From Sensing to Acclimation: The Role of Membrane Lipid Remodeling in Plant Responses to Low Temperatures.

Low temperatures pose a dramatic challenge to plant viability. Chilling and freezing disrupt cellular processes, forcing metabolic adaptations reflected in alterations to membrane compositions. Understanding the mechanisms of plant cold tolerance is increasingly important due to anticipated increases in the frequency, severity, and duration of cold events. This review synthesizes current knowledge on the adaptive changes of membrane glycerolipids, sphingolipids, and phytosterols in response to cold stress. We delve into key mechanisms of low temperature membrane remodeling, including acyl editing and headgroup exchange, lipase activity, and phytosterol abundance changes, focusing on their impact at the subcellular level. Furthermore, we tabulate and analyze current gycerolipidomic data from cold treatments of Arabidopsis, maize, and sorghum. This analysis highlights congruencies of lipid abundance changes in response to varying degrees of cold stress. Ultimately, this review aids in rationalizing observed lipid fluctuations and pinpoints key gaps in our current capacity to fully understand how plants orchestrate these membrane responses to cold stress.

All members of the Arabidopsis DGAT and PDAT acyltransferase families operate during high and low temperatures.

The accumulation of triacylglycerol (TAG) in vegetative tissues is necessary to adapt to changing temperatures. It has been hypothesized that TAG accumulation is required as a storage location for maladaptive membrane lipids. The TAG acyltransferase family has five members (DIACYLGLYCEROL ACYLTRANSFERSE1/2/3 and PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1/2), and their individual roles during temperature challenges have either been described conflictingly or not at all. Therefore, we used Arabidopsis (Arabidopsis thaliana) loss of function mutants in each acyltransferase to investigate the effects of temperature challenge on TAG accumulation, plasma membrane integrity, and temperature tolerance. All mutants were tested under one high- and two low-temperature regimens, during which we quantified lipids, assessed temperature sensitivity, and measured plasma membrane electrolyte leakage. Our findings revealed reduced effectiveness in TAG production during at least one temperature regimen for all acyltransferase mutants compared to the wild type, resolved conflicting roles of pdat1 and dgat1 by demonstrating their distinct temperature-specific actions, and uncovered that plasma membrane integrity and TAG accumulation do not always coincide, suggesting a multifaceted role of TAG beyond its conventional lipid reservoir function during temperature stress.

Characterization of Soybean Events with Enhanced Expression of the Microtubule-Associated Protein 65-1 (MAP65-1).

Microtubule-associated protein 65-1 (MAP65-1) protein plays an essential role in plant cellular dynamics through impacting stabilization of the cytoskeleton by serving as a crosslinker of microtubules. The role of MAP65-1 in plants has been associated with phenotypic outcomes in response to various environmental stresses. The Arabidopsis MAP65-1 (AtMAP65-1) is a known virulence target of plant bacterial pathogens and is thus a component of plant immunity. Soybean events were generated that carry transgenic alleles for both AtMAP65-1 and GmMAP65-1, the soybean AtMAP65-1 homolog, under control of cauliflower mosaic virus 35S promoter. Both AtMAP65-1 and GmMAP65-1 transgenic soybeans are more resistant to challenges by the soybean bacterial pathogen Pseudomonas syringae pv. glycinea and the oomycete pathogen Phytophthora sojae, but not the soybean cyst nematode, Heterodera glycines. Soybean plants expressing AtMAP65-1 and GmMAP65-1 also display a tolerance to the herbicide oryzalin, which has a mode of action to destabilize microtubules. In addition, GmMAP65-1-expressing soybean plants show reduced cytosol ion leakage under freezing conditions, hinting that ectopic expression of GmMAP65-1 may enhance cold tolerance in soybean. Taken together, overexpression of AtMAP65-1 and GmMAP65-1 confers tolerance of soybean plants to various biotic and abiotic stresses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Rhythmic lipid and gene expression responses to chilling in panicoid grasses.

