The calcium-dependent protein kinase CPK16 regulates hypoxia-induced ROS production by phosphorylating the NADPH oxidase RBOHD in Arabidopsis.

Reactive oxygen species (ROS) production is a key event in modulating plant responses to hypoxia and post-hypoxia reoxygenation. However, the molecular mechanism by which hypoxia-associated ROS homeostasis is controlled remains largely unknown. Here, we showed that the calcium-dependent protein kinase CPK16 regulates plant hypoxia tolerance by phosphorylating the plasma membrane-anchored NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) to regulate ROS production in Arabidopsis (Arabidopsis thaliana). In response to hypoxia or reoxygenation, CPK16 was activated through phosphorylation of its Ser274 residue. The cpk16 knockout mutant displayed enhanced hypoxia tolerance, whereas CPK16-overexpressing (CPK16-OE) lines showed increased sensitivity to hypoxic stress. In agreement with these observations, hypoxia and reoxygenation both induced ROS accumulation in the rosettes of CPK16-OEs more strongly than in rosettes of the cpk16-1 mutant or the wild type. Moreover, CPK16 interacted with and phosphorylated the N terminus of RBOHD at four serine residues (Ser133, Ser148, Ser163, and Ser347) that were necessary for hypoxia- and reoxygenation-induced ROS accumulation. Furthermore, the hypoxia-tolerant phenotype of cpk16-1 was fully abolished in the cpk16 rbohd double mutant. Thus, we have uncovered a regulatory mechanism by which the CPK16-RBOHD module shapes ROS production during hypoxia and reoxygenation in Arabidopsis.

14-3-3 proteins contribute to autophagy by modulating SINAT-mediated degradation of ATG13.

In multicellular eukaryotes, autophagy is a conserved process that delivers cellular components to the vacuole or lysosome for recycling during development and stress responses. Induction of autophagy activates AUTOPHAGY-RELATED PROTEIN 1 (ATG1) and ATG13 to form a protein kinase complex that initiates autophagosome formation. However, the detailed molecular mechanism underlying the regulation of this protein complex in plants remains unclear. Here, we determined that in Arabidopsis thaliana, the regulatory proteins 14-3-3λ and 14-3-3κ redundantly modulate autophagy dynamics by facilitating SEVEN IN ABSENTIA OF ARABIDOPSIS THALIANA (SINAT)-mediated proteolysis of ATG13a and ATG13b. 14-3-3λ and 14-3-3κ directly interacted with SINATs and ATG13a/b in vitro and in vivo. Compared to wild-type (WT), the 14-3-3λ 14-3-3κ double mutant showed increased tolerance to nutrient starvation, delayed leaf senescence, and enhanced starvation-induced autophagic vesicles. Moreover, 14-3-3s were required for SINAT1-mediated ubiquitination and degradation of ATG13a. Consistent with their roles in ATG degradation, the 14-3-3λ 14-3-3κ double mutant accumulated higher levels of ATG1a/b/c and ATG13a/b than the WT upon nutrient deprivation. Furthermore, the specific association of 14-3-3s with phosphorylated ATG13a was crucial for ATG13a stability and formation of the ATG1-ATG13 complex. Thus, our findings demonstrate that 14-3-3λ and 14-3-3κ function as molecular adaptors to regulate autophagy by modulating the homeostasis of phosphorylated ATG13.

Phosphatidic acid modulates MPK3- and MPK6-mediated hypoxia signaling in Arabidopsis.

