Showing papers in "Plant Journal in 1994"

Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

Abstract: Summary A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons Calli induced from scutella were very good starting materials A strain of A tumefaciens that carried a so-called ‘super-binary’ vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture

Extensin:repetitive motifs, functional sites, post-translational codes, and phylogeny

Abstract: Homologous hydroxyproline-rich glycoproteins (HRGPs) of the plant extracellular matrix include extensins, repetitive proline-rich proteins (RPRPs), some nodulins, gum arabic glycoprotein (GAGP), arabinogalactan-proteins (AGPs), and chimeric proteins such as potato lectin which contain an extensin module fused to a lectin. The key to the role of HRGPs in cell wall self-assembly and cell extension lies in their chemistry, which is dependent on extensive post-translational modifications (PTMs): hydroxylation, glycosylation, and cross-linking. Repetitive peptide motifs characterize HRGPs. One or more repetitive peptide motifs and their variants, singly or in combination, may constitute functional sites involved in various aspects of cell wall assembly, as follows: (i) X-Hypn including Ser-Hyp4 (arabinosylation site, molecular rigidity, and reptation). (ii) Pro-Hyp-Val-Tyr-Lys and variants (putative intermolecular cross-links, adhesion, cohesion, and possible beta-turns). (iii) Tyr-X-Tyr-Lys (intramolecular isodityrosine [IDT] cross-links increase molecular rigidity and hydrophobicity). (iv) (Glyco)peptide palindromes (centrosymmetric domains: putative self-assembly nucleation sites). (v) Ionic interaction sites (protein-protein and protein-carbohydrate cross-links). (vi) Hyp and Ser glycosylation sites (enhance conformational stability and molecular recognition). (vii) Extensin modules in chimeric proteins (e.g. solanaceous lectins). Rules for the post-translational modifications are emerging: (i) Hydroxylation of proline residues may depend on multiple, sequence-specific prolyl hydroxylases rather than on a single (polyproline-II) conformation-dependent enzyme. Furthermore, Lys-Pro, Tyr-Pro, and Phe-Pro are not hydroxylated, while Pro-Val is always. (ii) Contiguity of Hyp residues probably determines the extent of Hyp glycosylation, blocks of tetrahydroxyproline (Hyp4) being the most highly arabinosylated, while single non-contiguous Hyp residues are rarely arabinosylated, although they are likely attachment sites for the larger arabinogalactan substituents of gum arabic glycoprotein and arabinogalactan-proteins. (iii) While intramolecular cross-links involve IDT, unidentified intermolecular cross-links most likely involve the Val-Tyr-Lys motif (perhaps also Val-Lys-Pro-Tyr-His-Pro), probably as an adduct between Tyr and Lys catalyzed in vitro by a pI 4.6 extensin cross-linking peroxidase. Thus, we can classify HRGPs functionally as either cross-linking or non-cross-linking, i.e. CL- or NCL-extensins. Their protistan origin obscures the phylogenetic affinities of a single extensin-HRGP family due to their sequence divergence. We propose a phylogenetic series ranging from the minimally glycosylated basic RPRPs to the highly glycosylated acidic AGPs. Furthermore, based on similarities between dicots and gymnosperm extensins, and their marked difference from graminaceous monocot extensins, graminaceous monocot and dicot lines may have diverged as early as the progymnosperms.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of pea cytosolic ascorbate peroxidase and other antioxidant enzymes during the progression of drought stress and following recovery from drought.

Abstract: Summary The molecular mechanism underlying the regulation of the expression of Apxl, the gene encoding cytosolic ascorbate peroxidase, as well as other antioxidant enzymes, was studied during the progression of drought stress and following recovery from drought. Increase in steady-state transcript levels of the cytosolic isozymes of ascorbate peroxidase and Cu/Zn-superoxide dismutase paralleled the increase in stomatal resistance during drought stress, but was even more dramatically enhanced following recovery from drought. Cytosolic Cu/Zn-superoxide dismutase and ascorbate peroxidase and chloroplastic Cu/Zn-superoxide dismutase protein and activity increased during drought stress and following recovery. In contrast, catalase activity increased during drought stress but returned to normal levels following recovery. During recovery from drought stress, cytosolic ascorbate peroxidase expression was regulated post-transcriptionally at the level of protein synthesis. The transcription rate of the Apxl gene, as determined by nuclear run-on assay, increased during drought stress and at 10 h following rewatering.

