according to the current evidence, what is the major function of cytokinin in plants?
Proc Natl Acad Sci U S A. 2001 Aug 28; 98(eighteen): 10487–10492.
Plant Biology
Regulation of plant growth by cytokinin
Tomáš Werner
*Centre for Found Molecular Biology (ZMBP)/Allgemeine Genetik, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany; †Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 135, CZ-16502 Prague 6, Czech Republic; and ‡Palacký University and Institute of Experimental Botany ASCR, Section of Botany, Laboratory of Growth Regulators, lechtitelů 11, CZ-78371 Olomouc, Czech republic
Václav Motyka
*Centre for Plant Molecular Biology (ZMBP)/Allgemeine Genetik, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany; †Institute of Experimental Botany, Academy of Sciences of the Czech Commonwealth, Rozvojová 135, CZ-16502 Prague half dozen, Czech Republic; and ‡Palacký University and Institute of Experimental Phytology ASCR, Department of Botany, Laboratory of Growth Regulators, lechtitelů 11, CZ-78371 Olomouc, Czechia
Miroslav Strnad
*Centre for Plant Molecular Biological science (ZMBP)/Allgemeine Genetik, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany; †Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 135, CZ-16502 Prague 6, Czech Republic; and ‡Palacký Academy and Establish of Experimental Botany ASCR, Department of Botany, Laboratory of Growth Regulators, lechtitelů 11, CZ-78371 Olomouc, Czech Republic
Thomas Schmülling
*Centre for Plant Molecular Biology (ZMBP)/Allgemeine Genetik, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany; †Institute of Experimental Botany, Academy of Sciences of the Czechia, Rozvojová 135, CZ-16502 Prague 6, Czechia; and ‡Palacký University and Institute of Experimental Botany ASCR, Department of Phytology, Laboratory of Growth Regulators, lechtitelů eleven, CZ-78371 Olomouc, Czech republic
Received 2001 February 15; Accepted 2001 Jun 15.
Abstract
Cytokinins are a class of institute-specific hormones that play a fundamental role during the cell cycle and influence numerous developmental programs. Because of the lack of biosynthetic and signaling mutants, the regulatory roles of cytokinins are not well understood. We genetically engineered cytokinin oxidase expression in transgenic tobacco plants to reduce their endogenous cytokinin content. Cytokinin-deficient plants adult stunted shoots with smaller upmost meristems. The plastochrone was prolonged, and leaf cell production was merely 3–4% that of wild type, indicating an absolute requirement of cytokinins for leafage growth. In dissimilarity, root meristems of transgenic plants were enlarged and gave ascension to faster growing and more than branched roots. These results suggest that cytokinins are an of import regulatory factor of plant meristem activity and morphogenesis, with opposing roles in shoots and roots.
Cytokinins were discovered during the 1950s because of their ability to induce plant cell segmentation (i). Shortly after their discovery, Skoog and Miller coined the auxin–cytokinin hypothesis of institute morphogenesis (2). The hypothesis predicted that cytokinin, together with auxin, plays an essential function in plant morphogenesis, having a profound influence on the germination of roots and shoots and their relative growth.
Chemically, natural cytokinins are N6-substituted purine derivatives. Isopentenyladenine (iP), zeatin (Z), and dihydrozeatin (DZ) are the predominant cytokinins found in higher plants. The free bases and their ribosides (iPR, ZR, DZR) are thought to be the biologically agile compounds. Glycosidic conjugates play a role in cytokinin transport, protection from degradation, and reversible and irreversible inactivation (3).
Numerous reports accredit a stimulatory or inhibitory function to cytokinins in dissimilar developmental processes such as root growth and branching, control of apical dominance in the shoot, chloroplast evolution, and leaf senescence (four). Conclusions about the biological functions of cytokinins have mainly been derived from studies on the consequences of exogenous cytokinin awarding or endogenously enhanced cytokinin levels (five, 6). Upwards to now, information technology has not been possible to address the contrary question: what are the consequences for plant growth and development if the endogenous cytokinin concentration is decreased? Plants with a reduced cytokinin content are expected to yield more precise data about processes cytokinins limit and, therefore, might regulate. Dissimilar other plant hormones such as abscisic acid, gibberellins, and ethylene, no cytokinin biosynthetic mutants have been isolated (7).
