A plant DJ-1 homolog is essential for Arabidopsis thaliana chloroplast development

Protein superfamilies can exhibit considerable diversification of function among their members in various organisms. The DJ-1 superfamily is composed of proteins that are principally involved in stress response and are widely distributed in all kingdoms of life. The model flowering plant Arabidopsis thaliana contains three close homologs of animal DJ-1, all of which are tandem duplications of the DJ-1 domain. Consequently, the plant DJ-1 homologs are likely pseudo-dimeric proteins composed of a single polypeptide chain. We report that one A. thaliana DJ-1 homolog (AtDJ1C) is the first DJ-1 homolog in any organism that is required for viability. Homozygous disruption of the AtDJ1C gene results in non-viable, albino seedlings that can be complemented by expression of wild-type or epitope-tagged AtDJ1C. The plastids from these dj1c plants lack thylakoid membranes and granal stacks, indicating that AtDJ1C is required for proper chloroplast development. AtDJ1C is expressed early in leaf development when chloroplasts mature, but is downregulated in older tissue, consistent with a proposed role in plastid development. In addition to its plant-specific function, AtDJ1C is an atypical member of the DJ-1 superfamily that lacks a conserved cysteine residue that is required for the functions of most other superfamily members. The essential role for AtDJ1C in chloroplast maturation expands the known functional diversity of the DJ-1 superfamily and provides the first evidence of a role for specialized DJ-1-like proteins in eukaryotic development.     

A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence

We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2–3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1 mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl₂ which delayed loss of chlorophyll but not that of photochemical efficiency.     

Characterization of a CENP-C homolog in Arabidopsis thaliana

Centromere protein C (CENP-C) is a component of the kinetochore essential for correct segregation of sister chromatids in mammals. In Arabidopsis thaliana, a single-copy gene encoding a protein homologous to CENP-C has been found by homology in the whole-genome sequence. To investigate the CENP-C homolog (AtCENP-C), we cloned cDNAs by RT-PCR and determined its full-length coding sequence. Antibodies against the synthetic peptide for the C-terminal residues of AtCENP-C detected a polypeptide in Arabidopsis cell extracts on western blots. Immunofluorescence labeling with the antibodies and fluorescence in situ hybridization demonstrated clearly that AtCENP-C is present at the centromeric regions throughout the cell cycle.     

A kinetic analysis of hyponastic growth and petiole elongation upon ethylene exposure in Rumex palustris

Background and Aims

Complete submergence is an important stress factor for many terrestrial plants, and a limited number of species have evolved mechanisms to deal with these conditions. Rumex palustris is one such species and manages to outgrow the water, and thus restore contact with the atmosphere, through upward leaf growth (hyponasty) followed by strongly enhanced petiole elongation. These responses are initiated by the gaseous plant hormone ethylene, which accumulates inside plants due to physical entrapment. This study aimed to investigate the kinetics of ethylene-induced leaf hyponasty and petiole elongation.

Methods

Leaf hyponasty and petiole elongation was studied using a computerized digital camera set-up followed by image analyses. Linear variable displacement transducers were used for fine resolution monitoring and measurement of petiole growth rates.

Key Results

We show that submergence-induced hyponastic growth and petiole elongation in R. palustris can be mimicked by exposing plants to ethylene. The petiole elongation response to ethylene is shown to depend on the initial angle of the petiole. When petiole angles were artificially kept at 0°, rather than the natural angle of 35°, ethylene could not induce enhanced petiole elongation. This is very similar to submergence studies and confirms the idea that there are endogenous, angle-dependent signals that influence the petiole elongation response to ethylene.

Conclusions

Our data suggest that submergence and ethylene-induced hyponastic growth and enhanced petiole elongation responses in R. palustris are largely similar. However, there are some differences that may relate to the complexity of the submergence treatment as compared with an ethylene treatment.     

Expression of the cytokinin-induced type-A response regulator gene ARR9 is regulated by the circadian clock in Arabidopsis thaliana

In Arabidopsis thaliana, a set of type-A authentic response regulator (ARR) genes, consisting of 10 homologous members, is induced primarily in response to the phytohormone cytokinin. Among these, we found that the expression of ARR9 is uniquely regulated through the circadian clock in a cytokinin-independent manner. This finding appears to be compatible to the current idea that some ARR genes (namely, ARR3, ARR4, ARR8, and ARR9) are implicated in an additional level of regulation of the circadian clock. Hence, the result of this study provided us with a new insight into the complex molecular mechanisms underlying both the cytokinin signaling and circadian rhythm.     

