Purification and complete amino acid sequence of a new type of sweet protein taste-modifying activity, curculin

A new taste-modifying protein named curculin was extracted with 0.5 M NaCl from the fruits of Curculigo latifolia and purified by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and gel filtration. Purified curculin thus obtained gave a single band having a Mr of 12,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 8 M urea. The molecular weight determined by low-angle laser light scattering was 27,800. These results suggest that native curculin is a dimer of a 12,000-Da polypeptide. The complete amino acid sequence of curculin was determined by automatic Edman degradation. Curculin consists of 114 residues. Curculin itself elicits a sweet taste. After curculin, water elicits a sweet taste, and sour substances induce a stronger sense of sweetness. No protein with both sweet-tasting and taste-modifying activities has ever been found. There are five sets of tripeptides common to miraculin (a taste-modifying protein), six sets of tripeptides common to thaumatin (a sweet protein), and two sets of tripeptides common to monellin (a sweet protein). Anti-miraculin serum was not immunologically reactive with curculin. The mechanism of the taste-modifying action of curculin is discussed.     

Neoculin as a new taste-modifying protein occurring in the fruit of Curculigo latifolia

A unique taste-modifying activity that converts the sense of sourness to the sense of sweetness occurs in the fruit of the plant Curculigo latifolia, intrinsic to West Malaysia. The active component, known as curculin, is a protein consisting of two identical subunits. We have found a new taste-modifying protein, named neoculin, of the same origin. Both chemical analysis and cDNA cloning characterized neoculin as a heterodimeric protein consisting of an acidic, glycosylated subunit of 113 amino acid residues and a basic subunit that is the monomeric curculin itself.     

Curculin exhibits sweet-tasting and taste-modifying activities through its distinct molecular surfaces

Curculin isolated from Curculigo latifolia, a plant grown in Malaysia, has an intriguing property of modifying sour taste into sweet taste. In addition to this taste-modifying activity, curculin itself elicits a sweet taste. Although these activities have been attributed to the heterodimeric isoform and not homodimers of curculin, the underlying mechanisms for the dual action of this protein have been largely unknown. To identify critical sites for these activities, we performed a mutational and structural study of recombinant curculin. Based on the comparison of crystal structures of curculin homo- and heterodimers, a series of mutants was designed and subjected to tasting assays. Mapping of amino acid residues on the three-dimensional structure according to their mutational effects revealed that the curculin heterodimer exhibits sweet-tasting and taste-modifying activities through its partially overlapping but distinct molecular surfaces. These findings suggest that the two activities of the curculin heterodimer are expressed through its two different modes of interactions with the T1R2-T1R3 heterodimeric sweet taste receptor.     

Curculin,a sweet-tasting and taste-modifying protein,is a non-functional mannose-binding lectin

A three-dimensional model of curculin, a sweet-tasting and taste-modifying protein from the fruits of Curculigo latifolia, was built from the X-ray coordinates of GNA, a mannose-binding lectin from snowdrop (Galanthus nivalis). The three mannose-binding sites present in GNA were found in curculin but are devoid of mannose-binding activity as shown by docking experiments performed with mannose. Some regions well exposed on the surface of the three-dimensional model of curculin could act as epitopes responsible for the sweet-tasting properties of this protein.     

Activity and Stability of a New Sweet Protein with Taste-modifying Action, Curculin

Curculin elicited a sweet taste. After the sweetness of curculindiminished, application of deionized water or an acid to thetongue induced a sweet taste. The maximum sweetness of curculinitself was equivalent to thesweetness of 0.35 M sucrose. Themaximum sweetness induced by 0.02 M citric acid or deionizedwater after curculin dissolved in a buffer of pH 6.0 was heldin mouth for 3 min was also equivalent to that of 0.35 M sucrose.The sweetness induced by deionized water was completely suppressedby the presence of 1 mM CaCl₂ or MgCl₂, while that induced byan acid was not suppressed by the presence of divalent cations.Based on these results, the mechanism of the taste-modifyingactivity was discussed. Stability of curculin was examined undervarious conditions. The taste-modifying activity of curculinwas unchanged when curculin was incubated at 50°C for 1h between pH 3 and 11.     