Phosphatidic acid (PA) is an important lipid essential for several aspects of plant development and biotic and abiotic stress responses. We previously suggested that submergence induces PA accumulation in Arabidopsis thaliana; however, the molecular mechanism underlying PA-mediated regulation of submergence-induced hypoxia signaling remains unknown. Here, we showed that in Arabidopsis, loss of the phospholipase D (PLD) proteins PLDα1 and PLDδ leads to hypersensitivity to hypoxia, but increased tolerance to submergence. This enhanced tolerance is likely due to improvement of PA-mediated membrane integrity. PA bound to the mitogen-activated protein kinase 3 (MPK3) and MPK6 in vitro and contributed to hypoxia-induced phosphorylation of MPK3 and MPK6 in vivo. Moreover, mpk3 and mpk6 mutants were more sensitive to hypoxia and submergence stress compared with wild type, and fully suppressed the submergence-tolerant phenotypes of pldα1 and pldδ mutants. MPK3 and MPK6 interacted with and phosphorylated RELATED TO AP2.12, a master transcription factor in the hypoxia signaling pathway, and modulated its activity. In addition, MPK3 and MPK6 formed a regulatory feedback loop with PLDα1 and/or PLDδ to regulate PLD stability and submergence-induced PA production. Thus, our findings demonstrate that PA modulates plant tolerance to submergence via both membrane integrity and MPK3/6-mediated hypoxia signaling in Arabidopsis.

MYB30 integrates light signals with antioxidant biosynthesis to regulate plant responses during postsubmergence recovery.

Submergence is an abiotic stress that limits agricultural production world-wide. Plants sense oxygen levels during submergence and postsubmergence reoxygenation and modulate their responses. Increasing evidence suggests that completely submerged plants are often exposed to low-light stress, owing to the depth and turbidity of the surrounding water; however, how light availability affects submergence tolerance remains largely unknown. Here, we showed that Arabidopsis thaliana MYB DOMAIN PROTEIN30 (MYB30) is an important transcription factor that integrates light signaling and postsubmergence stress responses. MYB DOMAIN PROTEIN30 protein abundance decreased upon submergence and accumulated during reoxygenation. Under submergence conditions, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), a central regulator of light signaling, caused the ubiquitination and degradation of MYB30. In response to desubmergence, however, light-induced MYB30 interacted with MYC2, a master transcription factor involved in jasmonate signaling, and activated the expression of the VITAMIN C DEFECTIVE1 (VTC1) and GLUTATHIONE SYNTHETASE1 (GSH1) gene families to enhance antioxidant biosynthesis. Consistent with this, the myb30 knockout mutant showed increased sensitivity to submergence, which was partially rescued by overexpression of VTC1 or GSH1. Thus, our findings uncover the mechanism by which the COP1-MYB30 module integrates light signals with cellular oxidative homeostasis to coordinate plant responses to postsubmergence stress.

SINAT E3 Ubiquitin Ligases Mediate FREE1 and VPS23A Degradation to Modulate Abscisic Acid Signaling.

In plants, the ubiquitin-proteasome system, endosomal sorting, and autophagy are essential for protein degradation; however, their interplay remains poorly understood. Here, we show that four Arabidopsis (Arabidopsis thaliana) E3 ubiquitin ligases, SEVEN IN ABSENTIA OF ARABIDOPSIS THALIANA1 (SINAT1), SINAT2, SINAT3, and SINAT4, regulate the stabilities of FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FREE1) and VACUOLAR PROTEIN SORTING23A (VPS23A), key components of the endosomal sorting complex required for transport-I, to modulate abscisic acid (ABA) signaling. GFP-SINAT1, GFP-SINAT2, and GFP-SINAT4 primarily localized to the endosomal and autophagic vesicles. SINATs controlled FREE1 and VPS23A ubiquitination and proteasomal degradation. SINAT overexpressors showed increased ABA sensitivity, ABA-responsive gene expression, and PYRABACTIN RESISTANCE1-LIKE4 protein levels. Furthermore, the SINAT-FREE1/VPS23A proteins were codegraded by the vacuolar pathway. In particular, during recovery post-ABA exposure, SINATs formed homo- and hetero-oligomers in vivo, which were disrupted by the autophagy machinery. Taken together, our findings reveal a novel mechanism by which the proteasomal and vacuolar turnover systems regulate ABA signaling in plants.

