Heritable gene editing in tomato through viral delivery of isopentenyl transferase and single-guide RNAs to latent axillary meristematic cells.

Realizing the full potential of genome editing for crop improvement has been slow due to inefficient methods for reagent delivery and the reliance on tissue culture for creating gene-edited plants. RNA viral vectors offer an alternative approach for delivering gene engineering reagents and bypassing the tissue culture requirement. Viruses, however, are often excluded from the shoot apical meristem, making virus-mediated gene editing inefficient in some species. Here, we developed effective approaches for generating gene-edited shoots in Cas9-expressing transgenic tomato plants using RNA virus-mediated delivery of single-guide RNAs (sgRNAs). RNA viral vectors expressing sgRNAs were either delivered to leaves or sites near axillary meristems. Trimming of the apical and axillary meristems induced new shoots to form from edited somatic cells. To further encourage the induction of shoots, we used RNA viral vectors to deliver sgRNAs along with the cytokinin biosynthesis gene, isopentenyl transferase. Abundant, phenotypically normal, gene-edited shoots were induced per infected plant with single and multiplexed gene edits fixed in the germline. The use of viruses to deliver both gene editing reagents and developmental regulators overcomes the bottleneck in applying virus-induced gene editing to dicotyledonous crops such as tomato and reduces the dependency on tissue culture.

Moniliophthora perniciosa, the causal agent of witches' broom disease of cacao, interferes with cytokinin metabolism during infection of Micro-Tom tomato and promotes symptom development.

Moniliophthora perniciosa causes witches' broom disease of cacao and inflicts symptoms suggestive of hormonal imbalance. We investigated whether infection of the tomato (Solanum lycopersicum) model system Micro-Tom (MT) by the Solanaceae (S)-biotype of Moniliophthora perniciosa, which causes stem swelling and hypertrophic growth of axillary shoots, results from changes in host cytokinin metabolism. Inoculation of an MT-transgenic line that overexpresses the Arabidopsis CYTOKININ OXIDASE-2 gene (35S::AtCKX2) resulted in a reduction in disease incidence and stem diameter. RNA-sequencing analysis of infected MT and 35S::AtCKX2 revealed the activation of cytokinin-responsive marker genes when symptoms were conspicuous. The expression of an Moniliophthora perniciosa tRNA-ISOPENTENYL-TRANSFERASE suggests the production of isopentenyladenine (iP), detected in mycelia grown in vitro. Inoculated MT stems showed higher levels of dihydrozeatin and trans-zeatin but not iP. The application of benzyladenine induced symptoms similar to infection, whereas applying the cytokinin receptor inhibitors LGR-991 and PI55 decreased symptoms. Moniliophthora perniciosa produces iP that might contribute to cytokinin synthesis by the host, which results in vascular and cortex enlargement, axillary shoot outgrowth, reduction in root biomass and an increase in fruit locule number. This strategy may be associated with the manipulation of sink establishment to favour infection by the fungus.

Natural variation in cytokinin maintenance improves salt tolerance in apple rootstocks.

Plants experiencing salt-induced stress often reduce cytokinin levels during the early phases of stress-response. Interestingly, we found that the cytokinin content in the apple rootstock "robusta" was maintained at a high level under salt stress. Through screening genes involved in cytokinin biosynthesis and catabolism, we found that the high expression levels of IPT5b in robusta roots were involved in maintaining the high cytokinin content. We identified a 42 bp deletion in the promoter region of IPT5b, which elevated IPT5b expression levels, and this deletion was linked to salt tolerance in robusta×M.9 segregating population. The 42 bp deletion resulted in the deletion of a Proline Response Element (ProRE), and our results suggest that ProRE negatively regulates IPT5b expression in response to proline. Under salt stress, the robusta cultivar maintains high cytokinin levels as IPT5b expression cannot be inhibited by proline due to the deletion of ProRE, leading to improve salt tolerance.

Differential Gene Expression in the Meristem and during Early Fruit Growth of Pisum sativum L. Identifies Potential Targets for Breeding.

