Inhibition of aconitase in citrus fruit callus results in a metabolic shift towards amino acid biosynthesis

Citrate, a major determinant of citrus fruit quality, accumulates early in fruit development and declines towards maturation. The isomerization of citrate to isocitrate, catalyzed by aconitase is a key step in acid metabolism. Inhibition of mitochondrial aconitase activity early in fruit development contributes to acid accumulation, whereas increased cytosolic activity of aconitase causes citrate decline. It was previously hypothesized that the block in mitochondrial aconitase activity, inducing acid accumulation, is caused by citramalate. Here, we investigated the effect of citramalate and of another aconitase inhibitor, oxalomalate, on aconitase activity and regulation in callus originated from juice sacs. These compounds significantly increased citrate content and reduced the enzyme’s activity, while slightly inducing its protein level. Citramalate inhibited the mitochondrial, but not cytosolic form of the enzyme. Its external application to mandarin fruits resulted in inhibition of aconitase activity, with a transient increase in fruit acidity detected a few weeks later. The endogenous level of citramalate was analyzed in five citrus varieties: its pattern of accumulation challenged the notion of its action as an endogenous inhibitor of mitochondrial aconitase. Metabolite profiling of oxalomalate-treated cells showed significant increases in a few amino acids and organic acids. The activities of alanine transaminase, aspartate transaminase and aspartate kinase, as well as these of two γ-aminobutyrate (GABA)-shunt enzymes, succinic semialdehyde reductase (SSAR) and succinic semialdehyde dehydrogenase (SSAD) were significantly induced in oxalomalate-treated cells. It is suggested that the increase in citrate, caused by aconitase inhibition, induces amino acid synthesis and the GABA shunt, in accordance with the suggested fate of citrate during the acid decline stage in citrus fruit.     

光照对柑橘果皮类胡萝卜素和色泽形成的影响

以“红柿柑”为试材,在柑橘果实膨大末期通过套袋遮光处理以抑制果皮光合作用,研究光照对果皮糖、叶绿素、类胡萝卜素及果实外观色泽的影响。结果表明,遮光后果皮叶绿素含量迅速下降引起果实转色提早,但各种类胡萝卜素含量及总量并未提高,而是显著下降;至果实成熟时由于遮光与光照处理的果皮叶绿素均消失,遮光果实类胡萝卜素含量低颜色变淡,与光照处理相比,遮光前期果皮糖含量下降不大,而后期下降明显;若在后期去袋照光,果皮糖含量上升,与此相应,类胡萝卜素,尤其是β-隐黄质的积累增加,颜色加深,表明光对果皮类胡萝卜素合成尤其是β-隐黄质的积累有促进作用,其原因是光以环境信号的方式影响果皮的类胡萝卜素形成。     

Aconitase activity and expression during the development of lemon fruit

Citrus fruits are characterized by the accumulation of high levels of citric acid in the juice sac cells and a decline in acid level toward maturation. It has been suggested that changes in mitochondrial aconitase (EC 4.2.1.3) activity affect fruit acidity. Recently, a cytosolic aconitase (cyt-Aco) homologous to mammalian iron-regulated proteins was identified in plants, leading us to re-evaluate the role of aconitase in acid accumulation. Aconitase activity was studied in 2 contrasting citrus varieties, sweet lime ( Citrus limettioides Tan., low acid) and sour lemon ( Citrus limon var. Eureka, high acid). Two aconitase isozymes were detected. One declined early in sour lemon fruit development, but was constant throughout sweet lime fruit development. Its reduction in sour lemon was associated with a decrease in aconitase activity in the mitochondrial fraction. Another isozyme was detected in sour lemon toward maturation, and was associated with an increase in aconitase activity in the soluble fraction, suggesting a cytosolic localization. The cyt-Aco was cloned from lemon juice sac cells, but in contrast to the changes in isozyme activity, its expression was constant during fruit development. We present a model, which suggests that reduction of the mitochondrial aconitase activity plays a role in acid accumulation, while an increase in the cyt-Aco activity reduces acid level toward fruit maturation.     

杨梅果实发育进程中的碳水化合物代谢

以‘乌紫’和‘荸荠’两个杨梅品种为试材,测定了干鲜重、糖含量、可滴定酸含量、蔗糖和己糖代谢相关酶活性的动态变化。结果表明,杨梅果实的干鲜重、含糖量的快速增长和可滴定酸含量的快速下降均发生在果实发育后期。成熟‘乌紫’杨梅果实的蔗糖含量约占总糖的2／3以上,而‘荸荠’杨梅仅为总糖的49％。‘荸荠’杨梅的转化酶和蔗糖合酶分解活性随着果实发育呈上升趋势,‘乌紫’杨梅的则变化不大。两个品种的蔗糖磷酸合酶活性随着果实发育呈上升趋势,但蔗糖合酶合成活性到果实发育中期后下降。两个品种的己糖激酶活性变化相似,但果糖激酶活性的变化趋势不同。     

Differential effects of abscisic acid and ethylene on the fruit maturation of <Emphasis Type="Italic">Litchi chinensis</Emphasis> Sonn.

