Regulation of splicing by protein phosphorylation.

Most eukaryotic messenger RNAs are transcribed as precursors that necessitate specific and exact processing of intron boundaries. Furthermore, the choice of these boundaries appears to be fluid and adaptive to the rate of transcription and the developmental and physiological state of the cell. A central regulator of splicing reactions and choice are kinases that work through phosphorylation of specific factors like RNA polymerase II, which influences the pace of transcription and of SR splicing factors. While very different in their mechanisms both regulatory pathways will impact on splicing site choice. This chapter summarizes the biology of splicing-related phosphorylation activity, emphasizing plant-specific aspects in relation to the metazoan counterpart.

Organ-specific and light-induced expression of plant genes.

Light plays a pivotal role in the development of plants. The photoregulation of plant genes involves recognition of light quality and quantity by phytochrome and other light receptors. Two gene families, rbcS and Cab, which code for abundant proteins active in photosynthesis, the small subunit of ribulose bisphosphate carboxylase and the chlorophyll a/b binding protein, show a 20-to 50-fold increase in transcript abundance in the light. Analyses in calli and transgenic plants of deletions of the rbcS gene and of chimeric constructions has allowed localization of two regions involved in light-induced transcription. One element is confined to a 33-base pair region surrounding the TATA box. In addition, an enhancer-like element contained within a 240-base pair fragment can confer phytochrome-induced transcription and organ specificity on nonregulated promoters.

Role of ammonia secretion and pH modulation on pathogenicity of Colletotrichum coccodes on tomato fruit.

Colletotrichum coccodes was found to alkalinize the decaying tissue of tomato fruit via accumulation and secretion of ammonia. Alkalinization dynamics caused by ammonia secretion from growing hyphae was examined microscopically using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Values of pH of 7.9 observed in the host tissue close to the hyphal tips declined to pH 6.0 at 10 mm away from the hyphal tip, which was a value that was still higher than that detected in the healthy tissue, pH 4.2. Ammonia accumulation at the infection site depended on the initial environmental pH. Treatments with low (4.0) pH buffer at the infection site resulted in high levels of ammonia secretion and increased virulence of C. coccodes compared with similar treatments with buffer at pH 7.0. Significantly, mutants of C. coccodes defective in nitrogen utilization, nit-, and areA- were impaired in ammonia secretion and showed reduced decay development. The reduced infection rate of nit- mutants could be complemented by adding glutamine at the infection site. Thus, ammonia accumulation is a critical factor contributing to C. coccodes pathogenicity on tomato fruit. The results show that the initial acidic pH of the fruit is conducive to ammonia secretion and the subsequent alkalinization of the infection site, and facilitates fungal virulence and the transformation from the quiescent-biotrophic to active-necrotrophic state.

Calcium Requirement for Ethylene-Dependent Responses.

Ethylene, a gaseous plant hormone, plays a role in plant development, defense, and climacteric fruit ripening. Both genetic and biochemical evidence suggest that the response of plants to ethylene is mediated by a specific ethylene receptor. The signal emanating from the receptor-effector complex is then presumably transduced via an unknown cascade pathway. We have used the plant pathogenesis response, exemplified by the induction of the pathogenesis-related gene chitinase, as a paradigm to investigate ethylene-dependent signal transduction in the plant cell. We showed that calcium is necessarily involved in the ethylene-mediated pathogenesis response. Blocking calcium fluxes with chelators inhibited ethylene-dependent induction of chitinase accumulation, but not ethylene independent induction. Artificially increasing cytosolic calcium levels by treatments with the calcium ionophore ionomycin or the calcium pump blocker thapsigargin stimulated chitinase accumulation. Plants grown in calcium-poor soil showed a 10-fold reduction in leaf extractable calcium. Their leaves exhibited a reduced pathogenesis reaction to ethylene and were impaired in another hormone response mediated by calcium, i.e., abscisic acid-controlled closure of guard cells. The addition of calcium to leaves excised from calcium-deficient plants restored their sensitivity to ethylene. Ethylene participates in the control of seedling growth, promoting the so-called "triple response" that results in distinct morphological development, such as hypocotyl hook formation. This effect, similar to the ethylene-promoted pathogenesis response, was found to be calcium dependent. The results indicate that calcium is required for a variety of ethylene-dependent processes.

