Carolina Escobar

Early transcriptomic events in microdissected Arabidopsis nematode‐induced giant cells

Root-knot nematodes differentiate highly specialized feeding cells in roots (giant cells, GCs), through poorly characterized mechanisms that include extensive transcriptional changes. While global transcriptome analyses have used galls, which are complex root structures that include GCs and surrounding tissues, no global gene expression changes specific to GCs have been described. We report on the differential transcriptome of GCs versus root vascular cells, induced in Arabidopsis by Meloidogyne javanica at a very early stage of their development, 3 days after infection (d.p.i.). Laser microdissection was used to capture GCs and root vascular cells for microarray analysis, which was validated through qPCR and by a promoter-GUS fusion study. Results show that by 3 d.p.i., GCs exhibit major gene repression. Although some genes showed similar regulation in both galls and GCs, the majority had different expression patterns, confirming the molecular distinctiveness of the GCs within the gall. Most of the differentially regulated genes in GCs have no previously assigned function. Comparisons with other transcriptome analyses revealed similarities between GCs and cell suspensions differentiating into xylem cells. This suggests a molecular link between GCs and developing vascular cells, which represent putative GC stem cells. Gene expression in GCs at 3 d.p.i. was also found to be similar to crown galls induced by Agrobacterium tumefaciens, a specialized root biotroph.

Differential alterations of antioxidant defenses as bioindicators of mercury and cadmium toxicity in alfalfa.

Several physiological parameters related to oxidative stress, which is a characteristic of plants exposed to toxic metals, were studied in 3-week-old alfalfa plants treated with cadmium (Cd) or mercury (Hg) at doses of 0, 3, 10 and 30 microM for 7d. The concentrations of biothiols, glutathione (GSH), homoglutathione (hGSH) and phytochelatins (PCs) increased dramatically in metals-treated plants, in particular in the presence of Cd. This was accompanied by a remarkable up-regulation of gamma-glutamyl cysteine synthetase gene, probably in response to the higher demand for GSH|hGSH needed for PC synthesis. The presence of metals enhanced lipid peroxidation in shoots, while chlorophyll content declined in a concentration dependent manner. Ascorbate peroxidase (APX) activity increased moderately in roots of Cd-exposed plants, and a new basic root peroxidase isoform was found in both Cd- and Hg-treated plants. Glutathione reductase (GR) activity was enhanced in shoots of plants exposed to Cd and Hg. However, this enzymatic activity showed a metal dependent response in roots, and was enhanced in Cd-treated plants but was severely inhibited in roots of plants treated with Hg. Inhibition of GR by Hg was confirmed in vitro by incubating a commercially available GR and control shoot extracts with several doses of Hg and Cd. Ascorbate concentrations were elevated with treatments of 3 microM Hg, 10 microM Cd and 30 microM Cd, indicating that this compound is necessary for redox cellular homeostasis. The different responses observed with Cd and Hg treatments might be the basis for specific stress bioindicators.

Contribution of glutathione to the control of cellular redox homeostasis under toxic metal and metalloid stress

The accumulation of toxic metals and metalloids, such as cadmium (Cd), mercury (Hg), or arsenic (As), as a consequence of various anthropogenic activities, poses a serious threat to the environment and human health. The ability of plants to take up mineral nutrients from the soil can be exploited to develop phytoremediation technologies able to alleviate the negative impact of toxic elements in terrestrial ecosystems. However, we must select plant species or populations capable of tolerating exposure to hazardous elements. The tolerance of plant cells to toxic elements is highly dependent on glutathione (GSH) metabolism. GSH is a biothiol tripeptide that plays a fundamental dual role: first, as an antioxidant to mitigate the redox imbalance caused by toxic metal(loid) accumulation, and second as a precursor of phytochelatins (PCs), ligand peptides that limit the free ion cellular concentration of those pollutants. The sulphur assimilation pathway, synthesis of GSH, and production of PCs are tightly regulated in order to alleviate the phytotoxicity of different hazardous elements, which might induce specific stress signatures. This review provides an update on mechanisms of tolerance that depend on biothiols in plant cells exposed to toxic elements, with a particular emphasis on the Hg-triggered responses, and considering the contribution of hormones to their regulation.

