Cytokinin Regulation Research Articles

Cytokinin Regulation of Gene Expression in the AHP Gene Family in Arabidopsis thaliana

In higher plants histidine-aspartate phosphorelays are involved in hormone and stress signaling via a two-component system of signal transduction. In this system a histidine-containing phosphotransmitter (HPt) mediates signal transmission from a sensory histidine kinase to a response regulator, providing integration and/or branching of several different signaling pathways. Five genes encoding HPts, AHP1-5, have been identified in Arabidopsis. Histidine-aspartate phosphorelays involving HPts have been at least partly implicated in cytokinin signaling. We analyzed the regulation by cytokinins of AHP gene expression. We compared the effects on steady–state levels of AHP transcripts of a short-term treatment with an aromatic cytokinin and increase in endogenous isoprenoid cytokinin levels using an activable ipt system in 8-day-old Arabidopsis seedlings. Following ipt activation, a rapid and highly preferential increase in trans-zeatin-type cytokinins was observed, whereas other isoprenoid-type cytokinins showed no or only marginal increases. The levels of cytokinin metabolites under long-term ipt activation suggest that the seedlings may have difficulties in efficiently downregulating active forms of the hormone. Using real-time RT-PCR, transient increases in steady-state levels of AHP1-4 transcripts in response to both the short-term N 6-benzyladenine treatment and the increase in endogenous trans-zeatin-type cytokinin levels were observed. In contrast, both the full and the alternatively spliced AHP5 transcripts remained unaltered. On the other hand, increases in steady-state levels of AHP1-4 transcripts observed in seedlings cultivated continuously in the presence of exogenous N 6-benzyladenine were not paralleled in seedlings with constitutively increased endogenous trans-zeatin-type cytokinins, providing further indirect evidence for distinct functions of aromatic and isoprenoid cytokinins.

Feedback regulation of xylem cytokinin content is conserved in pea and Arabidopsis.

Increased-branching mutants of garden pea (Pisum sativum; ramosus [rms]) and Arabidopsis (Arabidopsis thaliana; more axillary branches) were used to investigate control of cytokinin export from roots in relation to shoot branching. In particular, we tested the hypothesis that regulation of xylem sap cytokinin is dependent on a long-distance feedback signal moving from shoot to root. With the exception of rms2, branching mutants from both species had greatly reduced amounts of the major cytokinins zeatin riboside, zeatin, and isopentenyl adenosine in xylem sap compared with wild-type plants. Reciprocally grafted mutant and wild-type Arabidopsis plants gave similar results to those observed previously in pea, with xylem sap cytokinin down-regulated in all graft combinations possessing branched shoots, regardless of root genotype. This long-distance feedback mechanism thus appears to be conserved between pea and Arabidopsis. Experiments with grafted pea plants bearing two shoots of the same or different genotype revealed that regulation of root cytokinin export is probably mediated by an inhibitory signal. Moreover, the signaling mechanism appears independent of the number of growing axillary shoots because a suppressed axillary meristem mutation that prevents axillary meristem development at most nodes did not abolish long-distance regulation of root cytokinin export in rms4 plants. Based on double mutant and grafting experiments, we conclude that RMS2 is essential for long-distance feedback regulation of cytokinin export from roots. Finally, the startling disconnection between cytokinin content of xylem sap and shoot tissues of various rms mutants indicates that shoots possess powerful homeostatic mechanisms for regulation of cytokinin levels.

The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti.

Legumes develop different types of lateral organs from their primary root, lateral roots and nodules, the latter depending on a symbiotic interaction with Sinorhizobium meliloti. Phytohormones have been shown to function in the control of these organogeneses. However, related signaling pathways have not been identified in legumes. We cloned and characterized the expression of Medicago truncatula genes encoding members of cytokinin signaling pathways. RNA interference of the cytokinin receptor homolog Cytokinin Response1 (Mt CRE1) led to cytokinin-insensitive roots, which showed an increased number of lateral roots and a strong reduction in nodulation. Both the progression of S. meliloti infection and nodule primordia formation were affected. We also identified two cytokinin signaling response regulator genes, Mt RR1 and Mt RR4, which are induced early during the symbiotic interaction. Induction of these genes by S. meliloti infection is altered in mutants affected in the Nod factor signaling pathway; conversely, cytokinin regulation of the early nodulin Nodule Inception1 (Mt NIN) depends on Mt CRE1. Hence, cytokinin signaling mediated by a single receptor, Mt CRE1, leads to an opposite control of symbiotic nodule and lateral root organogenesis. Mt NIN, Mt RR1, and Mt RR4 define a common pathway activated during early S. meliloti interaction, allowing crosstalk between plant cytokinins and bacterial Nod factors signals.

