Mitotic B-type cyclins are differentially regulated by
phytohormones and during yellow lupine nodule development
Joanna Jelen´ska
a, Joanna Deckert
b, Eva Kondorosi
c, Andrzej B. Legocki
a,*
aInstitute of Bioorganic Chemistry,Polish Academy of Sciences,Noskowskiego12/14,61-704Poznan´,Poland bLaboratory of Plant Ecophysiology,A.Mickiewicz Uni6ersity,Niepodlegl*os´ci14,61-713Poznan´,Poland
cInstitut des Sciences Ve´ge´tales,CNRS,A6enue de la Terrasse,91198Gif-sur-Y6ette,France
Received 27 May 1999; received in revised form 29 July 1999; accepted 29 July 1999
Abstract
The progression of cell cycle in eukaryotes is controlled by protein complexes composed of p34 protein kinase and cyclin subunits. Recently, we have described four B1 type mitotic cyclin in yellow lupine. The presence of several closely related cyclin genes within the same plant species raised the question about tissue specificity of respective cyclins or their different regulations by plant-specific signals. Therefore, we examined the expression pattern of four B1 cyclins in various lupine tissues, with special emphasis put on developing root nodules. We also studied the effect of phytohormones on the level of respective cyclin mRNAs. As expected, cyclin transcript accumulation was restricted to proliferating tissues. Detailed analysis by reverse transcription-PCR and using primers specific to each cyclin allow to establish that different genes are engaged in cell divisions of various meristematic tissues. All four genes were activated during nodule development, however, theCyc3 andCyc4 genes mostly at the early stages of nodulation, whereas the Cyc1 and Cyc2 genes within the mature nodule organs. The expression of cyclins was regulated differently by plant growth factors. Both auxin and cytokinin induced theCyc1 andCyc4 genes and their transcript level was also abundant within the root, shoot and floral meristems. The precise localisation of cyclin transcripts by in situ hybridisation revealed that lupine nodule meristem was active during the whole process of nodule development, even in old organs. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Cell cycle; Cyclins; Expression;Lupinus luteus
www.elsevier.com/locate/plantsci
1. Introduction
Plants, like other higher organisms, develop ow-ing to the processes of cellular division, cell expan-sion and differentiation. The formation of any new organ or tissue of defined structure and func-tion starts with cell proliferafunc-tion. In contrast to animals, plants grow during their whole lifetime. Their growth is observed mostly within the centers of dividing cells such as shoot and root apical meristems and within the cambium. Moreover, plant cells are totipotent and after differentiation they are capable to re-enter the cell cycle as a
result of developmental control or in response to plant-specific signals, such as phytohormones, or external factors like light, wounding, pathogenic or symbiotic interactions. In spite of differences in the development of higher plants and other eu-karyotes, the progression through the cell cycle and the key regulators of cell division are con-served throughout the evolution.
Cell cycle is controlled at G1/S and G2/M
tran-sition by complexes of cyclin-dependent kinases (CDKs) and their regulatory subunits: cyclins (re-viewed in: [1 – 4]). Different types of cyclins are expressed at specific stages and regulate transition through respective phases of cell cycle: mitotic
cyclins control the entry into mitosis (G2/M) and
G1 cyclins control the passage through G1 to S * Corresponding author.
E-mail address:[email protected] (A.B. Legocki)
phase. Plant cyclins have been recently classified based on their structure and putative function [5]. The nomenclature proposed by Renaudin et al. [5] is used throughout this paper.
Expression of cyclins is restricted to proliferat-ing tissues [6,7] and regulated at multiple levels of gene expression. Cyclin mRNA and proteins are both unstable molecules and are rapidly degraded at the end of the respective stages of the cell cycle [8]. Therefore, cyclins are good markers of divid-ing cells and are used in studies of plant develop-mental processes as well as in search of signaling pathways leading to the formation of new plant organs.
In our study, we are particularly interested in the regulation of cell-cycle genes during nodule development in lupine. Symbiotic soil bacteria of the Rhizobiaceae family induce the formation of nitrogen-fixing nodules on the roots of legume plants. Although many stages of this process were described in detail (reviewed in: [9 – 11]), the pre-cise chain of events leading to the appearance of a new organ still remains to be determined.
We have isolated four clones coding for putative
mitotic cyclins of B1 type from Lupinus luteus
nodule cDNA library [12]. Lupine cyclin genes have been named according to the proposed
nomenclature as Luplu;CycB1;1 (EMBL/
Gen-Bank accession number U24192), Luplu;CycB1;2
(U24193), Luplu;CycB1;3 (U24194),
Lu-plu;CycB1;4 (U44857) [12 – 14], but here they are
referred as Cyc1, Cyc2, Cyc3 and Cyc4 for
sim-plicity. The presence of a family of closely related cyclins, belonging to the same subgroup, may suggest their different function during plant devel-opmental processes or their various regulation by plant-specific factors, such as phytohormones or signals released by symbiotic bacteria. The multi-ple variants of the same type of plant cell cycle regulators may be necessary to assure flexible reac-tion to internal signals and variable environment conditions.
