Impaired Chloroplast Biogenesis in Immutans, an Arabidopsis Variegation Mutant, Modifies Developmental Programming, Cell Wall Composition and Resistance to Pseudomonas syringae.

The immutans (im) variegation mutation of Arabidopsis has green- and white- sectored leaves due to action of a nuclear recessive gene. IM codes for PTOX, a plastoquinol oxidase in plastid membranes. Previous studies have revealed that the green and white sectors develop into sources (green tissues) and sinks (white tissues) early in leaf development. In this report we focus on white sectors, and show that their transformation into effective sinks involves a sharp reduction in plastid number and size. Despite these reductions, cells in the white sectors have near-normal amounts of plastid RNA and protein, and surprisingly, a marked amplification of chloroplast DNA. The maintenance of protein synthesis capacity in the white sectors might poise plastids for their development into other plastid types. The green and white im sectors have different cell wall compositions: whereas cell walls in the green sectors resemble those in wild type, cell walls in the white sectors have reduced lignin and cellulose microfibrils, as well as alterations in galactomannans and the decoration of xyloglucan. These changes promote susceptibility to the pathogen Pseudomonas syringae. Enhanced susceptibility can also be explained by repressed expression of some, but not all, defense genes. We suggest that differences in morphology, physiology and biochemistry between the green and white sectors is caused by a reprogramming of leaf development that is coordinated, in part, by mechanisms of retrograde (plastid-to-nucleus) signaling, perhaps mediated by ROS. We conclude that variegation mutants offer a novel system to study leaf developmental programming, cell wall metabolism and host-pathogen interactions.

Quantitative analysis of the chloroplast molecular chaperone ClpC/Hsp93 in Arabidopsis reveals new insights into its localization, interaction with the Clp proteolytic core, and functional importance.

The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import.

Changes in endogenous gene transcript and protein levels in maize plants expressing the soybean ferritin transgene.

Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, we tested effects of soybean ferritin transgene (SoyFer1, M64337) on transcript and protein levels of endogenous genes in maize. Results showed that the transgene was successfully introduced and expressed in the maize seed endosperm. mRNA abundance of seven tested iron homeostasis genes and seed storage protein genes differed significantly between seed samples positive and negative for the transgene. The PCR negative samples had higher zein and total protein content compared to the positive samples. However, PCR positive samples had significantly higher concentrations of calcium, magnesium, and iron. We have shown that the soybean ferritin transgene affected the expression of native iron homeostasis genes in the maize plant. These results underscore the importance of taking a holistic approach to the evaluation of transgenic events in target plants, comparing the transgenic plant to the untransformed controls.

SUPPRESSOR OF VARIEGATION4, a new var2 suppressor locus, encodes a pioneer protein that is required for chloroplast biogenesis.

VAR2 is an integral thylakoid membrane protein and a member of the versatile FtsH class of metalloproteases in prokaryotes and eukaryotes. Recessive mutations in the VAR2 locus give rise to variegated plants (var2) that contain white sectors with abnormal plastids and green sectors with normal-appearing chloroplasts. In a continuing effort to isolate second-site suppressors of var2 variegation, we characterize in this report ems2505, a suppressor strain that has a virescent phenotype due to a missense mutation in At4g28590, the gene for a pioneer protein. We designated this gene SVR4 (for SUPPRESSOR OF VARIEGATION4) and the mutant allele in ems2505 as svr4-1. We demonstrate that SVR4 is located in chloroplasts and that svr4-1 single mutants are normal with respect to chloroplast anatomy and thylakoid membrane protein accumulation. However, they are modestly impaired in several aspects of photochemistry and have enhanced non-photochemical quenching (NPQ) capacity. A T-DNA insertion allele of SVR4, svr4-2, is seedling-lethal due to an early blockage of chloroplast development. We conclude that SVR4 is essential for chloroplast biogenesis, and hypothesize that SVR4 mediates some aspect of thylakoid structure or function that controls NPQ. We propose that in the suppressor strain, photoinhibitory pressure caused by a lack of VAR2 is ameliorated early in chloroplast development by enhanced NPQ capacity caused by reduced SVR4 activity. This would result in an increase in the number of chloroplasts that are able to surmount a threshold necessary to avoid photo-damage and thereby develop into functional chloroplasts.

