Selective induction and subcellular distribution of ACONITASE 3 reveal the importance of cytosolic citrate metabolism during lipid mobilization in Arabidopsis.

Arabidopsis thaliana has three genes that encode distinct aconitases (ACO), but little is known about the function of each isoenzyme during plant development. In newly emerged seedlings of Arabidopsis, transcript and protein levels for ACO3 were selectively induced to yield more than 80% of total aconitase activity. Characterization of knockout mutants for each of the three ACOs suggests a major role for only ACO3 in citrate metabolism. The aco3 mutant showed delayed early seedling growth, altered assimilation of [14C]acetate feeding and elevated citrate levels, which were nearly 4-fold greater than in wild-type, aco1 or aco2. However, both ACO1 and ACO2 are active in seedlings as shown by inhibition of aco3 growth by the toxin monofluoroacetate, and altered [14C]acetate assimilation and metabolite levels in aco1 and aco2. Relative levels of fumarate and malate differed between aco2 and aco3, indicating metabolically isolated pools of these metabolites in seedlings. Our inability to enrich ACO protein through mitochondria isolation, and the reduced cytosolic ACO activity of the iron-sulfur centre assembly mutant atm3-1, indicated a cytosolic localization of ACO3 in 3-day-old seedlings. Subsequently, we determined that more than 90% of ACO3 was cytosolic. We conclude that ACO3 is cytosolic in young seedlings and functions in citrate catabolism consistent with the operation of the classic glyoxylate and not direct catabolism of citrate within mitochondria.

Evidence that ACN1 (acetate non-utilizing 1) prevents carbon leakage from peroxisomes during lipid mobilization in Arabidopsis seedlings.

ACN1 (acetate non-utilizing 1) is a short-chain acyl-CoA synthetase which recycles free acetate to acetyl-CoA in peroxisomes of Arabidopsis. Pulse-chase [2-(13)C]acetate feeding of the mutant acn1-2 revealed that acetate accumulation and assimilation were no different to that of wild-type, Col-7. However, the lack of acn1-2 led to a decrease of nearly 50% in (13)C-labelling of glutamine, a major carbon sink in seedlings, and large decreases in primary metabolite levels. In contrast, acetyl-CoA levels were higher in acn1-2 compared with Col-7. The disappearance of eicosenoic acid was slightly delayed in acn1-2 indicating only a small effect on the rate of lipid breakdown. A comparison of transcript levels in acn1-2 and Col-7 showed that induced genes included a number of metabolic genes and also a large number of signalling-related genes. Genes repressed in the mutant were represented primarily by embryogenesis-related genes. Transcript levels of glyoxylate cycle genes also were lower in acn1-2 than in Col-7. We conclude that deficiency in peroxisomal acetate assimilation comprises only a small proportion of total acetate use, but this affects both primary metabolism and gene expression. We discuss the possibility that ACN1 safeguards against the loss of carbon as acetate from peroxisomes during lipid mobilization.

Modelling the peroxisomal carbon leak during lipid mobilization in Arabidopsis.

Mutation of the ACN1 (acetate non-utilizing 1) locus of Arabidopsis results in altered acetate assimilation into gluconeogenic sugars and anapleurotic amino acids and leads to an overall depression in primary metabolite levels by approx. 50% during seedling development. Levels of acetyl-CoA were higher in acn1 compared with wild-type, which is counterintuitive to the activity of ACN1 as a peroxisomal acetyl-CoA synthetase. We hypothesize that ACN1 recycles free acetate to acetyl-CoA within peroxisomes in order that carbon remains fed into the glyoxylate cycle. When ACN1 is not present, carbon in the form of acetate can leak out of peroxisomes and is reactivated to acetyl-CoA within the cytosol. Kinetic models incorporating estimates of carbon input and pathway dynamics from a variety of literature sources have proven useful in explaining how ACN1 may prevent the carbon leak and even contribute to the control of peroxisomal carbon metabolism.

Incorporating clickers into an enzymology course improves student performance.

