Differential expression of anthocyanin biosynthetic genes in relation to anthocyanin accumulation in the pericarp of Litchi chinensis Sonn

Litchi has diverse fruit color phenotypes, yet no research reflects the biochemical background of this diversity. In this study, we evaluated 12 litchi cultivars for chromatic parameters and pigments, and investigated the effects of abscisic acid, forchlorofenron (CPPU), bagging and debagging treatments on fruit coloration in cv. Feizixiao, an unevenly red cultivar. Six genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) were isolated from the pericarp of the fully red litchi cv. Nuomici, and their expression was analyzed in different cultivars and under the above mentioned treatments. Pericarp anthocyanin concentration varied from none to 734 mg m⁻² among the 12 litchi cultivars, which were divided into three coloration types, i.e. non-red (‘Kuixingqingpitian’, ‘Xingqiumili’, ‘Yamulong’and ‘Yongxing No. 2′), unevenly red (‘Feizixiao’ and ‘Sanyuehong’) and fully red (‘Meiguili’, ‘Baila’, Baitangying’ ’Guiwei’, ‘Nuomici’ and ‘Guinuo’). The fully red type cultivars had different levels of anthocyanin but with the same composition. The expression of the six genes, especially LcF3H, LcDFR, LcANS and LcUFGT, in the pericarp of non-red cultivars was much weaker as compared to those red cultivars. Their expression, LcDFR and LcUFGT in particular, was positively correlated with anthocyanin concentrations in the pericarp. These results suggest the late genes in the anthocyanin biosynthetic pathway were coordinately expressed during red coloration of litchi fruits. Low expression of these genes resulted in absence or extremely low anthocyanin accumulation in non-red cultivars. Zero-red pericarp from either immature or CPPU treated fruits appeared to be lacking in anthocyanins due to the absence of UFGT expression. Among these six genes, only the expression of UFGT was found significantly correlated with the pericarp anthocyanin concentration (r = 0.84). These results suggest that UFGT played a predominant role in the anthocyanin accumulation in litchi as well as pericarp coloration of a given cultivar.     

早红考密斯及其芽变果实中花青素含量与相关酶活性变化的研究

以西洋梨早红考密斯及其绿色芽变果实为材料,研究了果实发育期间果皮色泽、花青苷含量及其相关酶活性变化.结果显示:(1)早红考密斯果皮色泽从成熟前的暗红色逐渐变为成熟时的浅红色,并在色泽分布不均匀的地方显出黄色底色,色泽指数(a*)值从花后45 d的16.4降低到成熟时的7.4,降低54.9%;花青苷含量从成熟前的258.4μg?g-1降到成熟时的118.3μg?g-1;早红考密斯果皮色泽和果皮花青苷含量具有密切的相关性.(2)早红考密斯的绿色芽变在果实发育的前期检测不到花青苷,发育后期果实向阳部出现浅红晕,但花青苷含量极低,与亲本差异极显著.(3)果实发育期间,两品种间苯丙氨酸解氨酶(PAL)变化趋势相似,总体呈下降趋势,且早红考密斯的活性总体低于其绿色芽变;两品种查耳酮异构酶(CHI)活性总体变化趋势基本一致,均呈现缓慢上升的趋势,在前期绿色芽变的CHI活性高于其亲本,后期低于亲本;类黄酮3-O-葡(萄)糖基转移酶(UFGT)活性在两品种间的差异较大,在整个果实发育期间早红考密斯的UFGT活性远高于其绿色芽变.研究表明,早红考密斯果皮色泽变化主要由花青苷的含量不同引起;PAL和CHI不是绿色芽变的直接原因;UFGT与花青苷合成密切相关,绿色芽变果皮中UFGT活性显著降低.     

