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Cell Cycle Control in Plants

A. S. N. Reddy

Colorado State Universir, Fort Collins. Colorado


Cell division is one of the fundaei processes of growth and development of plants and

animals. The time and place of cell division in an organism play a critical role in many

developmental processes. The development of a complex organism with a defined form and

structure requires tightly regulated cell growth and proliferation as well as transitions from

cycling state to quiescent state and vice versa. To duplicate the genetic material and produce

two daughter cells, the cell goes through a set of orderly events generally referred to as cell

cycle. The cell cycle consists of four distinct phases called gapi (01), synthetic phase (S), gap2

(G2), and mitosis (M). In the G phase cells prepare for S phase, during which DNA synthesis

takes place and the cell replicates its chromosomes [1,21. The completion of S phase leads into

another gap phase (02). Upon completion of 02, cells enter mitosis (M phase), where the

duplicated chromosomes segregate into two daughter cells [3]. However, it should be pointed

out that in some rare instances cycling cells have only two phases (M and S) without intervening

gap phases (Gi and G2). For example, the first 13 nuclear division cycles during Drosophila

embryo development do not have any gap phases (4]. Similarly, nuclear division cycles during

early endosperm development in plants seem to lack gap phases [5].

Normal proliferating cells in G can continue to cycle or revert to quiescent (03) state. The

decision to undergo another round of DNA synthesis and continue to cycle, or to exit the cell

cycle to enter into a quiescent state (Ga, is made during 01 phase [I]. Cells in G0 state either

terminally differentiate or can be activated to reenter into cell cycle. These switches in and out

of 01 are primarily controlled by extracellular factors such as hormones and other mitogens
[1]. However, once the cells have entered into S phase, the cell cycle events become independent

of extracellular factors, leading to mitosis and production of two daughter cells. These events

are mostly regulated by internal controls. Stringent control of decision points in the cell cycle

is vital for normal growth and development of organisms (1,4,6,7]. Deregulation of the

regulatory mechanisms that control decision points in the cell cycle results in uncontrolled cell


148 Reddy
division, leading to abnormal growth. The biochemical and molecular mechanisms that regulate the cell cycle are of
great interest, not only to help us understand how cells divide during normal growth and development of organisms
but also to get insight into abnormal growth processes such as cancer. Knowledge derived from cell cycle regulation
in plants should enhance our ability to manipulate growth and developmental processes in plants and could have
practical implications. For instance, regeneration of plants is very critical for crop improvement through genetic
engineering [8,9]. However, the ability to regenerate a whole plant from differentiated somatic tissues varies
considerably from species to species [10]. The induction of cell division in differentiated cells (GdG1 transition) is
the first critical step in the regeneration process. Hence, the analysis of cell cycle regulation and differentiated state
of regenerable and non regenerable (recalcitrant) tissues in terms of the cell cycle stage may provide some clues or
correlation between cell cycle phase and cells ability to divide [11,12].
In recent years, considerable advances with yeast and animal systems have led to our understanding of the control of
different phases of the cell cycle (1315]. The combination of genetic, biochemical, and molecular approaches has
resulted in the identification of decision points in the cell cycle and of key regulatory proteins that control
progression through the decision points of cell cycle. A number of excellent reviews describing the cell cycle
regulation in fungi [14,16,17], insects [4], and mammalian cells [13,15,18] have appeared. Cell cycle research in
plants is in its very early stages. However, research during the last 3 years shows that at least some of the key cell
cycle regulatory proteins arc structurally and functionally conserved among plants and other unicellular and
multicellular eukaryotes. The gob. here is to summarize what is known about cell cycle regulation in plants and
some of the unique aspects of the cell cycle in plants. Because our information about plant systems is limited, and
because there is considerable similarity in cell cycle regulation across phylogenetically divergent species, it is
necessary to present an overview of cell cycle regulation in fungi and animal systems.
Largely based on genetic analysis in yeast, the eukaryotic cell cycle is believed to be regulated at two major decision
points: a point late in G1 called START, which marks a cells commitment to DNA replication, and GM phase
transition [16]. Studies with fungi and animal systems indicate that both these transitions, as well as progression of
cells through the S phase, are controlled by protein kinases whose activity is regulated in a very complex manner L
A. Key Proteins Involved in GzM Phase Transition
1. p.34 Protein Kinase
Research in the past several years with yeast and mammalian systems using various approaches indicates that the
onset of M phase is regulated by a mechanism that is common to all eukaryotic cells [15]. Central to this mechanism
is a serine/threonine protein kinase, p34t2 (thereafter referred to as p34 protein kinase); in the active form, this
kinase acts as a mitotic trigger. The p34 protein kinase was identified genetically in the fission yeast
(Saccharomyces pombe) as the product of cell division cycle gene (cdc2) that encodes for a 34 Id) protein [1921].
Homologues of this gene have been found in budding yeast (p34C8) (2224], several vertebrates (2527],
invertebrates [28], and plants [2935] and are shown to be highly conserved both structurally and functionally
among all eukaryotes. Genes for p34 protein kinase from evolutionarily distant multicellular organisms, including
vertebrates and plants, have been shown to complement yeast mutants in this gene [25,3133]. Hence, it is
considered to be a universal regulator of mitosis in eukaryotic cells [14,15].
Cell Cycle Control lit Plants 149
2. Cyclists and Regulation of p34 Protein Kinase Activity
Genetic and biochemical studies indicate that the activity of the p34 kinase is required for entry into mitosis, and its
inactivation is required for exit from mitosis [13,15]. The level of p34 protein kinase is fairly constant during the cell
cycle of dividing cells in yeast [13,361. However, the activity of this kinase increases significantly prior to the onset
of M phase [1315]. Extensive studies from various laboratories indicate that the activity of p34 kinase is regulated
in a very complex manner by several positive and negative regulators. The p34 kinase by itself is inactive and is
activated by its association with certain members of a family of proteins called cyclins, which are accumulated and
degraded at very precise stages of the cell cycle [13]. Cyclins were first discovered in clams and sea urchins as a
class of proteins that accumulate to high levels in interphase and are abruptly destroyed at the end of M phase (37
39). Later it was found that the cyclins associate with p34 protein kinase and function as regulatory subunit of a
maturation-promoting factor (MPF) [4043]. First discovered in oocytcs as a factor responsible for GIM transition,
MPF activity was subseq uently found in all dividing cells (131.
Cyclins associate with p34 kinase to forTn active MPF. Cyclins that associate with p34 protein kinase during G2
phase are called mitotic cyclins and have been identified in a number of organisms ranging from yeast to man
including plants (39,4449). Recent studies indicate that each major decision point in thee Il cycle (START, G2/M,
and progression through S) is regulated by a distinct set of cyclins (50,511. The activity of MPF is also regulated by
phosphorylation and dephosphorylation of Thr14, Tyrl5, and Thr161 of p34 protein kinase [15,5256]. Two of
the phosphorylation sites (Thr14 and Tyrl5)are located in the ATP-binding region of the kinase [57). In yeast, p34
protein kinase is inactivated by phosphorylation of a tyrosine (Tyrl5) (53,54). The products of wee! and mikl genes
are implicated in phosphorylation of this residue (5861). Both these genes code for protein kinases and the
product of wee! (pl07t) has been shown to possess serine/tyrosine kinase activity in vitro (60,61).
During G2/M transition, a phosphotyrosine phosph4tase coded by a cdc25 gene product (p8Cfck2S) in fission yeast
and its homologues in other systems specifically dephosphorylates and activates MPF [6165]. In animal cells, the
p34 kinase is maintained in inactive state by phosphorylation of both tyrosine (Tyrl5) and threonine (Thr14)
residues. This phosphorylation is carried out by a protein kinase, probably a homologue ofweel kinase (56].
Dephosphorylation of Tyrl5 alone is not sufficient to activate inactive MPF in animal cells. It has been shown that
dephosphorylation of both residues is required for complete activation of p34 kinase and entry of cells into mitosis
[15]. These studies indicate that the dephosphorylation of both tyrosine and threonine residues is important in animal
cells. In animal cells, cdc25 is capable of both serine/threonine and tyrosine dephosphorylation (65]. The genes wed
and cdc25 in animal cells are unique in that they are capable of dual threonylltyrosyl phosphorylacion and
dephosphorylat ion, respectively [65]. Homologues of cdc25 have been identified from a number of eukaryotic
species and they are highly divergent outside catalytic domains [6670]. However, in spite of this divergence,
Drosophila and human cdc25 genes have been shown to rescue yeast cdc25 mutations [66,69,70]. A ccLc2S
homologue has not yet been reported from plants.
Both in yeast and in vertebrates, phosphorylation of a threonine (Thr167 in fission yeast or Thrl6l in Xenopus)
residue of p34 protein kinase seems to be involved in the stabilization of p34 protein kinase and cyclin subunits,
thereby activating the protein kinase complex [5456]. The regulation of the activity of MPF by phosphorylation
and dephospho?1ation events in animal cells is shown schematically in Figure 1. Other modes of regulation of p34
kinase include proteins (e.g., pl3 or p40) that bind to p34 kinase and inhibit its activity



