Abstract
The Arabidopsis (Arabidopsis thaliana) CYCD2;1 gene introduced in genomic form increased cell formation in the Arabidopsis root apex and leaf, while generating full-length mRNA, raised CDK/CYCLIN enzyme activity, reduced G1-phase duration, and reduced size of cells at S phase and division. Other cell cycle genes, CDKA;1, CYCLIN B;1, and the cDNA form of CYCD2;1 that produced an aberrantly spliced mRNA, produced smaller or zero increases in CDK/CYCLIN activity and did not increase the number of cells formed. Plants with a homozygous single insert of genomic CYCD2;1 grew with normal morphology and without accelerated growth of root or shoot, not providing evidence that cell formation or CYCLIN D2 controls growth of postembryonic vegetative tissues. At the root apex, cells progressed normally from meristem to elongation, but their smaller size enclosed less growth and a 40% reduction in final size of epidermal and cortical cells was seen. Smaller elongated cell size inhibited endoreduplication, indicating a cell size requirement. Leaf cells were also smaller and more numerous during proliferation and epidermal pavement and palisade cells attained 59% and 69% of controls, whereas laminas reached normal size. Autonomous control of expansion was therefore not evident in abundant cell types that formed tissues of root or leaf. Cell size was reduced by a greater number formed in a tissue prior to cell and tissue expansion. Initiation and termination of expansion did not correlate with cell dimension or number and may be determined by tissue-wide signals acting across cellular boundaries.
Highlights
Final cell size in the root apex was strongly influenced by partitioning of growth between division and cell expansion, which is determined by the boundaries of meristem and elongation zones
The depression of cell size removed a stimulus for endoreduplication from a high cytoplasmto-DNA ratio and provides novel evidence that the endoreduplication process requires adequate cell size for its initiation
It has previously seemed likely that reduplication is the cause of cell enlargement (Cebolla et al, 1999), but current evidence tracking cells through normal development terminating at smaller size (Fig. 7 below) suggests that, large cell size is a requirement for reduplication
Summary
Plant Material and TreatmentsSurface-sterilized seeds of Arabidopsis (Arabidopsis thaliana) Columbia background were germinated in continuous light (80 mmol m22 s21) at 20°C on agar-solidified modified Hoagland solution supplemented with 0.5% Suc, in 90-mm-diameter petri dishes, sealed with porous tape, and sloped at 85° to obtain root growth along the surface of the agar. Care was taken in growth comparisons to use seed with equal reserves produced by plants uncrowded and control and test plants were grown in parallel within the same containers. Cyclin-dependent kinase activity was assayed from whole 7-d-old seedlings with a high proportion of dividing cells, in fractions purified with SUC1 (Zhang et al, 1996), or with antibody raised against the unique carboxyterminal sequence of CDKA; CALEHEYFKDLGGMP, as described (Zhang et al, 2005). First true leaves were taken from 6-d-old seedlings where proliferating epidermal cells were observed in identical areas in the basal lamina of control and transgenic leaves. Expanded leaves were sampled at 22 d and images were taken from identical areas in midleaf between midrib and margin. Cell dimensions and areas were estimated and statistically analyzed with ImageJ software from the National Institutes of Health (NIH)
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