The catabolic enzyme cytokinin oxidase (CKX, ref. viii) plays maybe the principal part in controlling cytokinin levels in institute tissues. CKX activity has been found in a smashing number of higher plants and in dissimilar plant tissues (8). The enzyme is a FAD-containing oxidoreductase that catalyzes the degradation of cytokinins begetting unsaturated isoprenoid side bondage. The complimentary bases, iP and Z, and their corresponding ribosides are the preferred substrates. The reaction products of iP catabolism are adenine and the unsaturated aldehyde 3-methyl-ii-butenal (viii). Recently, a cytokinin oxidase gene from Zea mays has been isolated (9, 10). The manipulation of CKX gene expression could partially overcome the lack of cytokinin biosynthetic mutants and might be used equally a powerful tool to report the relevance of iP- and Z-type cytokinins during the whole life wheel of higher plants. In this article, we report the cloning of four putative CKX genes from Arabidopsis thaliana and the results of their systemic overexpression in transgenic tobacco plants. Our data indicate an important office for cytokinins in plant growth regulation via a differential influence on the number and/or duration of cell sectionalisation cycles in the root and shoot meristems.
Materials and Methods
Gene Cloning.
The genomic sequences of the AtCKX1, AtCKX2, AtCKX3, and AtCKX4 genes were amplified by PCR from Dna of A. thaliana accession Col-0. Oligonucleotide primers were designed co-ordinate to the published genomic sequences of AtCKX genes [GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"AC002510","term_id":"20196946","term_text":"AC002510"}}AC002510 (AtCKX1), {"type":"entrez-nucleotide","attrs":{"text":"AC005917","term_id":"20197478","term_text":"AC005917"}}AC005917 (AtCKX2), {"type":"entrez-nucleotide","attrs":{"text":"AB024035","term_id":"4519194","term_text":"AB024035"}}AB024035 (AtCKX3), and {"type":"entrez-nucleotide","attrs":{"text":"AL079344","term_id":"5123543","term_text":"AL079344"}}AL079344 (AtCKX4)] and had 5′ and 3′ overhangs with SalI or KpnI restriction sites, which permitted subcloning in the vector pUC19. The length of the amplified sequences were 2,235 bp (AtCKX1), 3,104 bp (AtCKX2), three,397 bp (AtCKX3), and 2,890 bp (AtCKX4). Genes were sequenced and inserted into vector pBINHygTx under the transcriptional command of a constitutively expressed 35S promoter (11). The cDNA of AtCKX2 was cloned past reverse transcription–PCR from total RNA of AtCKX2 transgenic plant tissue with the OneStep reverse transcription–PCR kit (Qiagen, Chatsworth, CA). The PCR products were sequenced and positioned under control of the GAL1 promoter in the yeast expression vector pYES2. The control strain harbored only the empty vector. Induction of gene expression past galactose was carried out for vi h equally suggested by Invitrogen.
Constitute Transformation and Plant Civilisation.
Nicotiana tabacum 50. cv. Samsun NN leaf explants were transformed and regenerated as described (12). At least fifteen contained transformants showing very like phenotypes were obtained for each of the four genes. Plants were cultured in vitro on MS medium or in a glass firm with 15-h light/9-h dark cycles, twenty°C during the dark flow and 24°C during the light period. Characterizations of the transgenic tobacco were carried out on Tii progeny obtained by selfing. Phenotypic changes noted for the independent transformants were very similar and differed just gradually. Independent transformants, confirmed past Northern blot assay and/or by measuring the cytokinin oxidase activeness, looked similar to the transformants shown in Fig. 2 B. Quantitative growth parameters were obtained from at least 10 individuals of two independent clones (AtCKX1-28, AtCKX1-l and AtCKX2-38, AtCKX2-40, respectively). Segregation analyses of the hygromycin resistance cistron indicated one or two (AtCKX2-forty) chromosomal T-DNA insertion loci. For the sake of clarity, the results for only one clone are shown in Figs. 2 (C and D) and 3 (C and D).
Shoot phenotype of AtCKX1-expressing tobacco plants. (A) Top view of six-week-quondam plants. (B) Tobacco plants at the flowering stage. (C) Kinetics of stem elongation. Arrows mark the onset of flowering. Age of plants (days afterwards germination) and leafage number at that stage are indicated above the arrows. Bars indicate SD; n = 12. (D) Number of leaves (due north = 12) formed between day 68 and twenty-four hours 100 after germination and last surface area of these leaves (100% of wild type is 3646 ± 144 cm2; northward = 3). (E) Comparing of foliage size and senescence. Leaves were from nodes number four, 9, 12, sixteen, and xx from the top (from left to right).