Field evaluation of three-component solid food-based dispenser versus torula yeast for capturing Mediterranean and oriental fruit flies (Diptera: Tephritidae)

Certain species of true fruit flies (Diptera: Tephritidae) are worldwide pests of fruits and vegetables, and many countries operate trapping programs to detect and monitor invasions. Torula yeast-borax (TYB) solution is a widely used food bait, although its effective field longevity is relatively short (1–2 weeks). This led to the development of a synthetic cone-shaped food dispenser that contains ammonium acetate, putrescine, and trimethylamine. The food cone has received limited testing, and the objective of this study was to compare captures of the Mediterranean fruit fly (Ceratitis capitata (Wiedemann)) and the oriental fruit fly (Bactrocera dorsalis (Hendel)) in traps baited with the fresh TYB solution versus traps baited with fresh or weathered food cones. Captures showed a female bias for both baits and both species. For C. capitata, fresh food cones generally attracted more individuals than fresh TYB. Weathered food cones attracted similar or greater numbers of medflies than TYB for the initial two weeks, but food cones weathered for 4 or 6 weeks attracted fewer flies than TYB. For B. dorsalis, TYB-baited traps captured more females than fresh or weathered food cone-baited traps in each of six weekly sampling periods, and the differences were statistically significant in weeks 1–4. In a release-recapture experiment, both C. capitata and B. dorsalis were captured in higher numbers in traps baited with fresh TYB than those baited with food cones weathered for 0 (fresh) to 6 weeks. The implications of these results for fruit fly surveillance are discussed.     

Cell Wall Changes in Nectarines (Prunus persica) : Solubilization and Depolymerization of Pectic and Neutral Polymers during Ripening and in Mealy Fruit

Nectarine fruit (Prunus persica L. Batsch var nectarina [Ait] maxim) cultivar Fantasia were either ripened immediately after harvest at 20°C or stored for 5 weeks at 2°C prior to ripening. Fruit ripened after 5 weeks of storage did not soften to the same extent as normally ripened fruit, they lacked juice, and had a dry, mealy texture. Pectic and hemicellulosic polysaccharides were solubilized from the mesocarp of the fruit using phenol:acetic acid:water (PAW) treatment to yield PAW-soluble material and cell wall material (CWM). The carbohydrate composition and relative molecular weight (M_r) of polysaccharide fractions released from the CWM by sequential treatment with cyclohexane-trans-1,2-diamine tetra-acetate, 0.05 m Na₂CO₃, 6 m guanidinium thiocyanate, and 4 m KOH were determined. Normal ripening of nectarines resulted in solubilization of pectic polymers of high M_r from CWM during the first 2 d at ripening temperatures. Concurrently, galactan side chains were removed from pectic polymers. Solubilized pectic polymers were depolymerized to lower M_r species during the latter stages of ripening. Upon removal from cool storage, fruit had undergone some pectic polymer solubilization, and after ripening, pectins were not depolymerized and were of high M_r. Side chains did not appear to be removed from insoluble pectic polymers and branched pectins accumulated in the CWM. The molecular weight profiles obtained by gel filtration of the hemicellulosic fractions from normally ripening and mealy fruit were similar. The results suggest that mealiness results as a consequence of altered pectic polymer breakdown, including that associated with neutral side chains.     

Changes in fatty acid composition of the lipid classes in developing oil palm mesocarp

During fruit development of oil palm (Elaeis guineensis) oil deposition in the mesocarp startedca12–13 weeks after flowering (WAF) and continued until the fruit ripened at 20 WAF. Over the next 1–2 weeks oil continued to be deposited but the fruit became loose and readily detached from the bunch. The lipids extracted at this stage contained over 50 % free fatty acids andca6%, polar lipids. The major fatty acids in the storage triacylglycerols were 16:0,18:1 and 18:2. The fatty acid composition of the neutral lipid classes and polar lipids during oil deposition were similar except that the latter also contained a high proportion of 18:3. Longer chain acids (20:3 and 22:0) were detected in certain lipid classes at 8 and 12 WAF.     

