositional changes.

Deciphering the roles of Arabidopsis LPCAT and PAH in phosphatidylcholine homeostasis and pathway coordination for chloroplast lipid synthesis.

Phosphatidylcholine (PC) is a key intermediate in the metabolic network of glycerolipid biosynthesis. Lysophosphatidylcholine acyltransferase (LPCAT) and phosphatidic acid phosphatase (PAH) are two key enzymes of PC homeostasis. We report that LPCAT activity is markedly induced in the Arabidopsis pah mutant. The quadruple pah lpcat mutant, with dual defects in PAH and LPCAT, had a level of lysophosphatidylcholine (LPC) that was much higher than that in the lpcat mutants and a PC content that was higher than that in the pah mutant. Comparative molecular profile analysis of monogalactosyldiacylglycerol and digalactosyldiacylglycerol revealed that both the pah and pah lpcat mutants had increased proportions of 34:6 from the prokaryotic pathway despite differing levels of LPCAT activity. We show that a decreased representation of the C16:0 C18:2 diacylglycerol moiety in PC was a shared feature of pah and pah lpcat, and that this change in PC metabolic profile correlated with the increased prokaryotic contribution to chloroplast lipid synthesis. We detected increased PC deacylation in the pah lpcat mutant that was attributable at least in part to the induced phospholipases. Increased LPC generation was also evident in the pah mutant, but the phospholipases were not induced, raising the possibility that PC deacylation is mediated by the reverse reaction of LPCAT. We discuss possible roles of LPCAT and PAH in PC turnover that impacts lipid pathway coordination for chloroplast lipid synthesis.

Metabolic interactions between the Lands cycle and the Kennedy pathway of glycerolipid synthesis in Arabidopsis developing seeds.

It has been widely accepted that the primary function of the Lands cycle is to provide a route for acyl remodeling to modify fatty acid (FA) composition of phospholipids derived from the Kennedy pathway. Lysophosphatidylcholine acyltransferase (LPCAT) is an evolutionarily conserved key enzyme in the Lands cycle. In this study, we provide direct evidence that the Arabidopsis thaliana LPCATs, LPCAT1 and LPCAT2, participate in the Lands cycle in developing seeds. In spite of a substantially reduced initial rate of nascent FA incorporation into phosphatidylcholine (PC), the PC level in the double mutant lpcat1 lpcat2-2 remained unchanged. LPCAT deficiency triggered a compensatory response of de novo PC synthesis and a concomitant acceleration of PC turnover that were attributable at least in part to PC deacylation. Acyl-CoA profile analysis revealed complicated metabolic alterations rather than merely reduced acyl group shuffling from PC in the mutant. Shifts in FA stereo-specific distribution in triacylglycerol of the mutant seed suggested a preferential retention of saturated acyl chains at the stereospecific numbering (sn)-1 position from PC and likely a channeling of lysophosphatidic acid, derived from PC, into the Kennedy pathway. Our study thus illustrates an intricate relationship between the Lands cycle and the Kennedy pathway.