RNA Grooming and Blot Analysis.
Full RNA extraction from leaf tissue and Northern blot analysis, with l μg of total RNA, was carried out essentially every bit described (13).
Histological Analysis.
Establish tissue was fixed and embedded in LR White (Plano, Wetzlar, Germany) according to ref. 14, and 2.5 μM thin sections were stained with 0.1% toluidine blueish. For Deoxyribonucleic acid staining, roots were fixed in ethanol:acetic acrid (6:1), incubated for 15 min in a solution of 4′,6-diamidino-ii-phenylindole (DAPI, 1 μg/ml HiiO), and washed three times with water. Scanning electron microscopy was carried out according to ref. xv.
Quantitative Analysis of Cytokinin Oxidase Action.
The standard assay for CKX action was based on the conversion of [2-3H]iP to adenine equally described (16).
Quantitative Assay of Cytokinin Content.
Cytokinin extraction, immunopurification, HPLC separation, and quantification by ELISA methods were carried out as described (13).
Results
The Construction of the AtCKX Genes.
A Blast search of the GenBank database identified half-dozen cytokinin oxidase-like genes in Arabidopsis that code for enzymes with 32–41% amino acid identity to the maize poly peptide and 33–66% amino acid identity between private family members. The gene structure is partially conserved betwixt maize and Arabidopsis. The predicted Arabidopsis genes have five exons and four introns, whereas the maize gene has only 2 introns that are at identical positions every bit ii of the Arabidopsis introns. Common motifs of these CKX genes are putative N-terminal betoken peptides, which point for most of the corresponding proteins transport to the secretory pathway, and a ≈lxx-aa-long FAD-bounden domain in the N-terminal region (information non shown; ref. 17).
Transgenic Plants Bear witness Increased Cytokinin Oxidase Activity.
We cloned the AtCKX1, AtCKX2, AtCKX3, and AtCKX4 genes, positioned them under the control of a constitutive 35S promoter, transformed tobacco plants individually with these genes, and selected overexpressing transgenic clones by Northern blot assay (Fig. i A). The leaves of expressing transgenic tobacco lines showed a 2.half-dozen-fold to 10.4-fold increase in cytokinin oxidase activity (Fig. one B). Besides, cells of Saccharomyces cerevisiae expressing AtCKX2 showed a higher cytokinin oxidase activeness than the control strain (Fig. 1 C). The majority of the enzyme activity accumulated in the yeast culture medium (Fig. ane C). This upshot, and like observations in fission yeast and Physcomitrella patens cells expressing the cytokinin oxidase of maize (ix, 10), indicates that the cytokinin deposition pathway might be, at to the lowest degree partially, located extracellularly. The apparent K one thousand values for the cytokinin oxidases analyzed in this study are in the range of 0.3 to 9.5 μM with iP as a substrate. This is like to or even lower than the maize enzyme, which has an apparent K thousand of 19.ii μM in kernels (xviii) and a native K yard of ii.eight μM (17) with iP as a substrate. These results demonstrate that the proteins encoded by these four AtCKX genes do indeed have cytokinin oxidase action and could exist used as a tool to study the relevance of cytokinins during the whole life cycle of higher plants.
AtCKX gene expression and enzyme action in transgenic tobacco plants and yeast. (A) Northern blots (fifty μg full RNA) of private transformants were probed with cistron-specific probes that covered the whole genomic sequences. But clones with detectable AtCKX transcripts showed a phenotype, and no cantankerous-hybridization with untransformed tobacco (WT) was detected. 25S, hybridization with 25S rRNA every bit control for loading. (B) Cytokinin oxidase activity in leaves of tobacco plants. Specific activeness in extracts of wild blazon (100%) was 8 ± 0.9 pmol adenine × mg protein−ane × h−1. Bars testify SD; due north = 3. (C) Cytokinin oxidase activity in yeast cells and medium. Specific activity of the control strain (100%) was 1.16 nmol adenine × mg protein−1 × h−1.
Transgenic Plants Accept a Reduced Cytokinin Content.
