Distribution and metabolism of ascorbic acid in apple fruits (Malus domestica Borkh cv. Gala)

The objective of this study was to determine ascorbic acid (AsA) distribution, biosynthesis and recycling in different tissues of young and mature fruit of cv. Gala apple (Malus domestica Borkh). Our results showed that the peel of ‘Gala’ apple had the highest AsA levels among all the tissue types, which resulted from a combination of, lower ascorbate peroxidase (APX, EC 1.11.1.11) activity consuming AsA, and higher dehydroascorbate reductase (DHAR, EC 1.8.5.1) and monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activities used to recycle AsA. Exogenous feeding of AsA synthesis precursors demonstrated that the peel was capable of de nono AsA biosynthesis via l-galactose and d-galacturonic acid pathways whereas the flesh and seed were only able to synthesize AsA via l-galactose pathway. The young fruit had higher AsA concentration and stronger capability of AsA biosynthesis and recycling. The sun-exposed peel had higher AsA concentration and stronger capability of recycling AsA than the shaded peel, while there was no difference in the flesh between the sun-exposed side and the shaded side. Abundant AsA was found in fruit vascular tissue, which suggests that AsA can be transported to vascular tissues of fruit or vascular tissues could synthesize AsA itself in ‘Gala’ apple.     

Influence of light on ascorbate formation and metabolism in apple fruits

To further understand the regulatory mechanism of light on the formation of ascorbic acid (AsA) in the sink organs of plants, a systematical investigation on AsA levels, activities of two key biosynthsis enzymes and their mRNA expression as well as the recycling was performed in the fruits of apple (Malus domestica Borkh), under different levels of shade. After the whole trees were shaded with the sun-light about 50–55% for 20 days, AsA levels were significantly decreased in fruit peel, flesh and leaves, while mRNA expression levels and activities of l-galactose dehydrogenase (l-GalDH, EC 1.1.1.117) and l-galactono-1,4-lactone dehydrogenase (l-GalLDH, EC 1.3.2.3) as well as activities of recycling enzymes was clearly declined in the leaf and peel but not in the flesh. By shading fruits only for 20 days, AsA levels, relative mRNA levels and activities of l-GalDH and l-GalLDH as well as activities of recycling enzymes all showed obvious decrease in the peel, but not in the flesh. However, their levels in the peel were markedly increased after the full shade was removed and re-exposed these fruits on natural light for 5 days. It is concluded that light affects AsA biosynthesis and recycling in the peel and leaf, but did not in the fresh. Results also suggest that apple fruit is potential to biosynthesize AsA via the l-galactose pathway, and AsA content in the fruits may depend partly on levels of AsA or other photochemistry controlled by light in the leaves.     

Ascorbate biosynthesis during early fruit development is the main reason for its accumulation in kiwi

Background

Ascorbic acid (AsA) is a unique antioxidant as well as an enzyme cofactor. Although it has multiple roles in plants, it is unclear how its accumulation is controlled at the expression level, especially in sink tissues. Kiwifruit (Actinidia) is well-known for its high ascorbate content. Our objective was to determine whether AsA accumulates in the fruits primarily through biosynthesis or because it is imported from the foliage.

Methodology/Principal Findings

We systematically investigated AsA levels, biosynthetic capacity, and mRNA expression of genes involved in AsA biosynthesis in kiwi (A. deliciosa cv. Qinmei). Recycling and AsA localization were also monitored during fruit development and among different tissue types. Over time, the amount of AsA, with its capacity for higher biosynthesis and lower recycling, peaked at 30 days after anthesis (DAA), and then decreased markedly up to 60 DAA before declining more slowly. Expression of key genes showed similar patterns of change, except for L-galactono-1,4-lactone dehydrogenase and L-galactose-1-phosphate phosphatase (GPP). However, GPP had good correlation with the rate of AsA accumulation. The expression of these genes could be detected in phloem of stem as well as petiole of leaf and fruit. Additionally, fruit petioles had greater ascorbate amounts, although that was the site of lowest expression by most genes. Fruit microtubule tissues also had higher AsA. However, exogenous applications of AsA to those petioles did not lead to its transport into fruits, and distribution of ascorbate was cell-specific in the fruits, with more accumulation occurring in larger cells.

Conclusions

These results suggest that AsA biosynthesis in kiwi during early fruit development is the main reason for its accumulation in the fruits. We also postulate here that GPP is a good candidate for regulating AsA biosynthesis whereas GDP-L-galactose-1-phosphate phosphorylase is not.     