Figure 1 Regulation of p34 protein kinase activity during G2IM transition in animal cells by protein phosphorylationl
dephOsphOrYlatiOfl of Thrl4. Tyrl4. and Thr161. Gene products that have a stimulatory effect on p34 kiriase are indicated by
pointed arrows; those that have an inhibitory effect arc shown by blunt arrows. PP2A is protein phosphatase 2A; for other details,
see text.
3. Mode of Action of p34 Kirzase in GM Transition arid Exit from M Phase
A number of changes take place in the cell as it proceeds from G2to M phase (3,6,73]. These
include chromosome condensation, nuclear envelope breakdown, reorganization of microtubules
into a mitotic spindle and transient inhibition of membrane traffic RNA and protein synthesis. in
addition, the M phase is characterized by the appearance of newly phosphorylated proteins (14,74]. At the time of exit from M
phase, these changes are reversed as the cell returns to
interphase. Numerous studies indicate that in all eukaryotes, entry into M phase involves the activation of p34 protein kinase and
exit from mitosis involves inactivation of p34 protein kinase [13,15]. As discussed above, the activation of p34 protein kinase
prior to GM transition is very complex, involving cyclins and phosphorylation and dephosphorylation events. Once the p34
protein kinase has been activated, it initiates various events of M phase such as disassembly of nucleus, cytoskeletal
reorganization. and chromosome condensation. Understanding the mechan ism(s) by which these M-phase events are initiated
requires the identification of physiological substrates of p34 kinase and the role of these phosphorylated substrates in bringing
about M-phase events. Although only a few proteins have been identified as in vivo substrates of p34 protein kinase [7578], a
number of substrates have been shown to be phosphorylated in vitro by p34 protein kinase (Table 1) (79100]. At least some of
the substrates listed in Table 1 (e.g., lamins, histone , nucleolin, pp60C, GTP-binding proteins, caldesmon) are among the
physiological substrates of p34 protein kinase (79,81,82,93]. it has been shown that the nuclear disassembly during mitosis
involves the phosphorylation of the larnin component of the nuclear envelope by p34 protein kinase [101,102]. Destruction of
cyclins in M phase inactivates p34 protein kinase and is required for the transition from mitosis to interphase 11315]. Sudden
destruction of cyclins just prior to anaphase is mediated by the ubiquitin pathway of protein degradation (103105]. in the
amino-terminal region, mitotic cyclins have a conserved stretch of amino acids (RXXLXXIX.N followed by a lysine-rich stretch)
called the destruction box. In addition to inactivation of p34 protein kinase, reentry into the interphase requires
dephosphorylation of proteins involving protein phosphatase action. Protein phosphatases that are required in late mitosis have
been identified in yeast (defective in sister chromatid disjoining, or dLcI, and bypass of wee suppression, or bwsl) and
Aspergillus (blocked in mitosis, or bimG) (106].


Cell Cycle Control in Plants 151

Table I Substrates for p34 Kinase

Substrate Nature of the protein Possible function in M phase Ref.

