Progress 11/15/01 to 05/14/06
Outputs
Evidence indicating the operation of alternative AsA biosynthetic pathways Vitamin C (AsA) has important antioxidant and metabolic functions in both plants and animals, but humans have lost the ability to synthesize it. Vegetables are the major source of AsA in the human diet yet limited information on AsA synthesis in plants. In contrast, the animal AsA pathway has been known since the 1960s. Two biosynthetic pathways for AsA in plants were known prior to our work on this project. The D-mannose pathway appears to be predominant in leaf tissue, but a D-galacturonic acid pathway operates in developing fruits. We have previously shown that transforming lettuce and tobacco with a cDNA encoding the terminal enzyme of the animal pathway, L-gulono-1,4-lactone oxidase (GLOase, EC. 1.1.3.8), increased the AsA leaf content between 4 and 7-fold. Additionally, we found that tobacco plants had elevated AsA levels when fed L-gulono-1,4-lactone, the animal precursor. These data
suggest that at least part of the animal pathway may be present in plants. To further investigate this possibility, wild type and vitamin C deficient Arabidopsis thaliana (vtc) plants were transformed with a 35S:GLOase constructand AsA levels were compared to untransformed controls. Wild-type plants transformed with the construct showed up to a 2-fold increase in AsA leaf content compared to controls. All five vtc mutant lines expressing GLOase had a rescued AsA leaf content equal or higher (up to 3-fold) than wt leaves. These data and the current knowledge about the identity of genes mutated in the vtc lines suggested that an alternative pathway is present in plants, which can bypass the deficiency of GDP-mannose production of the vtc1-1 mutant and possibly circumvent other steps in the D-mannose pathway to synthesize vitamin C. Discovery of an alternative plant AsA biosynthetic pathway from myo-inositol We isolated a PCR fragment that encodes a MI oxygenase (MIOX, EC 1.13.99.1) from
Arabidopsis genome based on sequence similarities to a pig MIOX cDNA. The identity of the MIOX4 gene, on chromosome 4, was confirmed by expressing the ORF in bacteria. MIOX specific activity obtained from the orcinol-based assay was 2174nmol GlcUA min/mg protein. To our knowledge, this was the first MIOX gene of plant origin that had been cloned and characterized. Based on sequence similarity and domain organization, this miox appears to be part of a four-member gene family in the Arabidopsis genome. To study the contribution of MI to AsA biosynthesis, the miox4 ORF was expressed under the control of the strong constitutive 35S promoter in Arabidopsis plants. Analysis of three independent homozygous lines (L1, L2 and L3) revealed a 2 to 3-fold increase in the AsA content of leaves compared to wt, and a line transformed with the empty vector grown under identical conditions. There are just two enzymatic reactions required for the conversion of glucuronic acid (GlcUA), the MIOX4
product, to ascorbic acid. Future work will be directed toward cloning and characterizing the remaining enzymes in the MI-ascorbic acid pathway and determining their role in overall flux of this important antioxidant.
Impacts
Identification of a new vitamin C pathway in plants has led to genetic approaches for increasing the amount of this important antioxidant in plants. Not only will this benefit consumer health, but as a natural preservative food with elevated vitamin C will remain fresher longer extending shelf life and reducing waste.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
Ascorbate (AsA) biosynthesis in plants occurs through a complex, interconnected network with mannose, myo-inositol and galacturonate as principle entry points. Regulation within and between pathways in the network is largely uncharacterized. A putative F-box gene (VCF1, vitamin C F-box protein 1) was identified in Arabidopsis thaliana from an activation-tagged (AT), ozone-sensitive mutant that had 60 percent less leaf AsA than wild type (wt) plants. In contrast, two independent knockout lines disrupting VCF1 accumulated 2- to 3-fold greater foliar AsA and were more ozone tolerant than wt plants. Constitutive expression of VCF1 in the wt Col-0 genetic background resulted in AsA levels similar to those in the AT mutant, whereas expression of VCF1 in the knockouts reduced AsA content to wt levels. RT-PCR analysis of steady-state transcripts of genes involved in AsA biosynthesis showed that VCF1 negatively affected the expression of GDP-mannose pyrophosphorylase,
GDP-mannose- 3,5-epimerase, L-galactose-1-phosphate phosphatase, and L-galactose dehydrogenase, early and late enzymes of the mannose/galactose pathway to AsA. VCF1 expression appears to be developmentally controlled, since, as leaf tissue aged, VCF1 transcripts accumulated with a concomitant decrease in AsA. These findings provide the first evidence of an F-box gene regulating expression of multiple enzymes in a major plant biosynthetic pathway.
Impacts
Identification of an F-box gene that regulates the D-mannose/L-galactose vitamin C pathway will facilitate the identification of a master regulatory protein that can be used to increase the level of this critical nutrient and natural preservative in plants. In addition this compound will help protect crops from environmental stress and thus increase yields for producers.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/29/04
Outputs
There is evidence of the operation of three biosynthetic pathways for vitamin C (ascorbate, AsA) in plants: the mannose/L-galactose pathway, a D-galacturonic acid pathway and a route that resembles the pathway in animals. We have published molecular and biochemical evidence for an additional route using myo-inositol (MI) as the initial substrate. A MI oxygenase (MIOX) gene was identified in chromosome 4 (miox4) of Arabidopsis. AsA levels increased 2- to 3-fold in homozygous Arabidopsis lines over-expressing the miox4 open reading frame thus suggesting the role of MI in AsA biosynthesis and the potential for using this gene for the agronomic and nutritional enhancement of crops. According to sequence similarity analysis, miox4 (ORF At4g26260) belongs to a family of four members in the Arabidopsis genome. Its homologs are located in chromosomes 1, 2 and 5 (named miox1, miox2 and miox5, ORFs At1g14520, At2g19800, At5g56640, respectively). Analysis of the protein
structure showed that Miox4/Miox5 and Miox1/Miox2 are closely related. We have cloned all ORFs and confirmed the enzymatic activity of a truncated Miox2, containing the catalytic domain that is common to all members, in bacterially expressed recombinant protein. RT-PCR experiments performed with RNA extracted from 5-week old plants showed that miox1 is predominantly expressed in cauline leaves and siliques, and miox5 in flowers, while miox2 is highly expressed in all tissues. Assays performed with homozygous T-DNA knockout lines of miox1, 2, 4 and 5 under different light conditions revealed that miox1 is the member of the family that contributes the most to the AsA content of leaf tissue in Arabidopsis. Silencing of all miox genes caused an arrest of growth in the cotyledonary stage of the T1 generation plants transformed with an RNAi construct indicating the importance of the miox family not only for AsA biosynthesis, but also to normal growth and development.