In this study we describe a detailed expression pattern of four cyclin genes in various tissues of lupine, with special emphasis put on developing root nodule and the effect caused by phytohor-mones. Cyclin expression is analyzed by Northern hybridization, reverse transcription-PCR with the
use of 3%-end primers unique to respective cyclin,
as well as by in situ hybridization.
2. Materials and methods
2.1. Plant material
The seeds of yellow lupine (Lupinus luteus cv.
Ventus) were surface sterilized and germinated for 2 days as described [15]. Seedlings were transferred to sterile plastic growth pouches [16] containing
mineral solution [17] and inoculated with
Bradyrhizobium sp. (Lupinus) strain USDA 3045
or treated with 1 mM phytohormone solutions:
IAA (indole-3-acetic acid), kinetin, GA3 (gibbere-llin A3) or ABA (abscisic acid). Roots, from which the apical and lateral root meristems had been removed, were collected 3 days after either hor-mones addition or symbiotic bacteria infection. For long term experiments, lupine seeds were
im-bibed for 24 h, inoculated withBradyrhizobiumsp.
and plants were growing in sterile perlite at 23°C
with 16 h day/8 h night photoperiod.
2.2. Isolation of RNA
Total RNA was isolated using guanidinum thio-cyanate [18] from various lupine organs, develop-ing nodule and roots treated with phytohormones.
2.3. Northern blot analysis
Each RNA preparation (20mg) was fractionated
on 1% agarose gel containing 2.2 M formaldehyde [19]. Relative loading was confirmed by subse-quent running of the samples and using UV fluorescence of ethidium bromide stain. The RNA was blotted onto nylon filters (Hybond-N, Amer-sham) using standard procedures [19] and
hy-bridized to lupine Cyc1 cDNA. The probe was
labeled with Random Primed DNA Labelling Kit
(Boehringer Mannheim) and [a32P]ATP.
Hy-bridization was carried out in the presence of 50% formamide, at 42°C for 48 h.
2.4. RT-PCR
A sample containing 2 mg of total RNA was
used for reverse transcription by M-MuLV reverse
transcriptase (Boehringer Mannheim) with (dT)15
reaction were as follows: 95°C, 5 min, {94°C for 30 s, 52°C for 30 s, 72°C for 40 s} 30 times and final extension at 72°C for 5 min. Preliminary studies showed that 20-cycle PCR amplification of cyclin transcripts followed by hybridization with radiolabeled probe gave comparable results as 30-cycle reaction and staining the products with ethidium bromide (data not shown). Primers spe-cific for four cyclins were designed for unique
regions at 3% end of respective cDNA clones:
Cyc1: 3%aF-AGCTTCATTTTCTTGATGGGT,
3%aR-GATTCGCCCAATATCATTCA,
(ex-pected PCR product is 148 bp);
Cyc2: 3%bF-GGCTCTAGAGTTTGAGGGGA,
3%bR-ACAACAATCATCAATAATGCCA (261
bp);
Cyc3: 3%cF-TCCGGCACTAGAGTTTCAAA,
3%cR-CATTGACTTGAGTTGTCCTGG (216
bp);
Cyc4: 3%dF- AGGGGGAGGGATTGATTTA,
3%dR-ACAAAACAATAACTTCCACACATG
(177 bp).
Simultaneously, PCR amplification of a consti-tutively expressed gene: Gln-tRNA synthetase was performed as a control, using primers:
syntB-F-AAAGGAGTATAGGGAGAAGA,
syntF-R-CCGGAGAAGGTTGAGAA (986 bp) [20]. For each cyclin, the amplification of respective cDNA clone (in pBluescript vector) was used as a positive control and amplification of plant RNA, without reverse transcription, served as a negative control. All RT-PCRs were repeated twice or more, using two independent RNA preparations.
2.5. In situ hybridization
Uninfected roots and nodules at different stages of development, were fixed in 50% ethanol, 5% acetic acid, 10% formaldehyde and embedded in
Paraplast Plus (Sherwood Medical) at 60°C for
several days [13]. The 10-mm thick sections were
attached to Superfrost Plus slides (Menzel-Glaser). The procedure of in situ hybridization was essen-tially the same as the method developed by
McK-hann and Hirsch [21]. Digoxigenin-labeled
antisense and sense (negative control) RNA probes were synthesised using T7 (Boehringer Mannheim) or T3 (Stratagene) RNA polymerase.
