Laser capture microdissection (LCM) is a technique by which individual cells can be harvested from tissue sections while they are viewed under the microscope, by tacking selected cells to an adhesive film with a laser beam. Harvested cells can provide DNA, RNA, and protein for the profiling of genomic characteristics, gene expression, and protein spectra from individual cell types. We have optimized LCM for a variety of plant tissues and species, permitting the harvesting of cells from paraffin sections that maintain histological detail. We show that RNA can be extracted from LCM-harvested plant cells in amount and quality that are sufficient for the comparison of RNAs among individual cell types. The linear amplification of LCM-captured RNA should permit the expression profiling of plant cell types.

No abstract

Within the root meristem of flowering plants is a group of mitotically inactive cells designated the quiescent center (QC). Recent work links the quiescent state to high levels of the growth regulator auxin that accumulates in the QC via polar transport. This in turn results in elevated levels of the enzyme ascorbic acid oxidase (AAO), resulting in a reduction of ascorbic acid (AA) within the QC and mitotic quiescence. We present evidence for additional interactions between auxin, AAO, and AA, and report that, in vitro, AAO oxidatively decarboxylates auxin, suggesting a mechanism for regulating auxin levels within the QC. We also report that oxidative decarboxylation occurs at the root tip and that an intact root cap must be present for this metabolic event to occur. Finally, we consider how interaction between auxin and AAO may influence root development by regulating the formation of the QC.

No abstract

A new hypothesis for the formation of the quiescent center is presented. Reported data support a mechanism for the establishment and maintenance of the quiescent center. The quiescent center is located at the most distal part of the root proper, the most terminal location in the root proper on the path of polar transport from the shoot. Of the many substances polarly transported in the root, auxin is one of the best studied and has been shown to affect root meristem organization. In our mechanism, polar auxin is directly linked to quiescence through the action of ascorbate oxidase and ascorbic acid. Immunolocalization of auxin in the root tip of Zea mays showed that auxin levels in the quiescent center were high compared to the levels in the immediately surrounding meristematic cells. Isolated quiescent centers were shown to have high levels of ascorbate oxidase mRNA and ascorbate oxidase activity relative to proximal meristem tissue. Exogenous auxin caused an increase in ascorbate oxidase mRNA levels and ascorbate oxidase enzyme activity in cultured root tissue. Immunolocalization of ascorbate oxidase in Zea root tips showed high levels of the protein in the quiescent center relative to surrounding cells. This is the first report of a positive marker and activity for the quiescent center. Histochemical detection of ascorbic acid in Zea root tips showed that quiescent center cells have low or undetectable levels of ascorbic acid, presumably due to the high levels of ascorbate oxidase in the quiescent center. As ascorbic acid is a compound known to be necessary for the transition from G1 to S in the cell cycle, its low levels in the quiescent center may be directly responsible for holding these rarely dividing cells in the extended G1 state in which they are mainly found. We propose that our mechanism complements published mathematical modeling of the anatomical structure of root apices, and further propose that the control of relative growth rates in this focal region of the root apex by this mechanism is a determining aspect in generating anatomical patterning in the root apex.