Overexpression of the Arabidopsis MACPF Protein AtMACP2 Promotes Pathogen Resistance by Activating SA Signaling.

Immune response in plants is tightly regulated by the coordination of the cell surface and intracellular receptors. In animals, the membrane attack complex/perforin-like (MACPF) protein superfamily creates oligomeric pore structures on the cell surface during pathogen infection. However, the function and molecular mechanism of MACPF proteins in plant pathogen responses remain largely unclear. In this study, we identified an Arabidopsis MACP2 and investigated the responsiveness of this protein during both bacterial and fungal pathogens. We suggest that MACP2 induces programmed cell death, bacterial pathogen resistance, and necrotrophic fungal pathogen sensitivity by activating the biosynthesis of tryptophan-derived indole glucosinolates and the salicylic acid signaling pathway dependent on the activity of enhanced disease susceptibility 1 (EDS1). Moreover, the response of MACP2 mRNA isoforms upon pathogen attack is differentially regulated by a posttranscriptional mechanism: alternative splicing. In comparison to previously reported MACPFs in Arabidopsis, MACP2 shares a redundant but nonoverlapping role in plant immunity. Thus, our findings provide novel insights and genetic tools for the MACPF family in maintaining SA accumulation in response to pathogens in Arabidopsis.

Brassinosteroids Antagonize Jasmonate-Activated Plant Defense Responses through BRI1-EMS-SUPPRESSOR1 (BES1).

Brassinosteroids (BRs) and jasmonates (JAs) regulate plant growth, development, and defense responses, but how these phytohormones mediate the growth-defense tradeoff is unclear. Here, we identified the Arabidopsis (Arabidopsis thaliana) dwarf at early stages1 (dwe1) mutant, which exhibits enhanced expression of defensin genes PLANT DEFENSIN1.2a (PDF1.2a) and PDF1.2b The dwe1 mutant showed increased resistance to herbivory by beet armyworms (Spodoptera exigua) and infection by botrytis (Botrytis cinerea). DWE1 encodes ROTUNDIFOLIA3, a cytochrome P450 protein essential for BR biosynthesis. The JA-inducible transcription of PDF1.2a and PDF1.2b was significantly reduced in the BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1) gain-of-function mutant bes1- D, which was highly susceptible to S. exigua and B. cinerea BES1 directly targeted the terminator regions of PDF1.2a/PDF1.2b and suppressed their expression. PDF1.2a overexpression diminished the enhanced susceptibility of bes1- D to B. cinerea but did not improve resistance of bes1- D to S. exigua In response to S. exigua herbivory, BES1 inhibited biosynthesis of the JA-induced insect defense-related metabolite indolic glucosinolate by interacting with transcription factors MYB DOMAIN PROTEIN34 (MYB34), MYB51, and MYB122 and suppressing expression of genes encoding CYTOCHROME P450 FAMILY79 SUBFAMILY B POLYPEPTIDE3 (CYP79B3) and UDP-GLUCOSYL TRANSFERASE 74B1 (UGT74B1). Thus, BR contributes to the growth-defense tradeoff by suppressing expression of defensin and glucosinolate biosynthesis genes.

TRAF Family Proteins Regulate Autophagy Dynamics by Modulating AUTOPHAGY PROTEIN6 Stability in Arabidopsis.