Chilling stress threatens plant growth and development, particularly affecting membrane fluidity and cellular integrity. Understanding plant membrane responses to chilling stress is important for unraveling the molecular mechanisms of stress tolerance. Whereas core transcriptional responses to chilling stress and stress tolerance are conserved across species, the associated changes in membrane lipids appear to be less conserved, as which lipids are affected by chilling stress varies by species. Here, we investigated changes in gene expression and membrane lipids in response to chilling stress during one 24 hour cycle in chilling-tolerant foxtail millet (Setaria italica), and chilling-sensitive sorghum (Sorghum bicolor), and Urochloa (browntop signal grass, Urochloa fusca, lipids only), leveraging their evolutionary relatedness and differing levels of chilling-stress tolerance. We show that most chilling-induced lipid changes are conserved across the three species, while we observed distinct, time-specific responses in chilling-tolerant foxtail millet, indicating the presence of a finely orchestrated adaptive mechanism. We detected rhythmicity in lipid responses to chilling stress in the three grasses, which were also present in Arabidopsis (Arabidopsis thaliana), suggesting the conservation of rhythmic patterns across species and highlighting the importance of accounting for time of day. When integrating lipid datasets with gene expression profiles, we identified potential candidate genes that showed corresponding transcriptional changes in response to chilling stress, providing insights into the differences in regulatory mechanisms between chilling-sensitive sorghum and chilling-tolerant foxtail millet.

Predicting transcriptional responses to cold stress across plant species.

Although genome-sequence assemblies are available for a growing number of plant species, gene-expression responses to stimuli have been cataloged for only a subset of these species. Many genes show altered transcription patterns in response to abiotic stresses. However, orthologous genes in related species often exhibit different responses to a given stress. Accordingly, data on the regulation of gene expression in one species are not reliable predictors of orthologous gene responses in a related species. Here, we trained a supervised classification model to identify genes that transcriptionally respond to cold stress. A model trained with only features calculated directly from genome assemblies exhibited only modest decreases in performance relative to models trained by using genomic, chromatin, and evolution/diversity features. Models trained with data from one species successfully predicted which genes would respond to cold stress in other related species. Cross-species predictions remained accurate when training was performed in cold-sensitive species and predictions were performed in cold-tolerant species and vice versa. Models trained with data on gene expression in multiple species provided at least equivalent performance to models trained and tested in a single species and outperformed single-species models in cross-species prediction. These results suggest that classifiers trained on stress data from well-studied species may suffice for predicting gene-expression patterns in related, less-studied species with sequenced genomes.

Oligogalactolipid production during cold challenge is conserved in early diverging lineages.

Severe cold, defined as a damaging cold beyond acclimation temperatures, has unique responses, but the signaling and evolution of these responses are not well understood. Production of oligogalactolipids, which is triggered by cytosolic acidification in Arabidopsis (Arabidopsis thaliana), contributes to survival in severe cold. Here, we investigated oligogalactolipid production in species from bryophytes to angiosperms. Production of oligogalactolipids differed within each clade, suggesting multiple evolutionary origins of severe cold tolerance. We also observed greater oligogalactolipid production in control samples than in temperature-challenged samples of some species. Further examination of representative species revealed a tight association between temperature, damage, and oligogalactolipid production that scaled with the cold tolerance of each species. Based on oligogalactolipid production and transcript changes, multiple angiosperm species share a signal of oligogalactolipid production initially described in Arabidopsis, namely cytosolic acidification. Together, these data suggest that oligogalactolipid production is a severe cold response that originated from an ancestral damage response that remains in many land plant lineages and that cytosolic acidification may be a common signaling mechanism for its activation.

Parallels between natural selection in the cold-adapted crop-wild relative Tripsacum dactyloides and artificial selection in temperate adapted maize.

Artificial selection has produced varieties of domesticated maize that thrive in temperate climates around the world. However, the direct progenitor of maize, teosinte, is indigenous only to a relatively small range of tropical and subtropical latitudes and grows poorly or not at all outside of this region. Tripsacum, a sister genus to maize and teosinte, is naturally endemic to the majority of areas in the western hemisphere where maize is cultivated. A full-length reference transcriptome for Tripsacum dactyloides generated using long-read Iso-Seq data was used to characterize independent adaptation to temperate climates in this clade. Genes related to phospholipid biosynthesis, a critical component of cold acclimation in other cold-adapted plant lineages, were enriched among those genes experiencing more rapid rates of protein sequence evolution in T. dactyloides. In contrast with previous studies of parallel selection, we find that there is a significant overlap between the genes that were targets of artificial selection during the adaptation of maize to temperate climates and those that were targets of natural selection in temperate-adapted T. dactyloides. Genes related to growth, development, response to stimulus, signaling, and organelles were enriched in the set of genes identified as both targets of natural and artificial selection.

