Spatial distribution of chitinases and β-1,3-glucanase transcripts in bean arbuscular mycorrhizal roots under low and high soil phosphate conditions

Arbuscular mycorrhizal (AM) fungi extensively colonize the root cortex under low-soil-phosphate (P) conditions, whereas infection is limited under high-P conditions. Fungal growth under both P conditions might be influenced by plant defence-related gene expression. In this study, we used in situ hybridization methods to compare the cellular localization of three defence-related mRNAs in non-infected bean roots and in relation to fungal infection units. In non-infected and infected roots, mRNAs encoding acidic and basic endochitinases were generally most abundant in the vascular cylinder. High-P-grown mycorrhizal roots showed localized accumulation of the acidic endochitinase mRNA in cortical cells containing arbuscules and in their immediate vicinity (one to five cell layers). The pattern of accumulation of the basic endochitinase mRNA was not affected by P or AM fungal infection. At the low P concentration, the β-1,3-glucanase mRNA accumulated predominantly in the vascular cylinder of non-infected roots. Suppression of β-1,3-glucanase mRNA accumulation in these tissues was observed in non-infected roots at the high-P and in mycorrhizal roots at both P concentrations. The observed suppression extends at least several mm from fungal infection units, characterizing a systemic effect. Beta-1,3-glucanase mRNA accumulated also around a number of cortical cells containing arbuscules only at the low P concentration. The localized accumulations of the endochitinase and β-1,3-glucanase mRNAs suggest that the encoded proteins might be involved in the control of intraradical fungal growth.     

Coordinate Expression of Rubisco Activase and Rubisco during Barley Leaf Cell Development

We have utilized the cellular differentiation gradient and photomorphogenic responses of the first leaf of 7-day-old barley (Hordeum vulgare L.) to examine the accumulation of mRNA and protein encoded by the ribulose-1,5-biphosphate carboxylase holoenzyme (rubisco) activase gene (rca). Previous studies have revealed a pattern of coordinate expression of rubisco subunit polypeptides during development. We compared the expression of rubisco polypeptides and mRNAs with those encoded by rca. The mRNAs encoding both rubisco activase and rubisco are expressed exclusively in leaf tissue of 7-day-old barley seedlings; mRNAs and polypeptides of rca accumulate progressively from the leaf base in a pattern that is qualitatively similar to that of rubisco subunit mRNAs and polypeptides. The parallel pattern of rca protein and mRNA accumulation indicate that a primary control of rca gene expression in this system lies at the level of mRNA production. Light-induced expression of rca in etiolated barley follows a different pattern from that of the acropetal barley leaf gradient, however. Etiolated, 7-day-old barley seedlings contain levels of rca mRNA near the limit of detection in Northern blot hybridization assays. White light induces a 50- to 100-fold accumulation of rca mRNA, which is detectable within 30 min after the onset of illumination. In contrast, steady state levels of mRNAs encoding the small rubisco subunit are affected little by light, and mRNAs encoding the large subunit accumulate about 5-fold in response to illumination. While rca mRNA levels are low in etiolated barley leaves, levels of the protein are approximately 50 to 75% of those found in fully green leaves.     

Adenovirus early gene products may control viral mRNA accumulation and translation in vivo

The mechanisms controlling early adenovirus gene expression in vivo have been studied using inhibitors of protein synthesis. When inhibitors were added shortly before or at the onset of infection, viral mRNA from all early regions was transcribed, spliced and accumulated over a 7 hr period. After longer pretreatment, accumulation of several early mRNAs were suppressed. Addition of inhibitors 1 hr after infection enhanced the accumulation of viral mRNA in the cytoplasm. Translation of early mRNA selected on adenovirus DNA in a cell-free system reflected the amount of viral mRNA present. A viral coded product may therefore control accumulation of viral mRNA.A different pattern emerged when inhibitors of protein synthesis were removed at 5 hr postinfection and cells were pulse-labeled in vivo. If inhibitors were introduced at or before infection, early viral proteins were synthesized only after a lag of 1–3 hr. However, if treatment was introduced 1 hr post-infection, reversion of the protein synthesis block was instantaneous. It appears that protein synthesis inhibitors reveal an in vivo translational block for viral mRNA. This block could be overcome by preinfection with a related virus. Furthermore, no block was observed in a virus-transformed human embryonic kidney cell line (293) which expresses early region 1 of the viral genome. Viral gene product(s) encoded in early region 1 may control translation of early adenovirus messenger RNA in vivo.     

