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Mee-len Chye

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Papers by Mee-len Chye

Plant Acyl-CoA-Binding Proteins—Their Lipid and Protein Interactors in Abiotic and Biotic Stresses

Cells, Apr 30, 2021

Plants are constantly exposed to environmental stresses during their growth and development. Owin... more Plants are constantly exposed to environmental stresses during their growth and development. Owing to their immobility, plants possess stress-sensing abilities and adaptive responses to cope with the abiotic and biotic stresses caused by extreme temperatures, drought, flooding, salinity, heavy metals and pathogens. Acyl-CoA-binding proteins (ACBPs), a family of conserved proteins among prokaryotes and eukaryotes, bind to a variety of acyl-CoA esters with different affinities and play a role in the transport and maintenance of subcellular acyl-CoA pools. In plants, studies have revealed ACBP functions in development and stress responses through their interactions with lipids and protein partners. This review summarises the roles of plant ACBPs and their lipid and protein interactors in abiotic and biotic stress responses.

The overexpression of OsACBP5 protects transgenic rice against necrotrophic, hemibiotrophic and biotrophic pathogens

Scientific Reports, Sep 10, 2020

The overexpression of OsACBP5 protects transgenic rice against necrotrophic, hemibiotrophic and b... more The overexpression of OsACBP5 protects transgenic rice against necrotrophic, hemibiotrophic and biotrophic pathogens Saritha panthapulakkal narayanan, Shiu-cheung Lung, pan Liao, clive Lo & Mee-Len chye * the most devastating diseases in rice (Oryza sativa) are sheath blight caused by the fungal necrotroph Rhizoctonia solani, rice blast by hemibiotrophic fungus Magnaporthe oryzae, and leaf blight by bacterial biotroph Xanthomonas oryzae (Xoo). it has been reported that the class iii acyl-coA-binding proteins (AcBps) such as those from dicots (Arabidopsis and grapevine) play a role in defence against biotrophic pathogens. of the six Arabidopsis (Arabidopsis thaliana) ACBPs, AtACBP3 conferred protection in transgenic Arabidopsis against Pseudomonas syringae, but not the necrotrophic fungus, Botrytis cinerea. Similar to Arabidopsis, rice possesses six AcBps, designated osAcBps. the aims of this study were to test whether OsACBP5, the homologue of AtACBP3, can confer resistance against representative necrotrophic, hemibiotrophic and biotrophic phytopathogens and to understand the mechanisms in protection. Herein, when OsACBP5 was overexpressed in rice, the OsACBP5overexpressing (OsACBP5-OE) lines exhibited enhanced disease resistance against representative necrotrophic (R. solani & Cercospora oryzae), hemibiotrophic (M. oryzae & Fusarium graminearum) and biotrophic (Xoo) phytopathogens. Progeny from a cross between OsACBP5-OE9 and the jasmonate (JA)-signalling deficient mutant were more susceptible than the wild type to infection by the necrotroph R. solani. In contrast, progeny from a cross between OsACBP5-OE9 and the salicylic acid (SA)-signalling deficient mutant was more susceptible to infection by the hemibiotroph M. oryzae and biotroph Xoo. Hence, enhanced resistance of OsACBP5-OEs against representative necrotrophs appears to be JA-dependent whilst that to (hemi)biotrophs is SA-mediated. Despite the existence of natural defence mechanisms in plants, crops can become susceptible to bacterial and fungal pathogens which cause negative impacts on food security and food safety worldwide 1 . This happens when the virulence factors of a pathogen overwhelm the defence responses of a specific host plant 2 . Phytopathogens are classified into necrotrophs, hemibiotrophs and biotrophs based on their interaction with the host. Necrotrophic pathogens rapidly kill host tissues by secreting toxins and survive on the dead remains, whereas biotrophs derive nutrients from living cells and therefore sustain host viability . Hemibiotrophic pathogens exhibit both forms of nutrient acquisition, starting from the suppression of host immune system in a biotrophic phase, followed by a later necrotrophic phase during which the pathogen induces host cell death by its toxin production 5 . Thus, necrotrophic pathogens tend to exert a more damaging effect on host plants than biotrophic pathogens and are deemed more detrimental to crop productivity 4 . Necrotrophic soil-borne phytopathogenic fungi such as Fusarium and Rhizoctonia infect prominent economically important monocot crops such as rice (Oryza sativa), wheat and maize . Fusarium solani infection resulted in a 10-30% drop in yield for pulse crops following severe root-rot 8 . In Japan, soybean foliar blight arising from the necrotroph Rhizoctonia solani AG-1 culminated to around 70% decrease in yield 9 . It has been reported that necrotrophic pathogens have a higher economic impact on agriculture over biotrophic pathogens 10 .