Mutations at two new Arabidopsis ABA response loci are similar to the abi3 mutations

Abstract: Summary Two new Arabidopsis loci involved in ABA response, ABA-insensitive (ABI)4 and ABI5, have been identified by mutation. The abi4 and abi5 mutants were characterized in terms of ABA sensitivity of seed germination, dormancy, seed-specific gene expression and stomatal regulation. Their phenotypes are similar to mutations in ABI3, which is thought to encode a seed-specific transcriptional activator. Analysis of double mutants combining abi4 and abi5 mutations with abi1, abi2 or abi3 mutations also suggests that ABI4 and ABI5 act in the same transduction pathway as ABI3.

Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression.

Abstract: An improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protoplasts support high levels of expression of the bacterial reporter gene coding for beta-glucuronidase (GUS), under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient expression of GUS activity was monitored spectrophotometrically and reached a maximum between 18 and 48 h after polyethylene glycol (PEG)-mediated DNA uptake. Histochemical staining for GUS activity revealed reproducible transformation frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expression system, the subcellular localization of GUS proteins tagged with different nuclear polypeptides was studied in transfected mesophyll protoplasts, revealing nuclear compartmentalization of the chimeric GUS enzymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin-mediated induction of chloramphenicol acetyl-transferase (CAT) activity when transfected with a transcriptional fusion between the CAT reporter gene and the early auxin-inducible PS-IAA4/5 promoter. Hence, the method allows in vivo analysis of promoter activity and subcellular localization of fusion proteins in a homologous transformation system.

SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein

Abstract: Summary An important, most likely essential step for the long distance transport of sucrose in higher plants is the energy-dependent, uncoupler-sensitive loading into phloem cells via a sucrose-H+ symporter. This paper describes functional expression in Saccharomyces cerevisiae of two cDNAs encoding energy-dependent sucrose transporters from the plasma membrane of Arabidopsis thaliana, SUC1 and SUC2. Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene (PMA1) transport sucrose, and to a lesser extent also maltose, across their plasma membranes in an energy-dependent manner. The KM-values for sucrose transport are 0.50 mM and 0.77 mM, respectively, and transport by both proteins is strongly inhibited by uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or SH-group inhibitors. The VMAX but not the KM-values of sucrose transport depend on the energy status of transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much more active at neutral and slightly acidic pH values than SUC2. The proteins share 78% identical amino acids, their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 putative transmembrane helices. A modified SUC1-His6 cDNA encoding a histidine tag at the SUC1 C-terminus was also expressed in S. cerevisiae. The tagged protein is fully active and is shown to migrate at an apparent molecular weight of 45 kDa on 10% SDS—polyacrylamide gels.

Fertile transgenic wheat from microprojectile bombardment of scutellar tissue.

Abstract: A reproducible transformation system for hexaploid wheat was developed based on particle bombardment of scutellar tissue of immature embryos. Particle bombardment was carried out using a PDS 1000/He gun. Plant material was bombarded with the plasmid pDB1 containing the beta-glucuronidase gene (uidA) under the control of the actin-1 promoter of rice, and the selectable marker gene bar (phosphinothricin acetyltransferase) under the control of the CaMV 35S promoter. Selection was carried out using the herbicide Basta (Glufosinate-ammonium). From a total number of 1050 bombarded immature embryos, in seven independent transformation experiments, 59 plants could be regenerated. Putative transformants were screened for enzyme activity by the histochemical GUS assay using cut leaf material and by spraying the whole plants with an aqueous solution of the herbicide Basta. Twelve regenerants survived Basta spraying and showed GUS-activity. Southern-blot analysis indicated the presence of introduced foreign genes in the genomic DNA of the transformants and both marker genes were present in all plants analysed. To date, four plants have been grown to maturity and set seed. Histochemically stained pollen grains showed a 1:1 segregation of the uidA gene in all plants tested. A 3:1 segregation of the introduced genes was demonstrated by enzyme activity tests and Southern blot analysis of R1 plants.

