苹果和猕猴桃抗坏血酸形成与积累的生理和分子机理研究
摘要
抗坏血酸(Ascorbic acid,AsA),又名维生素C(Vitamin C,Vc),是生物体内重要的抗氧化剂和许多酶的辅因子。植物体内的AsA不仅与自身生长发育和逆境的抗性密切相关,而且由于人类自身不能合成AsA,它又是人体重要的天然AsA来源,与人类的健康密切相关。目前,对AsA在作物可食部分积累机制及其AsA含量差异的原因尚不清楚。本文以低AsA含量的苹果(Malus domestica Borkh‘Gala’)和高AsA含量的猕猴桃(Actinidia deliciosa cv. Qinmei)为材料,从AsA的组织分配、生物合成、再生、运输及光调控等方面对AsA形成和积累机理进行了研究。获得的主要结果如下:
1.从苹果果实中克隆获得了AsA再生酶MDHAR和DHAR2及合成酶GME和GalUR基因cDNA全长或部分序列。其中,MDHAR长1400 bp,包含了1305 bp的完整开放阅读框,编码一个膜结合MDHAR;DHAR2长933 bp,包含了798 bp的完整开放阅读框,属于GSH依赖性胞质DHAR;GME长1323 bp,包含了1131 bp的完整开放阅读框,序列分析发现其编码一个NAD依赖性胞质酶。而GalUR长668 bp,与草莓GalUR氨基酸同源性高达80%,为GalUR基因的cDNA部分序列。
2.在‘嘎啦’苹果果实的果皮、果肉中检测到了AsA合成关键酶GalDH和GalLDH基因mRNA的表达和活性;通过不同AsA合成前体物饲喂发现,参与L-半乳糖途径的L-Gal和L-GL能引起果皮和果肉AsA含量的明显增加,D-GalUA也能引起果皮AsA含量的明显增加,且幼果的增加幅度高于成熟果。在果实的不同组织中,高AsA含量的果皮有高的AsA合成和再生酶基因的表达量和活性;果皮中AsA含量受光的调控,与阴面果皮相比,阳面果皮具有高的GalDH、GalLDH、GalUR及MDHAR、DHAR mRNA表达量和活性,而阳面和阴面果肉间则无明显差异。对苹果AsA运输的初步研究发现,在果实微管组织中存在大量AsA的积累,且在具有AsA合成能力的果柄和叶柄韧皮部及其卸载液中也存在大量AsA,但果柄施加外源AsA并不能引起果实AsA含量的增加。这表明在苹果中AsA可能能从叶片运输到果实,但在果实中不能很好的卸载从而在维管束中积累;或者这些组织中自身合成的AsA仅在果柄或叶柄中发挥它的抗氧化等功能,而与果实和叶片间AsA运输关系不大。这些结果表明生物合成是苹果AsA形成的主要原因。
3.光直接影响苹果叶片和果皮中的AsA合成和代谢。在弱光条件下,苹果叶片和果皮中AsA合成酶GalDH和GalLDH mRNA表达量与活性及再生能力明显降低,AsA含量下降。果实或整个树体遮光后,果肉组织中GalDH和GalLDH转录水平及其活性和再生酶活性均无明显变化。果实遮光对果肉AsA水平无明显影响,但整个树体遮光20 d后果肉中T-AsA和AsA含量明显降低,且树冠底部内膛果实果肉的AsA含量也较低。这说明苹果果肉组织中AsA的合成和代谢能力不直接受光的调控,但光可通过影响叶片间接地对果肉中AsA含量起到调控作用。
4.在苹果果实生长发育过程中,单位鲜重果实中T-AsA和AsA含量在花后0 d的子房中最高,之后随着生长发育逐渐下降,在花后60 d后至果实成熟基本保持不变。从单个果实中AsA的积累量来看,在整个果实生长发育过程中AsA积累一直在发生,即在单个果实中AsA合成速率一直大于DHA的降解速率。在果实生长发育过程中,AsA合成酶基因表达量表现出不同的变化模式。GME、GGP和GalUR mRNA表达量在果实生长发育过程中显著地增加与AsA含量的变化不一致,表明它们对AsA含量不起关键调控作用。GPP在果实发育过程中与单位鲜重中AsA含量的变化模式存在相似性。在幼果期果实高的GalDH和GalLDH mRNA表达量和酶活性与幼果期由L-Gal和L-GL合成AsA的能力相一致。在AsA再生系统中,MDHAR mRNA表达量和酶活性随果实的生长表现出明显的下降趋势,与AsA/DHA比值呈负相关。
5.为了探索叶片AsA形成与调控和果实的异同点,研究了不同叶龄苹果叶片AsA形成及其与合成和再生的调控关系。结果表明,在不同叶龄的苹果叶片中,AsA的积累主要发生在幼叶向成熟叶的转变期,该时期叶片有高的AsA合成和再生能力,而衰老期AsA合成和再生能力下降,AsA含量降低。在AsA合成中,GPP的转录调控可能对苹果叶片AsA水平起着重要的调控作用,而GMP、GGP、GalLDH表达量的变化模式表明它们对AsA水平不具有调控功能。此外,GGP和GalUR在老叶中高的表达量可能与衰老有关。在再生系统中,MDHAR表达量和活性与苹果叶片中DHA含量相关,表明MDHAR可能在维持植物AsA氧化还原状态中起重要作用。而在AsA水平稳定的成熟叶中高的DHAR表达量和活性表明DHAR在维持AsA合成和氧化降解之间的平衡中可能起重要作用。
6.从美味猕猴桃品种‘秦美’果实中克隆获得了MDHAR、DHAR2和GME基因cDNA部分序列及DHAR1和GPP基因的cDNA全长序列。其中,MDHAR长1019 bp,属于膜结合MDHAR基因的cDNA部分序列; DHAR1长821 bp,包含了639 bp的完整开放阅读框,而DHAR2长542 bp,包含5′端起始密码子ATG,分别与已报道的两种植物胞质DHAR有高度的同源性,均为GSH依赖性胞质DHAR;GME长846 bp,与其它植物GME基因氨基酸同源性多在90%以上,为美味猕猴桃GME基因的cDNA部分序列;GGP为1542 bp,包含1353 bp的完全开放阅读框,对其编码蛋白结构分析发现,它具有螺旋跨膜区,是一个膜结合蛋白。
7.在美味猕猴桃品种‘秦美’果实中也检测到了AsA合成酶GalUR、GalDH和GalLDH mRNA表达及GalDH和GalLDH活性。不同AsA合成前体物饲喂发现,不仅参与L-半乳糖途径的L-Gal和L-GL能引起果实AsA含量的显著增加,而且L-GulL和D-GalUA也能引起AsA含量的明显增加,且幼果高于成熟果。这说明猕猴桃果实具有AsA合成能力,且可能存在多个途径。在果实的不同组织中,有高AsA合成和再生酶基因表达和活性的果肉及种子区有最高的AsA含量,而合成酶基因表达量和活性水平较低的中轴AsA含量很低。同时,猕猴桃果实AsA的分布也存在细胞特异性,果皮细胞中几乎观察不到AsA的存在;在果肉细胞中,大细胞不仅胞内有大量的AsA积累,而且在其细胞壁上也有AsA的存在;中轴细胞中AsA主要分布在细胞壁等质外体;维管束的导管细胞中也存在大量的AsA。对猕猴桃AsA运输能力的研究表明,果柄和叶柄中自身合成的AsA可能仅在果柄或叶柄中发挥抗氧化等功能,而与果实与叶片间AsA运输关系无关。但幼果期果柄施加外源蔗糖能引起幼果AsA含量的明显增加,表明猕猴桃果实中的AsA可能与叶片糖源的供应能力有关。这些结果也说明猕猴桃果实中AsA形成的主要原因是自身合成,同时叶片的糖源供应能力可能调控着幼果期果肉中AsA合成速率。
