Regulation of carotenoid biosynthesis during fruit maturation in the red-fleshed orange mutant Cara Cara

Cara Cara is a spontaneous bud mutation of Navel orange (Citrus. sinensis L. Osbeck) characterized by developing fruits with a pulp of bright red coloration due to the presence of lycopene. Peel of mutant fruits is however orange and indistinguishable from its parental. To elucidate the basis of lycopene accumulation in Cara Cara, we analyzed carotenoid profile and expression of three isoprenoid and nine carotenoid genes in flavedo and pulp of Cara Cara and Navel fruits throughout development and maturation. The pulp of the mutant accumulated high amounts of lycopene, but also phytoene and phytofluene, from early developmental stages. The peel of Cara Cara also accumulated phytoene and phytofluene. The expression of isoprenoid genes and of carotenoid biosynthetic genes downstream PDS (phytoene desaturase) was higher in the pulp of Cara Cara than in Navel. Not important differences in the expression of these genes were observed between the peel of both oranges. Moreover, the content of the plant hormone ABA (abscisic acid) was lower in the pulp of Cara Cara, but the expression of two genes involved in its biosynthesis was higher. The results suggest that an altered carotenoid composition may conduct to a positive feedback regulatory mechanism of carotenoid biosynthesis in citrus fruits. Increased levels of isoprenoid precursors in the mutant that could be channeled to carotenoid biosynthesis may be related to the red-fleshed phenotype of Cara Cara.     

外源ABA和GA3对红肉脐橙果皮主要色素含量变化和果实着色的影响

测定了红肉脐橙(Citrus sinensis Osbeskcv.CaraCara)果实发育期间和果实转色前用不同浓度的外源ABA和GA₃处理后果皮叶绿素和类胡萝卜素的动态含量,并测定了外源ABA和GA₃处理后成熟红肉脐橙果皮色泽的表现。结果表明:红肉脐橙果皮叶绿素含量于9月20日出现最大值,为0.1469mg·g^-1FW;类胡萝卜素含量于12月20日达到最大值,为0.0321mg·g^-1FW;果实转色前,用外源ABA处理后加速了果皮叶绿素的降解,但也抑制了果皮类胡萝卜素的积累,用GA₃处理后延缓了果皮叶绿素的降解,同样抑制了果皮类胡萝卜素的积累,严重阻碍了果皮类胡萝卜素的合成;外源ABA或GA₃处理均不利于果实色泽品质的提高。     

Construction and Characterization of a cDNA Library from the Pulp of Cara Cara Navel Orange （Citrus sinensis Osbeck）

A cDNA library was constructed end characterized from the pulp of Cera Care navel orange （Citrus sinensis Osbeck） at different stages of ripening. Tittering results revealed that approximately 5.086×10^5 independent clones were included in this library. Electrophoresls gel results of 15 randomly selected clones revealed that the size of the insertion fragments ranged from 400 bp to 2 kb, with an average size of 900 bp. Sequencing results of 150 randomly picked clones showed that the recombination rate was 94%. During subsequent sequence analysis, 41 of 139 clones failed to be identified end the amino sequence of 71 clones shared less than 30% identity with related plants in GenBank. Of 27 clones whose amino sequences shared more than 60% identity with other related plants in GenBenk, 17 clones showed an 80% identity with the corresponding candidate genes of citrus. The clone recognized as the type Ⅲ metallothlonein-llke （MT） gene was observed to occur 13 tlmes, Indlcatlng that the protein may play an important role In frult development and rlpenlng.     

Carotenoid Accumulation and Carotenogenic Genes Expression During Two Types of Persimmon Fruit (<Emphasis Type="Italic">Diospyros kaki</Emphasis> L.) Development

