Nucleotide sequence of a gene from Arabidopsis thaliana encoding a myb homologue

A gene encoding a proto-oncogene, a myb-related gene named Atmyb1, was cloned from Arabidopsis thaliana, and its nucleotide sequence was determined. The Atmyb1 gene contains an intron of 494 bp, and there are no highly homologous sequences present in the A. thaliana genome, but evidence was found that other myb-related genes exist. In the 5 flanking region, we found several typical cis-acting elements found in plant promoters. Sequence comparisons revealed that the ATMYB1 protein has a putative DNA-binding domain with two repeats of tryptophan clusters, which is common in MYB-related proteins in plants, while animal MYB-related proteins contain DNA-binding domains with three repeats of tryptophan clusters. The putative DNA-binding domain of the ATMYB1 protein has higher homology with that of the human c-MYB protein than with those of other plant MYB proteins.     

AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death.

In plants, cysteine protease inhibitors are involved in the regulation of protein turnover and play an important role in resistance against insects and pathogens. AtCYS1 from Arabidopsis thaliana encodes a protein of 102 amino acids that contains the conserved motif of cysteine protease inhibitors belonging to the cystatin superfamily (Gln-Val-Val-Ala-Gly). Recombinant A. thaliana cystatin-1 (AtCYS1) was expressed in Escherichia coli and purified. AtCYS1 inhibits the catalytic activity of papain (Kd = 4.0 x 10-2 micro m, at pH 7.0 and 25 degrees C), generally taken as a molecular model of cysteine proteases. The molecular bases for papain inhibition by AtCYS1 have been analysed taking into account the three-dimensional structure of the papain-stefin B complex. AtCYS1 is constitutively expressed in roots and in developing siliques of A. thaliana. In leaves, AtCYS1 is strongly induced by wounding, by challenge with avirulent pathogens and by nitric oxide (NO). The overexpression of AtCYS1 blocks cell death activated by either avirulent pathogens or by oxidative and nitrosative stress in both A. thaliana suspension cultured cells and in transgenic tobacco plants. The suppression of the NO-mediated cell death in plants overexpressing AtCYS1 provides the evidence that NO is not cytotoxic for the plant, indicating that NO functions as cell death trigger through the stimulation of an active process, in which cysteine proteases and theirs proteinaceous inhibitors appear to play a crucial role.     

A functional OGG1 homologue from Arabidopsis thaliana

One of the major mutagenic base lesions in DNA caused by exposure to reactive oxygen species is 7,8-dihydro-8-oxoguanine (8-oxoG). Genes coding for DNA repair enzymes that recognise 8-oxoG have been reported in bacteria, yeast, mammals and plants. The prokaryotic and eukaryotic genes are functional homologues but differ in their primary sequence. We have cloned, sequenced, and expressed a new Arabidopsis thaliana cDNA that shows sequence homology to the eukaryotic genes coding for 8-oxoG DNA N-glycosylases (OGG1). The 40.3-kDa enzyme it encodes (AtOGG1) introduces a chain break in a double-stranded oligonucleotide specifically at an 8-oxoG residue. In addition, AtOGG1 can form a Schiff base with 8-oxoG in the presence of NaBH₄, suggesting that it is a bifunctional DNA N-glycosylase. Furthermore, expression of AtOGG1 in an Escherichia coli strain that is deficient in the repair of 8-oxoG in DNA suppresses its spontaneous-mutator phenotype. Thus, we have demonstrated that AtOGG1 is not only a structural but also a functional eukaryotic OGG1 homologue.     

Isolation of Arabidopsis thaliana mutants hypersensitive to gamma radiation

A screening method for mutants of Arabidopsis thaliana hypersensitive to -radiation has been devised. Plants grown from ethyl methanesulfonate (EMS)-treated seeds were irradiated at the seedling stage, which is highly radiosensitive due to extensive cell division. Severe growth inhibition of mutant plants by a -ray dose which only slightly affects wild-type plants was the selective criterion. Twelve true-breeding hyper-sensitive lines were isolated from a total of 3394 screened plants. Genetic analysis of five of the lines revealed five new genes, designated RAD1-RAD5. These Arabidopsis RAD mutants are phenotypically similar to mutants in the RAD52 epistasis group of Saccharomyces cerevisiae, which are highly sensitive to ionizing radiation but not hypersensitive to UV light. One possibility is that the Arabidopsis mutants are defective in a nonhomologous or illegitimate recombination mechanism used by plants for repair of chromosome breaks.     