Characterization and function of a sunflower (Helianthus annuus L.) Class II acyl-CoA-binding protein

Plant Science, Nov 1, 2020

Acyl-CoA-binding proteins (ACBP) bind to long-chain acyl-CoA esters and phospholipids, enhancing ... more Acyl-CoA-binding proteins (ACBP) bind to long-chain acyl-CoA esters and phospholipids, enhancing the activity of different acyltransferases in animals and plants. Nevertheless, the role of these proteins in the synthesis of triacylglycerols (TAGs) remains unclear. Here, we cloned a cDNA encoding HaACBP1, a Class II ACBP from sunflower (Helianthus annuus), one of the world's most important oilseed crop plants. Transcriptome analysis of this gene revealed strong expression in developing seeds from 16 to 30 days after flowering. The recombinant protein (rHaACBP1) was expressed in Escherichia coli and purified to be studied by in vitro isothermal titration calorimetry and for phospholipid binding. Its high affinity for saturated palmitoyl-CoA (16:0-CoA; K D 0.11 µM) and stearoyl-CoA (18:0-CoA; K D 0.13 µM) esters suggests that rHaACBP1 could act in acyl-CoA transfer pathways that involve saturated acyl derivatives. Furthermore, rHaACBP1 also binds to both oleoyl-CoA (18:1-CoA; K D 6.4 µM) and linoleoyl-CoA (18:2-CoA; K D 21.4 µM) esters, the main acyl-CoA substrates used to synthesise the TAGs that accumulate in sunflower seeds. Interestingly, rHaACBP1 also appears to bind to different species of phosphatidylcholines (dioleoyl-PC and dilinoleoyl-PC), glycerolipids that are also involved in TAG synthesis, and while it interacts with dioleoyl-PA, this is less prominent than its binding to the PC derivative. Expression of rHaACBP in yeast altering its fatty acid composition, as well as the composition and size of the host acyl-CoA pool. These results suggest that HaACBP1 may potentially fulfil a role in the transport and trafficking of acyl-CoAs during sunflower seed development.

Arabidopsis cytosolic acyl‐CoA‐binding proteins function in determining seed oil composition

Plant direct, Dec 1, 2019

As plant seed oils provide animals with essential fatty acids (FAs), genes that regulate plant li... more As plant seed oils provide animals with essential fatty acids (FAs), genes that regulate plant lipid metabolism have been used in genetic manipulation to improve dietary seed oil composition and benefit human health. Herein, the Arabidopsis thaliana cytosolic acyl-CoA-binding proteins (AtACBPs), AtACBP4, AtACBP5, and AtACBP6 were shown to play a role in determining seed oil content by analysis of atacbp (atacbp4, atacbp5, atacbp6, atacbp4atacbp5, atacbp4atacbp6, atacbp5atacbp6, and atacb-p4atacbp5atacbp6) seed oil content in comparison with the Col-0 wild type (WT). Triacylglycerol (TAG) composition in electrospray ionization-mass spectrometer (ESI-MS) analysis on atacbp6 seed oil showed a reduction (-50%) of C58-TAGs in comparison with the WT. Investigations on fatty acid composition of atacbp mutants indicated that 18:2-FA accumulated in atacbp6 and 18:3-FA in atacbp4, both at the expense of 20:1-FA. As TAG composition can be modified by acyl editing through phosphatidylcholines (PC) and lysophosphatidylcholines (LPC), total PC and LPC content in atacbp6 mature seeds was determined and ESI-MS analysis revealed that LPC had increased (+300%) at the expense of PC. Among all the 14 tested PC species,