High Incidence of Preharvest Colonization of Huanglongbing-Symptomatic Citrus sinensis Fruit by Lasiodiplodia theobromae (Diplodia natalensis) and Exacerbation of Postharvest Fruit Decay by That Fungus

Huanglongbing (HLB), presumably caused by the bacterium “Candidatus Liberibacter asiaticus,” is a devastating citrus disease associated with excessive preharvest fruit drop. Lasiodiplodia theobromae (diplodia) is the causal organism of citrus stem end rot (SER). The pathogen infects citrus fruit under the calyx abscission zone (AZ-C) and is associated with cell wall hydrolytic enzymes similar to plant enzymes involved in abscission. By means of DNA sequencing, diplodia was found in “Ca. Liberibacter asiaticus”-positive juice from HLB-symptomatic fruit (S) but not in “Ca. Liberibacter asiaticus”-negative juice. Therefore, the incidence of diplodia in fruit tissues, the impact on HLB-related postharvest decay, and the implications for HLB-related preharvest fruit drop were investigated in Hamlin and Valencia oranges. Quantitative PCR results (qPCR) revealed a significantly (P < 0.001) greater incidence of diplodia in the AZ-C of HLB-symptomatic (S; “Ca. Liberibacter asiaticus” threshold cycle [C_T] of <30) than in the AZ-C of in asymptomatic (AS; “Ca. Liberibacter asiaticus” C_T of ≥30) fruit. In agreement with the qPCR results, 2 weeks after exposure to ethylene, the incidences of SER in S fruit were 66.7% (Hamlin) and 58.7% (Valencia), whereas for AS fruit the decay rates were 6.7% (Hamlin) and 5.3% (Valencia). Diplodia colonization of S fruit AZ-C was observed by scanning electron microscopy and confirmed by PCR test and morphology of conidia in isolates from the AZ-C after surface sterilization. Diplodia C_T values were negatively correlated with ethylene production (R = −0.838 for Hamlin; R = −0.858 for Valencia) in S fruit, and positively correlated with fruit detachment force (R = 0.855 for Hamlin; R = 0.850 for Valencia), suggesting that diplodia colonization in AZ-C may exacerbate HLB-associated preharvest fruit drop.     

An antibody against a conserved C-terminal consensus motif from plant alternative oxidase (AOX) isoforms 1 and 2 label plastids in the explosive dwarf mistletoe (Arceuthobium americanum, Santalaceae) fruit exocarp

Dwarf mistletoes, genus Arceuthobium (Santalaceae), are parasitic angiosperms that spread their seeds by an explosive process. As gentle heating triggers discharge in the lab, we wondered if thermogenesis (endogenous heat production) is associated with dispersal. Thermogenesis occurs in many plants and is enabled by mitochondrial alternative oxidase (AOX) activity. The purpose of this study was to probe Arceuthobium americanum fruit (including seed tissues) collected over a 10-week period with an anti-AOX antibody/gold-labeled secondary antibody to determine if AOX could be localized in situ, and if so, quantitatively assess whether label distribution changed during development; immunochemical results were evaluated with Western blotting. No label could be detected in the mitochondria of any fruit or seed tissue, but was observed in fruit exocarp plastids of samples collected in the last 2 weeks of study; plastids collected in week 10 had significantly more label than week 9 (p?=?0.002). Western blotting of whole fruit and mitochondrial proteins revealed a signal at 30–36 kD, suggestive of AOX, while blots of whole fruit (but not mitochondrial fraction) proteins showed a second band at 40–45 kD, in agreement with plastid terminal oxidases (PTOXs). AOX enzymes are likely present in the A. americanum fruit, even though they were not labeled in mitochondria. The results strongly indicate that the anti-AOX antibody was labeling PTOX in plastids, probably at a C-terminal region conserved in both enzymes. PTOX in plastids may be involved in fruit ripening, although a role for PTOX in thermogenesis cannot be eliminated.     

Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection

Key message

We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics.

Abstract

Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12–16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1–2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying.     

DNA marker-assisted evaluation of fruit firmness at harvest and post-harvest fruit softening in a diverse apple germplasm

Several different genes have been proposed as responsible for fruit texture variability at harvest and/or after storage. We have analysed 127 apple cultivars for allelic composition in two key genes that are directly involved in the ethylene biosynthesis pathway, Md-ACS1 and Md-ACO1, and two other genes that are involved in cell wall degradation, Md-Exp7 and Md-PG1. Firmness was measured with a penetrometer at harvest and after 6 or 12 weeks (early- and late-maturing cultivars, respectively) of cold storage. Maturation time was positively correlated with firmness at harvest and negatively correlated with fruit softening rate (difference between firmness at harvest and after storage, divided by number of weeks in storage). Polyploid cultivars showed significantly higher firmness at harvest compared to diploids. Alleles previously described as responsible for good texture were associated with significantly lower softening for Md-ACS1 and Md-PG1, but the opposite was noted for Md-EXP7. Results were nonsignificant for Md-ACO1. Allele frequencies were very uneven in all four loci, with the three most common multi-locus configurations accounting for 64 % of the entire material. The predictive power of these genes was calculated with a partial least squares discriminant analysis, and these accounted for 15 % of the observed variation in initial firmness and 18 % for softening rate. Inclusion of maturation time, storage time (i.e. 6 or 12 weeks) and initial firmness into the model however increased the predictability of softening rate to 38 %. Dividing the material in modern (released after 1960) and old cultivars did not change the outcome of our analyses.     

Phytochemical composition and in vitro biological activities of Morinda citrifolia fruit juice

Morinda citrifolia is a plant with broad nutraceutical and therapeutic effects and used in the traditional treatment of several ailments. The objective of this work is to investigate the phytochemistry of the fruit juice of M. citrifolia on one hand and on other hand to evaluate its antiradical and antibacterial activity. The phytochemical investigation was carried out by tube staining tests of the extract of two types of fruit juice of M. citrifolia. The antioxidant activity of these juices was evaluated by reducing the DPPH radical method. Concerning the antibacterial activity, it was tested on the in vitro growth of 10 reference bacterial strains using the well diffusion method. Qualitative phytochemistry of M. citrifolia fruit juices revealed the presence of large groups of secondary metabolites including polyphenols, reducing compounds, mucilage and terpernoids. The antioxidant activity of M. citrifolia fruit juices is dose-dependent and higher than that of ascorbic acid. Antimicrobial activity on other hand revealed that fruit juices inhibit growth inhibitory activity of Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, S. epidermidis, Proteus vulgaris, Streptococcus oralis, Enterococcus faecalis and Escherichia coli. This observed difference is significant for each juices on the strains (p < 0.001). These results support the use of M. citrifolia in traditional medicine and are the starting points for the development of a new drug to combat both dietary conditions and chronic conditions associated with oxidative stress.     

In Vivo Biochemical and Gene Expression Analyses of the Antioxidant Activities and Hypocholesterolaemic Properties of Tamarindus indica Fruit Pulp Extract

Background

Tamarindus indica (T. indica) is a medicinal plant with many biological activities including anti-diabetic, hypolipidaemic and anti-bacterial activities. A recent study demonstrated the hypolipidaemic effect of T. indica fruit pulp in hamsters. However, the biochemical and molecular mechanisms responsible for these effects have not been fully elucidated. Hence, the aims of this study were to evaluate the antioxidant activities and potential hypocholesterolaemic properties of T. indica, using in vitro and in vivo approaches.