A distinctly regulated protein repair L-isoaspartylmethyltransferase from Arabidopsis thaliana

Protein-L-isoaspartate (D-aspartate) O-methyltransferases (EC 2.1.1.77) that catalyze the transfer of methyl groups from S-adenosylmethionine to abnormal L-isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely distributed in procaryotes and eucaryotes. These enzymes participate in the repair of spontaneous protein damage by facilitating the conversion of L-isoaspartyl and D-aspartyl residues to normal L-aspartyl residues. In this work, we have identified an L-isoaspartyl methyltransferase activity in Arabidopsis thaliana, a dicotyledonous plant of the mustard family. The highest levels of activity were detected in seeds. Using degenerate oligonucleotides corresponding to two highly conserved amino acid regions shared among the Escherichia coli, wheat, and human enzymes, we isolated and sequenced a full-length genomic clone encoding the A. thaliana methyltransferase. Several methyltransferase cDNAs were also characterized, including ones that would encode full-length polypeptides of 230 amino acid residues. Messenger RNAs for the A. thaliana enzyme were found in a variety of tissues that did not contain significant amounts of active enzyme suggesting the possibility of translational or posttranslational controls on methyltransferase levels. We have identified a putative abscisic acid-response element (ABRE) in the 5-untranslated region of the A. thaliana L-isoaspartyl methyltransferase gene and have shown that the expression of the mRNA is responsive to exogenous abscisic acid (ABA), but not to the environmental stresses of salt or drought. The expression of the A. thaliana enzyme appears to be regulated in a distinct fashion from that seen in wheat or in animal tissues.     

An RNA-binding protein, AtRBP1, is expressed in actively proliferative regions in Arabidopsis thaliana

A cDNA clone, named XF41, that encodes an RNA-binding protein was isolated from Arabidopsis thaliana. The deduced protein, named AtRBP1, contains two conserved consensus sequence-type RNA-binding domains (CS-RBDs) in the N-terminal half, a putative PY motif (a target of a WW domain) in the center, and uncharacterized C-terminal domain. A binding assay demonstrated that the AtRBP1 can bind to single-stranded nucleic acids in vitro. Analysis of localization of the GUS activity of transgenic Arabidopsis thaliana plants that have the chimeric gene containing the upstream sequence of the AtRBP1 gene and GUS gene demonstrated that the AtRBP1 gene is expressed in meristematic tissues such as the vegetative shoot apex and root tips, developing organs such as floral buds and pistils of young flowers, abscission layers of immature siliques and junctions of pedicels. Considering the specificity of the expression, AtRBP1 may be required in the progress of cell proliferation.     

Genomic sequence of a calnexin homolog from Arabidopsis thaliana.

Calnexin is a membrane-bound protein of the ER in animal cells (Wada et al., 1991). It shows considerable similarity to the major calcium-sequestering protein of the ER lumen, calreticulin, with two calcium-binding regions--a high-affinity, low-capacity region in the ER lumen and a low-affinity, high-capacity region in the cytoplasm. The protein is postulated to act as a calcium-regulated chaperone during protein maturation (Ou et al., 1993). We have isolated a genomic sequence showing significant homology to the animal gene over the predicted coding sequence (Table I). A partial cDNA from Zea mays was isolated from an expression library made from 6-d coleoptiles (Clontech, Palo Alto, CA). The library was screened using a monoclonal antibody raised against a small number of microsomal proteins resulting from a partial purification of plasma membrane Ca2+ ATPase (Briars et al., 1988). The partial cDNA showed sequence homology to the calcium-binding region common to calreticulin and calnexin. The fragment was used to screen a genomic library constructed from Arabidopsis thaliana (cv Larasbonerecta), and a 15-kb fragment was isolated and subcloned and the relevant subfragments were sequenced. The coding region contains five introns, two in the N-terminal region and three in the C-terminal region. The predicted amino acid sequence shows a high level of homology with the animal calnexin, although the terminal highly acidic calcium-binding region is shorter. A cDNA for a putative homolog of calnexin was isolated from A. thaliana (cv Columbia) by Huang et al.(1993); our coding sequence shows 85% identity and 92% similarity determined by FASTA (Wisconsin Genetics Computer Group package); however, the differences are greater than would be expected between cultivars of the same species. A Southern blot probed with DNA from the central calcium-binding region shows multiple bands. This, combined with the sequence heterogeneity, suggests that calnexin belongs to a family of related genes.     