Flexibility in photosynthetic electron transport: the physiological role of plastoquinol terminal oxidase (PTOX).

Oxygenic photosynthesis depends on a highly conserved electron transport system, which must be particularly dynamic in its response to environmental and physiological changes, in order to avoid an excess of excitation energy and subsequent oxidative damage. Apart from cyclic electron flow around PSII and around PSI, several alternative electron transport pathways exist including a plastoquinol terminal oxidase (PTOX) that mediates electron flow from plastoquinol to O(2). The existence of PTOX was first hypothesized in 1982 and this was verified years later based on the discovery of a non-heme, di-iron carboxylate protein localized to thylakoid membranes that displayed sequence similarity to the mitochondrial alternative oxidase. The absence of this protein renders higher plants susceptible to excitation pressure dependant variegation combined with impaired carotenoid synthesis. Chloroplasts, as well as other plastids (i.e. etioplasts, amyloplasts and chromoplasts), fail to assemble organized internal membrane structures correctly, when exposed to high excitation pressure early in development. While the role of PTOX in plastid development is established, its physiological role under stress conditions remains equivocal and we postulate that it serves as an alternative electron sink under conditions where the acceptor side of PSI is limited. The aim of this review is to provide an overview of the past achievements in this field and to offer directions for future investigative efforts. Plastoquinol terminal oxidase (PTOX) is involved in an alternative electron transport pathway that mediates electron flow from plastoquinol to O(2). This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.

Photosynthetic redox imbalance governs leaf sectoring in the Arabidopsis thaliana variegation mutants immutans, spotty, var1, and var2.

We hypothesized that chloroplast energy imbalance sensed through alterations in the redox state of the photosynthetic electron transport chain, measured as excitation pressure, governs the extent of variegation in the immutans mutant of Arabidopsis thaliana. To test this hypothesis, we developed a nondestructive imaging technique and used it to quantify the extent of variegation in vivo as a function of growth temperature and irradiance. The extent of variegation was positively correlated (R(2) = 0.750) with an increase in excitation pressure irrespective of whether high light, low temperature, or continuous illumination was used to induce increased excitation pressure. Similar trends were observed with the variegated mutants spotty, var1, and var2. Measurements of greening of etiolated wild-type and immutans cotyledons indicated that the absence of IMMUTANS increased excitation pressure twofold during the first 6 to 12 h of greening, which led to impaired biogenesis of thylakoid membranes. In contrast with IMMUTANS, the expression of its mitochondrial analog, AOX1a, was transiently upregulated in the wild type but permanently upregulated in immutans, indicating that the effects of excitation pressure during greening were also detectable in mitochondria. We conclude that mutations involving components of the photosynthetic electron transport chain, such as those present in immutans, spotty, var1, and var2, predispose Arabidopsis chloroplasts to photooxidation under high excitation pressure, resulting in the variegated phenotype.

Conserved active site sequences in Arabidopsis plastid terminal oxidase (PTOX): in vitro and in planta mutagenesis studies.

The plastid terminal oxidase (PTOX) is distantly related to the mitochondrial alternative oxidase (AOX). Both are members of the diiron carboxylate quinol oxidase (DOX) class of proteins. PTOX and AOX contain 20 highly conserved amino acids, six of which are Fe-binding ligands. We have previously used in vitro and in planta activity assays to examine the functional importance of the Fe-binding sites. In this report, we conduct alanine-scanning mutagenesis on the 14 other conserved sites using our in vitro and in planta assay procedures. We found that the 14 sites fall into three classes: (i) Ala-139, Pro-142, Glu-171, Asn-174, Leu-179, Pro-216, Ala-230, Asp-287, and Arg-293 are dispensable for activity; (ii) Tyr-234 and Asp-295 are essential for activity; and (iii) Leu-135, His-151, and Tyr-212 are important but not essential for activity. Our data are consistent with the proposed role of some of these residues in active site conformation, substrate binding, and/or catalysis. Titration experiments showed that down-regulation of PTOX to approximately 3% of wild-type levels did not compromise plant growth, at least under ambient growth conditions. This suggests that PTOX is normally in excess, especially early in thylakoid membrane biogenesis.