Two alleles of the single-copy chalcone synthase gene in parsley differ by a transposon-like element

We have analysed three nearly full-length cDNAs complementary to mRNAs encoding two PR1 (pathogenesis-related, class 1) proteins in parsley (Petroselinum crispum). Furthermore, one selected genomic clone containing the PcPR1-1 gene was investigated in detail. The structural organization and possible regulatory elements in the 5' flanking region of this gene are presented. In situ RNA hybridization in fungus-infected parsley leaf tissue demonstrated rapid and massive PR1 mRNA accumulation around infection sites.     

Light-regulated gene expression during maize leaf development

We have established schedules of expression during maize leaf development in light and darkness for the messenger RNAs (mRNAs) and polypeptides for ribulose 1,5-bisphosphate carboxylase (RuBPCase) subunits, phosphoenolpyruvate carboxylase (PEPCase), and the light- harvesting chlorophyll a/b-binding protein (LHCP). Levels of mRNAs were measured by hybridization with cloned probes, and proteins were measured by immunodetection on protein gel blots. The initial synthesis in leaves of all four mRNAs follows a light-independent schedule; illumination influences only the level to which each mRNA accumulates. The synthesis of RuBPCase small and large subunits and of PEPCase polypeptides also follows a light-independent schedule which is modified quantitatively by light. However, the accumulation of LHCP polypeptides absolutely requires illumination. The accumulation of each protein closely follows the accumulation of its mRNA during growth in light. Higher ratios of PEPCase and RuBPCase protein to mRNA occur during dark growth.     

Two differentially regulated class II chitinases from parsley

Two distinct cDNA clones, PcCHI1 and PcCHI2, with high sequence similarity to plant chitinases were isolated from parsley (Petroselinum crispum), expressed in Escherichia coli, and the encoded proteins functionally identified as endochitinases. Different expression patterns of the corresponding mRNAs and proteins in infected and uninfected parsley plants indicated distinct roles of the two isoforms in both pathogen defense and plant development. Infection of parsley leaf buds with Phytophthora sojae resulted in the rapid, transient and highly localized accumulation of PcCHI1 mRNA and protein around infection sites, whereas PcCHI2 mRNA and protein were systemically induced at later infection stages. Similar differences in the timing of induction were observed in elicitor-treated, suspension-cultured parsley cells. In uninfected plants, PcCHI1 mRNA was particularly abundant in the transmitting tract of healthy flowers, suggesting a role in the constitutive protection of susceptible transmitting tissue of the style against pathogen ingress and/or in the fertilization process, possibly by affecting pollen tube growth. Localization of PcCHI2 mRNA and protein in the parenchymatic collenchyme of young pedicels may indicate a function in the constitutive protection of this tissue. In addition to such distinct roles of PcCHI1 and PcCHI2 in preformed and induced pathogen defense, both chitinases may have endogenous regulatory functions in plant development.     

Photosynthetic gene expression and cellular differentiation in developing maize leaves

We have exploited the positional gradient of cellular differentiation in Zea mays leaves to study the accumulation of mRNAs encoding subunits of the two CO₂-fixing enzymes and the major chlorophyll-binding protein. These three proteins are differentially compartmentalized in the two photosynthetically active cell types of the leaf. Previous studies have shown that accumulation of the two carboxylases commences 2 to 4 cm from the base of the leaf (Mayfield SP, WC Taylor Planta 161: 481-486) at a position where bundle sheath and mesophyll cells show morphological evidence of maturation. The light-harvesting chlorophyll a/b protein accumulates progressively from the leaf base, as does its mRNA, in spite of its localization in mesophyll cells after cellular differentiation occurs. While small quantities of phosphoenolpyruvate carboxylase mRNA are detectable in the basal region of the leaf, significant mRNA accumulation is coincident with that of the polypeptide at 4 to 6 cm from the leaf base, the region where bundle sheath and mesophyll cells exhibit fully differentiated morphologies. mRNAs encoding the small and large subunits of ribulose 1,5-bisphosphate carboxylase accumulate to significant levels before bundle sheath cells are fully differentiated and before their polypeptides are detectable. Cytological examination indicates that this is the position at which the maturation of intermediate vascular bundles is first evident. Cytosolically localized small subunit mRNA and chloroplast-localized large subunit mRNA are complexed with polyribosomes at all positions of the leaf.     