Nuclear lamins Cytoskeletal protein Nuclear lamin disassembly 79,80

Caldesmon Cytoskeletal protein Microfilanient contraction 81

Vimentin Cytoskeletal protein Intermediate filament disassembly 82

Neuro filament H Cytoskeleta] protein Unknown 83

Myosin regulatory Cytoskelecat protein Contractile ring activation 84

light chain

Histone Hi Chromalin-associate protein Chromosome condensation 85

HMG I, Y, Pt Chromatin-associate protein Chromosome condensation 86

N038. nucleolin Chromatin-associate protein Nuclear disassembly 75

SW 15 Transcription factor Chromosome condensation 87

c-myb Transcription factor Chromosome condensation 88

csrc Protein kinase Cytoskeletal rearrangements 89

pl50abl Protein kinase Unknown 90

Casein kinase LI (aJl3) Protein kinase Chromosome condensation 91

RNA polymerase LI Transcription enzyme Transcription inhibition 92

RablfRab4 GTP-binding proteins Inhibition of endomembrane traffic 93

p58 lamin B-receptor Nuclear membrane protein Dissociation of laniins 94

EF-ly Elongation factor Translation inhibition 95

SV4O large T antigen DNA binding protein DNA replication 96

DNA polymerase a Replication enzyme DNA replication 97

pRb polymerases Retinoblastoma gene product Unknown 98

Cyclin B Subunit of p34 kinase Regulates p34 protein kinase 99

Cdc25 Protein phosphacase Activates 34 protein kinase 100

B. M-Phase Regulatoty Proteins in Plants

Cell cycle research in plants at the biochemical and molecular level started very recently and is greatly benefiting
from the tools arid information obtained with fungal and animal systems. The obvious first step was to find out
which of the known cell cycle regulatory components are conserved in plants. Research itt the 1990s has yielded
some information indicating that at least some of the key cell cycle regulatory proteins (e.g., p34 protein kinase and
cyclins, mitogen-activated protein kinase) are present and highly conserved, whereas the presence of various other
proteins is yet to be explored. The availability of these genes will help in studying the detailed regulation of various
components involved in cell cycle. The presence of the p34 protein kinase homologue in a number of plants was
first reported by John et at. [29], using antipeptide antibodies made to a stretch of amino acids that is highly
conserved in all yeast and animal p34 protein kinases and the antibodies raised against p34 protein kinase from
yeast. A 34 Ic]) protein was detected with these antibodies in different plant systems. In Chlwr.ydomonas, it has
been shown that the phosphorylation of p34 protein increases at the time the cells commit themselves to cell division
[29]. The highly conserved nature of cell cycle regulatory proteins is enabling plant scientists to isolate plant
counterparts. Primers made to conserved regions of key cell cycle regulatory proteins have helped in isolating partial
sequences of p34 protein kinase by the polymerase chain reaction (PCR) [3035,48]. Screening of libraries with
such PCR generated probes resulted in isolation of full-length p34 protein kinase complementary DNA clones from
Arabidopsis [32,34], rice [107,108], maize [31], soybean [35], and mothbean [109]. The isolated cDNAs showed
extensive homology with yeast and animal p34 protein kinases (6166% sequence identity at the amino acid level),
indicating that the plant p34 protein kinase is structurally similar to other eukaryotic p34 protein kinases (Figure 2).
It has been

152 Reddy
shown that p34 protein kinase cDNAs from Arabidopsis, corn, alfalfa, and soybean complement yeast cdc2ICDC28 mutation (31,33,35].
These cross-species complementation studies indicate a high degree of functional conservation between plant and yeast p34 protein kinase.
Currently very little is known about the regulation of the p34 protein kinase activity in plants. However, the amino acids that are known to be
involved in the regulation of p34 protein kinase activity at the posttranslational level (viz., Thrl4, Tyrl5, and Thrl6l) are present in all known plant
V ____________________
mbea:cdc2 :1 -E R
rnzekinaa :1 -E L.--A N.
ricecdc2 :1 -EE R H.
yspcdc2 :1 -EN-Q H-LSGRIV-H D-S VNd
hscdc2 :1 -ED-T-! G-Il-T-GQVV-M S-E LR.
mbeancdc2 . . . -R---R S-E-V-. . -PRQV-HF C
mzekinaa -G---R-H I F---C-E-A-. . .NPTL--S H ricecdc2 : .. . -G---R-H--I I F---C-E-A-. . NPTL--S
yspcdc2 ennR--C-R-L-IL-A-SK F--H----Y--RISETGAts1-PRLVQKFT--LVN
hscdc2 . . . -P---S----I2IQDS----I--F-SM----YL--IPGQYm. . -SSL,V-S Q
mzekinaa -v A
ricecdc2 -v A
yspcdc2 : -VNF---R-II K- . S--V-L-NY---I
hscdc2 --VF---R DKGT.I 1-VY
nibeancdc2 : R V VNRR E
mzeP.jnaa ----ARQ V VN E -L---N-QS-P--SC
ricecdc2 : RQ H VN E VL---N-QS-P--S spcdc2 : V----R----G RRS EI----QVL---N-EV-P---L,
h.scdc2 V----AR I-T----LAT----H R---AL---NNEV-PE-E t hp3 4 cdc2
mb.ancdc2 : ---F--T----P-K--A-V AA-LN---S--CL--S---T--X-V IKFV
mzekjnaa -- -F-I-- -R-QAQ--A-V A-!.. RYE-S---T--Q EVV
ricecdc2 QAQ--A-I--T---A-L RYE-N---T--Q
yspcdc2 : -Q----T--R--RH--HKv---GEE-AIE---A--Vy--AN--S-KR--QQN-LR-FH..
hscdc2 : -Q---NT GS-ASH-K---EN-i, IY--A---SGKN--N-P--N--LQ
athp3dcdc2 P... 294
mbeancdc2 - 294
mzekinaa : Q 294
ricecdc2 : Q 294
yspcdc2 297
hscdc2 : IKKM 297
Figure 2 Amino acid sequence comparison of p3.4 protein kinase homologues from plants with yeast and human p34 protein kinase.
Dashes indicate aligned identical amino acids, uppercase letters denote aligned nonidentical amino acids, and lowercase letters indicate
unaligned amino acids. Gaps are denoted by dots. The solid line shows a highly conserved stretch of amino acids (PSTAIRE region),
characteristic of p34 protein kinases. Arrows indicate the amino acids (Thrl4, Tyrt5, and Thr161)that have been shown to be involved in
regulating the activity of p34 protein kinase by phhoiylation and dephosphorylation events in animal cells. The amino acid sequence of
Ara.bi4ospsis p34 (athp34cdc2, Ref. 32) is aligned with mothbean (mbeancdc2, Ref. 109). corn (m.zekinaa, Ref. 31), rice (ricecdc2. Ref. 108),
fission yeast (yspcdc2, Ref. 21). and human (hscdc2, Ref. 25) p34 protein kinases.