金花茶类黄酮糖基转移酶基因的克隆和功能初步研究

类黄酮糖基转移酶(UDP flavonoid glycosyltransferase, UFGT)催化黄酮醇、花青素等形成稳定的糖苷,是黄酮醇苷、花青素苷合成的最后一步反应的关键。该研究以金花茶的花瓣为材料,采用PCR扩增的方法,获得了2个金花茶转录组中筛选到的类黄酮糖基转移酶基因。结果显示：(1)CnUFGT14基因(登录号为MT370521)全长1 562 bp,开放阅读框1 380 bp,编码459个氨基酸;CnUFGT15基因(登录号为MT370520)全长1 546 bp,开放阅读框1 368 bp,编码455个氨基酸;两个蛋白序列均具有UFGT蛋白特有的 PSPG 保守区域。(2)系统进化树分析发现,CnUFGT14和CnUFGT15分别与茶树UFGT78A14和UFGT78A15亲缘关系最近。(3) 荧光定量PCR分析发现,CnUFGT14基因的表达量与多种多酚组分的含量呈正相关,CnUFGT15基因的表达量与花瓣黄酮醇、多酚等的含量相关性不显著。(4)亚细胞定位研究发现,CnUFGT14、CnUFGT15蛋白在细胞核膜、细胞质、细胞膜部位均呈现明显的定位。(5)叶盘法转化烟草发现,CnUFGT14基因表达量较高的转基因株系中总多酚含量及多种多酚组分含量升高,而CnUFGT15基因的转基因株系中黄酮、多酚组分变化不显著。研究表明,CnUFGT14基因具有促进多酚合成的作用,而CnUFGT15基因对类黄酮通路不具有明显作用。     

Expression of anthocyanin biosynthesis pathway genes in red and white grapes

The expression of seven genes from the anthocyanin biosynthesis pathway was determined in different tissues of Shiraz grapevines. All of the tissues contained proanthocyanidins, but only the berry skin accumulated anthocyanins. In most tissues, all of the flavonoid genes except UDP glucose-flavonoid 3-o-glucosyl transferase (UFGT) were expressed, but UFGT expression was only detected in berry skin. Similar patterns of expression were observed in the skin of other red grapes. In white grapes, UFGT expression was not detected. White grape cultivars appear to lack anthocyanins because they lack UFGT, although they also had decreased expression of other flavonoid pathway genes.     

Accumulation of Flavonoid Glycosides and UFGT Gene Expression in Mulberry Leaves (Morus alba L.) before and after Frost

In order to determine the molecular mechanism underlying the influence of frost on chemical changes in mulberry leaves, the UFGT activity, expression level, and accumulation of flavonoid glycosides in mulberry leaves (Morus alba L.) were studied. The expression of UFGT gene was investigated by quantitative real‐time PCR (qRT‐PCR) and the UFGT activity, accumulation of flavonoid glycosides was studied by high performance liquid chromatography. Then, the correlation between the expression level of UFGT, the UFGT activity, and the flavonoid glycosides accumulation with temperature was explored. The accumulation of isoquercitrin and astragalin is significantly positively correlated with UFGT gene expression and UFGT activity. On the contrary, the average temperature was significantly negatively correlated with the level of UFGT gene expression and UFGT activity. The results show that after frost, low temperature can induce the expression of UFGT gene in mulberry leaves, resulting in the accumulation of flavonoid glycosides.     

Expression analysis of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene in an interspecific hybrid grape between Vitis ficifolia var. ganebu and Vitis vinifera cv. Muscat of Alexandria

Kadainou R-1, an interspecific hybrid grape derived from red (Vitis ficifolia var. ganebu) and white (V. vinifera cv. Muscat of Alexandria) grapes, accumulates high concentrations of anthocyanin in the berry skin. Hence, the expression of uridine 5′-diphosphate (UDP)-glucose:flavonoid 3-O-glucosyltransferase (UFGT), the key enzyme of the anthocyanin pathway, was examined in the berry skin of Kadainou R-1. As information on gene sequences of V. ficifolia var. ganebu and other wild grape species was unavailable, we performed GeneChip hybridization using biotin-labeled genomic deoxyribonucleic acid (DNA) to investigate how the genomic sequences of V. vinifera varieties and that of V. ficifolia var. ganebu differ. The study showed a lower correlation coefficient between V. vinifera cultivars and V. ficifolia var. ganebu than that among V. vinifera cultivars. The sequences of the UFGT gene derived from both parents of the red and white cultivars were sequenced in Kadainou R1 and revealed that both were expressed irrespective of the fact that it was not expressed in the white grape (male parent).     