KNOX Action in Arabidopsis Is Mediated by Coordinate Regulation of Cytokinin and Gibberellin Activities

The shoot apical meristem (SAM) is a pluripotent group of cells that gives rise to the aerial parts of higher plants. Class-I KNOTTED1-like homeobox (KNOX) transcription factors promote meristem function partly through repression of biosynthesis of the growth regulator gibberellin (GA). However, regulation of GA activity cannot fully account for KNOX action. Here, we show that KNOX function is also mediated by cytokinin (CK), a growth regulator that promotes cell division and meristem function. We demonstrate that KNOX activity is sufficient to rapidly activate both CK biosynthetic gene expression and a SAM-localized CK-response regulator. We also show that CK signaling is necessary for SAM function in a weak hypomorphic allele of the KNOX gene SHOOTMERISTEMLESS (STM). Additionally, we provide evidence that a combination of constitutive GA signaling and reduced CK levels is detrimental to SAM function. Our results indicate that CK activity is both necessary and sufficient for stimulating GA catabolic gene expression, thus reinforcing the low-GA regime established by KNOX proteins in the SAM. We propose that KNOX proteins may act as general orchestrators of growth-regulator homeostasis at the shoot apex of Arabidopsis by simultaneously activating CK and repressing GA biosynthesis, thus promoting meristem activity.

(289) GUS Expression in LEACO10.92kb-GUS Tobacco Plants Suggests That Auxin and Ethylene Are Involved in LEACO10.92kb Promoter Induction

A 920 bp fragment of the ACC oxidase gene promoter from tomato (LEACO1) was used to drive GUS gene expression. The LEACO10.92kb fragment contained two stress-responsive short motifs; a 10 bp TCA motif (5'-TCATCTTCTT-3') twice (allowing two substitutions) and an 8 bp element (5'-AA/TTTCAAA-3') once. The TCA motif is found in over 30 stress- and pathogen-inducible genes while the 8 bp element is necessary for ethylene-response in the carnation GST1 and the tomato E4 gene promoters. Previously in chrysanthemum, cytokinin regulation with LEACO10.92kb produced dramatic increases in lateral branching and bud initiation. Tobacco plants carrying LEACO10.92kb–GUS were used to examine the response of the LEACO10.92kb promoter to various hormones and hormone inhibitors. GUS activity in LEACO10.92kb–GUS plants was detected in leaves and stems, but not roots. High expression was detected in shoots with the apical bud intact, but GUS activity decreased with the apical bud removed. Applying IAA to the shoot apex after removing the apical bud, restored GUS activity. However, the IAA transport inhibitor TIBA reduced GUS activity in shoots with intact apical buds, and in IAA-treated shoots with excised buds. In shoots with excised apical buds, GUS activity increased when the ethylene precursor ACC was applied, but decreased in intact shoots when the ethylene biosynthesis inhibitor AOA was applied. These data suggest that auxins produced in the apical meristem are capable of regulating LEACO10.92kb activity, probably through auxin-induced ethylene biosynthetic pathway activity.