Cell Cycle Control in Plants 153

p34 protein kinases (see Figure 2), indicating that they could be involved in regulating the activity of plant p34
protein kinase by protein phosphorylationidephosphorylation. However. presence of the protein kinases (e.g.,
weel/miki) or protein phospharases (cdc25) that are responsible for these phosphorylationldephosphorylacion
events has not been shown in plants.
1. Expression of p.34 Protein Kinase
In yeast, the level of protein kinase remains constant throughout the cell cycle, although the activity peaks at G4M
transition (14,36). In animal systems, noncycling cells (terminally differentiated cells or senescent cells) do not
express p34 protein kinase in response to mitogen stimulation. Fluctuations in the level of the p34 protein kinase
messenger RNA and protein are observed in synchronized human cells [110, Ill). In other animal systems also p34
protein kinase mRNA level is correlated with the proliferative state of the cell [27 .281. In plants, the expression of
p34 protein kinase at the mRNA level is detected in all the tissues including differentiated tissues [31,321.
However, the highest level of expression is found in meristematic tissues such as apical meristem, and immature
leaf [31.112115). Furthermore, the expression of plant p34 protein kinase genes has been shown to be induced by
external factors that are known to induce cell division, such as phytohormones (112.114] and Rhizobium [351
infection. Two distinct p34 protein kinase genes from soybean showed differential expression pattern [351. One of
the genes (cdc2-S5) is highly expressed in roots, whereas the other one (cdc2-S6) is active in aerial parts.
Furthermore, infection of roots with Rhirnbium resulted in enhanced expression of cdc2-S5 but not cdc2-S6,
indicating that the two genes function in different developmental programs. Furthermore, these two genes differed in
their response to auxin. These results suggest that in a multicellular organism, cell division in different tissues may
be controlled by different p34 protein kiriases and may respond to different signals. By in situ hybridization studies,
Martinez et al. [1121 have shown that p34 protein kinase expression at the mRNA level in the root and shoot apex
parallels the pattern of mitotic activity. In addition, strong expression is observed in pericycle and perivascular
parenchyma cells that are competent to divide but are not actively dividing. When the level of p34 protein kinase
during cell differentiation in wheat leaf was studied using antibodies, it was found to be developmentally regulated
[1131. There is about 20 times less p34 protein kinase in the differentiated cells that have ceased to divide than in
cells in the meristematic region. In carrot, cessation of cell division during cotyledon development is shown to
correlate with a decrease (16 times less than dividing cells) in the level of p34 protein kinase (1141. Furthermore,
induction of cell division in explants from mature cotyledons by auxin resulted in a tenfold increase in the amount of
p34 protein kinase (114]. This and other studies [35,112,113) with plant systems also indicate that the relative level
of p34 kinase is important in determining whether a cell undergoes a division and its capacity to resume cell
In corn, two p34 protein kinase cDNAs (96% identical at the nucleotide sequence) have been isolated, and Southern
analysis with PCR-generated probes under high stringency conditions suggests more cdc2 genes [31J. Miao et al.
[35) also isolated two cDNA clones that code for p34 protein kinase from soybean. These two clones shared 90%
sequence identity in the coding region, whereas 5 and 3 untranslated sequences did not show significant sequence
Like yeast and mammalian p34 protein kinase. plant p34 kinase has affinity for pl3. In yeast, p13 modulates p34
kinase activity and is shown to be necessary for mitosis [15,71]. Using antibodies raised to yeast pl3, a plant
homologue of identical size has been detected in wheat, pea, and Chlamydomonas [115]. Unlike p34 protein kinase,
a high level of p13 has been found in differentiated cells of wheat.
2. Cydllns
Using yeast complementation and PCR approaches, several different types of cyclins have been isolated and
characterized from mammalian systems [50,51, 116,117). Of the different animal

154 Reddy
cyclins, the A, B, and E types share considerable similarity in the cyclin box, whereas other cyclins (C, D. and E) do not have
much homology among themselves or with the A. B. and E types (116,1171. Hata et al. [47] isolated plant cyclins from carrot
and soybean by screening the cDNA libraries with degenerate oligonucleotides corresponding to conserved regions of mitotic
cychns. The carrot cDNA and one of the soybean cDNAs were partial, whereas the other soybean clones contained the entire
coding region. The deduced amino acid sequence from the soybean clone is more similar to B-type cyclins, whereas carrot cyclin
resembles A-type cyclin. However, because of the divergent amino acid sequence of plant cyclins. these cyclins could not be
classified into any of the known cyclin sequences.
A full-length cyclin cDNA from Arabidopsis [48] and two partial cDNAs from alfalfa cycMsi and cycMs2, [49] have also
been isolated. Alfalfa cyclins were found to be expressed in dividing suspension-cultured cells but not in the cells in stationary
phase. Analysis of the expression of cycMsl and cycMs2 during different stages of the cell cycle showed maximal expression of
both these genes in the 02 and M phases. However, cycMsl transcripts appeared early in 02 whereas cycMs2 expression was
found only in late G2 and M phase cells (49]. Microinjection of synthetic mRNA from soybean and Arabidopsis into Xenopus
oocytes has been shown to induce maturation, indicating the functional conservation of mitotic cyclins between plants and
animals [47,48]. On the basis of the expression pattern of cyclAt and its homology with conserved regions of mitotic cyclins, it
was concluded that cyclAt is a mitotic yclin. We used primers designed to two stretches of conserved regions in A, B, and E type
cyclins to amplify corresponding cyclin sequences from Arabidopsis using PCR. Among the 25 clones sequenced, three
different cyclin sequences were detected. Southern analysis of genomic DNA from Arabidopsis with two of the PCR-generated
probes indicated five to six hybridizing bands under high stringency conditions. Amplification of cyclin sequences with these
primers using genomic DNA as a template yielded same-size product as cDNA, indicating the absence of an intron in this region
[118). Hence, each of the bands that is observed on Southern blots may represent a cyclin sequence, indicating the existence of
several cyclin sequences. Screening of a cDNA library prepared from flower meristem with a mixture of two of the PCR-
generated probes resulted in isolation of several eDNA clones. Restriction enzyme pattern and sequence analysis of 5 and 3
ends of these clones revealed the presence of four different cyclin sequences. indicating that there is a family of cyclins in
Arabidopsis (1181. In maize also, four distinct cyclins have been isolated (119].
Compared to recent progress made in understanding the regulation of the onset of M phase, little is known about the mechanisms
that control G1IS transition (16,50,51]. In yeast, the same protein kinase (p34218) is responsible for regulating START, a point in
G at which a cell commits itself to DNA synthesis. The G cyclins (CLN genes) interact with 34cdc2lCDC28 to drive the cell through
this G1 restriction point (START) to enter S phase 116]. In multicellular organisms, however, progression through and S phases
seems to be much more complex and to be controlled by a family of protein kinases that are structurally related to p34 kinase
(50,51 .120123]. These protein kinases, like p34 protein kinase, require specific cyclins to become activated; hence they are
designated as cyclin-dependent protein kinases (cdks) (50,51] and are numbered in the order of their discovery. Several new cdks
(e.g., cdk2, cdk4, cdk5the p34 cdc2 is sometimes referred to as cdki) that are involved in cell cycle regulation have been
identified in animal systems (120123]. Each of the cdks seems to associate with a specific type of cyclin to be activated and to
be involved in a specific phase of the cell cycle [50,51.116.122,124,125] (Table 2). Using different approaches such as yeast