8.猕猴桃果实套袋遮光不影响果实中AsA含量,但幼果中AsA水平明显受日变化的影响,清晨6:00比中午和下午的AsA含量明显低。在果实不同发育阶段树体遮光处理中,花后0-40 d间遮光能显著降低幼果中T-AsA和AsA含量,而花后40 d后遮光对AsA含量无明显影响。这表明光不直接影响猕猴桃果实中AsA含量,但能通过叶片影响幼果中AsA含量。猕猴桃树体遮光显著降低了叶片AsA合成和再生酶基因的表达量和活性,引起了AsA含量显著下降和DHA增加。尽管如此,花后0-40 d树体遮光也显著降低了花后40 d猕猴桃果实中AsA合成酶GalLDH、GalDH、GPP、GME和GalUR及再生酶MDHAR和DHAR mRNA表达量,并降低了GalLDH、GalDH、MDHAR和DHAR活性,幼果AsA含量和积累量下降,40 d后果实生长速率下降,果实变小。而花后40-120 d遮光虽引起了糖含量的显著下降,但对AsA含量、合成和再生无明显影响。这表明花后0-40 d树体遮光降低了猕猴桃幼果AsA合成能力。外源蔗糖对幼果AsA含量的增加表明花后0-40 d遮光处理对猕猴桃幼果AsA合成的影响可能与淀粉、可溶性总糖和蔗糖含量的明显降低有关。这些结果表明光不直接影响猕猴桃果实AsA含量和合成,但能通过影响叶片对果实的糖供应能力或其它信号调控幼果AsA合成和再生能力,间接调控着幼果中AsA含量和果实中AsA积累量,且这种对幼果AsA含量的影响可能与后期果实膨大有关。
9.在猕猴桃果实生长发育过程中,单位鲜重果实中T-AsA和AsA含量在花后迅速增加,并在花后30 d达到最高,之后逐渐下降,在花后60 d后至果实成熟基本保持不变。从单个果实中AsA的积累量来看,T-AsA和AsA积累量在花后伴随果实的生长迅速增加,在花后45 d达到最高,之后至果实成熟期保持不变。这说明猕猴桃果实中AsA的积累主要发生在花后45 d前的幼果期。在果实生长发育过程中,GPP和GGP在转录水平上对果实AsA合成调控的可能性最大,特别是GPP,而从GalLDH、GalDH、GMP和GME转录水平的变化模式来看,它们对AsA合成调控的可能性很小。在AsA再生系统中,MDHAR和DHAR表达量及活性与幼果期AsA的快速积累关系不大。但45 d后高的表达和活性(尤其MDHAR)可能与AsA水平的维持有关。在碳水化合物中,可溶性总糖、还原糖和淀粉含量与AsA的变化模式均不存在相关性,但在花后45 d之前的幼果期蔗糖和淀粉的变化模式与AsA含量有着相似性,说明蔗糖可能与幼果期猕猴桃果实中AsA的合成有一定的关系。
总之,苹果和猕猴桃果实中AsA形成的主要原因是自身的生物合成。与苹果相比,猕猴桃不仅有着高的AsA合成能力,还可能存在D-半乳糖醛酸等支路途径。叶片可通过影响糖源的供应或其它物质调控着果实中AsA的合成能力,进而不可逆的影响着果实中AsA含量和积累量。从整个果实中AsA积累模式来看,‘嘎啦’苹果在整个果实发育过程中均有AsA的积累发生,但积累速率低;猕猴桃果实中AsA积累主要发生在花后45 d前,之后至果实成熟基本维持不变,且在花后30 d前幼果中的AsA积累量明显大于果实变大对细胞中AsA的稀释作用,是猕猴桃高AsA积累的主要时期。从AsA水平的维持来看,虽然MDHAR和DHAR对苹果和猕猴桃果实AsA的积累量不起关键作用,但它们在维持AsA氧化还原状态及AsA合成和氧化降解的平衡中起着重要作用。与苹果相比,猕猴桃果实有显著高的MDHAR和DHAR活性,低的AsA氧化程度,这也是猕猴桃果实高AsA含量的另一原因。通过对苹果和猕猴桃果实发育过程中、不同叶龄的苹果叶片中及猕猴桃树体遮光下AsA合成和代谢酶基因转录水平与AsA含量和积累量的关系研究发现,在AsA合成的L-半乳糖途径中GPP的转录调控对AsA的合成与积累起着重要的调控作用,它可能是L-半乳糖途径合成AsA的关键调控基因。
L-Ascorbic acid (AsA), also known as vitamin C or ascorbate, is one of the most abundant antioxidants and cofactor for several enzymes. It has been reported that AsA is necessary for plant growth and development, and is also well known to have an important role in resistance to oxidative stress. Moreover, it is the main source of natural AsA to human because humans are incapable of synthesizing AsA and must secure it by means of dietary uptake to be healthy. However, results from the present study can’t fully answer the reasons why there are clear differences in AsA content among different fruits. In the present study, to understand mechanism of AsA formation and accumulation, a systematical investigation on distribution, biosynthesis, recycling, transportation and accumulation of AsA as well as their light regulation was performed on physiological and molecular level in apple fruits (Malus domestica Borkh‘Gala’.) and kiwifruits (Actinidia deliciosa cv. Qinmei), which has huge difference in AsA content. The main results were as follows.
