Catalase is encoded by a multigene family in Arabidopsis thaliana (L.) Heynh.

The catalase multigene family in Arabidopsis includes three genes encoding individual subunits that associate to form at least six isozymes that are readily resolved by nondenaturing gel electrophoresis. CAT1 and CAT3 map to chromosome 1, and CAT2 maps to chromosome 4. The nucleotide sequences of the three coding regions are 70 to 72% identical. The amino acid sequences of the three catalase subunits are 75 to 84% identical and 87 to 94% similar, considering conservative substitutions. Both the individual isozymes and the individual subunit mRNAs show distinct patterns of spatial (organ-specific) expression. Six isozymes are detected in flowers and leaves and two are seen in roots. Similarly, mRNA abundance of the three genes varies among organs. All three mRNAs are highly expressed in bolts, and CAT2 and CAT3 are highly expressed in leaves.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Endogenous gibberellin levels influence in-vitro shoot regeneration in Arabidopsis thaliana (L.) Heynh.

The regeneration of shoot buds from callus cells in vitro is an important technique in modern plant genetic manipulation. Whilst it is clear that genetic factors play a major role in determining the ability of callus cells to become organized into regenerating shoot buds, the precise nature of these factors remains unknown. Here we show that callus derived from mutants of Arabidopsis thaliana which have reduced levels of endogenous bioactive gibberellins (GAs), or reduced responsivity to GAs, regenerates shoot buds more readily than does callus derived from wild-type controls. In addition, exogenous GA reduces, and exogenous paclobutrazol (a GA-biosynthesis inhibitor) increases, the frequency of shoot bud regeneration from wild-type callus. These results show that GA levels play a role in regulating shoot bud regeneration from callus, and suggest that variation in endogenous GA levels or responsivity may account for a major component of the genetic variation in shoot bud regeneration frequency described in other species.     

The role of the hexose transporter in the chloroplasts of Arabidopsis thaliana L.

The metabolism of wild-type Arabidopsis thaliana L. and its mutant TC265 were compared in order to reveal the role of the chloroplast glucose transporter. Plants were grown in a 12-h photoperiod. From 20 to 40 days after germination, starch per gram fresh weight of shoot in the mutant was four times that in the wild type. The extent of this difference did not alter during this period. Stereological analysis showed that the chloroplasts in the mutant were larger than those in the wild type; the thylakoids appeared to be distorted by the high starch content. [U-¹⁴C]Glucose and [U-¹⁴C]glycerol were supplied, separately, to excised leaves in the dark. [U-¹⁴C]Glucose was a good precursor of sucrose in the wild type and mutant; [U-¹⁴C]glycerol was a poor precursor of sucrose in both. The distribution of ¹⁴C in the wild type was used to calculate that the net flux was from hexose monophosphates to triose phosphates, not vice versa. During the first 4 h of the night the sugar content (75% sucrose, 20% glucose) of the leaves of the mutant dropped sharply, and at all times during the night it was less than that of the wild-type leaves. This drop in sugar coincided with a decrease in the rate of respiration. The growth rate of the mutant was less than that of the wild type. Addition of sucrose restored the rate of respiration at night and increased the rate of growth. It is argued that a major function of the glucose transporter in Arabidopsis chloroplasts is export of the products of starch breakdown that are destined for sucrose synthesis at night.We thank Professor C.R. Somerville for his generous gift of seed of the Arabidopsis mutant TC265. We are also grateful to Mr B. Chapman for assistance with the preparation of the sections for electron microscopy. R.N.T. thanks the Science and Engineering Research Council for a studentship.     

Control of the appearance of ascorbate peroxidase (EC 1.11.1.11) in mustard seedling cotyledons by phytochrome and photooxidative treatments

