, DINA F . MANDOLI AND WINSLOW R . BRIGGS Carnegie Institution of Washington , Stanford , California 94305 - 1297

Red light-induced cell elongation and division in intact, etiolated oat (Avena sativa cv Lodi) seedlings have been assessed. The middle of coleoptile was especialy responsive in the very low fluence range whereas the region immediately below the coleoptile tip and the two regions just above the coleoptilar node were more responsive than the entire organ in the low fluence range. These responses in the coleoptile are both the result of an increase in cell elongation. Coleoptile cel division is slightly inhibited in the very low and slightly stimulated by red Ught in the low fluence range. The one-sixth of the mesocotyl closest to the node is more suppressed in its growth than is any other region in the very low fluence range. However, the low fluence response involved the entire mesocotyl equally. In the apical one-sixth of the mesocotyl, a strong suppression of cell division and a weak suppression of cell elongation occurs. In the lower five regions of the mesocotyl, red light in both fluence ranges suppresses only cell elongation. Apparently, the difference between red light-induced oat growth stimulation and suppression primarily involves differences in the response of the cell elongation process. Mandoli and Briggs (17) characterized two phytochrome-mediated photoresponses in etiolated oat tissues. The VLF2 response resulted in 45% mesocotyl growth suppresstion and 30%Yo coleoptile growth stimulation over the 24 h subsequent to a brief pulse of R. The VLF was detected at 10-4 umol m and became saturated at 3 x 10-2 umol m-2. The LF response resulted in 80%o mesocotyl suppression and 60% coleoptile stimulation. The LF response was detected at 100 ,tmol m-2 and became saturated at 3 x 102 ,umol m-2 of R. Phytochrome controls organ morphogenesis, but reliable data on cellular morphogenesis are scarce. The use of unsafe working lights (R or green light) for seedling manipulations (7, 8, 11, 23), fluences far above saturation (white light, Ref. 2; R, Refs. 1, 7, 8, 20, and 22), and the analysis of cellular morphogenesis only through experiments that average cellular responses over the entire organ (20) are limitations of the studies available concerning the effect ofphytochrome photoconversion on cellular morphogenesis in seedlings. None of the studies available reports reciprocity data even for extremely long exposures. Several studies have analyzed and compared large regions (thirds) of organs (oat, Ref. 8, ctf Ref. 7; wheat, Refs. 20 and 22; corn, Refs. 13 and 14) and/or assessed cell morphogenesis at a given position relative to an anatomical structure (e.g. coleoptile tip or node) (20, 23). The former studies do not provide very high resolution and the latter assumed that cells in an organ were 'Carnegie Institution of Washington, Department of Plant Biology Publication 798. 2 Abbreviations: FR, far-red light; LF, low fluence; R, red light; VLF, very low fluence. stationary (10). Although Huisinga (12) performed experiments in total darkness and analyzed very small regions of both the oat coleoptile and mesocotyl, his study was confined to excised seedlings and presents data only for individual seedlings given fluences in the LF range. The data collectively indicate that changes both in cell division and elongation affect organ morphogenesis. Unfortunately, the evidence often cited as the strongest (for intact Rirradiated seedlings; Refs. 2 and 9), employed unquantitated 'safe' R and only calculated changes in cell number from data on mean cell length from whole organs (8) on the assumption that cellular packing and cell position in an organ were constant. An additional problem is that, although phytochrome has been found throughout the etiolated oat seedling (4, 6), the sites of photoperception in oat seedlings (1.5-2.5 mm above the node and 4.5-6.5 mm below the node; Ref. 17), do not match the areas where phytochrome is most heavily concentrated: the coleoptilar node and tip (4). Thus, the phytochrome absolute concentration in a given cell may be less important than that cell's ability to respond to R and to affect the particular response being assayed, in this case, stimulation of coleoptile and suppression ofmesocotyl growth. A technique has been developed which allows 54-h-old, intact etiolated seedlings to be marked at 1-mm intervals in total darkness (12). These marks can be used to monitor growth responses of specific regions of the coleoptile and the mesocotyl to R. The response of these regions in each organ can then be compared to the sites of photoperception and the sites at which the highest concentrations of phytochrome have been found. The relative contributions of cell elongation and cell division to these responses in each region have been assessed for R in the VLF and LF response ranges. MATERIALS AND METHODS Growth of Seedlings. Seedlings of oats (Avena sativa cv Lodi) were grown according to Mandoli and Briggs (17) with the following two modifications. Seeds were dehusked and imbibed for 0.5 h by laying the glass, paper, and tape assembly in a pan of water 0.5 cm deep, such that none of the seeds were covered, but the paper was allowed to soak completely. Seeds were then transferred to racks and grown as by Mandoli and Briggs (17). The entire imbibition process and all manipulations were done in total darkness. Seedlings were used at 54 h postimbibition since organ growth, cell elongation, and cell division are maximal at this time (26). Analysis of growth of whole organs was completed five times (I plate/fluence in each experiment, each plate with about 20 usable seedlings). Illumination and Marking. Seeds were grown in darkness for 54 h, and then illuminated with R according to Mandoli and Briggs (17). Fluence rates were varied with Balzer's neutral density filters, and measured with a Licor Quantum Radiometer (model LI0185A). A device for marking seedlings in the dark was utilized (Fig. 1). Seedlings, still adhering to the masking tape on which they were grown, were laid on a flat Plexiglas plate. A Plexiglas bridge with thin fishing lines (0.25 mm in diameter) stretched