The probes corresponding to EcoRI –HindIII
fragment of Cyc4 cDNA were used in all in situ
experiments. After hybridization, slides were
washed twice for 1 h in 2×SSC, once for 1 h in
1×SSC and 30 min in 0.5×SSC. The
digoxi-genin-labeled probe was detected with anti-digoxi-genin alkaline phosphatase antibody (Boehringer
Mannheim) diluted 1:200 and using NBT/BCIP
(Boehringer Mannheim) as a substrate. The reac-tion was terminated with TE buffer, then the slides were viewed and photographed with a bright field microscope. In control in situ hybridization, using the sense probe, the alkaline phosphatase activity was at the background level.
3. Results
3.1. Expression of cyclin genes in 6arious tissues
Cyclin expression in various tissues of 14-day-old infected plant was analyzed by Northern
hy-bridization and usingCyc1 cDNA as a probe (Fig.
1). The level of cyclin mRNA was much higher in both root and shoot apical meristems than in lateral root region. The cyclin transcript was visi-ble in a nodule-forming zone, whereas in expanded leaf, hypocotyl and root elongation zone, the cy-clin expression was not detected. This confirmed that cyclin expression was correlated with prolifer-ating tissues.
To follow the cyclin expression during early stages of nodulation, RNA was isolated from root segments located above lateral root zone, where lupine nodules usually appear. Both infected and control plants were analyzed on day 1, 2, 4, 8, 12
and 16 after inoculation with Bradyrhizobium sp.
(Lupinus) (Fig. 2). From the 4th day after inocula-tion, the cyclin level was significantly higher in the inoculated root than in uninfected plants. The highest cyclin expression was observed 12 – 16 days after infection.
Fig. 2. The expression of B-type cyclins during early stages of nodulation ofLupinus luteus. Total RNAs were isolated on day 1, 2, 4, 8, 12 and 16 from root segments of lupine uninoculated (A) or inoculated (B) withBradyrhizobiumsp. (Lupinus). The blotted RNA was hybridized to cyclinCyc1 cDNA probe.
3.2. Differential expression of four cyclin genes in lupine organs and in response to phytohormones
To gain further insight into the regulation of cyclin expression in plants, the pattern of relative transcript level of four previously identified lupine
B1 mitotic cyclin genes: Cyc1, Cyc2, Cyc3 and
Cyc4 [12 – 14] was established by RT-PCR. Re-verse transcription of RNA isolated from lupine shoot, floral and root meristems, leaf, stem, pods, root fragment located above lateral root zone, root segments with developing nodules on day 3,
6, 9, 13, 21 and 30 after inoculation with
Bradyrhi-zobium and root treated with auxin
(indole-3-acetic acid, IAA), cytokinin (kinetin-K),
gibberellin (GA3) or abscisic acid (ABA) was fol-lowed by PCR amplification of gene-specific se-quences (Fig. 3). Expression of four cyclin genes was monitored relative to transcript level of con-stitutive Gln-tRNA synthetase gene [20].
The Cyc1 expression appeared to be much
higher within the shoot (SM) and root meristems
(RM) than in floral meristem (FM). The Cyc1
transcript was undetectable in leaf (L), stem (S) and pod (P). During nodule development, the Cyc1 mRNA level increased and the maximum was observed between the 9th and 21st day after inoculation with symbiotic bacteria. The 3-day-long treatment of roots with phytohormones
re-sulted in highly elevated expression of Cyc1 in the
presence of either auxin (IAA) or cytokinin (K). The Cyc2 mRNA level was relatively high in flower and root meristems (FM and RM,
respec-tively). The expression ofCyc2 was very low both
in leaf (L) and stem (S) and undetectable in lupine
pod (P). However, infection with Bradyrhizobium
sp. (Lupinus) increased strongly the Cyc2 mRNA
level and was the highest at the 21st day after
inoculation. TheCyc2 mRNA level was also
stim-ulated by auxin (IAA).
The Cyc3 gene was expressed in all examined
tissues at relatively low level, except for nodules,
where a high amount of mRNA was observed.
The maximal level of the Cyc3 transcript was
detected already at 3 days after inoculation, then decreased with aging of the nodule. The level of
Cyc3 mRNA is stimulated by phytohormones
treatment and increased in the presence of auxin (IAA), cytokinin (K) and gibberellin (GA3).
The Cyc4 gene was mostly expressed in floral
meristem and within nodules. Similarly to Cyc3,
the transcript level of Cyc4 was highest during the
early stages of nodule development (3rd day after inoculation), but it was regulated differently by phytohormones. Only auxin (IAA) and kinetin (K)
strongly activated the Cyc4 gene expression.
