Zhi-Yan Du

Depletion of the membrane-associated acyl-coenzyme A-binding protein ACBP1 enhances the ability of cold acclimation in Arabidopsis.

In Arabidopsis (Arabidopsis thaliana), a family of six genes encodes acyl-coenzyme A-binding proteins (ACBPs). A member of this family, ACBP1, contains an amino-terminal transmembrane domain that targets it to the plasma membrane and the endoplasmic reticulum. To investigate ACBP1 function, ACBP1-overexpressing transgenic Arabidopsis plants were characterized using lipid analysis. ACBP1 overexpressors showed reduction in several species of diunsaturated phosphatidylcholine (PC), prompting us to investigate if they were altered in response to freezing stress. ACBP1 overexpressors demonstrated increased freezing sensitivity accompanied by a decrease in PC and an increase in phosphatidic acid (PA), while acbp1 mutant plants showed enhanced freezing tolerance associated with PC accumulation and PA reduction. We also showed binding of a recombinant eukaryotic ACBP (ACBP1) to PA, indicative of the possibility of enhanced PA interaction in ACBP1 overexpressors. Since phospholipase Dα1 (PLDα1) is a major enzyme promoting the hydrolysis of PC to PA, PLDα1 expression was examined and was observed to be higher in ACBP1 overexpressors than in acbp1 mutant plants. In contrast, the expression of PLDδ, which plays a positive role in freezing tolerance, declined in the ACBP1 overexpressors but increased in acbp1 mutant plants. Given that ACBP1 is localized to the endoplasmic reticulum and plasma membrane, it may regulate the expression of PLDα1 and PLDδ by maintaining a membrane-associated PA pool through its ability to bind PA. Moreover, both genotypes showed no alterations in proline and soluble sugar content or in cold-regulated (COR6.6 and COR47) gene expression, suggesting that the ACBP1-mediated response is PLD associated and is independent of osmolyte accumulation.

Stress-induced neutral lipid biosynthesis in microalgae - Molecular, cellular and physiological insights.

Photosynthetic microalgae have promise as biofuel feedstock. Under certain conditions, they produce substantial amounts of neutral lipids, mainly in the form of triacylglycerols (TAGs), which can be converted to fuels. Much of our current knowledge on the genetic and molecular basis of algal neutral lipid metabolism derives mainly from studies of plants, i.e. seed tissues, and to a lesser extent from direct studies of algal lipid metabolism. Thus, the knowledge of TAG synthesis and the cellular trafficking of TAG precursors in algal cells is to a large extent based on genome predictions, and most aspects of TAG metabolism have yet to be experimentally verified. The biofuel prospects of microalgae have raised the interest in mechanistic studies of algal TAG biosynthesis in recent years and resulted in an increasing number of publications on lipid metabolism in microalgae. In this review we summarize the current findings on genetic, molecular and physiological studies of TAG accumulation in microalgae. Special emphasis is on the functional analysis of key genes involved in TAG synthesis, molecular mechanisms of regulation of TAG biosynthesis, as well as on possible mechanisms of lipid droplet formation in microalgal cells. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Arabidopsis acyl-CoA-binding protein ACBP1 participates in the regulation of seed germination and seedling development.

