Arabidopsis ACYL CARRIER PROTEIN4 and RHOMBOID LIKE10 act independently in chloroplast phosphatidate synthesis.

ACYL CARRIER PROTEIN4 (ACP4) is the most abundant ACP isoform in Arabidopsis (Arabidopsis thaliana) leaves and acts as a scaffold for de novo fatty acid biosynthesis and as a substrate for acyl-ACP-utilizing enzymes. Recently, ACP4 was found to interact with a protein-designated plastid RHOMBOID LIKE10 (RBL10) that affects chloroplast monogalactosyldiacylglycerol (MGDG) biosynthesis, but the cellular function of this interaction remains to be explored. Here, we generated and characterized acp4 rbl10 double mutants to explore whether ACP4 and RBL10 directly interact in influencing chloroplast lipid metabolism. Alterations in the content and molecular species of chloroplast lipids such as MGDG and phosphatidylglycerol were observed in the acp4 and rbl10 mutants, which are likely associated with the changes in the size and profiles of diacylglycerol (DAG), phosphatidic acid (PA), and acyl-ACP precursor pools. ACP4 contributed to the size and profile of the acyl-ACP pool and interacted with acyl-ACP-utilizing enzymes, as expected for its role in fatty acid biosynthesis and chloroplast lipid assembly. RBL10 appeared to be involved in the conversion of PA to DAG precursors for MGDG biosynthesis as evidenced by the increased 34:x PA and decreased 34:x DAG in the rbl10 mutant and the slow turnover of radiolabeled PA in isolated chloroplasts fed with [14C] acetate. Interestingly, the impaired PA turnover in rbl10 was partially reversed in the acp4 rbl10 double mutant. Collectively, this study shows that ACP4 and RBL10 affect chloroplast lipid biosynthesis by modulating substrate precursor pools and appear to act independently.

Expression of malic enzyme reveals subcellular carbon partitioning for storage reserve production in soybeans.

Central metabolism produces amino and fatty acids for protein and lipids that establish seed value. Biosynthesis of storage reserves occurs in multiple organelles that exchange central intermediates including two essential metabolites, malate, and pyruvate that are linked by malic enzyme. Malic enzyme can be active in multiple subcellular compartments, partitioning carbon and reducing equivalents for anabolic and catabolic requirements. Prior studies based on isotopic labeling and steady-state metabolic flux analyses indicated malic enzyme provides carbon for fatty acid biosynthesis in plants, though genetic evidence confirming this role is lacking. We hypothesized that increasing malic enzyme flux would alter carbon partitioning and result in increased lipid levels in soybeans. Homozygous transgenic soybean plants expressing Arabidopsis malic enzyme alleles, targeting the translational products to plastid or outside the plastid during seed development, were verified by transcript and enzyme activity analyses, organelle proteomics, and transient expression assays. Protein, oil, central metabolites, cofactors, and acyl-acyl carrier protein (ACPs) levels were quantified overdevelopment. Amino and fatty acid levels were altered resulting in an increase in lipids by 0.5-2% of seed biomass (i.e. 2-9% change in oil). Subcellular targeting of a single gene product in central metabolism impacts carbon and reducing equivalent partitioning for seed storage reserves in soybeans.

Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves.

The metabolic plasticity of tobacco leaves has been demonstrated via the generation of transgenic plants that can accumulate over 30% dry weight as triacylglycerols. In investigating the changes in carbon partitioning in these high lipid-producing (HLP) leaves, foliar lipids accumulated stepwise over development. Interestingly, non-transient starch was observed to accumulate with plant age in WT but not HLP leaves, with a drop in foliar starch concurrent with an increase in lipid content. The metabolic carbon tradeoff between starch and lipid was studied using 13CO2-labeling experiments and isotopically nonstationary metabolic flux analysis, not previously applied to the mature leaves of a crop. Fatty acid synthesis was investigated through assessment of acyl-acyl carrier proteins using a recently derived quantification method that was extended to accommodate isotopic labeling. Analysis of labeling patterns and flux modeling indicated the continued production of unlabeled starch, sucrose cycling, and a significant contribution of NADP-malic enzyme to plastidic pyruvate production for the production of lipids in HLP leaves, with the latter verified by enzyme activity assays. The results suggest an inherent capacity for a developmentally regulated carbon sink in tobacco leaves and may in part explain the uniquely successful leaf lipid engineering efforts in this crop.