Eukaryotic cells use autophagy to recycle cellular components. During autophagy, autophagosomes deliver cytoplasmic contents to the vacuole or lysosome for breakdown. Mammalian cells regulate the dynamics of autophagy via ubiquitin-mediated proteolysis of autophagy proteins. Here, we show that the Arabidopsis thaliana Tumor necrosis factor Receptor-Associated Factor (TRAF) family proteins TRAF1a and TRAF1b (previously named MUSE14 and MUSE13, respectively) help regulate autophagy via ubiquitination. Upon starvation, cytoplasmic TRAF1a and TRAF1b translocated to autophagosomes. Knockout traf1a/b lines showed reduced tolerance to nutrient deficiency, increased salicylic acid and reactive oxygen species levels, and constitutive cell death in rosettes, resembling the phenotypes of autophagy-defective mutants. Starvation-activated autophagosome accumulation decreased in traf1a/b root cells, indicating that TRAF1a and TRAF1b function redundantly in regulating autophagosome formation. TRAF1a and TRAF1b interacted in planta with ATG6 and the RING finger E3 ligases SINAT1, SINAT2, and SINAT6 (with a truncated RING-finger domain). SINAT1 and SINAT2 require the presence of TRAF1a and TRAF1b to ubiquitinate and destabilize AUTOPHAGY PROTEIN6 (ATG6) in vivo. Conversely, starvation-induced SINAT6 reduced SINAT1- and SINAT2-mediated ubiquitination and degradation of ATG6. Consistently, SINAT1/SINAT2 and SINAT6 knockout mutants exhibited increased tolerance and sensitivity, respectively, to nutrient starvation. Therefore, TRAF1a and TRAF1b function as molecular adaptors that help regulate autophagy by modulating ATG6 stability in Arabidopsis.

Polyunsaturated linolenoyl-CoA modulates ERF-VII-mediated hypoxia signaling in Arabidopsis.

In plants, submergence from flooding causes hypoxia, which impairs energy production and affects plant growth, productivity, and survival. In Arabidopsis, hypoxia induces nuclear localization of the group VII ethylene-responsive transcription factor RELATED TO AP2.12 (RAP2.12), following its dissociation from the plasma membrane-anchored ACYL-COA BINDING PROTEIN1 (ACBP1) and ACBP2. Here, we show that polyunsaturated linolenoyl-CoA (18:3-CoA) regulates RAP2.12 release from the plasma membrane. Submergence caused a significant increase in 18:3-CoA, but a significant decrease in 18:0-, 18:1-, and 18:2-CoA. Application of 18:3-CoA promoted nuclear accumulation of the green fluorescent protein (GFP) fusions RAP2.12-GFP, HYPOXIA-RESPONSIVE ERF1-GFP, and RAP2.3-GFP, and enhanced transcript levels of hypoxia-responsive genes. Plants with decreased ACBP1 and ACBP2 (acbp1 ACBP2-RNAi, produced by ACBP2 RNA interference in the acbp1 mutant) had reduced tolerance to hypoxia and impaired 18:3-CoA-induced expression of hypoxia-related genes. In knockout mutants and overexpression lines of LONG-CHAIN ACYL-COA SYNTHASE2 (LACS2) and FATTY ACID DESATURASE 3 (FAD3), the acyl-CoA pool size and 18:3-CoA levels were closely related to ERF-VII-mediated signaling and hypoxia tolerance. These findings demonstrate that polyunsaturation of long-chain acyl-CoAs functions as important mechanism in the regulation of plant hypoxia signaling, by modulating ACBP-ERF-VII dynamics.

DIACYLGLYCEROL ACYLTRANSFERASE and DIACYLGLYCEROL KINASE Modulate Triacylglycerol and Phosphatidic Acid Production in the Plant Response to Freezing Stress.

