pressed from late S to G 2 phase, suggesting a B- and A-type function for these cyclins, respectively [167].
A third soybean cyclin gene’s (Glyma;CycA3;1) expression was limited to S phase, suggesting that it is
a novel class of plant cyclin which correlates to its A3 group classification [167]. Human mitotic cyclin
B antibodies recognized two proteins in synchronized Allium cepaL. root meristem cells that were ex-
pressed during G 2 with a maximum at late G 2 to early M phase, and degraded in the late hours of mitosis,
suggesting a cyclin B–like pattern of expression [172].
Expression of the cyclins has also been studied as a function of the cell cycle using cell cycle in-
hibitors to halt the cells at a particular stage and then either testing their mRNA expression or letting them
divide synchronously following inhibition. Various plant cyclins have been correlated with specific cell
cycle stages. In alfalfa, Medsa;CycB2;1 and Medsa;CycB2;2 mRNA showed maximal expression during
G 2 and M phase with Medsa;CycB2;1 appearing earlier than Medsa;CycB2;2 in G 2 [148]. Expression of
Medsa;CycB2;1 and Medsa;CycB2;2 was also correlated with the growth phase of cell suspension cul-
tures, being expressed during the logarithmic stage but not the stationary phase [148]. The Arabidopsis
cyclin Arath;CycB1;1 was expressed in higher amounts in G 2 nuclei than in G 0 -G 1 nuclei separated by
flow cytometry [124]. Arath;CycB1;1 expression was decreased in roots treated with the cell cycle in-
hibitor hydroxyurea, which holds cells at G 1 /S, indicating that its expression is during G 2 /M as found in
nuclei from G 2 cells. The Arath;CycB2;1 and Arath;CycB2;2 messages were identified in S to metaphase,
and the Arath;CycA2;1 and Arath;CycA2;2 messages were identified in late G 1 to metaphase [151]. Pro-
moter analysis of Arath;CycB1;1 showed an increase in the rate of transcription upon exit of the S phase,
a peak at the G 2 -to-M transition and during mitosis, and a decrease upon exit from the M phase; similar
analysis of Arath;A;2;1 showed low transcription during G 1 with a slow increase in S, a peak at the G 2
and G 2 -to-M transition, and down-regulation before early metaphase [173]. Tobacco cyclin mRNA for
Nicta;CycA1;1 and Nicta;CycA2;1 was detectable through S, G 2 , and M phases (A type–like expression),
and Nicta;CycB;2 was detectable from G 2 to M (B type–like expression) [169]. Reichheld et al. [174] iso-
lated five cDNA clones for A-type cyclins in tobacco that have multiple expression patterns through the
cell cycle, suggesting different roles for different cyclins. Promoter analysis of Nicsy;CycB1;1, which is
homologous to Nicta:B1;1, showed that the 1149-bp 5 -flanking region is sufficient to regulate expres-
sion in a cell cycle regulated manner [175]. The reporter gene exhibited the same pattern of a peak just
before mitosis and disappearance immediately after anaphase as did endogenous Nicsy;CycB1;1 and its
homologue Nicta;CycB1;1 [175,176]. These studies substantiate the classification of A- and B-type cy-
clins by sequence and indicate that cyclins in plants have roles similar to those of their counterparts in an-
imals. A few exceptions were also noted. Adica;CYCA1;1, while phylogenetically falling in the A-type
cyclins, was not expressed at the onset of S phase of the first cycle of germinating spores and became de-
tectable after S phase and accumulated during the second G 1 phase [147]. Also, Medsa;CycA2;1, al-
though mitotic-like in sequence, was found to be present in all cell cycle stages [149]. This suggests that
plants may have some different roles than their animal counterparts.
Promoter analysis of the Catharanthus roseusB-type cyclin Catro;CycB1;1 (CYM) showed that the
promoter could direct M phase–specific transcription of a -glucuronidase reporter gene [158]. Muta-
tional analysis of the promoter showed that a 9-bp element is essential for M phase-specific promoter ac-
tivity [177]. The promoter contained three similar elements, and when these elements were fused to a het-
erologous promoter, they were sufficient for M phase–specific expression. Similar elements (called
M-specific activators, MSAs) were found in other B-type cyclin promoters including Glyma;CycB1;3,
Nicta;CycB1;3 (Nt-CYM), Arath;CycB1;1, and Arath;CycB2;1. MSA-like sequences were also found in
two other M phase–specific tobacco kinesin-like proteins, suggesting that MSA may be a common cis-
acting promoter element that controls M phase–specific expression of cell cycle–related genes in plants
[177].
Cell cycle–dependent proteolysis of the mitotic cyclins has been demonstrated in yeast and animals
[42,47,89,90,178]. Plant ubiquitin/proteasome-mediated degradation of mitotic-like A- and B-type cy-
clins in tobacco was studied using the N-terminal domains of the cyclins containing the destruction box
motif [179]. Fusions of the domains to chloramphenicol acetyltransferase (CAT) reporter gene caused an
oscillation of the fusion proteins in a cell cycle–specific manner. Mutations in the destruction box abol-
ished cell cycle–specific proteolysis. Cyclin A-CAT proteolysis was turned off during S phase, whereas
cyclin B-CAT proteolysis was turned off during late G 2 phase. As further evidence, a known proteasome
inhibitor, MG132, blocked the cells during metaphase and the cyclin-CAT fusion proteins remained sta-
ble [179].
240 REDDY AND DAY