Here, we describe how poor exam results of undergraduate students enrolled in an enzymology course at the University of Bordeaux were improved through the introduction of 'clickers' as an audience response system. By using clickers only in a small-group tutorial element of a large theoretical course, we observed an improvement in exam scores that resulted in a lower failure rate for the course. Furthermore, students of all abilities were found to benefit from their use. Students reported better retention of both lecture and tutorial content. An analysis of how clickers were employed within the tutorials indicated that the use of clickers to promote discussion and impart knowledge likely resulted in a moderate improvement of exam scores. We hypothesize that students were more prepared for exams through greater reflection of exam questions, resulting in an enhanced ability to retrieve memorized information and apply it within a time-limited exam setting.

The Arabidopsis ALDP protein homologue COMATOSE is instrumental in peroxisomal acetate metabolism.

The Arabidopsis acn (acetate non-utilizing) mutants were isolated by fluoroacetate-resistant germination and seedling establishment. We report the characterization of the acn2 mutant. Physiological analyses of acn2 showed that it possessed characteristics similar to those of the mutants cts (COMATOSE)-1 and pxa [peroxisomal ABC (ATP-binding-cassette) transporter]1. The acn2 locus was mapped to within 3 cM of the CTS gene on the bottom arm of chromosome IV using CAPS (cleavage amplification polymorphism) and SSLP (simple sequence-length polymorphism) markers. Crossing acn2 and cts-1 failed to restore the fluoroacetate-sensitive phenotype, suggesting that these mutations were allelic. Sequencing of the ACN2 locus revealed a C-->T nonsense mutation in exon 13, which would have resulted in the elimination of the C-terminal hemitransporter domain of the encoded protein. Neither the full-length CTS protein nor the truncated protein was detected on immunoblots using either C-terminal- or N-terminal-specific anti-CTS antibodies respectively, demonstrating the absence of the entire CTS protein in acn2 mutants. Emerged seedlings of both cts-1 and pxa1 alleles displayed increased resistance to FAc (monofluoroacetic acid) compared with the corresponding wild-type seedlings. Complementation studies showed that mutation of the CTS gene was responsible for the FAc-resistant phenotype, as when the wild-type protein was expressed in both the cts-1 and pxa1 mutant lines, the strains became FAc-sensitive. Feeding studies confirmed that both acn2 and cts-1 mutants were compromised in their ability to convert radiolabelled acetate into soluble carbohydrate. These results demonstrate a role for the ABC protein CTS in providing acetate to the glyoxylate cycle in developing seedlings.

Components of Arabidopsis defense- and ethylene-signaling pathways regulate susceptibility to Cauliflower mosaic virus by restricting long-distance movement.

We analyzed the susceptibility of Arabidopsis mutants with defects in salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling to infection by Cauliflower mosaic virus (CaMV). Mutants cpr1-1 and cpr5-2, in which SA-dependent defense signaling is activated constitutively, were substantially more resistant than the wild type to systemic infection, implicating SA signaling in defense against CaMV. However, SA-deficient NahG, sid2-2, eds5-1, and pad4-1 did not show enhanced susceptibility. A cpr5 eds5 double mutant also was resistant, suggesting that resistance in cpr5 may function partially independently of SA. Treatment of cpr5 and cpr5 eds5, but not cpr1, with salicyl-hydroxamic acid, an inhibitor of alternative oxidase, partially restored susceptibility to wild-type levels. Mutants etr1-1, etr1-3, and ein2-1, and two mutants with lesions in ET/JA-mediated defense, eds4 and eds8, also showed reduced virus susceptibility, demonstrating that ET-dependent responses also play a role in susceptibility. We used a green fluorescent protein (GFP)-expressing CaMV recombinant to monitor virus movement. In mutants with reduced susceptibility, cpr1-1, cpr5-2, and etr1-1, CaMV-GFP formed local lesions similar to the wild type, but systemic spread was almost completely absent in cpr1 and cpr5 and was substantially reduced in etr1-1. Thus, mutations with enhanced systemic acquired resistance or compromised ET signaling show diminished long-distance virus movement.