苹果中磷酸蔗糖合酶家族基因的表达特性及其与蔗糖含量的关系

磷酸蔗糖合酶(sucrose phosphate synthase,SPS)是植物中蔗糖合成的主要限速酶,影响植物的生长发育和果实中蔗糖的含量。为探明苹果中SPS基因家族特性及其在蔗糖合成中的作用,该研究从苹果基因组中分离了MdSPS家族基因,分析了它们的进化关系以及mRNA表达特性与酶活性和蔗糖含量的关系。结果显示:(1)在苹果基因组中有8个SPS家族基因表达,它们分别属于双子叶植物的3个SPS亚家族。(2)荧光定量PCR分析显示,苹果C类的MdSPS6基因和A类的MdSPS1a/b基因是苹果中表达丰度最高的SPS基因成员,其中MdSPS6在苹果成熟果中表达丰度最高,其次是成熟叶片,而MdSPS1a/b在不积累蔗糖的幼果中表达丰度最高。(3)在果实发育过程中,除MdSPS1a/b之外,其它5个苹果MdSPS家族基因均随果实的生长表达丰度增加,与SPS活性和蔗糖含量明显呈正相关关系。研究表明,C类家族MdSPS6是苹果果实发育后期和叶片中蔗糖合成的主要SPS基因。     

High levels of expression of multiple enzymes in the Smirnoff-Wheeler pathway are important for high accumulation of ascorbic acid in acerola fruits

ABSTRACT

Acerola fruits contain abundant ascorbic acid (AsA). The gene expression levels of three upstream enzymes in the primary AsA biosynthesis pathway were correlated with AsA contents in the fruits of two acerola cultivars. Multiple overexpression of the enzymes increased AsA contents, suggesting their high expression is important for high AsA accumulation in acerola fruits and the breeding of AsA-rich plants.

Abbreviations: AsA: ascorbic acid; PMI: phosphomannose isomerase; PMM: phosphomannomutase; GMP: GDP-d-mannose pyrophosphorylase; GME: GDP-d-mannose 3?,5?-epimerase; GGP: GDP-l-galactose phosphorylase; GPP: l-galactose-1-phosphate phosphatase; GDH: l-galactose dehydrogenase; GLDH: l-galactono-1,4-lactone dehydrogenase     

Detergent extraction of enzymes from tobacco leaves varying in maturity

Enzyme activities of tobacco leaves were compared in detergent extracts. Highest levels of chlorogenic acid oxidase, malate-NAD oxidoreductase, and acid phosphatase were obtained from bud tissues. Peroxidase activity was least in young leaves and highest in senescent leaves yellowed with Ethrel. Peroxidase zymograms obtained by means of disc electrophoresis showed differences in isozyme composition among all five samples examined. Although protease was found in material extracted from buds, upper, middle, and lower leaf positions, none could be demonstrated in Ethrel-treated mature leaves.     

Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola (Malpighia glabra)

The Smirnoff-Wheeler (SW) pathway has been proven to be the only significant source of l-ascorbic acid (AsA; vitamin C) in the seedlings of the model plant Arabidopsis thaliana. It is yet uncertain whether the same pathway holds for all other plants and their various organs as AsA may also be synthesized through alternative pathways. In this study, we have cloned some of the genes involved in the SW-pathway from acerola (Malpighia glabra), a plant containing enormous amount of AsA, and examined the expression patterns of these genes in the plant. The AsA contents of acerola leaves were about 8-fold more than that of Arabidopsis with 5-700-fold higher mRNA abundance in AsA-biosynthesizing genes. The unripe fruits have the highest AsA content but the accumulation was substantially repressed as the fruit transitions to maturation. The mRNAs encoding these genes showed correlation in their expression with the AsA contents of the fruits. Although very little AsA was recorded in the seeds the mRNAs encoding all the genes, with the exception of the mitochondrially located L-galactono-1,4-lactone dehydrogenase, were clearly detected in the seeds of the unripe fruits. In young leaves of acerola, the expression of most genes were repressed by the dark and induced by light. However, the expression of GDP-D-mannose pyrophosphorylase similar to that encoded by A. thaliana VTC1 was induced in the dark. The expressions of all the genes surged after 24h following wounding stress on the young leaves. These findings will advance the investigation into the molecular factors regulating the biosynthesis of abundant AsA in acerola.     

Sorbitol metabolism and sink-source interconversions in developing apple leaves

In apple (Malus domestica Borkh.) sorbitol is the primary product of photosynthesis, the major translocated form of carbon, and a common fruit constituent and storage compound. Previous work on sorbitol metabolism has revealed a NADPH-dependent aldose 6-phosphate reductase (A6PR) in green tissues, and a NAD-dependent sorbitol dehydrogenase in nongreen tissues. Results here show a decrease in sorbitol dehydrogenase activity and an increase in A6PR activity as leaves developing in the spring undergo the transition from sink to source. Sorbitol dehydrogenase activity reached a minimum as A6PR peaked. These changes were related to increases in leaf carbohydrate levels, especially sorbitol, and to increases in rates of net photosynthesis. Studies conducted in the autumn on senescing leaves also showed changes in enzyme activites, leaf carbohydrate levels, and photosynthesis. At this time, however, sorbitol dehydrogenase increased in specific activity, whereas A6PR activity, leaf carbohydrates, and photosynthetic rates all decreased substantially. Other experiments showed differences in the ability of young and mature leaves to metabolize sorbitol and in the distribution of sorbitol enzymes in leaves at transitional developmental stages. The results suggest that sorbitol metabolism in apple is tightly controlled and may be related to mechanisms regulating partitioning or source and sink activity.     