For successful molecular breeding it is important to identify targets to the gene family level, and in the specific species of interest, in this case Pisum sativum L. The cytokinins have been identified as a key breeding target due to their influence on plant architecture, and on seed size and sink activity. We focused on the cytokinin biosynthetic gene family (the IPTs) and the gene family key to the destruction of cytokinins (the CKXs), as well as other gene families potentially affected by changing cytokinin levels. These included key meristem genes (WUS and BAM1) and the transporter gene families, sucrose transporters (SUTs) and amino acid permeases (AAPs). We used reverse transcription quantitative PCR (RT-qPCR) to monitor gene expression in the vegetative meristem and in pre- and post-fertilisation young pea fruits. PsWUS expression was specific to the shoot apical meristem while PsBAM1 was highly expressed in the shoot apical meristem (SAM) but was also expressed at a low level in the young fruit. Differential expression was shown between genes and within gene families for IPT, CKX, SUT, and AAP. PsCKX7 showed strong gene family member-specific expression in the SAM, and was also expressed in young pea fruits. We suggest that PsCKX7 is a potential target for downregulation via molecular breeding or gene editing.

Cytokinin: a key driver of seed yield.

The cytokinins have been implicated in many facets of plant growth and development including cell division and differentiation, shoot and root growth, apical dominance, senescence, fruit and seed development, and the response to biotic and abiotic stressors. Cytokinin levels are regulated by a balance between biosynthesis [isopentenyl transferase (IPT)], activation [Lonely Guy (LOG)], inactivation (O-glucosyl transferase), re-activation (ß-glucosidase), and degradation [cytokinin oxidase/dehydrogenase (CKX)]. During senescence, the levels of active cytokinins decrease, with premature senescence leading to a decrease in yield. During the early stages of fruit and seed development, cytokinin levels are transiently elevated, and coincide with nuclear and cell divisions which are a determinant of final seed size. Exogenous application of cytokinin, ectopic expression of IPT, or down-regulation of CKX have, on occasions, led to increased seed yield, leading to the suggestion that cytokinin may be limiting yield. However, manipulation of cytokinins is complex, not only because of their pleiotropic nature but also because the genes coding for biosynthesis and metabolism belong to multigene families, the members of which are themselves spatially and temporally differentiated. Previous research on yield of rice showed that plant breeders could directly target the cytokinins. Modern genome editing tools could be employed to target and manipulate cytokinin levels to increase seed yield with the concurrent aim of maintaining quality. However, how the cytokinin level is modified and whether IPT or CKX is targeted may depend on whether the plant is considered to be in a source-limiting environment or to be sink limited.

Expression patterns of Brassica napus genes implicate IPT, CKX, sucrose transporter, cell wall invertase, and amino acid permease gene family members in leaf, flower, silique, and seed development.

Forage brassica (Brassica napus cv. Greenland) is bred for vegetative growth and biomass production, while its seed yield remains to be improved for seed producers without affecting forage yield and quality. Cytokinins affect seed yield by influencing flower, silique and seed number, and seed size. To identify specific cytokinin gene family members as targets for breeding, as well as genes associated with yield and/or quality, a B. napus transcriptome was obtained from a mixed sample including leaves, flower buds and siliques of various stages. Gene families for cytokinin biosynthesis (BnIPT1, 2, 3, 5, 7, 8 and 9), cytokinin degradation (BnCKX1 to BnCKX7), cell wall invertase (BnCWINV1 to BnCWINV6), sugar transporter (BnSUT1 to BnSUT6) and amino acid permease (BnAAP1 to BnAAP8) were identified. As B. napus is tetraploid, homoeologues of each gene family member were sought. Using multiple alignments and phylogenetic analysis, the parental genomes of the two B. napus homoeologues could be differentiated. RT-qPCR was then used to determine the expression of gene family members and their homoeologues in leaves, flowers, siliques and seeds of different developmental stages. The expression analysis showed both temporal and organ-specific expression profiles among members of these multi-gene families. Several pairs of homoeologues showed differential expression, both in terms of level of expression and differences in temporal or organ-specificity. BnCKX2 and 4 were identified as targets for TILLING, EcoTILLING and MAS.

Knockdown of LjIPT3 influences nodule development in Lotus japonicus.