1. The full-length cDNA of MDHAR, DHAR2 and GME as well as partial cDNA of GalUR were cloned by RT-PCR from apple fruits. Homology analysis of these cDNA showed high homology with the cDNA from other plants. The full-length of MDHAR cDNA is 1400 bp and contains a complete open reading frame (ORF) of 1305 bp, which encodes a membrane bound MDHAR. The full-length of DHAR2 cDNA is 933 bp and contains an ORF of 798 bp, which encodes a GSH dependent-cytoplasmic DHAR. The full-length of GME cDNA is 1323 bp and contains an ORF of 1131, which encodes a NAD dependent-GME. The cDNA of GalUR is 668 bp. Homology analysis of GalUR cDNA showed 80% homology with the cDNA from strawberry, and it is a partial cDNA of apple GalUR contained 5’-terminal start codon.
2. It was detected on gene expression and enzyme activities of GalLDH, GalDH and GalUR which were involved in AsA biosynthesis in apple fruits. Moreover, when different tissues of fruit were incubated with non-labeled putative substrates used to synthesize AsA, incubations with L-Gal and L-GL invoved in L-galactose panthway clearly increased AsA levels in peel and flesh, while D-GalUA was also able to stimulate AsA levels in peel. And the young fruits had stronger capability of AsA biosynthesis. These results suggest that apple fruit is able to synthesize AsA. In different tissues of fruit, the peel with higher AsA content showed higher gene expression level and enzyme activities of GalLDH, GalDH and GalUR as well as MDHAR and DHAR cpmpared with that in the flesh. Additionally, the sun-exposed peel with higher AsA concentration had stronger capability of AsA synthesizing and recycling than the shaded peel, while there was no difference in between the flesh of the sun-exposed side and the shaded side. In transportation, abundant AsA was found in vascular tissues of fruit, and the capability of AsA synthesis can be detected in pedicels of fruit and leaf, but there was no change in AsA level when exogenous AsA was added to fruit pedicels in vitro or vivo. It suggested that AsA could be transported to vascular tissues of fruit but could not well unload in fruits or the AsA in pedicels and vascular tissues might be synthesized by self to simply supply theses tissue’s AsA requirement for ROS detoxification. Therefore, biosynthesis was a major pathway of AsA formation in apple fruits.
3. Light directly influenced on capability of AsA biosynthesis and recycling in apple leaves and peel. After the whole trees were shaded for 20 days, AsA levels were significantly decreased in fruit peel and leaves, with clear decline of the mRNA expression levels and activities of GalLDH and GalDH as well as activities of recycling enzymes. Both of fruit and whole tree shading could not lead to clear changes of the mRNA expression levels and activities of GalLDH and GalDH as well as activities of recycling enzymes in the flesh, but the shading of whole tree could lead to decreasing of AsA content in the flesh but shading of fruit could not. Moreover, the fruit located on sun–exposed side of a tree top had higher AsA accumulation level compared with those for the inner shaded side of a tree. It was concluded that light directly affect AsA biosynthesis and recycling in the peel and leaves, but it indirectly affect AsA content via the leaves in the flesh of apple.
4. During growth and development of apple fruits, T-AsA and AsA content based on per fresh weight in fruits were the highest in ovaries of 0 DAA, followed by a continuous decreasing and nearly reach a constant after 60 DAA. However, AsA accumulation level based on per fruit was continuous increased with growth of fruits, which demonstrated that AsA accumulation was occurred, and the rate of AsA synthesis in a fruit was higher than rate of DHA degradation through fruit growth and development in apple fruits. Expression levels of genes involved in AsA synthesis showed different patterns during growth and development of apple fruits. Markedly up-regulated mRNA expression level of GME, GGP and GalUR did not lead to changes of AsA content, which indicated that GME, GGP and GalUR did not play important roles in controlling AsA synthesis in apple fruits, while expression level of GPP was consistent with AsA content. And higher mRNA expression levels and enzyme activities of GalLDH and GalDH were agreeable with high capability of conversion from L-GL and L-Gal to AsA. In recycling system, mRNA expression levels and enzyme activities of MDHAR showed a decreased pattern with growth of fruits, and it was negative correlation with AsA/DHA.
5. To understand the difference in controlling pattern of AsA synthesis and accumulation between fruit and leaves, it was investigated on AsA formation and its regulation in biosynthesis and recycling in apple leaves with different age. The results suggested that AsA accumulation in apple leaves mainly occurred during the transition phase from young to mature leaves with high rates of synthesis and recycling, while AsA content was clearly declined in senescent leaves with dropping of AsA synthesis and recycling. The datas also suggested that GPP could play an important role in controlling AsA biosynthesis via the L-galactose pathway in apple leaves, while patterns of GMP, GGP and GalLDH expression showed no roles in different age leaves of apple. Increased expression levels of GGP and GalUR were found in senescent leaves, which suggested that they might be correlative with leaf senescence. In recycling system, mRNA expression level and activity of MDHAR were relative to DHA content in leaves, which implied that MDHAR had important roles in maintaining redox state of AsA. Activity and the mRNA expression level of DHAR were clearly correlated with AsA content in leaves of different ages, which suggested that DHAR could play an important role in maintaining balance of AsA redox state, especially in mature leaves with low MDHAR activity.
6. The full-length cDNA of DHAR2 and GGP as well as partial cDNA of MDHAR, DHAR1 and GME were cloned by RT-PCR from kiwi fruits. MDHAR cDNA is 1019 bp and partial cDNA of a membrane bound MDHAR. The full-length of DHAR1 cDNA is 821 bp and contains an ORF of 639 bp, while DHAR2 cDNA is 542 bp and partial cDNA of DHAR, both of which encode GSH dependent-cytoplasmic DHAR. The cDNA of GME is 846 bp and encodes a NAD dependent-GME. Homology analysis of GME cDNA showed high homology with the cDNA from other plants, and it is a partial cDNA of kiwi GME. The full-length of GGP cDNA is 1542 bp and contains an ORF of 1353 bp, which encodes a membrane bound GGP protein.