벼의 엽신 및 캘러스에서 Cytokinin 유도성 A-type 및 C-type Cyclin 유전자의 발현 분석

세포 주기의 조절에 있어 중요한 역할을 담당하고 있는 cyclin 유전자들을의 발현 양상을 벼에서 유도한 캘러스와 엽신을 이용하여 관찰하였다. 특히 캘러스를 이용한 분석결과 2,4-D와 kinetin이 조합된 배지 러에 7일 동안 암 배양한 후 이를 cytokinin만이 첨가된 배치로 옮겼을 때 A-, B-, C-type cyclin 유전자들의 발현이 더 증가되었다. 또한 벼실생의 각 기관에서 cyclin 유전자의 발현양을 조사한 결과 다른 기관들에 비해 잎에서 A-, B, C-type cyclin 유전자들이 더 많이 발현하였으나 잎에 cytokinin의 종류와 농도를 다르게 처리하여 24시간동안 배양한 결과에서는 zeatin을 제외하고는 cytokinin의 종류와 양이 cyclin 유전자들의 발현에 큰 영향을 미치지 못함을 알 수 있었다. 본 실험에서는 벼 cyclin유전자의 발현과 식물생장조절제 처리와의 상관 관계에 대해서 조사하였다. The expression patterns of cyclin genes, which play a crucial role on cell cycle control, were analyzed with rice calli and leaf blades from seedlings. When callus was transferred from media containing the combinations of 2,4-D and kinetin under the dark conditions to medium supplemented with cytokinin-only on 7 days after the cultures, the expression levels of A-, B- and C-type cyclins from callus were increased significantly. Despite the fact that cyclin genes were well expressed on leaf blades rather than other organs in rice seedlings, rice leaf blades grown on the medium containing various combinations and concentrations of cytokinin for 24 hours had no major effect on the expression patterns of cyclins except zeatin. The relation between cytokinin regulation and the expression of cyclins of rice is discussed.

One-Two Punch to Ethylene Production

Ethylene gas functions as a hormone in plants and is produced in response to stress or the growth regulator cytokinin. The rate-limiting step of ethylene biosynthesis is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid synthase (ACS), and in some instances, ethylene production is regulated through control of ACS expression. However, posttranscriptional regulation also appears to be important, and Wang et al. have clarified a new mechanism by which such regulation may occur. They characterized eto1 , a gene that decreases ACS activity and production of ethylene. The association of ETO1 protein with ACS was confirmed by yeast two-hybrid assays, in vitro experiments, and immunoprecipitation from plant cells. In vitro, ETO1 directly inhibited ACS activity. But ETO also contains a BTB (Broad-complex, Tramtrack, Bic-a-brac) domain that is associated with adaptor proteins that link substrates to ubiquitin ligase complexes that mark proteins for proteasomal degradation. ETO1 interacted with the CUL3A protein, which functions as a scaffold in ubiquitin E3 ligase complexes. Overexpression of ETO1 in cultured cells caused increased degradation of ACS, an effect that was blocked by a proteasome inhibitor, MG132. Thus, the authors propose that ETO1 has a two-pronged inhibitory effect on ACS, directly inhibiting the enzyme's activity and also targeting ACS for degradation. K. L.-C. Wang, H. Yoshida, C. Lurin, J. R. Ecker, Regulation of ethylene gas biosynthesis by the Arabidopsis ETO1 protein. Nature 428 , 945-950 (2004). [Online Journal]

Regulation of ethylene gas biosynthesis by the Arabidopsis ETO1 protein.

Ethylene gas is used as a hormone by plants, in which it acts as a critical growth regulator. Its synthesis is also rapidly evoked in response to a variety of biotic and abiotic stresses. The Arabidopsis ethylene-overproducer mutants eto2 and eto3 have previously been identified as having mutations in two genes, ACS5 and ACS9, respectively; these encode isozymes of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), which catalyse the rate-limiting step in ethylene biosynthesis. Here we report that another ethylene-overproducer mutation, eto1, is in a gene that negatively regulates ACS activity and ethylene production. The ETO1 protein directly interacts with and inhibits the enzyme activity of full-length ACS5 but not of a truncated form of the enzyme, resulting in a marked accumulation of ACS5 protein and ethylene. Overexpression of ETO1 inhibited induction of ethylene production by the plant growth regulator cytokinin, and promoted ACS5 degradation by a proteasome-dependent pathway. ETO1 also interacts with CUL3, a constituent of ubiquitin ligase complexes in which we propose that ETO1 serves as a substrate-specific adaptor protein. ETO1 thus has a dual mechanism, inhibiting ACS enzyme activity and targeting it for protein degradation. This permits rapid modulation of the concentration of ethylene.

Discoveries and Dilemmas Concerning Cytokinin Metabolism.

Natural cytokinin compounds that function as plant hormones are adenine molecules connected to an N6 side chain. Metabolic pathways produce various structural modifications to the adenine moiety and/or the side chain that affect cytokinin function, activity, stability, and transport in plants. This review focuses on recent research that is elucidating the genes and biochemical pathways involved in cytokinin conjugation, deconjugation, and interconversion. One area of new discovery concerns the process of O-glucoside conjugate formation. Genes have been identified for O-glucosyltransferase enzymes in Phaseolus (ZOG1 and ZOX1) and maize (cisZOG1) and have been used to study enzyme activity, structure, and function in plant development. New information about the genetic basis of the adenine salvage pathway and its relationship to cytokinin interconversion and regulation has also become available. However, various dilemmas about cytokinin metabolism remain and need to be resolved using a combination of research approaches.

