Fibronectin (FN), a large dimeric glycoprotein, functions primarily as a connecting molecule in the extracellular matrices of tissues by mediating both cell-matrix and matrix-matrix interactions. All members of the FN family are products of a single FN gene; heterogeneity arises from the alternative splicing of at least three regions (IIIB, IIIA, and V) during processing of a common primary transcript. During chick embryonic limb chondrogenesis, FN structure changes from B+ A(+) in precartilage mesenchyme to B+ A(-) in differentiated cartilage, and exon IIIA has been shown to be necessary for the process of mesenchymal cellular condensation, a requisite event that precedes overt expression of chondrocyte phenotype. This study aims to investigate the mechanistic action of the FN isoforms in mesenchymal chondrogenesis and, in particular, to identify the specific cellular function in mesenchymal condensation mediated by the mesenchymal (B+ A(+) ) FN isoform. Full-length cDNAs corresponding to four splice variants (B+ A(+) , B+ A(-) , B- A(+) , B- A(-) ) of FN were constructed, and expressed the corresponding proteins using a baculovirus expression vector system. Cell adhesion assays with purified proteins showed that, although the relative levels of cell attachment were approximately the same, chick limb-bud mesenchymal cells spread up to 40 % less on mesenchymal (B+ A(+) ) FN than on cartilage (B+ A(-) ) FN, (B- A(+) ) FN, or plasma (B- A(-) ) FN. Cellular condensation and chondrogenic differentiation were also promoted in high-density micromass cultures of limb mesenchymal cells plated onto B+ A(+) FN. These observations suggest that the process of mesenchymal condensation is mediated at least in part by the enhanced ability of chondrogenic mesenchymal cells to migrate and aggregate as a consequence of residing in and interacting with mesenchymal FN. Our findings are consistent with and provide a mechanistic basis for previous observations that rounding of limb mesenchymal cells precedes the onset of chondrogenesis.

The rice blast resistance (R) gene Pi-ta mediates gene-for-gene resistance against strains of the fungus Magnaporthe grisea that express avirulent alleles of AVR-Pita. Using a map-based cloning strategy, we cloned Pi-ta, which is linked to the centromere of chromosome 12. Pi-ta encodes a predicted 928-amino acid cytoplasmic receptor with a centrally localized nucleotide binding site. A single-copy gene, Pi-ta shows low constitutive expression in both resistant and susceptible rice. Susceptible rice varieties contain pi-ta(-) alleles encoding predicted proteins that share a single amino acid difference relative to the Pi-ta resistance protein: serine instead of alanine at position 918. Transient expression in rice cells of a Pi-ta(+) R gene together with AVR-Pita(+) induces a resistance response. No resistance response is induced in transient assays that use a naturally occurring pi-ta(-) allele differing only by the serine at position 918. Rice varieties reported to have the linked Pi-ta(2) gene contain Pi-ta plus at least one other R gene, potentially explaining the broadened resistance spectrum of Pi-ta(2) relative to Pi-ta. Molecular cloning of the AVR-Pita and Pi-ta genes will aid in deployment of R genes for effective genetic control of rice blast disease.

Rice expressing the Pi‐ta gene is resistant to strains of the rice blast fungus, Magnaporthe grisea, expressing AVR‐Pita in a gene‐for‐gene relationship. Pi‐ta encodes a putative cytoplasmic receptor with a centrally localized nucleotide‐binding site and leucine‐rich domain (LRD) at the C‐terminus. AVR‐Pita is predicted to encode a metalloprotease with an N‐terminal secretory signal and pro‐protein sequences. AVR‐Pita176 lacks the secretory and pro‐protein sequences. We report here that transient expression of AVR‐Pita176 inside plant cells results in a Pi‐ta‐dependent resistance response. AVR‐Pita176 protein is shown to bind specifically to the LRD of the Pi‐ta protein, both in the yeast two‐hybrid system and in an in vitro binding assay. Single amino acid substitutions in the Pi‐ta LRD or in the AVR‐Pita176 protease motif that result in loss of resistance in the plant also disrupt the physical interaction, both in yeast and in vitro. These data suggest that the AVR‐Pita176 protein binds directly to the Pi‐ta LRD region inside the plant cell to initiate a Pi‐ta‐mediated defense response.