A family of six genes encoding acyl-CoA-binding proteins (ACBPs), ACBP1-ACBP6, has been characterized in Arabidopsis thaliana. In this study, we demonstrate that ACBP1 promotes abscisic acid (ABA) signaling during germination and seedling development. ACBP1 was induced by ABA, and transgenic Arabidopsis ACBP1-over-expressors showed increased sensitivity to ABA during germination and seedling development, whereas the acbp1 mutant showed decreased ABA sensitivity during these processes. Subsequent RNA assays showed that ACBP1 over-production in 12-day-old seedlings up-regulated the expression of PHOSPHOLIPASE Dα1 (PLDα1) and three ABA/stress-responsive genes: ABA-RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1), RESPONSE TO DESICCATION29A (RD29A) and bHLH-TRANSCRIPTION FACTOR MYC2 (MYC2). The expression of AREB1 and PLDα1 was suppressed in the acbp1 mutant in comparison with the wild type following ABA treatment. PLDα1 has been reported to promote ABA signal transduction by producing phosphatidic acid, an important lipid messenger in ABA signaling. Using lipid profiling, seeds and 12-day-old seedlings of ACBP1-over-expressing lines were shown to accumulate more phosphatidic acid after ABA treatment, in contrast to lower phosphatidic acid in the acbp1 mutant. Bimolecular fluorescence complementation assays indicated that ACBP1 interacts with PLDα1 at the plasma membrane. Their interaction was further confirmed by yeast two-hybrid analysis. As recombinant ACBP1 binds phosphatidic acid and phosphatidylcholine, ACBP1 probably promotes PLDα1 action. Taken together, these results suggest that ACBP1 participates in ABA-mediated seed germination and seedling development.

Overexpression of Arabidopsis acyl‐CoA‐binding protein ACBP2 enhances drought tolerance

Arabidopsis thaliana acyl-CoA-binding protein 2 (ACBP2) is a stress-responsive protein that is also important in embryogenesis. Here, we assign a role for ACBP2 in abscisic acid (ABA) signalling during seed germination, seedling development and the drought response. ACBP2 was induced by ABA and drought, and transgenic Arabidopsis overexpressing ACBP2 (ACBP2-OXs) showed increased sensitivity to ABA treatment during germination and seedling development. ACBP2-OXs also displayed improved drought tolerance and ABA-mediated reactive oxygen species (ROS) production in guard cells, thereby promoting stomatal closure, reducing water loss and enhancing drought tolerance. In contrast, acbp2 mutant plants showed decreased sensitivity to ABA in root development and were more sensitive to drought stress. RNA analyses revealed that ACBP2 overexpression up-regulated the expression of Respiratory Burst Oxidase Homolog D (AtrbohD) and AtrbohF, two NAD(P)H oxidases essential for ABA-mediated ROS production, whereas the expression of Hypersensitive to ABA1 (HAB1), an important negative regulator in ABA signalling, was down-regulated. In addition, transgenic plants expressing ACBP2pro:GUS showed beta-glucuronidase (GUS) staining in guard cells, confirming a role for ACBP2 at the stomata. These observations support a positive role for ACBP2 in promoting ABA signalling in germination, seedling development and the drought response.

Rapid labeling of intracellular His-tagged proteins in living cells

Significance The hexahistidine-Ni2+-NTA system is used extensively in protein purification, and large numbers of His-tagged protein libraries exist worldwide. The application of this tagging system to image proteins in live cells would offer significant opportunities to track cellular events with minimal spatial and functional perturbation on a protein of interest. However, previously reported Ni-NTA–based probes suffer from poor membrane permeability and have been limited to label membrane proteins only. Here we report, to our knowledge, the first small fluorescent probe, Ni-NTA-AC, that can rapidly cross cell membranes to specifically target His-tagged proteins in various types of live cells, even in plant tissues. The probe will provide new opportunities for in situ analysis of various cellular events. Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni2+-NTA–based probes. Unfortunately, previous Ni-NTA–based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni2+ ions. The probe, driven by Ni2+-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

Triacylglycerol Accumulation in Photosynthetic Cells in Plants and Algae

Plant and algal oils are some of the most energy-dense renewable compounds provided by nature. Triacylglycerols (TAGs) are the major constituent of plant oils, which can be converted into fatty acid methyl esters commonly known as biodiesel. As one of the most efficient producers of TAGs, photosynthetic microalgae have attracted substantial interest for renewable fuel production. Currently, the big challenge of microalgae based TAGs for biofuels is their high cost compared to fossil fuels. A conundrum is that microalgae accumulate large amounts of TAGs only during stress conditions such as nutrient deprivation and temperature stress, which inevitably will inhibit growth. Thus, a better understanding of why and how microalgae induce TAG biosynthesis under stress conditions would allow the development of engineered microalgae with increased TAG production during conditions optimal for growth. Land plants also synthesize TAGs during stresses and we will compare new findings on environmental stress-induced TAG accumulation in plants and microalgae especially in the well-characterized model alga Chlamydomonas reinhardtii and a biotechnologically relevant genus Nannochloropsis.