Transgene‐mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover

Abstract: Summary Co-suppression of the pigmentation gene chalcone synthase (chs) in Petunia hybrida by chs transgenes leads to white or variegated flowers and is characterized by a reduction in steady-state mRNA levels. To determine the level at which suppression occurs different petunia transformants were analysed containing CaMV-35S RNA promoter-driven hybrid genes consisting of the β-glucuronidase gene (uidA) linked to the full-length chsA cDNA, the 5′ half or to the 3′ half. With these transgenes one out of 12–15 primary transformants showed suppression of the transgenes and of the resident chs genes throughout the flower or in sectors. The reduction in steady-state chs mRNA was not the result of a transcriptional inactivation event. As determined by nuclear run-on experiments, transcription of suppressed chs genes was similar to that of non-suppressed genes. This indicates that co-suppression occurs post-transcriptionally. Among individual transformants the transgenes were transcribed at different levels but neither a high nor a low level correlated with a particular degree or pattern of suppression. Surprisingly, even a promoterless chs transgene construct was found to suppress the endogenous chs genes in three out of 15 transformants. It remains, however, unknown whether or not transcription of the transgene locus is required to induce co-suppression. The data suggest that properties of the chs transgene locus other than the expression level are important for inducing co-suppression. The possible role of antisense RNA, which was detected in all transformants, ectopic pairing and the structure of the integrated T-DNAs in the mechanism of the selective increase in chs RNA turnover are discussed.

Manipulation of lignin quality by downregulation of cinnamyl alcohol dehydrogenase

Abstract: The composition of lignin in tobacco stems has been altered by genetic engineering. Antisense expression of sequences encoding cinnamyl alcohol dehydrogenase (CAD), the enzyme catalysing the final step in lignin precursor synthesis, leads to the production of a modified lignin in otherwise normal plants. Although Klason and acetyl bromide lignin determinations show little quantitative change in lignin deposition in CAD antisense plants, a number of qualitative changes have been identified. The lignin is altered in both composition and structure and is more susceptible to chemical extraction. Consistent with a block in CAD activity, antisense plants incorporate less cinnamyl alcohol monomers and more cinnamyl aidehyde monomers into lignin than corresponding control plants. Antisense plants with very low levels of CAD activity also show a novel phenotype with the appearance of a red-brown colour in xylem tissues. A similar phenotype is correlated with altered lignification and improved digestibility in brownmidrib mutants of maize and sorghum. The improved chemical extractability of lignin in CAD antisense plants supports a role for this technology in improving the pulp and paper-making value of forest trees while the similarity with brown-midrib mutants suggests a route to more digestible forage crops.

Water channels in the plant plasma membrane cloned by immunoselection from a mammalian expression system

Abstract: Summary Expression in mammalian COS cells and an efficient microtiter-based strategy for immunoselection was used in a novel approach to identify genes encoding plant membrane proteins. COS cells were transfected with an Arabidopsis thaliana root cDNA library constructed in a bacterial mammalian shuttle vector and screened with an antiserum raised against purified deglycosylated integral plasma membrane proteins from A. thaliana roots. Antibodies directed against a prominent 27 kDa antigen led to the identification of five different genes. They comprised two subfamilies related to the major intrinsic protein (MIP) superfamily and were named plasma membrane intrinsic proteins, PIP1 and PIP2, since the cellular localization of PIP1 and most probably PIP2 proteins in the plasma membrane was independently confirmed by their co-segregation with marker enzymes during aequeous two-phase partitioning. Surprisingly, expression in Xenopus laevis oocytes revealed that all five PIP mRNAs coded for Hg2+-sensitive water transport facilitating activities. There had been no previous evidence of the existence of water channels in the plasma membrane of plant cells and the high diffusional water permeability of the lipid bilayer was considered to be sufficient for water exchange. Nevertheless, Northern and Western analyses showed that the PIP genes are constitutively and possibly even redundantly expressed from the small A. thaliana genome.

