The Arabidopsis FILAMENTOUS FLOWER gene is required for flower formation.

A screen for mutations affecting flower formation was carried out and several filamentous flower (fil) alleles were identified. In fil mutants, floral primordia occasionally give rise to pedicels lacking flowers at their ends. This defect is dramatically enhanced in fil rev double mutants, in which every floral primordium produces a flowerless pedicel. These data suggest that the FIL and REV genes are required for an early step of flower formation, possibly for the establishment of a flower-forming domain within the floral primordium. The FIL gene is also required for establishment of floral meristem identity and for flower development. During flower development, the FIL gene is required for floral organ formation in terms of the correct numbers and positions; correct spatial activity of the AGAMOUS, APETALA3, PISTILLATA and SUPERMAN genes; and floral organ development.     

Zinc release from the CH2C6 zinc finger domain of FILAMENTOUS FLOWER protein from Arabidopsis thaliana induces self-assembly

The FILAMENTOUS FLOWER gene from Arabidopsis thaliana is a member of a gene family whose role is to specify abaxial cell fate in lateral organs. Analysis of the amino-terminal region of the FILAMENTOUS FLOWER protein suggests that seven cysteine residues at positions 14, 26, 30, 33, 54, 56, and 57, and two histidine residues at positions 18 and 24 contribute to a putative zinc finger motif, Cys-X(3)-His-X(5)-His-X-Cys-X(3)-Cys-X(2)-Cys-X(20)-Cys-X-Cys-Cys. Zinc determination experiments revealed that the FILAMENTOUS FLOWER protein binds two zinc ions per molecule. Chemical modification was required to release one zinc ion, whereas the other was released spontaneously or more rapidly in the presence of metallochromic indicator. The loss of a zinc ion and the subsequent structural change of the zinc finger domain were correlated with the multimerization of the FILAMENTOUS FLOWER protein. A cysteine residue at position 56 in the FILAMENTOUS FLOWER protein potentially interferes with zinc ligation within the zinc finger and causes this zinc release. In support of this, substitution of the Cys(56) by alanine suppressed both the zinc release and the multimerization of the FILAMENTOUS FLOWER protein. Deletion analysis showed that the region between positions 45 and 107 functions in the intermolecular contacts between FILAMENTOUS FLOWER proteins. This region corresponds to the carboxyl-terminal half of the zinc finger domain and the following hydrophobic region containing two putative alpha-helices. Our results suggest that the FILAMENTOUS FLOWER protein forms a range of different conformers. This attribute may lead to a greater degree of functional flexibility that is central to its role as an abaxial cell fate regulator.     

Genetic Control by Arabidopsis Genes LEUNIG and FILAMENTOUS FLOWER in Gynoecium Fusion

Arabidopsis gene FILAMENTOUS FLOWER (FIL) has been demonstrated to control the formation and development of inflorescence and floral meristems. This includes an early step in the establishment of a flower-forming domain within the floral primordium and the establishment of floral meristem identity. Another Arabidopsis gene LEUNIG (LUG) was previously found to specify the identity of the floral organ and control gynoecium fusion. In this paper, we describe floral phenotypes of a newly isolated fil allele, fil-21, and the phenotypic comparison of gynoecia between the fil-21 single mutant and fil-21 lug-101 double mutant. The gynoecium of fil-21 displays a well-fused structure, while that of the strong lug allele, lug-101, is unfused except at the gynoecium apex. However, gynoecia are markedly affected in the fil-21 lug-101 double mutant, being unfused. In late-appearing flowers of the double mutant, the gynoecia can even separate completely into several parts. These results suggest that LUG and FIL have a functional domain that is partially redundant in flower development, and synergistically regulate the gynoecium fusion. Received 18 June 2001/ Accepted in revised form 1 October 2001     

Sequence-specific binding property of Arabidopsis thaliana telomeric DNA binding protein 1 (AtTBP1)