In photosynthetic cells the plastidic ascorbate-glutathione pathway is considered the major sequence involved in the elimination of active oxygen species. Ascorbate peroxidase (APO; EC 1.11.1.11) is an essential constituent of this pathway. In the present paper control of the appearance of APO was studied in the cotyledons of mustard (Sinapis alba L.) seedlings with the following results: (i) Two isoforms of APO (APO I, APO II) could be separated by anion-exchange chromatography; APO I is a plastidic protein, while APO II is extraplastidic, very probably cytosolic. (ii) The appearance of APO is regulated by light via phytochrome. This control is observed with both isoforms. Moreover, a strong positive control over APO II appearance (very probably over APO II synthesis) is exerted by photooxidative treatment of the plastids. (iii) Additional synthesis of extraplastidic APO II is induced by a signal created by intraplastidic pigment-photosensitized oxidative stress. The response is obligatorily oxygen-dependent and abolished by quenchers of singlet oxygen such as -tocopherol and p-benzoquinone. (iv) A short-term (4 h) photooxidative treatment suffices to saturate the signal. Signal transduction cannot be abolished or diminished by replacing the plants in non-photooxidizing conditions. Several observations indicate that control of APO synthesis by active oxygen is not an experimental artifact but a natural phenomenon.Abbreviations APO ascorbate-specific peroxidase (EC 1.11.1.11) - D darkness - FPLC fast protein liquid chromatography - FR far-red light (3.5 W · m^–2) - NF Norflurazon - R red light (6.8 W · m^–2) This research was supported by a grant from the Deutsche For-schungsgemeinschaft. B. Th. was the recipient of a stipend from the Studienstiftung des Deutschen Volkes.     

Characterization of eight new members of the calmodulin-like domain protein kinase gene family from Arabidopsis thaliana

A family of calcium-responsive protein kinases is abundant in plant cell extracts but has not been identified in animals and fungi. These enzymes have a unique structure consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. In this report, we present the amino acid sequences for eight new Arabidopsis cDNA clones encoding isoforms of this enzyme. Three isoforms were expressed as fusion proteins in Escherichia coli and exhibited calcium-stimulated protein kinase activity. We propose CPK as the gene designation for this family of enzymes and describe a phylogenetic analysis for all known isoforms.     

Identification of a copper transporter family in Arabidopsis thaliana

Despite copper ions being crucial in proteins participating in plant processes such as electron transport, free-radical elimination and hormone perception and signaling, very little is known about copper inward transport across plant membranes. In this work, a five-member family (COPT1–5) of putative Arabidopsis copper transporters is described. We ascertain the ability of these proteins to functionally complement and transport copper in the corresponding Saccharomyces cerevisiae high-affinity copper transport mutant. The specific expression pattern of the Arabidopsis COPT1–5 mRNA in different tissues was analyzed by RT-PCR. Although all members are ubiquitously expressed, differences in their relative abundance in roots, leaves, stem and flowers have been observed. Moreover, steady-state COPT1 and COPT2 mRNA levels, the members that are most efficacious in complementing the S. cerevisiae high-affinity copper transport mutant, are down-regulated under copper excess, consistent with a role for these proteins in copper transport in Arabidopsis cells.     

Antisense reduction of thylakoidal ascorbate peroxidase in <Emphasis Type="Italic">Arabidopsis</Emphasis> enhances Paraquat-induced photooxidative stress and Nitric Oxide-induced cell death

The production and characterization of Arabidopsis plants containing a transgene in which the Arabidopsis tAPX is inserted in antisense orientation, is described. tAPX activity in these transgenic tAPX plants is around 50% of control level. The tAPX antisense plants are phenotypically indistinguishable from control plants under normal growth conditions; they show, however, enhanced sensitivity to the O₂^–-generating herbicide, Paraquat. Interestingly, the tAPX antisense plants show enhanced symptoms of damage when cell death is triggered through treatment with the nitric oxide-donor, SNP. These results are in accordance with the ones recently obtained with transgenic plants overexpressing tAPX; altogether, they suggest that tAPX, besides the known ROS scavenging role, is also involved in the fine changes of H₂O₂ concentration during signaling events.     

Effect of temperature on ascorbate peroxidase activity and flowering of Arabidopsis thaliana ecotypes under different light conditions

Four Arabidopsis thaliana ecotypes were grown at 14 degrees C and 22 degrees C under two light conditions (300 microE m-2 s-1 or 150 microE m-2 s-1) and the effect of temperature on their growth and flowering time was studied. Flowering occurred within 31 days (experimental period) at 22 degrees C, whereas a decrease in growth temperature resulted in a delay in flowering (63 days) under both light conditions. At 14 degrees C, membrane-bound APX (tAPX) activity decreased and total chlorophyll (Chl) content increased with growth under both light conditions. However, at 22 degrees C, the tAPX activity increased and total Chl content decreased with growth under both light conditions. These results suggest that at 22 degrees C oxidative stress was high under both light conditions and consequently Chl content decreased under stressful conditions or vice versa for all the four A. thaliana ecotypes studied. Under both the temperature and light conditions, soluble APX activity showed an irregular pattern of growth. The increase in tAPX activity, with growth only at 22 degrees C but not at 14 degrees C, suggests increased H2O2 formation in flowering plants at 22 degrees C for all the four A. thaliana ecotypes studied. Before flowering, the tAPX activity showed a significantly negative correlation with flowering time. Higher oxidative stress in the lower-latitude ecotypes might induce earlier flowering than the higher-latitude ecotypes. From these results, we propose a hypothesis that H2O2 is one of the possible factors in flower induction.     