Oxylipin signaling in salt-stressed soybean is modulated by ligand-dependent interaction of Class II acyl-CoA-binding proteins with lipoxygenase

The Plant Cell, 2021

Plant lipoxygenases (LOXs) oxygenate linoleic and linolenic acids, creating hydroperoxy derivativ... more Plant lipoxygenases (LOXs) oxygenate linoleic and linolenic acids, creating hydroperoxy derivatives, and from these, jasmonates and other oxylipins are derived. Despite the importance of oxylipin signaling, its activation mechanism remains largely unknown. Here, we show that soybean ACYL-COA-BINDING PROTEIN3 (ACBP3) and ACBP4, two Class II acyl-CoA-binding proteins, suppressed activity of the vegetative LOX homolog VLXB by sequestering it at the endoplasmic reticulum. The ACBP4–VLXB interaction was facilitated by linoleoyl-CoA and linolenoyl-CoA, which competed with phosphatidic acid (PA) for ACBP4 binding. In salt-stressed roots, alternative splicing produced ACBP variants incapable of VLXB interaction. Overexpression of the variants enhanced LOX activity and salt tolerance in Arabidopsis and soybean hairy roots, whereas overexpressors of the native forms exhibited reciprocal phenotypes. Consistently, the differential alternative splicing pattern in two soybean genotypes coincided ...

Plant Acyl-CoA-Binding Proteins—Their Lipid and Protein Interactors in Abiotic and Biotic Stresses

Cells, 2021

Plants are constantly exposed to environmental stresses during their growth and development. Owin... more Plants are constantly exposed to environmental stresses during their growth and development. Owing to their immobility, plants possess stress-sensing abilities and adaptive responses to cope with the abiotic and biotic stresses caused by extreme temperatures, drought, flooding, salinity, heavy metals and pathogens. Acyl-CoA-binding proteins (ACBPs), a family of conserved proteins among prokaryotes and eukaryotes, bind to a variety of acyl-CoA esters with different affinities and play a role in the transport and maintenance of subcellular acyl-CoA pools. In plants, studies have revealed ACBP functions in development and stress responses through their interactions with lipids and protein partners. This review summarises the roles of plant ACBPs and their lipid and protein interactors in abiotic and biotic stress responses.

Characterization and function of a sunflower (Helianthus annuus L.) Class II acyl-CoA-binding protein

Plant Science, 2020

Acyl-CoA-binding proteins (ACBP) bind to long-chain acyl-CoA esters and phospholipids, enhancing ... more Acyl-CoA-binding proteins (ACBP) bind to long-chain acyl-CoA esters and phospholipids, enhancing the activity of different acyltransferases in animals and plants. Nevertheless, the role of these proteins in the synthesis of triacylglycerols (TAGs) remains unclear. Here, we cloned a cDNA encoding HaACBP1, a Class II ACBP from sunflower (Helianthus annuus), one of the world's most important oilseed crop plants. Transcriptome analysis of this gene revealed strong expression in developing seeds from 16 to 30 days after flowering. The recombinant protein (rHaACBP1) was expressed in Escherichia coli and purified to be studied by in vitro isothermal titration calorimetry and for phospholipid binding. Its high affinity for saturated palmitoyl-CoA (16:0-CoA; K D 0.11 µM) and stearoyl-CoA (18:0-CoA; K D 0.13 µM) esters suggests that rHaACBP1 could act in acyl-CoA transfer pathways that involve saturated acyl derivatives. Furthermore, rHaACBP1 also binds to both oleoyl-CoA (18:1-CoA; K D 6.4 µM) and linoleoyl-CoA (18:2-CoA; K D 21.4 µM) esters, the main acyl-CoA substrates used to synthesise the TAGs that accumulate in sunflower seeds. Interestingly, rHaACBP1 also appears to bind to different species of phosphatidylcholines (dioleoyl-PC and dilinoleoyl-PC), glycerolipids that are also involved in TAG synthesis, and while it interacts with dioleoyl-PA, this is less prominent than its binding to the PC derivative. Expression of rHaACBP in yeast altering its fatty acid composition, as well as the composition and size of the host acyl-CoA pool. These results suggest that HaACBP1 may potentially fulfil a role in the transport and trafficking of acyl-CoAs during sunflower seed development.