We have identified an Arabidopsis thaliana cDNA, designated as AtTBP1, encoding a protein with a predicted size of 70.6 kDa that specifically binds to the plant telomeric repeat sequence TTTAGGG. AtTBP1 is present as a single-copy gene in Arabidopsis genome and is expressed ubiquitously in various organs. AtTBP1 has a single Myb telomeric DNA binding domain at the C-terminus and an extensive homology with other known telomere-binding proteins. The isolated C-terminus of AtTBP1 is capable of sequence-specific DNA binding to plant duplex telomeric DNA. These results suggest that AtTBP1 may play important roles in plant telomere function in vivo.     

Interaction of the PHD-finger homeodomain protein HAT3.1 from Arabidopsis thaliana with DNA. Specific DNA binding by a homeodomain with histidine at position 51

HAT3.1 is a member of the PHD-finger homeodomain protein family. The HAT3.1 homeodomain is highly divergent in sequence even at positions that are almost invariable among homeodomains. In this work, we have applied the random oligonucleotide selection technique to investigate if the HAT3.1 homeodomain is able to recognize specific DNA sequences. Analysis of the selected molecules followed by hydroxyl radical footprinting experiments and yeast one-hybrid assays indicated that HAT3.1 shows a preference for the sequence T(A/G)(A/C)ACCA, different from those bound by other homeodomains. Binding was dependent on homeodomain residues located at positions 47, 50, 51, and 54, the same positions that usually participate in DNA binding in most homeodomains. The study of the interaction of mutants at these positions with DNA carrying nucleotide changes at specific sites suggested that H51 and K50 most likely interact with nucleotides 2 to 4 and 5 to 6, respectively, while W54 would establish contacts with position 4. The presence of H51 and W54 represents an innovation among homeodomain structures. The fact that the HAT3.1 homeodomain is able to interact with specific DNA sequences is evidence of the inherent plasticity of the homeodomain as a DNA binding unit.     

Arabidopsis thaliana TERMINAL FLOWER2 is involved in light-controlled signalling during seedling photomorphogenesis

Plants respond to changes in the environment by altering their growth pattern. Light is one of the most important environmental cues and affects plants throughout the life cycle. It is perceived by photoreceptors such as phytochromes that absorb light of red and far-red wavelengths and control, for example, seedling de-etiolation, chlorophyll biosynthesis and shade avoidance response. We report that the terminal flower2 (tfl2) mutant, carrying a mutation in the Arabidopsis thaliana HETEROCHROMATIN PROTEIN1 homolog, functions in negative regulation of phytochrome dependent light signalling. tfl2 shows defects in both hypocotyl elongation and shade avoidance response. Double mutant analysis indicates that mutants of the red/far-red light absorbing phytochrome family of plant photoreceptors, phyA and phyB, are epistatic to tfl2 in far-red and red light, respectively. An overlap between genes regulated by light and by auxin has earlier been reported and, in tfl2 plants light-dependent auxin-regulated genes are misexpressed. Further, we show that TFL2 binds to IAA5 and IAA19 suggesting that TFL2 might be involved in regulation of phytochrome-mediated light responses through auxin action.     