Two litchi cultivars, a well-coloured ‘Nuomici’ and a poorly coloured ‘Feizixiao’, were used to investigate changes in endogenous abscisic acid (ABA) concentration and ethylene production during fruit maturation and to test the effects of exogenous growth regulators on litchi fruit maturation. Abscisic acid concentration in both the aril and pericarp increased with fruit maturation. Transfusion of ABA into the fruit 3 weeks before harvest accelerated, whereas transfusion of 6-benzyl aminopurine (6-BA) retarded sugar accumulation and pigmentation. The effect of 6-BA was assumed to link with the resultant decrease in ABA. In contrast, 1-aminocyclopropane-1-carboxylic acid (ACC) concentration and ACC oxidase (ACO) activities in the aril remained relatively constant during sugar accumulation. Transfusion of aminooxyacetic acid (AOA) significantly decreased ACC concentration but had no effect on sugar accumulation in the aril. These results suggested that endogenous ABA, but not ethylene, was critical for the sugar accumulation. However, the roles of ABA and ethylene in pericarp pigmentation were rather complicated. Application of exogenous ABA promoted anthocyanin synthesis significantly, but had very little effect on chlorophyll degradation. Ethylene production in litchi fruit decreased with development, but a transient increase of endogenous ethylene production was detected just around the colour break in ‘Nuomici’. Enhanced ACO activity in the pericarp was detected during pigmentation. Ethrel at 400 mg l⁻¹ showed no effect on pericarp coloration, but accelerated chlorophyll degradation and anthocyanin synthesis at a much higher concentration (800 mg l⁻¹). Fruit dipped in ABA solution alone yielded no effect on chlorophyll degradation, but the combined use of ABA and Ethrel at 400 mg l⁻¹ enhanced both chlorophyll degradation and anthocyanin synthesis. These results indicated the possible synergistic action of ethylene and ABA during litchi fruit colouration. ABA is suggested to play a more crucial role in anthocyanin synthesis, while ethylene is more important in chlorophyll degradation. ABA can increase the sensitivity of pericarp tissue to ethylene.     

Sugar regulation of plastid interconversions in epicarp of citrus fruit

Seasonal transformations between chloroplasts and chromoplasts, as measured by changes in chlorophyll content, in the epicarp of degreening and regreening Citrus sinensis (L.) Osbeck cv Valencia fruit closely parallelled the accumulation and later loss of soluble sugars. At any stage of development, reversing the relative soluble sugar content in the epicarp by culturing pericarp segments on agar media with low (15 millimolar) or high (150 millimolar) sucrose concentrations reversed the direction of change in chlorophyll content. Fruit of C. madurensis Lour., which mature year around and do not regreen, also accumulated soluble sugars in the pericarp as degreening was initiated.

The epicarp of C. sinensis fruit accumulated nitrogen, but total nitrogen concentrations and amino acid concentrations changed little, during degreening and regreening of C. sinensis fruit. Cessation of nitrogen fertilization reduced the tendency of pericarp segments to regreen in vitro during subsequent years, but regreening tendency was restored by inclusion of KNO₃ in the media.

It is concluded that chloroplasts become chromoplasts and citrus fruit degreen partially in response to the accumulation of sugars in the epicarp and that the reverse transformation accompanying regreening of certain citrus species occurs when accumulated sugars disappear. Change in nitrogen flux to the fruit is probably not a factor in regulating seasonal transformations, but an abundance of nitrogen in the epicarp diminishes the effects of high sugar concentrations in inducing transformation of chloroplasts to chromoplasts, thereby retarding degreening and promoting regreening.

     

Girdling affects carbohydrate-related gene expression in leaves,bark and roots of alternate-bearing citrus trees

Effects of girdling on carbohydrate status and carbohydrate-related gene expression in citrus trees were investigated. Alternate-bearing 'Murcott' (a Citrus reticulata hybrid of unknown origin) trees were girdled during autumn (25 Sep. 2001) and examined 10 weeks later. Girdling brought about carbohydrate (soluble sugar and starch) accumulation in leaves and shoot bark above the girdle, in trees during their fruitless, 'off' year. Trees during their heavy fruit load, 'on' year did not accumulate carbohydrates above the girdle due to the high demand for carbohydrates by the developing fruit. Girdling caused a strong decline in soluble sugar and starch concentrations in organs below the girdle (roots), in both 'on' and 'off' trees. Expression of STPH-L and STPH-H (two isoforms of starch phosphorylase), Agps (ADP-glucose pyrophosphorylase, small subunit), AATP (plastidic ADP/ATP transporter), PGM-C (phosphoglucomutase) and CitSuS1 (sucrose synthase), all of which are associated with starch accumulation, was studied. It was found that gene expression is related to starch accumulation in all 'off' tree organs. RNA levels of all the genes examined were high in leaves and bark that accumulated high concentrations of starch, and low in roots with declining starch concentrations. It may be hypothesized that changes in specific sugars signal the up- and down-regulation of genes involved in starch synthesis.     