Differentially Regulated Orthologs in Sorghum and the Subgenomes of Maize.

Identifying interspecies changes in gene regulation, one of the two primary sources of phenotypic variation, is challenging on a genome-wide scale. The use of paired time-course data on cold-responsive gene expression in maize (Zea mays) and sorghum (Sorghum bicolor) allowed us to identify differentially regulated orthologs. While the majority of cold-responsive transcriptional regulation of conserved gene pairs is species specific, the initial transcriptional responses to cold appear to be more conserved than later responses. In maize, the promoters of genes with conserved transcriptional responses to cold tend to contain more micrococcal nuclease hypersensitive sites in their promoters, a proxy for open chromatin. Genes with conserved patterns of transcriptional regulation between the two species show lower ratios of nonsynonymous to synonymous substitutions. Genes involved in lipid metabolism, known to be involved in cold acclimation, tended to show consistent regulation in both species. Genes with species-specific cold responses did not cluster in particular pathways nor were they enriched in particular functional categories. We propose that cold-responsive transcriptional regulation in individual species may not be a reliable marker for function, while a core set of genes involved in perceiving and responding to cold stress are subject to functionally constrained cold-responsive regulation across the grass tribe Andropogoneae.

Correction: Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779.

[This corrects the article DOI: 10.1371/journal.pgen.1003064.].

Recovery from N Deprivation Is a Transcriptionally and Functionally Distinct State in Chlamydomonas.

Facing adverse conditions such as nitrogen (N) deprivation, microalgae enter cellular quiescence, a reversible cell cycle arrest with drastic changes in metabolism allowing cells to remain viable. Recovering from N deprivation and quiescence is an active and orderly process as we are showing here for Chlamydomonas reinhardtii We conducted comparative transcriptomics on this alga to discern processes relevant to quiescence in the context of N deprivation and recovery following refeeding. A mutant with slow recovery from N deprivation, compromised hydrolysis of triacylglycerols7 (cht7), was included to better define the regulatory processes governing the respective transitions. We identified an ordered set of biological processes with expression patterns that showed sequential reversal following N resupply and uncovered acclimation responses specific to the recovery phase. Biochemical assays and microscopy validated selected inferences made based on the transcriptional analyses. These comprise (1) the restoration of N source preference and cellular bioenergetics during the early stage of recovery; (2) flagellum-based motility in the mid to late stage of recovery; and (3) recovery phase-specific gene groups cooperating in the rapid replenishment of chloroplast proteins. In the cht7 mutant, a large number of programmed responses failed to readjust in a timely manner. Finally, evidence is provided for the involvement of the cAMP-protein kinase A pathway in gating the recovery. We conclude that the recovery from N deprivation represents not simply a reversal of processes directly following N deprivation, but a distinct cellular state.

Synthesis and transfer of galactolipids in the chloroplast envelope membranes of Arabidopsis thaliana.

Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] are the hallmark lipids of photosynthetic membranes. The galactolipid synthases MGD1 and DGD1 catalyze consecutive galactosyltransfer reactions but localize to the inner and outer chloroplast envelopes, respectively, necessitating intermembrane lipid transfer. Here we show that the N-terminal sequence of DGD1 (NDGD1) is required for galactolipid transfer between the envelopes. Different diglycosyllipid synthases (DGD1, DGD2, and Chloroflexus glucosyltransferase) were introduced into the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions (NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction of DGDG synthesis in the outer envelope membrane was observed only with diglycosyllipid synthase fusion proteins carrying NDGD1, indicating that NDGD1 enables galactolipid translocation between envelopes. NDGD1 binds to phosphatidic acid (PA) in membranes and mediates PA-dependent membrane fusion in vitro. These findings provide a mechanism for the sorting and selective channeling of lipid precursors between the galactolipid pools of the two envelope membranes.

Lipid transport required to make lipids of photosynthetic membranes.