BBX16, a B‐box protein,positively regulates light‐induced anthocyanin accumulation by activating MYB10 in red pear

The red coloration of pear (Pyrus pyrifolia) results from anthocyanin accumulation in the fruit peel. Light is required for anthocyanin biosynthesis in pear. A pear homolog of Arabidopsis thaliana BBX22, PpBBX16, was differentially expressed after fruits were removed from bags and may be involved in anthocyanin biosynthesis. Here, the expression and function of PpBBX16 were analysed. PpBBX16's expression was highly induced by white‐light irradiation, as was anthocyanin accumulation. PpBBX16's ectopic expression in Arabidopsis increased anthocyanin biosynthesis in the hypocotyls and tops of flower stalks. PpBBX16 was localized in the nucleus and showed trans‐activity in yeast cells. Although PpBBX16 could not directly bind to the promoter of PpMYB10 or PpCHS in yeast one‐hybrid assays, the complex of PpBBX16/PpHY5 strongly trans‐activated anthocyanin pathway genes in tobacco. PpBBX16's overexpression in pear calli enhanced the red coloration during light treatments. Additionally, PpBBX16's transient overexpression in pear peel increased anthocyanin accumulation, while virus‐induced gene silencing of PpBBX16 decreased anthocyanin accumulation. The expression patterns of pear BBX family members were analysed, and six additional BBX genes, which were differentially expressed during light‐induced anthocyanin biosynthesis, were identified. Thus, PpBBX16 is a positive regulator of light‐induced anthocyanin accumulation, but it could not directly induce the expression of the anthocyanin biosynthesis‐related genes by itself but needed PpHY5 to gain full function. Our work uncovered regulatory modes for PpBBX16 and suggested the potential functions of other pear BBX genes in the regulation of anthocyanin accumulation, thereby providing target genes for further studies on anthocyanin biosynthesis.     

High‐temperature inhibition of biosynthesis and transportation of anthocyanins results in the poor red coloration in red‐fleshed Actinidia chinensis

In plants, the role of anthocyanins trafficking in response to high temperature has been rarely studied, and therefore poorly understood. Red‐fleshed kiwifruit has stimulated the world kiwifruit industry owing to its appealing color. However, fruit in warmer climates have been found to have poor flesh coloration, and the factors responsible for this response remain elusive. Partial correlation and regression analysis confirmed that accumulative temperatures above 25°C (T25) was one of the dominant factors inhibiting anthocyanin accumulation in red‐fleshed Actinidia chinensis, ‘Hongyang’. Expression of structural genes, AcMRP and AcMYB1 in inner pericarp sampled from the two high altitudes (low temperature area), was notably higher than the low altitude (high temperature area) during fruit coloration. AcMYB1 and structural genes coordinate expression supported the MYB–bHLH (basic helix‐loop‐helix)–WD40 regulatory complex mediated downregulation of anthocyanin biosynthesis induced by high temperatures in kiwifruit. Moreover, cytological observations using the light and transmission electronic microscopy showed that there were a series of anthocyanic vacuolar inclusion (AVI)‐like structures involved in their vacuolization process and dissolution of the pigmented bodies inside cells of fruit inner pericarp. Anthocyanin transport was inhibited by high temperature via retardation of vacuolization or reduction in AIV‐like structure formation. Our findings strongly suggested that complex multimechanisms influenced the effects of high temperature on red‐fleshed kiwifruit coloration.     

石榴PgUFGT基因克隆及表达分析

利用RT-PCR和RACE方法,从石榴(Punica granatum L.)果皮中克隆到一个类黄酮糖基转移酶(UFGT)基因(PgUFGT)全长cDNA序列(GenBank登录号为KF841620)。PgUFGT基因编码区1 476bp,编码491个氨基酸。PgUFGT蛋白具有保守PSPG基序、UDP-糖基转移酶家族结构域和UDP-葡萄糖醛酸基/葡萄糖基转移酶保守域(UDPGT),与其他植物UFGT蛋白一致性较高;系统进化树分析结果表明,PgUFGT属于类黄酮3-O-糖基转移酶类。荧光定量qRT-PCR结果表明,PgUFGT基因在‘红宝石’和‘水晶甜’2个石榴品种的发育期内具有不同的表达模式,PgUFGT在‘红宝石’石榴中有2个转录表达高峰,而在‘水晶甜’石榴中仅有1个表达高峰,表明PgUFGT可能在2个石榴品种中具有不同的催化作用。该研究结果为进一步研究石榴果实色泽形成的分子机制奠定了基础。