The endogenous concentrations of different cytokinin metabolites was significantly reduced in AtCKX1- and AtCKX2-expressing transgenic seedlings. The total content of iP and Z metabolites in individual transgenic clones was between 31% and 63% that of wild type (Table 1). Amidst the 16 different cytokinin metabolites that were measured, the greatest change occurred in the iP-type cytokinins in AtCKX2 overexpressers. Smaller alterations were noted for Z-type cytokinins, which could be due to different accessibility of the substrate or a lower substrate specificity of the protein. Interestingly, the cytokinin reserve pool of O-glucosides was also lowered in the transgenics (Table 1). The concentrations of N-glucosides and dihydrozeatin-type cytokinins were very low in wild-type plants and were not, or but marginally, altered in transgenic seedlings (data not shown). It is noteworthy that the overall subtract in the content of iP-type cytokinins is more than pronounced in AtCKX2-expressing plants than in AtCKX1 transgenics, which show a stronger phenotype in the shoot. The changes in concentration of Z and ZR were similar in both cases. It is not known which cytokinin metabolite is relevant for the different traits that were analyzed in the transgenic plants. It may exist that the different cytokinin forms take differing roles to play in the various developmental processes.
Table i
Cytokinin content of AtCKX transgenic plants
| Line | Wild type | AtCKX1-2 | AtCKX1-28 | AtCKX2-38 | AtCKX2-40 | ||||
|---|---|---|---|---|---|---|---|---|---|
| Cytokinin metabolite | Concentration | Concentration | % of WT | Concentration | % of WT | Concentration | % of WT | Concentration | % of WT |
| iP | 5.90 ± one.80 | 4.76 ± 0.82 | 81 | 4.94 ± 2.62 | 84 | 1.82 ± 0.44 | 31 | 2.85 ± 0.62 | 48 |
| iPR | 2.36 ± 0.74 | 1.53 ± 0.xiv | 65 | 0.75 ± 0.27 | 32 | 0.55 ± 0.39 | 23 | 0.89 ± 0.07 | 38 |
| iPRP | three.32 ± 0.73 | 0.87 ± 0.26 | 28 | 1.12 ± 0.13 | 34 | 0.80 ± 0.48 | 24 | 1.68 ± 0.45 | 51 |
| Z | 0.24 ± 0.06 | 0.17 ± 0.02 | 71 | 0.22 ± 0.03 | 92 | 0.21 ± 0.06 | 88 | 0.22 ± 0.02 | 92 |
| ZR | 0.60 ± 0.13 | 0.32 ± 0.12 | 53 | 0.34 ± 0.03 | 57 | 0.34 ± 0.15 | 57 | 0.32 ± 0.05 | 53 |
| ZRP | 0.39 ± 0.17 | 0.42 ± 0.11 | 107 | 0.28 ± 0.15 | 72 | 0.06 ± 0.01 | 15 | 0.17 ± 0.06 | 44 |
| ZOG | 0.46 ± 0.20 | 0.32 ± 0.09 | 70 | 0.26 ± 0.13 | 57 | 0.20 ± 0.07 | 43 | 0.12 ± 0.02 | 26 |
| ZROG | 0.48 ± 0.17 | 0.thirty ± 0.06 | 63 | 0.47 ± 0.02 | 98 | 0.23 ± 0.05 | 48 | 0.xxx ± 0.13 | 63 |
| | | | | | | | | | |
| Full | thirteen.75 | viii.69 | 63 | viii.38 | 61 | 4.21 | 31 | 6.55 | 48 |
Transgenic Plants Have an Altered Phenotype.
AtCKX gene overexpression caused striking developmental alterations in the plant shoot and root organization. The alterations were very like, but not identical, for the unlike genes. AtCKX1 and AtCKX3 overexpressers were alike every bit were AtCKX2 and AtCKX4 transgenics. By and large, the ii quondam showed higher expression of the traits, peculiarly in the shoot. Because of these similarities, the phenotypic changes of two contained clones expressing AtCKX1 or AtCKX2 were analyzed in greater detail. Most information shown refer to the stronger phenotype of the AtCKX1 transgenics.
