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.     

Biochemical and molecular characterization and expression of the 11S-type storage protein from Coffea arabica endosperm

In order to define better the endosperm protein content of commercial coffee species Coffea arabica (Arabica) and C. canephora (Robusta), the principal storage protein of coffee grains has been analysed by 2-dimensional electrophoresis (2DE) and amino acid microsequencing. The most abundant polypeptide spots observed on mature coffee grain 2DE profiles were found to be subunits of the same protein, which exists as multiple isoforms with varying pIs. Strong sequence similaritywas found to the 11S family of plant storage proteins. The structure is typical of the 11S type, which occurs as a precursor of 55 kDa, and is observed under denaturing and reducing conditions on 2DE profiles in the form of cleavage products at approximately 20 kDa (β arms) and 32 kDa (α arms). Differences between Arabica and Robusta 2DE profiles indicate a secondary 11S protein family in some varieties of the latter. The existence of multiple pI forms may indicate that a multigene family encodes for these proteins. We estimate that the protein accounts for approximately 45 % of total grain protein. A cloned full-length cDNA of 1 706 bp coding for one of the isoforms is described and discussed in relation to other coffee storage protein sequences.     

Construction and characterization of a Coffea canephora BAC library to study the organization of sucrose biosynthesis genes

The first bacterial artificial chromosome (BAC) library of Robusta coffee (Coffea canephora) was constructed, with the aim of developing molecular resources to study the genome structure and evolution of this perennial crop. Clone 126, which is highly productive and confers good technological and organoleptic qualities of beverage, was chosen for development of this library. The BAC library contains 55,296 clones, with an average insert size of 135 Kb per plasmid, therefore representing theoretically nine haploid genome equivalents of C. canephora. Its validation was achieved with a set of 13 genetically anchored single-copy and 4 duplicated RFLP probes and yielded on average 9 BAC clones per probe. Screening of this BAC library was also carried out with partial cDNA probes coding for enzymes of sugar metabolism like invertases and sucrose synthase, with the aim of characterizing the organization and promoter structure of this important class of genes. It was shown that genes for both cell wall and vacuolar forms of invertases were probably unique in the Robusta genome whereas sucrose synthase was encoded by at least two genes. One of them (CcSUS1) was cloned and sequenced, showing that our BAC library is a valuable tool to rapidly identify genes of agronomic interest or linked to cup quality in C. canephora.     

Micro-collinearity and genome evolution in the vicinity of an ethylene receptor gene of cultivated diploid and allotetraploid coffee species (Coffea)

Arabica coffee (Coffea arabica L.) is a self-compatible perennial allotetraploid species (2n=4x=44), whereas Robusta coffee (C. canephora L.) is a self-incompatible perennial diploid species (2n=2x=22). C. arabica (C(a) C(a) E(a) E(a) ) is derived from a spontaneous hybridization between two closely related diploid coffee species, C. canephora (CC) and C. eugenioides (EE). To investigate the patterns and degree of DNA sequence divergence between the Arabica and Robusta coffee genomes, we identified orthologous bacterial artificial chromosomes (BACs) from C. arabica and C. canephora, and compared their sequences to trace their evolutionary history. Although a high level of sequence similarity was found between BACs from C. arabica and C. canephora, numerous chromosomal rearrangements were detected, including inversions, deletions and insertions. DNA sequence identity between C. arabica and C. canephora orthologous BACs ranged from 93.4% (between E(a) and C(a) ) to 94.6% (between C(a) and C). Analysis of eight orthologous gene pairs resulted in estimated ages of divergence between 0.046 and 0.665 million years, indicating a recent origin of the allotetraploid species C. arabica. Analysis of transposable elements revealed differential insertion events that contributed to the size increase in the C(a) sub-genome compared to its diploid relative. In particular, we showed that insertion of a Ty1-copia LTR retrotransposon occurred specifically in C. arabica, probably shortly after allopolyploid formation. The two sub-genomes of C. arabica, C(a) and E(a) , showed sufficient sequence differences, and a whole-genome shotgun approach could be suitable for sequencing the allotetraploid genome of C. arabica.     

