Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 2017 Jan 17;173(3):1783–1797. doi: 10.1104/pp.16.01903

Gain-of-Function Mutants of the Cytokinin Receptors AHK2 and AHK3 Regulate Plant Organ Size, Flowering Time and Plant Longevity1

Isabel Bartrina 1,2,3,2, Helen Jensen 1,2,3,2, Ondřej Novák 1,2,3, Miroslav Strnad 1,2,3, Tomáš Werner 1,2,3,*, Thomas Schmülling 1,2,3,*
PMCID: PMC5338655  PMID: 28096190

Gain-of-function variants of two Arabidopsis cytokinin receptors show the impact of cytokinin on regulating shoot organ size, flowering time, plant longevity, and seed yield.

Abstract

The phytohormone cytokinin is a regulator of numerous processes in plants. In Arabidopsis (Arabidopsis thaliana), the cytokinin signal is perceived by three membrane-located receptors named ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and AHK4/CRE1. How the signal is transmitted across the membrane is an entirely unknown process. The three receptors have been shown to operate mostly in a redundant fashion, and very few specific roles have been attributed to single receptors. Using a forward genetic approach, we isolated constitutively active gain-of-function variants of the AHK2 and AHK3 genes, named repressor of cytokinin deficiency2 (rock2) and rock3, respectively. It is hypothesized that the structural changes caused by these mutations in the sensory and adjacent transmembrane domains emulate the structural changes caused by cytokinin binding, resulting in domain motion propagating the signal across the membrane. Detailed analysis of lines carrying rock2 and rock3 alleles revealed how plants respond to locally enhanced cytokinin signaling. Early flowering time, a prolonged reproductive growth phase, and, thereby, increased seed yield suggest that cytokinin regulates various aspects of reproductive growth. In particular, it counteracts the global proliferative arrest, a correlative inhibition of maternal growth by seeds, an as yet unknown activity of the hormone.

The phytohormone cytokinin regulates numerous developmental processes, including cell proliferation and differentiation, shoot and root growth, seed germination, and leaf senescence (Werner and Schmülling, 2009; Kieber and Schaller, 2014; Zürcher and Müller, 2016). Cytokinin signaling is mediated by a phosphorelay system that resembles bacterial two-component signaling systems (Hwang and Sheen, 2001; Müller and Sheen, 2007). In Arabidopsis (Arabidopsis thaliana), there are three membrane-spanning His protein kinases (AHKs) that serve as cytokinin receptors, AHK2, AHK3, and AHK4/CRE1 (named AHK4 in the following; Inoue et al., 2001; Ueguchi et al., 2001; Yamada et al., 2001). The hormone is recognized and bound by an ∼270-amino acid extracytoplasmic binding domain called the CHASE domain (for cyclin His kinase-associated sensory). This domain is flanked by two transmembrane domains and followed toward the C-terminal end on the cytoplasmic side by a His kinase and a receiver domain (Steklov et al., 2013). The three-dimensional structure of the CHASE domain of AHK4 has been resolved by X-ray crystallography (Hothorn et al., 2011). This has revealed that the receptor acts as a dimer and that the hormone is bound by a Per-Arnt-Sim (PAS)-like domain. A simple size-exclusion mechanism allows only for the binding of the free cytokinin bases, which are isopentenyladenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin. Binding of bulkier metabolites such as ribosides and nucleotides is prohibited (Hothorn et al., 2011). This result has been confirmed by an in planta cytokinin receptor-binding assay (Lomin et al., 2015). However, it is clear that the three cytokinin receptors have different affinities for the different cytokinin bases (Suzuki et al., 2001; Spíchal et al., 2004; Romanov et al., 2006; Stolz et al., 2011; Lomin et al., 2015). Interestingly, the ligand affinity of AHK2 resembles that of AHK4, while AHK3 differs from these in its lower affinity to iP (Romanov et al., 2006; Stolz et al., 2011).

The bulk of cytokinin receptors is located in the endoplasmic reticulum (Caesar et al., 2011; Wulfetange et al., 2011; Lomin et al., 2015). Upon ligand binding, the signal is transmitted via an as yet unknown mechanism across the membrane, the cytoplasmic kinase domain is activated, and the protein autophosphorylates at the His residue. The high-energy phosphoryl group is next transferred within the same molecule to the Asp residue of the receiver domain and from there to His phosphotransfer proteins. These translocate to the nucleus, where they transfer the phosphoryl group to B-type response regulators, which act as transcription factors and orchestrate downstream responses (Müller and Sheen, 2007). In Arabidopsis, the transcript abundance of numerous genes is altered within minutes in response to a cytokinin stimulus (Brenner et al., 2012; Bhargava et al., 2013; Brenner and Schmülling, 2015).