Plants accumulate the lipids phosphatidic acid (PA), diacylglycerol (DAG), and triacylglycerol (TAG) during cold stress, but how plants balance the levels of these lipids to mediate cold responses remains unknown. The enzymes ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) and DIACYLGLYCEROL KINASE (DGK) catalyze the conversion of DAG to TAG and PA, respectively. Here, we show that DGAT1, DGK2, DGK3, and DGK5 contribute to the response to cold in Arabidopsis (Arabidopsis thaliana). With or without cold acclimation, the dgat1 mutants exhibited higher sensitivity upon freezing exposure compared with the wild type. Under cold conditions, the dgat1 mutants showed reduced expression of C-REPEAT/DRE BINDING FACTOR2 and its regulons, which are essential for the acquisition of cold tolerance. Lipid profiling revealed that freezing significantly increased the levels of PA and DAG while decreasing TAG in the rosettes of dgat1 mutant plants. During freezing stress, the accumulation of PA in dgat1 plants stimulated NADPH oxidase activity and enhanced RbohD-dependent hydrogen peroxide production compared with the wild type. Moreover, the cold-inducible transcripts of DGK2, DGK3, and DGK5 were significantly more up-regulated in the dgat1 mutants than in the wild type during cold stress. Consistent with this observation, dgk2, dgk3, and dgk5 knockout mutants showed improved tolerance and attenuated PA production in response to freezing temperatures. Our findings demonstrate that the conversion of DAG to TAG by DGAT1 is critical for plant freezing tolerance, acting by balancing TAG and PA production in Arabidopsis.

Jasmonate Regulates Plant Responses to Postsubmergence Reoxygenation through Transcriptional Activation of Antioxidant Synthesis.

Submergence induces hypoxia in plants; exposure to oxygen following submergence, termed reoxygenation, produces a burst of reactive oxygen species. The mechanisms of hypoxia sensing and signaling in plants have been well studied, but how plants respond to reoxygenation remains unclear. Here, we show that reoxygenation in Arabidopsis (Arabidopsis thaliana) involves rapid accumulation of jasmonates (JAs) and increased transcript levels of JA biosynthesis genes. Application of exogenous methyl jasmonate improved tolerance to reoxygenation in wild-type Arabidopsis; also, mutants deficient in JA biosynthesis and signaling were very sensitive to reoxygenation. Moreover, overexpression of the transcription factor gene MYC2 enhanced tolerance to posthypoxic stress, and myc2 knockout mutants showed increased sensitivity to reoxygenation, indicating that MYC2 functions as a key regulator in the JA-mediated reoxygenation response. MYC2 transcriptionally activates members of the VITAMIN C DEFECTIVE (VTC) and GLUTATHIONE SYNTHETASE (GSH) gene families, which encode rate-limiting enzymes in the ascorbate and glutathione synthesis pathways. Overexpression of VTC1 and GSH1 in the myc2-2 mutant suppressed the posthypoxic hypersensitive phenotype. The JA-inducible accumulation of antioxidants may alleviate oxidative damage caused by reoxygenation, improving plant survival after submergence. Taken together, our findings demonstrate that JA signaling interacts with the antioxidant pathway to regulate reoxygenation responses in Arabidopsis.

Unsaturation of very-long-chain ceramides protects plant from hypoxia-induced damages by modulating ethylene signaling in Arabidopsis.

Lipid remodeling is crucial for hypoxic tolerance in animals, whilst little is known about the hypoxia-induced lipid dynamics in plants. Here we performed a mass spectrometry-based analysis to survey the lipid profiles of Arabidopsis rosettes under various hypoxic conditions. We observed that hypoxia caused a significant increase in total amounts of phosphatidylserine, phosphatidic acid and oxidized lipids, but a decrease in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Particularly, significant gains in the polyunsaturated species of PC, PE and phosphatidylinositol, and losses in their saturated and mono-unsaturated species were evident during hypoxia. Moreover, hypoxia led to a remarkable elevation of ceramides and hydroxyceramides. Disruption of ceramide synthases LOH1, LOH2 and LOH3 enhanced plant sensitivity to dark submergence, but displayed more resistance to submergence under light than wild type. Consistently, levels of unsaturated very-long-chain (VLC) ceramide species (22:1, 24:1 and 26:1) predominantly declined in the loh1, loh2 and loh3 mutants under dark submergence. In contrast, significant reduction of VLC ceramides in the loh1-1 loh3-1 knockdown double mutant and lacking of VLC unsaturated ceramides in the ads2 mutants impaired plant tolerance to both dark and light submergences. Evidence that C24:1-ceramide interacted with recombinant CTR1 protein and inhibited its kinase activity in vitro, enhanced ER-to-nucleus translocation of EIN2-GFP and stabilization of EIN3-GFP in vivo, suggests a role of ceramides in modulating CTR1-mediated ethylene signaling. The dark submergence-sensitive phenotypes of loh mutants were rescued by a ctr1-1 mutation. Thus, our findings demonstrate that unsaturation of VLC ceramides is a protective strategy for hypoxic tolerance in Arabidopsis.