GFP-tagging of Arabidopsis acyl-activating enzymes raises the issue of peroxisome-chloroplast import competition versus dual localization.

Protein sequence analysis of a subfamily of 18 Arabidopsis acyl-activating enzymes (AAE) for organelle targeting signals revealed that eight of them possessed putative peroxisomal targeting signals (PTS1), five of which belonged to Clade VI of the AAE superfamily. Peroxisomal localization was confirmed by confocal microscopy of green fluorescent protein (GFP)-AAE fusion proteins co-localizing with peroxisomal RFP. The sequence analysis also revealed that all enzymes of Clade VI possess N-terminal regions indicative of chloroplast transit peptides (cTP). Among the five Clade VI peroxisomal enzymes tested, masking the PTS1 signal with GFP redirected three to plastids. In addition, three other peroxisomal AAEs appeared to be redirected to plastids in AAE-GFP fusion constructs. Due to the lack of evidence supporting plastid localization, we propose that competition dictates the exclusive localization to peroxisomes. AAE2 of Clade VI was the only enzyme with a putative mitochondrial targeting sequence, and it appeared to be targeted to mitochondria. The remainder of the AAEs appeared to be localized to plastids or cytosol. The AAE9-GFP fusion protein appeared to be located within discreet structures within plastids that may be plastoglobules. AAE15-GFP, but not AAE16-GFP appeared to be located in the chloroplast envelope. The number of examples is increasing whereby proteins located within other compartments contribute to plastid function. We provide an example of this through the light-sensitive phenotype of mutants of AAE2.

Correlation Network Analysis reveals a sequential reorganization of metabolic and transcriptional states during germination and gene-metabolite relationships in developing seedlings of Arabidopsis.

BACKGROUND: Holistic profiling and systems biology studies of nutrient availability are providing more and more insight into the mechanisms by which gene expression responds to diverse nutrients and metabolites. Less is known about the mechanisms by which gene expression is affected by endogenous metabolites, which can change dramatically during development. Multivariate statistics and correlation network analysis approaches were applied to non-targeted profiling data to investigate transcriptional and metabolic states and to identify metabolites potentially influencing gene expression during the heterotrophic to autotrophic transition of seedling establishment. RESULTS: Microarray-based transcript profiles were obtained from extracts of Arabidopsis seeds or seedlings harvested from imbibition to eight days-old. 1H-NMR metabolite profiles were obtained for corresponding samples. Analysis of transcript data revealed high differential gene expression through seedling emergence followed by a period of less change. Differential gene expression increased gradually to day 8, and showed two days, 5 and 7, with a very high proportion of up-regulated genes, including transcription factor/signaling genes. Network cartography using spring embedding revealed two primary clusters of highly correlated metabolites, which appear to reflect temporally distinct metabolic states. Principle Component Analyses of both sets of profiling data produced a chronological spread of time points, which would be expected of a developmental series. The network cartography of the transcript data produced two distinct clusters comprising days 0 to 2 and days 3 to 8, whereas the corresponding analysis of metabolite data revealed a shift of day 2 into the day 3 to 8 group. A metabolite and transcript pair-wise correlation analysis encompassing all time points gave a set of 237 highly significant correlations. Of 129 genes correlated to sucrose, 44 of them were known to be sucrose responsive including a number of transcription factors. CONCLUSIONS: Microarray analysis during germination and establishment revealed major transitions in transcriptional activity at time points potentially associated with developmental transitions. Network cartography using spring-embedding indicate that a shift in the state of nutritionally important metabolites precedes a major shift in the transcriptional state going from germination to seedling emergence. Pair-wise linear correlations of transcript and metabolite levels identified many genes known to be influenced by metabolites, and provided other targets to investigate metabolite regulation of gene expression during seedling establishment.

Long-distance movement of Cauliflower mosaic virus and host defence responses in Arabidopsis follow a predictable pattern that is determined by the leaf orthostichy.