7. It was detected on gene expression and enzyme activities of GalLDH, GalDH and GalUR in kiwifruit. When the flesh was incubated with non-labeled putative substrates used to synthesize AsA, not only incubations with L-Gal and L-GL could clearly increased AsA levels but also L-GulL and D-GalUA were also able to stimulate AsA levels. And the expression level of genes and enzyme activities as well as capability of AsA biosynthesis in young fruits were much higher than that in ripening fruits. Existing results indicated that not only kiwifruit could synthesize AsA by self via L-galactose but also alternative AsA biosynthetic pathways (e.g. D-galacturonic acid) pathway might be operated. In different tissues, the flesh and seed zone, which had higher gene expression level and enzyme activities of GalLDH, GalDH, MDHAR and DHAR, showed the highest AsA content, while the core was very low in AsA content with lower gene expression level and activities of these enzymes. Moreover, AsA distribution in kiwifruit showed cell diversities. AsA content was almost observed in peel cell. In cells of flesh, the big size of cell had abundant AsA not only in intracellular zone but also in cell wall while the AsA was hardly observed in small size of cell. Moreover, abundant AsA could be observed in wall of the core cell but not in intracellular, and AsA was also detected in ductal cell of vascular bundle. As in apple, AsA content and AsA synthesis can be detected in pedicels of fruit and leaf, but there was no influence on AsA levels when exogenous AsA, DHA and L-GL were added to pedicels of fruit in vitro and vivo. It also suggested that the AsA in pedicels and vascular tissue of kiwi might be synthesized by self and simply supplied AsA requirement for ROS detoxification in these tissues. However, AsA content in young fruit was clearly increased when exogenous sucrose was used to feed fruit pedicels at stage of young fruit, but not at stage of mature fruit. These results indicated that biosynthesis was a major pathway of AsA formation in kiwifruits, and supply of sugar source from leaves might influence on AsA synthesis rate in young fruit.
8. Shading of fruit with bags also had no influence on AsA content and accumulation in kiwifruit, but AsA levels in young fruits were clearly regulated by diurnal variation, and the fruits at 6:00 had lower T-AsA and AsA content compared with that in noon or afternoon. Moreover, AsA content could be significantly decreased when the trees were shaded before 40 DAA. These results indicated that light indirectly influenced on AsA content via the leaves in kiwifruit but not directly. Shading of whole trees led to significant down-regulation of gene expression levels and enzyme activities related with AsA synthesis and recycling, which led to marked decrease of AsA content and increase of DHA content. When the trees were shaded at stage of young fruits (0-40 DAA), expression levels of genes (including GalLDH, GalDH, GPP, GME, GalUR, MDHAR and DHAR) and enzyme activities (including GalLDH, GalDH, MDHAR and DHAR) showed a huge decline in 40 DAA fruits, which led to significant decrease of AsA content and accumulation in 40 DAA fruits. But there was no influence on these indexes when the trees were shaded after 40 DAA caompared with the control. These results indicated that the capability of AsA biosynthesis was inhibited while the trees were shaded before 40 DAA. Although decreased sugar content did not result in the changes of AsA content as well as its synthesis and recycling in fruit after 40 DAA, given the fact that exogenous sucrose could increase AsA content in young fruits, decreased sugar content (especially sucrose) might be contributed to decline of AsA synthesis capability in young fruits while the trees were shaded before 40 DAA. These results suggested that light did not directly influence on AsA content and synthesis in kiwifruit, but indirectly controlled AsA synthesis and recycling with changes capability of sugar supply or other signal from leaf to fruit, which led to decline of AsA content in young fruits. And this decline of AsA content might be relative to growth of fruits after 40 DAA.
9. During growth and development of kiwifruits, T-AsA and AsA content based on per fresh weight in fruits were rapidly increased after anthesis and reached the highest at 30 DAA, followed by a continuous decrease until it nearly maintained a constant after 60 DAA. Based on per fruit, T-AsA and AsA accumulation levels also showed significant increase after anthesis and reached the highest at 45 DAA, then it nearly maintained a constant until fruit maturate. These results demonstrated that AsA accumulation in kiwifruit mainly occurred at stage of young fruit (before 45 DAA). Expression pattern of GPP and GGP showed high agreement with AsA accumulation rate, which implied that they might have control roles in AsA biosynthesis and accumulation rate of kiwi fruits. But expression patterns of GalLDH, GalDH, GMP and GME showed that they did not play roles in regulating on AsA accumulation. In AsA recycling system, mRNA expression level and enzyme activities of MDHAR and DHAR had little role in accumulating at stage of young fruits, but higher mRNA expression and activities of MDHAR and DHAR implied that they play an important role in maintaining AsA level in fruits after 45 DAA. In carbohydrate, change patterns of AsA level were no clear correlative with total soluble sugar, reduced sugar, starch, fructose, sucrose and glucose content during growth and development of fruits, but changes patterns of sucrose and starch content were agreement with AsA content at stage of young fruit (before 45 DAA), which indicated that sucrose or starch might be contributed to AsA synthesis in young fruit of kiwi.
In conclusion, biosynthesis is a main reason why AsA can be accumulated in apple and kiwi fruits. Compared with apple, kiwi fruits have much higher capability of AsA synthesis, and there are other pathways (e.g. D-galacturonate pathway) used to synthesize AsA in kiwifruit. Leaf could control AsA biosynthesis and recycling of fruits with changes of supply of sugar source or other substances to fruits, which regulates AsA content and accumulation in fruits. On patterns of AsA accumulation of each fruit, AsA accumulation is occurred through growth and development of apple fruits, but the rate is very slow compared with young kiwi fruits. However, AsA accumulation in kiwi fruits mainly occurs at stage of young fruit (before 45 DAA) with high rate before 30 DAA, and then it nearly maintains a constant until fruit maturate. In recycling system, MDHAR and DHAR do not play important roles in controlling accumulation levels of AsA in apple and kiwi fruits, but they have essential roles in maintaining redox state of AsA and balance between synthesis and degradation. Compared with apple fruits, kiwi fruits have much higher activities of MDHAR and DHAR during growth and development of fruits and lower rate of oxidized AsA, which is another reason of much higher AsA content in kiwi fruits. Based on analysis of relationship of expression levels of genes to AsA content and accumulation levels during growth and development of apple and kiwi fruits, among different age leaves of apple as well as under different light condition, it is indicated that GPP could play an important role in controlling AsA biosynthesis via L-galactose pathway.