A retrospective comparative study evaluating the efficacy of adding intra-arterial methotrexate infusion to uterine artery embolisation followed by curettage for cesarean scar pregnancy.

PURPOSE: The study aimed to compare the efficacy of intra-arterial methotrexate (MTX) infusion combined with uterine artery embolisation (UAE) and uterine curettage with that of UAE and curettage without MTX infusion for the treatment of cesarean scar pregnancy (CSP). METHODS: In this retrospective study, data of CSP patients admitted from January 2011 to July 2015 were obtained from electronic patient records. Clinical information at baseline and after treatment were extracted and analyzed. RESULTS: A total of 93 CSP patients were included, with 57 patients receiving UAE followed by curettage (UC) and 36 patients receiving intra-arterial MTX infusion followed by UAE and curettage (MUC). The baseline characteristics were not significantly different between the two groups. Without additional intervention, 32 (88.9%) patients were successfully treated by MUC, and 49 (86.0%) patients were successfully treated by UC, defined by discontinued ectopic conceptus growth, normalized serum ß-human chorionic gonadotropin (ß-hCG) level, ceased vaginal bleeding and preservation of uterus, with no significant difference between the two groups. Additionally, intra-operative blood loss volume and post-operative bleeding events were not significantly different between the two groups. However, serum ß-hCG decline on the first day after surgery was significantly promoted, and the hospitalization length and the time needed for serum ß-hCG normalization were significantly shortened by addition of intra-arterial MTX infusion. CONCLUSIONS: Adding intra-arterial MTX to UAE and curettage significantly promoted post-operative recovery, though success rate and bleeding events were not significantly affected, suggesting that addition of intra-arterial MTX might not be necessary.

Arabidopsis acyl-CoA-binding protein ACBP3 participates in plant response to hypoxia by modulating very-long-chain fatty acid metabolism.

In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by a family of six genes (ACBP1 to ACBP6), and are essential for diverse cellular activities. Recent investigations suggest that the membrane-anchored ACBPs are involved in oxygen sensing by sequestration of group VII ethylene-responsive factors under normoxia. Here, we demonstrate the involvement of Arabidopsis ACBP3 in hypoxic tolerance. ACBP3 transcription was remarkably induced following submergence under both dark (DS) and light (LS) conditions. ACBP3-overexpressors (ACBP3-OEs) showed hypersensitivity to DS, LS and ethanolic stresses, with reduced transcription of hypoxia-responsive genes as well as accumulation of hydrogen peroxide in the rosettes. In contrast, suppression of ACBP3 in ACBP3-KOs enhanced plant tolerance to DS, LS and ethanol treatments. By analyses of double combinations of OE-1 with npr1-5, coi1-2, ein3-1 as well as ctr1-1 mutants, we observed that the attenuated hypoxic tolerance in ACBP3-OEs was dependent on NPR1- and CTR1-mediated signaling pathways. Lipid profiling revealed that both the total amounts and very-long-chain species of phosphatidylserine (C42:2- and C42:3-PS) and glucosylinositolphosphorylceramides (C22:0-, C22:1-, C24:0-, C24:1-, and C26:1-GIPC) were significantly lower in ACBP3-OEs but increased in ACBP3-KOs upon LS exposure. By microscale thermophoresis analysis, the recombinant ACBP3 protein bound VLC acyl-CoA esters with high affinities in vitro. Further, a knockout mutant of MYB30, a master regulator of very-long-chain fatty acid (VLCFA) biosynthesis, exhibited enhanced sensitivities to LS and ethanolic stresses, phenotypes that were ameliorated by ACBP3-RNAi. Taken together, these findings suggest that Arabidopsis ACBP3 participates in plant response to hypoxia by modulating VLCFA metabolism.