Acetolactate synthase (ALS) is the first committed step of branched-chain amino acid biosynthesis in plants and bacteria. The bacterial holoenzyme has been well characterized and is a tetramer of two identical large subunits (LSUs) of 60 kDa and two identical small subunits (SSUs) ranging in molecular mass from 9 to 17 kDa depending on the isozyme. The enzyme from plants is much less well characterized. Attempts to purify the protein have yielded an enzyme which appears to be an oligomer of LSUs, with the potential existence of a SSU for the plant enzyme remaining a matter of considerable speculation. We report here the discovery of a cDNA clone that encodes a SSU of plant ALS based upon the homology of the encoded peptide with various bacterial ALS SSUs. The plant ALS SSU is more than twice as large as any of its prokaryotic homologues and contains two domains that each encode a full-length copy of the prokaryotic SSU polypeptide. The cDNA clone was used to express Nicotiana plumbaginifolia SSU in Escherichia coli. Mixing a partially purified preparation of this SSU with the LSU of ALS from either N. plumbaginifolia or Arabidopsis thaliana results in both increased specific activity and increased stability of the enzymic activity. These results are consistent with those observed for the bacterial enzyme in similar experiments and represent the first functional demonstration of the existence of a SSU for plant ALS.

Fructans are linear or branched polymers containing a single sucrose and repeating fructose residues. An early model for fructan biosynthesis in higher plants suggested that partial synthesis of the polymer occurred in the cell cytosol. The current model suggests that synthesis requires the interaction of two separate fructosyltransferases located in the vacuole. Tobacco lines containing a chemically induced promoter, directing expression of the Bacillus amyloliquefaciens SacB gene in the present study, provided an opportunity to regulate and target fructan synthesis to the cytosol of transgenic plants. Induced expression of the gene led to rapid destruction of leaf tissue. Amino acid substitution at a highly conserved site (Arg(331)) in the SacB gene reduced the fructosyltransferase efficiency without reducing the invertase activity of the enzyme. Expression of the mutant gene in transgenic tobacco also resulted in leaf damage. However, the appearance of necrotic tissue was greatly delayed. The results suggest that the phenotype is due to accumulation of fructan in the cytosol. Fructan metabolism in the cytosol of potato tubers was also detrimental to development. Tuber size and starch synthesis was significantly reduced in lines containing the untargeted gene. Transgenic tobacco and potato containing the SacB gene offer an opportunity to study the metabolism of fructan and the effect of accumulation on plant cell development.

A series of seven carboxy-terminal deletion mutants of oat phytochrome A were stably expressed in transgenic tobacco to localize phytochrome domains involved in chromophore attachment, spectral integrity, photoreversibility between the red light (Pr)- and far-red light (Pfr)-absorbing forms, dimerization, and biological activity. Amino acids necessary for chromophore attachment in vivo were localized to the amino-terminal 398 residues because mutant proteins this small had covalently bound chromophore. Deletion mutants from the carboxy terminus to residue 653 were spectrally indistinguishable from the full-length chromoprotein. In contrast, further truncation to residue 399 resulted in a chromoprotein with a bleached Pfr absorbance spectrum, Pr and Pfr absorbance maxima shifted toward shorter wavelengths, and reduced Pfr to Pr phototransformation efficiency. Thus, residues between 399 ad 652 are required for spectral integrity but are not essential for chromophore attachment. The sequence(s) between residues 919 and 1093 appears to be necessary for dimerization. Carboxy-terminal mutants containing this region behaved as dimers under nondenaturing conditions in vitro, whereas truncations without this region behaved as monomers. None of the plants expressing high levels of deletion mutants lacking the 35 carboxy-terminal amino acids displayed the light-exaggerated phenotype characteristic of plants expressing biologically active phytochrome A, even when the truncated phytochromes were expressed at levels 6- to 15-fold greater than that effective for the full-length chromoprotein. Collectively, these data show that the phytochrome protein contains several separable carboxy-terminal domains required for structure/function and identify a domain within 35 residues of the carboxy terminus that is critical for the biological activity of the photoreceptor in vivo.

