Contribution of C3 carboxylation to the circadian rhythm of carbon dioxide uptake in a Crassulacean acid metabolism plant Kalanchoe daigremontiana.
During the endogenous circadian rhythm of carbon dioxide uptake in continuous light by a Crassula cean acid metabolism plant, Kalanchoe daigremontiana, the two carboxylating enzymes, phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco), are active simultaneously, although, until now, only the role of PEPC in generating the rhythm has been acknowledged. According to the established model, the rhythm is primarily regulated at the PEPC activity level, modulated by periodic compartmentation of its inhibitor, malate, in the vacuole and controlled by tension/relaxation of the tonoplast. However, the circadian accumulation of malic acid (the main indicator of PEPC activity) dampened significantly within the first few periods without affecting the rhythm's amplitude. Moreover, the amount of malate accumulated during a free-running oscillation was several-fold lower than the amount expected if PEPC were the key carboxylating enzyme, based on a 1:1 stoichiometry of CO(2) and malate. Together with the observation that rates of CO(2) uptake under continuous light were higher than in darkness, the evidence shows that C(3) carboxylation greatly contributes to the generation of rhythmic CO(2) uptake in continuous light in this 'obligate' CAM plant. Because the shift from predominantly CAM to predominantly C(3) carboxylation is smooth and does not distort the trajectory of the rhythm, its control probably arises from a robust network of oscillators, perhaps also involving stomata. (+info)
Functional subsets of the virB type IV transport complex proteins involved in the capacity of Agrobacterium tumefaciens to serve as a recipient in virB-mediated conjugal transfer of plasmid RSF1010.
The virB-encoded type IV transport complex of Agrobacterium tumefaciens mediates the transfer of DNA and proteins into plant cells, as well as the conjugal transfer of IncQ plasmids, such as RSF1010, between Agrobacterium strains. While several studies have indicated that there are physical interactions among the 11 VirB proteins, the functional significance of the interactions has been difficult to establish since all of the proteins are required for substrate transfer. Our previous studies, however, indicated that although all of the VirB proteins are required for the capacity of a strain to serve as an RSF1010 donor, only a subset of these proteins in the recipient is necessary to increase the conjugal frequency by 3 to 4 logs. The roles of particular groups of VirB proteins in this increased recipient activity were examined in the study reported here. Examination of the expression of subgroups of virB genes revealed that translation of virB6 is necessary for expression of downstream open reading frames. Expression of limited subsets of the VirB proteins in a recipient strain lacking the Ti plasmid revealed that the VirB7 to VirB10 proteins yield a subcomplex that is functional in the recipient assay but that the VirB1 to VirB4 proteins, as a group, dramatically increase this activity in strains expressing VirB7 to VirB10. Finally, the membrane distribution and cross-linking patterns of VirB10, but not of VirB8 or VirB9, in a strain expressing only VirB7 to VirB10 are significantly altered compared to the patterns of the wild type. These characteristics are, however, restored to the wild-type status by coexpression of VirB1 to VirB3. Taken together, these results define subsets of type IV transport complex proteins that are critical in allowing a strain to participate as a recipient in virB-mediated conjugal RSF1010 transfer. (+info)
Movement of water from old to young leaves in three species of succulents.
A hypothetical adaptive response of succulent plants to drought-stress is the redistribution of water from old to young leaves. We examined the effects of possible movement of water from old to young leaves in three succulent species, Carpobrotus edulis (weak CAM-inducible), Kalanchoe tubiflora (CAM) and Sedum spectabile (possibly a CAM-cycler or CAM-inducible). Old leaves were removed from plants, and photosynthesis, transpiration, f. wt : d. wt ratios, diurnal acid fluctuations, stomatal conductance and internal CO2 concentrations of the remaining young leaves were measured during drought-stress. Comparison was made with plants retaining old leaves. There was no evidence that water moved from old to young leaves during drought-stress as previously hypothesized. Only in drought-stressed plants of K. tubiflora, were photosynthetic and transpiration rates of young leaves greater on shoots with old leaves removed compared with attached. There was a trend in all species for greater fluctuations in acidity in young leaves on shoots that lacked older leaves. For two of the three species studied, the f. wt : d. wt ratios of young leaves were greater under drought-stress, on shoots with old leaves removed than with them attached. Absence of old leaves may reduce competition for water with young leaves, which consequently have higher water content and greater photosynthetic rates. (+info)
Day-night changes of energy-rich compounds in crassulacean acid metabolism (CAM) species utilizing hexose and starch.
