Phosphorylation of signaling phospholipids in Coffea arabica cells

Membrane preparations of Coffea arabica suspension cells were incubated in the presence of 〚³²P〛γ-ATP. After lipid extraction and separation by thin layer chromatography, the following phosphorylated lipids were detected: phosphatidylinositol 4,5 bis-phosphate (PtdIns4,5P₂), lyso-phosphatidylinositol 4-phosphate (LPtdIns4P), phosphatidylinositol 4-phosphate (PtdIns4P), diacylglycerol pyrophosphate (DGPP), lyso-phosphatidic acid (LPA) and phosphatidic acid (PA). This suggests the presence of phosphatidylinositol (EC 2.7.1.67), phosphatidylinositol 4 phosphate (EC 2.7.1.68), diacylglycerol (EC 2.7.1.107) and monoacylglycerol (EC 2.1.1.94) kinases. The activities of these lipid kinases changed during the culture period of the C. arabica cells reaching peak at day 7 of culture; however, enzymatic activities were very low before and after day 7. The behavior of these lipid kinases in the presence of their respective substrates and exogenous substrates such as ATP was characterized. The apparent K_m values for ATP of all the lipid kinase activities were lower than 30 μM. All kinase activities assayed were totally dependent on the presence of Mg²⁺ and were unable to use Mn²⁺ or Ca²⁺ which produced a strong inhibition of all the lipid kinase activities. By using polyclonal antibodies against PtdIns 4-kinase and PtdInsP 5-kinase, we were able to identify at least two putative isoforms for the PtdIns 4-kinase and one for the PtdInsP 5-kinase. In both cases, the correlation of the amount of these proteins with their respective kinase activities depended on the culture cycle. The present work describes for the first time the characterization of the lipid kinases of C. arabica suspension cells, and the correlation of these activities with the culture cycle.     

Chilling-induced changes in membrane fluidity and antioxidant enzyme activities in Coffea arabica L. roots

Exposure of coffee to low temperatures caused growth inhibition, changes in metabolic rates, and membrane alterations. Root tissue exposed to 10 °C evolved significantly lower rates of metabolic heat compared with controls grown at 25 °C, and the values were closely associated with the observed root growth inhibition. Electron paramagnetic resonance spectra of intact tissues showed that the spin probe 5-doxylstearic acid was capable to intercalate within the cellular membrane lipids. Indeed, at the depth of the 5th carbon atoms of the alkyl chains, the nitroxide radical detected more rigid membranes in seedlings exposed to 10 °C compared with 25 °C treated samples. Ascorbate peroxidase and catalase activities did not show appreciable changes under chilling conditions, while guaiacol peroxidase activity increased 55 % compared to the control. On the other hand, glutathione reductase activity decreased, in parallel to a significant decline in the capacity to reduce triphenyl-tetrazolium. Our results showed a marked correlation between lipid peroxidation and root tissue damage, which seemed to be associated with increased membrane rigidity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Factors influencing protoplast isolation from Coffea arabica cells

Cultured plant cells such as Coffea arabica L. cells, accumulate low concentration of secondary metabolites. One way to obtain high-producing plant cell cultures is to prepare single cell clones by using protoplast systems. Identification of limiting factors should facilitate the development of an isolation procedure that can generate adequate yields of intact and viable protoplasts Coffea arabica L. suspension cells. The most suitable conditions for protoplasting were as follows: 6 g of fresh tissue were plasmolysed in 100 ml of K ₃ salts (Nagy & Maliga 1976) containing 0.5 M sucrose for 1 h at 24°C. Then, 1 g of preplasmolysed cells were incubated in 10 ml of cellulase R10 (1%), macerozyme R10 (0.8%) and driselase (0.5%) in preplasmolysis medium. The protoplasts were collected and purified after 15 h of lytic reaction in the dark, at 28°C. More than 75% and 95% of the cells were converted into protoplasts when 5 and 8 day-old suspensions respectively were used for the release step. A number of viable protoplasts ranging from 3.5×10⁶ to 4.6×10⁶ P g^-1 fresh weight was obtained corresponding to an increase by a factor 10 to 15 of the protoplast yield obtained by Acuna & De Pena (1991).Abbreviations BAP 6-benzylamino purine - BSA Bovine Serum Albumin - 2,4-d 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - MES 2-(N-morpholino)ethanesulfonic acid - NAA naphthalene acetic acid - PI propidium iodide - PCV Packed Cell Volume - fw fresh weight     