Cell Cycle Control in Plants


Table 2 p34 Protein Kinase Family Members and Their Cyclin Partners in Animal Systems

Cyclin partner for cdk3 has not been idernified. However, it was called cdiJ because of its ability to complement CDC28 in yeast.

and polymerase chain reaction, several different types of cyclins and cdks have been identified. There are seven
cyclin types: A. B, C, D. E. F and G. Of these, types A, B, C, D, and E are implicated in regulating different control
points in the cell cycle (Figure 3), whereas the functions of F and 0 arc unknown (50J. About 10 different protein
kinases that are related to the cdc2 family have been reported. Some of these associate with a specific ype of cyclin,
whereas cyclin partners for some of the p34-related protein kinases are not identified L1211281. Other cdc2-
related kinases with no known cyclin partners are named after the amino acid sequences

Figure 3 Schematic diagram of the cell cycle, indicating various cell cycle regulatory proteins and their involvement
during different phases of the cell cycle in mammalian systems. MAP kinase, mitogen-activ ated protein kinase; cdk,
cyclin-dependent protein kinasc.

Type of protein kinase Sequence in PSTALRE motif Cyclin partner Ref.

cdkl(cdc2) PSTAIRE B types 25

cdk2 PSTAIRE A. D. and E types 50. 122. 125

Cdk3a PSTAIRE Not known 123

cdk4 PV/ISTVRE D types 124

cdk5 PSSALRE D types 124

cdk PLSTIRE Dtype 50. 126






156 Reddy
in the PSTAIRE motif of cdc2. Known cdc2-related protein kinases and their cyclin partners are summarized in
Table 2.
in plants very little is known about progression through G phase and G 1/S transition. Recent studies indicate that
plants have several distinct p34-like protein kinases [129,130] and a family of cyclins (31,1181. Whether plants have
G1 -specific cyclins and their corresponding protein kinase partners remains to be seen. Two cdc2 homologues from
alfalfa (cdc2A and cdc2B) appear to regulate different phases of cell cycle. The cdc2A could complement only
G2IM, transition whereas cdc2B complemented G1IS function [130].
To study gene expression during the cell cycle in plants, Kodama ci al. 1131] analyzed phase-specific mRNAs and
polypeptides using synchronized cultures. The rate of synthesis of 13 different polypeptides was found to change
during the cell cycle. In addition, the levels of a small population of mRNAs (four) varied during the cell cycle,
suggesting that the majority of the polypeptides whose synthesis varied during the cell cycle could be due to
posttranscript ional and posttranslational regulation. By differential screening of a cDNA library prepared from the
cell cycle S phase, two genes (cycO2 and cycO7). which are preferentially expressed at the G1!S boundary, have
been isolated [1321. The deduced amino acid sequence of cycO2. which is 101 amino acids long, did not show
homology with the sequences in the databases. Neither the identity nor the function of these genes is known.
Using auxin-dependent tobacco suspension cultures, seven different auxin-inducible cDNA clones have been
isolated and characterized 1133.1341. Messenger RNA corresponding to these clones is rapidly induced when
quiescent cells are triggered to undergo cell division by exogenously supplied auxin. Takahashi er a!. (135.136]
isolated two auxin-induced cDNAs named parA and parB (protoplast auxin regulated) from tobacco mesophyll
protoplasts. Addition of auxins and cytokinins can induce cell division in tobacco mesophyll protoplasts, which are
differentiated cells that have ceased to divide. Expression of the par genes was not detected in differentiated cells,
whereas it is expressed in protoplasts cultured in the presence of auxin. Both parA and parB genes are expressed
during transition from G0 to S phase of in vitro cultured protoplasts 1135.136]. Furthermore, the expression of par
genes was observed prior to initiation of DNA synthesis. The gene parB has been identified as glutathione S-
transferase 1136]. Although this enzyme is mostly known to be involved in the detoxification of-xenobiotics, recent
studies implicate its involvement in cell proliferation 1137139). The role ofaparB-coded enzyme in tobacco
mesophyll protoplasts is not yet known.
Proliferating-cell nuclear antigen (PCNA)an acidic, nonhistone nuclear protein and an auxiliary protein of DNA
polymerase-6is shown to be present only in proliferating mammalian cells, not in nondividing cells [140]. A
gene that codes for a plant homologue of PCNA has been cloned in rice and Catharanthus (1411. Like its animal
counterpart, plant PCNA is preferentially expressed in proliferating cells and was not detectable in quiescent cells,
in synchronized population of cells, PCNA was highly expressed in S phase 1141).
it is somewhat inznguing tha none of the auxin-regulated genes implicated in cell division nor any cell cycle phase
specific genes showed any homology to known key cell cycle regulatory genes 1132,133,135.136]. in recent studies
using p34 protein kinase cDNAs and antibodies, it has been shown that auxin induces p34 protein kinase mRNA and
protein 135,112,113115]. However, it should be noted that the effect of auxin on p34 protein kinase mRNA and
protein was studied a long time after the auxin treatment (the earliest time point is one day), whereas most of the
auxin-regulated cDNAs that are implicated in cell division [133,135,136] 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 corresponding 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.