Expression of a maize proteinase inhibitor gene is induced in response to wounding and fungal infection: systemic wound-response of a monocot gene

The isolation and characterization of cDNA and genomic clones encoding a proteinase inhibitor protein (MPI) in maize is reported. Accumulation of the MPI mRNA is induced in response to fungal infection in germinating maize embryos. The expression pattern of the MPI gene, in healthy and fungal infected maize tissues, was examined and compared with the expression pattern of a gene that codes for a pathogenesis-related protein (the PRms protein) from maize. These two genes are induced by fungal infection, however different signals trigger their activation. Accumulation of the proteinase inhibitor mRNA is more a consequence of the wound produced by the penetration and colonization of the host tissues by the pathogen, than the result of a direct molecular recognition of the pathogen by the plant, as is the case for the induction of the PRms gene. Wounding, or treatment with abscisic acid or methyl jasmonate, stimulate MPI mRNA accumulation, but not PRms mRNA accumulation. Local and systemic induction of the MPI gene expression in response to wounding occurs in maize plants. To the authors' knowledge, this is the first example of a gene from a monocotyledonous species that clearly shows a systemic wound response. The possible functional implications for the existence of different signal transduction pathways that simultaneously activate a battery of defense mechanisms against potential pathogens are discussed.     

Molecular and cytological characterization of ZTL in <Emphasis Type="Italic">Ipomoea nil</Emphasis>

The ZEITLUPE (ZTL) protein is involved in the control of circadian period, hypocotyl elongation and flowering time in Arabidopsis thaliana. The aim of the present work was the identification of the InZTL gene and localization of its mRNA in the model short-day plant Ipomoea nil. The deduced InZTL protein of 622 amino acid residues contained a LOV domain at the N-terminal part, followed by an F-box domain and six carboxy terminal kelch repeats. Amino acid sequence of InZTL showed 84 % homology with Mesembryanthemum crystallinum ZTL (McZTL) and 83 % with Arabidopsis thaliana ZTL (AtZTL). Fluorescence in situ hybridization (FISH) to InZTL mRNA showed its high accumulation in the vascular bundles as well in the guard cells of the cotyledon. Immunolocalization of ZTL protein indicated a similar distribution pattern of ZTL protein as InZTL mRNAs.     

VP22 of herpes simplex virus 1 promotes protein synthesis at late times in infection and accumulation of a subset of viral mRNAs at early times in infection

VP22, encoded by the U_L49 gene, is one of the most abundant proteins of the herpes simplex virus 1 (HSV-1) tegument. In the present study we show VP22 is required for optimal protein synthesis at late times in infection. Specifically, in the absence of VP22, viral proteins accumulated to wild-type levels until ~6 h postinfection. At that time, ongoing synthesis of most viral proteins dramatically decreased in the absence of VP22, whereas protein stability was not affected. Of the individual proteins we assayed, VP22 was required for optimal synthesis of the late viral proteins gE and gD and the immediate-early protein ICP0 but did not have discernible effects on accumulation of the immediate-early proteins ICP4 or ICP27. In addition, we found VP22 is required for the accumulation of a subset of mRNAs to wild-type levels at early, but not late, times in infection. Specifically, the presence of VP22 enhanced the accumulation of gE and gD mRNAs until ~9 h postinfection, but it had no discernible effect at later times in infection. Also, VP22 did not significantly affect ICP0 mRNA at any time in infection. Thus, the protein synthesis and mRNA phenotypes observed with the U_L49-null virus are separable with regard to both timing during infection and the genes affected and suggest separate roles for VP22 in enhancing the accumulation of viral proteins and mRNAs. Finally, we show that VP22's effects on protein synthesis and mRNA accumulation occur independently of mutations in genes encoding the VP22-interacting partners VP16 and vhs.     

Induced systemic resistance in wounded rice plants

Wounding of one leaf of young rice plants caused a strong and transient accumulation of non-conjugated (–)-JA, followed by induction of a number of pathogenesis-related genes, in the treated leaf. The non-treated leaf of wounded plants that emerged after the treatment was more resistant to challenge infection by the rice blast fungus Magnaporthe grisea (Hebert) Barr. The systemic leaf also showed a transient, but delayed accumulation of jasmonic acid. Unlike the wounded leaf, there was no accumulation of pathogenesis-related mRNAs or proteins in the systemically protected leaf. Local application of jasmonic acid, the putative signal of the wound response, resulted in a similar degree of systemic disease resistance as wounding. The results suggest the operation of systemic, wound-related signalling processes in rice that induce systemic disease resistance.     