Nucleotide sequence of a cDNA clone encoding the precursor of ribulose-1,5-bisphosphate carboxylase small subunit from Hevea brasiliensis (rubber tree)

Plant Molecular Biology, 1991

... Mee-Len Chye, Sian Tan, Chio-Tee Tan, Anil Kush and Nam-Hai Chua 1 Institute of Molecular and... more

Overexpression of the Arabidopsis 10-Kilodalton Acyl-Coenzyme A-Binding Protein ACBP6 Enhances Freezing Tolerance

Plant Physiology, Jul 11, 2008

Small 10-kD acyl-coenzyme A-binding proteins (ACBPs) are highly conserved proteins that are preva... more Small 10-kD acyl-coenzyme A-binding proteins (ACBPs) are highly conserved proteins that are prevalent in eukaryotes. In Arabidopsis (Arabidopsis thaliana), other than the 10-kD ACBP homolog (designated Arabidopsis ACBP6), there are five larger forms of ACBPs ranging from 37.5 to 73.1 kD. In this study, the cytosolic subcellular localization of Arabidopsis ACBP6 was confirmed by analyses of transgenic Arabidopsis expressing autofluorescence-tagged ACBP6 and western-blot analysis of subcellular fractions using ACBP6-specific antibodies. The expression of Arabidopsis ACBP6 was noticeably induced at 48 h after 4°C treatment by northern-blot analysis and western-blot analysis. Furthermore, an acbp6 T-DNA insertional mutant that lacked ACBP6 mRNA and protein displayed increased sensitivity to freezing temperature (28°C), while ACBP6-overexpressing transgenic Arabidopsis plants were conferred enhanced freezing tolerance. Northern-blot analysis indicated that ACBP6-associated freezing tolerance was not dependent on the induction of cold-regulated COLD-RESPONSIVE gene expression. Instead, ACBP6 overexpressors showed increased expression of mRNA encoding phospholipase Dd. Lipid profiling analyses of rosettes from cold-acclimated, freezingtreated (28°C) transgenic Arabidopsis plants overexpressing ACBP6 showed a decline in phosphatidylcholine (236% and 246%) and an elevation of phosphatidic acid (73% and 67%) in comparison with wild-type plants. From our comparison, the gain in freezing tolerance in ACBP6 overexpressors that was accompanied by decreases in phosphatidylcholine and an accumulation of phosphatidic acid is consistent with previous findings on phospholipase Dd-overexpressing transgenic Arabidopsis. In vitro filterbinding assays indicating that histidine-tagged ACBP6 binds phosphatidylcholine, but not phosphatidic acid or lysophosphatidylcholine, further imply a role for ACBP6 in phospholipid metabolism in Arabidopsis, including the possibility of ACBP6 in the cytosolic trafficking of phosphatidylcholine.

Depletion of Arabidopsis ACYL-COA-BINDING PROTEIN3 Affects Fatty Acid Composition in the Phloem

Frontiers in Plant Science, Jan 25, 2018

Oxylipins are crucial components in plant wound responses that are mobilised via the plant vascul... more 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 woundresponsive 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 wildtype 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 K D 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

New Phytologist, Dec 30, 2017

Fatty acids (FAs) and sterols constitute building blocks of eukaryotic membranes and lipid signal... more 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 elementbinding 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 twohybrid, co-localization, pull-down, co-immunoprecipitation and b-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 PLDa1) were misregulated, causing glabra2resembling 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.