Arabidopsis acyl-CoA-binding protein ACBP6 localizes in the phloem and affects jasmonate composition.

Arabidopsis thaliana ACYL-COA-BINDING PROTEIN6 (AtACBP6) encodes a cytosolic 10-kDa AtACBP. It confers freezing tolerance in transgenic Arabidopsis, possibly by its interaction with lipids as indicated by the binding of acyl-CoA esters and phosphatidylcholine to recombinant AtACBP6. Herein, transgenic Arabidopsis transformed with an AtACBP6 promoter-driven ß-glucuronidase (GUS) construct exhibited strong GUS activity in the vascular tissues. Immunoelectron microscopy using anti-AtACBP6 antibodies showed AtACBP6 localization in the phloem especially in the companion cells and sieve elements. Also, the presence of gold grains in the plasmodesmata indicated its potential role in systemic trafficking. The AtACBP6 protein, but not its mRNA, was found in phloem exudate of wild-type Arabidopsis. Fatty acid profiling using gas chromatography-mass spectrometry revealed an increase in the jasmonic acid (JA) precursor, 12-oxo-cis,cis-10,15-phytodienoic acid (cis-OPDA), and a reduction in JA and/or its derivatives in acbp6 phloem exudates in comparison to the wild type. Quantitative real-time PCR showed down-regulation of COMATOSE (CTS) in acbp6 rosettes suggesting that AtACBP6 affects CTS function. AtACBP6 appeared to affect the content of JA and/or its derivatives in the sieve tubes, which is consistent with its role in pathogen-defense and in its wound-inducibility of AtACBP6pro::GUS. Taken together, our results suggest the involvement of AtACBP6 in JA-biosynthesis in Arabidopsis phloem tissues.

Expression of Arabidopsis acyl-CoA-binding proteins AtACBP1 and AtACBP4 confers Pb(II) accumulation in Brassica juncea roots.

In Arabidopsis thaliana, the expression of two genes encoding acyl-CoA-binding proteins (ACBPs) AtACBP1 and AtACBP4, were observed to be induced by lead [Pb(II)] in shoots and roots in qRT-PCR analyses. Quantitative GUS (ß-glucuronidase) activity assays confirmed induction of AtACBP1pro::GUS by Pb(II). Electrophoretic mobility shift assays (EMSAs) revealed that Pas elements in the 5'-flanking region of AtACBP1 were responsive to Pb(II) treatment. AtACBP1 and AtACBP4 were further compared in Pb(II) uptake using Brassica juncea, a potential candidate for phytoremediation given its rapid growth, large roots, high biomass and good capacity to accumulate heavy metals. Results from atomic absorption analyses on transgenic B. juncea expressing AtACBP1 or AtACBP4 indicated Pb(II) accumulation in roots. Subsequent Pb(II)-tracing assays demonstrated Pb(II) accumulation in the cytosol of root tips and vascular tissues of transgenic B. juncea AtACBP1-overexpressors (OXs) and AtACBP4-OXs and transgenic Arabidopsis AtACBP1-OXs. Transgenic Arabidopsis AtACBP1-OXs sequestered Pb(II) in the trichomes and displayed tolerance to hydrogen peroxide (H2 O2 ) treatment. In addition, AtACBP1 and AtACBP4 were H2 O2 -induced in the roots of wild-type Arabidopsis, while lipid hydroperoxide (LOOH) measurements of B. juncea AtACBP1-OX and AtACBP4-OX roots suggested that AtACBP1 and AtACBP4 can protect lipids against Pb(II)-induced lipid peroxidation.

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.

Transgenic Arabidopsis flowers overexpressing acyl-CoA-binding protein ACBP6 are freezing tolerant.