Cytokinin growth responses in Arabidopsis involve the 26S proteasome subunit RPN12.

The 26S proteasome is an ATP-dependent eukaryotic protease responsible for degrading many important cell regulators, especially those conjugated with multiple ubiquitins. Bound on both ends of the 20S core protease is a multisubunit regulatory particle that plays a crucial role in substrate selection by an as yet unknown mechanism(s). Here, we show that the RPN12 subunit of the Arabidopsis regulatory particle is involved in cytokinin responses. A T-DNA insertion mutant that affects RPN12a has a decreased rate of leaf formation, reduced root elongation, delayed skotomorphogenesis, and altered growth responses to exogenous cytokinins, suggesting that the mutant has decreased sensitivity to the hormone. The cytokinin-inducible genes CYCD3 and NIA1 are upregulated constitutively in rpn12a-1, indicating that feedback-inhibitory mechanisms also may be altered. rpn12a-1 seedlings also showed changes in auxin-induced growth responses, further illustrating the close interaction between auxin and cytokinin regulation. In yeast, RPN12 is necessary for the G1/S and G2/M transitions of the cell cycle, phases that have been shown to be under cytokinin control in plants. We propose that RPN12a is part of the Arabidopsis 26S proteasome that controls the stability of one or more of the factors involved in cytokinin regulation.

Increased seed cytokinin levels in transgenic tobacco influence embryo and seedling development.

Seed-specific expression of a heterologous vicilin-isopentenyl transferase (ipt) gene has been determined in transgenic tobacco lines 7 and 16. Genetic analysis of self-fertilized progeny showed that a single copy of the vicilin-ipt gene had been integrated, and T2 progeny had become homozygous for the transgene. Stable inheritance of the vicilin-ipt gene in T3 progeny was verified by polymerase chain reaction analysis. Seed cytokinin levels were 2-3-fold higher than control levels. Cytological analyses revealed a significant increase in the number of plerome cell layers and enlargement of diameter in transgenic embryos compared with controls. Dry weight of mature seeds and subsequent seedling growth was increased significantly. This correlated with the level of vicilin-ipt overexpression and increased cytokinin levels in transgenic tobacco seeds. The results suggest a crucial role for cytokinins in regulation of tobacco embryo development.

Degradation of cytokinins by cytokinin oxidases in plants

The degradation metabolism of cytokinins is an important process that controls the levels of cytokinin active forms and their distribution in plant tissues. It appears to be due, in large part, to the activity of a specific enzyme, cytokinin oxidase. This review attempts to collate the limited information available about this enzyme and introduce new facts, obtained in our laboratory, concerning the mechanism of degradation of cytokinins bearing unsaturated isoprene side chains. However, complete clarification of the effects of cytokinin oxidase on cytokinin regulation and its molecular and biochemical properties will be dependent upon the purification of the protein with cytokinin oxidase activity to homogeneity and progress in the development of requisite molecular probes.

Anatomy of shoots and tumors of in vitro habituated rhododendron ‘montego’ (Ericaceae) cultures with tissue proliferation

Tissue proliferation (TP) is characterized primarily by the formation of galls or tumors at the crown of container-grown rhododendrons that were initially propagated in vitro. In the cultivar 'Montego', TP-like symptoms are first observed in vitro as shoot clusters with small leaves and nodal tumors. In addition, unlike the normal in vitro non-TP (TP-) shoots, in vitro TP (TP+) shoots proliferate rapidly without the presence of the plant growth regulator cytokinin in the tissue culture medium. Comparisons of the anatomy of TP+ and TP- shoot tips showed that TP+ shoots had a less developed vascular system, longer cells in the pith and cortex, and altered internodal elongation at the shoot apex. In addition, TP+ axillary buds were abnormal in that they were displaced onto the stem above the leaf axil, and a small group of proliferating cells replaced the shell zone at the base of the bud. Initiation of tumor formation began with the expansion of this region of cell proliferation (RCP) and shoot growth from the abnormal axillary bud (tumor bud). Organization of the tumor bud and extension of the RCP characterized the further development of two types of tumors. In polar shoot tumors, shoot growth continued from the persistent tumor bud and the tumor at the base of the shoot remained small in size. The RCP extends downward to the vascular junction of the subtending leaf and the stem of the TP+ shoot. In nonpolar tumors, continuous de novo meristem formation led to the development of large tumors with or without shoots. The RCP is present throughout the tumor and is associated with de novo meristem formation. Comparisons to the anatomy of other tumor-like structures showed that TP tumors of Rhododendron 'Montego' are most similar to tobacco genetic tumors.