The gene encoding Arabidopsis acyl-CoA-binding protein 3 is pathogen inducible and subject to circadian regulation

Journal of Experimental Botany, Feb 15, 2012

In Arabidopsis thaliana, acyl-CoA-binding protein 3 ( ACBP3), one of six ACBPs, is unique in term... more In Arabidopsis thaliana, acyl-CoA-binding protein 3 ( ACBP3), one of six ACBPs, is unique in terms of the C-terminal location of its acyl-CoA-binding domain. It promotes autophagy-mediated leaf senescence and confers resistance to Pseudomonas syringae pv. tomato DC3000. To understand the regulation of ACBP3, a 1.7 kb 5'-flanking region of ACBP3 and its deletion derivatives were characterized using b-glucuronidase (GUS) fusions. A 374 bp minimal fragment (-151/+223) could drive GUS expression while a 1698 bp fragment (-1475/+223) conferred maximal activity. Further, histochemical analysis on transgenic Arabidopsis harbouring the largest (1698 bp) ACBP3pro::GUS fusion displayed ubiquitous expression in floral organs and vegetative tissues (vascular bundles of leaves and stems), consistent with previous results showing that extracellularly localized ACBP3 functions in plant defence. A 160 bp region (-434/-274) induced expression in extended darkness and caused down-regulation in extended light. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay showed that the DNA-binding with one finger box (Dof-box, -341/-338) interacted specifically with leaf nuclear proteins from dark-treated Arabidopsis, while GT-1 (-406/-401) binds both dark-and light-treated Arabidopsis, suggesting that Dof and GT-1 motifs are required to mediate circadian regulation of ACBP3. Moreover, GUS staining and fluorometric measurements revealed that a 109 bp region (-543/-434) was responsive to phytohormones and pathogens. An S-box of AT-rich sequence (-516/-512) was identified to bind nuclear proteins from pathogen-infected Arabidopsis leaves, providing the basis for pathogeninducible regulation of ACBP3 expression. Thus, three cis-responsive elements (Dof, GT-1, and the S-box) in the 5'-flanking region of ACBP3 are proven functional in the regulation of ACBP3.

The Arabidopsis Cytosolic Acyl-CoA-Binding Proteins Play Combinatory Roles in Pollen Development

Plant and Cell Physiology, Dec 22, 2014

In Arabidopsis, six acyl-CoA-binding proteins (ACBPs) have been identified and they have been dem... more In Arabidopsis, six acyl-CoA-binding proteins (ACBPs) have been identified and they have been demonstrated to function in plant stress responses and development. Three of these AtACBPs (AtACBP4-AtACBP6) are cytosolic proteins and all are expressed in floral organs as well as in other tissues. The roles of cytosolic AtACBPs in floral development were addressed in this study. To this end, a T-DNA insertional knockout mutant of acbp5 was characterized before use in crosses with the already available acbp4 and acbp6 T-DNA knockout mutants to examine their independent and combinatory functions in floral development. The single-gene knockout mutations did not cause any significant phenotypic changes, while phenotypic deficiencies affecting siliques and pollen were observed in the double mutants (acbp4acbp6 and acbp5acbp6) and the acbp4acbp5acbp6 triple mutant. Vacuole accumulation in the acbp4acbp6, acbp5acbp6 and acbp4acbp5acbp6 pollen was the most severe abnormality occurring in the double and triple mutants. Furthermore, scanning electron microscopy and transmission electron microscopy revealed exine and oil body defects in the acbp4acbp5acbp6 mutant, which also displayed reduced ability in in vitro pollen germination. Transgenic Arabidopsis expressing b-glucuronidase (GUS) driven from the various AtACBP promoters indicated that AtACBP6pro::GUS expression overlapped with AtACBP4pro::GUS expression in pollen grains and with AtACBP5pro::GUS expression in the microspores and tapetal cells. Taken together, these results suggest that the three cytosolic AtACBPs play combinatory roles in acyl-lipid metabolism during pollen development.