Distinct and conserved transcriptomic changes during nematode‐induced giant cell development in tomato compared with Arabidopsis: a functional role for gene repression

Root-knot nematodes (RKNs) induce giant cells (GCs) from root vascular cells inside the galls. Accompanying molecular changes as a function of infection time and across different species, and their functional impact, are still poorly understood. Thus, the transcriptomes of tomato galls and laser capture microdissected (LCM) GCs over the course of parasitism were compared with those of Arabidopsis, and functional analysis of a repressed gene was performed. Microarray hybridization with RNA from galls and LCM GCs, infection-reproduction tests and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) transcriptional profiles in susceptible and resistant (Mi-1) lines were performed in tomato. Tomato GC-induced genes include some possibly contributing to the epigenetic control of GC identity. GC-repressed genes are conserved between tomato and Arabidopsis, notably those involved in lignin deposition. However, genes related to the regulation of gene expression diverge, suggesting that diverse transcriptional regulators mediate common responses leading to GC formation in different plant species. TPX1, a cell wall peroxidase specifically involved in lignification, was strongly repressed in GCs/galls, but induced in a nearly isogenic Mi-1 resistant line on nematode infection. TPX1 overexpression in susceptible plants hindered nematode reproduction and GC expansion. Time-course and cross-species comparisons of gall and GC transcriptomes provide novel insights pointing to the relevance of gene repression during RKN establishment.

Role of hydrogen peroxide and the redox state of ascorbate in the induction of antioxidant enzymes in pea leaves under excess light stress

In this work we used two different pea cultivars, JI281 is a semidomesticated land race of pea from Ethiopia whereas JI399 is a typical domesticated pea variety. Exposure of pea leaves to excess light (EL) for 1 h caused a reversible photoinhibition of photosynthesis as showed by changes in Fv / Fm. Although little difference existed between the two pea genotypes with respect to photoinhibition, after 60 min of EL the decline in Fv / Fm was higher in JI281 than in JI399 leaves. As a consequence of EL, H2O2 increased in both pea cultivars, whereas lipid peroxidation and protein oxidation slightly increased, although differences between cultivars were minimal. The redox state of ascorbate shifted towards its oxidized form under EL stress in both cultivars. Transcript levels of genes coding antioxidant enzymes varied with EL in both cultivars, but the response was more pronounced in JI399. The induction observed during EL was maintained or increased after the stress period, as occurred for cytGR and chlMDHAR. GR protein accumulation and activity correlated with the transcript accumulation in JI399, but not in JI288. In this work, a possible role for H2O2 and redox status of ascorbate in the photoxidative stress signalling is discussed.

Two closely related members of Arabidopsis 13-lipoxygenases (13-LOXs), LOX3 and LOX4, reveal distinct functions in response to plant-parasitic nematode infection

The responses of two closely related members of Arabidopsis 13-lipoxygenases (13-LOXs), LOX3 and LOX4, to infection by the sedentary nematodes root-knot nematode (Meloidogyne javanica) and cyst nematode (Heterodera schachtii) were analysed in transgenic Arabidopsis seedlings. The tissue localization of LOX3 and LOX4 gene expression using β-glucuronidase (GUS) reporter gene constructs showed local induction of LOX3 expression when second-stage juveniles reached the vascular bundle and during the early stages of plant-nematode interaction through gall and syncytia formation. Thin sections of nematode-infested knots indicated LOX3 expression in mature giant cells, and high expression in neighbouring cells and those surrounding the female body. LOX4 promoter was also activated by nematode infection, although the GUS signal weakened as infection and disease progressed. Homozygous insertion mutants lacking LOX3 were less susceptible than wild-type plants to root-knot nematode infection, as reflected by a decrease in female counts. Conversely, deficiency in LOX4 function led to a marked increase in females and egg mass number and in the female to male ratio of M. javanica and H. schachtii, respectively. The susceptibility of lox4 mutants was accompanied by increased expression of allene oxide synthase, allene oxide cyclase and ethylene-responsive transcription factor 4, and the accumulation of jasmonic acid, measured in the roots of lox4 mutants. This response was not found in lox3 mutants. Taken together, our results reveal that LOX4 and LOX3 interfere differentially with distinct metabolic and signalling pathways, and that LOX4 plays a major role in controlling plant defence against nematode infection.

Isolation of RNA from laser-capture-microdissected giant cells at early differentiation stages suitable for differential transcriptome analysis