1.从苹果果实中克隆获得了AsA再生酶MDHAR和DHAR2及合成酶GME和GalUR基因cDNA全长或部分序列。其中,MDHAR长1400 bp,包含了1305 bp的完整开放阅读框,编码一个膜结合MDHAR;DHAR2长933 bp,包含了798 bp的完整开放阅读框,属于GSH依赖性胞质DHAR;GME长1323 bp,包含了1131 bp的完整开放阅读框,序列分析发现其编码一个NAD依赖性胞质酶。而GalUR长668 bp,与草莓GalUR氨基酸同源性高达80%,为GalUR基因的cDNA部分序列。
2.在‘嘎啦’苹果果实的果皮、果肉中检测到了AsA合成关键酶GalDH和GalLDH基因mRNA的表达和活性;通过不同AsA合成前体物饲喂发现,参与L-半乳糖途径的L-Gal和L-GL能引起果皮和果肉AsA含量的明显增加,D-GalUA也能引起果皮AsA含量的明显增加,且幼果的增加幅度高于成熟果。在果实的不同组织中,高AsA含量的果皮有高的AsA合成和再生酶基因的表达量和活性;果皮中AsA含量受光的调控,与阴面果皮相比,阳面果皮具有高的GalDH、GalLDH、GalUR及MDHAR、DHAR mRNA表达量和活性,而阳面和阴面果肉间则无明显差异。对苹果AsA运输的初步研究发现,在果实微管组织中存在大量AsA的积累,且在具有AsA合成能力的果柄和叶柄韧皮部及其卸载液中也存在大量AsA,但果柄施加外源AsA并不能引起果实AsA含量的增加。这表明在苹果中AsA可能能从叶片运输到果实,但在果实中不能很好的卸载从而在维管束中积累;或者这些组织中自身合成的AsA仅在果柄或叶柄中发挥它的抗氧化等功能,而与果实和叶片间AsA运输关系不大。这些结果表明生物合成是苹果AsA形成的主要原因。
3.光直接影响苹果叶片和果皮中的AsA合成和代谢。在弱光条件下,苹果叶片和果皮中AsA合成酶GalDH和GalLDH mRNA表达量与活性及再生能力明显降低,AsA含量下降。果实或整个树体遮光后,果肉组织中GalDH和GalLDH转录水平及其活性和再生酶活性均无明显变化。果实遮光对果肉AsA水平无明显影响,但整个树体遮光20 d后果肉中T-AsA和AsA含量明显降低,且树冠底部内膛果实果肉的AsA含量也较低。这说明苹果果肉组织中AsA的合成和代谢能力不直接受光的调控,但光可通过影响叶片间接地对果肉中AsA含量起到调控作用。
4.在苹果果实生长发育过程中,单位鲜重果实中T-AsA和AsA含量在花后0 d的子房中最高,之后随着生长发育逐渐下降,在花后60 d后至果实成熟基本保持不变。从单个果实中AsA的积累量来看,在整个果实生长发育过程中AsA积累一直在发生,即在单个果实中AsA合成速率一直大于DHA的降解速率。在果实生长发育过程中,AsA合成酶基因表达量表现出不同的变化模式。GME、GGP和GalUR mRNA表达量在果实生长发育过程中显著地增加与AsA含量的变化不一致,表明它们对AsA含量不起关键调控作用。GPP在果实发育过程中与单位鲜重中AsA含量的变化模式存在相似性。在幼果期果实高的GalDH和GalLDH mRNA表达量和酶活性与幼果期由L-Gal和L-GL合成AsA的能力相一致。在AsA再生系统中,MDHAR mRNA表达量和酶活性随果实的生长表现出明显的下降趋势,与AsA/DHA比值呈负相关。
5.为了探索叶片AsA形成与调控和果实的异同点,研究了不同叶龄苹果叶片AsA形成及其与合成和再生的调控关系。结果表明,在不同叶龄的苹果叶片中,AsA的积累主要发生在幼叶向成熟叶的转变期,该时期叶片有高的AsA合成和再生能力,而衰老期AsA合成和再生能力下降,AsA含量降低。在AsA合成中,GPP的转录调控可能对苹果叶片AsA水平起着重要的调控作用,而GMP、GGP、GalLDH表达量的变化模式表明它们对AsA水平不具有调控功能。此外,GGP和GalUR在老叶中高的表达量可能与衰老有关。在再生系统中,MDHAR表达量和活性与苹果叶片中DHA含量相关,表明MDHAR可能在维持植物AsA氧化还原状态中起重要作用。而在AsA水平稳定的成熟叶中高的DHAR表达量和活性表明DHAR在维持AsA合成和氧化降解之间的平衡中可能起重要作用。
6.从美味猕猴桃品种‘秦美’果实中克隆获得了MDHAR、DHAR2和GME基因cDNA部分序列及DHAR1和GPP基因的cDNA全长序列。其中,MDHAR长1019 bp,属于膜结合MDHAR基因的cDNA部分序列; DHAR1长821 bp,包含了639 bp的完整开放阅读框,而DHAR2长542 bp,包含5′端起始密码子ATG,分别与已报道的两种植物胞质DHAR有高度的同源性,均为GSH依赖性胞质DHAR;GME长846 bp,与其它植物GME基因氨基酸同源性多在90%以上,为美味猕猴桃GME基因的cDNA部分序列;GGP为1542 bp,包含1353 bp的完全开放阅读框,对其编码蛋白结构分析发现,它具有螺旋跨膜区,是一个膜结合蛋白。
7.在美味猕猴桃品种‘秦美’果实中也检测到了AsA合成酶GalUR、GalDH和GalLDH mRNA表达及GalDH和GalLDH活性。不同AsA合成前体物饲喂发现,不仅参与L-半乳糖途径的L-Gal和L-GL能引起果实AsA含量的显著增加,而且L-GulL和D-GalUA也能引起AsA含量的明显增加,且幼果高于成熟果。这说明猕猴桃果实具有AsA合成能力,且可能存在多个途径。在果实的不同组织中,有高AsA合成和再生酶基因表达和活性的果肉及种子区有最高的AsA含量,而合成酶基因表达量和活性水平较低的中轴AsA含量很低。同时,猕猴桃果实AsA的分布也存在细胞特异性,果皮细胞中几乎观察不到AsA的存在;在果肉细胞中,大细胞不仅胞内有大量的AsA积累,而且在其细胞壁上也有AsA的存在;中轴细胞中AsA主要分布在细胞壁等质外体;维管束的导管细胞中也存在大量的AsA。对猕猴桃AsA运输能力的研究表明,果柄和叶柄中自身合成的AsA可能仅在果柄或叶柄中发挥抗氧化等功能,而与果实与叶片间AsA运输关系无关。但幼果期果柄施加外源蔗糖能引起幼果AsA含量的明显增加,表明猕猴桃果实中的AsA可能与叶片糖源的供应能力有关。这些结果也说明猕猴桃果实中AsA形成的主要原因是自身合成,同时叶片的糖源供应能力可能调控着幼果期果肉中AsA合成速率。