Temporal and spatial patterns of 1, 3-β-glucanase and chitinase induction in potato leaves infected by Phytophthora infestans

Infection of potato (Solarium tuberosum L.) leaves with the fungal pathogen Phytophthora infestans caused a similar, strong and coordinated induction of 1, 3-β-glucanases and chitinases in compatible (plant susceptible) and incompatible (plant resistant) interactions of two selected plant cultivars with appropriate races of the fungus. The temporal and spatial patterns of 1, 3-β-glucanase induction were studied in further detail by immunohistochemical and in-situ hybridization methods. Accumulation of the protein was preceded by progressive activation of the corresponding gene, commencing near infection sites and spreading rapidly throughout the whole infected leaf as well as to adjacent, non-infected leaves. Protein and mRNA distribution patterns were nearly identical in compatible and incompatible interactions. In comparison with 1, 3-β-glucanase mRNA, phenyl-alanine ammonia-lyase mRNA accumulated more rapidly and remained restricted to the vicinity of fungal infection sites, in addition to its constitutive occurrence in the vascular bundles. Even more rapid than any detectable mRNA induction was the accumulation of auto fluorescing material in plant cells immediately surrounding fungal structures, particularly and invariably in incompatible interactions and less frequently in compatible interactions. It is concluded that cultivar-race-specific resistance is established early in the interaction of potato leaves with P. infestans and hence the observed massive accumulation of 1, 3-β-glucanase and chitinase is presumably not involved in determining this specificity.     

Isolation of a flax (Linum usitatissimum) gene induced during susceptible infection by flax rust (Melampsora lini)

In a susceptible infection of flax ( Linum usitatissimum ), the obligate rust pathogen ( Melampsora lini ) can grow in the leaf without triggering the hypersensitive resistance response. The rust establishes specialized structures (haustoria) in plant mesophyll cells and induces changes in plant subcellular organization. Subtraction hybridization methods were used to isolate cDNA clones of mRNAs that have altered expression in infected leaves. Most of the cDNAs recovered were of fungal origin, but one clone, pFIS1 ( f lax i nducible s equence No. 1), recovered from several independent experiments, was a plant-specified mRNA that showed a 10-fold increase in steady-state levels during susceptible growth. The increase in fis1 mRNA levels was not seen in the resistant reaction (hypersensitive reaction) and the predicted protein sequence (551 amino acids with a predicted molecular weight of 61 kDa) has no similarity to known pathogenesis-related proteins. Searches of sequence data bases showed that fis1 encodes a protein which contains amino acid sequence motifs that are conserved in all previously characterized aldehyde dehydrogenases.     

Spatial and temporal expression of a maize lipid transfer protein gene.

We studied the temporal and spatial pattern of lipid transfer protein (LTP) gene expression, as well as the localization of this protein, in maize. Using an LTP gene, we observed an accumulation of LTP mRNA in embryos and endosperms during seed maturation. LTP gene expression was also investigated in young seedlings. After germination, the level of LTP mRNA in the coleoptile increased, with a maximum at 7 days, whereas LTP mRNA levels were low in the scutellum and negligible in roots. The high levels of LTP mRNA found in coleoptiles and embryos were confirmed by in situ hybridization. Moreover, LTP gene expression appeared to be localized in the external cellular layers and around the leaf veins. Using immunogold methods, we also observed that LTP was distributed heterogeneously in the different cells of coleoptiles and leaves. The highest concentrations of LTP were found in the outer epidermis of the coleoptiles as well as the leaf veins. Together, our observations indicate that LTP gene expression is not only organ specific and time specific but also cell specific.     

In situ localization of storage protein mRNAs in developing meristems of Brassica napus embryos

Probes derived from cDNA clones of napin and cruciferin, the major storage proteins of Brassica napus, and in situ hybridization techniques were used to examine changes in the spatial and temporal distribution of storage protein messages during the course of embryogeny, with a special emphasis on the developing apical meristems. Napin mRNAs begin to accumulate in the cortex of the axis during late heart stage, in the outer faces of the cotyledons during torpedo stage and in the inner faces of the cotyledons during cotyledon stage. Cruciferin mRNAs accumulate in a similar pattern but approximately 5 days later. Cells in the apical regions where root and shoot meristems develop do not accumulate storage protein messages during early stages of embryogeny. In the upper axis, the boundary between these apical cells and immediately adjacent cells that accumulate napin and cruciferin mRNAs is particularly distinct. Our analysis indicates that this boundary is not related to differences in tissue or cell type, but appears instead to be coincident with the site of a particular set of early cell divisions. A major change in the mRNA accumulation patterns occurs halfway through embryogeny, as the embryos enter maturation stage and start drying down. Final maturation of the shoot apical meristem is associated with the development of leaf primordia and the accumulation of napin mRNAs in the meristem, associated leaf primordia and vascular tissue. Cruciferin mRNAs accumulate only in certain zones of the shoot apical meristem and on the flanks of leaf primordia. Neither type of mRNA accumulates in the root apical meristem at any stage.