Recent Advances in the Regulation of Citric Acid Metabolism in Citrus Fruit

The regulation of citric acid metabolism during fruit ripening has a major impact on the production of high-quality fruit. The impact of citric acid on organoleptic fruit quality attributes, fruit storage performance and the synthesis of several secondary metabolites has led to an exponential increase in research efforts during the last two decades. Recent research has focused on the relationship among citric acid biosynthesis, transportation, storage, and utilization. Among citrate metabolic processes, activities of a proton pump, especially the plasma membrane H⁺-ATPase on tonoplast and citrate catabolism in cytosol play important roles in the regulation of citrate accumulation in cell vacuoles. Moreover, we highlight recent advances and provide an overview of citrate metabolism, postharvest physiology of citrate metabolism, and the influence of agro-climatic factors on citrus fruits. It is the first review that provides a comprehensive model for citrate metabolism in citrus fruit juice sacs. We anticipate that this model for the regulation of citrate metabolism will facilitate the study of fruit acidity in citrus and other nonclimacteric fruits.     

宁夏枸杞果实遮光处理对果实糖积累和相关酶活性的影响

在宁夏枸杞盛花期对果实进行遮光处理,以自然照光为对照,通过测定枸杞果实生长指标、果实叶绿素含量、蔗糖代谢糖分含量及其蔗糖代谢相关酶活性,以研究枸杞果实光合作用在枸杞果实糖积累中的效应及对枸杞果实多糖和总糖含量积累的影响。结果表明:(1)遮光后,果实单粒重和果实中叶绿素含量均降低,体积却有所增加,遮光处理主要影响果实着色期和成熟期的糖含量,对果实发育初期影响不大;(2)遮光处理不同程度增加了果实转化酶、蔗糖磷酸合成酶和蔗糖合成酶活性。枸杞果实多糖的形成与果实光照具有一定的关系,而总糖含量的积累与光照关系不大。     

套袋对梨果实发育过程中糖组分及其相关酶活性的影响

以翠冠和黄金梨为试材,测定套袋和未套袋(对照)梨果实发育时期果实中蔗糖、葡萄糖、果糖和山梨醇含量以及蔗糖代谢相关酶酸性转化酶(AI)、中性转化酶(NI)、蔗糖合成酶(SS)和蔗糖磷酸合成酶(SPS)的活性,并对果实中糖组分积累与酶活性的关系进行了分析.结果表明:(1)两梨品种套袋果实在发育过程中蔗糖、葡萄糖、果糖、山梨醇和糖代谢相关酶活性变化趋势与对照基本一致,套袋果实糖含量均低于对照但差异不显著,而各相关酶活性在两类果实间差异表现各异.(2)在梨果实发育早期,果实中以分解酶类为主,糖分积累低;发育后期以合成酶类为主,糖分积累多.(3)两品种套袋和对照果实AI活性与葡萄糖含量均呈显著或极显著正相关,SS合成方向活性与蔗糖含量均为极显著正相关,且翠冠对照果SPS活性与蔗糖含量呈极显著正相关.可见,套袋通过提高果实发育早期转化酶(Inv)活性,降低果实后期蔗糖磷酸合成酶(SPS)、蔗糖合成酶(SS)的活性来影响糖分积累,从而影响梨果品质.     

Novel expression patterns of carotenoid pathway-related genes in citrus leaves and maturing fruits

Carotenoids are abundant in citrus fruits and vary among cultivars and species. In the present study, high performance liquid chromatography (HPLC) and real-time polymerase chain reaction (PCR) were used to investigate the expression patterns of 23 carotenoid biosynthesis gene family members and their possible relation with carotenoid accumulation in fresh flavedo, juice sacs and leaves of Valencia orange during fruit maturation. Violaxanthin and lutein mainly accumulated in fruit (flavedo and juice sacs) and leaves (young and mature), respectively, accounting for nearly 79 %, 57 %, 53 % and 70 % of corresponding total carotenoids in February. Violaxanthin content quickly began to increase in flavedo in December, but the increase in juice sacs began later in January. In mature leaves, lutein content was three times that in young leaves; α-carotene and β-carotene were also much higher in mature leaves than in flavedo or juice sacs. Generally most of the carotenoid biosynthesis gene members were expressed at higher levels in flavedo than in juice sacs, and the expression of some continued to increase in flavedo during fruit maturation. All CHYB members expressed at high levels and had similar patterns in juice sacs. Interestingly, the capsanthin capsorubin synthase (CCS) members had similar expression levels and patterns in flavedo and juice sacs. Differences in gene expression between leaf and fruit tissues were noted, pointing to some tissue specificity for certain members of the gene families associated with carotenogenesis. The expression patterns of these 23 citrus carotenoid biosynthesis gene members were also compared with their expression patterns in other plants. Taken together, these first-hand expression data will be useful to define the tissue-specific roles of each gene member in accumulation of different carotenoids in citrus leaves and maturing fruits.