Four closely related genes coding for mitotic cyclins are expressed at various level in different plant tissues. In every particular tissue more than one cyclin is active. The transcription of all of them is simulated during nodule development. The
Cyc3 and Cyc4 genes are activated during the
initial stages of nodulation, whereas the Cyc1 and
Cyc2 genes are activated at the later phases. The
Cyc3 gene may be regarded as nodule-specific one,
as in all the other tissues, including apical meris-tems, the amount of corresponding transcript was much lower than in the nodules. The analysis of cyclin expression after phytohormone treatment, proved that auxin increased the transcript level of all examined genes and abscisic acid did not ex-erted perceptible effect. None of the phytohor-mones decreased cyclin mRNA amount beneath the level observed in control plants treated with water.
3.3. Localization of cyclin transcripts by in situ hybridization
The expression of lupine mitotic cyclins was studied further by precise localization of tran-scripts by in situ hybridization and the use of
digoxigenin-labeled antisense Cyc4 RNA probe
seen in lupine nodules during hybridization, seems to be characteristic to these organs, as it is not observed in other tissues or other plant species. However, this disadvantage does not in-terfere with distinguishing the proper
hybridiza-tion signals. The in situ hybridizahybridiza-tion does not distinguish expression of various cyclin mRNAs, since the long probe, consisting of a sequence conserved in all lupine cyclins, was used in our study. The signal derived from the short probe is too weak to be distinguishable from the back-ground (data not shown). Moreover, the diffu-sion of the alkaline phosphatase substrate did not allow the cell-specific detection but could lo-calize the region where B-type cyclins are ex-pressed.
As expected, high cyclin expression was ob-served in numerous dividing cells (Fig. 4). Strong expression was detected in shoot meristem (Fig. 4B), emerging lateral root primordia (Fig. 4D), apical meristematic region of lateral roots (Fig. 4F), nodule primordia (Fig. 4H). Despite signifi-cant background observed in symbiotic cells, strongly stimulated expression of cyclin was de-tected within nodule meristem (Fig. 4J).
We particularly wished to visualize the spatial and temporal pattern of cyclin expression in de-veloping root nodule, as little is known about the activity of nodule meristem in lupine. By means of in situ hybridization cyclin transcripts were detected 2 – 3 days after inoculation with Bradyrhizobium sp. (Lupinus) within dividing cells of root cortex that form nodule primordium (Fig. 4H). Cyclin mRNA was localised in the central, globular part of young, 7-day-old nod-ules, as well as in their developing vascular bun-dles (Fig. 4J). Hybridization pattern showed that in later stages of nodule development, 12 days after infection, the nodule meristems were lo-cated distally and surrounded the cortex part of the organ (Fig. 4L). In mature nodules, 20 days after inoculation, the cyclin transcripts were still detected in a lateral meristematic zone, which enclosed the central bacteroid tissue (Fig. 4N). The regions expressing cyclin genes were contin-ually present even in older nodules, 30 days after infection, however, most meristematic cells lost their proliferating activity (Fig. 4P). The earlier morphological studies have shown that meris-tems are located in the lateral part of lupine root nodules [22]. In situ hybridization confirmed the previous microscope observations and proved that meristem regions are active from the first cell division till the late stages of lupine nodule development.
Fig. 3. Expression of four lupine cyclins, Cyc1, Cyc2, Cyc3 andCyc4, in different plant organs, during various stages of nodule formation and in roots treated with phytohormones. Two mg of total RNA from each tissue sample was used in
RT-PCR. First strand cDNAs were used to amplify Cyc1,
Cyc2, Cyc3, Cyc4 and Gln-tRNA synthetase gene-specific sequences as described in Section 2. The amplified products were separated on 2% agarose gel and stained with ethidium bromide. The products were obtained by RT-PCR from RNA isolated from: shoot meristem (SM), floral meristem (FM), root meristem (RM), leaf (L), stem (S), pod (P), uninoculated root fragments located above the lateral root region (R) and inoculated root fragments or nodules at 3, 6, 9, 13, 21 and 30 days after infection withBradyrhizobium sp. (Lupinus) (3, 6, 9, 13, 21 and 30), roots of 5-day-old seedlings with removed meristems, treated for 3 days with: auxin (IAA), cytokinin (K), gibberellin (GA3), abscisic acid (ABA),Bradyrhizobium
sp. (Lupinus) (B.l.), and water (control without phytohor-mones) (H2O). RNA without reverse transcription was a
4. Discussion
We have previously characterized cDNA clones corresponding to four different mitotic cyclins [12 – 14]. The comparison of amino acid sequences of lupine cyclins showed that three of them were very closely related to each other, whereas the Cyc1 gene was more distinct. Despite that, all four lupine cyclins belong to the same group of mitotic cyclin of type B1 [5]. The studies on expression pattern of respective lupine cyclins may help to explain the phenomenon of existence of several, very similar genes within the same plant species. Although numerous reports discuss cyclin expres-sion in various plant species [6,7,23 – 26], only lim-ited data are available on the expression pattern of cyclin genes of the same subgroup within one plant [27].