Chloroplast photooxidation-induced transcriptome reprogramming in Arabidopsis immutans white leaf sectors.

Arabidopsis (Arabidopsis thaliana) immutans (im) has green and white sectoring due to the action of a nuclear recessive gene, IMMUTANS. The green sectors contain normal-appearing chloroplasts, whereas the white sectors contain abnormal chloroplasts that lack colored carotenoids due to a defect in phytoene desaturase activity. Previous biochemical and molecular characterizations of the green leaf sectors revealed alterations suggestive of a source-sink relationship between the green and white sectors of im. In this study, we use an Affymetrix ATH1 oligoarray to further explore the nature of sink metabolism in im white tissues. We show that lack of colored carotenoids in the im white tissues elicits a differential response from a large number of genes involved in various cellular processes and stress responses. Gene expression patterns correlate with the repression of photosynthesis and photosynthesis-related processes in im white tissues, with an induction of Suc catabolism and transport, and with mitochondrial electron transport and fermentation. These results suggest that energy is derived via aerobic and anaerobic metabolism of imported sugar in im white tissues for growth and development. We also show that oxidative stress responses are largely induced in im white tissues; however, im green sectors develop additional energy-dissipating mechanisms that perhaps allow for the formation of green sectors. Furthermore, a comparison of the transcriptomes of im white and norflurazon-treated white leaf tissues reveals global as well as tissue-specific responses to photooxidation. We conclude that the differences in the mechanism of phytoene desaturase inhibition play an important role in differentiating these two white tissues.

Alterations in photosynthesis in Arabidopsis lacking IMMUTANS, a chloroplast terminal oxidase.

Green and white variegation in the Arabidopsis immutans (im) mutant is caused by a nuclear recessive gene. The green sectors contain cells with normal-appearing chloroplasts, while cells in the white sectors have photooxidized plastids lacking organized lamellae. In the present experiments, we found that the green im sectors have enhanced rates of carbon assimilation (monitored by (14)CO(2) uptake) and that there are corresponding increases in the activities of Rubisco and SPS, elevated starch and sucrose pool sizes, and an altered pattern of carbohydrate partitioning that favors sucrose over starch. We hypothesize that these increases are due, at least in part, to interactions with white sectors, perhaps to compensate for reductions in total source tissue. Consistent with this idea, the im white sectors accumulate low levels of sucrose and acid invertase activities are markedly increased in the white versus green cells. This suggests that there is a sucrose gradient between the green and white sectors, and that sucrose is transported from the green to white cells in response to sink demand. The expression of photosynthetic genes is not appreciably altered in the green im sectors versus wild type, but rather there is an up-regulation of genes involved in defense against oxidative stress and down-regulation of genes involved in cell wall biosynthesis. We postulate that changes in photosynthesis in the im green cells are driven by a need for photoprotection (especially early in chloroplast biogenesis) and due to source-sink interactions.

IMMUTANS does not act as a stress-induced safety valve in the protection of the photosynthetic apparatus of Arabidopsis during steady-state photosynthesis.

IMMUTANS (IM) encodes a thylakoid membrane protein that has been hypothesized to act as a terminal oxidase that couples the reduction of O(2) to the oxidation of the plastoquinone (PQ) pool of the photosynthetic electron transport chain. Because IM shares sequence similarity to the stress-induced mitochondrial alternative oxidase (AOX), it has been suggested that the protein encoded by IM acts as a safety valve during the generation of excess photosynthetically generated electrons. We combined in vivo chlorophyll fluorescence quenching analyses with measurements of the redox state of P(700) to assess the capacity of IM to compete with photosystem I for intersystem electrons during steady-state photosynthesis in Arabidopsis (Arabidopsis thaliana). Comparisons were made between wild-type plants, im mutant plants, as well as transgenics in which IM protein levels had been overexpressed six (OE-6 x) and 16 (OE-16 x) times. Immunoblots indicated that IM abundance was the only major variant that we could detect between these genotypes. Overexpression of IM did not result in increased capacity to keep the PQ pool oxidized compared to either the wild type or im grown under control conditions (25 degrees C and photosynthetic photon flux density of 150 micromol photons m(-2) s(-1)). Similar results were observed either after 3-d cold stress at 5 degrees C or after full-leaf expansion at 5 degrees C and photosynthetic photon flux density of 150 micromol photons m(-2) s(-1). Furthermore, IM abundance did not enhance protection of either photosystem II or photosystem I from photoinhibition at either 25 degrees C or 5 degrees C. Our in vivo data indicate that modulation of IM expression and polypeptide accumulation does not alter the flux of intersystem electrons to P(700)(+) during steady-state photosynthesis and does not provide any significant photoprotection. In contrast to AOX1a, meta-analyses of published Arabidopsis microarray data indicated that IM expression exhibited minimal modulation in response to myriad abiotic stresses, which is consistent with our functional data. However, IM exhibited significant modulation in response to development in concert with changes in AOX1a expression. Thus, neither our functional analyses of the IM knockout and overexpression lines nor meta-analyses of gene expression support the model that IM acts as a safety valve to regulate the redox state of the PQ pool during stress and acclimation. Rather, IM appears to be strongly regulated by developmental stage of Arabidopsis.

Functional redundancy of AtFtsH metalloproteases in thylakoid membrane complexes.

FtsH is an ATP-dependent metalloprotease found in bacteria, mitochondria, and plastids. Arabidopsis (Arabidopsis thaliana) contains 12 AtFtsH proteins, three in the mitochondrion and nine in the chloroplast. Four of the chloroplast FtsH proteins are encoded by paired members of closely related genes (AtFtsH1 and 5, and AtFtsH2 and 8). We have previously reported that AtFtsH2 and 8 are interchangeable components of AtFtsH complexes in the thylakoid membrane. In this article, we show that the var1 variegation mutant, which is defective in AtFtsH5, has a coordinate reduction in the AtFtsH2 and 8 pair, and that the levels of both pairs are restored to normal in var1 plants that overexpress AtFtsH1. Overexpression of AtFtsH1, but not AtFtsH2/VAR2, normalizes the pattern of var1 variegation, restoring a nonvariegated phenotype. We conclude that AtFtsH proteins within a pair, but not between pairs, are interchangeable and functionally redundant, at least in part. We further propose that the abundance of each pair is matched with that of the other pair, with excess subunits being turned over. The variegation phenotype of var1 (as well as var2, which is defective in AtFtsH2) suggests that a threshold concentration of subunits is required for normal chloroplast function. AtFtsH1, 2, 5, and 8 do not show evidence of tissue or developmental specific expression. Phylogenetic analyses revealed that rice (Oryza sativa) and Arabidopsis share a conserved core of seven FtsH subunit genes, including the AtFtsH1 and 5 and AtFtsH2 and 8 pairs, and that the structure of the present-day gene families can be explained by duplication events in each species following the monocot/dicot divergence.

Mutation of a UDP-glucose-4-epimerase alters nematode susceptibility and ethylene responses in Arabidopsis roots.

In Arabidopsis, mutation of RHD1, a UDP-glucose-4-epimerase, causes root-specific phenotypes, including hypersusceptibility to the cyst nematode Heterodera schachtii, increased root hair elongation, decreased root length, and root epidermal bulging. Previous experiments suggested that increased ethylene sensitivity or production mediated the rhd1-4 phenotypes. In the present study, double mutant analyses revealed that only rhd1-4 hypersusceptibility to H. schachtii and increased root hair elongation were dependent upon the ethylene signaling genes EIN2 and EIN3 but not upon ethylene signaling mediated by the auxin efflux carrier EIR1. In contrast, the rhd1-4 short root and root epidermal bulging phenotypes did not require EIN2, EIN3, or EIR1. A time-course analysis of RHD1 transcript levels in wild-type plants treated with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid showed a root-specific downregulation of RHD1 expression by ethylene. This observation was corroborated by our finding of increased RHD1 transcript levels in roots of the ethylene-insensitive mutants etr1 and ein2. In addition to ethylene, auxin strongly influences H. schachtii susceptibility and root hair elongation. Therefore, we investigated the sensitivity of rhd1-4 roots to indole-3-acetic acid (IAA). Equivalent IAA concentrations caused a greater reduction in rhd1-4 root elongation compared with wild-type roots. Finally, H. schachtii parasitism was found to strongly downregulate RHD1 expression in the root 3 days after inoculation. We conclude that RHD1 is a likely target of root-specific negative regulation by ethylene and that loss of RHD1 function results in a heightened sensitivity of root tissues to both ethylene and auxin.