Arabidopsis thaliana CENTRORADIALIS homologue (ATC) acts systemically to inhibit floral initiation in Arabidopsis

Floral initiation is orchestrated by systemic floral activators and inhibitors. This remote‐control system may integrate environmental cues to modulate floral initiation. Recently, FLOWERING LOCUS T (FT) was found to be a florigen. However, the identity of systemic floral inhibitor or anti‐florigen remains to be elucidated. Here we show that Arabidopsis thaliana CENTRORADIALIS homologue (ATC), an Arabidopsis FT homologue, may act in a non‐cell autonomous manner to inhibit floral initiation. Analysis of the ATC null mutant revealed that ATC is a short‐day‐induced floral inhibitor. Cell type‐specific expression showed that companion cells and apex that express ATC are sufficient to inhibit floral initiation. Histochemical analysis showed that the promoter activity of ATC was mainly found in vasculature but under the detection limit in apex, a finding that suggests that ATC may move from the vasculature to the apex to influence flowering. Consistent with this notion, Arabidopsis seedling grafting experiments demonstrated that ATC moved over a long distance and that floral inhibition by ATC is graft transmissible. ATC probably antagonizes FT activity, because both ATC and FT interact with FD and affect the same downstream meristem identity genes APETALA1, in an opposite manner. Thus, photoperiodic variations may trigger functionally opposite FT homologues to systemically influence floral initiation.     

A novel plastid-targeted J-domain protein in Arabidopsis thaliana

Arabidopsis cDNAs encoding ATJ11, the smallest known J-domain protein, have been isolated and characterized. The precursor protein of 161 amino acid residues was synthesized in vitro and imported by isolated pea chloroplasts where it was localized to the stroma and cleaved to a mature protein of 125 amino acid residues. The mature protein consists of an 80 amino acid J-domain, and N- and C-terminal extensions of 24 and 21 amino acid residues, respectively, which show no similarity to regions in other DnaJ-related proteins. ATJ11 produced in Escherichia coli stimulated the weak ATPase activity of E. coli DnaK, but was unable to stimulate refolding of firefly luciferase by DnaK, and inhibited refolding by DnaK, DnaJ and GrpE. ATJ11 is encoded by a single-copy gene on chromosome 4, and is expressed in all plant organs examined. A paralogue of ATJ11, showing 72% identity, is encoded in a 4.5 Mb duplication of chromosome 4 on chromosome 2. These proteins represent a novel class of J-domain proteins.     

Direct delivery of bacterial avirulence proteins into resistant Arabidopsis protoplasts leads to hypersensitive cell death

Many bacterial avirulence (Avr) proteins, including the Pseudomonas syringae proteins, AvrRpt2 and AvrB, appear to be recognized inside the host plant cell by resistance mechanisms mediated by the cognate resistance (R) genes. It is thought that Avr proteins are either delivered directly into the host cell via the bacterial type III secretion system (TTSS) or taken up by the plant cell following secretion into the apoplast through the TTSS. Recently, it was shown that the Xanthomonas campestris AvrBs2 protein can be delivered directly into the host plant cell by the TTSS. However, it is not known whether other type III effectors of phytopathogens behave similarly. Here, using a novel protein transfection method, we demonstrate that AvrRpt2 and AvrB must enter the plant cell to be recognized by R gene-mediated mechanisms. First, we established a hypersensitive cell death assay for protoplasts using the membrane-impermeable, nuclear-staining dye, YO-PRO-1, and transgenic Arabidopsis plants that carry an inducible avrRpt2 gene. Second, we transfected E. coli-produced AvrRpt2 or AvrB proteins into Arabidopsis protoplasts using a protein transfection kit based on the carrier peptide Pep-1, and demonstrated that hypersensitive cell death occurs in a gene-for-gene-specific manner. In contrast, these Avr proteins failed to elicit hypersensitive cell death when they were applied to protoplasts without the carrier peptide. We conclude that our preparations of E. coli-produced AvrRpt2 and AvrB are active, that AvrRpt2 and AvrB must be delivered into the plant cell to be recognized, and that a method based on a carrier peptide can be used to introduce proteins into plant cells.     