Cell Cycle Control lit Plants 157

In animal cells, entry of quiescent state (Go) into the cell cycle by mitogens or other external factors is accompanied
by rapid changes in the status of phosphorylacion of a number of cellular proteins. These protein phosphorylation
changes are believed to be involved in changes in gene expression that trigger cells into cell cycle or a new
developmental pathway. A key enzyme called mitogen-activated protein kinase (MAP kinase). which is involved in
mitocic stimulation in animals [142,143], has been cloned from plant systems [144.145]. In pea. MAP kinase is
expressed in dividing cells and quiescent cells; hence there is no correlation between the MAP kinase mRNA and
cell proliferation (1451. However, it is likely that the activity of this enzyme could be reguicd posttranslationally by
protein phosphorylation [144.146].
/ f
CIcium, a key intracellular messenger in both plants and animals, has been shown to regulate many different
processes in plants [147149). Calmodulin, a calcium-binding protein found in all eukaryoces, is one of the
primary mediators of calcium action (see Chapter 34. Calcium as a Messenger in Stress Signals Transduction, for
more information on calmodulin). For more than a decade, calcium and calmodulin have been implicated in
controlling cell proliferation in eukaryotic cells, including plant cells (147,150152). Calcium is essential for the
growth of all eukaryotic cells (153). It has been shown that cells require the presence of millimolar level of
extracellular calcium to proliferate (154,1551.
Progression of normal cells through the cell cycle is found to be associated with transient changes in intracellular
calcium concentration [150,151,156]. Neoplastic cells, which can proliferate in the absence of external calcium,
contain higher levels of intracellular calcium than normal cells (157). Manipulation of cytosolic calcium
concentration has been shown to affect cell cycle events (158160). By determining the level of intracellular
calcium during different stages of the cell cycle, it has been demonstrated that rapid and transient increases in
intracellular calcium occur at specific stages of the cell cycle in plant and animal cells [161 [64]. Calcium
transients are observed at G2/M transition, as the cells completed mitosis, and on both sides of the G1/S boundary
[151]. Mitotic events such as the breakdown of the nuclearenvelope. chromatjn condensation, and the onset of
anaphase have been correlated with a transient increase in intracellular calcium [160,161]. Furthermore, these
mitotic events could be induced prematurely by artificially elevating cytosolic calcium, whereas chelation of
intracellular calcium by calcium chelating agents blocked the nuclear envelope breakdown and the
metaphase/anaphase transition, suggesting that an increase in cytosolic calcium is required for these mitotic events
to take place [158160]. Blocking of intracellular calcium prior to the G1IS boundary results in inhibition of DNA
synthesis (151]. This finding suggests that calcium transients are critical for the progression of cells from G1 to S
phase of the cell cycle.
Studies with both plant and animal tissues have revealed higher levels of calmodulin in dividing cells than in
nondividing cells [147,151,165]. Increased levels of calmodulin mRNA, protein, and activity are observed in
meristematic tissues of the plants [147,166,167]. In vertebrates and lower eukaryotic cells, a twofold increase in the
intracellular calmodulin concentration is the G1/S boundary [168170]. Stimulation of quiescent cells
to reenter the proliferative state elevated the amount of calmodulin. Furthermore, transformed mammalian cell lines
have been shown to contain elevated levels of calmodulin [171 .1721. To study the effect of altered levels of
calmodulin on the cell cycle, Rasmussen and Means [173,174] manipulated the levels of calmodulin level by stably
transforming the mouse cell lines with vectors that constitutively or inducibly express either calmodulin sense or
antisense RNA. A transient increase in calmodulin resulted in acceleration of proliferation, while a decrease in
calmodulin caused a transient cell cycle arrest. Constitutive elevation of intracellular


calmodulin levels in these cells shortened G1, and, in turn, the cell cycle. Calcium and calmodulin level determinations during
different stages of the cell cycle and the data on the effect of elevated or reduced levels of calmodulin on the cell cycle indicate
that three specific points in the cell cycle (G,/S, G2/M, and metaphaselanapbase) are sensitive to calcium and calmodulin (Figure
4). Overexpression of calmodulin in Aspergillus niduIons increased growth rate by decreasing cell cycle time, whereas reduced
levels of calmodulin prevented entry into mitosis 11511.
Calcium and calmodulin have multiple functions and regulate a variety of processes, including some housekeeping functions
(147,165.175]. Hence, it has been argued that the observed effects of calcium and calmodulin manipulations on the cell cycle
may not affect specific control points but could be due to the requirement for calcium and calmodulin to carry out many
housekeeping functions. Recent studies with unicellular fungi (yeast and Aspergillus nidulans) that are amenable to genetic
manipulations indicate that calcium and calmodulin regulate specific decision points during the cell cycle 1151). However, the
mechanisms by which calcium and calmodulin control the cell cycle are beginning to be elucidated.
A. Mode of Calcium and Calmodulin Action in Regulating G2/M Transition
Repression of calmodulin synthesis, and thereby calmodulin levels, or reduced extracellular cakum in Aspergiltus cells, blocked
entry into mitosis 1176,177]. Under these conditions tyrosine dephosphorylation of p34 protein kinase, which is needed for its
activation, is blocked:
and the activity of NIMA protein kinase, a protein kinase required for G2IM transition in Aspergillus, is also reduced. The
effects of reduced calmodulin and calcium could be reversed by elevating their levels. The activation of p34 kinase and NIMA
protein kinase by calcium and calmodulin could be due to direct interaction of NIMA protein kinase and the enzyme responsible
for tyrosine dephosphorylation of p34 kinase with the calciumlcalmodulin complex. or it could be indirect, through proteins that
bind to this complex. The NIMA protein kinase and tyrosine phosphatase involved in p34 activation did not bind to
calciumlcalmodulin. and the activity of immunoprecipitated NIMA kinase was not affected by calcium and calmodulin.
Figure 4 The phases of the cell cycle that require calcium and calmodulin; arrows indicate the control points that are regulated by
calcium and calmodulin.