Arabidopsis thaliana organelles mimic the T7 phage DNA replisome with specific interactions between Twinkle protein and DNA polymerases Pol1A and Pol1B.

BACKGROUND: Plant chloroplasts and mitochondria utilize nuclear encoded proteins to replicate their DNA. These proteins are purposely built for replication in the organelle environment and are distinct from those involved in replication of the nuclear genome. These organelle-localized proteins have ancestral roots in bacterial and bacteriophage genes, supporting the endosymbiotic theory of their origin. We examined the interactions between three of these proteins from Arabidopsis thaliana: a DNA helicase-primase similar to bacteriophage T7 gp4 protein and animal mitochondrial Twinkle, and two DNA polymerases, Pol1A and Pol1B. We used a three-pronged approach to analyze the interactions, including Yeast-two-hybrid analysis, Direct Coupling Analysis (DCA), and thermophoresis. RESULTS: Yeast-two-hybrid analysis reveals residues 120-295 of Twinkle as the minimal region that can still interact with Pol1A or Pol1B. This region is a part of the primase domain of the protein and slightly overlaps the zinc-finger and RNA polymerase subdomains located within. Additionally, we observed that Arabidopsis Twinkle interacts much more strongly with Pol1A versus Pol1B. Thermophoresis also confirms that the primase domain of Twinkle has higher binding affinity than any other region of the protein. Direct-Coupling-Analysis identified specific residues in Twinkle and the DNA polymerases critical to positive interaction between the two proteins. CONCLUSIONS: The interaction of Twinkle with Pol1A or Pol1B mimics the minimal DNA replisomes of T7 phage and those present in mammalian mitochondria. However, while T7 and mammals absolutely require their homolog of Twinkle DNA helicase-primase, Arabidopsis Twinkle mutants are seemingly unaffected by this loss. This implies that while Arabidopsis mitochondria mimic minimal replisomes from T7 and mammalian mitochondria, there is an extra level of redundancy specific to loss of Twinkle function.

Depleting Cationic Lipids Involved in Antimicrobial Resistance Drives Adaptive Lipid Remodeling in Enterococcus faecalis.

The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly, and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human ß-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalis mprF. We analyzed the lipid profiles of parental wild-type and mprF mutant strains and show that while ΔmprF2 and ΔmprF1 ΔmprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the ΔmprF1 mutant synthesized ~70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in ΔmprF2 and ΔmprF1 ΔmprF2. In the mprF mutants, particularly ΔmprF1 ΔmprF2, the decrease in L-PG and phosphatidylglycerol (PG) is compensated by an increase in a phosphorus-containing lipid, glycerophospho-diglucosyl-diacylglycerol (GPDGDAG), and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the ΔmprF1 ΔmprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both ΔmprF2 and ΔmprF1 ΔmprF2, compared to the wild type and ΔmprF1. Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF. IMPORTANCE The cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion. Here we demonstrate that L-PG depletion has several unexpected consequences in Enterococcus faecalis, including a reduction of phosphatidylglycerol (PG), enrichment of a phosphorus-containing lipid, reduced fatty acid synthesis accompanied by an accumulation of long-chain acyl-acyl carrier proteins (long chain acyl-ACPs), lower membrane fluidity, and impaired secretion. These changes are not deleterious to the organism as long as exogenous fatty acids are available for uptake from the culture medium. Our findings suggest an adaptive mechanism involving compensatory changes across the entire lipidome upon removal of a single phospholipid modification. Such adaptations must be considered when devising antimicrobial strategies that target membrane lipids.

