ABA inhibits embryo cell expansion and early cell division events during coffee (Coffea arabica 'Rubi') seed germination

Background and Aims

Coffee seed germination represents an interplay between the embryo and the surrounding endosperm. A sequence of events in both parts of the seed determines whether germination will be successful or not. Following previous studies, the aim here was to further characterize the morphology of endosperm degradation and embryo growth with respect to morphology and cell cycle, and the influence of abscisic acid on these processes.

Methods

Growth of cells in a fixed region of the axis was quantified from light micrographs. Cell cycle events were measured by flow cytometry and by immunocytochemistry, using antibodies against β-tubulin. Aspects of the endosperm were visualized by light and scanning electron microscopy.

Key Results

The embryonic axis cells grew initially by isodiametric expansion. This event coincided with reorientation and increase in abundance of microtubules and with accumulation of β-tubulin. Radicle protrusion was characterized by a shift from isodiametric expansion to elongation of radicle cells and further accumulation of β-tubulin. Early cell division events started prior to radicle protrusion. Abscisic acid decreased the abundance of microtubules and inhibited the growth of the embryo cells, the reorganization of the microtubules, DNA replication in the embryonic axis, the formation of a protuberance and the completion of germination. The endosperm cap cells had smaller and thinner cell walls than the rest of the endosperm. Cells in the endosperm cap displayed compression followed by loss of cell integrity and the appearance of a protuberance prior to radicle protrusion.

Conclusions

Coffee seed germination is the result of isodiametric growth of the embryo followed by elongation, at the expense of integrity of endosperm cap cells. The cell cycle, including cell division, is initiated prior to radicle protrusion. ABA inhibits expansion of the embryo, and hence subsequent events, including germination.Key words: Abscisic acid, β-tubulin, Coffea arabica, coffee seed, cell morphology, germination, microtubules     

Abscisic acid controls embryo growth potential and endosperm cap weakening during coffee (<Emphasis Type=Italic>Coffea arabica</Emphasis> cv. Rubi) seed germination

The mechanism and regulation of coffee seed germination were studied in Coffea arabica L. cv. Rubi. The coffee embryo grew inside the endosperm prior to radicle protrusion and abscisic acid (ABA) inhibited the increase in its pressure potential. There were two steps of endosperm cap weakening. An increase in cellulase activity coincided with the first step and an increase in endo--mannanase (EBM) activity with the second step. ABA inhibited the second step of endosperm cap weakening, presumably by inhibiting the activities of at least two EBM isoforms and/or, indirectly, by inhibiting the pressure force of the radicle. The increase in the activities of EBM and cellulase coincided with the decrease in the force required to puncture the endosperm and with the appearance of porosity in the cell walls as observed by low-temperature scanning electronic microscopy. Tissue printing showed that EBM activity was spatially regulated in the endosperm. Activity was initiated in the endosperm cap whereas later during germination it could also be detected in the remainder of the endosperm. Tissue printing revealed that ABA inhibited most of the EBM activity in the endosperm cap, but not in the remainder of the endosperm. ABA did not inhibit cellulase activity. There was a transient rise in ABA content in the embryo during imbibition, which was likely to be responsible for slow germination, suggesting that endogenous ABA also may control embryo growth potential and the second step of endosperm cap weakening during coffee seed germination.     

Germination of Coffee Seeds (Coffea arabica L. cv. Mundo Novo)

The life-span of coffee seeds is extended when seeds are storedwith high moisture content. Germination in darkness is alwayshigher than in the light. Exogenous gibberellic acid and abscisicacid inhibit germination while kinetin reverses this inhibitoryeffect. Low levels of endogenous gibberellin- and abscisic acid-likeand high levels of cytokinin-like substances favour germinationwhile the opposite combination of regulators delays germination.     