The mitochondrion--an organelle commonly involved in programmed cell death in Arabidopsis thaliana

Plant cells undergoing programmed cell death (PCD) at late stages typically show chromatin condensation and endonucleolytic cleavage prior to obvious membrane or organelle ultrastructural changes. To investigate possible early PCD-associated events, we used microscopic observations and flow cytometry to quantitate mitochondrial membrane potential (DeltaPsim) changes during PCD at the single cell and population levels using Arabidopsis protoplasts. A DeltaPsim loss was commonly induced early during plant PCD and was important for PCD execution, as evidenced by the concomitant reduction of the change in DeltaPsim and PCD by cyclosporin A, which inhibits mitochondrial permeability transition pores in animal cells. DeltaPsim loss occurred prior to nuclear morphological changes and was only associated with mitochondrial cytochrome c release (an apoptotic trigger in animals) in response to one of three PCD elicitors. Three different stimuli in wild type implicated DeltaPsim changes in PCD: ceramide, protoporphyrin IX, and the hypersensitive response elicitor AvrRpt2. Additionally, the behavior of the conditional ectopic cell death mutant accelerated cell death2 and ACD2-overproducing plants also implicated DeltaPsim alteration as key for PCD execution. Because ACD2 is largely a chloroplast component in mature plants, the observation that the cell death in acd2 mutants requires changes in mitochondrial functions implicates communication between chloroplasts and mitochondria in mediating PCD activation. We suggest that DeltaPsim loss is a common early marker in plant PCD, similar to what has been documented in animals. However, unlike in animal cells, in plant cells, mitochondrial cytochrome c release is not an obligatory step in PCD control.     

Proteomic analysis of S-nitrosylated proteins in Arabidopsis thaliana undergoing hypersensitive response

Nitric oxide (NO) has a fundamental role in the plant hypersensitive disease resistance response (HR), and S-nitrosylation is emerging as an important mechanism for the transduction of its bioactivity. A key step toward elucidating the mechanisms by which NO functions during the HR is the identification of the proteins that are subjected to this PTM. By using a proteomic approach involving 2-DE and MS we characterized, for the first time, changes in S-nitrosylated proteins in Arabidopsis thaliana undergoing HR. The 16 S-nitrosylated proteins identified are mostly enzymes serving intermediary metabolism, signaling and antioxidant defense. The study of the effects of S-nitrosylation on the activity of the identified proteins and its role during the execution of the disease resistance response will help to understand S-nitrosylation function and significance in plants.     

A TMV-Cg mutant with a truncated coat protein induces cell death resembling the hypersensitive response in Arabidopsis

Tobacco mosaic virus (TMV)-Cg is able to propagate and multiply systemically to high levels in Arabidopsis thaliana ecotype Col-0. In this study, we obtained a Cg mutant, Cgk1, which expresses a coat protein with a truncated carboxyl terminus. Interestingly, Cgk1 induced necrosis that resembled the hypersensitive response and caused more pronounced disease symptoms than wild type Cg in Arabidopsis Col-0 plants. A reactive oxidative burst occurred prior to this necrosis. We found that expression of the pathogenesis-related gene PR-1 was induced by Cgk1 infection, and also by infection with wild type Cg, but only in npr1-2 mutant plants, not in NahG transgenic plants. These results suggested that PR-1 expression is dependent on the salicylic acid signaling pathway, but is independent of NPR1.     