Methodology/Principal Findings

The in vitro study demonstrated that T. indica fruit pulp had significant amount of phenolic (244.9±10.1 mg GAE/extract) and flavonoid (93.9±2.6 mg RE/g extract) content and possessed antioxidant activities. In the in vivo study, hamsters fed with high-cholesterol diet for ten weeks showed elevated serum triglyceride, total cholesterol, HDL-C and LDL-C levels. Administration of T. indica fruit pulp to hypercholesterolaemic hamsters significantly lowered serum triglyceride, total cholesterol and LDL-C levels but had no effect on the HDL-C level. The lipid-lowering effect was accompanied with significant increase in the expression of Apo A1, Abcg5 and LDL receptor genes and significant decrease in the expression of HMG-CoA reductase and Mtp genes. Administration of T. indica fruit pulp to hypercholesterolaemic hamsters also protected against oxidative damage by increasing hepatic antioxidant enzymes, antioxidant activities and preventing hepatic lipid peroxidation.

Conclusion/Significance

It is postulated that tamarind fruit pulp exerts its hypocholesterolaemic effect by increasing cholesterol efflux, enhancing LDL-C uptake and clearance, suppressing triglyceride accumulation and inhibiting cholesterol biosynthesis. T. indica fruit pulp has potential antioxidative effects and is potentially protective against diet-induced hypercholesterolaemia.     

Host status of ‘Scifresh’ apples to the invasive fruit fly species Bactrocera dorsalis,Zeugodacus cucurbitae,and Ceratitis capitata (Diptera: Tephritidae)

We conducted no-choice cage and field infestation studies to determine if the fruit of apples (Malus x domestica L., ‘Scifresh’) are hosts for three invasive tephritid fruit fly species that may enter New Zealand or other apple growing areas. In screen cage tests, punctured and unpunctured (intact) fruit of ‘Scifresh’ apples were exposed to gravid females of Bactrocera dorsalis (Hendel) (Oriental fruit fly), Zeugodacus (Bactrocera) cucurbitae (Coquillet) (melon fly), or Ceratitis capitata (Wiedemann) (Mediterranean fruit fly), outdoors for 24 h and then held on sand in the laboratory for four weeks for pupal development and adult emergence. Unpunctured fruit produced an average of 269.4, 4.3 and 70.1 puparia per kg of fruit for B. dorsalis, Z. cucurbitae and C. capitata, respectively. Punctured fruit produced an average of 619.4, 0.8 and 129.5 puparia per kg of fruit for B. dorsalis, Z. cucurbitae and C. capitata, respectively. By comparison, unpunctured and punctured papaya fruit (Carica papaya, ‘Rainbow’, a preferred host) produced 206–675 and 464–735 puparia per kg of fruit, respectively, across all species. In general, the average weight of individual fruit fly puparia from apple was significantly less (41–71%) than that of puparia reared from papaya, and development times were slower on apple than on papaya. Overall, ‘Scifresh’ apples were a moderately good host for B. dorsalis and C. capitata, and a very poor host for Z. cucurbitae in cage tests. Field exposure of ‘Scifresh’ apples suspended from papaya trees resulted in no infestation by B. dorsalis or Z. cucurbitae under natural conditions. This information will help to inform decisions about quarantine restrictions and potential crop loss in the event of incursions of these fruit flies into apple-producing countries.     

Status of pollinators and their efficiency in coffee fruit set in a fragmented landscape mosaic in South India