Ethylene- and pathogen-inducible Arabidopsis acyl-CoA-binding protein 4 interacts with an ethylene-responsive element binding protein

Journal of Experimental Botany, Oct 9, 2008

Six genes encode proteins with acyl-CoA-binding domains in Arabidopsis thaliana. They are the sma... more Six genes encode proteins with acyl-CoA-binding domains in Arabidopsis thaliana. They are the small 10-kDa cytosolic acyl-CoA-binding protein (ACBP), membrane-associated ACBP1 and ACBP2, extracellularly-targeted ACBP3, and kelch-motif containing ACBP4 and ACBP5. Here, the interaction of ACBP4 with an A. thaliana ethylene-responsive element binding protein (AtEBP), identified in a yeast two-hybrid screen, was confirmed by co-immunoprecipitation. The subcellular localization of ACBP4 and AtEBP, was addressed using an ACBP4:DsRed red fluorescent protein fusion and a green fluorescent protein (GFP):AtEBP fusion. Transient expression of these autofluoresence-tagged proteins in agroinfiltrated tobacco leaves, followed by confocal laser scanning microscopy, indicated their co-localization predominantly at the cytosol which was confirmed by FRET analysis. Immuno-electron microscopy on Arabidopsis sections not only localized ACBP4 to the cytosol but also to the periphery of the nucleus upon closer examination, perhaps as a result of its interaction with AtEBP. Furthermore, the expression of ACBP4 and AtEBP in Northern blot analyses was induced by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, methyl jasmonate treatments, and Botrytis cinerea infection, suggesting that the interaction of ACBP4 and AtEBP may be related to AtEBPmediated defence possibly via ethylene and/or jasmonate signalling.

Arabidopsis ACBP6 is an acyl-CoA-binding protein associated with phospholipid metabolism

Plant Signaling & Behavior, Nov 1, 2008

The overexpression of rice <scp>ACYL</scp> ‐ <scp>CoA</scp> ‐ <scp>BINDING PROTEIN</scp> 2 increases grain size and bran oil content in transgenic rice

Plant Journal, Sep 21, 2019

As Oryza sativa (rice) seeds represent food for over three billion people worldwide, the identifi... more As Oryza sativa (rice) seeds represent food for over three billion people worldwide, the identification of genes that enhance grain size and composition is much desired. Past reports have indicated that Arabidopsis thaliana acyl-CoA-binding proteins (ACBPs) are important in seed development but did not affect seed size. Herein, rice OsACBP2 was demonstrated not only to play a role in seed development and germination, but also to influence grain size. OsACBP2 mRNA accumulated in embryos and endosperm of germinating seeds in qRT-PCR analysis, while b-glucuronidase (GUS) assays on OsACBP2pro::GUS rice transformants showed GUS expression in embryos, as well as the scutellum and aleurone layer of germinating seeds. Deletion analysis of the OsACBP2 5'-flanking region revealed five copies of the seed cis-element, Skn-I-like motif (À1486/À1482, À956/À952, À939/À935, À826/À822, and À766/À762), and the removal of any adversely affected expression in seeds, thereby providing a molecular basis for OsACBP2 expression in seeds. When OsACBP2 function was investigated using osacbp2 mutants and transgenic rice overexpressing OsACBP2 (OsACBP2-OE), osacbp2 was retarded in germination, while OsACBP2-OEs performed better than the wild-type and vector-transformed controls, in germination, seedling growth, grain size and grain weight. Transmission electron microscopy of OsACBP2-OE mature seeds revealed an accumulation of oil bodies in the scutellum cells, while confocal laser scanning microscopy indicated oil accumulation in OsACBP2-OE aleurone tissues. Correspondingly, OsACBP2-OE seeds showed gain in triacylglycerols and long-chain fatty acids over the vector-transformed control. As dietary rice bran contains beneficial bioactive components, OsACBP2 appears to be a promising candidate for enriching seed nutritional value.

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

Plant Physiology, May 12, 2017

Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as struct... more 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 Da1 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+/2smo1-1 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+/2smo1-1. Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling.