Cytokinins play important roles in legume-rhizobia symbiosis. Here we report isolation of six genes encoding isopentenyl transferase (IPT) from Lotus japonicus, which catalyze the rate-limiting step of cytokinin biosynthesis. The LjIPT3 gene was found to be up-regulated in infected roots and mature nodules. Histochemical analysis demonstrated expression of Pro(LjIPT3):GUS (ß-glucuronidase) in vegetative and reproductive organs, and was especially high in the vascular bundles of roots. When inoculated with Mesorhizobium loti MAFF303099, LjIPT3 was undetectable in the nodule primordia and developing nodules, and later it was expressed only in the vascular bundles of mature nodules. In addition, knockdown of LjIPT3 (LjIPT3i) by RNA interference reduced levels of endogenous cytokinins, affected plant development and accelerated Chl degradation during dark-induced leaf senescence. Compared with the wild type, LjIPT3i plants produced fewer infection threads and nodules. In addition, expression of downstream nodulation-related transcription factor genes LjNSP1, LjNSP2 and LjNIN decreased dramatically in LjIPT3i plants. These results suggest that LjIPT3 regulates the CRE1-dependent cytokinin pathway, affecting nodule initiation and thereby influencing the number of infection threads and nodules. Detection of nitrogenase activity and observation of nodule structure showed that endogenous cytokinins are required for full development of the infected cells in mature nodules by preventing early senescence. Therefore, our results indicate that the LjIPT3 gene product is required for nodule initiation and development, and does not appear to be involved in early infection events.

Proteome and metabolome profiling of cytokinin action in Arabidopsis identifying both distinct and similar responses to cytokinin down- and up-regulation.

In plants, numerous developmental processes are controlled by cytokinin (CK) levels and their ratios to levels of other hormones. While molecular mechanisms underlying the regulatory roles of CKs have been intensely researched, proteomic and metabolomic responses to CK deficiency are unknown. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S>GR>HvCKX2) and agrobacterial isopentenyl transferase (CaMV35S>GR>ipt) constructs were profiled to elucidate proteome- and metabolome-wide responses to down- and up-regulation of CK levels, respectively. Proteome profiling identified >1100 proteins, 155 of which responded to HvCKX2 and/or ipt activation, mostly involved in growth, development, and/or hormone and light signalling. The metabolome profiling covered 79 metabolites, 33 of which responded to HvCKX2 and/or ipt activation, mostly amino acids, carbohydrates, and organic acids. Comparison of the data sets obtained from activated CaMV35S>GR>HvCKX2 and CaMV35S>GR>ipt plants revealed unexpectedly extensive overlaps. Integration of the proteomic and metabolomic data sets revealed: (i) novel components of molecular circuits involved in CK action (e.g. ribosomal proteins); (ii) previously unrecognized links to redox regulation and stress hormone signalling networks; and (iii) CK content markers. The striking overlaps in profiles observed in CK-deficient and CK-overproducing seedlings might explain surprising previously reported similarities between plants with down- and up-regulated CK levels.

MdIPT1, an adenylate isopentenyltransferase coding gene from Malus domestica, is involved in branching and flowering regulation.

Flowering and shoot branching are significant agricultural traits for apple tree breeding. Cytokinin metabolism and signaling pathways play a crucial role in plant development. However, little is known about cytokinin biosynthetic molecular mechanism and function involved in apple flowering and branching. In this study, an adenylate isopentenyl transferase coding gene MdIPT1, homologous to AtIPT3/AtIPT5 in Arabidopsis thaliana, was identified. MdIPT1 was highly expressed in apple floral and axillary buds and was dramatically up-regulated during floral induction and axillary bud outgrowth. The promoter of MdIPT1 showed high activity in multiple tissues and responded to different hormone treatments. The MdIPT1-overexpressing Arabidopsis showed a multi-branching and early-flowering phenotype, with elevated endogenous cytokinin levels and altered expression of genes related to branching and flower formation. Overexpression of MdIPT1 confers the growth vigor of transgenic apple callus on a CKs-deficient medium. Our findings suggest that MdIPT1 is a positive regulator involved in branching and flowering. The data presented herein provide extensive research results on MdIPT1 and will contribute to molecular breeding for new apple varieties.

Comparative analysis of adenylate isopentenyl transferase genes in plant growth-promoting bacteria and plant pathogenic bacteria.