The region of Kodagu, South India, comprises a fragmented landscape with a high density of remnant forest patches dispersed within a mosaic dominated by shaded coffee agro-forests. We evaluated the role of self, wind and insect pollination to Coffea canephora production in this landscape. The giant Asian honeybee, Apis dorsata, which nests in remnant forests, was the main pollinator of coffee (accounting for 58% of the floral visitors). The proportion of flowers that developed into fruits was highest when hand cross-pollinated (44%), followed by open- (insect and wind combined; 33%) and wind- (22.1%) pollination treatments. Pollination by bees therefore increases fruit production by 50% over that achieved by wind. Self-pollination (1.7%) and no pollination (1%) treatments produced very low fruit set, emphasizing the importance of cross-pollination in C. canephora. Unlike measures of pollination success, initial fruit set (five weeks after flowering) proved an unreliable proxy for final fruit set. Size of adjoining forest fragments (mostly 0.3–20 ha, with a few exceeding 200 ha) positively influenced pollinator visitation to coffee flowers, but distance to such fragments had no influence on pollination. This study demonstrates the importance of cross-pollination for crop production in C. canephora, the important contribution that pollinating insects make to coffee production, and the benefits of relatively large forest fragments within the landscape mosaic to support insect pollinators of coffee. A comparison of pollinator composition to that of 100 years ago indicated that coffee pollination services remained intact despite changes in pollinator community composition.     

An increase of lycopene content in tomato fruit is associated with a novel Cyc-B allele isolated through TILLING technology

Tomato Cyc-B gene encodes a chromoplast-specific lycopene β-cyclase that converts lycopene to β-carotene during ripening of the fruit. By screening the tomato Red Setter mutant population with the TILLING method, we identified eight new alleles at the Cyc-B locus. Results of greenhouse phenotypic analysis revealed that the novel A949G Cyc-B allele produced modifications in the carotenoid profile and content of tomato petals and fruit. The cyc-b7 genotype, carrying the A949G Cyc-B allele, was therefore evaluated in an open field trial for standard agronomic traits as well as carotenoid content of the fruit. Results of the field trial confirmed that the induced A949G missense mutation favored the accumulation of lycopene in the fruit with no detrimental effects on the yield or on other agronomic and technological properties such as fruit firmness and Brix degree of fruit juice. On the basis of these results, it can be affirmed that the A949G Cyc-B allele constitutes a useful new genetic variant which can be used for improving carotenoid content in tomato fruit and for the development of new tomato commercial lines. Finally, the results presented here furthermore demonstrate that TILLING is a powerful methodology not only as a confirmatory system for gene functional analysis but also for selecting new gene variants useful for genetic improvement of important crops.     

Morphological and histological impacts of the laurina mutation on fructification and seed characteristics in Coffea arabica L.

Key message

The comparison between the cultivar Bourbon and its mutant, the Bourbon pointu, of Coffea arabica led to five novel findings on fruit development and three main impacts of the mutation.

Abstract

Coffea arabica ‘Laurina’ (Bourbon pointu) is a natural mutant of Coffea arabica ‘Bourbon’. Relative to the ‘Bourbon’ cultivar, it is characterized by internode dwarfism, a Christmas tree shape, and lower caffeine content. The effects of the laurina mutation on fructification over time, the fruit structure and seed characteristics were studied here. Fruits of ‘Bourbon’ and ‘Bourbon pointu’ were monitored. The trees were grown in the same plot and flowered on the same day. Harvesting was done every 2 weeks from the 6th to the 26th week after flowering. Histological observations were carried out using multiphoton and conventional microscopes. The measurements concerned the fruit, parchment and seed. Five novel findings on fructification development were obtained: (1) a sigmoid model and non-linear regression efficiently described the phenomenon; (2) a precise relationship was defined between the qualitative stages of fructification and quantitative observations, thus revealing key weeks in this process; (3) the parchment had a mesocarpic origin; (4) a meristematic zone was present close to the parchment; and (5) an endocarp with three cell layers was visible in young fruits. Three effects of the laurina mutation were highlighted: (1) fruit growth ended 1 week earlier in ‘Bourbon’, but without difference in fruit length. In contrast, fruits were wider on average in ‘Bourbon’; (2) the parchment of narrow seeds in ‘Bourbon pointu’ was thicker than in other ‘Bourbon pointu’ and ‘Bourbon’ seeds; and (3) the narrow seed frequency in ‘Bourbon pointu’ depended on environmental conditions.     

Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.)

A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar “White Winter Pearmain”. When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4 °C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.