Serine proteinase inhibitor proteins: Exogenous and endogenous functions

In Vitro Cellular & Developmental Biology – Plant, Mar 1, 2006

Proteinase inhibitor II (PIN2) proteins from the Solanaceae family have been previously used in p... more Proteinase inhibitor II (PIN2) proteins from the Solanaceae family have been previously used in plant transformation to acquire protection against caterpillars. Some of these PIN2 proteins have been shown to exhibit exogenous activities against trypsin and/or chymotrypsin in vitro. Despite their application in conferring insect resistance in transgenic plants, the endogenous roles of this family of proteins in various plant species have not been well defined. To investigate the exogenous and endogenous functions of PIN2 proteins, cDNAs encoding PIN2 proteins from the weed Solanum americanum (American black nightshade), designated SaPIN2a and SaPIN2b, were cloned and characterized. The localization of S. americanum SaPIN2a and SaPIN2b mRNAs and proteins in the reproductive tissues destined to undergo developmental programmed cell death subsequently led to investigations into their function during seed development. Using plant transformation of lettuce and S. americanum, it was evident that: (1) the expression of SaPIN2a in transgenic lettuce conferred resistance to cabbage looper (Trichoplusia ni) caterpillars; and (2) the expression of siRNAs from a PIN2-RNAi construct resulted in transgenic S. americanum that were impaired in seed development. These results suggest that S. americanum PIN2 proteins not only enhance resistance to caterpillars (when expressed exogenously) but they function in inhibiting endogenous proteases that are expressed during seed development. Specifically, the aborted seeds of PIN2-RNAi lines showed abnormal endothelium that subsequently affected endosperm and embryo development.

Arabidopsis acyl-CoA-binding proteins ACBP4 and ACBP5 are subcellularly localized to the cytosol and ACBP4 depletion affects membrane lipid composition

Plant Molecular Biology, Sep 5, 2008

In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by six genes, and they dis... more In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by six genes, and they display varying affinities for acyl-CoA esters. Recombinant ACBP4 and ACBP5 have been shown to bind oleoyl-CoA esters in vitro. In this study, the subcellular localizations of ACBP4 and ACBP5 were determined by biochemical fractionation followed by western blot analyses using anti-ACBP4 and anti-ACBP5 antibodies and immuno-electron microscopy. Confocal microscopy of autofluorescence-tagged ACBP4 and ACBP5, expressed transiently in onion epidermal cells and in transgenic Arabidopsis, confirmed their expression in the cytosol. Taken together, ACBP4 and ACBP5 are available in the cytosol to bind and transfer cytosolic oleoyl-CoA esters. Lipid profile analysis further revealed that an acbp4 knockout mutant showed decreases in membrane lipids (digalactosyldiacylglycerol, monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol) while acbp4-complemented lines attained levels similar to wild type, suggesting that ACBP4 plays a role in the biosynthesis of membrane lipids including galactolipids and phospholipids.

The <i>Arabidopsis acbp1acbp2</i> double mutant lacking acyl‐CoA‐binding proteins ACBP1 and ACBP2 is embryo lethal

New Phytologist, Mar 22, 2010

In Arabidopsis thaliana, the amino acid sequences of membrane-associated acyl-CoAbinding proteins... more In Arabidopsis thaliana, the amino acid sequences of membrane-associated acyl-CoAbinding proteins ACBP1 and ACBP2 are highly-conserved. We have previously shown that in developing seeds, ACBP1 accumulates in the cotyledonary cells of embryos and ACBP1 was proposed to be involved in lipid transfer. We show here by immunolocalization using ACBP2-specific antibodies, that ACBP2 is also expressed in the embryos at various stages of seed development in Arabidopsis. • Phenotypic analyses of acbp1 and acbp2 single mutants revealed that knockout of either ACBP1 or ACBP2 alone did not affect their life cycle since both single mutants exhibited normal growth and development similar to wild type. However, the acbp1acbp2 double mutant was embryo lethal and was also defective in callus induction. • On lipid and acyl-CoA profiling analyses, siliques but not leaves of the acbp1 mutant were shown to accumulate galactolipid monogalactosyldiacylglycerol (MGDG) and 18:0-CoA but the levels of most polyunsaturated species of phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) had declined. • Since recombinant ACBP1 and ACBP2 bind unsaturated PC and acyl-CoA esters in vitro, we propose that ACBP1 and ACBP2 are essential in lipid transfer during early embryogenesis in Arabidopsis.