Changes in some characteristics between the wild and Al-tolerant coffee (Coffea arabica L.) cell line

An aluminium (Al)-tolerant cell line (LAMt) of coffee (Coffea arabica L.) was obtained from a cell suspension culture and biochemically and molecularly characterized in an MS medium at half ionic strength and low pH. LAMt grew 30% more than the control line (susceptible to Al) in the presence of different concentrations of Al, showed a lower free Al concentration in the medium and had higher phospholipase C specific activity (80%). Membrane integrity of the LAMt was 50% greater than the control line when both were incubated in the presence of different Al concentrations (measured by Evans Blue uptake). Finally, the use of microsatellite primers revealed no difference in the DNA pattern of both cell lines.     

Biochemical and genomic analysis of sucrose metabolism during coffee (Coffea arabica) fruit development

Sucrose metabolism and the role of sucrose synthase were investigated in the fruit tissues (pericarp, perisperm, and endosperm) of Coffea arabica during development. Acid invertase, sucrose phosphate synthase, and sucrose synthase activities were monitored and compared with the levels of sucrose and reducing sugars. Among these enzymes, sucrose synthase showed the highest activities during the last stage of endosperm and pericarp development and this activity paralleled closely the accumulation of sucrose in these tissues at this stage. Carbon partitioning in fruits was studied by pulse-chase experiments with (14)C-sugars and revealed high rates of sucrose turnover in perisperm and endosperm tissues. Additional feeding experiments with (14)CO(2) showed that leaf photosynthesis contributed more to seed development than the pericarp in terms of photosynthate supply to the endosperm. Sugar analysis, feeding experiments, and histological studies indicated that the perisperm plays an important role in this downloading process. It was observed that the perisperm presents a transient accumulation of starch which is degraded as the seed develops. Two full-length cDNAs (CaSUS1 and CaSUS2) and the complete gene sequence of the latter were also isolated. They encode sucrose synthase isoforms that are phylogenetically distinct, indicating their involvement in different physiological functions during cherry development. Contrasting expression patterns were observed for CaSUS1 and CaSUS2 in perisperm, endosperm, and pericarp tissues: CaSUS1 mRNAs accumulated mainly during the early development of perisperm and endosperm, as well as during pericarp growing phases, whereas those of CaSUS2 paralleled sucrose synthase activity in the last weeks of pericarp and endosperm development. Taken together, these results indicate that sucrose synthase plays an important role in sugar metabolism during sucrose accumulation in the coffee fruit.     

Brevipalpus mites associated with coffee plants (Coffea arabica and C. canephora) in Chiapas,Mexico

Experimental and Applied Acarology - Tenuipalpid mites of the genus Brevipalpus are of significant economic and quarantine importance in agriculture. They can damage and vector phytopathogenic...     

Inhibition of caffeine biosynthesis in tea (Camellia sinensis) and coffee (Coffea arabica) plants by ribavirin

The effects of ribavirin, an inhibitor of inosine-5'-monophosphate (IMP) dehydrogenase, on [8-(14)C]inosine metabolism in tea leaves, coffee leaves and coffee fruits were investigated. Incorporation of radioactivity from [8-(14)C]inosine into purine alkaloids, such as theobromine and caffeine, guanine residues of RNA, and CO(2) was reduced by ribavirin, while incorporation into nucleotides, including IMP and adenine residues of RNA, was increased. The results indicate that inhibition of IMP dehydrogenase by ribavirin inhibits both caffeine and guanine nucleotide biosynthesis in caffeine-forming plants. The use of IMP dehydrogenase-deficient plants as a potential source of good quality caffeine-deficient tea and coffee plants is discussed.     