Metabolic synergy in Camelina reproductive tissues for seed development.

Photosynthesis in fruits is well documented, but its contribution to seed development and yield remains largely unquantified. In oilseeds, the pods are green and elevated with direct access to sunlight. With 13C labeling in planta and through an intact pod labeling system, a unique multi-tissue comprehensive flux model mechanistically described how pods assimilate up to one-half (33 to 45%) of seed carbon by proximal photosynthesis in Camelina sativa. By capturing integrated tissue metabolism, the studies reveal the contribution of plant architecture beyond leaves, to enable seed filling and maximize the number of viable seeds. The latent capacity of the pod wall in the absence of leaves contributes approximately 79% of seed biomass, supporting greater seed sink capacity and higher theoretical yields that suggest an opportunity for crop productivity gains.

Suppression of SDP1 Improves Soybean Seed Composition by Increasing Oil and Reducing Undigestible Oligosaccharides.

In developing soybean seeds, carbon is partitioned between oil, protein and carbohydrates. Here, we demonstrate that suppression of lipase-mediated turnover of triacylglycerols (TAG) during late seed development increases fatty acid content and decreases the presence of undigestible oligosaccharides. During late stages of embryo development, the fatty acid content of soybean seed decreases while the levels of the oligosaccharides raffinose and stachyose increase. Three soybean genes orthologous to the Arabidopsis lipase gene SUGAR-DEPENDENT1 (SDP1) are upregulated at this time. Suppression of these genes resulted in higher oil levels, with lipid levels in the best lines exceeding 24% of seed weight. In addition, lipase-suppressed lines produced larger seeds compared to wild-type plants, resulting in increases of over 20% in total lipid per seed. Levels of raffinose and stachyose were lower in the transgenic lines, with average reductions of 15% in total raffinose family oligosaccharides observed. Despite the increase in oil, protein content was not negatively impacted and trended higher in the transgenic lines. These results are consistent with a role for SDP1 in turning over TAG to supply carbon for other needs, including the synthesis of oligosaccharides, and offer new strategies to further improve the composition of soybean seeds.

Plant Organelle Genome Replication.

Mitochondria and chloroplasts perform essential functions in respiration, ATP production, and photosynthesis, and both organelles contain genomes that encode only some of the proteins that are required for these functions. The proteins and mechanisms for organelle DNA replication are very similar to bacterial or phage systems. The minimal replisome may consist of DNA polymerase, a primase/helicase, and a single-stranded DNA binding protein (SSB), similar to that found in bacteriophage T7. In Arabidopsis, there are two genes for organellar DNA polymerases and multiple potential genes for SSB, but there is only one known primase/helicase protein to date. Genome copy number varies widely between type and age of plant tissues. Replication mechanisms are only poorly understood at present, and may involve multiple processes, including recombination-dependent replication (RDR) in plant mitochondria and perhaps also in chloroplasts. There are still important questions remaining as to how the genomes are maintained in new organelles, and how genome copy number is determined. This review summarizes our current understanding of these processes.

Plant mitochondrial DNA.

Plants possess mitochondrial genomes that are large and complex compared to animals. Nearly all animal mitochondrial genomes are about 16.5. kbp in length, whereas plant mitochondrial genomes range between 200-2,000 kbp. This is curious if we assume modern mitochondria originated from a common alpha-proteobacterial ancestor. Despite their size, plant mitochondrial genomes do not contain significantly more genes than their animal counterparts. Most of the additional DNA found in plant mitochondrial genomes consists of large introns, repeats and non-coding regions. Furthermore, plant mtDNA does not exist as large circular DNA molecules but mostly as a collection of linear DNA with combinations of smaller circular and branched molecules. Studies into these highly fragmented genomes heavily imply that recombination is the main mechanism driving replication of plant mtDNA.

Chloroplast DNA Copy Number Changes during Plant Development in Organelle DNA Polymerase Mutants.

Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles.