Light-grown 7-day-old wheat seedlgs (Trticum aestavum, var. Maris Dove) showed an increase of 200% in plastids per ceUl between 1.7 and 4.5 centimeters from the leaf base. This inacrease was the result of divisions of young chbiolasts at various stages of development, and was well separated in distance, and therefore in time from the region of ceO division in the basal meristem. 13HIThymide was incorporated into plastid DNA throughout the zone of plastid division, but not above it.During leaf development, proplastids differentiate into chloroplasts and their differentiation is accompanied by a large increase in the number of plastids per cell, caused by the division of both proplastids and young chloroplasts (11). In spinach leaves the division of young grana-containing, photosynthetic chloroplasts is the most important factor determining the number of chloroplasts per mature leaf cell: Possingham and Saurer (19) have shown that up to 90%o of the final number of plastids can be accounted for by the division of young chloroplasts. Chloroplast division has also been observed to occur in vitro (8,22) but no subsequent increase in size has been observed.For ease of experimentation, the effects of exogenous factors on chloroplast division have been examined mainly by using discs excised from leaf tissue (4,14,(16)(17)(18)20); but in view of the large biochemical changes which occur rapidly after the excision of leaf tissue (15), the results of investigations of the control of plastid division in leaf discs should be interpreted with some caution. The division of young chloroplasts and its significance in the development and function of the leaf would be examined least equivocally in an intact, growing plant.The aim of the present investigation was to use an intact plant in which a large increase in the number of plastids per cell occurred during leaf development and to examine the sequence of biochemical events associated with this increase. To follow the division process sequentially, we required plants from which large numbers of cells and plastids could be harvested at each stage of development. Dicotyledonous leaves are not an ideal tissue in which to study plastid division since the leaf is a mosaic of cells at different stages of differentiation. The advantage of many monocotyledonous plants is that in the leaves of their young seedlings all cell divisions occur in a basal meristem, resulting in a developmental sequence of cells from the base to the tip of each leaf. This has already been exploited in maize (2,5,6,12) (27) to study plastid and cell differentiation.In the present paper we demonstrate how a similar sequential developmental system in wheat leaves can be used to investigate the replication process of young chloroplasts and the relationship between the timing of DNA synthesis and plastid division. Wheat was chosen because many varieties and special lines are available whose genetics are well understood, and the complication of chloroplast dimorphism, as found in maize, is avoided. MATERIALS ...

A method is described for relating the age of a cereal leaf cell to its distance from the leaf base. The rates of chlorophyll synthesis per plastid in the first leaf of llght-grown and of greening etiolated seedlngs of wheat (Triicun aestivum, var. Maris Dove) increase with cel age. Normally developing plastids of light-grown wheat take over 24 hours to reach the chlorophyll a/b ratio characteristic of mature wheat chloroplasts (4.5), but mature etioplasts need only 8 hours light to achieve this a/b ratio. Plastid greening potental depends only on cell age, whereas the chlorophyl a/b ratio is influenced both by cel age and by light. This paper presents the results of a study of this kind. We have examined plastid development in wheat in order to avoid dealing with dimorphic chloroplasts, and because we already have a good background knowledge of the development of wheat seedlings (5). Methods are described for measuring the leaf tip growth rate over several days before harvest, and the growth rate at any position in the leaf relative to the growth rate at its tip, for both etiolated and light-grown wheat seedlings. Using these measurements it has been possible to convert distance from the leaf base into cell age, and so to make direct comparisons between chloroplasts and etiochloroplasts in cells of the same age in light-grown and greening etiolated seedlings.The seedlings of many cereals are particularly well suited to the study of chloroplast development, since the developing chloroplasts are in a linear series, with the youngest in cells near the base and the oldest in cells near the tip of the leaf (3, 5, 6, 8, 10-12, 14, 15). Robertson and Laetsch (14) took advantage of this arrangement to study Chl synthesis during the greening of etiolated maize seedlings, and demonstrated that the age ofdeveloping etiolated tissue had a considerable effect on the rates of Chl synthesis following illumination. They reported that the rate of greening, per g fresh weight, was greater in regions distant from the leafbase than in the younger regions near the base. Mackender (11) found a similar increase in greening potential with cell age when he measured the levels ofprotochlorophyllide/g fresh weight in sections cut at various distances from the bases of etiolated barley leaves.A greening etiolated leaf is far from being a natural phenome- MATERIALS AND METHODS Plant Material. Seeds of wheat, Triticum aestivum, var. Mans Dove (Dickson, Brown & Tait Ltd., Altrincham, U.K.) were soaked in running tap water at 20 C and surface-sterilized in sodium hypochlorite solution (13% free chlorine) after 1 h. After being soaked for an additional 16 h, the seeds were sown in Levington Universal Compost (Fisons, U.K.) at a depth of 1 cm. Light-grown seedlings were given a photoperiod of 16 h at 20 C with a 5 C night depression and 70%o RH. The light intensity at the level of the seedlings, measured with a solarimeter (Kipp & Zonen), was 4.0 mw.cm2. Dark-grown seedlings were grown in total darkness at 25 C, about 100%1o RH. After 5...

Plastids at different stages of development were isolated from lightgrown wheat ( Triticum aestivum, var. Mans Dove) seedling leaves, and the average chloroplast DNA (cpDNA) The leaf cells of different species of higher plants differ markedly in their complement of chloroplasts (6). At the extremes of the range are species such as cocoa (Theobroma cacao) and Peperomia metallica with an average of three plastids per cell and radish (Raphanus sativus) with over 300. The photosynthetic rate of leaf tissue will clearly be influenced by the number and morphology of the chloroplasts in its mesophyll cells and factors determining the final numbers of cells and of chloroplasts per cell will be particularly important determinants of leaf function. As yet none of the factors determining chloroplast number has been experimentally identified, but microscopical observations of leaves of polyploid series of crop plants, particularly sugar beet (5) have indicated that the amount of nuclear DNA per cell, and to a lesser extent cell size (7), may be correlated with the final number of chloroplasts in the mature leaf cell.In order to examine in detail the relationship between cpDNA and nDNA3 synthesis and chloroplast number, we chose young seedlings of a modern hexaploid wheat, Triticum aestivum var. Maris Dove. As in other graminaceous monocotyledons, the chloroplast-containing cells of young wheat leaves are produced in a basal meristem and the leaf cells are in a linear array with the oldest nearest the tip and the youngest nearest the base of the leaf (3). We have previously shown (3) that nDNA synthesis is restricted to the zone below I cm from the base of the leaf blade

This book contains many chapters describing methods for isolating and modifying DNA molecules. The most usual way of checking the success of such procedures is by looking at the products using electrophoresis in agarose gels. This process separates DNA molecules by size, and the molecules are made visible using the fluorescent dye ethidium bromide. In this way DNA can be checked for size, intactness, homogeneity, and purity. The method is rapid and simple, yet capable of high resolution, and is so sensitive that usually little of the sample is needed for analysis.

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