Photosynthetic membranes provide much of the usable energy for life on earth. To produce photosynthetic membrane lipids, multiple transport steps are required, including fatty acid export from the chloroplast stroma to the endoplasmic reticulum, and lipid transport from the endoplasmic reticulum to the chloroplast envelope membranes. Transport of hydrophobic molecules through aqueous space is energetically unfavorable and must be catalyzed by dedicated enzymes, frequently on specialized membrane structures. Here, we review photosynthetic membrane lipid transport to the chloroplast in the context of photosynthetic membrane lipid synthesis. We independently consider the identity of transported lipids, the proteinaceous transport components, and membrane structures which may allow efficient transport. Recent advances in lipid transport of chloroplasts, bacteria, and other systems strongly suggest that lipid transport is achieved by multiple mechanisms which include membrane contact sites with specialized protein machinery. This machinery is likely to include the TGD1, 2, 3 complex with the TGD5 and TGD4/LPTD1 systems, and may also include a number of proteins with domains similar to other membrane contact site lipid-binding proteins. Importantly, the likelihood of membrane contact sites does not preclude lipid transport by other mechanisms including vectorial acylation and vesicle transport. Substantial progress is needed to fully understand all photosynthetic membrane lipid transport processes and how they are integrated.

Chloroplast Membrane Remodeling during Freezing Stress Is Accompanied by Cytoplasmic Acidification Activating SENSITIVE TO FREEZING2.

Low temperature is a seasonal abiotic stress that restricts native plant ranges and crop distributions. Two types of low-temperature stress can be distinguished: chilling and freezing. Much work has been done on the mechanisms by which chilling is sensed, but relatively little is known about how plants sense freezing. Recently, Arabidopsis (Arabidopsis thaliana) SENSITIVE TO FREEZING2 (SFR2) was identified as a protein that responds in a nontranscriptional manner to freezing. Here, we investigate the cellular conditions that allow SFR2 activation. Using a combination of isolated organelle, whole-tissue, and whole-plant assays, we provide evidence that SFR2 is activated by changes in cytosolic pH and Mg(2+) Manipulation of pH and Mg(2+) in cold-acclimated plants is shown to cause changes similar to those of freezing. We conclude that pH and Mg(2+) are perceived as intracellular cues as part of the sensing mechanism for freezing conditions. This evidence provides a specific molecular mechanism to combat freezing.

Arabidopsis lipins, PDAT1 acyltransferase, and SDP1 triacylglycerol lipase synergistically direct fatty acids toward ß-oxidation, thereby maintaining membrane lipid homeostasis.

Triacylglycerol (TAG) metabolism is a key aspect of intracellular lipid homeostasis in yeast and mammals, but its role in vegetative tissues of plants remains poorly defined. We previously reported that PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is crucial for diverting fatty acids (FAs) from membrane lipid synthesis to TAG and thereby protecting against FA-induced cell death in leaves. Here, we show that overexpression of PDAT1 enhances the turnover of FAs in leaf lipids. Using the trigalactosyldiacylglycerol1-1 (tgd1-1) mutant, which displays substantially enhanced PDAT1-mediated TAG synthesis, we demonstrate that disruption of SUGAR-DEPENDENT1 (SDP1) TAG lipase or PEROXISOMAL TRANSPORTER1 (PXA1) severely decreases FA turnover, leading to increases in leaf TAG accumulation, to 9% of dry weight, and in total leaf lipid, by 3-fold. The membrane lipid composition of tgd1-1 sdp1-4 and tgd1-1 pxa1-2 double mutants is altered, and their growth and development are compromised. We also show that two Arabidopsis thaliana lipin homologs provide most of the diacylglycerol for TAG synthesis and that loss of their functions markedly reduces TAG content, but with only minor impact on eukaryotic galactolipid synthesis. Collectively, these results show that Arabidopsis lipins, along with PDAT1 and SDP1, function synergistically in directing FAs toward peroxisomal ß-oxidation via TAG intermediates, thereby maintaining membrane lipid homeostasis in leaves.

Lipid trafficking in plant cells.

Plant cells contain unique organelles such as chloroplasts with an extensive photosynthetic membrane. In addition, specialized epidermal cells produce an extracellular cuticle composed primarily of lipids, and storage cells accumulate large amounts of storage lipids. As lipid assembly is associated only with discrete membranes or organelles, there is a need for extensive lipid trafficking within plant cells, more so in specialized cells and sometimes also in response to changing environmental conditions such as phosphate deprivation. Because of the complexity of plant lipid metabolism and the inherent recalcitrance of membrane lipid transporters, the mechanisms of lipid transport within plant cells are not yet fully understood. Recently, several new proteins have been implicated in different aspects of plant lipid trafficking. While these proteins provide only first insights into limited aspects of lipid transport phenomena in plant cells, they represent exciting opportunities for further studies.

Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii.

In plants, neutral lipids are frequently synthesized and stored in seed tissues, where the assembly of lipid droplets (LDs) coincides with the accumulation of triacylglycerols (TAGs). In addition, photosynthetic, vegetative cells can form cytosolic LDs and much less information is known about the makeup and biogenesis of these LDs. Here we focus on Chlamydomonas reinhardtii as a reference model for LDs in a photosynthetic cell, because in this unicellular green alga LD dynamics can be readily manipulated by nitrogen availability. Nitrogen deprivation leads to cellular quiescence during which cell divisions cease and TAGs accumulate. The major lipid droplet protein (MLDP) forms a proteinaceous coat surrounding mature LDs. Reducing the amount of MLDP affects LD size and number, TAG breakdown and timely progression out of cellular quiescence following nitrogen resupply. Depending on nitrogen availability, MLDP recruits different proteins to LDs, tubulins in particular. Conversely, depolymerization of microtubules drastically alters the association of MLDP with LDs. LDs also contain select chloroplast envelope membrane proteins hinting at an origin of LDs, at least in part, from chloroplast membranes. Moreover, LD surface lipids are rich in de novo synthesized fatty acids, and are mainly composed of galactolipids which are typical components of chloroplast membranes. The composition of the LD membrane is altered in the absence of MLDP. Collectively, our results suggest a mechanism for LD formation in C. reinhardtii involving chloroplast envelope membranes by which specific proteins are recruited to LDs and a specialized polar lipid monolayer surrounding the LD is formed.

Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue.

In plants, lipids of the photosynthetic membrane are synthesized by parallel pathways associated with the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Lipids derived from the two pathways are distinguished by their acyl-constituents. Following this plant paradigm, the prevalent acyl composition of chloroplast lipids suggests that Chlamydomonas reinhardtii (Chlamydomonas) does not use the ER pathway; however, the Chlamydomonas genome encodes presumed plant orthologues of a chloroplast lipid transporter consisting of TGD (TRIGALACTOSYLDIACYLGLYCEROL) proteins that are required for ER-to-chloroplast lipid trafficking in plants. To resolve this conundrum, we identified a mutant of Chlamydomonas deleted in the TGD2 gene and characterized the respective protein, CrTGD2. Notably, the viability of the mutant was reduced, showing the importance of CrTGD2. Galactoglycerolipid metabolism was altered in the tgd2 mutant with monogalactosyldiacylglycerol (MGDG) synthase activity being strongly stimulated. We hypothesize this to be a result of phosphatidic acid accumulation in the chloroplast outer envelope membrane, the location of MGDG synthase in Chlamydomonas. Concomitantly, increased conversion of MGDG into triacylglycerol (TAG) was observed. This TAG accumulated in lipid droplets in the tgd2 mutant under normal growth conditions. Labeling kinetics indicate that Chlamydomonas can import lipid precursors from the ER, a process that is impaired in the tgd2 mutant.

Structural determinants allowing transferase activity in SENSITIVE TO FREEZING 2, classified as a family I glycosyl hydrolase.

SENSITIVE TO FREEZING 2 (SFR2) is classified as a family I glycosyl hydrolase but has recently been shown to have galactosyltransferase activity in Arabidopsis thaliana. Natural occurrences of apparent glycosyl hydrolases acting as transferases are interesting from a biocatalysis standpoint, and knowledge about the interconversion can assist in engineering SFR2 in crop plants to resist freezing. To understand how SFR2 evolved into a transferase, the relationship between its structure and function are investigated by activity assay, molecular modeling, and site-directed mutagenesis. SFR2 has no detectable hydrolase activity, although its catalytic site is highly conserved with that of family 1 glycosyl hydrolases. Three regions disparate from glycosyl hydrolases are identified as required for transferase activity as follows: a loop insertion, the C-terminal peptide, and a hydrophobic patch adjacent to the catalytic site. Rationales for the effects of these regions on the SFR2 mechanism are discussed.

Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779.

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica-specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.