The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation

Abstract: Two stimuli that have been associated with nutrient remobilization in plants are phosphate (P(i)) starvation and senescence. Little is known about how the nutrient remobilization machinery is induced at the molecular level, but in the case of P(i) starvation, ribonucleases are considered to play important roles in the remobilization process. Here, the control of two closely related ribonuclease genes of Arabidopsis, RNS1 and RNS3 is investigated. The RNS1 gene is sharply induced during starvation for P(i), an effect specific among the major macronutrients, whereas RNS3 transcript levels remain relatively constant. RNS1 and RNS3 produced in yeast co-migrate with Arabidopsis ribonuclease activities that exhibit the same induction properties as the transcripts in both wild-type plants and the pho1 mutant, which is defective in xylem loading of P(i). In contrast to what occurs during P(i) starvation, both RNS1 and RNS3 are modestly induced during senescence, indicating that the two stimuli could trigger different signal transduction pathways. The characterization of RNS1, in particular, provides an important first step towards elucidating the mechanisms by which plants sense and respond to P(i) limitation, a prominent condition in many soil types.

Self‐fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs

Abstract: A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R0 and R1 progeny plants. Segregation of the PAT activity and herbicide resistance in R1 progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R1 progeny.

The late-flowering phenotype of FRIGIDA and mutations in LUMINIDEPENDENS is suppressed in the Landsberg erecta strain of Arabidopsis

Abstract: Late-flowering phenotype of mutations in the LUMINIDEPENDENS (LD) gene and the late flowering caused by the naturally occurring dominant gene FRIGIDA (FRI) are suppressed in the Landsberg erecta (Ler) strain of Arabidopsis thaliana. This suppression is dependent on a locus on chromosome 5 designated FLC. Of the ecotypes tested only the Ler strain contains the suppressor allele of FLC; ld mutations and FRI cause late flowering in the other genetic backgrounds. The allele at FLC also has a moderate effect on flowering time in the absence of FRI or ld mutations. The flowering time effects of FLC are gene dosage dependent.

Real-time imaging of phloem unloading in the root tip of Arabidopsis

Abstract: Summary Confocal laser scanning microscopy (CLSM) has been used to image phloem transport and unloading in the root tip of Arabidopsis. The fluorescent probe 5(6) carboxyfluorescein (CF) was ester loaded into a single cotyledon and the entire seedling placed within an observation chamber under the microscope. Translocation of CF to the root tip was rapid, followed by unloading into discrete concentric files of cells. The position of the prominent unloading ‘zone’ corresponded precisely with that of the two protophloem files of sieve elements, demonstrating a functional role of these cells in symplastic sieve-element unloading. Symplastic transport following unloading was confined to the elongating zone of the root with little basipetal transport to more mature cells. Following photobleaching of the unloading zone, phloem transport was restored immediately into the protophloem sieve elements, followed rapidly by lateral, symplastic sieve-element unloading. The results demonstrate that phloem transport processes can now be imaged in real time, and non-invasively, within an intact plant system.

A chimeric transactivator allows tetracycline-responsive gene expression in whole plants

Abstract: Summary The chimeric transcriptional activator tTA, a fusion between the Tn 10 encoded Tet repressor and the activation domain of the Herpes simplex virion protein VP16, was stably expressed in transgenic tobacco plants. It stimulates transcription of the β-glucuronidase (gus) gene from an artificial promoter consisting of 7 tet operators and a TATA-box. Tetracycline, which Interferes with binding of tTA to operator DNA, reduces gus expression over several orders of magnitude. This stringency of regulation suggests that the system can be used to construct transgenic plants encoding a potentially lethal gene product. Furthermore, the specific and fast inactivation of tTA allows study of the stability of RNAs and proteins.