8.猕猴桃果实套袋遮光不影响果实中AsA含量,但幼果中AsA水平明显受日变化的影响,清晨6:00比中午和下午的AsA含量明显低。在果实不同发育阶段树体遮光处理中,花后0-40 d间遮光能显著降低幼果中T-AsA和AsA含量,而花后40 d后遮光对AsA含量无明显影响。这表明光不直接影响猕猴桃果实中AsA含量,但能通过叶片影响幼果中AsA含量。猕猴桃树体遮光显著降低了叶片AsA合成和再生酶基因的表达量和活性,引起了AsA含量显著下降和DHA增加。尽管如此,花后0-40 d树体遮光也显著降低了花后40 d猕猴桃果实中AsA合成酶GalLDH、GalDH、GPP、GME和GalUR及再生酶MDHAR和DHAR mRNA表达量,并降低了GalLDH、GalDH、MDHAR和DHAR活性,幼果AsA含量和积累量下降,40 d后果实生长速率下降,果实变小。而花后40-120 d遮光虽引起了糖含量的显著下降,但对AsA含量、合成和再生无明显影响。这表明花后0-40 d树体遮光降低了猕猴桃幼果AsA合成能力。外源蔗糖对幼果AsA含量的增加表明花后0-40 d遮光处理对猕猴桃幼果AsA合成的影响可能与淀粉、可溶性总糖和蔗糖含量的明显降低有关。这些结果表明光不直接影响猕猴桃果实AsA含量和合成,但能通过影响叶片对果实的糖供应能力或其它信号调控幼果AsA合成和再生能力,间接调控着幼果中AsA含量和果实中AsA积累量,且这种对幼果AsA含量的影响可能与后期果实膨大有关。
9.在猕猴桃果实生长发育过程中,单位鲜重果实中T-AsA和AsA含量在花后迅速增加,并在花后30 d达到最高,之后逐渐下降,在花后60 d后至果实成熟基本保持不变。从单个果实中AsA的积累量来看,T-AsA和AsA积累量在花后伴随果实的生长迅速增加,在花后45 d达到最高,之后至果实成熟期保持不变。这说明猕猴桃果实中AsA的积累主要发生在花后45 d前的幼果期。在果实生长发育过程中,GPP和GGP在转录水平上对果实AsA合成调控的可能性最大,特别是GPP,而从GalLDH、GalDH、GMP和GME转录水平的变化模式来看,它们对AsA合成调控的可能性很小。在AsA再生系统中,MDHAR和DHAR表达量及活性与幼果期AsA的快速积累关系不大。但45 d后高的表达和活性(尤其MDHAR)可能与AsA水平的维持有关。在碳水化合物中,可溶性总糖、还原糖和淀粉含量与AsA的变化模式均不存在相关性,但在花后45 d之前的幼果期蔗糖和淀粉的变化模式与AsA含量有着相似性,说明蔗糖可能与幼果期猕猴桃果实中AsA的合成有一定的关系。
总之,苹果和猕猴桃果实中AsA形成的主要原因是自身的生物合成。与苹果相比,猕猴桃不仅有着高的AsA合成能力,还可能存在D-半乳糖醛酸等支路途径。叶片可通过影响糖源的供应或其它物质调控着果实中AsA的合成能力,进而不可逆的影响着果实中AsA含量和积累量。从整个果实中AsA积累模式来看,‘嘎啦’苹果在整个果实发育过程中均有AsA的积累发生,但积累速率低;猕猴桃果实中AsA积累主要发生在花后45 d前,之后至果实成熟基本维持不变,且在花后30 d前幼果中的AsA积累量明显大于果实变大对细胞中AsA的稀释作用,是猕猴桃高AsA积累的主要时期。从AsA水平的维持来看,虽然MDHAR和DHAR对苹果和猕猴桃果实AsA的积累量不起关键作用,但它们在维持AsA氧化还原状态及AsA合成和氧化降解的平衡中起着重要作用。与苹果相比,猕猴桃果实有显著高的MDHAR和DHAR活性,低的AsA氧化程度,这也是猕猴桃果实高AsA含量的另一原因。通过对苹果和猕猴桃果实发育过程中、不同叶龄的苹果叶片中及猕猴桃树体遮光下AsA合成和代谢酶基因转录水平与AsA含量和积累量的关系研究发现,在AsA合成的L-半乳糖途径中GPP的转录调控对AsA的合成与积累起着重要的调控作用,它可能是L-半乳糖途径合成AsA的关键调控基因。
L-Ascorbic acid (AsA), also known as vitamin C or ascorbate, is one of the most abundant antioxidants and cofactor for several enzymes. It has been reported that AsA is necessary for plant growth and development, and is also well known to have an important role in resistance to oxidative stress. Moreover, it is the main source of natural AsA to human because humans are incapable of synthesizing AsA and must secure it by means of dietary uptake to be healthy. However, results from the present study can’t fully answer the reasons why there are clear differences in AsA content among different fruits. In the present study, to understand mechanism of AsA formation and accumulation, a systematical investigation on distribution, biosynthesis, recycling, transportation and accumulation of AsA as well as their light regulation was performed on physiological and molecular level in apple fruits (Malus domestica Borkh‘Gala’.) and kiwifruits (Actinidia deliciosa cv. Qinmei), which has huge difference in AsA content. The main results were as follows.
