To prepare tissues for analysis of NAD', NADH, NADPI, and NADPH, common practice is to freeze samples in liquid nitrogen, often followed by freeze-drying, before Nicotinamide adenine dinucleotide coenzymes are involved in functions of about 200 dehydrogenases (9). To extract these nucleotides for quantitative analysis, liquid N2 is usually used to arrest enzyme activity (1, 3, 6-9), sometimes followed by freezedrying (6-8 mg (mean and SD).A motor-driven Teflon pestle that fit tightly in a 15-ml Corex centrifuge tube was used for tissue homogenization. For NADP+ and NAD+, 3.0 ml of 0.2 N HCI were used to homogenize each group of five cotyledons; for NADPH and NADH, 3.0 ml of 0.2 N NaOH were used. Each homogenate was made to 10 ml with the respective HCl or NaOH solution, heated in a boiling water bath for 5 min, cooled in an ice bath, then centrifuged at 14,000g at 0 to 4°C for 10 min. Supernatant solutions were transferred to separate tubes and kept on ice for coenzyme assay.Assay of Coenzymes. Enzyme cycling assays were employed with MTT as the terminal electron acceptor. Procedures for these assays were described by Matsumura and Miyachi (6). A cycling mixtube of MTT, PES catalyst, ethanol and alcohol dehydrogenase was used to determine LAD and NADH; for NADP+ and NADPH, glucose-6-P and glucose-6-P dehydrogenase replaced ethanol and alcohnl dehydrofenase.

The effect of light on the abundance of j0-tubulin mRNA was measured in etiolated Avena sativa L. and Hordeum vulgare L.seedlings. Slot blot analysis employing an oat #-tubulin cDNA clone was used to measure f-tubulin mRNA levels. White light induced a 45% decrease in oat,-tubulin mRNA abundance by 2 hours after transfer. A saturating red light pulse induced 40 and 55% decreases in ,j-tubulin mRNA levels in oats and barley, respectively. Recovery of ,@-tubulin mRNA levels was observed after a red light pulse but not after transfer to continuous white light. The red light induced decrease in oat fI-tubulin mRNA abundance was not reversible by a subsequent far-red light treatment. The mesocotyl portion of etiolated oat seedlings exhibited a more dramatic decrease in fB-tubulin mRNA abundance in response to red light than did the coleoptile portion. The results indicate that the well-documented effects of red light on the growth of etiolated seedlings are accompanied by changes in the expression of the j-tubulin genes.3-Tubulin is one of the two subunits of tubulin, the major structural protein of microtubules (10). The abundance of ,Btubulin mRNA has been shown to depend on the concentration of unpolymerized /3-tubulin subunits in cultured animal cells. A twofold increase in the level of unpolymerized tubulin subunits in Chinese hamster ovary cells results in a fivefold decrease in #-tubulin mRNA abundance (2). The /-tubulin mRNA level in mouse cells is also decreased in response to microtubule depolymerization (9, 31). 3-Tubulin mRNA levels are down-regulated during the cell cycle in Physarum (23). The down-regulation in Physarum correlates with depolymerization of microtubules after metaphase. In contrast, an increase in /-tubulin mRNA abundance can be induced by deflagellation of Chlamydomonas cells, a treatment that stimulates polymerization of tubulin and concomittant production of microtubules (24).Light, acting through phytochrome, induces major changes in the growth pattern of etiolated seedlings. For example, red (3,12,14,15,28) is down-regulated by phytochrome. Given that phytochrome can modulate both gene expression and growth events involving microtubules, we have investigated the effect of light on the expression of the f3-tubulin genes in etiolated oat and barley seedlings. Our results indicate that light treatment induces a decrease in fl-tubulin mRNA abundance in etiolated seedlings of both these species. MATERIALS AND METHODS Growth and Irradiation of PlantsOat (Avena sativa L. cv "Garry") and barley (Hordeum vulgare L. cv "Harrington") seeds were imbibed in distilled water and planted in moist vermiculite in 250 mL disposable polyethylene beakers. Up to 12 beakers were placed in larger polyethylene containers. About 1.5 L of water was added to the container and the containers were covered with aluminum foil. The seedlings were grown for 4 d at 25°C in total darkness. The mean length of the shoot portion of the 4-d-old etiolated oat seedlings grown under our conditions was 64 mm (n = 201,...

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