Spatial patterns of expression of flavonoid/isoflavonoid pathway genes during interactions between roots of Medicago truncatula and the mycorrhizal fungus Glomus versiforme

Abstract: Summary A combination of Northern blot analysis and in situ hybridization was used to measure and localize changes in the levels of transcripts encoding five enzymes of phenylpropanoid/flavonoid/isoflavonoid metabolism in Medicago truncatula roots following colonization with the mycorrhizal fungus Glomus versiforme. In colonized roots, elevated levels of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) transcripts were detected specifically in the cortical cells containing arbuscules. This localization was discrete, and elevated levels of transcripts were not observed in adjacent, non-colonized cells. In contrast, isoflavone reductase (IFR) transcripts, which were detected at relatively high levels in the cortical cells of non-colonized roots, were almost undetectable in the colonized cortical cells containing arbuscules and were also lowered in adjacent cells. The expression of chalcone isomerase (CHI) and isoliquiritigenin 2′-O-methyltransferase (ChalOMT) was unaffected by colonization, and the tissue-specific patterns of gene expression were the same as in non-colonized roots. It is concluded that the establishment of the mycorrhizal interaction results in cell type-specific differential expression of genes of phenylpropanoid/flavonoid/isoflavonoid biosynthesis, which may be causally related to arbuscule development.

Methyl jasmonate vapor increases the developmentally controlled synthesis of alkaloids in Catharanthus and Cinchona seedlings

Abstract: Summary Shortly after germination, alkaloids are rapidly synthesized in seedlings of both Catharanthus roseus L.G. Don and Cinchona ledgeriana Moens. The effect of low-level, atmospheric methyl jasmonate on this developmentally controlled process was studied. In both species, about 1 p.p.m. of methyl jasmonate vapor significantly enhanced alkaloid synthesis during germination, resulting in a doubling of alkaloid content in seedlings. Treatment with methyl jasmonate resulted in increased allocation of alkaloid precursors and in enhanced enzyme activities in alkaloid biosynthesis. The ability of methyl jasmonate to increase alkaloid biosynthesis decreased with age of the seedlings. Susceptibility of the process to methyl jasmonate was confined to a narrow time interval where the developmentally regulated onset of alkaloid synthesis occurred. When methyl jasmonate was applied at later developmental stages, its ability to enhance alkaloid content in the seedlings declined sharply.

LAT52 protein is essential for tomato pollen development: pollen expressing antisense LAT52 RNA hydrates and germinates abnormally and cannot achieve fertilization

Abstract: The LAT52 gene of tomato is expressed in a pollen-specific manner. It is shown that LAT52 encodes a heat-stable, glycosylated protein that traverses the secretory pathway when expressed in a baculovirus expression system. The LAT52 protein shows some similarity with Kunitz trypsin inhibitors and with pollen proteins from maize, rice and olive, but the biological function of these pollen proteins is unknown. To test whether the LAT52 protein plays an important role during pollen development, tomato plants were transformed with an antisense LAT52 gene driven by the LAT52 promoter. Because the LAT52 gene is expressed gametophytically, only 50% of the pollen of the primary transformants would be expected to express the antisense construct. Selfprogeny of 19 of the primary transformants showed the predicted 3:1 segregation for a single locus insertion of the linked kanamycin-resistance gene. However, the self-progeny of the other 32 primary transformants showed a 1:1 segregation pattern and could not transmit the linked kanamycin-resistance gene through the male. A subset of these 1:1 segregation class plants was examined in detail. The pollen showed lower levels of LAT52 mRNA and LAT52 protein when compared with wild-type. In vitro, approximately 50% of the pollen grains appear to hydrate abnormally; this anomaly is not present when the same pollen grains are incubated in a medium with higher water potential. In vivo pollination experiments showed that the growth of around 50% of the pollen tubes is arrested in the style. The 3:1 segregation class plants showed no significant differences from untransformed control plants. Taken together, the results show a direct correlation between the reduced expression of LAT52 protein and abnormal pollen function, and suggest that the LAT52 protein plays a role in pollen hydration and/or pollen germination.

hsr203J, a tobacco gene whose activation is rapid, highly localized and specific for incompatible plant/pathogen interactions