Cytokinin is a major phytohormone that has been used in agriculture as a plant-growth stimulating compound since its initial discovery in the 1960s. Isopentenyl transferase (IPT) is a rate-limiting enzyme for cytokinin biosynthesis, which is produced by plants as well as bacteria including both plant pathogenic species and plant growth-promoting bacteria (PGPB). It has been hypothesized that there may be differences in IPT function between plant pathogens and PGPB. However, a comprehensive comparison of IPT genes between plant pathogenic and PGPB species has not been performed. Here, we performed a global comparison of IPT genes across bacteria, analyzing their DNA sequences, codon usage, phyletic distribution, promoter structure and genomic context. We found that adenylate type IPT genes are highly specific to plant-associated bacteria and subdivide into two major clades: clade A, largely composed of proteobacterial plant pathogens; and clade B, largely composed of actinomycete PGPB species. Besides these phylogenetic differences, we identified several genomic features that suggest differences in IPT regulation between pathogens and PGPB. Pathogen-associated IPTs tended to occur in predicted virulence loci, whereas PGPB-associated IPTs tended to co-occur with other genes involved in cytokinin metabolism and degradation. Pathogen-associated IPTs also showed elevated gene copy numbers, significant deviation in codon usage patterns, and extended promoters, suggesting differences in regulation and activity levels. Our results are consistent with the hypothesis that differences in IPT regulation and activity exist between plant pathogens and PGPB, which determine their effect on plant host phenotypes through the control of cytokinin levels.

MiaA (Rv2727c) mediated tRNA isopentenylation of Mycobacterium tuberculosis H37Rv.

tRNA modifications play a significant role in the structural stability as well as translational fidelity in all organisms from bacteria to humans. They also play a major role in bacterial physiology by regulating translation in response to various environmental stresses. Modifications coming at the anticodon-stem loop (ASL) are particularly important as they stabilize codon-anticodon interactions, ensuring accuracy and speed in decoding mRNAs Addition of isopentenyl group (i6A) at A37 position by tRNA isopentenyltransferase (MiaA) is a well conserved modification from bacteria to human. We studied M. tuberculosis MiaA from strain H37Rv and identified the target tRNAs for this modification based on the A36A37A38 motif. i6A modification of target tRNAs tRNALeuCAA, tRNAPheGAA, tRNATrpCCA and tRNASerCGA were further confirmed by isopentenyltransferase assay providing the substrate DMAPP and recombinant MiaA enzyme.

Genome-Wide Identification and Expression Analysis of Isopentenyl transferase Family Genes during Development and Resistance to Abiotic Stresses in Tea Plant (Camellia sinensis).

The tea plant is an important economic crop and is widely cultivated. Isopentenyl transferase (IPT) is the first and rate-limiting enzyme of cytokinin (CK) signaling, which plays key roles in plant development and abiotic stress. However, the IPT gene family in tea plants has not been systematically investigated until now. The phylogenetic analyses, gene structures, and conserved domains were predicted here. The results showed that a total of 13 CsIPT members were identified from a tea plant genome database and phylogenetically classified into four groups. Furthermore, 10 CsIPT members belonged to plant ADP/ATP-IPT genes, and 3 CsIPTs were tRNA-IPT genes. There is a conserved putative ATP/GTP-binding site (P-loop motif) in all the CsIPT sequences. Based on publicly available transcriptome data as well as through RNA-seq and qRT-PCR analysis, the CsIPT genes which play key roles in the development of different tissues were identified, respectively. Furthermore, CsIPT6.2 may be involved in the response to different light treatments. CsIPT6.4 may play a key role during the dormancy and flush of the lateral buds. CsIPT5.1 may play important regulatory roles during the development of the lateral bud, leaf, and flower. CsIPT5.2 and CsIPT6.2 may both play key roles for increased resistance to cold-stress, whereas CsIPT3.2 may play a key role in improving resistance to high-temperature stress as well as drought-stress and rewatering. This study could provide a reference for further studies of CsIPT family's functions and could contribute to tea molecular breeding.

Biochemical and Structural Aspects of Cytokinin Biosynthesis and Degradation in Bacteria.

It has been known for quite some time that cytokinins, hormones typical of plants, are also produced and metabolized in bacteria. Most bacteria can only form the tRNA-bound cytokinins, but there are examples of plant-associated bacteria, both pathogenic and beneficial, that actively synthesize cytokinins to interact with their host. Similar to plants, bacteria produce diverse cytokinin metabolites, employing corresponding metabolic pathways. The identification of genes encoding the enzymes involved in cytokinin biosynthesis and metabolism facilitated their detailed characterization based on both classical enzyme assays and structural approaches. This review summarizes the present knowledge on key enzymes involved in cytokinin biosynthesis, modifications, and degradation in bacteria, and discusses their catalytic properties in relation to the presence of specific amino acid residues and protein structure.