The physiological responses of transgenic tobacco (Nicotiana tabacum L.) plants that express high levels of an introduced oat (Avena sativa L.) phytochrome (phyA) gene to various light treatments are compared with those of wild-type (WT) plants. Seeds, etiolated seedlings, and light-grown plants from a homozygous transgenic tobacco line (9A4) constructed by Keller et al. (EMBO J, 8, 1005–1012, 1989) were treated with red (R), far-red (FR), or white light (WL) with or without supplemental FR light, revealing major perturbations of the normal photobiological responses. White light stimulated germination of both WT and transgenic seed, but addition of FR to the WL treatment suppressed germination. In the WT, all fluence rates tested inhibited germination, but in the transgenics, reduction effluence rate partially relieved germination from the FR-mediated inhibition. It is suggested that the higher absolute levels of the FR-absorbing form of phytochrome (Pfr) in the irradiated transgenics, compared to the WT, may be responsible for the reduced FR-mediated inhibition of germination in the former. Hypocotyl extension of dark-grown seedlings of both WT and transgenic lines was inhibited by continuous R or FR irradiation, typical of the high-irradiance response (HIR). After 2 d of de-etiolation in WL, the WT seedlings had lost the FR-mediated inhibition of hypocotyl extension, whereas it was retained in the transgenics. The FR-mediated inhibition of hypocotyl extension in the transgenic seedlings after de-etiolation may reflect the persistence of an, FR-HIR response mediated by the overexpressed oat PhyA phytochrome. Light-grown WT seedlings exhibited typical shade-avoidance responses when treated with WL supplemented with high levels of FR radiation. Internode and petiole extension rates were markedly increased, and the chlorophyll a∶b ratio decreased, in the low-R: FR treatment. The transgenics, however, showed no increases in extension growth under low-R: FR treatments, and at low fluence rates both internode and petiole extension rates were significantly decreased by low R ∶ FR. Interpretation of these data is difficult. The depression of the chlorophyll a∶b ratio by low R ∶ FR was identical in WT and transgenic plants, indicating that not all shade-avoidance responses of light-grown plants were disrupted by the over-expression of the introduced oat phyA gene. The results are discussed in relation to the proposal that different members of the phytochrome family may have different physiological roles.

During the 30 years since its initial isolation, a great body of information has accumulated concerning the structure of phytochrome, the physiological responses it controls, and the genes whose expression it affects, yet little is known about the molecular mechanisms of phytochrome action. The recent advent of technologies allowing the expression of heterologous phytochrome genes in transgenic plants provide an important new method for research into the mechanisms of phytochrome action (Keller et al., 1989; Boylan and Quail, 1989; Kay et al., 1989). In the first report of this approach, Keller et al. (1989) described the expression of a functional oat phytochrome in tobacco. Transgenic plants expressing the oat protein have a radically altered phenotype characterized by decreased stem elongation, increased leaf chlorophyll content, reduced apical dominance, and delayed leaf senescence. Exploiting this “light-exaggerated” phenotype as an assay, it is now possible to identify and examine domains involved in phytochrome structure and function by in vitro mutagenesis.

The fundamental notion that phytochrome controls plant development through differential regulation of gene expression (Mohr, 1966) is now well supported by direct experimental evidence (Benfey and Chua, 1989; Gilmartin et al., 1990; Kuhlemeier et al., 1987; Nagy et al., 1988; Tobin and Silverthorne, 1985). However, the molecular mechanism by which the photoreceptor transduces its regulatory signal to genes under its control remains unknown. For some time we have approached this question by simultaneously studying the properties of the photoreceptor molecule and the negative autoregulation of phytochrome (phy) genes as a paradigm of phytochrome-regulated gene expression (Colbert, 1988; Lissemore and Quail, 1988; Quail et al., 1987b, 1990). Recent molecular-genetic studies have revealed that phytochrome is encoded by a small family of divergent and differentially regulated genes (Dehesh et al., 1990b; Sharrock and Quail, 1989); have shown that overexpression of the photoreceptor in heterologous, transgenic plants provides a system for directed mutational analysis of functional regions of the polypeptide (Boylan and Quail, 1989; Kay et al., 1989c; Keller et al., 1989); and have begun to provide insight into the cis-regulatory elements and trans-acting factors involved in phy gene transcription (Brace et al., 1989, 1990; Dehesh et al., 1990a).