BACKGROUND AND AIMS: Plants with crassulacean acid metabolism (CAM) can be divided into two groups according to the major carbohydrates used for malic acid synthesis, either polysaccharide (starch) or monosaccharide (hexose). This is related to the mechanism and affects energy metabolism in the two groups. In Kalanchoe pinnata and K. daigremontiana, which utilize starch, ATP-dependent phosphofructokinase (tonoplast inorganic pyrophosphatase) activity is greater than inorganic pyrophosphate-dependent phosphofructokinase (tonoplast adenosine triphosphatase) activity, but the reverse is the case in pineapple (Ananas comosus) utilizing hexose. To test the hypothesis that the energy metabolism of the two groups differs, day-night changes in the contents of ATP, ADP, AMP, inorganic phosphate (Pi), phosphoenolpyruvate (PEP) and inorganic pyrophosphate (PPi) in K. pinnata and K. daigremontiana leaves and in pineapple chlorenchyma were analysed. METHODS: The contents of energy-rich compounds were measured spectrophotometrically in extracts of tissue sampled in the light and dark, using potted plants, kept for 15 d before the experiments in a growth chamber. KEY RESULTS: In the three species, ATP content and adenylate energy charge (AEC) increased in the dark and decreased in the light, in contrast to ADP and AMP. Changes in ATP and AEC were greater in Kalanchoe leaves than in pineapple chlorenchyma. PPi content in the three species increased in the dark, but on illumination it decreased rapidly and substantially, remaining little changed through the rest of the light period. Pi content of Kalanchoe leaves did not change between dark and light, whereas Pi in pineapple chlorenchyma increased in the dark and decreased in the light, and the changes were far greater than in Kalanchoe leaves. Light-dark changes in PEP content in the three species were similar. CONCLUSIONS: These results corroborate our hypothesis that day-night changes in the contents of energy-rich compounds differ between CAM species and are related to the carbohydrate used for malic acid synthesis. (+info)
Purification and partial characterization of a lectin from the fresh leaves of Kalanchoe crenata (Andr.) Haw.
A haemagglutinating protein from the saline extracts of Kalanchoe crenata leaves, which agglutinate all human blood types, was purified to homogeneity by ion-exchange chromatography on a DEAE-Cellulose column followed by gel filtration on a Sephadex G-100 column. The purified protein showed one band, both in non-denaturing PAGE and SDS-PAGE. The M(r) that was determined by SDS-PAGE was 44,000 Da and that estimated from gel filtration was 47,000. Treatment of the haemagglutinating protein with 5 mM EDTA diminished the haemagglutinating activity to 50% of the original level. The addition of divalent cations, 10 mM Mg(2+), 10 mM Mn(2+), or 10mM Ba(2+), totally restored and enhanced the activity. The protein showed maximum activity over the 3-7 pH range and was heat-resistant. It was also a glycoprotein containing about 1.5% carbohydrate. (+info)
Osa protein constitutes a strong oncogenic suppression system that can block vir-dependent transfer of IncQ plasmids between Agrobacterium cells and the establishment of IncQ plasmids in plant cells.