Vacuolar processing enzyme is essential for mycotoxin-induced cell death in Arabidopsis thaliana

Some compatible pathogens secrete toxins to induce host cell death and promote their growth. The toxin-induced cell death is a pathogen strategy for infection. To clarify the executioner of the toxin-induced cell death, we examined a fungal toxin (fumonisin B1 (FB1))-induced cell death of Arabidopsis plants. FB1-induced cell death was accompanied with disruption of vacuolar membrane followed by lesion formation. The features of FB1-induced cell death were completely abolished in the Arabidopsis vacuolar processing enzyme (VPE)-null mutant, which lacks all four VPE genes of the genome. Interestingly, an inhibitor of caspase-1 abolished FB1-induced lesion formation, as did a VPE inhibitor. The VPE-null mutant had no detectable activities of caspase-1 or VPE in the FB1-treated leaves, although wild-type leaves had the caspase-1 and VPE activities, both of which were inhibited by a caspase-1 inhibitor. gammaVPE is the most essential among the four VPE homologues for FB1-induced cell death in Arabidopsis leaves. Recombinant gammaVPE recognized a VPE substrate with Km = 30.3 microm and a caspase-1 substrate with Km = 44.2 microm, which is comparable with the values for mammalian caspase-1. The gammaVPE precursor was self-catalytically converted into the mature form exhibiting caspase-1 activity. These in vivo and in vitro analyses demonstrate that gammaVPE is the proteinase that exhibits a caspase-1 activity. We show that VPE exhibiting a caspase-1 activity is a key molecule in toxin-induced cell death. Our findings suggest that a susceptible response of toxin-induced cell death is caused by the VPE-mediated vacuolar mechanism similar to a resistance response of hypersensitive cell death (Hatsugai, N., Kuroyanagi, M., Yamada, K., Meshi, T., Tsuda, S., Kondo, M., Nishimura, M., and Hara-Nishimura, I. (2004) Science 305, 855-858).     

The natural compound trans-chalcone induces programmed cell death in Arabidopsis thaliana roots

Chalcone (1,3-diphenyl-2-propen-1-one) is an aromatic ketone precursor of important molecules in plants such as flavonoids or anthocyanins. Its phytotoxicity has been demonstrated on different plant species, but to date little is known about the mechanisms of action of this secondary metabolite at plant cellular level. Detailed analysis by light and transmission electron microscopy (TEM) was conducted to examine the root meristems' ultrastructure of control and chalcone-treated Arabidopsis seedlings. Mitochondrial dysfunction was analysed by measuring mitochondrial membrane potential with JC-1 fluorochrome. Finally, acridine orange/ethidium bromide staining was used for the detection of programmed cell death. Microscopy revealed tissue alterations, inhibition of root hair formation and important changes after 7 and 14 d at the chalcone IC(50) value. Chalcone-treated cells showed signs of programmed cell death such as mitochondrial condensation, disruption of organelles and chromatin fragmentation. Acridine orange/ethidium bromide staining confirmed the programmed cell death, which could be induced by the reduction of mitochondrial transmembrane potential (ΔΨ(m)) that was detected after chalcone treatment. These results confirm the phytotoxic activity of chalcone on Arabidopsis seedlings, the alteration of mitochondrial membrane potential and the induction of programmed cell death.     

Preexisting systemic acquired resistance suppresses hypersensitive response-associated cell death in Arabidopsis hrl1 mutant

The hypersensitive response (HR) displayed by resistant plants against invading pathogens is a prominent feature of plant-pathogen interactions. The Arabidopsis hypersensitive response like lesions1 (hrl1) mutant is characterized by heightened defense responses that make it more resistant to virulent pathogens. However, hrl1 suppresses avirulent pathogen-induced HR cell death. Furthermore, the high PR-1 expression observed in hrl1 remains unaltered after avirulent and virulent pathogen infections. The suppressed HR phenotype in hrl1 is observed even when an elicitor is expressed endogenously from an inducible promoter, suggesting that an impaired transfer of avirulent factors is not the reason. Interestingly, the lack of HR phenotype in hrl1 is reversed if the constitutive defense responses are compromised either by a mutation in NON EXPRESSOR OF PR-1 (NPR1) or by depleting salicylic acid due to the expression of the nahG gene. The rescue of HR cell death in hrl1 npr1 and in hrl1 nahG depends on the extent to which the constitutive systemic acquired response (SAR) is compromised. Pretreating Arabidopsis wild-type plants with SAR-inducers, before pathogen infection resulted in a significant decrease in HR cell death. Together, these results demonstrate that the preexisting SAR may serve as one form of negative feedback loop to regulate HR-associated cell death in hrl1 mutant and in the wild-type plants.