Senescence-induced increases in intracellular oxidative stress and enhancement of the need for ascorbic acid in human fibroblasts

Many studies have suggested that there is a close correlation among declines in internal ascorbic acid (AsA) levels, various disorders, and senescence. To clarify the relationships between age-associated changes in intracellular AsA levels and the effects of AsA administration on intracellular reactive oxygen species (ROS) levels, we investigated aging-related changes in AsA uptake, ROS levels, and the effects of AsA administration on intracellular ROS levels in young and old (senescent) human fibroblasts. Our results demonstrated that AsA uptake was increased in old cells compared with young cells, although mRNA and protein expression of sodium-dependent vitamin C transporter 2 was barely altered between the young and old cells. We also demonstrated that the intracellular superoxide anion level was higher in young cells, whereas the level of intracellular peroxides was significantly increased in old cells under both normal and oxidative stress conditions. Moreover, AsA administration markedly decreased the augmentation of intracellular peroxides in old cells, whereas there was no effect of AsA treatment in young cells under both normal and oxidative stress conditions. Therefore, our results also indicate that AsA could play an important role in regulating the intracellular ROS levels in senescent cells and that the need for AsA is enhanced by cellular senescence.     

Engineering ascorbic acid biosynthetic pathway in Arabidopsis leaves by single and double gene transformation

Six genes, which encode enzymes involved in ascorbic acid (AsA) biosynthesis, including guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP), GDP-mannose-3′,5′-epimerase (GME), GDP-galactose guanylyltransferase (GGT), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH) and L-galactono-1,4-lactone dehydrogenase (GLDH) were transformed into Arabidopsis thaliana, to evaluate the contribution of each gene to AsA accumulation. Additionally, two combinations, GGT-GPP and GGT-GLDH, were co-transformed into Arabidopsis with a reliable double-gene transformation system. AsA content of GGT transgenic lines was 2.9-fold higher as compared to the control, and co-transformation led up to 4.1-fold AsA enhancement. These results provided further evidence that GGT is the key enzyme in plant AsA biosynthesis.     

Long-chain free fatty acids from Momordica cochinchinensis leaves as attractants to its insect pest,Aulacophora foveicollis Lucas (Coleoptera: Chrysomelidae)

Extraction, thin layer chromatography, and gas chromatography–mass spectrometry of young, mature, and senescent leaves of Momordica cochinchinensis Spreng revealed 13 free fatty acids, representing a total of 82.29, 91.30, and 68.52% of fatty acids in young, mature, and senescent leaves, respectively. Palmitic acid was the predominant fatty acid followed by stearic acid in three types of leaves. The free fatty acids from young, mature, and senescent leaves attracted female Aulacophora foveicollis Lucas (Coleoptera: Chrysomelidae) at the minimal concentrations of 4, 2, and 8 μg, respectively; whereas the mixtures of synthetic fatty acids mimicking free fatty acids of young, mature, and senescent leaves showed attraction at the minimal concentrations of 4, 2, and 10 μg, respectively, in Y-shaped glass tube olfactometer bioassay under laboratory condition. The results indicate that A. foveicollis may employ long-chain free fatty acids as an olfactory cue for host location. The individual synthetic fatty acids mimicking the proportions detected in three types of leaves were also evaluated through olfactometer bioassay. Only synthetic palmitic acid at the minimal amount of 2.17 μg attracted the insect. A synthetic blend of fatty acids mimicking 8 μg free fatty acid concentration of mature leaves or an amount of 5.42 μg palmitic acid produced the highest attraction of the insect. Hence 5.42 μg palmitic acid might be used for insect pest management program such as baited traps.     

The Occurence of delta-Tocopherylquinone in Higher Plants and Its Relation to Senescence

delta-Tocopherylquinone (deltaTQ) content was determined in tobacco and yellow maple leaves, green ivy leaves and cactus tissues. It was found that the concentration of delta-TQ was highest in mature or senescent tissues, such as white tobacco leaves (0.02 mumole/g dry wt) while its detection was uncertain in young, green leaves from the apex of tobacco plants. Fractionation by centrifugation of senescent tobacco leaves showed that the osmiophilic globule fraction was enriched in delta-TQ. Electron microscope studies of young, mature and senescent tobacco tissues showed progressive changes in the size and number of osmiophilic globules. After chloroplast breakdown in senescent tobacco leaves, these globules became the predominant constituents of the organelle. delta-TQ which is associated with osmiophilic globules may play a role in the development of plants, particularly during senescence.