A simple method to measure effective catalase activities: optimization, validation, and application in green coffee

Oxidative metabolism in coffee cherries during maturation appears to be regulated by the timely expression of redox enzymes such as catalase (CAT), peroxidase (POD), and polyphenoloxidase (PPO). Among these enzymes, CAT is suspected to contribute significantly in setting the redox status of the healthy cherry and the processed bean. The initial redox status of the green bean might further control the nature and dynamics of reactions induced by roasting and eventually quality aspects of the end product. In this respect, Arabica (Coffea arabica) and Robusta (Coffea canephora) typically differ by their cup coffee flavor profiles. We developed an assay that allowed us to screen numerous green coffee samples for effective CAT activities. The proposed assay, which monitors CAT activities by online oxygen sensing in green coffee crude suspensions incubated with H2O2, seeks to integrate potential effects of endogenous inhibitors and activators. After optimization and validation of the assay, 23 Arabicas, 23 Robustas, and 8 Arabustas were analyzed. Nearly all Arabicas (22 of 23) harbored high CAT activity levels, whereas all Robustas harbored low ones. Arabustas performed like Arabicas of the lower CAT activity range. The traditional spectrophotometric assay did not reveal these specificities. Because of its simplicity, our assay might be valuable for assessing effective CAT activities in various plant tissues.     

Polysaccharides of green Arabica and Robusta coffee beans

Two independent procedures for the quantitative determination of the polysaccharide content of Arabica Caturra (Coffea arabica var. Caturra) and Robusta ROM (Coffea canephora var. ROM) green coffee beans showed that they both contained identical amounts of polysaccharide. Cell wall material (CWM) was prepared from the beans and partial solubilisation of component polysaccharides was effected by sequential extraction with water, 1 M KOH, 0.3% NaClO2, 4 M KOH and 8 M KOH. The monosaccharide compositions of the CWMs were similar, although Arabica beans contained slightly more mannose than Robusta. In the latter, more arabinogalactan was solubilised during preparation of the CWM and the water-soluble fraction of the CWM contained higher amounts of galactomannan than in Arabica. Linkage analysis indicated that the galactomannans possessed unbranched to branched mannose ratios between 14:1 and 30:1 which is higher than previously reported. No major difference in the structural features of the galactomannans between species was found. The arabinogalactans were heterogeneous both with regard to the degree of branching and the degree of polymerisation of their arabinan side-chains. Compared to Arabica, Robusta appeared to contain greater amounts of arabinogalactans with longer side chains. It is concluded that there was no detectable difference between the Arabica and Robusta varieties of this study in their absolute polysaccharide content or in the gross structural features of their galactomannans. Differences were apparent both in the structural features and ease of solubility of the arabinogalactans but a more detailed study of several varieties of Arabica and Robusta will be required to determine whether these differences occur consistently between species.     

Somatic embryogenesis: A valuable alternative for propagating selected robusta coffee (Coffea canephora) clones

In Vitro Cellular &; Developmental Biology - Plant - The behavior of four coffee (Coffea canephora cv. Robusta) clones was evaluated in this work to define the conditions that would allõw...     

宁夏枸杞果实糖积累和蔗糖代谢相关酶活性的关系

通过对枸杞果实发育过程中果实生长模式、蔗糖、果糖、葡萄糖和淀粉含量及糖代谢相关酶活性的测定,研究了宁夏枸杞果实生长发育过程中糖的代谢积累与相关酶活性的关系.结果表明:(1)宁夏枸杞果实发育呈双S"曲线,果实主要以积累己糖为主.(2)蔗糖磷酸合成酶(SPS)活性在果实发育初期处于下降的趋势,在花后19d开始上升,果实转色后又逐渐下降;蔗糖合成酶(SS)活性总体表现为SS分解方向的活性大于SS合成方向的活性,说明枸杞果实发育过程中,SS的活性主要以分解方向的为主;酸性转化酶(AI)和中性转化酶(NI)的活性随果实发育呈上升趋势,但在果实成熟后期有所下降,且AI和NI活性高于合成酶类的活性,较高的转化酶类活性促进了果实内部己糖的积累.(3)在枸杞果实生长发育中,葡萄糖和果糖含量与AI和NI均呈极显著正相关,而与其它酶不具有相关性.说明AI和NI在宁夏枸杞果实的糖代谢中起着主要的调控作用.     

