in maize endosperm gives evidence that two types of Cdks are involved in cell cycle regulation [220].
H1K activity was associated with a protein fraction recovered from a p13suc1-agarose column known to
bind cdc2 kinases. Activity of this kinase correlated with early endosperm development when mitosis is
still taking place. Addition of extract from endosperm with low cdc2 H1K activity caused the inactiva-
tion of kinase from early stage endosperm, indicating the presence of an inhibitor in the extract [220]. A
second H1K activity in maize endosperm was associated with a maize cdc2-related protein precipitated
with GST (glutathione-S-transferase) fusions to E2F-1 and E1A. This suggests that different cyclin-de-
pendent kinase complexes are involved in cell cycle regulation in maize: an M phase kinase that can in-
teract with p13suc1and an S phase kinase that can interact with the human transcription factor E2F-1 and
the adenovirus E1A proteins. In alfalfa cell extracts, two temporally separable S-phase H1K peaks could
be resolved by precipitation with anti–human cyclin antibodies (early S peak) or p13suc1(later S peak)
[136].
A new group of plant cyclins have been identified in Arabidopsis[155], alfalfa [221], pea [222], and
tobacco [223]. In Arabidopsis, three cDNA clones (Arath;CycD1;1, Arath;CycD2;1, Arath;CycD3;1)
were identified that could complement G 1 cyclin function in the yeast mutants [155]. Sequence analysis
revealed that they did have cyclin characteristics but not the mitotic destruction box motif. Rather, like
animal G 1 cyclins, they have PEST sequences. When compared with databases, they showed the greatest
relatedness to mammalian D-type cyclins. Cell cycle inhibitor studies indicated that Arath;CycD3;1 is ex-
pressed prior to DNA synthesis and Arath;CycD1;1 and Arath;CycD2;1 are expressed in G 1. Using cD-
NAs in DNA hybridization studies, hybridizing bands were found in tobacco, Jerusalem artichoke,
cauliflower, and Antirrhinum[155]. In situ hybridization in alfalfa detected a D-type cyclin
(Medsa;CycD3;1) in meristem tissue in a subset of interphase cells [221]. Of particular note was the pres-
ence of a conserved motif, LXCXE, for Rb binding [224]. This domain, which is present in animal G 1 cy-
clins, was found in all three Arabidopsisclones and in the alfalfa Medsa;CycD3;1 clone but not in plant
mitotic-like cyclin sequences [155,221]. Three tobacco D-type cyclins isolated using cyclin D cDNA
probes also contain the LXCXE motif [223]. One of these, Nicta;CycD3;2, was induced in G 1 following
a stationary phase and then remained at a constant level in synchronous cells. The other two
(Nicta;CycD2;1 and Nicta;CycD3;1) accumulated during mitosis, which is not typical of D-type cyclins
and suggests a mitotic role for these cyclins. A fourth cyclin D (Arath;CycD4;1) was isolated from Ara-
bidopsisin a two-hybrid interaction screen using a Cdk, CDC2aAt, as a bait [156]. This was significant
as cyclin/Cdk pairs have not previously been identified in plants. In situ hybridization studies suggested
a role for this D-type cyclin in developmental processes [156]. Based on sequence similarity, a C-type cy-
clin was isolated from rice, but expression and functional analyses have not been performed [225].
Human Rb protein can bind in vitro to translated Arabidopsiscyclin D() [226] and Rb-related pro-
teins have been isolated in maize [227–230], tobacco [231], and Arabidopsis[232]. The maize Rb pro-
teins have been shown to bind to plant cyclin D and to be phosphorylatable by mammalian Rb-kinases
and seem to be involved in developmental processes [227–230]. The tobacco Rb-like protein was shown
to be phosphorylated in insect cells by a tobacco Cdc2-kinase/cyclin D complex [231]. Another player in
the cyclin D/Cdk, Rb regulation of the cell cycle has been isolated from a plant. Using the tobacco Rb
protein ZmRb1 in a yeast two-hybrid screen of a wheat cDNA library, an interacting clone was identified
that showed homology to E2F family members [233]. The plant E2F was shown to be expressed in pro-
liferating cultured cells and in differentiated tissues and was up-regulated early in S phase. These data
suggest that control of the G 1 /S transition in plants is similar to animal G 1 /S cell cycle control.
Proliferating cell nuclear antigen (PCNA), which associates with cyclin D/Cdk complexes in ani-
mals, has been isolated in rice and Catharanthusand peas [234,235]. Like its animal counterpart, plant
PCNA is also preferentially expressed in proliferating cells and was not detectable in quiescent cells
[234]. In a synchronized population of cells, PCNA expression was highly expressed in S phase. Shimizu
and Mori [235] found that PCNA associates with a pea cyclin D (Pissa;CycD3;1) during dormancy but
not in growing buds, suggesting a possible mechanism for arrest at G 1.
Inhibitors of the cell cycle are also being isolated in plants. A yeast two-hybrid screen with Ara-
bidopsisCdc2a as a bait identified a cyclin-dependent kinase inhibitor called ICK1 [236,237]. It contains
an important consensus sequence found in the mammalian Cdk inhibitor p27 (Kip1), but the rest of the
sequence shows little similarity to any known Cdk inhibitor. ICK1 was also identified in a two-hybrid
screen with the ArabidopsisCycD3 and was shown to be induced by abscisic acid, and along with its in-
duction there was a decrease in cdc2-like histone H1 kinase activity [236].
244 REDDY AND DAY