Identification of two cytosolic ascorbate peroxidase cDNAs from soybean leaves and characterization of their products by functional expression in E. coli

Screening of a cDNA library from soybean (Glycine max (L.) Merr. cv. Century) with probes based upon cytosolic ascorbate peroxidase (APx; EC 1.11.1.11) genes identified two full-length clones (SOYAPx1, SOYAPx2) apparently encoding for different soybean leaf cytosolic APxs. The deduced amino acid sequences of the two APx cDNA products differed in 13 of the 250 amino acids. The SOYAPx1 cDNA was identical to the cytosolic APx cDNA previously found in soybean root nodules. Escherichia coli expression systems were developed using both soybean APx cDNAs. Recombinant SOYAPx1 and SOYAPx2 were then utilized to characterize the enzymatic properties of the two APx cDNA products. Received: 10 May 1997 / Accepted: 19 June 1997     

In Arabidopsis thaliana, 1% of the genome codes for a novel protein family unique to plants

In the sequences released by the Arabidopsis Genome Initiative (AGI), we discovered a new and unexpectedly large family of orphan genes (127 genes by 01.08.99), named AtPCMP. The distribution of the AtPCMP genes on the five chromosomes suggests that the genome of Arabidopsis thaliana contains more than 200 genes of this family (1% of the whole genome). The deduced AtPCMP proteins are characterized by a surprising combinatorial organization of sequence motifs. The amino-terminal domain is made of a succession of three conserved motifs which generate an important diversity. These proteins are classified into three subfamilies based on the length and nature of their carboxy-terminal domain constituted by 1–6 motifs. All the motifs characterized have an important level of conservation in both sequence and spacing. A specific signature of this large family is defined. The presence of ESTs in databases and the detection of clones in A. thaliana cDNA libraries indicate that most of the genes of this family are expressed. The absence of similar sequences outside the plant kingdom strongly suggests that this unusually large orphan family is unique to plants. Features, the genesis, the potential function and the evolution of this plant combinatorial and modular protein family are discussed.     

Homologues of wheat ubiquitin-conjugating enzymes - TaUBC1 and TaUBC4 are encoded by small multigene families in Arabidopsis thaliana

Covalent attachment of ubiquitin to other cellular proteins has been implicated in a multitude of diverse physiological processes in eukaryotes including selective protein degradation. This attachment is carried out by a multi-enzyme pathway consisting of three classes of enzymes: ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin-protein ligases (E3s). E2s accept activated ubiquitin from E1 and conjugate it to target proteins with or without the participation of specific E3s. Previously, we have isolated wheat cDNAs encoding 16 and 23 kDa E2s, TaUBC1 and TaUBC4, respectively. TaUBC1 shows structural homology to the yeast RAD6 E2 that is essential for DNA repair whereas TaUBC4 is related to the yeast ScUBC8 E2, both of which effectively conjugate ubiquitin to histones in vitro but as yet are without a known in vivo function. Here, we report the isolation of genomic and cDNA homologues of these genes from Arabidopsis thaliana. In Arabidopsis, both of these E2s are encoded by three member gene families. Members of the AtUBC1 gene family, comprising AtUBC1, 2 and 3, encode 150–152 amino acid proteins that are 83–99% identical to each other and TaUBC1 and contain four introns that are conserved with respect to position. Members of the AtUBC4 gene family, comprising AtUBC4, 5 and 6, encode 187–191 amino acid proteins that are 73–88% identical to each other and TaUBC4 and contain five introns that are conserved with respect to position. In contrast, AtUBC1-3 gene products are only 31–36% identical to those derived from AtUBC4-6. mRNA for each family was detected in Arabidopsis roots, leaves, stems, and flowers indicating that members of each family are expressed in most if not all tissues.