Stress-induced expression of IPT gene in transgenic wheat reduces grain yield penalty under drought.

BACKGROUND: The heterologous expression of isopentenyl transferase (IPT) under the transcriptional control of the senescence-associated receptor-like kinase (SARK) promoter delayed cellular senescence and, through it, increased drought tolerance in plants. To evaluate the effect of pSARK::IPT expression in bread wheat, six independent transgenic events were obtained through the biolistic method and evaluated transgene expression, phenology, grain yield and physiological biomass components in plants grown under both drought and well-irrigating conditions. Experiments were performed at different levels: (i) pots and (ii) microplots inside a biosafety greenhouse, as well as under (iii) field conditions. RESULTS: Two transgenic events, called TR1 and TR4, outperformed the wild-type control under drought conditions. Transgenic plants showed higher yield under both greenhouse and field conditions, which was positively correlated to grain number (given by more spikes and grains per spike) than wild type. Interestingly, this yield advantage of the transgenic events was observed under both drought and well-watered conditions. CONCLUSIONS: The results obtained allow us to conclude that the SARK promoter-regulated expression of the IPT gene in bread wheat not only reduced the yield penalty produced by water stress but also led to improved productivity under well-watered conditions.

Plant Growth Regulators INCYDE and TD-K Underperform in Cereal Field Trials.

Using plant growth regulators to alter cytokinin homeostasis with the aim of enhancing endogenous cytokinin levels has been proposed as a strategy to increase yields in wheat and barley. The plant growth regulators INCYDE and CPPU inhibit the cytokinin degrading enzyme cytokinin oxidase/dehydrogenase (CKX), while TD-K inhibits the process of senescence. We report that the application of these plant growth regulators in wheat and barley field trials failed to enhance yields, or change the components of yields. Analyses of the endogenous cytokinin content showed a high concentration of trans-zeatin (tZ) in both wheat and barley grains at four days after anthesis, and statistically significant, but probably biologically insignificant, increases in cisZ-O-glucoside, along with small decreases in cZ riboside (cZR), dihydro Z (DHZ), and DHZR and DHZOG cytokinins, following INCYDE application to barley at anthesis. We discuss possible reasons for the lack of efficacy of the three plant growth regulators under field conditions and comment on future approaches to manipulating yield in the light of the strong homeostatic mechanisms controlling endogenous cytokinin levels.

Employing phytosulfokine α (PSKα) for delaying broccoli florets yellowing during cold storage.

The yellowing of florets limits the economic and nutritional value of broccoli during postharvest. We investigated mechanisms of action of 150 nM phytosulfokine α (PSKα) for delaying florets yellowing in broccoli during cold storage. Our results showed that SUMO E3 ligase (SIZ1) gene expression was higher in florets treated with PSKα, which may prevent endogenous H2O2 accumulation, resulting from the higher activity of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase. Besides, higher expression of methionine sulfoxide reductase and cysteine peroxiredoxin genes, concomitant with higher expression of heat shock proteins 70/90 genes, may arise from higherexpression of SIZ1 gene. Lower expression and activity of phospholipase D and lipoxygenase may be liable for membrane integrity protection featured by lower malondialdehyde accumulation in florets treated with PSKα. Additionally,florets treated with PSKα exhibited higher endogenous cytokinin accumulation which may arise from higher expression of isopentenyl transferase gene, concomitant with lower expression of cytokinin oxidase gene.

The Hulks and the Deadpools of the Cytokinin Universe: A Dual Strategy for Cytokinin Production, Translocation, and Signal Transduction.

Cytokinins are plant hormones, derivatives of adenine with a side chain at the N6-position. They are involved in many physiological processes. While the metabolism of trans-zeatin and isopentenyladenine, which are considered to be highly active cytokinins, has been extensively studied, there are others with less obvious functions, such as cis-zeatin, dihydrozeatin, and aromatic cytokinins, which have been comparatively neglected. To help explain this duality, we present a novel hypothesis metaphorically comparing various cytokinin forms, enzymes of CK metabolism, and their signalling and transporter functions to the comics superheroes Hulk and Deadpool. Hulk is a powerful but short-lived creation, whilst Deadpool presents a more subtle and enduring force. With this dual framework in mind, this review compares different cytokinin metabolites, and their biosynthesis, translocation, and sensing to illustrate the different mechanisms behind the two CK strategies. This is put together and applied to a plant developmental scale and, beyond plants, to interactions with organisms of other kingdoms, to highlight where future study can benefit the understanding of plant fitness and productivity.