Abstract: Summary A novel plant defense gene, hsr203J, whose corresponding mRNA accumulates preferentially during the incompatible interaction of tobacco (Nicotiana tabacum L.) with a pathogenic bacterium, Pseudomonas solanacearum, has been isolated and sequenced. No sequence homology of the putative product of this gene has been found in data bases. Evidence is presented here that the hsr203J gene promoter, when fused to the GUS reporter gene, is selectively expressed in response to the hypersensitive response (HR)-inducing bacteria in tobacco protoplasts and that the sequences responsible for this response are contained within 1.4 kb of the 5′ noncoding region. The temporal and spatial patterns of hsr203J activation in leaves and roots inoculated with P. solanacearum indicate that the hsr 203J promoter exhibits a rapid (3–6 h post-inoculation) and high level of induction only in plant cells inoculated with the HR-inducing bacterial isolate. In addition, this gene promoter which does not respond to various stress conditions and is only very weakly induced during compatible interactions, is strongly dependent on hrp (hypersensitive response and pathogenicity) genes of P. solanacearum. These data indicate that the hsr 203J gene promoter exhibits new and original characteristics of activation with regard to the plant defense genes studied so far; its spatial and temporal program of activation together with its specific induction during the HR underline the importance of this gene as a molecular tool for studying the establishment and regulation of the HR.

A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity

Abstract: Summary A novel chitinese gene of tobacco was isolated and characterized by DNA sequence analysis of a genomic clone and a cDNA clone. Comparative sequence analysis of both clones showed an identity of 94%. The proteins encoded by these sequences do not correspond to any of the previously characterized plant chitinases of classes I-IV and are designated as class V chitinaeas. Comparison of the chitinese class V paptide sequence with sequences in the Swiss Protein databank revealed significant sequence similarity with bacterial exo-chitinases from Bacillus circulans, Serratla marcescens and Streptomyces plicatus. It was demonstrated that class V chitinase gene expression is Induced after treatment of tobacco with different forms of stress, like TMV-infection, ethylene treatment, wounding or ultraviolet irradiation. Two related chitinsse class V proteins of 41 and 43 kDa were purified from Samsun NN tobacco leaves inoculated with tobacco mosaic virus. The proteins were purified by Chelating Suparose chromatography and gel filtration. In vitro assays demonstrated that class V chitinaeas have endo-chitinase activity and exhibit antifungal activity toward Trichoderma viride and Alternaria radicina. In addition, it was shown that class V chitlnase acts synergistically with tobacco class I 1~-l,3-glucanase agalnat Fusarlum solani germlings.

Characterization of two cDNAs that encode MAP kinase homologues in Arabidopsis thaliana and analysis of the possible role of auxin in activating such kinase activities in cultured cells.

Abstract: Summary Two cDNA clones, cATMPK1 and CATMPK2, encoding MAP kinases (mitogen-activated protein kinases) have been cloned from Arabidopsis thaliana and their nucleotide sequences have been determined. Putative proteins encoded by ATMPK1 and ATMPK2 genes, designated ATMPK1 and ATMPK2, contain 370 and 376 amino acid residues, respectively, and are 88.7% identical at the amino acid sequence level. ATMPK1 and ATMPK2 exhibit significant similarity to rat ERK2 (49%) and Xenopus MAP kinase (50%). The amino acid residues corresponding to the sites of phosphorylation (Thr-Glu-Tyr) that are involved in the activation of MAP kinases are conserved in ATMPK1 and ATMPK2. Northern blot analysis indicates that the ATMPK1 and ATMPK2 mRNAs are significantly present in all the organs except seeds. Genomic Southern blot analysis suggests that there are a few additional genes that are related to ATMPK1 and ATMPK2 in the Arabidopsis genome. Purified Xenopus MAP kinase kinase (MAPK kinase) phosphorylates ATMPK1 and ATMPK2 proteins that have been expressed in Escherichia coli, activating these enzymes. A rapid and transient activation of 46-kDa protein kinase activity that phosphorylated myelin basic protein (MBP) was detected when auxinstarved tobacco BY-2 cells were treated with synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). Protein kinase activities which phosphorylated the recombinant ATMPK2 protein also increased rapidly after auxin treatment in the auxin-starved BY-2 cells. These results suggest that auxin may function as an activator of plant MAP kinase homologues, as do various mitogens in animal systems.

Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene

Abstract: The expression of genes encoding patatin, a major tuber protein, is highly tissue-specific but is also modulated by exogenous sucrose. The patterns of transcription observed in potato plants could be due to mechanisms conferring tuber-specificity or they could reflect the concentrations of sucrose found in different tissues. To distinguish between these possibilities, a detailed examination was made of the function of a region of the promoter previously implicated in conferring tissue-specific and sucrose-inducible expression. Internal deletions of this region revealed three separate functional domains regulating expression. The B repeat region acted as a positive activator of transcription in the tuber and was also responsible for a degree of sucrose-inducibility. The distal region of the A repeat repressed transcription in leaf and tuber tissue, while the proximal region of the A repeat was able to confer sucrose-responsiveness. Each of these regions specifically bound nuclear proteins which may be putative transcription factors involved in conferring these responses. The region found to confer sucrose-inducible expression was conserved among some other genes that are also regulated by exogenous sucrose.

Biologically induced systemic acquired resistance in Arabidopsis thaliana

Abstract: Summary Local infection with a necrotizing pathogen can render plants resistant to subsequent infection by normally virulent pathogens. A system for biological induction of such systemic acquired resistance (SAR) in Arabidopsis thaliana is reported. When plants were immunized by local inoculation of a single leaf with avirulent Pseudomonas syringae pv. tomato (Pst) carrying the avrRpt2 avirulence gene, after 2 days other leaves became resistant, as measured symptomatically and by in planta bacterial growth, to challenge with a virulent Pst strain lacking this avirulence gene. Resistance was systemic and protected the plants against infection by other virulent pathogens including P. syringae pv. maculicola. Low-dose inoculation induced a strong SAR and double immunizations did not increase the level of protection indicating that the response of only a few cells to the immunizing bacteria is required. SAR was not induced by the virulent strain of Pst lacking avrRpt2. However, experiments with the Arabidopsis RPS2 disease resistance gene mutant rps2-201, which does not exhibit a local hypersensitive response to Pst carrying the corresponding avirulence gene avrRpt2, indicate that a hypersensitive response contributes to, but is not essential for, the induction of SAR. Thus, avrRpt2 activates either a branching signal pathway or separate parallel pathways for induction of localized hypersensitive resistance and SAR, with downstream potentiation of the systemic response by the local response. Using this system for the biological induction of SAR in Arabidopsis, it should be possible to dissect the molecular genetics of SAR by the isolation of mutants affected in the production, transmission, perception and transduction of the systemic signal(s).

Co‐suppression of the petunia homeotic gene fbp2 affects the identity of the generative meristem

Abstract: Summary The function of the petunia MADS box gene fbp2 in the control of floral development has been investigated. Inhibition of fbp2 expression in transgenic plants by a co-suppression approach resulted in the development of highly aberrant flowers with modified whorl two, three and four organs. This mutant flower phenotype inherited as a single Mendelian trait. The flowers possess a green corolla which is reduced in size. Furthermore, the stamens are replaced by green petaloid structures and the inner gynoecial whorl is dramatically reduced. No ovules or placenta are formed and instead two new inflorescences developed in the axils of the carpels. These homeotic transformations are accompanied by a complete down-regulation of the petunia MADS box gene fbp6 which is highly homologous to the Arabidopsis and Antirrhinum genes agamous (ag) and plena (ple). In contrast to this, two other petunia MADS box genes, exclusively expressed in whorls two and three, are still transcribed. Our results indicate that the fbp2 gene belongs to a new class of morphogenesis genes involved in the determination of the central part of the generative meristem.

Role of cytosolic free calcium in the reorientation of pollen tube growth

Abstract: Summary Growing pollen tubes of Agapanthus umbellatus exhibited a tip-to-base gradient in cytosolic free calcium ([Ca2+]c). Although this gradient is believed to be involved in pollen tube growth, its role in specifying reorientation is unknown. The direction of pollen tube growth could be modified by iontophoretic micro-injection, electrical fields (EFs) or photolysis of caged Ca2+. Iontophoretic injection resulted in a temporary cessation of growth, an increase in [Ca2+]c and, upon recovery, reoriented growth. Weak EFs also elevated [Ca2+]c, reduced growth rates and resulted in the reorientation of pollen tubes towards the cathode. Treatment with very low concentrations of the Ca2+-channel blocker lanthanum chloride, prior to exposure to an EF, inhibited both the increase in [Ca2+]c and reorientation whilst only slightly affecting growth rates. The responses of growth inhibition and reorientation were mimicked when [Ca2+]c was artificially elevated by photoactivating caged Ca2+ (Nitr-5). Our data suggest that [Ca2+]c is part of a transduction mechanism which enables growing pollen tubes to successfully reorient to directional signals in the style and thus accomplish eventual fertilization of the egg.