Genetic and developmental control of nuclear accumulation of COP1, a repressor of photomorphogenesis in Arabidopsis.

Using a beta-glucuronidase (GUS) reporter-COP1 fusion transgene, it was shown previously that Arabidopsis COP1 acts within the nucleus as a repressor of seedling photomorphogenic development and that high inactivation of COP1 was accompanied by a reduction of COP1 nuclear abundance (A.G. von Arnim, X.-W. Deng [1994] Cell 79: 1035-1045). Here we report that the GUS-COP1 fusion transgene can completely rescue the defect of cop1 mutations and thus is fully functional during seedling development. The kinetics of GUS-COP1 relocalization in a cop1 null mutant background during dark/light transitions imply that the regulation of the functional nuclear COP1 level plays a role in stably maintaining a committed seedling's developmental fate rather than in causing such a commitment. Analysis of GUS-COP1 cellular localization in mutant hypocotyls of all pleiotropic COP/DET/FUS loci revealed that nuclear localization of GUS-COP1 was diminished under both dark and light conditions in all mutants tested, whereas nuclear localization was not affected in the less pleiotropic cop4 mutant. Using both the brassinosteroid-deficient mutant det2 and brassinosteroid treatment of wild-type seedlings, we have demonstrated that brassinosteroid does not control the hypocotyl cell elongation through regulation nuclear localization of COP1. The growth regulator cytokinin, which also dramatically reduced hypocotyl cell elongation in the absence of light, did not prevent GUS-COP1 nuclear localization in dark-grown seedlings. Our results suggest that all of the previously characterized pleiotropic COP/DET/FUS loci are required for the proper nuclear localization of the COP1 protein in the dark, whereas the less pleiotropic COP/DET loci or plant regulators tested are likely to act either downstream of COP1 or by independent pathways.

Understanding the Role of Cytokinins in Tissue Proliferation of Rhododendron `Montego'

Tissue proliferation (TP) is characterized primarily by the formation of galls or tumors at the crown of container-grown rhododendrons propagated in vitro. However, TP of Rhododendron `Montego' is observed initially in in vitro shoot cultures and it is characterized by the formation of multiple shoots with small leaves and nodal tumors. The formation of shoots in `Montego' TP (TP+) shoot cultures occurs without the presence of exogenous cytokinin in the medium, unlike normal `Montego' (TP–) shoot cultures, which require cytokinin for shoot growth. Structural studies have shown that tumors are composed of many adventitious buds and parenchyma cells, suggesting that TP is a result of abnormal cytokinin regulation that is controlling tumor and shoot formation. Two approaches are being used to determine if differences in cytokinin concentration and/or metabolism exist between TP+ and TP– shoot cultures. In the first approach, shoot cultures are grown in vitro for 1 week in the presence of tritiated isopentenyladenine (iP). Cytokinin uptake and metabolism are analyzed using HPLC and other analytical methods. Experiments suggest that extensive degradation and N-glucoside conjugation occur in TP+ and TP– shoots, resulting in the removal of most of the exogenous iP. In the second approach, the levels of endogenous cytokinins such as iP, isopentenyladenosine, zeatin, and zeatin riboside, are being measured in TP+ tumors and shoots and in TP– shoots by an ELISA method.

Induction of Anthocyanin Accumulation by Cytokinins in Arabidopsis thaliana.