Shade adaptation of photosynthesis in Coffea arabica

The effect of irradiance on the rate of net photosynthesis was measured for mature leaves of coffee grown under five levels of radiation from 100% to 5% daylight. The rate of light-saturated photosynthesis per unit leaf area (P_Nmax) increased from 2 mol CO₂ m^-2 s^-1 under 5% daylight to 4.4 mol CO₂ m^-2 s^-1 under 100% daylight. The photon flux density (PAR, photosynthetically active radiation) needed for 50% saturation of photosynthesis, as well as the light compensation point, also increased with increasing levels of irradiation during growth. The quantum efficiency of photosynthesis (), measured by the initial slope of the photosynthetic response to increasing irradiance, was greater under shaded growth conditions. The rate of dark respiration was greatest for plants grown in full daylight. On the basis of the increase in the quantal efficiency of photosynthesis and the low light compensation point when grown under shaded conditions, coffee shows high shade adaptation. Plants adjusted to shade by an increased ability to utilize short-term increases in irradiance above the level of the growth irradiance (measured by the difference between photosynthesis at the growth irradiance, P_Ng, and P_Nmax).     

N-methyltransferase activities in suspension cultures of Coffea arabica L.

Suspension cultures of Coffea arabica L. are a useful source for methyltransferase preparations of high activity catalysing the transfer of methylgroups from S-adenosyl-L-methionine to 7-methylxanthine and to theobromine producing theobromine and caffeine respectively. Surprisingly, these enzyme activities are not correlated with the availability of precursors during a culture cycle. They are highest in the growth phase when supply of precursors is reduced. Mixed substrate experiments and time dependent changes in the enzyme activity ratio provide indirect evidence for the existence of two separate enzymes catalysing the final methylations in caffeine biosynthesis.     

Modification of the embryogenic response of Coffea arabica by the nitrogen source

Somatic embryogenesis was induced in coffee from in vitro cultured plants as starting material and the faster response obtained allowed lines from selected plants to be generated more quickly. In contrast to other systems, where embryos take 2 or 3 months to develop, globular embryos were obtained after 3 weeks. The optimum nitrogen concentrations for embryogenesis were between 3.75 and 15 mM nitrogen with a nitrate/ammonium molar ratio of 2:1 or 1:2.     

Polyamines modify the components of phospholipids-based signal transduction pathway in Coffea arabica L. cells.

Recent results, fundamentally obtained from animal tissues, suggest that polyamines (Pas), essential compounds for the growth and development of all life organisms, may interact with a signal transduction cascade. Because Pas are highly positive charged compounds, their binding with phospholipids involved in signal transduction is likely to be the case. In this work, the in vivo effect of Pas on some important components of phospholipid signal transduction pathway was studied, by the first time, in plant tissue. Endogenous Pas content varied during the culture cycle of Coffea arabica cells: putrescine (Put) levels increased at the end of the stationary phase, both spermidine (Spd) and spermine (Spm) accumulated at the beginning of the linear growth phase. Cells that were incubated with Put presented a significant increase in phospholipase D (PLD) (EC: 3.1.4.4) activity, phospholipase C (PLC) (EC: 3.1.4.3) activity decreased, and the effect on lipid kinases was less marked. However, the incubation of the cells with Spd and Spm significantly stimulated the lipid kinases activities, fundamentally increased the formation of phosphatidyl inositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2), while the effect on PLC and PLD activities was minor when compared with the cells treated with Put. The results presented here suggest that Pas may modulate the cellular signal of C. arabica cells by differentially affecting components of the phospholipid cascade.     