The most noticeable changes in the shoot were a severely retarded development with shorter internodes leading to a dwarfed growth habit, the germination of lanceolate epinastic leaves, and the formation of a reduced number of flowers (Fig. ii A and B). The time betwixt the initiation of new leaves (plastochrone) at the borders of the shoot meristem was on boilerplate 2.6 ± 0.1 days in wild blazon and 4.four ± 0.1 days in AtCKX1 transgenics (Fig. 2 C). The surface surface area of leaves formed by the transgenics during a defined time period was ≈15% that of wild type (Fig. 2 D). The width-to-length ratio of mature leaves was lowered from 1:2 in wild blazon to 1:three in AtCKX1 transgenics. The vasculature of AtCKX1 transgenic leaves was less developed, the spacing between veins was larger, and the veins were flat and non raised as in wild type. In contrast to wild-type leaves, leaf parenchyma cells continued to expand in transgenic clones in the transverse direction, resulting in thicker and rigid old leaves. A prominent difference was too noted for progression of leaf senescence. In tobacco, leafage senescence starts in the about basal leaves and leads to a compatible reduction of leaf pigment content. In contrast, aging leaves of AtCKX1 transgenic plants developed chlorotic intercostal regions but retained chlorophyll along the leafage veins (Fig. 2 Eastward). Leafage aging was like in AtCKX2-expressing plants, but chlorosis was less pronounced. Transgenic plants started to flower up to iii months later than wild-type plants (Fig. 2 C) and produced only 5–10 normal-sized flowers compared with >100 flowers in the wild types. The last leafage number at the onset of flowering was like in wild type and the transgenic clones, supporting the notion that foliage number is a determinant for blossom consecration in day-neutral tobacco (Fig. 2 C). Lateral buds in the leaf axils of transgenic plants developed 2 to 3 tiny leaves early on during vegetative development, in contrast to lateral buds of wild type, which remained completely inhibited. This indicates incomplete upmost potency in the transgenic plants.
In contrast to the inhibited shoot evolution of AtCKX transgenic tobacco, their root growth was enhanced (Fig. three A and B). Elongation of the master root was more rapid, primordia of lateral roots were noted closer to the root apex than in wild-type plants, and the number of lateral branches, as well as adventitious roots, increased (Fig. iii C). Enhanced root growth led to a 60% increment in root dry weight in transgenic plants grown in hydroponic solution (information not shown). These results suggest that cytokinins are involved in controlling both root growth rate and the generation of new root meristems. The dose-response bend of root growth inhibition by exogenous cytokinin showed the transgenic roots to accept cytokinin resistance (Fig. three D). Interestingly, the resistance of AtCKX1 transgenics to iPR was less marked than for AtCKX2, which is consequent with the smaller changes in iP-type cytokinins in the latter (Table 1).
Root phenotype of AtCKX-expressing transgenic tobacco plants. (A) Seedlings 17 days after germination. (B) Root organisation of soil-grown plants at the flowering stage. (C) Root length, number of lateral roots (LR), and adventitious roots (AR) on day 10 subsequently germination. (D) Dose-response curve of root growth inhibition by exogenous cytokinin. Seeds were sown on MS medium containing 3% sucrose and the indicated concentration of iPR. The length of chief roots was adamant after x days of tillage in the dark on vertically positioned plates. Bars signal ± SD; due north = xxx.
Histology of the Shoot Meristem, Shoot Organs, and Root Meristems.
A decreased or increased organ growth rate equally a consequence of a reduced cytokinin content could exist due to a changed prison cell division rate in the meristematic regions, a different population size of dividing cells, or contradistinct cell growth. In the AtCKX transgenics, the terminal length of cells in the stem was not reduced, and the concluding length of root cells was slightly decreased (149.7 ± 31.vii μM in clone AtCKX1-50 versus 167.0 ± 32.0 μM in wild blazon; n = 100), indicating that differences in jail cell growth did not contribute to, or fifty-fifty partially compensate for, altered growth of stalk and roots. Nonetheless, microscopic inspection of the shoot apical meristem (SAM), leaf, and the root meristem revealed that the morphological changes described to a higher place were reflected in distinct changes in cell number and charge per unit of jail cell formation in the AtCKX transgenics.