Production of Fertile Transgenic Maize Plants by Silicon Carbide Whisker-Mediated Transformation

Abstract: A simple and inexpensive system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed using silicon carbide whiskers to deliver plasmid DNA carrying the bacterial bar and uidA (gus) genes. Transformed cells were selected on medium containing the herbicide bialaphos. Integration of the bar gene and activity of the enzyme phosphinothricin acetyl transferase (PAT) were confirmed in all bialaphos-resistant callus lines analysed. Fertile transgenic maize plants were regenerated. Herbicide spraying of progeny plants revealed that the bar gene was transmitted in a Mendelian fashion.

Xanthophyll biosynthesis in chromoplasts: isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6-epoxycarotenoid into ketocarotenoid

Abstract: Summary The late steps of carotenoid biosynthesis in plants involve the formation of xanthophyUs. Little is known about the enzymology of these steps. This paper reports the purification to homogeneity of a xantho- phyll biosynthetic enzyme from Capsicum annuum chromoplasts, which catalyzes the conversion of the ubiquitous 5,6.epoxycarotenoids, antheraxanthin and violaxanthin, into capsanthin and capsorubin, respectively. Owing to its bifunctionallty, the name capsanthin-capsorubin synthase is proposed for this new enzyme. The purified enzyme is a monomer with a molecular mass of 50 kDa. Antibodies raised against this enzyme allowed the isolation of a full- length cDNA clone encoding a capsanthin capserubin synthase high molecular weight precursor. The primary deduced structure reveals the presence of a consensus nucleotide binding site. The capeanthin- capsorubin synthase gene is specifically expressed during chromoplast development in fruits accumu- lating ketocarotenoids, but not in mutants impaired in this biosynthetic step. phytofluene desaturase (Hugueney

Toward cataloguing all rice genes: large‐scale sequencing of randomly chosen rice cDNAs from a callus cDNA library

Abstract: A large-scale sequence analysis of rice cDNA was performed for a library from rice callus cultured in a medium containing 1 p.p.m. of 2,4-dichlorophenoxyacetic acid. Random sequencing of 2778 cDNA clones generated 2259 non-redundant expressed sequence tags (ESTs). The strategy of sequencing cDNAs can yield quickly a large number of novel genes. After translation, 690 sequences showed a significant amino acid sequence similarity to sequences already known from PIR. The source of known proteins ranged from bacteria to human. In this report, the non-redundant set of 280 identified ESTs is analyzed in detail.

Identification of nitrate transporter genes in Chlamydomonas reinhardtii

Abstract: Summary The Chlamydomonas reinhardtii nar-2, nar-3, and nar-4 genes, which are within a nitrate-regulated gene cluster containing the nitrate reductase structural gene nit-1, have been related to nitrate transport. Mutant strains defective in nitrate transport and having an active nitrate reductase have been genetically constructed. Their nitrate non-utilizing phenotype has been directly complemented by transformation using the pCO-5 plasmid which carries the nar-2, nar-3, and nar-4 clustered genes. Integration of pCO-5 DNA in the genome of nitrate transport mutants resulted in the expression of these nar transcripts and the recovery of a high affinity nitrate transport activity. Complementation of the nitrate non-utilizing phenotype of the constructed strains was also achieved by co-transformation with plasmids containing nar-2 and nar-3 genes or nar-2 and nar-4, but not with single plasmids containing each individual gene. In addition, DNA sequences of a practically complete cDNA of nar-3 and a partial one of nar-4 have been generated and the deduced amino acid sequences showed a very significant identity with that of the nitrate transporter gene (crnA) from Aspergillus nidulans. These data strongly support the hypothesis that the nitrate transport system in C. reinhardtii contains at least two protein components encoded by the nar-2 and nar-3 genes. The nar-4 gene would produce a protein with a high identity to that of nar-3.