Cell Cycle Control in Plants 159
These results indicate that the activation of p34 and NIMA kinases could be mediated by the proteins that bind to the
calciuin/calmodulin complex. About 20 calmodulin-binding proteins have been identified in animal systems [178].
Some preliminary results suggest that a calmodulin-dependent protein kinase, a multifunctional enzyme that requires
calcium and calmodulin for its activation, could be a likely candidate in mediating the calciumcalmodulin effect
on NIMA protein kinase and NIMT (a cdc25 homologue) of Aspergillus [151]. The purified calmodulin-dependent
protein kinase has been shown to phosphorylate NIMA kinase and NIMT in vitro in a calciuni/calmodulin-
dependent manner. Furthermore, B-type cyclins that are known to associate with cdc25 proteins and regulate their
activity (70] have been found to act as substrates for calcium/calmodulin-dependent protein kinase in vitro [151].
However, the effect of such phosphorylation on the activity of these enzymes is not known.
Studies with plants indicate that there are a number of calmodulin-binding proteins in plants [1481, although in
many cases their identity and function are not known [1791. Studies using the antibodies raised to animal
calciumlcalmodulin-dependent protein kinase suggest that the plants contain a homologue of calciunvcalmodulin-
dependen protein kinase [1 [180]. A cDNA that encodes a calcium/calmodulin-dependent protein kinase has been
recently isolated from plants [181].
In addition to calciumlcalmodulin-dependent protein kinase, plants contain a unique calcium.regulated protein
kinase that requires calcium but not calmodulin. This enzyme.calc ium-dependenc and calmodulin-independent
protein kinase, also called calcium-dependent protein kinase (CDPK) [182,1 831appears to be present in all
plants. Whether any of the calcium, calciurrt/calmodulin-regulated protein kinases, and calmodulin-binding proteins
are involved in plant cell cycle regulation is not known.
B. CalciumlCalmodulin in Metaphase/Anaphase Transition
Several lines of evidence indicate that calcium and calmodulin are required for the metap hase/anaphase transition
[161164]. A transient increase in cytosolic free calcium at the onset of anaphase has been demonstrated. As
indicated earlier, one of the critical events during inetaphase/anaphase transition is inactivation of p34 kiriase due to
the degradation of cyclins, which according to recent studies involves calcium and calmodulin (151]. It has been
demonstrated that micromolar concentrations of calcium induce cyclin B degradation in metaphase-arrested
Xenopus egg extracts [184]. The addition of a synthetic peptide that binds to the calcium/calmodulin complex, prior
to raising calcium level in the extract, blocked cyclin degradation and inactivation of p34 kinase [1841. The
inhibition of cyclin degradation by micromolar concentrations of calcium with calciure/calmodulin-binding peptide
could be reversed by adding calmodulin, suggesting that the calcium action is mediated by calmodulin. Furthermore,
by using appropriate inhibitors, the involvement of calpain, a calcium-dependent protease. and protein kinase C was
eliminated. These results indicate that calcium and calrnodulin are involved in cyclin degradation in Xenopus eggs.
It is known that cyclins are degraded by ubiquitin-dependent proteolysis [1031. The targets for calcium and
calmodulin in cyclin degradation pathways remain to be identified. Also, further studies are needed to see whether
calcium-induced cyclin degradation is a universal phenomenon.
Phytohormones, especially auxins and cytokinins, have been shown to be intimately involved in the control of cell
division [185]. In many plants these hormones, singly or in combination, induce cell division in dedifferentiated
noncycling cells. It has been well established from plant

160 Reddy
tissue culture studies that auxin and cytokinin are necessary for inducing cell division. Also, apical meristems, which
contain the cycling 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 1186,1871. In a few plant
systems that have been tested, the addition of auxin has been shown to induce the expression of p34 protein
kinase, both at the mRJ4A and the protein level (35,112,115]. A severalfold increase in p34 protein kinase was
observed during auxin-induced cell division in carrot cotyledons 1115]. As described in Section III, suspension
cultures or protoplasts that require an exogenous supply of auxin for cell division have been used to isolate auxin-
regulated genes and eventually to understand the role of these genes in cell division. Several different auxin-
inducible cDNA clones have been isolated and characterized from auxin-dependent tobacco suspension cultures and
protoplasts that require auxin for cell division (133135].
Other phytohormones such as abscisic acid (ABA) and gibberelic acid (GA) have been implicated in cell division
control in certain plant systems 11881921. In deepwater rice, GA induces growth, and part of this growth is found
to be due to stimulation of cell division (188]. Abscisic acid is implicated in inhibiting cell division in endosperm of
cultured maize kernels, maize root tips, pea buds, and in pollen mother cells (189192].
Synchronized cell populations arc essential to study biochemical and molecular events that take place during
different phases of the cell cycle. Much of our information about cell cycle regulatory proteins in animals was
obtained by studying the level or activity of a given protein during different phases of the cell cycle. Cells in plant
meristems have different cell cycle times and are highly asynchronous (5]. However, 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 divisions in the embryo and free nuclear divisions in
endosperm are also synchronous. Natural synchrony, which occurs rarely, was found to be inappropriate for
biochemical studies for various reasons (193]. Hence, several methods have been developed to obtain synchronized
popultins of cells in plant tissues and cultured cells. These methods include growing of cultured cells in the
presence of DNA synthesis inhibitors (e.g., aphidicolin, hydroxyurea. 5-aminouracil, fluorodeoxyuridine) or in some
nutrient-limiting medium (193,1941. However, only a few methods have been found to be effective in inducing
synchronization in plant cells; in the majority of cases, either the methods were partially effective or the agents that
cause synchrony were found to have toxic effects on cell metabolism.
Among the DNA synthesis inhibitors, aphidicolin is the most effective in inducing synchronous growth in
suspension cultures, as well as in differentiated tissues. However, because of endogenous aphidicolin-inactivating
activity in plant cells, which varies among cell types and plants, the concentration of aphidicolin and length of the
incubation should be determined empirically in each case. Treatment of bells with aphidicolin, a mycotoxin that
specifically blocks nuclear DNA replication by inhibiting DNA polymerase a 1195). causes accumulation of cells at
the G1/S boundary of the cell cycle. The effect of this inhibitor is reversible; hence removal of aphidicolin from the
medium results in synchronous resumption of DNA synthesis. in several plant cells, aphidicolin is shown to arrest
about 8095% of cells in G1 that were found to move synchronously through the first round of mitosis after G 1!S
In suspension cultures of Catharanthus roseus, double phosphate starvation effectively induces synchrony 1193].
This system is already helping to identify some of the phase-specific