Arabidopsis thaliana plants treated with exogenous cytokinins accumulate anthocyanin pigments. We have characterized this response because it is potentially useful as a genetic marker for cytokinin responsiveness. Levels of mRNAs for four genes of the anthocyanin biosynthesis pathway, phenylalanine ammonia lyase 1 (PAL1), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) were shown to increase coordinately in response to benzyladenine (BA). However, nuclear run-on transcription experiments suggested that although CHS and DFR are controlled by BA at the transcriptional level, PAL1 and CHI are controlled by BA posttranscriptionally. CHS mRNA levels increased within 2 h of BA spray application, and peaked by 3 h. Levels of PAL1 mRNA did not increase within 6 h of BA spray. We also showed that PAL1, CHS, CHI, and DFR mRNA levels fluctuate during a 24-h period and appear to be controlled by a circadian clock. The relation between cytokinin regulation and light regulation of CHS gene transcription is discussed.

Production of High Solids Tomatoes Through Molecular Modification of Levels of the Plant Growth Regulator Cytokinin

Production of High Solids Tomatoes Through Molecular Modification of Levels of the Plant Growth Regulator Cytokinin

A family of cyclin D homologs from plants differentially controlled by growth regulators and containing the conserved retinoblastoma protein interaction motif.

A new family of three related cyclins has been identified in Arabidopsis by complementation of a yeast strain deficient in G1 cyclins. Individual members show tissue-specific expression and are conserved in other plant species. They form a distinctive group of plant cyclins, which we named delta-type cyclins to indicate their similarities with mammalian D-type cyclins. The sequence relationships between delta and D cyclins include the N-terminal sequence LXCXE. This motif was originally identified in certain viral oncoproteins and is strongly implicated in binding to the retinoblastoma protein pRb. By analogy to mammalian cyclin D, these plant homologs may mediate growth and phytohormonal signals into the plant cell cycle. In support of this hypothesis, we show that, on restimulation of suspension-cultured cells, cyclin delta 3 is rapidly induced by the plant growth regulator cytokinin and cyclin delta 2 is induced by carbon source.

Evidence for isopentenyladenine modification on a cell cycle-regulated protein

We have prepared antibodies that recognize isopentenyladenosine (i6A), a modified nucleoside derived from mevalonic acid (MVA). In immunoblot assays, affinity-purified anti-i6 A antibodies specifically bound to a 26-kDa protein (i6A26) in Chinese hamster ovary cells. Anti-i6A recognition of i6A26 was blocked with i6A but not adenosine or isopentenol. Employing immunoblot analysis we have quantitated the level of i6A26 in cells expressing various rates of DNA synthesis. The cellular content of i6A26 was reduced 4-fold in quiescent cells cultured in the absence of serum. When serum-deprived cells were stimulated to enter the cell cycle, the amount of i6A26 increased in the cells during the G1 phase. However, when synchronized cells were stimulated with serum-containing medium in the presence of mevinolin (an inhibitor of cellular MVA synthesis), we observed impaired G1 expression of i6A26 and delayed onset of S phase DNA synthesis. Mevinolin addition to asynchronously growing cells resulted in low rates of cellular DNA synthesis and suppressed levels of i6A26 which were reversed by coincubation with MVA. The ability of MVA to restore DNA synthesis and the cellular content of i6A26 in mevinolin-treated cells showed similar MVA concentration and time dependences. Regenerating liver tissue also exhibited elevated levels of i6A26. Thus, the expression of i6A26 correlates with cellular proliferation and growth. We speculate that i6A26 contains isopentenyladenine moieties and mediates isoprenoid regulation of DNA synthesis. Isopentenyladenylated proteins may also function in cytokinin regulation of proliferation and differentiation in plants.

Cytokinin enhancement of the light induction of nitrate reductase transcript levels in etiolated barley leaves

To investigate the molecular mechanism of cytokinin regulation of nitrate reductase (NR) activity, the influence of benzyladenine (BA) on the level of NR transcript was studied in etiolated barley leaves using a barley NR cDNA as a probe. Northern blot analyses of the levels of NR poly (A)+ RNA indicate that the amount present is proportional to the concentration of BA (2 x 10(-8) to 2 x 10(-4) M) applied to the leaves. Enhancement of NR mRNA by 2 x 10(-5) M BA was clearly detected after 15 minutes of exposure of the leaves to light. The enhancement is cytokinin-specific and adenine is ineffective. Brief treatment with the protein synthesis inhibitor, cycloheximide, inhibited BA-enhanced NR activity but did not inhibit BA-enhanced NR transcript level, thus the enhancement was independent of concurrent protein synthesis. Nuclear runoff transcription studies showed that the enhancement of NR mRNA was at least partially due to increased transcription rates.