Similarly to other plants, accumulation of cyclin transcripts in lupine correlates with proliferation activity of tissue. The expression pattern of four genes in different plant organs revealed that mi-totic cyclins in lupine exerted tissue specificity. The Cyc1 gene is mostly induced within root and shoot
apical meristems, while theCyc4 gene is primarily
activated in flower bud. The two other genes: Cyc2 and Cyc3 do not appear to be specifically expressed at any of meristematic tissues. The genes coding for cyclins in other plants also tend to be expressed in a tissue or organ restricted manner [23,25].
The cyclin genes belong to plant genes that are induced by lipo-oligosaccharide signals released by symbiotic bacteria during formation of nitrogen-fixing nodule [28,29]. Increased level of cyclin ex-pression in emerging nodules was observed in
soybean [27], alfalfa [28,30], pea [29] and Sesbania
[31]. In lupine, the transcripts of mitotic cyclins were present in dividing cells of primary root cortex which subsequently formed nodule pri-mordium (Fig. 4H). During subsequent days after infection both cell proliferation and cyclin expres-sion took place in circular nodule cortex, which is
build up by infected cells and arising vascular bundles (Fig. 4J). When nodules were already visible on the root surface, the induction of cyclin genes was restricted to meristematic zone sur-rounding the kidney-shape bacteroid tissue (Fig. 4L). Within an old nodule, the active meristems were smaller than in expanding organ and some of them lost their proliferating activity (Fig. 4N).
Root nodule, which is an organ created de novo, represents a good model to analyze the processes of differentiation and development in plants. We were interested to know whether cell
divisions induced by bacteria of Bradyrhizobium
genus engage nodule-specific cyclins, or cyclins which are active during formation of any other organ. The results of detailed studies performed with an aid of RT-PCR technique suggested the presence of a nodule-specific cyclin gene. The
cy-clin Cyc3 may be considered as characteristic for
root nodule. Tissue or organ specificity should not be considered as the occurrence of gene products at one particular place but rather as preference of gene expression in a particular type of cells or tissue, without excluding its activation in other parts of organism.
The RT-PCR analysis showed that in cell divi-sions occurring at relatively early stages of nodule development mainly cyclins Cyc3 and Cyc4 partic-ipated, whereas Cyc1 and Cyc2 were predomi-nantly active within mature nodules. Such result
suggests that different kinds of regulatory
molecules were involved within young and older nodule tissue during the progression of cell cycle. The first cell divisions within the inner root cortex take place before the infection thread reaches this region and they are probably directly induced by bacterial Nod factor [28,29]. Moreover, Nod fac-tor causes cytoskeleton rearrangement in cells of inner cortex which then forms the pre-infection thread [32]. The formation of cytoplasmic bridges reminds some processes occurring during mitosis and was shown to be regulated by cell cycle genes
[29]. Expression of Cyc3 or/and Cyc4 is likely to
Fig. 4. (on pages34 – 35) Localization ofCyc4 cyclin transcripts by in situ hybridization in shoot and lateral root meristems and at different stages of nodule development. The paraffin-embedded sections (10 mm thick) were probed with digoxigenin-labeled
be stimulated specifically by lipo-oligosaccharide bacterial factors, either directly or via other regu-latory molecules. However, in these initial pro-cesses other cyclin genes may be involved, which are likely to be expressed in lupine root
immedi-ately after inoculation with Bradyrhizobium. In
our study we screened a library containing cDNA from 7-, 14- and 21-day-old nodules. In situ hy-bridization has shown that, after infection thread reached the nodule primordium, all cells of the bacteroid tissue divide intensively (Fig. 4J). We suggest that cyclins Cyc3 and Cyc4 may function in this process.