Plant organ gene expression profile analyses are complicated by the various cell types, and therefore transcription patterns, present in each organ. For example, each gall formed in roots following root knot nematode infection contains between four and eight specialized feeding cells (giant cells, GCs) embedded within hypertrophied root tissues. A recent goal in plant science has been the isolation of nematode feeding cell mRNAs for subsequent gene expression analysis. By adapting current protocols for different plant species and cells, we have developed a simple and rapid method for obtaining GCs from frozen tissue sections of tomato with good morphology and preserved RNA. The tissue sections obtained were suitable for the laser capture microdissection of GCs 6-7 days post-infection, and even of very early developing GCs (48-72 h post-infection), by fixation of tissue with ethanol-acetic acid, infiltration with sucrose and freezing in isopentane with optimal cutting temperature medium. This process was also successful for obtaining control vascular cells from uninfected roots for direct comparison with GCs. A minimum of about 300 GCs and 600 control vascular cells was required for efficient linear RNA amplification through in vitro transcription. Laser capture microdissection-derived RNA, after two rounds of amplification, was successfully used for microarray hybridization and validated with several differentially expressed genes by quantitative polymerase chain reaction. Consistent with our results, 117 homologous genes were found to be co-regulated in a previous microarray analysis of Arabidopsis galls at the same developmental stage. Therefore, we conclude that our method allows the isolation of a sufficient quantity of RNA with a high quality/integrity, appropriate for differential transcriptome analysis.

Isolation of the LEMMI9 gene and promoter analysis during a compatible plant-nematode interaction

Plant-endoparasitic root-knot nematodes feed on specialized giant cells that they induce in the vascular cylinder of susceptible plants. Although it has been established that a number of plant genes change their expression pattern during giant cell differentiation, virtually no data are available about the mechanisms involved in that change. One possibility is differential promoter recognition by the transcription factor(s) responsible for the expression of specific genes. We have isolated and characterized a genomic clone from tomato containing the promoter region of LEMMI9, one of the few plant genes that have been reported to be highly expressed in galls (predominantly in giant cells). The analysis of transgenic potato plants carrying a LEMMI9 promoter-beta glucuronidase (GUS) fusion has demonstrated that the tomato promoter was activated in Meloidogyne incognita-induced galls in a heterologous system. We have located putative regulatory sequences in the promoter and have found that nuclear proteins from the galls formed specific DNA-protein complexes with the proximal region of the LEMMI9 promoter. The nuclear protein-binding sequence mapped to a region of 111 bp immediately upstream from the TATA box. This region contains a 12-bp repeat possibly involved in the formation of DNA-protein complexes, which might be related to the LEMMI9 transcriptional activation in the giant cells.

A role for LATERAL ORGAN BOUNDARIES‐DOMAIN 16 during the interaction Arabidopsis–Meloidogyne spp. provides a molecular link between lateral root and root‐knot nematode feeding site development

Plant endoparasitic nematodes induce the formation of their feeding cells by injecting effectors from the esophageal glands into root cells. Although vascular cylinder cells seem to be involved in the formation of root-knot nematode (RKN) feeding structures, molecular evidence is scarce. We address the role during gall development of LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16), a key component of the auxin pathway leading to the divisions in the xylem pole pericycle (XPP) for lateral root (LR) formation. Arabidopsis T-DNA tagged J0192 and J0121 XPP marker lines, LBD16 and DR5::GUS promoter lines, and isolated J0192 protoplasts were assayed for nematode-dependent gene expression. Infection tests in LBD16 knock-out lines were used for functional analysis. J0192 and J0121 lines were activated in early developing galls and giant cells (GCs), resembling the pattern of the G2/M-transition specific ProC yc B 1;1 :CycB1;1(NT)-GUS line. LBD16 was regulated by auxins in galls as in LRs, and induced by RKN secretions. LBD16 loss of function mutants and a transgenic line with defective XPP cells showed a significantly reduced infection rate. The results show that genes expressed in the dividing XPP, particularly LBD16, are important for gall formation, as they are for LR development.

Induction of the Hahsp17.7G4 Promoter by Root-Knot Nematodes: Involvement of Heat-Shock Elements in Promoter Activity in Giant Cells

Root-knot nematodes feed from specialized giant cells induced in the plants that they parasitize. We found that the promoter of the Hahsp17.7G4 gene, which encodes a small heat-shock protein involved in embryogenesis and stress responses, directed GUS expression in tobacco galls induced by the root-knot nematode Meloidogyne incognita. In roots containing a GUS reporter fusion to the Hahsp17.7G4 promoter, 10% of the galls stained for GUS expression 1 to 3 days after infection and the fraction stained increased to 60 to 80% 17 to 20 days after infection. A DNA fragment from -83 to +163, which contains heat-shock element (HSE) core sequences, is sufficient to support a promoter activity largely restricted to giant cells within the galls. Two-point mutations in HSE cores, previously reported to abolish the heat-shock response and to strongly reduce the embryogenesis response of the same promoter, did not reduce expression in giant cells. This suggests a distinct regulation of the promoter by nematodes. However, additional point mutations located at positions crucial for binding of heat-shock transcription factors (HSFs) caused a severe decrease in the nematode response. These results demonstrate that HSEs are involved in the promoter activation in giant cells and suggest that HSFs may mediate this response.