UV-B-Induced PR-1 Accumulation Is Mediated by Active Oxygen Species.

Depletion of the stratospheric ozone layer may result in an increase in the levels of potentially harmful UV-B radiation reaching the surface of the earth. We have found that UV-B is a potent inducer of the plant pathogenesis-related protein PR-1 in tobacco leaves. UV-B fluences required for PR-1 accumulation are similar to those of other UV-B-induced responses. The UV-B-induced PR-1 accumulation was confined precisely to the irradiated area of the leaf but displayed no leaf tissue specificity. A study of some of the possible components of the signal transduction pathway between UV-B and PR-1 induction showed that photosynthetic processes are not essential, and photoreversible DNA damage is not involved. Antioxidants and cycloheximide were able to block the induction of PR-1 by UV-B, and treatment of leaves with a generator of reactive oxygen resulted in the accumulation of PR-1 protein. These results demonstrate an absolute requirement for active oxygen species and protein synthesis in this UV-B signal transduction pathway. In contrast, we also show that other elicitors, notably salicylic acid, are able to elicit PR-1 via nonreactive oxygen species-requiring pathways.

Ethylene Signal Is Transduced via Protein Phosphorylation Events in Plants.

A plethora of abiotic and biotic environmental stresses exert their influence on plants via the gaseous hormone ethylene. In addition, aspects of plant development and climacteric fruit ripening are regulated by ethylene. Sensitivity to ethylene is presumably mediated by a specific ethylene receptor whose activation signal is then transduced via an unknown cascade pathway. We have used the plant pathogenesis response, exemplified by the induction of pathogenesis-related (PR) genes, as a paradigm to investigate ethylene-dependent signal transduction in the plant cell. Ethylene application induced very rapid and transient protein phosphorylation in tobacco leaves. In the presence of the kinase inhibitors H-7 and K-252a, the transient rise in phosphorylation and the induced expression of PR genes were abolished. Similarly, these inhibitors blocked the response induced by an ethylene-dependent elicitor, [alpha]-AB. Reciprocally, application of okadaic acid, a specific inhibitor of phosphatases type 1 and type 2A, enhanced total protein phosphorylation and by itself elicited the accumulation of PR proteins. In the presence of H-7 and K-252a, PR protein accumulation induced by okadaic acid was blocked. In contrast to the action of ethylene and [alpha]-AB, xylanase elicits the accumulation of PR protein by an ethylene-independent pathway. Xylanase-induced PR protein accumulation was not affected by H-7 and K-252a. The results indicate that responsiveness to ethylene in leaves is transduced via putative phosphorylated intermediates that are regulated by specific kinases and phosphatases.

Two Distinct Sources of Elicited Reactive Oxygen Species in Tobacco Epidermal Cells.

Reactive oxygen species (ROS) play a prominent role in early and later stages of the plant pathogenesis response, putatively acting as both cellular signaling molecules and direct antipathogen agents. A single-cell assay, based on the fluorescent probe dichlorofluorescein, was used to scrutinize the generation and movement of ROS in tobacco epidermal tissue. ROS, generated within cells, quickly moved apoplastically as H2O2 into neighboring cells. Two classes of rapidly elicited intracellular ROS, originating from distinct sources, were distinguished. Cryptogein, the fungal elicitor from Phytophthora cryptogea, induced ROS from a flavin-containing oxidase source. ROS accumulation could be inhibited by a number of pharmacological agents, suggesting induction through an active signal transduction pathway. The insensitivity of the increase in ROS to the external addition of enzymes that dissipate ROS suggests that this oxidative increase is primarily intracellular. In contrast, amines and polyamines, compounds that form during wounding and pathogenesis, induced ROS at an apoplastic site from peroxidase- or amine oxidase-type enzyme(s). Salicylic acid, a putative inhibitor of cellular catalases and peroxidases, did not induce cellular ROS, as measured by dichlorofluorescein fluorescence. The physiological relevance of ROS-generated signals was indicated by the rapid alteration of the epidermal cell glutathione pool and the cellular redox state. In addition, induction of ROS by all elicitors was correlated with subsequent cell death.