The formation of organized meristems, which surround a central bacteroid zone, was observed not earlier than about 10 days after infection (Fig. 4L). The expression pattern of four cyclins (Fig. 3) allows the conclusion that within lateral nodule meristems both Cyc1 and Cyc2 cyclins are active. Hence, cell division cycle regulated by these two proteins probably is not induced directly by Nod factor, but by other molecules connected with nodule developmental program. More precise studies with the use of purified
lipo-oligosaccha-ride molecules released by Bradyrhizobium may
determine if it is the case. It is possible that about 20 days after inoculation, the Cyc2 protein is
involved in any particular process asCyc2 mRNA
reaches the maximal level at that time. It appears that there are cyclins specific not only for nodules, but also for different types of dividing cells in nitrogen-fixing organs: primordium or bacteroid tissue (Cyc3 and Cyc4) and lateral meristems (Cyc1 and Cyc2). In old nodules, which have ceased to expand (over 30 days after infection), the expression of cyclins considerably decreases. The detection of cyclin transcripts by in situ hybridisa-tion during the whole development process of lupine collar-type nodule confirms its indetermi-nate character [22].
Phytohormones were shown to regulate many cell cycle genes (review: [33,34]). In our studies, we
observed high stimulation of Cyc1 and Cyc4 gene
expression in lupine roots treated with auxin
(IAA). In Arabidopsis this growth regulator
acti-vates Arath;CycB, 1 gene in root pericycle, which
leads to the formation of lateral root [7]. The
products of lupine genes Cyc1 and/or Cyc4 may
also participate in morphogenesis of lateral roots. The increase of cyclin B1 level is likely to be a direct effect of transcription activation of
corre-sponding gene (not a secondary effect of cell cycle induction by other means), as numerous auxin response elements (auxRE) were found in its
pro-moter in tobacco [35]. Lupine gene Cyc4 is
strongly induced by IAA and its promoter also contains as many as six auxRE sequences
(Jelen´-ska, data not published, EMBL/GenBank
acces-sion no AF126108). The regulatory region of gene
Cyc2, which is only slightly activated by auxin,
has no such elements (AF126106).
Cytokinin mostly induced the expression of
cy-clins Cyc1 and Cyc4 genes, the same which
re-sponse to auxin was the strongest. Interestingly, these are the genes that are activated in main plant meristems, where phytohormone concentration is the highest. However, simple conclusion could not be drawn as in spite of similar reaction to
respec-tive growth regulators, Cyc1 and Cyc4 are
in-duced in different meristems. The physiological effect depends on the phytohormone ratio rather than on their absolute concentration and various parts of a plant respond differently. The reaction of plants to respective growth regulators involves tissue or organ-specific factors and it is not ex-cluded that some of them are tissue-specific cell cycle genes.
Cytokinin is necessary to initiate mitosis and it is, at least in part, produced by the cell that is going to divide [34]. Cytokinins repress lateral root formation, but they may induce foci of dividing cells in the primary cortex [36]. Processes induced by these phytohormones resemble events that take
place after Rhizobium infection or treatment with
purified Nod factor. Moreover, roots produce pseudonodules in the presence of cytokinins [37,38] and nitrogen deficit makes the cells more sensitive to both morphogenes. It was suggested that cytokinin is a part of transduction pathway of a signal derived from symbiotic bacteria [36,39,40].
Lupine cyclin Cyc4 may be an element of this
that is going to divide [41]. In such case, the
recipient of bacterial signal could also be Cyc4
gene, which is strongly activated by auxin and contains many auxin response elements in its promoter.
Another growth regulator, gibberellin, slightly induced expression of one lupine cyclin gene.
Al-though gibberellin activates cyclin andcdc2 kinase
genes in rice internodes [42,43], it instead causes cell extension, and not proliferation. Abscisic acid, which is known to inhibit numerous genes, did not exert any effect on cyclin expression.
There is a lot of evidence that phytohormones do not determine plant developmental programs per se, but only ensure their proper execution. It still remains unknown which factors on a higher level of co-ordination regulate cell proliferation. Plants are not able to move and react to external stimuli, among others, by cell multiplication. Ex-tremely broad family of cell cycle controlling gene variants, that often play similar roles, ensures the required flexibility of plant development.
Acknowledgements
This work was partly supported by the Polish-French Biotechnology Centre (CNRS, KBN).
References
[1] T. Jacobs, Cell cycle control, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46 (1995) 317 – 339.
[2] R.W. King, P.K. Jackson, M.W. Kirschner, Mitosis in transition, Cell 79 (1994) 563 – 571.
[3] P. Nurse, Ordering S phase and M phase in the cell cycle, Cell 79 (1994) 547 – 550.
[4] J. Pines, Cyclins and cyclin dependent kinases: a bio-chemical view, Biochem. J. 308 (1995) 697 – 711. [5] J.P. Renaudin, J.H. Doonan, D. Freeman, J. Hashimoto,
H. Hirt, D. Inze´, T. Jacobs, H. Kouchi, P. Rouze´, M. Sauter, A. Savoure´, D.A. Sorrell, V. Sundaresan, A.H. Murray, Plant cyclins: a unified nomenclature for plant A-, B- and D-type cyclins based on sequence organisa-tion, Plant Mol. Biol. 32 (1996) 1003 – 1018.