1. The full-length cDNA of MDHAR, DHAR2 and GME as well as partial cDNA of GalUR were cloned by RT-PCR from apple fruits. Homology analysis of these cDNA showed high homology with the cDNA from other plants. The full-length of MDHAR cDNA is 1400 bp and contains a complete open reading frame (ORF) of 1305 bp, which encodes a membrane bound MDHAR. The full-length of DHAR2 cDNA is 933 bp and contains an ORF of 798 bp, which encodes a GSH dependent-cytoplasmic DHAR. The full-length of GME cDNA is 1323 bp and contains an ORF of 1131, which encodes a NAD dependent-GME. The cDNA of GalUR is 668 bp. Homology analysis of GalUR cDNA showed 80% homology with the cDNA from strawberry, and it is a partial cDNA of apple GalUR contained 5’-terminal start codon.
2. It was detected on gene expression and enzyme activities of GalLDH, GalDH and GalUR which were involved in AsA biosynthesis in apple fruits. Moreover, when different tissues of fruit were incubated with non-labeled putative substrates used to synthesize AsA, incubations with L-Gal and L-GL invoved in L-galactose panthway clearly increased AsA levels in peel and flesh, while D-GalUA was also able to stimulate AsA levels in peel. And the young fruits had stronger capability of AsA biosynthesis. These results suggest that apple fruit is able to synthesize AsA. In different tissues of fruit, the peel with higher AsA content showed higher gene expression level and enzyme activities of GalLDH, GalDH and GalUR as well as MDHAR and DHAR cpmpared with that in the flesh. Additionally, the sun-exposed peel with higher AsA concentration had stronger capability of AsA synthesizing and recycling than the shaded peel, while there was no difference in between the flesh of the sun-exposed side and the shaded side. In transportation, abundant AsA was found in vascular tissues of fruit, and the capability of AsA synthesis can be detected in pedicels of fruit and leaf, but there was no change in AsA level when exogenous AsA was added to fruit pedicels in vitro or vivo. It suggested that AsA could be transported to vascular tissues of fruit but could not well unload in fruits or the AsA in pedicels and vascular tissues might be synthesized by self to simply supply theses tissue’s AsA requirement for ROS detoxification. Therefore, biosynthesis was a major pathway of AsA formation in apple fruits.
3. Light directly influenced on capability of AsA biosynthesis and recycling in apple leaves and peel. After the whole trees were shaded for 20 days, AsA levels were significantly decreased in fruit peel and leaves, with clear decline of the mRNA expression levels and activities of GalLDH and GalDH as well as activities of recycling enzymes. Both of fruit and whole tree shading could not lead to clear changes of the mRNA expression levels and activities of GalLDH and GalDH as well as activities of recycling enzymes in the flesh, but the shading of whole tree could lead to decreasing of AsA content in the flesh but shading of fruit could not. Moreover, the fruit located on sun–exposed side of a tree top had higher AsA accumulation level compared with those for the inner shaded side of a tree. It was concluded that light directly affect AsA biosynthesis and recycling in the peel and leaves, but it indirectly affect AsA content via the leaves in the flesh of apple.
4. During growth and development of apple fruits, T-AsA and AsA content based on per fresh weight in fruits were the highest in ovaries of 0 DAA, followed by a continuous decreasing and nearly reach a constant after 60 DAA. However, AsA accumulation level based on per fruit was continuous increased with growth of fruits, which demonstrated that AsA accumulation was occurred, and the rate of AsA synthesis in a fruit was higher than rate of DHA degradation through fruit growth and development in apple fruits. Expression levels of genes involved in AsA synthesis showed different patterns during growth and development of apple fruits. Markedly up-regulated mRNA expression level of GME, GGP and GalUR did not lead to changes of AsA content, which indicated that GME, GGP and GalUR did not play important roles in controlling AsA synthesis in apple fruits, while expression level of GPP was consistent with AsA content. And higher mRNA expression levels and enzyme activities of GalLDH and GalDH were agreeable with high capability of conversion from L-GL and L-Gal to AsA. In recycling system, mRNA expression levels and enzyme activities of MDHAR showed a decreased pattern with growth of fruits, and it was negative correlation with AsA/DHA.
5. To understand the difference in controlling pattern of AsA synthesis and accumulation between fruit and leaves, it was investigated on AsA formation and its regulation in biosynthesis and recycling in apple leaves with different age. The results suggested that AsA accumulation in apple leaves mainly occurred during the transition phase from young to mature leaves with high rates of synthesis and recycling, while AsA content was clearly declined in senescent leaves with dropping of AsA synthesis and recycling. The datas also suggested that GPP could play an important role in controlling AsA biosynthesis via the L-galactose pathway in apple leaves, while patterns of GMP, GGP and GalLDH expression showed no roles in different age leaves of apple. Increased expression levels of GGP and GalUR were found in senescent leaves, which suggested that they might be correlative with leaf senescence. In recycling system, mRNA expression level and activity of MDHAR were relative to DHA content in leaves, which implied that MDHAR had important roles in maintaining redox state of AsA. Activity and the mRNA expression level of DHAR were clearly correlated with AsA content in leaves of different ages, which suggested that DHAR could play an important role in maintaining balance of AsA redox state, especially in mature leaves with low MDHAR activity.
6. The full-length cDNA of DHAR2 and GGP as well as partial cDNA of MDHAR, DHAR1 and GME were cloned by RT-PCR from kiwi fruits. MDHAR cDNA is 1019 bp and partial cDNA of a membrane bound MDHAR. The full-length of DHAR1 cDNA is 821 bp and contains an ORF of 639 bp, while DHAR2 cDNA is 542 bp and partial cDNA of DHAR, both of which encode GSH dependent-cytoplasmic DHAR. The cDNA of GME is 846 bp and encodes a NAD dependent-GME. Homology analysis of GME cDNA showed high homology with the cDNA from other plants, and it is a partial cDNA of kiwi GME. The full-length of GGP cDNA is 1542 bp and contains an ORF of 1353 bp, which encodes a membrane bound GGP protein.
