Identification of a Ca(2+)-pectate binding site on an apoplastic peroxidase.

An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca(2)+-induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca(2)+-pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca(2)+-pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.

Molecular cloning, spatial and temporal characterization of spinach SOGA1 cDNA, encoding an alpha subunit of G protein.

Heterotrimeric G proteins are an important component of signal transduction pathway in animals. Although these proteins have been described in plants, their exact function and action mode are not clearly defined. In order to analyze the relationship between these proteins and the transduction of light signals in spinach, we have isolated by 5' and 3' RACE-PCR a 1660bp cDNA clone called SOGA1. This codes for a 383aa protein, which reveals a very strong homology with other plant Galpha subunit sequences. Genomic analysis suggested that SOGA1 belonged to a small multiple gene family. Northern blots and in-situ hybridization analyses showed that SOGA1 transcripts accumulate in all organs tested with a specific high level associated with the apex, roots and hypocotyls. Finally, a time-course analysis performed on the green tissues showed that accumulation of SOGA1 transcripts follows a circadian rhythm. However, in-situ hybridization analysis of the apex suggested the opposite behavior, while no variation was observed in the hypocotyl.

Molecular cloning and tissue-specific expression of an anionic peroxidase in zucchini.

A calcium-pectate-binding anionic isoperoxidase (APRX) from zucchini (Cucurbita pepo) was purified and subjected to N-terminal amino acid microsequencing. The cDNA encoding this enzyme was obtained by reverse transcriptase polymerase chain reaction from a cDNA library. It encoded a mature protein of 309 amino acids exhibiting all of the sequence characteristics of a plant peroxidase. Despite the presence of a C-terminal propeptide, APRX was found in the apoplast. APRX protein and mRNA were found in the root, hypocotyls, and cotyledons. In situ hybridization showed that the APRX-encoding gene was expressed in many different tissues. The strongest expression was observed in root epidermis and in some cells of the stele, in differentiating tracheary elements of hypocotyl, in the lower and upper epidermis, in the palisade parenchyma of cotyledons, and in lateral and adventitious root primordia. In the hypocotyl hook there was an asymmetric expression, with the inner part containing more transcripts than the outer part. Treatment with 2,3,5-triiodobenzoic acid reduced the expression of the APRX-encoding gene in the lower part of the hypocotyl. Our observations suggest that APRX could be involved in lignin formation and that the transcription of its gene was related to auxin level.

Cytological comparison of leaves and stems of Prunus avium L. shoots cultured on a solid medium with agar or gelrite.

An axillary proliferating clone of Prunus avium L. was subcultured every four weeks on solid MS medium with agar as the gelling agent. Vitrification (hyperhydricity) of shoots was induced in one four week cycle with the same medium except that agar was replaced by gelrite. During culture on the vitrifying medium, the water content of the shoots progressively increased with a parallel decrease in chlorophyll content. Cytological differences between the leaves and stems of the vitrified and normal shoots were detected by light and electron (both transmission and scanning) microscopy. Leaves of vitrified shoots were characterized by lower number of chloroplasts in the palisade parenchyma and by a defective cuticle. The stems of vitrified shoots had a less developed and lignifled xylem tissue, lacked sclerenchymatic areas and showed hypertrophy of the cortical parenchyma. More intense vacuolar activity with evaginations of the chloroplast envelope into the vacuole was noted in cells of vitrified leaves.

Effects of a mechanical stimulation of localization of annexin-like proteins in Bryonia dioica internodes.

Mechanical stimulation exerted by rubbing a young internode of Bryonia dioica plants inhibits its growth. Previous cellular and biochemical studies showed that this growth inhibition is associated with Ca(2+) redistribution and profound modifications of plasma membrane characteristics. We extracted and purified Ca(2+)-dependent phospholipid-binding proteins from B. dioica internodes. Two main proteins, p33 and p35, and other minor bands were isolated and identified as annexin-like proteins because of their biochemical properties and their cross-reactions with antibodies against maize (Zea mays L.) annexins. Rabbit antiserum was obtained by injection of B. dioica p35. This antiserum was used for the immunocytolocalization of annexin-like proteins in internode parenchyma cells. It appeared that the distribution of annexin-like proteins was different before and 30 min after the mechanical stimulation. Western analysis of proteins in membrane fractions after separation by free-flow electrophoresis showed that p35 was present in most fractions, whereas p33 appeared mainly in plasmalemma-enriched fractions after the mechanical stimulation. It is hypothesized that a subcellular redistribution of these proteins might be involved in growth inhibition by mechanical stress.

Peroxidase activity in shoot apical meristem from Spinacia.

Peroxidase activity was localized in the shoot apical meristem (SAM) of Spinacia at the light and electron microscope level with the histochemical method employing H2O2 and 3,3'-diaminobenzidine. At the light microscopic level, peroxidase activity was examined in unfixed cryostat sections at different pHs. The enzyme was found to be more intense at low than at neutral and high pH. The activity was evenly distributed over the different regions of the meristem at low pH, whereas it was more intense in the rib meristem at pH 6.8 and 7.2. At the ultrastructural level, peroxidase activity was found at 3 cellular sites: the cell wall, the tonoplast and the plasmalemma. These results were discussed in relationship with the in situ distribution of peroxidases in plant cells and other enzymes in vegetative SAM.

Glucose-6-phosphate dehydrogenase activity in spinach as measured by image analysis: a new approach for plant enzyme histochemistry.

A relatively low-cost computer-assisted image analysis system is described. Software has been specifically written for the continuous monitoring of absorbance readings on cryostat sections of plant tissues incubated in media to reveal enzyme activities. The equipment was tested by quantifying glucose-6-phosphate dehydrogenase activity in cryostat sections from shoot apices of spinach plants. The reaction rate of the dehydrogenase activity was monitored at two incubation temperatures, 20 degrees C and 30 degrees C. Control incubations were performed in media lacking substrate. The specific test minus control reaction at 30 degrees C was twice that at 20 degrees C. Variation of the substrate concentration at 30 degrees C yielded a Km value of 0.37 mM. These preliminary results show that our image analysis system can be used for kinetic measurements of dehydrogenase activity in frozen tissue sections and constitute a new approach for enzyme histochemistry in the shoot apical meristem.

Cytological study on water stress during germination of Zea mays.

Kernels of Zea mays were subjected to dehydration treatment at various times during germination. Embryos from kernels dehydrated during the first 36 h of germination are resistant to dehydration and subsequently germinate earlier than controls. Dehydration of kernels germinated during 72h leads to an irreversible arrest of growth of the embryos. However, autoradiographic observations showed that these embryos are still able to incorporate [(3)H] uridine and probably [4-5-(3)H] lysine. Incorporation of [(3)H] thymidine does not occur. The effect of dehydration on root ultrastructure was studied. In embryos dehydrated after 24 h and 72 h of germination, condensation of chromatin is seen and association of elements of rough endoplasmic reticulum with vacuoles and glyoxysomes can be noted. These changes are reversible in drought-resistant embryos and irreversible in drought-sensitive embryos. However, more notable changes than those seen after 24 h can be observed in embryos dehydrated after 72 h of germination: mitochondria and proplastids can not be distinguished with certainty, glyoxysomes fuse and preferably dispose at the periphery of the cell. Rehydration of drought-sensitive embryos causes breakdown in plasma and nuclear membranes, which leads to the loss of cellular compartimentalization. Moreover, the chromatin remains definitively condensed and has lost its function of genetic regulation.