[6] H. Hirt, K. Mink, M. Pfosser, L. Bogre, J. Gyorgyey, C. Jonak, A. Gartner, D. Dudits, E. Heberle-Bors, Alfalfa cyclins: differential expression during the cell cycle and in plant organs, Plant Cell 4 (1992) 1531 – 1538.
[7] P.C.G. Ferreira, A.S. Hemerly, J. de Almeida Engler, M. Van Montagu, G. Engler, D. Inze´, Developmental ex-pression of theArabidopsiscyclin genecyc1At, Plant Cell 6 (1994) 1763 – 1774.
[8] M. Glotzer, A.W. Murray, M.W. Kirschner, Cyclin is degraded by the ubiquitin pathway, Nature 349 (1991) 132 – 138.
[9] M. Schultze, A. Kondorosi, Regulation of symbiotic root nodule development, Annu. Rev. Genet. 32 (1998) 33 – 57.
[10] E.D. Schmidt, A.J. de Jong, S.C. de Vries, Signal molecules involved in plant embryogenesis, Plant Mol. Biol. 26 (1994) 1305 – 1313.
[11] H.P. Spaink, B.J. Lugtenberg, Role of rhizobial lipo-chitin oligosaccharide signal molecules in root nodule organogenesis, Plant Mol. Biol. 26 (1994) (1994) 1413 – 1422.
[12] J. Deckert, J. Jelen´ska, E.A. Gwo´z´dz´, A.B. Legocki, Isolation and classification of lupine cDNA clone coding for putative cyclin protein, Biochimie 78 (1996) 90 – 94. [13] J. Deckert, J. Jelen´ska, Z:. Zaborowska, A.B. Legocki,
The isolation of a family of cyclin gene homologues in
Lupinus luteus, Acta Biochim. Pol. 44 (1997) 37 – 42. [14] J. Jelen´ska, A.B. Legocki, Isolation of 5%ends of cDNAs
coding for yellow lupine mitotic cyclins (PGR98-158), Plant Physiol. 118 (1998) 330.
[15] T.J.W. Stokkermans, N.K. Peters, Bradyrhizobium elka
-nii lipo-oligosaccharide signals induce complete nodule structures onGlycine soja, Planta 194 (1994) 413 – 420. [16] G. Caetano-Anolles, P.M. Gresshoff, Efficiency of
nod-ule initiation and autoregulatory response in a su-pernodulating soybean mutant, Appl. Environ. Microbiol. 57 (1991) 2205 – 2210.
[17] A. Konieczny, K. Szczyglowski, L. Boron´, M. Przybyl-ska, A.B. Legocki, Expression of lupine nodulin genes during root nodule development, Plant Sci. 55 (1988) 145 – 149.
[18] J.M. Chirgwin, A.E. Przybyla, R.J. MacDonald, W.J. Rutter, Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry 18 (1979) 5294 – 5299.
[19] T. Maniatis, J. Sambrook, E.F. Fritsch, Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbour Laboratory Press, New York, 1989.
[20] M. Siatecka, M. Roz;ek, M. Ignacak, J. Barciszewski, Cloning and Sequencing of the First Plant GlnRS and GluRS Genes. Nucleic Acid Symposium, Series no. 33, Oxford University Press, Oxford, 1995, pp. 160 – 162. [21] H.I. McKhann, A.M. Hirsch, In Situ Localisation of
Specific mRNAs in Plant Tissues. Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, FL, 1993, pp. 179 – 205.
[22] W. Golinowski, J. Kopcin´ska, W. Borucki, The morpho-genesis of lupine root nodules during infection byRhizo
-bium lupini, Acta Soc. Bot. Pol. 54 (1987) 687 – 703. [23] I.S. Day, A.S.N. Reddy, Isolation and characterisation
of two cyclin-like cDNAs fromArabidopsis, Plant Mol. Biol. 36 (1998) 451 – 461.
[24] J.P. Renaudin, J. Colasanti, H. Rime, Z. Yuan, V. Sunderesan, Cloning four cyclins from maize indicates that higher plants have three structurally distinct groups of mitotic cyclins, Proc. Natl. Acad. Sci. USA 91 (1994) 7375 – 7379.
homo-logues in Brassica napus, Plant Mol. Biol. 27 (1995) 263 – 275.
[26] H. Kouchi, M. Sekine, S. Hata, Distinct classes of mi-totic cyclins are differentially expressed in the soybean shoot apex during the cell cycle, Plant Cell 7 (1995) 1143 – 1155.