Cell Cycle Controi in Plants 161

changes in mRNA and proteins LI 31,1 32j, In suspension cultures of Datura, hydroxyurea, another inhibitor of
DNA synthesis, reversibly arrested the cells at the G1/S boundary [196]. So far, only a few cell culture systems have
been well characterized in terms of synchronization with either DNA synthesis inhibitors or nutrient-deficient media
Synchronization of plant cells with the methods above coupled with flow cytometry should greatly expedite the
progress in cell cycle research in plants [1971. During the last 10 years flow cytometry has been used increasingly in
analyzing plant cells. Protoplasts and isolated nuclei are amenable to flow cytometry. When protoplasts are used,
however, some modifications in methods and instrumentation are necessary because of their large (2075 iJ.m) size
and fragility [197,1981. Recent developments in the use of flow cytometry for plant protoplasts have opened new
avenues for the analysis of cell cycle regulatory proteins. Using multiparameter analysis, one could monitor the
levels of two or more desired proteins during different phases of the cell cycle (L99j.
Cell division is one of the primary determinants of development in multicellular eukaryotic organisms. The
regulatory mechanisms that determine various aspects of the cell cycle (e.g., which cells in an organism should
undergo c II division, the timing and the plane of cell division in these cells, which cells should remain quiescent
and reenter into cell cycle) play a critical role in such plant developmental processes as embryogenesis, seed
germination, and flowering. Hence investigating these regulatory mechanisms not only will help us to understand
cell cycle regulation but also will enable us to elucidate developmental programm ing in plains. Various
developmehtal processes that involve the cell cycle are unique to plants. Unlike animals, cell division in higher
plants is restricted to meristematic regions (shoot apical meristem, root apical meristem. and lateral meristem). The
primary meristems such as shoot and root apical meristems continuously divide and contribute to the production of
new organs and growth of the plants. Furthermore, shoot apical meristem can lose its indeterminate vegetative
growth to become determinate floral meristem. The transition from vegetative meristem to floral meristem involves
the shortening of the cell cycle time as well as the synchronization of the cell cycle [51.
In plants, during the course of normal development, quiescent cells become proliferative. For instance, lateral
meristems (pericycle and cambium), auxiliary buds, and cambium retain their ability to undergo cell, division and
enter into the cell cycle in response to developmental cues. The root apex in plants contains, in addition to dividing
cells, a group of cells called the quiescent center, which do not normally undergo cell division. However, if the root
meristem is damaged, cells in the quiescent center reenter the cell cycle and form new meristem. In addition, if cells
from the quiescent center are cultured in vitro in the presence of hormones, they can undergo cell division and
regenerate into whole plants. Pericycle cells retain the ability to divide and are responsible for the formation of
lateral roots at vascular poles. Analysis of p34 protein kinase mRNA in roots has shown high levels of p34 protein
kinase mRNA in meristem and in all pericyle cells, but not in the quiescent center. There is uniform expression of
p34 protein kinase mRNA in all the pericycle cells, although lateral roots are initiated only at the vascular poles
[1121. These results suggest that lateral root initiation opposite to vascular poles is controlled by a mechanism other
than p34 protein kinase transcription. Studies on cell cycle regulation in plants should help us understand the
mechanisms and factors that maintain the cells in root and shoot apical meristems in the proliferative state as well as
the factors that cause withdrawal of cells from the cell cycle in other parts of the plant. Cell cycle studies

162 Reddy
should also help in elucidating the mechanisms by which differentiated cells dedifferentiate to enter the cell cycle by external
factors such as hormones, wounding. and Rhizobwm infection.
Unlike animal cells, plant cells are unique in the sense that they are totipotent. in several plant systems, terminally differentiated,
nondividing somatic cells can dedifferentiate, divide, and regenerate into a whole plant. Reinitiation of cell division in
differentiated and nondividing cells is a central feature in plant regeneration. Cytokinesis, a process by which cytoplasm is
divided, is different in plants. In plant cells, cytokinesis is initiated by forming a phragmoplast (made of microtubules) between
daughter cells, which is followed by deposition of cell wall material.
Several major themes are emerging from investigations on cell cycle regulatory mechanisms that used different model systems
ranging from simple eukaryotes (yeast and Aspergillus) to complex metazoans including vertebrates, invertebrates, and plants.
First, it is increasingly evident that a few key proteins are critical in controlling the decision points in the cell cycle. and these key
proteins are highly conserved in all eukaryotesindicating the universality of these key components. Second, the activity of
certain protein kinases appears to play a key role in regulating the transition points between different phases of the cell cycle.
Third, the mode of ret, lation of these key proteins may vary across phylogenetically divergent species. Finally, the regulatory
mechanisms that control the cell cycle are far more complex in multicelluar organisms than in unicellular organisms.
Cell cycle research in plants is in its very early stages. Recent developments, and the tremendous progress made with fungi,
vertebrate, and invertebrate systems, and the highly conserved nature of some of the key cell cycle regulatory proteins, should
expedite progress in discovering similarities and differences in regulatory mechanisms among plants and other eukaryotic
organisms. Some of the key proteins, known to be involved in yeast and mammalian systems, such as p34 protein kinase, cyclins.
MAP kinases, PCNA, and calmodulin, have been identified in plants. Although the cell cycle is common to all eukaryotes, it is
controlled by different hormones or growth factors in plants and animals. Hence, although some key proteins arc highly
conserved across phylogenetically divergent species, it is likely that regulatory mechanisms differ between plants and animals.
Since cell division is so fundamental to growth and development, it is bound to be an exciting area of research in plant biology.
Recent advances in molecular and cell biology offer new approaches to the investigation of this very complex process.
Manipulation of cell cycle regulatory proteins in cultured cells and transgenic plants should provide more insights into cell cycle
regulation in plants as well as in plant development.
I thank irene Day and Dr. Farida Chamberlain for reading the manuscript and for their help in preparing figures. Work in my
laboratory is supported by Biomedical Research Support Grant, Plant Biotechnology Laboratory. Colorado Biotechnology
Research institute, Colorado RNA Center and Agricultural Experiment Station (Project no. 702).
1. A. B. Pardee, Science. 246: 603 (1989).
2. R. A. Laskey, M. P. Fairman. and J. J. Blow, Science, 246: 609-614 (1989).
3. 3. R. Mcintosh, and M. P. Koonce, Science. 246: 622 (1989).