Long-distance virus transport takes place through the vascular system and is dependent on the movement of photoassimilates. Here, patterns of symptom development, virus movement and gene expression were analysed in Arabidopsis following inoculation with Cauliflower mosaic virus (CaMV) on a single leaf. Virus accumulation and expression of markers for the salicylic acid (SA) and ethylene/jasmonate (Et/JA) defence pathways, PR-1 and PDF1.2, were analysed on a leaf-by-leaf basis by real-time reverse transcription polymerase chain reaction (qRT-PCR). Virus spread followed a strictly defined pattern identical to that of a source-sink relationship. This was exploited to study differences between local and systemic defence responses in a developmental and spatial manner. In infected plants, PR-1 transcripts accumulated primarily but not exclusively in leaves with a direct vascular connection to the inoculated leaf. Abundances fell significantly as virus accumulated. By contrast, PDF1.2 transcripts were significantly lower than in controls in all leaves at early stages of infection, but recovered as virus accumulated. Virus and PR-1 transcript abundances are negatively correlated, and SA- and Et/JA-mediated signalling of gene expression occurs independently of the presence of virus. Although SA-dependent signalling responses were mainly linked to the orthostichy, Et/JA-dependent responses were independent of vascular connections.

Characterization of Arabidopsis fluoroacetate-resistant mutants reveals the principal mechanism of acetate activation for entry into the glyoxylate cycle.

The toxic acetate analogue monofluoroacetic acid was employed to isolate Arabidopsis tDNA-tagged plants deficient in their ability to utilize or sense acetate. Several tDNA-tagged lines were isolated, including two that were determined to be allelic to an EMS-mutagenized line denoted acn1 for ac non-utilizing. Following conventions, the tDNA-tagged mutants were designated acn1-2 and acn1-3. Both mutants displayed identical behavior to acn1-1 on a variety of fluorinated and nonfluorinated organic acids, indicating that resistance was specific to fluoroacetate. Thermal asymmetric interlaced PCR identified the sites of tDNA insertion in both mutants to be within different exons in a gene, which encoded a protein containing an AMP-binding motif. Reverse transcription-PCR confirmed that the gene was not expressed in the mutants, and quantitative reverse transcription-PCR showed that the gene is expressed in imbibed seeds and increases in amount during establishment. The wild type AMP-binding protein cDNA was cloned and expressed in Escherichia coli, and the expressed protein was purified by nickel chelate chromatography. The enzyme was identified as an acyl-CoA synthetase that was more active with acetate than butyrate and was not active with fatty acids longer than C-4. The enzyme was localized to peroxisomes by enzymatic analysis of organellar fractions isolated by sucrose density gradient centrifugation. Labeling studies with [(14)C]acetate showed that acn1 seedlings, like those of the isocitrate lyase mutant icl-1 (isocitrate lyase), are compromised in carbohydrate synthesis, indicating that this enzyme is responsible for activating exogenous acetate to the coenzyme A form for entry into the glyoxylate cycle.

Distribution of actin gene isoforms in the Arabidopsis leaf measured in microsamples from intact individual cells.

The contents of single plant cells can be sampled using glass microcapillaries. By combining such single-cell sampling with reverse transcription-polymerase chain reaction (RT-PCR), transcripts of individual genes can be identified and, in principle, quantified. This provides a valuable technique for the analysis and quantification of the intercellular distribution of gene expression in complex tissues. In a proof-of-principle study, the cellular locations of the transcripts of the eight isoforms of actin ( ACT) expressed in Arabidopsis thaliana (L.) Heynh. were analyzed. Cell sap was extracted from epidermal and mesophyll cells of leaves of 3- to 4-week-old plants. Single-cell (SC)-RT-PCR was used to amplify the actin transcripts using specific primer pairs for ACT1, 2, 3, 4, 7, 8, 11 and 12. Only ACT2 and ACT8 were found in epidermal and in mesophyll cells. In individual trichomes, in addition to ACT2 and ACT8, ACT7 and ACT11 transcripts were detectable. By employing the already well-characterized actin system we demonstrate the practicality and power of SC-RT-PCR as a technique for analyzing gene expression at the ultimate level of resolution, the single cell.