Light Regulates the Cytokinin-Dependent Cold Stress Responses in Arabidopsis.

To elucidate the effect of light intensity on the cold response (5°C; 7 days) in Arabidopsis thaliana, we compared the following parameters under standard light (150 µmol m-2 s-1), low light (20 µmol m-2 s-1), and dark conditions: membrane damage, photosynthetic parameters, cytokinin oxidase/dehydrogenase (CKX) activity, phytohormone levels, and transcription of selected stress- and hormone-related genes and proteome. The impact of cytokinins (CKs), hormones directly interacting with the light signaling pathway, on cold responses was evaluated using transformants overexpressing CK biosynthetic gene isopentenyl transferase (DEX:IPT) or CK degradation gene HvCKX2 (DEX:CKX) under a dexamethasone-inducible promoter. In wild-type plants, cold treatment under light conditions caused down-regulation of CKs (in shoots) and auxin, while abscisic acid (ABA), jasmonates, and salicylic acid (SA) were up-regulated, especially under low light. Cold treatment in the dark strongly suppressed all phytohormones, except ABA. DEX:IPT plants showed enhanced stress tolerance associated with elevated CK and SA levels in shoots and auxin in apices. Contrarily, DEX:CKX plants had weaker stress tolerance accompanied by lowered levels of CKs and auxins. Nevertheless, cold substantially diminished the impact from the inserted genes. Cold stress in dark minimized differences among the genotypes. Cold treatments in light strongly up-regulated stress marker genes RD29A, especially in roots, and CBF1-3 in shoots. Under control conditions, their levels were higher in DEX:CKX plants, but after 7-day stress, DEX:IPT plants exhibited the highest transcription. Transcription of genes related to CK metabolism and signaling showed a tendency to re-establish, at least partially, CK homeostasis in both transformants. Up-regulation of strigolactone-related genes in apices and leaves indicated their role in suppressing shoot growth. The analysis of leaf proteome revealed over 20,000 peptides, representing 3,800 proteins and 2,212 protein families (data available via ProteomeXchange, identifier PXD020480). Cold stress induced proteins involved in ABA and jasmonate metabolism, antioxidant enzymes, and enzymes of flavonoid and glucosinolate biosynthesis. DEX:IPT plants up-regulated phospholipase D and MAP-kinase 4. Cold stress response at the proteome level was similar in all genotypes under optimal light intensity, differing significantly under low light. The data characterized the decisive effect of light-CK cross-talk in the regulation of cold stress responses.

Targeting Cytokinin Homeostasis in Rapid Cycling Brassica rapa with Plant Growth Regulators INCYDE and TD-K.

Modifying the cytokinin content in plants is a means of improving plant productivity. Here, we report the development and biological activity of compound TD-K (1-(furan-2-ylmethyl)-3-(1,2,3-thiadiazol-5-yl)urea)which is related to thidiazuron. TD-K-which exhibited extremely high antisenescence activity in the wheat leaf bioassay-and INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine)-a plant growth regulator reported to inhibit cytokinin oxidase/dehydrogenase (CKX), an enzyme involved in the degradation of the plant hormone cytokinin-were selected for investigation of their effects on the model plant Rapid Cycling Brassica rapa (RCBr). We monitored the expression of BrCKX and isopentenyl transferase (BrIPT), which codes for the key cytokinin biosynthesis enzyme, in developing leaves following INCYDE and TD-K application. Growth room experiments revealed that INCYDE increased RCBr seed yield per plant, but only when applied multiple times and when grown in 5 mM KNO3. Expression in control leaves showed transient, high levels of expression of BrCKX and BrIPT at true leaf appearance. Following INCYDE application, there was a rapid and strong upregulation of BrCKX3, and a transient downregulation of BrIPT1 and BrIPT3. Interestingly, the upregulation of BrCKX3 persisted in a milder form throughout the course of the experiment (16 days). TD-K also upregulated BrCKX3. However, in contrast to INCYDE, this effect disappeared after two days. These results suggest that both compounds (CKX inhibitor and cytokinin TD-K) influenced cytokinin homeostasis in RCBr leaves, but with different mechanisms.