AtFim1 is an actin filament crosslinking protein from Arabidopsis thaliana

ATFIM1 is a widely expressed gene in Arabidopsis thaliana that encodes a putative actin filament-crosslinking protein, AtFim1, belonging to the fimbrin/plastin class of actin-binding proteins. In this report we have used bacterially expressed AtFim1 and actin isolated from Zea mays pollen to demonstrate that AtFim1 functions as an actin filament-crosslinking protein. AtFim1 binds pollen actin filaments (F-actin) in a calcium-independent manner, with an average dissociation constant (Kd) of 0.55+/-0.21 microM and with a stoichiometry at saturation of 1:4 (mol AtFim1 : mol actin monomer). AtFim1 also crosslinks pollen F-actin by a calcium-independent mechanism, in contrast to crosslinking of plant actin by human T-plastin, a known calcium-sensitive actin-crosslinking protein. When micro-injected at high concentration into living Tradescantia virginiana stamen hair cells, AtFim1 caused cessation of both cytoplasmic streaming and transvacuolar strand dynamics within 2-4 min. Using the 'nuclear displacement assay' as a measure of the integrity of the actin cytoskeleton in living stamen hair cells, we demonstrated that AtFim1 protects actin filaments in these cells from Z. mays profilin (ZmPRO5)-induced depolymerization, in a dose-dependent manner. The apparent ability of AtFim1 to protect actin filaments in vivo from profilin-mediated depolymerization was confirmed by in vitro sedimentation assays. Our results indicate that AtFim1 is a calcium-independent, actin filament-crosslinking protein that interacts with the actin cytoskeleton in living plant cells.     

Two discrete cis elements control the Abaxial side-specific expression of the FILAMENTOUS FLOWER gene in Arabidopsis

Our previous studies showed that a member of the YABBY gene family, FILAMENTOUS FLOWER (FIL), plays a role in specifying the abaxial side tissues in the development of lateral organs such as cotyledons, leaves, young flower buds, and flower organs. We examined the expression pattern of FIL and found a temporal change of expression domains in the developmental process of the floral meristem. We also examined the cis control regions by constructing a series of transgenic plants that carry green fluorescent protein under the control of the FIL promoter with several types of deletions, base changes, and tandem repeats and showed that the unique expression pattern is dependent on at least two cis-acting elements in the 5' regulatory region. One element proximal to the FIL gene would be responsible for the expression of both the abaxial and adaxial sides, and the other element of the 12-bp sequence would work to repress expression on the adaxial side.     

Single-stranded nucleic acid binding in Arabidopsis thaliana cold shock protein is cold shock domain dependent

Cold shock proteins (CSPs) are ancient nucleic acid-binding proteins and well conserved from bacteria to animals as well as plants. In prokaryotes, CSPs possess a single cold shock domain (CSD) while animal CSPs, flanked by N- and C-terminal domains, are commonly named Y-box proteins. Interestingly, the plants CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. The CSPs have been shown to play important role in development and stress adaptation in various plant species. The objective of this study was to find out the possible nucleic acid-binding affinities of whole CSP as well as independent domains, so that role of each individual domain may be revealed in Arabidopsis thaliana, the model plant species. The structure of CSP 3 protein from A. thaliana was modeled by homology-based approach and docking was done with different nucleic acid types.     

DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli.

To study the phosphorylation of one of the G-box binding factors from Arabidopsis (GBF1), we have obtained large amounts of this protein by expression in Escherichia coli. Bacterial GBF1 was shown to be phosphorylated very efficiently by nuclear extracts from broccoli. The phosphorylation activity was partially purified by chromatography on heparin-Sepharose and DEAE-cellulose and was characterized. It showed the essential features of casein kinase II activity: utilization of GTP in addition to ATP as a phosphate donor, strong inhibition by heparin, preference for acidic protein substrates, salt-induced binding to phosphocellulose, and salt-dependent deaggregation. The very low Km value for GBF1 (220 nM compared to approximately 10 microM for casein) was in the range observed for identified physiological substrates of casein kinase II. Phosphorylation of GBF1 resulted in stimulation of the G-box binding activity and formation of a slower migrating protein-DNA complex. The conditions of this stimulatory reaction fully corresponded to the properties of casein kinase II, in particular its dependence on the known phosphate donors. The DNA binding activity of the endogenous plant GBF was shown to be reduced by treatment with calf alkaline phosphatase; this reduction was diminished by addition of fluoride and phosphate or incubation in the presence of casein kinase II and ATP.     

