Argelia Lorence

myo -Inositol Oxygenase Offers a Possible Entry Point into Plant Ascorbate Biosynthesis

Two biosynthetic pathways for ascorbate (l-ascorbic acid [AsA]; vitamin C) in plants are presently known, the mannose/l-galactose pathway and an l-GalUA pathway. Here, we present molecular and biochemical evidence for a possible biosynthetic route using myo-inositol (MI) as the initial substrate. A MI oxygenase (MIOX) gene was identified in chromosome 4 (miox4) of Arabidopsis ecotype Columbia, and its enzymatic activity was confirmed in bacterially expressed recombinant protein. Miox4 was primarily expressed in flowers and leaves of wild-type Arabidopsis plants, tissues with a high concentration of AsA. Ascorbate levels increased 2- to 3-fold in homozygous Arabidopsis lines overexpressing 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.

L-Gulono-1,4-lactone oxidase expression rescues vitamin C-deficient Arabidopsis (vtc) mutants

Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, humans have lost the ability to synthesize it. Fresh produce is the major source of vitamin C in the human diet yet only limited information is available concerning its route(s) of synthesis in plants. In contrast, the animal vitamin C biosynthetic pathway has been elucidated since the 1960s. Two biosynthetic pathways for vitamin C in plants are presently known. The D-mannose pathway appears to be predominant in leaf tissue, but a D-galacturonic acid pathway operates in developing fruits. Our group has 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 vitamin C leaf content between 4- and 7-fold. Additionally, we found that wild-type (wt) tobacco plants had elevated vitamin C 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 (L.) Heynh (vtc) plants were transformed with a 35S:GLOase construct, homozygous lines were developed, and vitamin C levels were compared to those in untransformed controls. Wild-type plants transformed with the construct showed up to a 2-fold increase in vitamin C leaf content compared to controls. All five vtc mutant lines expressing GLOase had a rescued vitamin C 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 suggest 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.

Gene transfer and expression in plants.

Until recently, agriculture and plant breeding relied solely on the accumulated experience of generations of farmers and breeders that is, on sexual transfer of genes between plant species. However, recent developments in plant molecular biology and genomics now give us access to knowledge and understanding of plant genomes and the possibility of modifying them. This chapter presents an updated overview of the two most powerful technologies for transferring genetic material (DNA) into plants: Agrobacterium-mediated transformation and microparticle bombardment (biolistics). Some of the topics that are discussed in detail are the main variables controlling the transformation efficiency that can be achieved using each one of these approaches; the advantages and limitations of each methodology; transient versus stable transformation approaches; the potential of some in planta transformation systems; alternatives to developing transgenic plants without selection markers; the availability of diverse genetic tools generated as part of the genome sequencing of different plant species; transgene expression, gene silencing, and their association with regulatory elements; and prospects and ways to possibly overcome some transgene expression difficulties, in particular the use of matrix-attachment regions (MARs).

Recombinant Gene Expression

Recombinant production has become an invaluable tool for supplying research and therapy with proteins of interest. The target proteins are not in every case soluble and/or correctly folded. That is why different production parameters such as host, cultivation conditions and co-expression of chaperones and foldases are applied in order to yield functional recombinant protein. There has been a constant increase and success in the use of folding promoting agents in recombinant protein production. Recent cases are reviewed and discussed in this chapter. Any impact of such strategies cannot be predicted and has to be analyzed and optimized for the corresponding target protein. The in vivo effects of the agents are at least partially comparable to their in vitro mode of action and have been studied by means of modern systems approaches and even in combination with folding/activity screening assays. Resulting data can be used directly for experimental planning or can be fed into knowledge-based modelling. An overview of such technologies is included in the chapter in order to facilitate a decision about the potential in vivo use of folding promoting agents.

Ontogenetic changes in vitamin C in selected rice varieties.

Vitamin C (L-ascorbic acid) is a key antioxidant for both plants and animals. In plants, ascorbate is involved in several key physiological processes including photosynthesis, cell expansion and division, growth, flowering, and senescence. In addition, ascorbate is an enzyme cofactor and a regulator of gene expression. During exposure to abiotic stresses, ascorbate counteracts excessive reactive oxygen species within the cell and protects key molecules, including lipids, proteins, and nucleic acids, from irreversible damage. In this study we focus on understanding how ascorbate levels are controlled in rice (Oryza sativa) during plant development and in response to light intensity and photoperiod. Our results indicate that in rice ascorbate metabolism follows a different pattern compared to other species. In the rice accessions we analyzed, total foliar ascorbate content increases during development and peaks at the vegetative 2-4 and the reproductive 4 stages, whereas other research has shown that in Arabidopsis thaliana and other dicots, ascorbate content declines with plant age. The pattern in rice does not seem to change when plants were grown under increasing light intensity: 150, 400 or 1200-1500 μmol m(-2) s(-1). We observed little diurnal variation in AsA content in rice and did not see a steady decline during the dark period as has been reported in other species such as Arabidopsis and tomato. The total foliar ascorbate content of twenty-three rice accessions from four major rice subgroups was compared. These genotypes differed as much as eight-fold in ascorbate content at the V2 stage indicating the potential to enhance vitamin C levels in genotypes of global interest via breeding approaches.