Comparative genomics of Mortierella elongata and its bacterial endosymbiont Mycoavidus cysteinexigens

Endosymbiosis of bacteria by eukaryotes is a defining feature of cellular evolution. In addition to well-known bacterial origins for mitochondria and chloroplasts, multiple origins of bacterial endosymbiosis are known within the cells of diverse animals, plants and fungi. Early-diverging lineages of terrestrial fungi harbor endosymbiotic bacteria belonging to the Burkholderiaceae. We sequenced the metagenome of the soil-inhabiting fungus Mortierella elongata and assembled the complete circular chromosome of its endosymbiont, Mycoavidus cysteinexigens, which we place within a lineage of endofungal symbionts that are sister clade to Burkholderia. The genome of M. elongata strain AG77 features a core set of primary metabolic pathways for degradation of simple carbohydrates and lipid biosynthesis, while the M. cysteinexigens (AG77) genome is reduced in size and function. Experiments using antibiotics to cure the endobacterium from the host demonstrate that the fungal host metabolism is highly modulated by presence/absence of M. cysteinexigens. Independent comparative phylogenomic analyses of fungal and bacterial genomes are consistent with an ancient origin for M. elongata - M. cysteinexigens symbiosis, most likely over 350 million years ago and concomitant with the terrestrialization of Earth and diversification of land fungi and plants.

Interactions between Arabidopsis acyl-CoA-binding proteins and their protein partners

Protein–protein interactions are at the core of cellular interactomics and are essential for various biological functions. Since proteins commonly function as macromolecular complexes, it is important to identify their interacting partners to better understand their function and the significance in these interactions. The acyl-CoA-binding proteins (ACBPs) of eukaryotes show conservation in the presence of a lipid-binding acyl-CoA-binding domain. In Arabidopsis thaliana, four of six members from the AtACBP family possess ankyrin repeats (AtACBP1 and AtACBP2) or kelch motifs (AtACBP4 and AtACBP5), which can potentially mediate protein–protein interactions. Through yeast two-hybrid screens, a dozen putative protein partners interacting with AtACBPs have been isolated from an Arabidopsis cDNA library. Investigations in the past decade on the interaction between AtACBPs and their protein partners have revealed novel roles for AtACBPs, including functions in mediating oxidative stress responses, heavy metal tolerance and oxygen sensing. Recent progress and current questions on AtACBPs and their interactors are discussed in this review.

Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts

BackgroundPhotosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail.ResultsBased on their expression profile, six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6) were chosen for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds.ConclusionsTaken together, our results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.

Plant acyl-CoA-binding proteins: An emerging family involved in plant development and stress responses

Acyl-CoA-binding protein (ACBP) was first identified in mammals as a neuropeptide, and was demonstrated to belong to an important house-keeping protein family that extends across eukaryotes and some prokaryotes. In plants, the Arabidopsis ACBP family consists of six AtACBPs (AtACBP1 to AtACBP6), and has been investigated using gene knock-out mutants and overexpression lines. Herein, recent findings on the AtACBPs are examined to provide an insight on their functions in various plant developmental processes, such as embryo and seed development, seed dormancy and germination, seedling development and cuticle formation, as well as their roles under various environmental stresses. The significance of the AtACBPs in acyl-CoA/lipid metabolism, with focus on their interaction with long to very-long-chain (VLC) acyl-CoA esters and their potential role in the formation of lipid droplets in seeds and vegetative tissues are discussed. In addition, recent findings on the rice ACBP family are presented. The similarities and differences between ACBPs from Arabidopsis and rice, that represent eudicot and monocot model plants, respectively, are analyzed and the evolution of plant ACBPs by phylogenetic analysis reviewed. Finally, we propose potential uses of plant ACBPs in phytoremediation and in agriculture related to the improvement of environmental stress tolerance and seed oil production.