Mutations in ClpC2/Hsp100 suppress the requirement for FtsH in thylakoid membrane biogenesis.

The Arabidopsis var2 variegation mutant defines a nuclear gene for a chloroplast FtsH metalloprotease. Leaf variegation is expressed only in homozygous recessive plants. The cells in the green leaf sectors of this mutant contain morphologically normal chloroplasts, whereas cells in the white sectors contain abnormal plastids lacking organized lamellar structures. var2 mutants are hypersusceptible to photoinhibition, and VAR2 degrades unassembled polypeptides and is involved in the D1 repair cycle of photosystem II, likely by affecting turnover of the photodamaged D1 polypeptide. A second-site suppressor screen of var2 yielded a normal-appearing, nonvariegated line. Map-based cloning revealed that the suppression of variegation in this line is due to a splice site mutation in ClpC2, a chloroplast Hsp100 chaperone, that results in sharply reduced ClpC2 protein accumulation. Isolation of clpC2 single mutants showed that clpC2 is epistatic to var2, and that a lack of ClpC2 does not markedly alter the composition of the thylakoid membrane. Suppression by clpC2 is not allele-specific. Our results suggest that clpC2 is a suppressor of thylakoid biogenesis and maintenance and that ClpC2 might act by accelerating photooxidative stress. Arabidopsis has two ClpC genes (ClpC1 and ClpC2), and mutants with down-regulated expression of both genes have a phenotype different from clpC2, suggesting that ClpC1 and ClpC2 act synergistically and/or that they are only partially redundant. The isolation of a clpC2 mutant represents an important advance in the generation of tools to understand Hsp100 function and insight into the mechanisms of protein quality control in plants.

Control of chloroplast redox by the IMMUTANS terminal oxidase.

Variegation mutants offer excellent opportunities to study interactions between the nucleus-cytoplasm, the chloroplast, and the mitochondrion. Variegation in the immutans (im) mutant of Arabidopsis is induced by a nuclear recessive gene and the extent of variegation can be modulated by light and temperature. Whereas the green sectors have morphologically normal chloroplasts, the white sectors are devoid of pigments and accumulate a colourless carotenoid, phytoene. The green sectors are hypothesized to arise from cells that have avoided irreversible photooxidative damage whereas the white sectors originate from cells that are photooxidized. Cloning of the IMMUTANS (IM) gene has revealed that IMMUTANS (IM) is a plastid homologue of the mitochondrial alternative oxidase. This finding suggested a model in which IM functions as a redox component of the phytoene desaturation pathway, which requires phytoene desaturase activity. Consistent with this idea, IM has quinol oxidase activity in vitro. Recent studies have revealed that IM plays a more global role in plastid metabolism. For example, it appears to be the elusive terminal oxidase of chlororespiration and also functions as a light stress protein.

The Arabidopsis FtsH metalloprotease gene family: interchangeability of subunits in chloroplast oligomeric complexes.

The Arabidopsis At filamentation temperature sensitive (FtsH) metalloprotease gene family comprises 12 members (AtFtsH1-AtFtsH12), including three pairs of closely related genes that are targeted to chloroplasts (AtFtsH2 and AtFtsH8; AtFtsH1 and AtFtsH5; and AtFtsH7 and AtFtsH9). Mutations in AtFtsH5 (var1) and AtFtsH2 (var2) give rise to variegated plants with green- and white-sectored leaves. Cells in the green sectors contain morphologically normal chloroplasts, whereas cells in the white sectors are blocked in chloroplast biogenesis. A major question is how chloroplasts arise in cells that have a mutant genotype. We have found by two-dimensional (2-D) green gel and gel filtration analyses that AtFtsH2/VAR2 forms oligomeric complexes. Two bands in the 2-D green gels that correspond to AtFtsH5/VAR1 + AtFtsH1 and AtFtsH2/VAR2 + AtFtsH8 have been identified, and these bands are coordinately reduced in amount in var1 and var2 thylakoids that lack AtFtsH5/VAR1 and AtFtsH2/VAR2, respectively. These reductions are not because of alterations in transcript abundance. Overexpression of AtFtsH8 in var2-4 (a putative null allele) normalizes the variegation phenotype of the mutant and restores the two bands to their wild-type levels. These results suggest that AtFtsH8 is interchangeable with AtFtsH2/VAR2 in AtFtsH-containing oligomers, and that the two proteins have redundant functions. Consistent with this hypothesis, AtFtsH2 and AtFtsH8 have similar expression patterns, as monitored by promoter-beta-glucuronidase (GUS) fusion and RT-PCR experiments. Based on our findings, we propose that AtFtsH1, AtFtsH2/VAR2, AtFtsH5/VAR1, and AtFtsH8 interact to form oligomeric structures, and that subunit stoichiometry is controlled post-transcriptionally in var1 and var2, perhaps by turnover. A threshold model is presented to explain the pattern of variegation in var2 in which AtFtsH8 provides a compensating activity in the green sectors of the mutant.

Homologous soybean and Arabidopsis genes share responsiveness to cyst nematode infection.

SUMMARY We previously isolated a partial soybean cDNA clone (D17.1) whose corresponding transcript increases in susceptible roots 1 day post inoculation (dpi) with the soybean cyst nematode, Heterodera glycines. Here we isolated the corresponding full-length cDNA from a soybean cDNA library and designated this gene of unknown function Gm17.1. Time course RNA gel blot analyses revealed that Gm17.1 mRNA steady-state levels were elevated in soybean roots following H. glycines infection up to at least 6 dpi. For further in-depth study we identified a homologous Arabidopsis thaliana gene and designated this gene At17.1. Arabidopsis is successfully infected by the sugar beet cyst nematode (H. schachtii), a close relative of H. glycines. We isolated the At17.1 promoter, fused it to the beta-glucuronidase (GUS) reporter gene, and transformed this construct into Arabidopsis plants as well as soybean hairy roots. Histochemical analysis of plant materials containing the At17.1::GUS construct revealed that the At17.1 promoter is functional in Arabidopsis as well as in soybean and that during normal plant development the At17.1 promoter directs GUS expression predominantly to the vascular tissues and root tips of both plant species. When At17.1::GUS Arabidopsis plants and soybean hairy roots were inoculated with cyst nematodes, strong GUS activity was detected within the cyst nematode-induced feeding structures. Further tests of At17.1 promoter activity in Arabidopsis revealed that this promoter was induced by auxin, jasmonic acid, mannitol and dehydration. Quantitative real-time reverse transcription-polymerase chain reaction assays of At17.1 expression confirmed the observed promoter characteristics. Based on our expression data and the observation that both the soybean and the Arabidopsis homologues behaved in a similar fashion following cyst nematode infection, it is likely that these genes are closely associated with cyst nematode parasitism of plants, potentially with hormone and osmotic changes occurring in the developing nematode feeding cells. Furthermore, these data provide additional insights into the strengths of the Arabidopsis-H. schachtii pathosystem to study cyst nematode-plant interactions in lieu of less tractable pathosystems. This finding is supported by the fact that the Arabidopsis promoter tested here produced similar results in Arabidopsis and soybean.

Arabidopsis gene expression changes during cyst nematode parasitism revealed by statistical analyses of microarray expression profiles.

