[155]. The levels of Arath;CycD3;1 did not change with withdrawal or readdition of all three substrates.
However, when cytokinin alone was added, the expression of Arath;CycD3;1 increased fourfold and this
was somewhat enhanced with addition of sucrose. Auxin was antagonistic to the increase induced by cy-
tokinin. The levels of Arath;CycD2;1 decreased on removal and increased with addition of sucrose but
were independent of hormone. These results suggest that these cyclins respond to growth stimulators and
carbon source much like animal D-type cyclins. D-cyclins may in turn form active kinase complexes tar-
geting Rb homologues, causing inactivation and dissociation from E2F, which could then up regulate ex-
pression of S phase–specific genes. A recent study confirmed that Arath;CycD3;1 is induced by cytokinin
and showed that constitutive expression of Arath;CycD3;1 allowed induction and maintenance of cell di-
vision in the absence of exogenous cytokinin [312]. Riou-Khamlichi et al. [312] suggest that cytokinin
activatesArabidopsiscell division through induction of Arath;CycD3;1 at the G 1 /S transition. In alfalfa,
expression of the D-type cyclin Medsa;CycD3;1 was induced 12 hr following addition of auxin and cy-
tokinin to pieces of fully differentiated leaves [221]. Expression of another alfalfa cyclin,
Medsa;CycA2;1, was induced only 4 hr after addition [149]. Medsa;CycA2;1, although mitotic-like in se-
quence, is expressed in G 1 and responds to growth regulator and so may have a G 1 function.
Cytokinin has also been implicated in the stimulation of the tyrosine dephosphorylation and activa-
tion of Cdc2-like H1 histone kinase [142]. Addition of auxin and cytokinin to pith parenchyma cells re-
sulted in a greater than 40-fold increase in a Cdc2-like protein with high H1 histone kinase activity. With-
out cytokinin the amount of protein increased, but it was inactive and contained a high amount of
phosphotyrosine. This inactive protein could be activated by addition of bacterially produced yeast
CDC25 phosphatase. Zeatin has been shown to be necessary for the G 2 /M transition in tobacco cells
[313]. An inhibitor of cytokinin biosynthesis inhibited mitosis at the G 2 /M transition and this block could
be overcome only by addition of zeatin. On the other hand, zeatin was not restrictive for the occurrence
of the G 1 /S transition in tobacco cells [314]. Furthermore, addition of cytokinin at early G 1 blocked the
cycle at G 1 /S, suggesting that down-regulation of the zeatin type of cytokinins is important for the G 1 /S
transition [314]. A somewhat conflicting report finds that both auxin (dichlorophenoxyacetic acid) and
cytokinin (N^6 -benzyladenine) are necessary for release from a block at G 1 /S in Petunia[310]. Auxin alone
could not stimulate CDC2Pettranscript accumulation but together with cytokinin there was an increase
in transcript. Different phytohormones may have different effects on different tissues. In legume lateral
root formation, auxin but not cytokinins causes cells in the G 2 phase to reenter the cell cycle giving rise
to a lateral root primordium, while cytokinin inhibits lateral root formation and mimics Nod factors by
activating inner root cortical cells to form a nodule primordia [315].
Other phytohormones such as abscisic acid (ABA) and gibberellic acid (GA) have been implicated
in cell division control in certain plant systems [316–320]. In deepwater rice, GA induces growth and part
of this growth is found to be due to stimulation of cell division [316]. GA induces cell division in the in-
tercalary meristem of rice internodes in cells that are arrested at G 2 [165]. ABA is implicated in inhibit-
ing cell division in endosperm of cultured maize kernels, maize root tips, pea buds, and in pollen mother
cells [317–320]. ABA was shown to induce ICK1 (a putative Cdk inhibitor) in Arabidopsis, which re-
sulted in a reduction of Cdc2-like H1 kinase activity [237].
VII. SYNCHRONIZATION OF PLANT CELLS
Synchronized cell populations are essential to study biochemical and molecular events that take place dur-
ing different phases of the cell cycle. Much of the information about cell cycle regulatory proteins in an-
imals was obtained by studying the level or activity of a given protein during different phases of the cell
cycle. Cells in meristems of plants have different cell cycle times and are highly asynchronous [5]. How-
ever, at a certain stage during the life cycle of a plant, cells divide synchronously for several cycles. For
instance, microspore mother cells in anthers progress through meiosis synchronously. The first few divi-
sions in the embryo and free nuclear divisions in endosperm are also synchronous. Natural synchrony,
which occurs rarely, was found to be not appropriate for biochemical studies for various reasons [321].
Hence, several methods have been developed to obtain synchronized populations of cells in plant tissues
and cultured cells. These methods include growing cultured cells after treating the cell with DNA syn-
thesis inhibitors (e.g., aphidicolin, hydroxyurea, 5-aminouracil and fluorodeoxyuridine) or growing cells
in some nutrient-limiting medium [321,322]. However, only a few methods have been found to be effec-
CELL CYCLE REGULATION IN PLANTS 249