starvation was reversed by inhibitors of the calcium-dependent protease calpain [298]. Conflicting data
from another study indicated that cyclin D1 was more likely degraded by the proteasome than calpain al-
though both could degrade it [299]. It was concluded that in human fibroblast cells the protease that me-
diates the progression from late G 1 to S phase is calpain, not the proteasome [300].
VI. PHYTOHORMONES AND CELL DIVISION
Phytohormones, especially auxins or cytokinins, have been shown to be intimately involved in cell divi-
sion control in plants [5]. In many plants these hormones, singly or in combination, induce cell division
in dedifferentiated noncycling cells. It has been well established from plant tissue culture studies that
auxin and cytokinins are necessary for inducing cell division. Also, apical meristems that contain the cy-
cling cells contain high levels of auxin. Addition of these hormones to differentiated cells that have ceased
to divide results in dedifferentiation and reentry of these cells into the cell cycle [301,302].
Using auxin-dependent tobacco suspension cultures, seven different auxin-inducible cDNA clones
have been isolated and characterized [303,304]. mRNA corresponding to these clones is rapidly induced
when quiescent cells are triggered to undergo cell division by an exogenous supply of auxin. Takahashi
et al. [301,305] isolated two auxin-induced cDNAs named parAandparB(protoplast auxin regulated)
from tobacco mesophyll protoplasts. Addition of auxins and cytokinins can induce cell division in to-
bacco mesophyll protoplasts, which are differentiated cells that have ceased to divide. Expression of par
genes was not detected in differentiated cells, whereas they are expressed in protoplasts that are cultured
in the presence of auxin. Both parAandparBgenes are expressed during the transition from G 0 to S phase
of in vitro cultured protoplasts [301,305]. Furthermore, the expression of pargenes was observed prior to
initiation of DNA synthesis. parBhas been identified as glutathione S-transferase [305]. Although this
enzyme is mostly known to be involved in detoxification of xenobiotics, studies have indicated its in-
volvement in cell proliferation [269,306,307]. The role of the parB-coded enzyme in tobacco mesophyll
protoplasts is not yet known. It is somewhat intriguing that none of the auxin-regulated genes are similar
to genes implicated in cell division, nor do cell cycle phase specific genes show any homology to known
key cell cycle regulatory genes [301,303,305,308].
Two genes responsible for auxin mutants have been identified as genes that are involved in auxin sig-
nal transduction and are homologous to enzymes known to be involved in regulating the stability of key
cell cycle regulatory proteins such as the cyclin-dependent kinase inhibitor SIC1p [309]. The gene prod-
ucts AXR1 and TIR1 are homologous to proteins involved in the ubiquitination of SIC1p, targeting it for
destruction and thereby the release of Cdk inhibition. Auxin has also been shown to induce a mitogen-ac-
tivated protein kinase in Petunia, PMEK1 [310].
In studies using p34 protein kinase cDNAs and antibodies, it has been shown that auxin induces p34
protein kinase mRNA and protein [111,122,123,134,311]. However, it should be noted that the auxin ef-
fect on p34 protein kinase mRNA and protein was studied after a long time following the auxin treatment
(the earliest time point is 1 day), whereas most of the auxin-regulated cDNAs that are implicated in cell
division [301,304,305] have been isolated from the libraries that are made after several hours of auxin
treatment. This and other factors such as posttranscriptional regulation and abundance of mRNA corre-
sponding to known key cell regulatory proteins in relation to other auxin-regulated genes could account
for the absence of known key cell cycle regulatory genes in the pool of auxin-induced cDNAs. In a few
plant systems that have been tested, addition of auxin has been shown to induce the expression of p34 pro-
tein kinase at both the mRNA and protein level [98,111,122,123]. A severalfold increase, in p34 protein
kinase was observed during auxin-induced cell division in carrot cotyledons [122]. In addition, a soybean
p34 kinase highly expressed in roots (cdc2-S6, Table 4) is up-regulated by Rhizobiuminfection, which
leads to cell division and nodulation in alfalfa [111].
The expression of mitotic-like cyclins Arath;CycA;2;1&2 and Arath;CycB2;1&2 was greatly de-
creased if auxin was withdrawn from cell cultures, but no effect was shown on the expression of
Arath;CycB1;1 [151]. In situ hybridization studies of Arath;CycB1;1 showed that the increase in expres-
sion was due to stimulation of cell division by the addition of hormone but not directly by the hormone
itself [171].
D-type cyclins in plants would be expected to respond to growth factors such as hormones. To in-
vestigate this, auxin, cytokinin, and sucrose were withdrawn from Arabidopsiscell suspension cultures
248 REDDY AND DAY