Since their discovery, both the cap and the poly(A) tail have been implicated as being involved in the regulation of translational efficiency and message stability (Furuichi et al.

H 2 O 2 serves an important stress signaling function and promotes stomatal closure, whereas ascorbic acid (Asc) is the major antioxidant that scavenges H 2 O 2 . Dehydroascorbate reductase (DHAR) catalyzes the reduction of dehydroascorbate (oxidized ascorbate) to Asc and thus contributes to the regulation of the Asc redox state. In this study, we observed that the level of H 2 O 2 and the Asc redox state in guard cells and whole leaves are diurnally regulated such that the former increases during the afternoon, whereas the latter decreases. Plants with an increased guard cell Asc redox state were generated by increasing DHAR expression, and these exhibited a reduction in the level of guard cell H 2 O 2 . In addition, a higher percentage of open stomata, an increase in total open stomatal area, increased stomatal conductance, and increased transpiration were observed. Guard cells with an increase in Asc redox state were less responsive to H 2 O 2 or abscisic acid signaling, and the plants exhibited greater water loss under drought conditions, whereas suppressing DHAR expression conferred increased drought tolerance. Our analyses suggest that DHAR serves to maintain a basal level of Asc recycling in guard cells that is insufficient to scavenge the high rate of H 2 O 2 produced in the afternoon, thus resulting in stomatal closure.

Vitamin C (ascorbic acid) is essential to prevent disease associated with connective tissue (e.g., scurvy), improves cardiovascular and immune cell functions, and is used to regenerate ␣-tocopherol (vitamin E). In contrast to most animals, humans lack the ability to synthesize ascorbic acid as a result of a mutation in the last enzyme required for ascorbate biosynthesis. Vitamin C, therefore, must be obtained from dietary sources and, because it cannot be stored in the body, it must be obtained regularly. Once used, ascorbic acid can be regenerated from its oxidized form in a reaction catalyzed by dehydroascorbate reductase (DHAR). To examine whether overexpression of DHAR in plants would increase the level of ascorbic acid through improved ascorbate recycling, a DHAR cDNA from wheat was isolated and expressed in tobacco and maize, where DHAR expression was increased up to 32-and 100-fold, respectively. The increase in DHAR expression increased foliar and kernel ascorbic acid levels 2-to 4-fold and significantly increased the ascorbate redox state in both tobacco and maize. In addition, the level of glutathione, the reductant used by DHAR, also increased, as did its redox state. These results demonstrate that the vitamin C content of plants can be elevated by increasing expression of the enzyme responsible for recycling ascorbate.ascorbic acid ͉ dehydroascorbate reductase ͉ redox state ͉ glutathione ͉ nutrition

The 5-cap and the poly(A) tail act synergistically to increase the translational efficiency of eukaryotic mRNAs, which suggests that these two mRNA elements communicate during translation. We report here that the cap-associated eukaryotic initiation factors (eIFs), i.e. the two isoforms of the cap-binding complex (eIF-4F and eIF-iso4F) and eIF-4B, bind to the poly(A)-binding protein (PABP) both in the presence and absence of poly(A) RNA. The interactions between PABP and eIF-4F, eIF-iso4F, and eIF-4B were measured in the absence of poly(A) RNA using far Western analysis and confirmed by direct fluorescence titration studies. The functional consequence of the interaction between these initiation factors and PABP was examined using RNA binding assays and RNA mobility shift analysis. eIF-4F, eIF-iso4F, and eIF-4B promoted PABP activity through a shift in its equilibrium affinity for poly(A). eIF-iso4G, the large subunit of eIF-iso4F, was the subunit responsible for the interaction between eIF-iso4F and PABP and was the subunit that promoted PABP RNA binding activity. Truncation analysis of eIF-iso4G indicated that a domain close to its N-terminal end appeared to be involved in binding PABP. These results suggest that the interaction between PABP and eIF-4B and eIF-iso4G may be involved in mediating the functional co-dependence observed between the cap and the poly(A) tail during translation.Most eukaryotic mRNAs contain a cap (m 7

A 67-nucleotide portion of the non-coding, 5'-leader sequence of tobacco mosaic virus RNA [defined as omega' (Gr. omega prime)] has been shown to enhance the translation of contiguous foreign gene transcripts both in vitro and in vivo. Chemically-synthesized omega', containing convenient linker sequences, was inserted into derivatives of an in vitro transcription plasmid (pSP64) between the bacteriophage-SP6 promoter and sequences coding for either chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII). Run-off in vitro transcripts, with or without a 5'-cap structure (G(5')ppp(5')G) and/or the omega' sequence, were tested in mRNA-dependent cell-free translation systems derived from rabbit reticulocyte lysate, wheat germ extract or Escherichia coli (MRE 600). In all cases, the presence of omega' increased the translational expression of both reporter genes, typically between 2- to 10-fold. Electroporation of isolated mesophyll protoplasts from Nicotiana tabacum cv. Xanthi, or microinjection of oocytes from Xenopus laevis, with SP6-transcripts containing the CAT-coding region confirmed and extended the value of omega' as a potential translational enhancer of gene expression in vivo.

L-Ascorbic acid (Asc) is the most abundant water-soluble antioxidant in plants. It serves as a cofactor for enzymes involved in photosynthesis, hormone biosynthesis, and the regeneration of antioxidants such as α-tocopherol. Once used, Asc can be recycled by several different mechanisms. The short-lived monodehydroascorbate (MDHA) radical, produced following Asc oxidation, can be recycled following reduction by ferredoxin or monodehydroascorbate reductase (MDAR). MDHA can also undergo disproportionation into dehydroascorbate (DHA) and Asc. DHA can be recycled into Asc by dehydroascorbate reductase (DHAR) before it undergoes irrevocable hydrolysis. Through its recycling, Asc content and its redox state are maintained, which is critical under conditions of high demand, for example during high light or other stress conditions that increase reactive oxygen species (ROS) production. This review provides an overview of research in the last decade revealing the role that Asc recycling plays during germination, growth, and reproduction, as well as in response to environmental stress. These findings highlight the importance of DHAR- and MDAR-mediated mechanisms of Asc recycling in maintaining ROS at non-damaging levels while modulating ROS signalling function.

We characterized the progression of programmed cell death during maize (Zea mays L.) endosperm development of starchy (Su; wild-type) and shrunken2 (sh2) genotypes and tested the involvement of ethylene in mediating this process. Histological and viability staining demonstrated that endosperm cell death was initiated earlier and progressed more rapidly in sh2 endosperm compared with Su endosperm. lnternucleosomal DNA fragmentation accompanied endosperm cell death and occurred more extensively in sh2 endosperm. 1 -Aminocyclopropane-1 -carboxylic acid levels peaked approximately 16 d after pollination (dap) in Su endosperm and gradually decreased during subsequent development, whereas two large 1 -aminocyclopropane-1 -carboxylic acid peaks were observed in sh2 endosperm, the first between 16 and 20 dap and the second at 36 dap. Ethylene levels were elevated in sh2 kernels compared with Su kernels, with an initial peak 20 dap approximately 3-fold higher than in Su kernels and a second peak 36 dap approximately 5-fold higher than that in Su kernels. Ethylene treatment of Su kernels resulted in earlier and more extensive endosperm cell death and DNA fragmentation. Aminoethoxyvinylglycine treatment of sh2 kernels reduced the extent of DNA fragmentation. We conclude that ethylene is involved in triggering programmed cell death in developing maize endosperm and is responsible for the aberrant phenotype of sh2 kernels.

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