Arabidopsis thaliana telomeric DNA-binding protein 1 is required for telomere length homeostasis and its Myb-extension domain stabilizes plant telomeric DNA binding

Telomeres are specific protein–DNA complexes that protect the ends of eukaryotic chromosomes from fusion and degradation and are maintained by a specialized mechanism exerted by telomerase and telomere-binding proteins (TBPs), which are evolutionarily conserved. AtTBP1 is an Arabidopsis thaliana protein that binds plant telomeric DNA in vitro. Here, we demonstrated that lack of AtTBP1 results in a deregulation of telomere length control, with mutant telomeres expanding steadily by the fourth generation. DNA-binding studies with mutant AtTBP1 proteins showed that the Myb-extension domain of AtTBP1 is required for binding to plant telomeric DNA. Our results suggest that AtTBP1 is involved in the telomere length mechanism in A. thaliana and that the Myb-extension domain of AtTBP1 may stabilize plant telomeric DNA binding.     

乙酰化修饰降低Ku蛋白的DNA结合活性

【目的】研究乙酰化修饰对Ku蛋白活性的影响。【方法】利用耻垢分枝杆菌为表达菌株,转入Ku蛋白表达质粒,纯化具有乙酰化修饰的Ku蛋白和无乙酰化的Ku蛋白突变体,比较两类蛋白的生化活性;分析氧化压力和酸性环境下耻垢分枝杆菌细胞内Ku蛋白乙酰化水平的变化。【结果】Ku蛋白过量表达的耻垢分枝杆菌比转入空质粒的对照菌株生长缓慢;乙酰化Ku蛋白比未发生乙酰化Ku蛋白修复断裂DNA的活性降低、DNA结合活性降低;氧化压力和酸性压力环境下,耻垢分枝杆菌细胞内Ku蛋白数量降低,乙酰化Ku蛋白数量变化不大。【结论】乙酰化修饰能够调节Ku蛋白的DNA结合活性,从而调节非同源末端连接修复系统的活性;Ku蛋白乙酰化程度升高是耻垢分枝杆菌对不良生长环境的反应。     

Structural comparison of oxidized and reduced FKBP13 from Arabidopsis thaliana

AtFKBP13, an immunophilin in the chloroplast thylakoid lumen, participates in redox-regulatory processes via a pair of conserved disulfide bonds that are present at the N- and C-termini of the protein. Characterization of this protein by structural and biochemical analysis has revealed a novel mechanism of redox regulation in the thylakoid lumen. The protein is active in its oxidized form but is inactivated after reduction by the thioredoxin system. This is in sharp contrast with the regulation of biosynthetic enzymes in the stroma of the chloroplast, where reduction of enzymes by thioredoxin activates their function. To understand how the reduced form of AtFKBP13 is stabilized and how reduction of the cysteine residues affects the molecular properties of the enzyme, we determined the crystal structure of reduced AtFKBP13 at 1.88 A. Comparison of the reduced structure and the oxidized form that we published earlier shows rearrangements in redox site regions, readjustments of hydrogen-bonding interactions and the secondary structure of the active site residues 50-53, and reduced accessibility of the catalytic residues involved in the peptidyl proline isomerase (PPIase) activity of this enzyme. We propose that redox-linked changes in the secondary structure of the PPIase domain are responsible for significant functional differences in this protein in the reduced and oxidized states.     

Isolation of mutants of Arabidopsis thaliana in which accumulation of tobacco mosaic virus coat protein is reduced to low levels.