The role of calcium and activated oxygens as signals for controlling cross-tolerance.

Plants are confronted on a regular basis with a range of environmental stresses. These include abiotic insults caused by, for example, extreme temperatures, altered water status or nutrients, and biotic stresses generated by a plethora of plant pathogens. Many studies have shown that the cellular responses to these environmental challenges are rather similar, which might be why plants resistant to one stress are sometimes cross-tolerant to others. To understand this phenomenon and to be able to take full advantage of it in agriculture, we must determine whether the individual biochemical pathways that make up the responses to each external stimulus are activated by unique, overlapping or redundant signalling systems. We discuss the potential role of signalling molecules, such as calcium and activated oxygen species, in underlying cross-tolerance.

Comparative genetics of nucleotide binding site-leucine rich repeat resistance gene homologues in the genomes of two dicotyledons: tomato and arabidopsis.

The presence of a single resistance (R) gene allele can determine plant disease resistance. The protein products of such genes may act as receptors that specifically interact with pathogen-derived factors. Most functionally defined R-genes are of the nucleotide binding site-leucine rich repeat (NBS-LRR) supergene family and are present as large multigene families. The specificity of R-gene interactions together with the robustness of plant-pathogen interactions raises the question of their gene number and diversity in the genome. Genomic sequences from tomato showing significant homology to genes conferring race-specific resistance to pathogens were identified by systematically "scanning" the genome using a variety of primer pairs based on ubiquitous NBS motifs. Over 70 sequences were isolated and 10% are putative pseudogenes. Mapping of the amplified sequences on the tomato genetic map revealed their organization as mixed clusters of R-gene homologues that showed in many cases linkage to genetically characterized tomato resistance loci. Interspecific examination within Lycopersicon showed the existence of a null allele. Consideration of the tomato and potato comparative genetic maps unveiled conserved syntenic positions of R-gene homologues. Phylogenetic clustering of R-gene homologues within tomato and other Solanaceae family members was observed but not with R-gene homologues from Arabidopsis thaliana. Our data indicate remarkably rapid evolution of R-gene homologues during diversification of plant families.

Aldehyde oxidase and xanthine dehydrogenase in a flacca tomato mutant with deficient abscisic acid and wilty phenotype

The flacca tomato (Lycopersicon esculentum) mutant displays a wilty phenotype as a result of abscisic acid (ABA) deficiency. The Mo cofactor (MoCo)-containing aldehyde oxidases (AO; EC 1.2.3.1) are thought to play a role in the final oxidation step required for ABA biosynthesis. AO and related MoCo-containing enzymes xanthine dehydrogenase (XDH; EC 1.2.1.37) and nitrate reductase (EC 1.6.6.1) were examined in extracts of the flacca tomato genotype and of wild-type (WT) roots and shoots. The levels of MoCo were found to be similar in both genotypes. No significant XDH or AO (MoCo-containing hydroxylases) activities were detected in flacca leaves; however, the mutant exhibited considerable MoCo-containing hydroxylase activity in the roots, which contained notable amounts of ABA. Native western blots probed with an antibody to MoCo-containing hydroxylases revealed substantial, albeit reduced, levels of cross-reactive protein in the flacca mutant shoots and roots. The ABA xylem-loading rate was significantly lower than that in the WT, indicating that the flacca is also defective in ABA transport to the shoot. Significantly, in vitro sulfurylation with Na2S reactivated preexisting XDH and AO proteins in extracts from flacca, particularly from the shoots, and superinduced the basal-level activity in the WT extracts. The results indicate that in flacca, MoCo-sulfurylase activity is impaired in a tissue-dependent manner.