Persimmon (Diospyros kaki L.), belonging to the Ebenaceae family, has been used not only as a fresh fruit, but also for many medicinal uses. Carotenoids are the main pigment in persimmon fruit, which contribute significantly to fruit color and nutritional quality due to their composition and content. In this study, fruit quality indices, carotenoid contents and expression of carotenogenic genes were analyzed in two types of persimmon fruit. The results demonstrated that there was a positive correlation between fruit color and the contents of main composition and total carotenoids. Carotenoid accumulation in persimmon fruit resulted from the interaction of carotenogenic genes, but the molecular mechanisms responsible for accumulation of carotenoids in two types of persimmon fruit had a few differences. As a complete unit, the relatively low expression level of phytoene synthase gene (DkPSY) in “Niuxinshi” resulted in low carotenoid contents or even under the detection limit at the early fruit developmental stages; but low carotenoid contents in “Nishimurawase” were due to the relatively low expression level of carotenogenic genes other than DkPSY. At the late fruit developmental stages, increased expression levels of DkPSY, phytoene desaturase gene and beta-carotene hydroxylase gene (DkBCH) induced elevated carotenoid contents; because all carotenogenic genes strongly expressed in “Nishimurawase”, a large amount of carotenoids were accumulated. In addition, β-cryptoxanthin was the main composition whose content increased with the fruit maturity changes, which was mainly because of DkBCH which might lead more conversion of β-carotene to β-cryptoxanthin.     

外源ABA和GA3对红肉脐橙果皮主要色素含量变化的影响

测定了红肉脐橙果实发育期间果皮叶绿素和总类胡萝卜素含量的变化动态,并在果实转色前用不同浓度的外源ABA和GA₃处理后测定果皮色素含量的变化动态。结果表明,红肉脐橙果皮叶绿素总量于9月20日出现最大值(0.1469mg/gfw),总类胡萝卜素含量于12月20日达到最大值(0.0321mg/gfw);转色前用ABA处理后加速果皮叶绿素的降解,但抑制果皮类胡萝卜素的积累;转色前用GA₃处理后延缓果皮叶绿素的降解,并同样抑制果皮类胡萝卜素的积累,严重阻碍了果皮类胡萝卜素的合成。     

Induced point mutations in the phytoene synthase 1 gene cause differences in carotenoid content during tomato fruit ripening

In tomato, carotenoids are important with regard to major breeding traits such as fruit colour and human health. The enzyme phytoene synthase (PSY1) directs metabolic flux towards carotenoid synthesis. Through TILLING (Targeting Induced Local Lesions IN Genomes), we have identified two point mutations in the Psy1 gene. The first mutation is a knockout allele (W180*) and the second mutation leads to an amino acid substitution (P192L). Plants carrying the Psy1 knockout allele show fruit with a yellow flesh colour similar to the r, r mutant, with no further change in colour during ripening. In the line with P192L substitution, fruit remain yellow until 3 days post-breaker and eventually turn red. Metabolite profiling verified the absence of carotenoids in the W180* line and thereby confirms that PSY1 is the only enzyme introducing substrate into the carotenoid pathway in ripening fruit. More subtle effects on carotenoid accumulation were observed in the P192L line with a delay in lycopene and β-carotene accumulation clearly linked to a very slow synthesis of phytoene. The observation of lutein degradation with ripening in both lines showed that lutein and its precursors are still synthesised in ripening fruit. Gene expression analysis of key genes involved in carotenoid biosynthesis revealed that expression levels of genes in the pathway are not feedback-regulated by low levels or absence of carotenoid compounds. Furthermore, protein secondary structure modelling indicated that the P192L mutation affects PSY1 activity through misfolding, leading to the low phytoene accumulation.     

In situ feeding tactics of short-nosed fruit bat (Cynopterus sphinx) on mango fruits: evidence of extractive foraging in a flying mammal

We report a sequence of behaviors exhibited by the short-nosed fruit bat Cynopterus sphinx while feeding on fruits of Mangifera indica. They peel off the outer skin to form a feeding area of about 3–6 cm diameter. Such food preparatory behaviors were more pronounced on larger mangoes. Bats competed among themselves to feed on the mangoes that had such feeding areas exposed. Individuals that spent a considerable amount of time on food preparatory behaviors actively secured the fruits. Altogether, these behaviors indicate that Cynopterus bats might have learnt, over evolutionary time, and developed behaviors that facilitate efficient processing and feeding of fruits such as mangoes. It appears that actions exhibited by C. sphinx in peeling off the outer skin of mangoes exemplify “extractive foraging”, a behavior that is prominently known in large-brained mammals. Thus, our findings will have implications on the distribution and evolution of extractive foraging and “technical intelligence” among mammalian lineages.     