网纹甜瓜发育果实糖分积累与蔗糖代谢参与酶的关系

随着网纹甜瓜果实的发育,果实中葡萄糖和果糖的含量增加,蔗糖的快速积累发生在果实发育的中后期,高蔗糖积累型果实中蔗糖积累速率明显快于低蔗糖积累型.蔗糖磷酸合成酶活性在果实发育的前期短暂下降, 而后稳步上升,在果实发育的中后期高蔗糖积累型果实中该酶的活性显著高于低蔗糖积累型果实;随着果实发育,蔗糖合成酶的分解活性降低而合成活性升高.酸性和中性转化酶在未成熟果实中活性较高,而在成熟果实中很低; 高蔗糖积累型果实中酸性转化酶活性显著低于同期低蔗糖积累型果实.合成蔗糖的酶活性小于分解蔗糖的酶活性时蔗糖几乎没有积累.根据这些结果推测,转化酶活性的下降、蔗糖磷酸合成酶活性的增加以及蔗糖合成酶分解活性的下降和合成活性的增加,是引起果实蔗糖积累的主要内在因子.     

Sucrose Phosphate Synthase and Acid Invertase as Determinants of Sucrose Concentration in Developing Muskmelon (Cucumis melo L.) Fruits

Fruits of orange-fleshed and green-fleshed muskmelon (Cucumis melo L.) were harvested at different times throughout development to evaluate changes in metabolism which lead to sucrose accumulation, and to determine the basis of differences in fruit sucrose accumulation among genotypes. Concentrations of sucrose, raffinose saccharides, hexoses and starch, as well as activities of the sucrose metabolizing enzymes sucrose phosphate synthase (SPS) (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. Sucrose synthase and neutral invertase activities were relatively low (1.7 ± 0.3 micromole per hour per gram fresh weight and 2.2 ± 0.2, respectively) and changed little throughout fruit development. Acid invertase activity decreased during fruit development, (from as high as 40 micromoles per hour per gram fresh weight) in unripe fruit, to undetectable activity in mature, ripened fruits, while SPS activity in the fruit increased (from 7 micromoles per hour per gram fresh weight) to as high as 32 micromoles per hour per gram fresh weight. Genotypes which accumulated different amounts of sucrose had similar acid invertase activity but differed in SPS activity. Our results indicate that both acid invertase and SPS are determinants of sucrose accumulation in melon fruit. However, the decline in acid invertase appears to be a normal function of fruit maturation, and is not the primary factor which determines sucrose accumulation. Rather, the capacity for sucrose synthesis, reflected in the activity of SPS, appears to determine sucrose accumulation, which is an important component of fruit quality.     

Microfloral contamination and hydrolytic enzyme differences between monsooned and non-monsooned coffees

AIMS: To analyse and compare, for the first time, fungal and bacterial populations and hydrolytic enzyme production in four monsooned and non-monsooned Arabica and Robusta coffee types. METHODS AND RESULTS: Overall, using serial dilution, the populations of bacteria (approximately 10(8) cfu g(-1)) were highest in monsooned coffees of both varieties. Fungal populations were lower (10(5) cfu g(-1)) and found predominantly on monsooned coffee beans. The major fungal species were Aspergillus terreus, A. restrictus and A. ochraceus. Of 19 semi-quantitative enzymes analysed, significantly higher concentrations of 2-naphthyl-butyrate, caprylate and 2-naphthyl-D-galactopyranosidase were present in Arabica monsooned coffee. CONCLUSIONS: The results suggest that speciality monsooned coffee has markedly different microbial and physiological characteristics from normally produced green coffees. SIGNIFICANCE AND IMPACT OF THE STUDY: Monsooned coffee may have a higher contamination with spoilage moulds, especially mycotoxigenic species.     

Projected Shifts in Coffea arabica Suitability among Major Global Producing Regions Due to Climate Change

Regional studies have shown that climate change will affect climatic suitability for Arabica coffee (Coffea arabica) within current regions of production. Increases in temperature and changes in precipitation patterns will decrease yield, reduce quality and increase pest and disease pressure. This is the first global study on the impact of climate change on suitability to grow Arabica coffee. We modeled the global distribution of Arabica coffee under changes in climatic suitability by 2050s as projected by 21 global circulation models. The results suggest decreased areas suitable for Arabica coffee in Mesoamerica at lower altitudes. In South America close to the equator higher elevations could benefit, but higher latitudes lose suitability. Coffee regions in Ethiopia and Kenya are projected to become more suitable but those in India and Vietnam to become less suitable. Globally, we predict decreases in climatic suitability at lower altitudes and high latitudes, which may shift production among the major regions that produce Arabica coffee.     