Identification of novel and conserved microRNAs in Coffea canephora and Coffea arabica

As microRNAs (miRNAs) are important regulators of many biological processes, a series of small RNAomes from plants have been produced in the last decade. However, miRNA data from several groups of plants are still lacking, including some economically important crops. Here microRNAs from Coffea canephora leaves were profiled and 58 unique sequences belonging to 33 families were found, including two novel microRNAs that have never been described before in plants. Some of the microRNA sequences were also identified in Coffea arabica that, together with C. canephora, correspond to the two major sources of coffee production in the world. The targets of almost all miRNAs were also predicted on coffee expressed sequences. This is the first report of novel miRNAs in the genus Coffea, and also the first in the plant order Gentianales. The data obtained establishes the basis for the understanding of the complex miRNA-target network on those two important crops.     

Homeologous gene expression in response to growing temperature in a recent Allopolyploid (Coffea arabica L.)

Allopolyploidy is considered as a major factor contributing to speciation, diversification, and plant ecological adaptation. In particular, the expression of duplicate genes (homeologs) can be altered leading to functional plasticity and to phenotypic novelty. This study investigated the influence of growing temperatures on homeologous gene expression in Coffea arabica L., a recent allopolyploid involving 2 closely related diploid parental species. The relative expression of homeologs of 13 genes all located in the same genomic region was analyzed using an SNP ratio quantification method based on dideoxy-terminated sequences of cDNA amplicons. The relative expression of homeologous genes varied depending on the gene, the organ, and the growing condition. Nevertheless, expression of both homeologs was always detected (i.e., no silencing). Although the growing conditions were suitable for one or other of the parental species, neither subgenome appeared preferentially expressed. Furthermore, relative homeologous expression showed moderate variations across organs and conditions and appeared uncorrelated between adjacent genes. These results indicate the absence of signs of subfunctionalization suggesting C. arabica has not undergone noticeable diploidization. Furthermore, these results suggest that the expression of homeologous genes in C. arabica is regulated by a shared trans-regulation mechanism acting similarly on the 2 subgenomes and that the observed biases in the relative homeolog expression may result from cis fine-scale factors.     

Aluminium differentially modifies lipid metabolism from the phosphoinositide pathway in Coffea arabica cells

The effect of aluminium (Al) on phosphoinositide-specific phospholipase C (PLC) and lipid kinase activities was examined in a cellular suspension of coffee. Two main effects were seen when cells were treated with AlCl3. In periods as short as 1 minute, Al-exposed cells increased the activity of PLC and IP3 formation up to two fold. Over longer periods PLC activity was inhibited by more than 50%. The activity of phosphatidylinositol 4-kinase (Pl 4-K), phosphatidylinositol phosphate 5-kinase (PIP 5-K) and diacylglycerol kinase increased when cells were incubated in the presence of different concentrations of AlCl3. The present study reports for the first time that Al may have different effects on the Pl-signaling pathway depending on the time of exposure. Our results strongly support the hypothesis that Al disrupts the metabolism of membrane phospholipids regulating not only PLC but also other enzymes that have key roles in signal-transduction pathways.     

Single-locus inheritance in the allotetraploid Coffea arabica L. and interspecific hybrid C. arabica x C. canephora

Molecular cytogenetic analysis has indicated that Coffea arabica is an amphidiploid formed from the hybridization between two closely related diploid progenitor species, C. canephora and C. eugenioides. Our aim was to determine the mode of inheritance in C. arabica and in a tetraploid interspecific hybrid (called arabusta) between C. arabica and C. canephora as revealed by segregation analyses of restriction fragment length polymorphism (RFLP) loci markers. The observed RFLP allele segregations in an F(2) progeny of C. arabica conform to disomic inheritance as expected, with regular bivalent pairing of homologous chromosomes in the F1 hybrid. In contrast, RFLP loci followed tetrasomic inheritance in the arabusta interspecific hybrid, although bivalents have been reported to predominate greatly at meiosis in its hybrid. These results suggest that homologous chromosomes do not pair in C. arabica, not as a consequence of structural differentiation, but because of the functioning of pairing regulating factors. Moreover, the arabusta hybrid seems to offer the possibility of gene exchange between the homologous genomes.     