With the availability of microarray technology, the expression profiles of thousands of genes can be monitored simultaneously to help determine the mechanisms of these biological processes. We conducted Affymetrix GeneChip microarray analyses of the Arabidopsis-cyst nematode interaction and employed a statistical procedure to analyze the resultant data, which allowed us to identify significant gene expression changes. Quantitative real-time RT-PCR assays were used to confirm the microarray analyses. The results of the expression profiling revealed 128 genes with altered steady-state mRNA levels following infection by the sugar beet cyst nematode (Heterodera schachtii; BCN), in contrast to only 12 genes that had altered expression following infection by the soybean cyst nematode (H. glycines; SCN). The expression of these 12 genes also changed following infection by BCN, i.e. we did not identify any genes regulated exclusively by SCN. The identification of 116 genes whose expression changes during successful cyst nematode parasitism by BCN suggests a potential involvement of these genes in the infection events starting with successful syncytium induction. Further characterization of these genes will permit the formulation of testable hypotheses to explain successful cyst nematode parasitism.

Expression of an Arabidopsis phosphoglycerate mutase homologue is localized to apical meristems, regulated by hormones, and induced by sedentary plant-parasitic nematodes.

We previously isolated a partial soybean cDNA clone whose transcript abundance is increased upon infection by the sedentary, endoparasitic soybean cyst nematode Heterodera glycines. We now isolated the corresponding full-length cDNA and determined that the predicted gene product was similar to the group of cofactor-dependent phosphoglycerate mutase/bisphosphoglycerate mutase enzymes (PGM/bPGM; EC 5.4.2.1/5.4.2.4). We designated the corresponding soybean gene GmPGM. PGM and bPGM are key catalysts of glycolysis that have been well characterized in animals but not plants. Using the GmPGM cDNA sequence, we identified a homologous Arabidopsis thaliana gene, which we designated AtPGM. Histochemical GUS analyses of transgenic Arabidopsis plants containing the AtPGM promoter ::GUS construct revealed that the AtPGM promoter directs GUS expression in uninfected plants only to the shoot and root apical meristems. In infected plants, GUS staining also is evident in the nematode feeding structures induced by the cyst nematode Heterodera schachtii and by the root-knot nematode Meloidogyne incognita. Furthermore, we discovered that the AtPGM promoter was down-regulated by abscisic acid and hydroxyurea, whereas it was induced by sucrose, oryzalin, and auxin, thereby revealing expression characteristics typical of genes with roles in meristematic cells. Assessment of the auxin-inducible AUX1 gene promoter (a gene coding for a polar auxin transport protein) similarly revealed feeding cell and meristem expression, suggesting that auxin may be responsible for the observed tissue specificity of the AtPGM promoter. These results provide first insight into the possible roles of PGM/bPGM in plant physiology and in plant-pathogen interactions.

Identification and characterization of a soybean ethylene-responsive element-binding protein gene whose mRNA expression changes during soybean cyst nematode infection.

Ethylene-responsive element-binding proteins (EREBPs) are members of a family of plant transcription factors. Conserved EREBP domains of these proteins bind to the GCC box, an ethylene-responsive promoter element found in many pathogenesis-related (PR) genes. Using degenerate primers to the EREBP domain from diverse plant species, an EREBP homolog was isolated from a soybean cDNA library. Gel mobility-shift assays revealed that the translation product of this cDNA bound specifically to GCC box sequences. We, therefore, named this gene Glycine max ethylene-responsive element-binding protein 1 (GmEREBP1), i.e., a gene coding for the first confirmed GCC box-binding protein of soybean. GmEREBP1 mRNA abundance was analyzed by RNA blot hybridizations in soybean roots and shoots of cultivars Corsoy 79 and Hartwig, which are susceptible and resistant, respectively, to the soybean cyst nematode (Heterodera glycines). These analyses revealed that GmEREBP1 is expressed in a root-preferential manner and that GmEREBP1 mRNA abundance is changed after H. glycines infection. GmEREBP1 mRNA abundance decreased in infected (susceptible) 'Corsoy 79' roots, whereas it increased in abundance in infected (resistant) 'Hartwig' roots. Furthermore, ethephon treatment repressed GmEREBP1 mRNA accumulation in both cultivars, whereas wounding increased expression in both cultivars. These changes in mRNA steady-state levels suggest that GmEREBP1 plays a role in soybean-H. glycines interactions.