[beta]-Aminobutyric Acid Induces the Accumulation of Pathogenesis-Related Proteins in Tomato (Lycopersicon esculentum L.) Plants and Resistance to Late Blight Infection Caused by Phytophthora infestans.

Tomato (Lycopersicon esculentum L.) plants were sprayed with aqueous solutions of isomers of aminobutyric acid and were either analyzed for the accumulation of pathogenesis-related (PR) proteins or challenged with the late blight fungal agent Phytophthora infestans. The [beta] isomer of aminobutyric acid induced the accumulation of high levels of three proteins: P14a, [beta]-1,3 glucanase, and chitinase. These proteins either did not accumulate or accumulated to a much lower level in [alpha]- or [gamma]-aminobutyric acid-treated plants. Plants pretreated with [alpha]-, [beta]-, and [gamma]-aminobutyric acid were protected up to 11 d to an extent of 35, 92, and 6%, respectively, against a challenge infection with P. infestans. Protection by [beta]-aminobutyric acid was afforded against the blight even when the chemical was applied 1 d postinoculation. Examination of ethylene evolution showed that [alpha]-aminobutyric acid induced the production of 3-fold higher levels of ethylene compared with [beta]-aminobutyric acid, whereas [gamma]-aminobutyric acid induced no ethylene production. In addition, silver thiosulfate, a potent inhibitor of ethylene action, did not abolish the resistance induced by [beta]-aminobutyric acid. The results are consistent with the possibility that [beta]-aminobutyric acid protects tomato foliage against the late blight disease by a mechanism that is not mediated by ethylene and that PR proteins can be involved in induced resistance.

Juvenile-Specific Localization and Accumulation of a Rhamnosyltransferase and Its Bitter Flavonoid in Foliage, Flowers, and Young Citrus Fruits.

1-2-Rhamnosyltransferase catalyzes the production of disaccharide-flavonoids that accumulate to 75% of dry weight. Vast energy is expended in a short time span to produce these flavonoids. The highest rhamnosyltransferase activities and immunodetected concentrations were observed in early development of Citrus grandis (pummelo), coinciding with up to 13% of fresh weight as naringin. The concentration of naringin in leaves, petals, receptacles, filaments, albedo, and flavedo drops drastically during development and correlates directly with a decrease in the activity and amounts of 1-2-rhamnosyltransferase. Anthers had minute rhamnosyltransferase activities and low concentrations of naringin. Conversely, high 1-2-rhamnosyltransferase activity and naringin concentrations appeared in both young and mature ovaries, as well as in young fruits. The total amounts of naringin in mature leaves decreased without detectable in vitro degradation of naringin in leaves. There was still a net accumulation of naringin in the albedo and flavedo of older fruit even though these tissues had only traces of 1-2-rhamnosyltransferase. Traces of enzyme synthesis in fruits, or import of the product from leaves, may explain the net accumulation of naringin in growing fruits. Unlike the late-expressed genes for glycosyltransferases in anthocyanin biosynthesis, the rhamnosyltransferases from Citrus are active only in juvenile stages of development.

Plant disease resistance: commonality and novelty in multicellular innate immunity.

Pathogen avirulence genes encode for effector molecules that play a crucial role in the process of pathogen colonization of plant tissue. Successful host defense requires rapid and efficient detection of the pathogen avirulence factors. In the last few years, much progress has been made in delineating the plant molecular sentinels that participate in pathogen identification. Because this ability is genetic information that is 'hard-wired' into the genome, it is called 'innate immunity' and it draws its origins from a phylogenetically ancient form of immunity common to plants and animals. Conservation is shown in many of the functional molecular motifs of innate genes such as the Toll/interleukin 1 receptor domains, nucleotide binding domains and structures that contain leucine rich repeats. Novel plant molecular surveillance domains also include pathogen pattern recognition by coiled-coil domains and specialized kinases. The rapid evolution of plant innate immunity genes is readily detected in their sequence polymorphism, by their massive amplification and appearance in the genome in a clustered organization. By comparative biology of highly diverged innate immunity systems we can enhance our appreciation of the truly basic forces that have shaped its evolution in mutlicellular organisms.