The osa (oncogenic suppressive activity) gene of the IncW group plasmid pSa is sufficient to suppress tumorigenesis by Agrobacterium tumefaciens. osa confers oncogenic suppression by inhibiting VirE2 protein export. This result is similar, but not identical, to that of oncogenic suppression by the IncQ plasmid RSF1010. We conducted a series of experiments to compare oncogenic suppression by these two systems. Agrobacterium strains harboring plasmids containing osa are more able to effect oncogenic suppression than are similar strains containing various RSF1010 derivatives. When osa is present within a donor Agrobacterium strain that also carries a derivative of RSF1010, the transfer of RSF1010 derivatives to recipient bacteria and their establishment in plants are blocked. Oncogenic suppression is still effected when the osa gene is integrated into the Agrobacterium chromosome, suggesting that it is the osa gene product that is active in suppression and that suppression does not require a protein-nucleic acid intermediate like that described for IncQ plasmids. Extracellular complementation experiments with tobacco leaf disks indicated that Osa blocks stable transfer of RSF1010 to plant cells by inhibiting transfer of VirE2, which is essential for the transfer of RSF1010 into plant cells, and not by inhibiting the actual transfer of RSF1010 itself. Our results suggest that Osa and RSF1010 cause oncogenic suppression by using different mechanisms. (+info)
Molecular characterization of the Agrobacterium tumefaciens DNA transfer protein VirB6.
The VirB proteins of Agrobacterium tumefaciens assemble a T-pilus and a type IV secretion (T4S) apparatus for the transfer of DNA and proteins to plant cells. VirB6 is essential for DNA transfer and is a polytopic integral membrane protein with at least four membrane-spanning domains. VirB6 is postulated to function in T-pilus biogenesis and to be a component of the T4S apparatus. To identify amino acids required for VirB6 function, random mutations were introduced into virB6, and mutants that failed to complement a deletion in virB6 in tumour formation assays were isolated. Twenty-one non-functional mutants were identified, eleven of which had a point mutation that led to a substitution in a single amino acid. Characterization of the mutants indicated that the N-terminal large periplasmic domain and the transmembrane domain TM3 are required for VirB6 function. TM3 has an unusual sequence feature in that it is rich in bulky hydrophobic amino acids. This feature is found conserved in the VirB6 family of proteins. Studies on the effect of VirB6 on other VirB proteins showed that the octopine Ti-plasmid VirB6, unlike its nopaline Ti-plasmid counterpart, does not affect accumulation of VirB3 and VirB5, but has a strong negative effect on the accumulation of the VirB7-VirB7 dimer. Using indirect immunofluorescence microscopy the authors recently demonstrated that VirB6 localizes to a cell pole in a VirB-dependent manner. Mutations identified in the present study did not affect polar localization of the protein or the formation of the VirB7-VirB7 dimer. A VirB6-GFP fusion that contained the entire VirB6 ORF did not localize to a cell pole in either the presence or the absence of the other VirB proteins. IMF studies using dual labelling demonstrated that VirB6 colocalizes with VirB3 and VirB9, and not with VirB4, VirB5 and VirB11. These results support the conclusion that VirB6 is a structural component of the T4S apparatus. (+info)
Lon protease of the alpha-proteobacterium Agrobacterium tumefaciens is required for normal growth, cellular morphology and full virulence.
The ATP-dependent Lon (La) protease is ubiquitous in nature and regulates a diverse set of physiological responses in bacteria. In this paper a lon mutant of the alpha-proteobacterium Agrobacterium tumefaciens C58 has been characterized. Unlike lon mutants of Escherichia coli, the lon mutant of A. tumefaciens grows very slowly, is not filamentous and exhibits normal resistance to UV irradiation. The mutant retains motility and chemotaxis, produces apparently normal amounts of exopolysacchride, but displays severe defects in cell morphology, with 80 % of the mutant cells appearing Y-shaped. Lon protease of A. tumefaciens shares high homology with its counterparts in E. coli and in Sinorhizobium meliloti, and functionally complements an E. coli lon mutant for defects in morphology and RcsA-mediated regulation of capsular polysaccharide production. Mutations at sites of Lon(At) corresponding to the ATP-binding site and the active site serine of the E. coli Lon protease abolish complementation of phenotypes of the A. tumefaciens and E. coli lon mutants. The nucleotide sequence upstream of A. tumefaciens lon contains an element similar to the consensus sigma(32) heat-shock promoter of E. coli. Northern and Western blot analyses indicated that expression of lon is induced by elevated temperature, albeit to a much lower level than that of groEL. The lon mutant is highly attenuated for virulence, suggesting that Lon may be required for the proper expression, assembly or function of the VirB/D4-mediated T-DNA transfer system. (+info)