Low temperature stress adversely affects plant growth. It has been shown that the overexpression of ACYL-COENZYME A-BINDING PROTEIN6 (ACBP6) resulted in enhanced freezing tolerance in seedlings and rosettes accompanied by a decrease in phosphatidylcholine (PC), an increase in phosphatidic acid (PA) and an up-regulation of PHOSPHOLIPASE Dδ(PLDδ) in the absence of COLD-RESPONSIVE (COR)-related gene induction. Unlike rosettes, ACBP6-overexpressor (OE) flowers showed elevations in PC and monogalactosyldiacylglycerol (MGDG) accompanied by a decline in PA. The increase in PC species corresponded to a decline in specific PAs. To better understand such differences, the expression of PC-, MGDG-, proline-, ABA- and COR-related genes, and their transcription factors [C-repeat binding factors (CBFs), INDUCER OF CBF EXPRESSION1 (ICE1) and MYB15] was analyzed by quantitative real-time PCR (qRT-PCR). ACBP6-conferred freezing-tolerant flowers showed induction of COR-related genes, CBF genes and ICE1, PC-related genes (PLDδ, CK, CK-LIKE1, CK-LIKE2, CCT1, CCT2, LPCAT1, PLA2α, PAT-PLA-IIß, PAT-PLA-IIIα, PAT-PLA-IIIδ and PLDζ2), MGDG-related genes (MGD genes and SFR2) and ABA-responsive genes. In contrast, ACBP6-conferred freezing-tolerant rosettes were down-regulated in COR-related genes, CBF1, PC-related genes (PEAMT1, PEAMT2, PEAMT3, CK1, CCT1, CCT2, PLA2α, PAT-PLA-IIIδ and PLDζ2), MGDG-related genes (MGD2, MGD3 and SFR2) and some ABA-responsive genes including KIN1 and KIN2. These results suggest that the mechanism in ACBP6-conferred freezing tolerance varies in different organs.

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.

Overexpression of Arabidopsis acyl-CoA binding protein ACBP3 promotes starvation-induced and age-dependent leaf senescence.

In Arabidopsis thaliana, a family of six genes (ACBP1 to ACBP6) encodes acyl-CoA binding proteins (ACBPs). Investigations on ACBP3 reported here show its upregulation upon dark treatment and in senescing rosettes. Transgenic Arabidopsis overexpressing ACBP3 (ACBP3-OEs) displayed accelerated leaf senescence, whereas an acbp3 T-DNA insertional mutant and ACBP3 RNA interference transgenic Arabidopsis lines were delayed in dark-induced leaf senescence. Acyl-CoA and lipid profiling revealed that the overexpression of ACBP3 led to an increase in acyl-CoA and phosphatidylethanolamine (PE) levels, whereas ACBP3 downregulation reduced PE content. Moreover, significant losses in phosphatidylcholine (PC) and phosphatidylinositol, and gains in phosphatidic acid (PA), lysophospholipids, and oxylipin-containing galactolipids (arabidopsides) were evident in 3-week-old dark-treated and 6-week-old premature senescing ACBP3-OEs. Such accumulation of PA and arabidopsides (A, B, D, E, and G) resulting from lipid peroxidation in ACBP3-OEs likely promoted leaf senescence. The N-terminal signal sequence/transmembrane domain in ACBP3 was shown to be essential in ACBP3-green fluorescent protein targeting and in promoting senescence. Observations that recombinant ACBP3 binds PC, PE, and unsaturated acyl-CoAs in vitro and that ACBP3 overexpression enhances degradation of the autophagy (ATG)-related protein ATG8 and disrupts autophagosome formation suggest a role for ACBP3 as a phospholipid binding protein involved in the regulation of leaf senescence by modulating membrane phospholipid metabolism and ATG8 stability in Arabidopsis. Accelerated senescence in ACBP3-OEs is dependent on salicylic acid but not jasmonic acid signaling.