7. It was detected on gene expression and enzyme activities of GalLDH, GalDH and GalUR in kiwifruit. When the flesh was incubated with non-labeled putative substrates used to synthesize AsA, not only incubations with L-Gal and L-GL could clearly increased AsA levels but also L-GulL and D-GalUA were also able to stimulate AsA levels. And the expression level of genes and enzyme activities as well as capability of AsA biosynthesis in young fruits were much higher than that in ripening fruits. Existing results indicated that not only kiwifruit could synthesize AsA by self via L-galactose but also alternative AsA biosynthetic pathways (e.g. D-galacturonic acid) pathway might be operated. In different tissues, the flesh and seed zone, which had higher gene expression level and enzyme activities of GalLDH, GalDH, MDHAR and DHAR, showed the highest AsA content, while the core was very low in AsA content with lower gene expression level and activities of these enzymes. Moreover, AsA distribution in kiwifruit showed cell diversities. AsA content was almost observed in peel cell. In cells of flesh, the big size of cell had abundant AsA not only in intracellular zone but also in cell wall while the AsA was hardly observed in small size of cell. Moreover, abundant AsA could be observed in wall of the core cell but not in intracellular, and AsA was also detected in ductal cell of vascular bundle. As in apple, AsA content and AsA synthesis can be detected in pedicels of fruit and leaf, but there was no influence on AsA levels when exogenous AsA, DHA and L-GL were added to pedicels of fruit in vitro and vivo. It also suggested that the AsA in pedicels and vascular tissue of kiwi might be synthesized by self and simply supplied AsA requirement for ROS detoxification in these tissues. However, AsA content in young fruit was clearly increased when exogenous sucrose was used to feed fruit pedicels at stage of young fruit, but not at stage of mature fruit. These results indicated that biosynthesis was a major pathway of AsA formation in kiwifruits, and supply of sugar source from leaves might influence on AsA synthesis rate in young fruit.
8. Shading of fruit with bags also had no influence on AsA content and accumulation in kiwifruit, but AsA levels in young fruits were clearly regulated by diurnal variation, and the fruits at 6:00 had lower T-AsA and AsA content compared with that in noon or afternoon. Moreover, AsA content could be significantly decreased when the trees were shaded before 40 DAA. These results indicated that light indirectly influenced on AsA content via the leaves in kiwifruit but not directly. Shading of whole trees led to significant down-regulation of gene expression levels and enzyme activities related with AsA synthesis and recycling, which led to marked decrease of AsA content and increase of DHA content. When the trees were shaded at stage of young fruits (0-40 DAA), expression levels of genes (including GalLDH, GalDH, GPP, GME, GalUR, MDHAR and DHAR) and enzyme activities (including GalLDH, GalDH, MDHAR and DHAR) showed a huge decline in 40 DAA fruits, which led to significant decrease of AsA content and accumulation in 40 DAA fruits. But there was no influence on these indexes when the trees were shaded after 40 DAA caompared with the control. These results indicated that the capability of AsA biosynthesis was inhibited while the trees were shaded before 40 DAA. Although decreased sugar content did not result in the changes of AsA content as well as its synthesis and recycling in fruit after 40 DAA, given the fact that exogenous sucrose could increase AsA content in young fruits, decreased sugar content (especially sucrose) might be contributed to decline of AsA synthesis capability in young fruits while the trees were shaded before 40 DAA. These results suggested that light did not directly influence on AsA content and synthesis in kiwifruit, but indirectly controlled AsA synthesis and recycling with changes capability of sugar supply or other signal from leaf to fruit, which led to decline of AsA content in young fruits. And this decline of AsA content might be relative to growth of fruits after 40 DAA.
9. During growth and development of kiwifruits, T-AsA and AsA content based on per fresh weight in fruits were rapidly increased after anthesis and reached the highest at 30 DAA, followed by a continuous decrease until it nearly maintained a constant after 60 DAA. Based on per fruit, T-AsA and AsA accumulation levels also showed significant increase after anthesis and reached the highest at 45 DAA, then it nearly maintained a constant until fruit maturate. These results demonstrated that AsA accumulation in kiwifruit mainly occurred at stage of young fruit (before 45 DAA). Expression pattern of GPP and GGP showed high agreement with AsA accumulation rate, which implied that they might have control roles in AsA biosynthesis and accumulation rate of kiwi fruits. But expression patterns of GalLDH, GalDH, GMP and GME showed that they did not play roles in regulating on AsA accumulation. In AsA recycling system, mRNA expression level and enzyme activities of MDHAR and DHAR had little role in accumulating at stage of young fruits, but higher mRNA expression and activities of MDHAR and DHAR implied that they play an important role in maintaining AsA level in fruits after 45 DAA. In carbohydrate, change patterns of AsA level were no clear correlative with total soluble sugar, reduced sugar, starch, fructose, sucrose and glucose content during growth and development of fruits, but changes patterns of sucrose and starch content were agreement with AsA content at stage of young fruit (before 45 DAA), which indicated that sucrose or starch might be contributed to AsA synthesis in young fruit of kiwi.
In conclusion, biosynthesis is a main reason why AsA can be accumulated in apple and kiwi fruits. Compared with apple, kiwi fruits have much higher capability of AsA synthesis, and there are other pathways (e.g. D-galacturonate pathway) used to synthesize AsA in kiwifruit. Leaf could control AsA biosynthesis and recycling of fruits with changes of supply of sugar source or other substances to fruits, which regulates AsA content and accumulation in fruits. On patterns of AsA accumulation of each fruit, AsA accumulation is occurred through growth and development of apple fruits, but the rate is very slow compared with young kiwi fruits. However, AsA accumulation in kiwi fruits mainly occurs at stage of young fruit (before 45 DAA) with high rate before 30 DAA, and then it nearly maintains a constant until fruit maturate. In recycling system, MDHAR and DHAR do not play important roles in controlling accumulation levels of AsA in apple and kiwi fruits, but they have essential roles in maintaining redox state of AsA and balance between synthesis and degradation. Compared with apple fruits, kiwi fruits have much higher activities of MDHAR and DHAR during growth and development of fruits and lower rate of oxidized AsA, which is another reason of much higher AsA content in kiwi fruits. Based on analysis of relationship of expression levels of genes to AsA content and accumulation levels during growth and development of apple and kiwi fruits, among different age leaves of apple as well as under different light condition, it is indicated that GPP could play an important role in controlling AsA biosynthesis via L-galactose pathway.
引文
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