The SAM of AtCKX1 transgenic plants was smaller than in wild-blazon plants and fewer cells occupied the space between the cardinal zone and the peripheral zone of lateral organ formation, merely the cells were of the same size and no obvious changes of the differentiation pattern occurred (Fig. 4 A). Also, the overall tissue blueprint of leaves in cytokinin oxidase overexpressers was unchanged. However, the sizes of both phloem and xylem were significantly reduced (Fig. 4 B). In contrast, the average cell size of leaf parenchyma and epidermal cells was increased 4- to v-fold (Fig. four C and D). New cells of AtCKX1 transgenic leaves are formed at 3–4% of the rate of wild-type leaves, and last leafage cell number is estimated to be in the range of 5–half-dozen% that of wild type. Like but less pronounced changes occurred in the shoot of AtCKX2-expressing plants (data non shown). In contrast to leaves, neither prison cell size nor cell grade of floral organs was altered in the transgenic lines. Also, seed weight was similar in wild type and AtCKX1 and AtCKX2 transgenic plants (data not shown).
Histology of shoot meristems, leaves, and root meristems. (A) Longitudinal median department through the vegetative SAM. P, leaf primordia. (B) Vascular tissue in second order veins of leaves. X, xylem, PH, a phloem bundle. (C) Cross sections of fully developed leaves. (D) Scanning electron microscopy of the upper foliage epidermis. (E) Root apices stained with 4′,6-diamidino-ii-phenylindole. RM, root meristem. (F) Longitudinal median sections of root meristems ten days afterwards germination. RC, root cap; PM, promeristem. (G) Transverse root sections 10 mm from the apex. E, epidermis, C1–C4, cortical cell layer; X, xylem; PH, phloem. The textile for the analysis of the SAM and the mature fully expanded leaves was from 38- and 100-day-old plants (clone AtCKX1-50), respectively, which were cultivated in a dark-green house. Root assay was performed with primary roots of seedlings 10 days after germination. Confined, 100 μm.
The cell population in root meristems in the AtCKX1 and AtCKX2 transgenic plants was enlarged approximately 4-fold, and the cell numbers in both the primal and lateral columnella were increased (Fig. 4 E and F). Final root bore was increased by sixty% due to the increased diameter of all root cell types and an increased number of cells in each prison cell file. The radial root pattern was identical in wild type and transgenics, with the exception that ofttimes a fourth layer of cortex cells was noted in transgenic roots (Fig. four G).
Give-and-take
This analysis of the consequences of reduced endogenous cytokinin content strongly indicates in which found processes cytokinins are limiting and might, therefore, have a regulatory role. The slowed formation of new cells in the SAM, besides as of leaf primordia, and the reduced size of the SAM indicates that cytokinins have a dual function in the command of SAM proliferation. They are required to maintain the cell division bike but might also be involved in promoting the transition from undifferentiated stem cells to differentiation. Earlier piece of work has shown that in unorganized growing cells, cytokinins induce the formation of shoot meristems, demonstrating that they have a office beyond maintaining the cell bicycle (two). Known analogous factors of cell proliferation and differentiation in the SAM are transmembrane receptor proteins (e.k., CLV1) and transcription factors of the homeodomain grade (e.g., WUS, STM, KNAT1), which interact in regulatory loops (nineteen). Recent data betoken that a reciprocal interaction between cytokinins and some of these transcription factors exists (20–22). A role for cytokinins in the regulation of SAM differentiation could be realized through local gradients of the hormone or differences in the distribution of dissimilar cytokinin metabolites. This might modify effector cistron expression quantitatively, which could in plow influence cellular fate. Developmental changes in the concentration and localization of different cytokinin metabolites accept been reported for the SAM of tobacco (23). The reduced activity of the SAM could as well be the cause of the incomplete upmost dominance, which was noted in transgenic plants, as the corporeality of auxin produced for the maintenance of apical authorization might be lowered.
The slowed formation of leaf cells and their reduced number indicates an accented requirement for cytokinins during leaf formation, both to drive the prison cell division cycle at normal speed and to obtain the required number of divisions for a normal leaf size. That cytokinins function equally a regulatory cistron in leaf jail cell formation is supported by the fact that transgenic Arabidopsis plants with an enhanced cytokinin content produced more leaf cells than control plants (20). Moreover, cytokinins announced to restrict leaf cell size equally the cells of transgenic leaves are larger than in command plants. Alternatively, a compensatory mechanism may be activated in transgenic plants to reach a genetically determined organ size, as has been reported for plants expressing a dominant-negative form of cdc2 (24). In either case, the leaf phenotype of AtCKX overexpressers supports the view that cell proliferation and growth in tobacco leaves are not coupled.
