Shiu-Cheung Lung

Arabidopsis membrane-associated acyl-CoA-binding protein ACBP1 is involved in stem cuticle formation

Summary The binding of recombinant AtACBP1 to very-long-chain acyl-CoA esters is related to AtACBP1 function in Arabidopsis stem cuticle metabolism. Loss-of-function mutation adversely affected stem cuticle composition and structure.

Deciphering the roles of acyl-CoA-binding proteins in plant cells

Lipid trafficking is vital for metabolite exchange and signal communications between organelles and endomembranes. Acyl-CoA-binding proteins (ACBPs) are involved in the intracellular transport, protection, and pool formation of acyl-CoA esters, which are important intermediates and regulators in lipid metabolism and cellular signaling. In this review, we highlight recent advances in our understanding of plant ACBP families from a cellular and developmental perspective. Plant ACBPs have been extensively studied in Arabidopsis thaliana (a dicot) and to a lesser extent in Oryza sativa (a monocot). Thus far, they have been detected in the plasma membrane, vesicles, endoplasmic reticulum, Golgi apparatus, apoplast, cytosol, nuclear periphery, and peroxisomes. In combination with biochemical and molecular genetic tools, the widespread subcellular distribution of respective ACBP members has been explicitly linked to their functions in lipid metabolism during development and in response to stresses. At the cellular level, strong expression of specific ACBP homologs in specialized cells, such as embryos, stem epidermis, guard cells, male gametophytes, and phloem sap, is of relevance to their corresponding distinct roles in organ development and stress responses. Other interesting patterns in their subcellular localization and spatial expression that prompt new directions in future investigations are discussed.

Acyl-CoA-Binding Proteins (ACBPs) in Plant Development

Acyl-CoA-binding proteins (ACBPs) play a pivotal role in fatty acid metabolism because they can transport medium- and long-chain acyl-CoA esters. In eukaryotic cells, ACBPs are involved in intracellular trafficking of acyl-CoA esters and formation of a cytosolic acyl-CoA pool. In addition to these ubiquitous functions, more specific non-redundant roles of plant ACBP subclasses are implicated by the existence of multigene families with variable molecular masses, ligand specificities, functional domains (e.g. protein-protein interaction domains), subcellular locations and gene expression patterns. In this chapter, recent progress in the characterization of ACBPs from the model dicot plant, Arabidopsis thaliana, and the model monocot, Oryza sativa, and their emerging roles in plant growth and development are discussed. The functional significance of respective members of the plant ACBP families in various developmental and physiological processes such as seed development and germination, stem cuticle formation, pollen development, leaf senescence, peroxisomal fatty acid β-oxidation and phloem-mediated lipid transport is highlighted.

Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis

Arabidopsis acyl-CoA-binding protein ACBP1 forms an endoplasmic reticulum-associated complex with STEROL C4-METHYL OXIDASE1-1 and negatively modulates sterol synthesis during embryogenesis. Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1. Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 (GL2), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1, smo1-1, and ACBP1+/−smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1+/−smo1-1. Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling.

The binding versatility of plant acyl-CoA-binding proteins and their significance in lipid metabolism.

Acyl-CoA esters are the activated form of fatty acids and play important roles in lipid metabolism and the regulation of cell functions. They are bound and transported by nonenzymic proteins such as the acyl-CoA-binding proteins (ACBPs). Although plant ACBPs were so named by virtue of amino acid homology to existing yeast and mammalian counterparts, recent studies revealed that ligand specificities of plant ACBPs are not restricted to acyl-CoA esters. Arabidopsis and rice ACBPs also interact with phospholipids, and their affinities to different acyl-CoA species and phospholipid classes vary amongst isoforms. Their ligands also include heavy metals. Interactors of plant ACBPs are further diversified due to the evolution of protein-protein interacting domains. This review summarizes our current understanding of plant ACBPs with a focus on their binding versatility. Their broad ligand range is of paramount significance in serving a multitude of functions during development and stress responses as discussed herein. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

The C-terminus of Bienertia sinuspersici Toc159 contains essential elements for its targeting and anchorage to the chloroplast outer membrane.

Most nucleus-encoded chloroplast proteins rely on an N-terminal transit peptide (TP) as a post-translational sorting signal for directing them to the organelle. Although Toc159 is known to be a receptor for specific preprotein TPs at the chloroplast surface, the mechanism for its own targeting and integration into the chloroplast outer membrane is not completely understood. In a previous study, we identified a novel TP-like sorting signal at the C-terminus (CT) of a Toc159 homolog from the single-cell C4 species, Bienertia sinuspersici. In the current study, we have extended our understanding of the sorting signal using transient expression of fluorescently-tagged fusion proteins of variable-length, and with truncated and swapped versions of the CT. As was shown in the earlier study, the 56 residues of the CT contain crucial sorting information for reversible interaction of the receptor with the chloroplast envelope. Extension of this region to 100 residues in the current study stabilized the interaction via membrane integration, as demonstrated by more prominent plastid-associated signals and resistance of the fusion protein to alkaline extraction. Despite a high degree of sequence similarity, the plastid localization signals of the equivalent CT regions of Arabidopsis thaliana Toc159 homologs were not as strong as that of the B. sinuspersici counterparts. Together with computational and circular dichroism analyses of the CT domain structures, our data provide insights into the critical elements of the CT for the efficient targeting and anchorage of Toc159 receptors to the dimorphic chloroplasts in the single-cell C4 species.