Impacts
Manipulation of the plant vitamin C biosynthetic pathway has led to a 3-fold increase in this important vitamin. Crops utilizing this technology will provide added value to farmers through cold tolerance, grocers through longer shelf life, and consumers with increased nutritional value.
Publications
- Lorence A, Chevone BI, Mendes P, Nessler CL. 2004. Myo-inositol oxygenase offers a possible entry point into plant vitamin C biosynthesis, Plant Physiol., 134:1200-1205.
- Radzio JA, Lorence A, Chevone BI, Nessler CL 2004. L-gulono-1,4-lactone oxidase expression rescues vitamin C deficient Arabidopsis (vtc) mutants. Plant Mol. Biol. 53:837-844.
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Progress 10/01/02 to 09/30/03
Outputs
We have extended our work with the animal enzyme, GLOase (gulono-gamma-lactone oxidase), which we had previously shown to increase vitamin C when expressed in tobacco and lettuce. The same gene construct expressing GLOase was transformed into the model plant Arabidopsis thaliana. The gene was transformed into normal, wild-type plants and vitamin C deficient mutants (provided by Dr. Patricia Conklin SUNY Cortland). Normal plants transformed with the GLOase construct showed a 2-3 fold increase in vitamin C lower than the increase seen in lettuce and tobacco plants expressing the same gene. Vitamin C levels in each one of the deficient mutants recovered to normal levels, or greater suggesting that more than one vitamin C pathway exists in plants. Our group has also obtained strong evidence for the existence of a new, yet undiscovered vitamin C pathway in plants that uses the sugar alcohol myo-inositol (MI) as the initial substrate. We used bioinformatics to identify a MI
oxygenase (MIOX) gene in chromosome 4 (miox4) of Arabidopis and verified its enzymatic activity in bacterially expressed recombinant protein. The expression pattern of miox4 was examined in wild type Arabidopsis plants and found that it is predominantly expressed in flowers and leaves, tissues with a high demand for vitamin C. We then expressed the miox4 open reading frame in Arabidopsis and found a 2-3 fold increase in vitamin C levels. Based on these results we have filed a patent on this gene as a novel method for improving vitamin C levels in plants. Patent filed entitled: MANIPULATION OF ASCORBIC ACID LEVELS IN PLANTS. Number PCT/US03/27779, with an international filing date of September 8, 2003, and priority claimed to December 17, 2002.
Impacts
We have discovered a new pathway for vitamin C biosynthesis in plants and shown that at least one of the genes in this pathway can be used to double the level of vitamin C in plants. It is expected that this gene or other genes in the pathway will be useful in increasing the stress tolerance of crops, prolong their shelf life and add nutritional value to the consumer.
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
Previous work in our laboratory has shown that lettuce and tobacco plants expressing an animal GLOase (gulono-g-lactone oxidase) cDNA contained up to seven-fold more vitamin C than controls and grew normally indicating that it is possible to increase natural levels of vitamin C through metabolic engineering. Paradoxically other laboratories have recently provided strong evidence that plants make vitamin C via a different pathway than animals. Our current project is designed to determine the biochemical basis for the increase of vitamin C in our GLOase transgenics; and find out if there are multiple routes to making this compound in plants. We are using Arabidopsis because of its sequenced genome and the large number of available seed stocks including activation tagged lines in which a strong promoter has been randomly inserted into the genome that can activate nearby genes. Not only do these lines have some genes switched on at high levels, but in some cases the tag
may land inside a gene and disrupt its function. Thus we can screen the activation tagged lines for an increase or decrease of vitamin C and by identifying where the tag has landed begin to unravel which genes are involved in controlling its synthesis. Rather than analyzing thousands of tagged lines chemically we have chosen to assess vitamin C levels indirectly by ozone fumigation. Since plants with reduced vitamin C are known to be more ozone sensitive we first screen the tagged seedlings with sub-lethal ozone levels to identify potential vitamin C mutants. Once these are removed the remaining plants are exposed to a normally lethal dose of ozone to identify potential ozone resistant vitamin C overproducers. Not all of the sensitive lines we have recovered have low vitamin C nor do all the resistant plants have higher levels. These lines are being saved for future studies that may help us understand how plants resist air pollution. To date we have identified 12 tagged lines with low
vitamin C and 5 with increased levels. We are now in the process of identifying where the tags have landed in the Arabidopsis genome which will give us candidates for biosynthetic and regulatory genes in the vitamin C pathway that can be used to metabolically engineer other plants.
Impacts
We have identified 17 genetically tagged lines of the model plant Arabidopsis that have altered levels of vitamin C. The location of these tags is currently being sought to isolate specific genes in the pathway toward the goal of engineering plants with increased nutritional characteristics and freshness.
Publications
- No publications reported this period
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