Interestingly, the bloom phenotype of the transgenic plants was unaltered. This suggests that the part of cytokinins in the regulation of development of reproductive organs might be less important than it is during the vegetative phase. It may be that once the plant has entered the reproductive cycle, a more than stringent mechanism operates in the meristem to ensure the proper course of the developmental programme.
Contrasting with the promotive role in the SAM, cytokinins have a negative regulatory function in root growth. The increased cell number in the transgenic root meristems and the slightly reduced final cell length in transgenic roots indicate that the enhanced root growth is because of an enhanced cycling of cells rather than increased cell growth. In the presence of lowered cytokinin content, root meristem cells accept a prolonged meristematic phase and somewhen undergo additional rounds of mitosis earlier they get out the meristem and start to elongate. We conclude that the action of the initials and/or the get out of cells from the root meristem is regulated past a mechanism that is sensitive to cytokinins.
Taken together, the investigation of cytokinin-deficient plants has shown that the influence of cytokinins on morphogenesis is primarily achieved through cell bike regulation. Multiple functions and several molecular targets of cytokinins during unlike phases of the cell cycle are known. The hormone is required for S-phase entry in leaf mesophyll protoplasts and tobacco pith explants, and S-phase progression is accelerated in the presence of cytokinins (25, 26). Several prison cell cycle genes are regulated past cytokinins, including cdc2, CycD3, and different B-type cyclins (27–29). There is evidence that regulatory genes of the prison cell cycle are expressed in a tissue-specific fashion and that cytokinin furnishings on the cell cycle vary between unlike cell types (30, 31). Distinct expression patterns of cytokinin targets could be a reason for the opposite effects seen in shoot and root meristems. The enhanced organ growth in plants overexpressing D- and B-type cyclins (32, 33) is consistent with the hypothesis that cytokinins act through the regulation of cell wheel progression.
What could the office of CKX proteins during establish growth and development exist? A likely function is the degradation of cytokinins that accumulate transiently during the Thou2/1000 transition of cycling cells to a level that is several orders of magnitude higher than during the other cell wheel phases (34). It is not known how these cytokinin levels are rapidly readjusted to normal levels. CKX enzymes could take a part in recycling this prison cell division-derived cytokinin. The expression of AtCKX genes in regions of active growth is consequent with the proposed function in cycling cells (unpublished results). Additional roles for the enzymes could be the maintenance of an optimal level of cytokinins for growth and/or resetting a cytokinin signaling system to a basal level.
To summarize, in this work we have obtained proof of the office of four cytokinin oxidases from A. thaliana and used these genes as tools to generate plants with a reduced cytokinin content. The data lend support to the auxin–cytokinin hypothesis for quantitative growth parameters and organ ratio in plants (two). Nonetheless, we note that some, but not all, phenotypic changes tin exist explained by an altered ratio of these two hormones, as several aspects of auxin-overproducing plants are distinct from plants with a reduced cytokinin content (35, 36). This indicates that the auxin–cytokinin residual determines only a subset of morphogenetic parameters. Natural cytokinin levels are inhibitory to the development of a maximal root system, and fine aligning of cytokinin levels is needed to reach the optimal growth of shoots (20, 37). Evidently, the targeted manipulation of CKX cistron expression can be an important and novel tool to modulate growth characteristics and yield parameters of crop plants.
Acknowledgments
We dedicate this article to C. O. Miller and the late F. South. Skoog, who discovered cytokinins about 50 years ago and coined the auxin–cytokinin hypothesis of institute growth. We are indebted to K. Lemcke for initial help with gene cloning and sequence analyses, Yard. Riefler for support with structural factor analysis, Yard. Lenhard and Y. Stierhof for suggest for microscopic analyses, and M. Kamínek for helpful comments on the manuscript. We thank H. Martínková and V. Lacmanová for excellent technical aid and M. J. Beech and C. Scott-Taggart for proofreading. We acknowledge financial support of the Deutsche Forschungsgemeinschaft (Schm 814/13-1), the Volkswagen-Stiftung (I/72076), the Grant Agency of the Czech Republic (522/00/1346), and the Czech Ministry of Educational activity (MSM 153100008).
Abbreviations
| CKX | cytokinin oxidase |
| iP | isopentenyladenine |
| Z | trans-zeatin |
| SAM | shoot apical meristem |
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