[27] S. Hata, H. Kouchi, I. Suzuka, T. Ishi, Isolation and characterisation of cDNA clones for plant cyclins, EMBO J. 10 (1991) 2681 – 2688.
[28] A. Savoure´, Z. Magyar, M. Pierre, S. Brown, M. Schultze, D. Dudits, A. Kondorosi, E. Kondorosi, Acti-vation of cell cycle machinery and the isoflavonoid biosynthesis pathway by active Rhizobium meliloti Nod signal molecules in Medicago microcallus suspensions, EMBO J. 13 (1994) 1093 – 1102.
[29] W.C. Yang, C. de Blank, I. Meskiene, H. Hirt, J. Bakker, A. van Kammen, H. Franssen, T. Bisseling,
Rhizobium Nod factors reactivate the cell cycle during infection and nodule primordium formation, but the cycle is only completed in primordium formation, Plant Cell 6 (1994) 1415 – 1426.
[30] A. Savoure´, A. Fehe´r, P. Kalo´, G. Petrovics, G. Csana´di, J. Sze´csi, G. Kiss, S. Brown, A. Kondorosi, E. Kon-dorosi, Isolation of a full-length mitotic cyclin cDNA clone CycIIIMs from Medicago sati6a: chromosomal mapping and expression, Plant Mol. Biol. 27 (1995) 1059 – 1070.
[31] S. Goormachtig, M. Alves-Ferreira, M. Van Montagu, G. Engler, M. Holsters, Expression of cell cycle genes duringSesbania rostratastem nodule development, Mol. Plant-Microbe Interact. 10 (1997) 316 – 325.
[32] A.A.N. van Brussel, R. Bakhuizen, P.C. van Spronsen, H.P. Spaink, T. Tak, B.J.J. Lugtenberg, J.W. Kijne, Induction of preinfection thread structures in the legumi-nous host plant by mitogenic lipo-oligosaccharides of
Rhizobium, Science 257 (1992) 70 – 71.
[33] P. Ferreira, A. Hemerly, M. Van Montagu, D. Inze´, Control of cell proliferation during plant development, Plant Mol. Biol. 26 (1994) 1289 – 1303.
[34] P.C.I. John, The plant cell cycle: conserved and unique features in mitotic control, Prog. Cell Cycle Res. 2 (1996) 59 – 72.
[35] C. Tre´hin, I.O. Ahn, C. Perennes, F. Couteau, E. Lalanne, C. Bergounioux, Cloning of upstream se-quences responsible for cell cycle regulation of theNico
-tiana sil6estris CycB1;1 gene, Plant. Mol. Biol. 35 (1997) 667 – 672.
[36] P. Bauer, P. Ratet, M.D. Crespi, M. Schultze, A. Kon-dorosi, Nod factors and cytokinins induce similar corti-cal cell division, amyloplast deposition andMsEnod12A
expression patterns in alfalfa roots, Plant J. 10 (1996) 91 – 105.
[37] N. Arora, F. Skoog, O.N. Allen, Kinetin-induced pseudonodules on tobacco roots, Am. J. Bot. 46 (1959) 610 – 613.
[38] J.B. Cooper, S.R. Long, Morphogenetic rescue ofRhizo
-bium meliloti nodulation mutants by trans-zeatin secre-tion, Plant Cell 6 (1994) 215 – 225.
[39] C. Dehio, F.J. de Bruijn, The early nodulin gene
SrEnod2 from Sesbania rostrata is inducible by cy-tokinin, Plant J. 2 (1992) 117 – 128.
[40] A.M. Hirsh, Y. Fang, H.I. McKhann, K.L. Wycoff, B. Niner, S. Asad, P.T. Asmann, M. Jacobs, Nodule devel-opment, early nodulins, and plant growth regulators, in: Proceedings of the 1st European Nitrogen Fixation Con-ference, Szeged, Officina Press, 1994, pp. 116 – 121. [41] K.J.M. Boot, A.N. van Brussel, H.P. Spaink, J.W. Kijne,
Role of auxin in root nodule formation, in: Carel Wijffel-man, Andre´ Wijfjes and Ben Lugtenberg (Eds.), Ab-stracts of the Third European Nitrogen Fixation Conference, Leiden, The Netherlands, 1998, p. 24. [42] M. Sauter, S.L. Mekhedov, H. Kende, Gibberellin
pro-motes histone H1 kinase activity and the expression of
cdc2 and cyclin genes during the induction of rapid growth in deepwater rice internodes, Plant J 7 (1995) 623 – 632.
[43] M. Sauter, Differential expression of a CAK (cdc 2-acti-vating kinase)-like protein kinase, cyclins andcdc2 genes from rice during the cell cycle and in response to gibbere-llin, Plant J. 11 (1997) 181 – 190.