Clone bank of Nicotiana tabacum chloroplast DNA: mapping of the alpha, beta and epsilon subunits of the ATPase coupling factor, the large subunit of ribulosebisphosphate carboxylase, and the 32-kDal membrane protein.

All of the PstI restriction fragments of the chloroplast DNA of Nicotiana tabacum have been cloned in the plasmid vector pBR322. The cloned fragment sizes range from 0.8 to 26 kb, are stable, and can be amplified by chloramphenicol with varying efficiencies. Using these clones we have detailed a PstI physical map of the tobacco chloroplast genome. Selected clones of SalI, BamHI and PstI fragments were used to localize the map positions of the alpha, beta, and epsilon subunits of the chloroplast ATPase coupling factor, the large subunit of ribulosediphosphate carboxylase and the 32-kDal membrane protein. The gene products of these clones were characterized by RNA transcript sizing, immunoprecipitation of maxicell-directed protein synthesis, and hybrid-arrested translation.

Regulation of nuclear genes encoding chloroplast proteins in transgenic plants.

Transgenic plants have been particularly useful in studying nuclear genes encoding for photosynthetic functions. The expression of these genes and their chimeric constructs in transgenic plants faithfully mimics their natural counterparts. The use of sensitive chimeric reporter genes has enabled localizing the activity of genes encoding photosynthetic proteins to individual cells. Cab and rbcS transgenes have been shown to retain sensitivity to light quality, which is modulated by phytochrome. Conditional light activation under the influence of a circadian rhythm has been shown for Cab transgenes. Transgenic plants containing truncated promoters have helped delineate cis-regulatory positive and negative elements involved in light-mediated transcriptional induction and tissue specificity.

Xylanase, a novel elicitor of pathogenesis-related proteins in tobacco, uses a non-ethylene pathway for induction.

Antisera to acidic isoforms of pathogenesis-related proteins were used to measure the induction of these proteins in tobacco (Nicotiana tabacum) leaves. Endo-(1-4)-beta-xylanase purified from culture filtrates of Trichoderma viride was a strong elicitor of pathogenesis-related protein synthesis in tobacco leaves. The synthesis of these proteins was localized to tissue at the area of enzyme application. The inhibitors of ethylene biosynthesis and ethylene action, 1-aminoethoxyvinylglycine and silver thiosulfate, inhibited accumulation of pathogenesis-related proteins induced by tobacco mosaic virus and alpha-aminobutyric acid, but did not inhibit elicitation by xylanase. Likewise, the induction of these proteins by the tobacco pathogen Pseudomonas syringae pv. tabaci was not affected by the inhibitors of ethylene biosynthesis and action. The leaf response to tobacco mosaic virus and alpha-aminobutyric acid was dependent on light in normal and photosynthetically incompetent leaves. In contrast, the response of leaves to xylanase was independent of light. Tobacco mosaic virus and alpha-aminobutyric acid induced concerted accumulation of pathogenesis-related proteins. However, xylanase elicited the accumulation of only a subset of these proteins. Specifically, the plant (1-3)-beta-glucanases, which are normally a part of the concerted response, were underrepresented. These experiments have revealed the presence of a novel ethylene-independent pathway for pathogenesis-related protein induction that is activated by xylanase.

The expression of an abundant transmitting tract-specific endoglucanase (Sp41) is promoter-dependent and not essential for the reproductive physiology of tobacco.