Optimised DNA delivery into Coffea arabica suspension culture cells by particle bombardment

An optimised bombardment protocol to introduce DNA into Coffea arabica suspension culture cells was developed. Osmotic preconditioning of cells and physical bombardment parameters including Helium pressure, gap and target distances affecting DNA delivery were evaluated by monitoring transient expression of the uidA gene driven by the CaMV35S promoter. The highest transient GUS expression was obtained when cells were subjected to a 0.5 M mannitol–sorbitol pre-treatment 4 h prior to bombardment and a Helium pressure of 1550 psi, a 9-mm gap distance and 12 cm target distance as physical bombardment parameters. The optimised protocol was tested with two coffee promoters: -tubulin and arabicin, which presented similar activity to the CaMV35S promoter in suspension culture cells by fluorometric GUS assays. GUS expression was reduced in bombarded tissue culture leaves, and only the CaMV35S and arabicin promoters showed histochemical activity in coffee endosperms. This is the first report of optimization of particle bombardment on coffee suspension culture cells, equivalent CaMV35S activity for a coffee promoter and transient -glucoronidase expression in coffee endo-sperms.     

Photoinhibition and recovery of photosynthesis in Coffea arabica and C. canephora

Photosynthetic parameters were determined in disks from leaves of C. arabica cv. Red Catuaí and C. canephora cv. Kouillou grown in the field. Kouillou showed a relatively higher irradiance requirement for saturating photosynthesis, lower chlorophyll (Chl) content, and higher Chl a/b ratio than Catuaí. Photoinhibition of photosynthesis under bright irradiance was manifested by decreases in maximum photochemical efficiency (evaluated by the variable to maximum fluorescence ratio, Fv/Fm), as a consequence of an increased initial and a quenched maximum fluorescence. Restoration of Fv/Fm following photoinhibition in low irradiance was faster in Kouillou than in Catuaí. Chloramphenicol both accelerated photoinhibition (mainly in Kouillou) and blocked its recovery for at least 190 min in either cultivar. Photosynthetic oxygen evolution under photoinhibitory conditions was decreased by chloramphenicol; in control leaf disks this decrease was only observed in C. arabica, but with a rapid recovery within 90 min of low irradiance exposure. In both coffee cultivars, the depressed photochemical efficiency of photosystem 2 was not accompanied by a concomitant lowering in oxygen evolution during reversal from photoinhibition.     

Photoinhibition and recovery of photosynthesis in Coffea arabica and C. canephora

Photosynthetic parameters were determined in disks from leaves of C. arabica cv. Red Catuaí and C. canephora cv. Kouillou grown in the field. Kouillou showed a relatively higher irradiance requirement for saturating photosynthesis, lower chlorophyll (Chl) content, and higher Chl a/b ratio than Catuaí. Photoinhibition of photosynthesis under bright irradiance was manifested by decreases in maximum photochemical efficiency (evaluated by the variable to maximum fluorescence ratio, Fv/Fm), as a consequence of an increased initial and a quenched maximum fluorescence. Restoration of Fv/Fm following photoinhibition in low irradiance was faster in Kouillou than in Catuaí. Chloramphenicol both accelerated photoinhibition (mainly in Kouillou) and blocked its recovery for at least 190 min in either cultivar. Photosynthetic oxygen evolution under photoinhibitory conditions was decreased by chloramphenicol; in control leaf disks this decrease was only observed in C. arabica, but with a rapid recovery within 90 min of low irradiance exposure. In both coffee cultivars, the depressed photochemical efficiency of photosystem 2 was not accompanied by a concomitant lowering in oxygen evolution during reversal from photoinhibition.