Expressed sequence tags from persimmon at different developmental stages

Persimmon (Diospyros kaki Thunb.) is an important fruit in Asian countries, where it is eaten as a fresh fruit and is also used for many other purposes. To understand the molecular mechanism of fruit development and ripening in persimmon, we generated a total of 9,952 expressed sequence tags (ESTs) from randomly selected clones of two different cDNA libraries. One cDNA library was derived from fruit of “Saijo” persimmon at an early stage of development, and the other from ripening fruit. These ESTs were clustered into 6,700 non-redundant sequences. Of the 6,700 non-redundant sequences evaluated, the deduced amino acid sequences of 4,356 (65%) showed significant homology to known proteins, and 2,344 (35%) showed no significant similarity to any known proteins in Arabidopsis databases. We report comparison of genes identified in the two cDNA libraries and describe some putative genes involved in proanthocyanidin and carotenoid synthesis. This study provides the first global overview of a set of genes that are expressed during fruit development and ripening in persimmon.     

Carotenoids synthesized in citrus callus of different genotypes

Eight carotenoids, such as phytoene, α-carotene, violaxanthin, etc., synthesized in citrus callus of 31 genotypes were identified and determined. Though varied with genotypes, the carotenoids composition of callus derived from a certain genotype was stable, while carotenoids contents altered between sub-cultures. Some specific carotenoids were produced in calluses of limited genotypes: β-citraurin was only synthesized in calluses of Nianju tangerine (Citrus reticulata Blanco) and Page tangelo (C. reticulata × C. paradisi); while 9-Z-violaxanthin was only detected in Nianju tangerine and Skaggs Bonanza navel orange (C. sinensis L. Osbeck). Notably, the only carotenoid detected in calluses of Natsudaidai (C. aurantium L.) and other two sweet oranges (C. sinensis L. Osbeck) was phytoene. It implied that citrus calluses could be employed to produce specific carotenoids in the future. To further elucidate the characters of callus carotenoids profile, comparisons of carotenoids profiles was made among calluses, fruit tissues and leaves of four selected citrus genotypes. Results showed that lycopene was not detected in leaves and calluses; nevertheless, both citrus fruits and calluses accumulated phytoene, whereas leaves did not except those of Cara Cara navel orange. It is postulated that citrus callus featured its carotenoids profile different from fruit tissues and leaves. In conclusion, the advantages of using citrus callus as an alternative model research system in understanding the regulation of carotenogenesis have been discussed.     

Pollen and resource limitations to lifetime seed production in a wild population of the endangered plant Disanthus cercidifolius var. longipes H. T. Chang (Hamamelidaceae)

Disanthus cercidifolius Maxim. var. longipes H. T. Chang, a plant species that only occurs in a few counties in Hunan, Jiangxi and Zhejiang Provinces and with a relatively small number of individuals, is ranked as a second Class endangered species for conservation in China. We have studied the effect of pollen and resources available to female reproduction, and the reproductive mechanism of “excess flowers with low fruit set” in Disanthus cercidifolius Maxim. var. longipes H. T. Chang was discussed. Results are as follows Pollen from different sources has significant effects on fruit set and seed set of Disanthus cercidifolius Maxim. var. longipes H. T. Chang. The pollen source rather than pollen numbers significantly affected reproduction of this species. In wild populations, producing one fruit needs about 54.8 flowers, and one satiation seed needs about 6.60 flowers or 83.19 ovules. After fertilizing, which was propitious to flower development, the abortion rate of flower buds was decreasing, but the flowering rate was increasing. The fruit set and seed set was also significantly increasing, while abortion rate of fruit was significantly decreasing. With the increasing percentages of cutting leaves, the fruit set decreased, but the abortion rate of fruit shows no significant differentiation among treatments. After cutting branches that were puny, broken and insectin-fested branches, the flower number seemed to be decreasing, but the fruit set and seed set all increased significantly. After removing some flowers, the fruit set was calculated with respect to the number of flowers remaining after the treatment increased with increasing of percentages of flower removal, whereas fruit set calculated with respect to the initial number of flowers remained constant, and the mean weights of per fruit and per seed all decreased significantly. Sufficient spatial or temporal heterogeneities in nutrient levels might allow limitation of seed set by resources and pollen in a natural population, while supplying resources may indirectly affect pollination by increasing attraction of the flowers to pollinators. There were very low fruit and seed sets in natural populations of Disanthus cercidifolius Maxim. var. longipes H. T. Chang. Different factors may have interacted to effect a low fruit set. A joint adoption of the “selection abortion hypothesis”, “ovary reserve hypothesis” and “male function hypothesis” seems to be the most likely explanation for the reproductive strategy of “excess flowers with few fruit sets” in Disanthus cercidifolius Maxim. var. longipes H. T. Chang. __________ Translated from Acta Ecologica Sinica, 2006, 26(2): 496–502 [译自: 生态学报]     

