Purification and characterisation of a jacalin-related, coleoptile specific lectin from Hordeum vulgare.

A plant lectin was isolated from barley (Hordeum vulgare) coleoptiles using acidic extraction and different chromatographic methods. Sequencing of more than 50% of the protein sequence by Edman degradation confirmed a full-length cDNA clone. The subsequently identified open reading frame encodes for a 15 kDa protein which could be found in the soluble fraction of barley coleoptiles. This protein exhibited specificity towards mannose sugar and is therefore, accordingly named as Horcolin (Hordeum vulgare coleoptile lectin). Database searches performed with the Horcolin protein sequence revealed a sequence and structure homology to the lectin family of jacalin-related lectins. Together with its affinity towards mannose, Horcolin is now identified as a new member of the mannose specific subgroup of jacalin-related lectins in monocot species. Horcolin shares a high amino acid homology to the highly light-inducible protein HL#2 and, in addition to two methyl jasmonic acid-inducible proteins of 32.6 and 32.7 kDa where the jasmonic acid-inducible proteins are examples of bitopic chimerolectins containing a dirigent and jacalin-related domain. Immunoblot analysis with a cross-reactive anti-HL#2 antibody in combination with Northern blot analysis of the Horcolin cDNA revealed tissue specific expression of Horcolin in the coleoptiles. The function of Horcolin is discussed in the context of its particular expression in coleoptiles and is then compared to other lectins, which apparently share a related response to biotic or abiotic stress factors.

Investigations on epiphytic living Pseudomonas species from Malus domestica with an antagonistic effect to Venturia inaequalis on isolated plant cuticle membranes.

In order to understand better the survival and mutual interaction of epiphytic bacteria and fungi on apple plants, bacteria collected from these plants were cultivated on intact adaxial, stoma free cuticle membranes originally obtained from apple. The bacteria were labelled with luciferase genes from Vibrio harveyi in order to follow up their development and activity on the isolated cuticles. Our finding was that the epiphytic bacteria can have access to nutrients below the cuticle without causing damage to these cuticular membranes. Bacterial proteins may enable this nutrient mobilization and we found, indeed, that more than 46 proteins that must have been delivered by the bacteria in response to interaction with the cuticles as they could be found below the cuticle membrane. Eight major representatives of this group of external proteins have been sequenced with electron spray quadrupole time of flight mass spectrometry and subsequently identified by data base homology search as a flagellin, a porin type protein and proteins that are involved in amino acid recruitment and metabolism.

Early light-induced proteins protect Arabidopsis from photooxidative stress.

The early light-induced proteins (ELIPs) belong to the multigenic family of light-harvesting complexes, which bind chlorophyll and absorb solar energy in green plants. ELIPs accumulate transiently in plants exposed to high light intensities. By using an Arabidopsis thaliana mutant (chaos) affected in the posttranslational targeting of light-harvesting complex-type proteins to the thylakoids, we succeeded in suppressing the rapid accumulation of ELIPs during high-light stress, resulting in leaf bleaching and extensive photooxidative damage. Constitutive expression of ELIP genes in chaos before light stress resulted in ELIP accumulation and restored the phototolerance of the plants to the wild-type level. Free chlorophyll, a generator of singlet oxygen in the light, was detected by chlorophyll fluorescence lifetime measurements in chaos leaves before the symptoms of oxidative stress appeared. Our findings indicate that ELIPs fulfill a photoprotective function that could involve either the binding of chlorophylls released during turnover of pigment-binding proteins or the stabilization of the proper assembly of those proteins during high-light stress.

Non-invasive determination of plant-associated bacteria in the phyllosphere of plants.

Epiphytic living Pseudomonas strains isolated from different Malus domestica cultivars were transformed with two reporter genes [green fluorescent protein (gfp) and luciferase (luxAB)]. The establishment and distribution of these bacteria on sterile, in vitro-propagated, and thus genetically identical, Malus domestica plants were continuously analysed with a cooled, back-illuminated, charge-coupled-device (CCD) camera system. The combination of the assessment of bioluminescence and the use of a CCD camera offer an intriguing method to study, non-invasively and in real time, plant-microbe interactions as well as the colonization of the phyllosphere by microorganisms. Here we report on the applicability and sensitivity of the method with the goal to investigate quantitatively the interaction of symbiotic and pathogenic microorganisms with the corresponding host plant. It will be shown that the three bacterial isolates of the genus Pseudomonas studied, differ considerably with respect to their establishment on the host plants. It will also be shown that the chosen host apple variety has an impact on the activity of the bacterial cultivars. Analysis by a laser scanning fluorescence microscope provides the first evidence for the mode by which the epiphytic microorganisms interact with the plant.

Double mutation cpSRP43--/cpSRP54-- is necessary to abolish the cpSRP pathway required for thylakoid targeting of the light-harvesting chlorophyll proteins.

Biochemical and genetic studies have established that the light-harvesting chlorophyll proteins (LHCPs) of the photosystems use the cpSRP (chloroplast signal recognition particle) pathway for their targeting to thylakoids. Previous analyses of single cpSRP mutants, chaos and ffc, deficient in cpSRP43 and cpSRP54, respectively, have revealed that half of the LHCPs are still integrated into the thylakoid membranes. Surprisingly, the effects of both mutations are additive in the double mutant ffc/chaos described here. This mutant has pale yellow leaves at all stages of growth and drastically reduced levels of all the LHCPs except Lhcb 4. Although the chloroplasts have a normal shape, the thylakoid structure is affected by the mutation, probably as a consequence of reduction of all the LHCPs. ELIPs (early light-inducible proteins), nuclear-encoded proteins related to the LHCP family and inducible by light stress, were also drastically reduced in the double mutant. However, proteins targeted by other chloroplastic targeting pathways (DeltapH, Sec and spontaneous pathways) accumulated to similar levels in the wild-type and the double mutant. Therefore, the near total loss of LHCPs and ELIPs in the double mutant suggests that cpSRP is the predominant, if not exclusive, targeting pathway for these proteins. Phenotypic analysis of the double mutant, compared to the single mutants, suggests that the cpSRP subunits cpSRP43 and cpSRP54 contribute to antenna targeting in an independent but additive way.