Depletion of Arabidopsis ACYL-COA-BINDING PROTEIN3 affects fatty acid composition in the phloem

Oxylipins are crucial components in plant wound responses that are mobilised via the plant vasculature. Previous studies have shown that the overexpression of an Arabidopsis acyl-CoA-binding protein, AtACBP3, led to an accumulation of oxylipin-containing galactolipids, and AtACBP3pro::BETA-GLUCURONIDASE (GUS) was expressed in the phloem of transgenic Arabidopsis. To investigate the role of AtACBP3 in the phloem, reverse transcription-polymerase chain reaction and western blot analysis of phloem exudates from the acbp3 mutant and wild type revealed that the AtACBP3 protein, but not its mRNA, was detected in the phloem sap. Furthermore, micrografting demonstrated that AtACBP3 expressed from the 35S promoter was translocated from shoot to root. Subsequently, AtACBP3 was localised to the companion cells, sieve elements and the apoplastic space of phloem tissue by immunogold electron microscopy using anti-AtACBP3 antibodies. AtACBP3pro::GUS was induced locally in Arabidopsis leaves upon wounding, and the expression of wound-responsive jasmonic acid marker genes (JASMONATE ZIM-DOMAIN10, VEGETATIVE STORAGE PROTEIN2, and LIPOXYGENASE2) increased more significantly in both locally wounded and systemic leaves of the wild type in comparison to acbp3 and AtACBP3-RNAi. Oxylipin-related fatty acid (FA) (C18:2-FA, C18:3-FA and methyl jasmonate) content was observed to be lower in acbp3 and AtACBP3-RNAi than wild-type phloem exudates using gas chromatography-mass spectrometry. Experiments using recombinant AtACBP3 in isothermal titration calorimetry analysis showed that medium- and long-chain acyl-CoA esters bind (His)6-AtACBP3 with KD values in the micromolar range. Taken together, these results suggest that AtACBP3 is likely to be a phloem-mobile protein that affects the FA pool and jasmonate content in the phloem, possibly by its binding to acyl-CoA esters.

Arabidopsis ACYL-COA-BINDING PROTEIN1 interacts with STEROL C4-METHYL OXIDASE1-2 to modulate gene expression of homeodomain-leucine zipper IV transcription factors

Fatty acids (FAs) and sterols constitute building blocks of eukaryotic membranes and lipid signals. Co-regulation of FA and sterol synthesis is mediated by sterol regulatory element-binding proteins in animals but remains elusive in plants. We reported recently that Arabidopsis ACYL-COA-BINDING PROTEIN1 (ACBP1) modulates sterol synthesis via protein-protein interaction with STEROL C4-METHYL OXIDASE1-1 (SMO1-1). Herein, ACBP1 was demonstrated to co-express and interact with SMO1-2 by yeast two-hybrid, co-localization, pull-down, co-immunoprecipitation and β-glucuronidase assays. SMO1-2 silenced in acbp1 was used in phenotyping, GC-MS and expression profiling. ACBP1 co-expressed with SMO1-2 in embryo sacs, pollen and trichomes, corroborating with cooperative tissue-specific functions unseen with SMO1-1. SMO1-2 silencing in acbp1 impaired seed development, male and female gamete transmission, and pollen function. Genes encoding homeodomain-leucine zipper IV transcription factors (HDG5, HDG10, HDG11 and GLABRA2), which potentially bind phospholipids/sterols, were transcribed aberrantly. GLABRA2 targets (MYB23, MUM4 and PLDα1) were misregulated, causing glabra2-resembling trichome, seed coat mucilage and oil-accumulating phenotypes. Together with altered sterol and FA compositions upon ACBP1 mutation and/or SMO1-2 silencing, ACBP1-SMO1 interaction appears to mediate homeostatic co-regulation of FAs and sterols, which serve as lipid modulators for gene expression of homeodomain-leucine zipper IV transcription factors.

Present Status and Future Prospects of Transgenic Approaches for Drought Tolerance

Drought causes severe stress on plants and constrains crop production. New strategies are essential to overcome water loss and stabilize crop yield in agriculture in view of global climate change. Conventional methods employing direct selection for high-yielding cultivars or indirect identification of secondary traits under stressful conditions are useful, but these procedures are time-consuming and labor-intensive. In contrast, the newer “omic” technologies can more quickly help us acquire a molecular and physiological understanding of drought tolerance in plants, whereas transgenic strategies hold promise in using specific transgenes to minimize the deleterious effects of water deficiency. A substantial amount of research utilizing mutant and transgenic plant lines exhibiting altered expression of drought-responsive genes has significantly contributed to promoting drought protection. These genes can be broadly categorized into three groups based on their functions: regulatory genes, genes related to metabolites and osmoprotectants, and those associated with posttranslational modification. This chapter focuses on current progress on some transgenic approaches for enhancing drought tolerance in plants. Examples of successful applications involving various transcription factors, and strategies related to abscisic acid and osmolyte metabolism, as well as protein phosphorylation and farnesylation, are discussed.

Isolation of Chloroplasts from Plant Protoplasts

Chloroplasts can be isolated from higher plants directly following homogenization; however, the resulting yield, purity, and intactness are often low, necessitating a large amount of starting material. This protocol is optimized to produce a high yield of pure chloroplasts from isolated Arabidopsis protoplasts. The two-part method is a simple, scaled-down, and low-cost procedure that readily provides healthy mesophyll protoplasts, which are then ruptured to release intact chloroplasts. Chloroplasts isolated using this method are competent for use in biochemical, cellular, and molecular analyses.