In angiosperms the interactions between the secretory matrix of the stylar transmitting tract and the growing pollen tubes have central roles in determining a successful fertilization. Sp41 is a major glycosylated component of the soluble proteins of the transmitting tract matrix and exhibits (1-3)-beta-glucanase activity. It is a member of the pathogenesis-related protein superfamily, but shows developmental regulation as opposed to pathogen induction. In order to investigate the mechanisms regulating Sp41 expression, we isolated and characterized genomic clones corresponding to the sp41 alpha gene. Sp41 alpha contains an intervening sequence localized between the sequences encoding for a putative signal peptide and the mature protein. A fragment of 2.5 kb that lies 5' to the coding region of the gene was sufficient to confer transmitting tract specific expression to a beta-glucuronidase reporter gene in transgenic tobacco plants. The sp41 transcripts have unusually long 5'-untranslated sequences. The leader sequences contain small open reading frames, include secondary structures, and may be involved in post-transcriptional regulation. A possible function for Sp41 in reproductive physiology was tested by monitoring tobacco plants transformed with antisense stylar sp41 alpha RNA: Transgenic antisense plants with immunologically and enzymatically undetectable levels of (1-3)-beta-glucanase were obtained and their offspring analyzed. The progeny plants did not show any detectable phenotypic modifications as they had a normal flower morphology and were fully fertile.

Superoxide production by plant homologues of the gp91(phox) NADPH oxidase. Modulation of activity by calcium and by tobacco mosaic virus infection.

Genes encoding homologs of the gp91(phox) subunit of the plasma membrane NADPH oxidase complex have been identified in plants and are hypothesized to be a source of reactive oxygen species during defense responses. However, the direct involvement of the gene products in superoxide (O(2)(-)) production has yet to be shown. A novel activity gel assay based on protein fractionation in native or sodium dodecyl sulfate (SDS)-denaturing polyacrylamide gels was developed. In native polyacrylamide gel electrophoresis, one or two major O(2)(-)-producing formazan bands were detected in tomato (Lycopersicum esculentum Mill. cv Moneymaker) and tobacco (Nicotiana tabacum var. Samsun, NN) plasma membranes, respectively. Denaturing fractionation of tomato and tobacco plasma membrane in SDS-polyacrylamide gel electrophoresis, followed by regeneration of the in-gel activity, revealed NADPH-dependent O(2)(-)-producing formazan bands of 106-, 103-, and 80- to 75-kD molecular masses. The SDS and native activity bands were dependent on NADPH and completely inhibited by diphenylene iodonium or CuZn- O(2)(-) dismutase, indicating that the formazan precipitates were due to reduction by O(2)(-) radicals catalyzed by an NADPH-dependent flavin containing enzyme. The source of the plasma membrane activity bands was confirmed by their cross-reaction with antibody prepared from the C terminus of the tomato gp91(phox) homolog. Membrane extracts as well as the in-gel NADPH oxidase activities were stimulated in the presence of Ca(2+). In addition, the relative activity of the gp91(phox) homolog was enhanced in the plasma membrane of tobacco mosaic virus-infected leaves. Thus, in contrast to the mammalian gp91(phox), the plant homolog can produce O(2)(-) in the absence of additional cytosolic components and is stimulated directly by Ca(2+).

Conservation of sequence arrangement among higher plant chloroplast DNAs: molecular cross hybridization among the Solanaceae and between Nicotiana and Spinacia.

Isolated, nick-translated Pvu II fragments of Nicotiana tabacum chloroplast DNA produce specific intra- and intergeneric hybridization signals with chloroplast DNA digests from several representatives of the Solanaceae. These data, along with similarities in restriction enzyme patterns, permit construction of physical maps for Nicotiana line 92 (a cytoplasmic substitution line), Atropa belladonna and Petunia parodii. Plastid-DNA map differences among the Solanaceae are shown to result from single base-pair substitutions as well as local deletions or insertions. Several of these differences of Nicotiana tabacum chloroplast DNA fragments to a chloroplast DNA digest of Spinacia oleracea defines a sequential arrangement of fragments for spinach DNA which is very similar to its published physical map. This is achieved although chloroplast-DNA restriction enzyme patterns from the two organisms are grossly dissimilar. Alignment differences which have been revealed involve the edges of the inverted repeat region where certain single copy stretches in tobacco have been duplicated in spinach.