In silico characterization of putative members of the coffee (Coffea arabica) ethylene signaling pathway

The plant hormone ethylene is involved in several developmental and physiological processes in plants, including senescence, fruit ripening and organ abscission, as well as in biotic and abiotic stress responses. Initiation of these processes involves complex regulation of both ethylene biosynthesis and the ability of cells to perceive the hormone and respond in an appropriate manner, a process which is regulated both spatially and temporally. Ethylene is a gaseous hormone whose sensitivity is a key factor to limiting its response in target cells. We made a search of the Coffee Expressed Sequence Tag (CAFEST) database for expressed sequence tags related to known elements of the ethylene signaling pathway. Sequences showing a reliable similarity were clusterized, annotated and analyzed for conserved domains. Multiple alignments comprising the sequences that we found and sequences of ethylene signaling elements from other species were made, and their phylogeny was assessed by phylogenetic trees constructed with the MEGA4 software. The expression profile was assessed by in silico Northern blot analysis performed using the Cluster and TreeView programs. The CAFEST database was found to have a large number of sequences related to previously described ethylene signaling pathway elements, allowing identification of putative members from almost every step of this pathway. The phylogenetic trees demonstrated high similarity between the sequences found in the CAFEST and those from other species, and the electronic Northern blot analysis detected their expression in various tissues, development stages and stress conditions.     

3-Methylbutanoyl and 3-methylbut-2-enoyl disaccharides from green coffee beans (Coffea arabica)

The aerial parts of Teucrium oliverianum yielded two neo-clerodane diterpenoids, teucrolin F and G, together with the known teucrolin E. The previously proposed structure for teucrolin E was revised so that it contains a tetrahydrofuran ring instead of an oxetane ring. This was based on analysis of the NMR spectroscopic data of its diacetate, including its NOE spectra. In addition, the structural assignments of the new diterpenoids were based on 1H and 13C NMR spectroscopic studies, mainly 2D NMR experiments, including homonuclear and heteronuclear correlations.     

Long term culture and caffeine production of immobilized coffee (Coffea arabica) L. cells in polyurethane foam

Coffee (Coffea arabica L.) cells could be immobilized in polyurethane foam and subcultured repeatedly for a long time. Four phases were observed for cell growth and caffeine production, I; immobilization, II; growth, III; caffeine production, IV; regrowth. Their periods were influenced by the number of foam particles. Especially in the phase III, the immobilized cells produced a relatively large amount of caffeine in the subculture numbers 5–8 (34 cubes) when the fresh weight of the immobilized cells decreased despite culture in growth medium (DK medium). Caffeine production appeared to have a negative correlation with the growth of the immobilized cells throughout the subcultures.This paper is Part 71 in the series of Studies on Plant Tissue Cultures. For Part 70, see Orihara Y, Furuya T. submitted for publication.     

Physicochemical and biological properties of the coffee (Coffea arabica) rhizosphere suppress the root-knot nematode Meloidogyne exigua

ABSTRACT

We investigated the properties of rhizospheric soils infested with root-knot nematode (RKN) Meloidogyne exigua in 17 coffee (Coffea arabica) farms from the Southern region of Minas Gerais, Brazil. Physicochemical (pH, clay and organic matter) and biological properties (RKN parasites and microbiota volatile toxicity on M. exigua) were correlated with the number of second-stage juveniles (J₂) and the egg hatching of M. exigua extracted from those rhizospheres. In the five most suppressive farms, the number of J₂ was less than 50/100?g of soil and the egg hatching was significantly low. The bacterium Pasteuria penetrans was found in four of the most suppressive farms with an average of 30% of J₂ infected with endospores. By using in vitro experiments the microbiota volatiles emitted from the most suppressive soils killed more than 83% of the J₂. Additionally, volatiles produced by Fusarium oxysporum, Cladosporium sp. and Syncephalastrum sp. isolated from M. exigua eggs, significantly killed the J₂. Identification of nematicidal compounds from the soils by GC-MS supported the strong involvement of the microbiota volatile toward RKN suppressiveness. Clay percentage and pH were similar in farms with the most suppressive soils (42.5% and 6.6%, respectively). Finally, the most suppressive soils came from farms with the highest coffee bean yields. Collectively, these results suggest the strong involvement of parasitic microorganisms, clay percentage and the pH suppressing RKN in soils from the major coffee production region in Brazil, and that volatiles emitted from total microbiota and exclusively from egg-isolated fungi are toxic to M. exigua.     