Changes in Photosynthesis and Fruit Characteristics in Olive in Response to Assimilate Availability

The source level in the olive cultivar Leccino was varied by girdling at different stages of fruit growth. Afterwards, the effects on gas exchange, fruit growth, and ripening and blooming were studied. Girdling during fruit growth did not significantly influence net photosynthetic rate (P _N) except in the last phase of fruit growth when the P _N was reduced. In the girdled branch, P _N began to decrease at the onset of starch accumulation because fruit growth ceased. In mid-November stomatal conductance (g _s) and transpiration rate (E) were also reduced by girdling, whereas sub-stomatal CO₂ concentration (C _i) increased in leaves from the girdled branches. The total chlorophyll content (Chl) tended to decrease in parallel with the reduced P _N. Girdling did not substantially influence the leaf and shoot water contents. The large availability of assimilates seems to cause an earlier fruit ripening. In general, girdling increased fruit dry mass. Healing before the time when the majority of pulp growth occurs reduced the effect of girdling. June girdling increased the pit dry mass. Girdling at the beginning of August and September, compared to the control, increased the pulp dry mass, but the pit dry mass did not differ with respect to the control. The percentage of oil in the fruit, on a dry mass basis, increased with August and September girdlings, but the percentage of oil in the pulp did not change. Girdling reduced shoot growth, but the internode length was unchanged. Girdling slightly stimulated differentiation of flower buds.     

Identification and Expression Pattern of a Novel NAM,ATAF, and CUC-Like Gene from <Emphasis Type="Italic">Citrus sinensis</Emphasis> Osbeck

A citrus NAM, ATAF, and CUC (NAC)-like gene (CitNAC) was isolated from fruit tissues of Citrus sinensis Osbeck using complementary DNA (cDNA) amplified fragment length polymorphism and rapid amplification of cDNA ends techniques. Its full length was 988 bp in which 781 bp form the open reading frame, coding for a protein of 264 amino acids. Sequence comparison revealed that CitNAC possesses the general structural features at the N terminus of the NAC domains. Phylogenetic analysis results showed that CitNAC was closely related to AtNAP and PeNAP, which are involved in plant organ senescence. Gene expression analysis showed that the messenger RNA level of CitNAC was just detected in fruit peel and pulp during fruit ripening or senescence stage. The observed expression pattern of CitNAC along with the result of phylogenetic analysis suggested that CitNAC is related to fruit development and senescence.     

The effect of chimeric transgene architecture on co-ordinated gene silencing

Two gene constructs were made consisting of a 244-bp sense fragment from the 5′ end of a polygalacturonase cDNA, the 3′ end of which was ligated to a 414-bp fragment from the 5′ end of a phytoene synthase cDNA. In the first construct, the phytoene synthase fragment was in a sense orientation (sense/sense chimeric gene) and in the second construct the phytoene synthase fragment was in an antisense orientation (sense/antisense chimeric gene). Both chimeric genes were inserted between a cauliflower mosaic virus promoter and terminator. Tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) plants transformed with each construct gave rise to transformants with three distinct phenotypes: plants with red fruit, plants with pure yellow fruit and plants with red and yellow sectored fruit. For both chimeric constructs, expression of the endogenous polygalacturonase and phytoene synthase genes were found to be co-ordinately suppressed in yellow tissue, but showed normal expression in red tissue. Data from microscopic analyses of fruit chromoplasts, from the three phenotypes, implied that phytoene synthase suppression from each construct predominantly had two states within a cell: on or off. Received: 31 July 1997 / Accepted: 28 August 1997