Carbohydrate metabolism in growing rice seedlings under arsenic toxicity

We studied in the seedlings of two rice cultivars (Malviya-36 and Pant-12) the effect of increasing levels of arsenic in situ on the content of sugars and the activity of several enzymes of starch and sucrose metabolism: alpha-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2), starch phosphorylase (EC 2.4.1.1), acid invertase (EC 3.2.1.26), sucrose synthase (EC 2.4.1.13) and sucrose phosphate synthase (EC 2.4.1.14). During a growth period of 10-20 d As2O3 at 25 and 50 microM in the growth medium caused an increase in reducing, non-reducing and total soluble sugars. An increased conversion of non-reducing to reducing sugars was observed concomitant with As toxicity. The activities of alpha-amylase, beta-amylase and sucrose phosphate synthase declined, whereas starch phosphorylase, acid invertase and sucrose synthase were found to be elevated. Results indicate that in rice seedlings arsenic toxicity causes perturbations in carbohydrate metabolism leading to the accumulation of soluble sugars by altering enzyme activity. Sucrose synthase possibly plays a positive role in synthesis of sucrose under As-toxicity.     

A role for 'futile cycles' involving invertase and sucrose synthase in sucrose metabolism of tomato fruit.

Current concepts of the factors determining sink strength and the subsequent regulation of carbohydrate metabolism in tomato fruit are based upon an understanding of the relative roles of sucrose synthase, sucrose phosphate synthase and invertase, derived from studies in mutants and transformed plants. These enzymes participate in at least four futile cycles that involve sugar transport between the cytosol, vacuole and apoplast. Key reactions are (1) the continuous rapid degradation of sucrose in the cytosol by sucrose synthase (SuSy), (2) sucrose re-synthesis via either SuSy or sucrose phosphate synthase (SPS), (3) sucrose hydrolysis in the vacuole or apoplast by acid invertase, (4) subsequent transport of hexoses to the cytosol where they are once more converted into sucrose, and (5) rapid synthesis and breakdown of starch in the amyloplast. In this way futile cycles of sucrose/hexose interchange govern fruit sugar content and composition. The major function of the high and constant invertase activity in red tomato fruit is, therefore, to maintain high cellular hexose concentrations, the hydrolysis of sucrose in the vacuole and in the intercellular space allowing more efficient storage of sugar in these compartments. Vacuolar sugar storage may be important in sustaining fruit cell growth at times when less sucrose is available for the sink organs because of exhaustion of the carbohydrate pools in source leaves.     

Calcium-mediated conversion of sucrose to starch in relation to the activities of amylases and sucrose-metabolizing enzymes in sorghum grains raised through liquid culture

Detached ears of sorghum (Sorghum vulgare) were cultured in complete liquid medium containing Ca2+(0, 3, 10 and 30 mM) and effect of this ion on the conversion of sucrose to starch with respect to the activities of amylases, sucrose synthase, sucrose phosphate synthase and soluble invertases were studied in developing grains. Presence of 3 mM Ca2+ in culture medium enhanced both accumulation of starch and activity of alpha-amylase in grain but without having any influence on the activity of beta-amylase. However, with 10 and 30 mM Ca2+, the accumulation of starch and activities of both amylases decreased and with advancement in culturing period, starch accumulation was further decreased. Irrespective of its concentration, Ca2+ enhanced the activities of sucrose synthase (synthesis), sucrose-phosphate synthase, soluble acid invertase and soluble-neutral invertase. Increase in the concentration of Ca2+ in culture medium was concomitant with an elevation in relative proportion of sucrose in the grain reflecting a net balance in per cent increase with Ca2+ in the activities of sucrose-synthesizing enzymes over sucrose-hydrolysing ones. Based on the results, it is suggested that assimilation of Ca2+ by grain is essential for maintaining high activity of alpha-amylase to generate starch primers required for the conversion of sucrose to starch during grain filling in sorghum.