Rapid Image Analysis Screening Procedure for Identifying Chloroplast Number Mutants in Mesophyll Cells of Arabidopsis thaliana (L.) Heynh.

Abstract

MATERIALS AND METHODSTo analyze the genetic control of the process of chloroplast division, a direct image analysis screening procedure has been developed in which mutants of Arabidopsis thaliana (L.) Heynh. var Landsberg erect are selected on the basis of abnormal chloroplast number. The selection procedure is based on image analysis thresholding after iodine staining, which facilitates the automatic counting of chloroplasts in isolated mesophyll cells. M2 seedlings are screened for significant deviation from the wild type relationship between mesophyll cell size and chloroplast number. Mutants with both abnormally high and abnormally low chloroplast numbers were identified. Of 3500 individual M2 seedlings screened, 18 mutant lines have been isolated and shown to be stably inherited in three subsequent generations. The most extreme phenotypes show an 80% reduction or a 50% increase in chloroplast number per mesophyll cell.Chloroplast replication is a ubiquitous characteristic of leaf mesophyll cell development (4, 6). Up to 90% of the chloroplasts in a mature leaf mesophyll cell are products of the division of young green chloroplasts in expanding leaf cells. It has not been possible to identify the essential control mechanisms involved in chloroplast division, and plants in which the process of chloroplast division is modified would be of great potential value. Ideal (Levington F2) and covered with plastic lids until they germinated (3-4 d). The trays were placed in a controlled environment cabinet at 20C with a 5TC night depression, 70% relative humidity and a light intensity of 60 W/m2 (Philips TLD 58W/35 tubes). The seeds were always sown at 10 AM and the first leaves harvested at 10 AM between 9 and 22 d later for the wild-type and at 20 d for the M2 mutants. So that individual mutant seedlings could be readily identified, the M2 seeds were sown in a numbered grid of 96 compartments produced by placing a grid of 1-inch square wire mesh on the surface ofthe compost. M2 seeds were sown in each of the 96 squares in each tray with the aid of a blue l-mL disposable pipette tip (Anachem). Seeds were placed inside the tip; by holding the tip in a horizontal position and tapping gently with a finger, a few seeds at a time were dispensed. Up to five seeds were sown in each square and were thinned to one seedling on germination. Each M2 seedling was identified by a seed tray number and a coordinate number within the tray (e.g. YK8 1 [Y = York]).

Hybrid mimics and hybrid vigor in Arabidopsis

“…We scored an increased number of chloroplasts per leaf where the cell size was increased and where there was an increased number of cells (16). Chloroplasts are more frequent in larger cells and the cells have a greater chlorophyll content (17,18).…”

“…These morphological features of the hybrid and hybrid mimics have a common basis of increased cell number, and in some cases, increased size of leaf cells. The increased number and size of the photosynthetic cells result in larger numbers of chloroplasts per leaf that produce more photosynthate than the parental lines (17,18). The increased sugar and starches provide the raw building materials for the increased size of the vegetative plants (17,18).…”

An Arabidopsis Homolog of the Bacterial Cell Division Inhibitor SulA Is Involved in Plastid Division[W]

“…Isolated cells were obtained and observed as described (Pyke and Leech, 1991), except that leaf sections were fixed in 2% paraformaldehyde for 2 h.…”

“…These semiautonomous organelles can divide, and their division is crucial for their maintenance after mitosis. Moreover, this seems to be a tightly regulated process because plastid number depends on the cell type (in the Landsberg ecotype of Arabidopsis thaliana, meristematic cells contain 10 to 15 plastids, whereas mesophyll cells contain 120) (Marrison et al, 1999) and is correlated with the cell area in mesophyll cells (Pyke and Leech, 1991).…”

A Chloroplast Protein Homologous to the Eubacterial Topological Specificity Factor MinE Plays a Role in Chloroplast Division

“…A series of nuclear recessive mutants of the higher plant Arabidopsis, in which the chloroplast number per cell is greater or fewer than that in the wild-type plant, have been well documented (Pyke and Leech, 1991, 1994 Pyke et al, 1994; Robertson et al, 1996;Marrison et al, 1999). Characterization of these mutants, referred to as accumulation and replication of chloroplasts (arc) mutants, indicates that chloroplast division is a complex process that involves multiple distinct steps, such as expansion, division site selection, division initiation, constriction, and scission of chloroplasts, which are controlled by different nuclear genes, although no ARC genes have yet been cloned.…”