Modification of the culture medium to produce aluminum toxicity in cell suspensions of coffee (Coffea arabica L.)

Coffee (Coffea arabica) plants are usually grown in soils containing high levels of organic materials. Under these conditions, aluminum (Al) is toxic because of the acidic nature of the soils. Al is the most abundant metal found in the earth's crust and occurs in a number of different forms in soil. In acid soils, Al toxicity is a global problem that limits crop productivity. A major problem in obtaining cellular lines displaying Al tolerance in culture is the composition of the medium. In the experiments presented here, we modified the composition of the culture medium for a C. arabica cell line to produce Al toxicity. Murashige-Skoog media was used, complete (MS) and half ionic strength (MSHIS), at either pH 5.8 or 4.3. We found that MSHIS and pH 4.3 provided the optimal conditions to obtain Al toxicity as measured by the ability to grow in a range of Al concentrations (25-1,000 µM). The lethal dose (LD₅₀) under these conditions was 25 µM. The concentrations of free Al in the culture medium were corroborated by the fluorescent compound Morin. Al was found to enter the cell after 30 min, and the signal was then retained for up to 2 h.     

Genetic diversity of forest arabica coffee (Coffea arabica L.) in Ethiopia as revealed by random amplified polymorphic DNA (RAPD) analysis

Genetic diversity within the forest Coffea arabica L. gene pool in Ethiopia has not been extensively examined with molecular markers. In the present study, a total of 75 polymorphic RAPD bands generated by twelve random primers were used to assess genetic diversity among 144 genotypes representing 16 C. arabica populations. The number of polymorphic bands detected with each primer ranged from 2 to 9 with a mean of 6.25 bands per primer. Banding patterns ranged in percentage polymorphism from 37% to 73% with an overall mean of 56% for the populations analyzed. The amount of genetic variation among populations estimated by Shannon-Weaver diversity index was (H = 0.30). The within population and between populations differentiation values were 0.65 and 0.35, respectively. Genetic differentiations within and between zones of sample collection sites were 0.80 and 0.20, respectively. Within population average similarities estimated by simple matching coefficients ranged from 0.72 to 0.85, with an overall average of 0.78. In the cluster analysis that used individual samples as operational taxonomic units, most of the representatives of the same population failed to cluster before they joined members of other populations. Nevertheless, most of the populations were clustered on the basis of their geographic closeness and an east west differentiation was observed at approximately 75% similarity. The results obtained provide information on how to select sites for in situ conservation of C. arabica germplasm.     

Inter-simple sequence repeat (ISSR) variation in forest coffee trees (Coffea arabica L.) populations from Ethiopia

Genetic variation of forest coffee trees (Coffea arabica L.) from four regions of Ethiopia was investigated using inter-simple sequence repeat (ISSR) markers. A total of 160 individuals representing 16 populations were sampled. Eleven ISSR primers amplified a total of 123 fragments of which 31 fragments (25%) were polymorphic. Estimate of total gene diversity (H _T), and the coefficient of genetic differentiation (G _ST) were 0.37 and 0.81, respectively. This indicates that most of the variability is between populations than within populations. The partitioning of genetic variation into within and between populations based on Shannon’s information index also revealed more differentiation between populations (0.80) than within populations (0.20). In the phenogram most of the coffee tree samples were clustered on the basis of their regions of origin but failed to cluster according to their respective populations, which could be attributed to the presence of substantial gene flow between adjacent populations in each region assisted by man in the process of transplantation or by wild animals such as monkeys, which eat the berries and defecate the seeds elsewhere. On the other hand, the inter-regional clustering of some coffee tree samples from Bale and Jimma regions could be due to the transport of coffee seeds across regions and their subsequent planting. Although ISSR markers detected lower polymorphic loci than previously reported results with random amplified polymorphic DNA (RAPD) markers on the same materials, it can be used as an alternative method for molecular characterization of C. arabica populations. The results may provide information to select sites for in situ conservation.