Metacaspase activity of Arabidopsis thaliana is regulated by S-nitrosylation of a critical cysteine residue

Nitric oxide (NO) regulates a number of signaling functions in both animals and plants under several physiological and pathophysiological conditions. S-Nitrosylation linking a nitrosothiol on cysteine residues mediates NO signaling functions of a broad spectrum of mammalian proteins, including caspases, the main effectors of apoptosis. Metacaspases are suggested to be the ancestors of metazoan caspases, and plant metacaspases have previously been shown to be genuine cysteine proteases that autoprocess in a manner similar to that of caspases. We show that S-nitrosylation plays a central role in the regulation of the proteolytic activity of Arabidopsis thaliana metacaspase 9 (AtMC9) and hypothesize that this S-nitrosylation affects the cellular processes in which metacaspases are involved. We found that AtMC9 zymogens are S-nitrosylated at their active site cysteines in vivo and that this posttranslational modification suppresses both AtMC9 autoprocessing and proteolytic activity. However, the mature processed form is not prone to NO inhibition due to the presence of a second S-nitrosylation-insensitive cysteine that can replace the S-nitrosylated cysteine residue within the catalytic center of the processed AtMC9. This cysteine is absent in caspases and paracaspases but is conserved in all reported metacaspases.     

An Arabidopsis thaliana root-specific kinase homolog is induced by dehydration, ABA, and NaCl

Genomic and cDNA clones have been isolated for an Arabidopsis thaliana gene, ARSK1, that encodes a protein with structural similarities to serine/threonine kinases. Expression of ARSK1 is root specific and is induced by exposing roots to air during growth or by treatment of roots with ABA or NaCl. ARSK1 gene expression in transgenic plants is confined to cells in the tissues of the root as measured by β-glucuronidase (GUS) expression from an ARSK1 gene promoter—GUS gene construct. Transverse sections of the stained roots further defined the tissue-specificity; high levels of expression in the epidermal, endoepidermal and cortex regions, but no or very little expression in the vascular system. Another feature of the expression pattern of the ARSK1 gene was a gradual increase in the expression level along the root with the highest level of expression in the region closest to the root meristem. These studies suggest that ARSK1 may have a role in the signal transduction pathway of osmotic stress.     

Aphid-induced accumulation of trehalose in Arabidopsis thaliana is systemic and dependent upon aphid density

Trehalose is a disaccharide sugar that is now considered to be widely distributed among higher plants. Trehalose has been attributed a number of roles, including control of basic plant processes, such as photosynthesis, and conferring tolerance to abiotic stresses, such as desiccation and high salinity. Trehalose is also a common storage sugar used by insects. In this study, we used laboratory investigations to examine various aspects of trehalose dynamics in an aphid–host plant system (Arabidopsis and the peach potato aphid, Myzus persicae). Trehalose concentrations were measured by [1-H]-NMR. Myzus persicae reared on Arabidopsis, but not on black mustard or spring cabbage, contained considerable quantities of trehalose (5 % w/w dry matter). In Arabidopsis foliage, feeding by aphids induced a density-dependent accumulation of trehalose up to 5 mg g^?1 dry weight. Leaves that were not challenged directly by aphids also exhibited increased trehalose concentrations, indicating that this accumulation was systemic. Trehalose was measured at high concentrations in the phloem sap of plants challenged by aphids, suggesting that aphid feeding induced the plant to produce significant quantities of trehalose, which moved through the plant and into the aphids via the phloem sap. Trehalose was also excreted in the aphid honeydew. Further work is required to clarify whether this trehalose accumulation in Arabidopsis has a direct role or a signalling function in plant tolerance of, or resistance to, aphid feeding, and if a similar accumulation of this sugar occurs when other species or genotypes of aphids are reared on this host plant.     

A new homeobox-leucine zipper gene from Arabidopsis thaliana

We have isolated a homeobox-containing gene from Arabidopsis thaliana using a degenerate oligonucleotide probe corresponding to the most conserved region of the homeodomain. This strategy has been used previously to isolate homeobox-containing genes from Caenorhabditis, and recently from A. thaliana. The Arabidopsis genes have an unusual structure in that they have a leucine zipper motif adjacent to the carboxy terminal region of the homeo domain, a feature not found in homeobox-containing genes isolated from animals. We report the isolation and primary structure of a new member of this Arabidopsis homeobox-leucine zipper gene family. This new member has the homeodomain and leucine-zipper motif similar to the two genes previously identified, but differs from these genes in the part corresponding to the carboxy terminus of the polypeptide, as well as in size and isoelectric point of the protein.