Summary We have found that Arahidopsis thaliana is susceptible to infection with a crucifer strain of tobacco mosaic virus (TMV-Cg); the coat protein of TMV-Cg accumulated to a high level in uninoculated rosette leaves several days after inoculation. As a first step in the search for host-coded factors that are involved in virus multiplication, we isolated mutants of A. thaliana in which the accumulation of TMV-Cg coat protein was reduced to low levels. Of 6000 M₂ plants descended from ethyl methanesulfonate-treated seeds, two such lines (PD 114 and PD378) were isolated. Genetic analyses suggested that the PD 114 phenotype was caused by a single nuclear recessive mutation, and that PD114 and PD378 belonged to the same complementation group. The coat protein accumulation of a tomato strain of TMV (TMVL) was also reduced in PD 114 plants compared to that in the wild-type plants. In contrast, PD114 plants infected with turnip crinkle or turnip yellow mosaic viruses, which belong to taxonomic groups other than Tobamovirus, expressed similar levels of these coat proteins as did infected wild-type plants.In this paper, we use the term multiplication (of a virus in a plant) to mean a substantial increase in virus concentration in the uninoculated leaves of the infected plant. Therefore, the efficiency of each process of invasion of the plant by the virus, uncoating, replication and degradation of the virus genome, formation and degradation of the virus particles, and spreading of the virus in the plant will affect the degree of multiplication     

Phosphatidylglycerophosphate synthases from Arabidopsis thaliana.

Two Arabidopsis thaliana genes were shown to encode phosphatidylglycerophosphate synthases (PGPS) of 25.4 and 32.2 kDa, respectively. Apart from their N-terminal regions, the two proteins exhibit high sequence similarity. Functional expression studies in yeast provided evidence that the 25.4 kDa protein is a microsomal PGPS while the 32.2 kDa protein represents a preprotein which can be imported into yeast mitochondria and processed to a mature PGPS. The two isozymes were solubilized and purified as fusion proteins carrying a His tag at their C-terminus. Enzyme assays with both membrane fractions and purified enzyme fractions revealed that the two A. thaliana isozymes have similar properties but differ in their CDP-diacylglycerol species specificity.     

WVD2 is a novel microtubule-associated protein in Arabidopsis thaliana

Arabidopsis WAVE-DAMPENED 2 (WVD2) was identified by forward genetics as an activation-tagged allele that causes plant and organ stockiness and inversion of helical root growth handedness on agar surfaces. Plants with high constitutive expression of WVD2 or other members of the WVD2-LIKE (WDL) gene family have stems and roots that are short and thick, have reduced anisotropic cell elongation, are suppressed in a root-waving phenotype, and have inverted handedness of twisting in hypocotyls and roots compared with wild-type. The wvd2-1 mutant shows aberrantly organized cortical microtubules in peripheral root cap cells as well as reduced branching of trichomes, unicellular leaf structures whose development is regulated by microtubule stability. Orthologs of the WVD2/WDL family are found widely throughout the plant kingdom, but are not similar to non-plant proteins with the exception of a C-terminal domain distantly related to the vertebrate microtubule-associated protein TPX2. in vivo, WVD2 and its closest paralog WDL1 are localized to interphase cortical microtubules in leaves, hypocotyls and roots. Recombinant glutathione-S-transferase:WVD2 or maltose binding protein:WVD2 protein bind to and bundle microtubules in vitro. We speculate that a C-terminal domain of TPX2 has been utilised by the WVD2 family for functions critical to the organization of plant microtubules.     

Plastid DNA polymerases from higher plants, Arabidopsis thaliana

Previously, we described a novel DNA polymerase, designated as OsPolI-like, from rice. The OsPolI-like showed a high degree of sequence homology with the DNA polymerase I of cyanobacteria and was localized in the plastid. Here, we describe two PolI-like polymerases, designated as AtPolI-like A and AtPolI-like B, from Arabidopsis thaliana. In situ hybridization analysis demonstrated expression of both mRNAs in proliferating tissues such as the shoot apical meristem. Analysis of the localizations of GFP fusion proteins showed that AtPolI-like A and AtPolI-like B were localized to plastids. AtPolI-like B expression could be induced by exposure to the mutagen H(2)O(2). These results suggested that AtPolI-like B has a role in the repair of oxidation-induced DNA damage. Our data indicate that higher plants possess two plastid DNA polymerases that are not found in animals and yeasts.