Loss-of-function mutations of single cytokinin receptor genes have no or only weak effects on plant growth, indicating strong functional redundancy (Higuchi et al., 2004; Nishimura et al., 2004; Riefler et al., 2006). Similarly, simultaneous mutation of AHK2 or AHK3 together with AHK4 has only mild effects on development, suggesting that both AHK2 and AHK3 alone are sufficient to mediate the central functions of cytokinin. In contrast, ahk2 ahk3 mutants are dwarfed plants showing stronger root growth, demonstrating the importance of AHK2 and AHK3 in regulating vegetative plant growth (Riefler et al., 2006). Additional comprehensive examination of the cytokinin receptor genes also revealed some specialization in cytokinin receptor function (Heyl et al., 2012). For example, it was shown that AHK4 alone plays a role in embryonic root development, phosphate starvation response, and sulfate assimilation (Mähönen et al., 2000; Maruyama-Nakashita et al., 2004; Franco-Zorrilla et al., 2005). Furthermore, AHK3 plays a predominant role in regulating leaf senescence and cell differentiation in the transition zone of the root meristem (Kim et al., 2006; Riefler et al., 2006; Dello Ioio et al., 2007). No specific function has been shown for AHK2 so far.

The functional overlap is particularly high for AHK2 and AHK3, which both contribute to mediate a large number of developmental cytokinin functions, from seed germination to the regulation of shoot growth, and also responses to abiotic stresses, including drought (Tran et al., 2007), cold (Jeon et al., 2010), and high light (Cortleven et al., 2014). The mostly redundant action of AHK2 and AHK3 is intriguing and raises the question of why both genes with apparently similar functions have been conserved during evolution. Consistently, AHK2 and AHK3 have largely overlapping expression domains, with both being expressed predominantly in shoots (Higuchi et al., 2004). Stolz et al. (2011) showed that both AHK2 and AHK3 activate the cytokinin response in leaf mesophyll cells (AHK4 does not) but that only AHK3 mediates a response in stomata cells. Interestingly, promoter-swap and domain-swap analyses have shown that AHK4 can functionally replace AHK2 but not AHK3 (Stolz et al., 2011).

In view of the mostly redundant action of the AHK2 and AHK3 receptors, it might be informative to study gain-of-function mutants to compare receptor activities. In this work, we report on novel gain-of-function mutants of AHK2 and AHK3 named repressor of cytokinin deficiency2 (rock2) and rock3, respectively. These mutants were identified in a screen for suppressor mutants of the cytokinin deficiency syndrome displayed by 35S:CKX1-overexpressing (CKX1ox) plants (Niemann et al., 2015). Reversion of the cytokinin deficiency phenotype was due to the constitutive activity of the mutant receptors. The receptor variants do have differential impacts on individual phenotypic traits and, thus, are informative about cytokinin signaling during plant development. These results yield new information on the functions of these evolutionarily closely related receptors and highlight their roles in regulating flowering time and plant longevity. Given the promoting effect of the rock2 and rock3 alleles on shoot organ growth and, in particular, seed yield, we propose their potential value for biotechnological approaches.

RESULTS

rock2 and rock3 Suppress the Cytokinin Deficiency Phenotype

To identify the molecular factors required for establishing the cytokinin deficiency syndrome displayed by CKX1ox plants (Werner et al., 2003), we searched for suppressor mutants reverting the dwarf shoot phenotype of CKX1ox plants (Niemann et al., 2015). Among others, two mutants named rock2 and rock3, which grew larger than CKX1ox (Fig. 1, A and C), were identified and selected for further study. Genetic analysis showed that rock2 and rock3 are two dominant second site mutations (Supplemental Table S1). The reversion of the cytokinin-deficient phenotype was already obvious early after germination. In the CKX1ox background, rock2 and rock3 developed strongly enlarged cotyledons with longer petioles, which even exceeded the size of wild-type cotyledons (Fig. 1B). rock2 CKX1ox and rock3 CKX1ox plants developed larger rosette leaves, grew taller inflorescence stems with more flowers (Fig. 1, A and C), and the flowers of both suppressor lines were enlarged (Fig. 1D). In addition, both mutations suppressed the late-flowering phenotype of CKX1ox plants under long-day conditions (Werner et al., 2003). The rescue was partial in the case of rock3 CKX1ox, whereas rock2 CKX1ox flowered even earlier than wild-type plants (Fig. 1E). Under short-day conditions, the flowering transition defect of CKX1ox plants was more severe as these plants remained in the vegetative stage. Interestingly, only the rock2 mutation was able to suppress the nonflowering phenotype of CKX1ox under short-day conditions (Fig. 1F). In contrast, rock3 CKX1ox still failed to flower under short-day conditions. This indicates that rock2 particularly regulates processes associated with the change from vegetative to reproductive development under different light periods.

Figure 1.

Figure 1.

Open in a new tab

The rock2 and rock3 mutations suppress the CKX1ox phenotype. A, Morphology of wild-type (WT), CKX1ox, rock2 CKX1ox, and rock3 CKX1ox plants at the rosette stage. Plants were grown for 25 d under long-day conditions. B, rock2 CKX1ox and rock3 CKX1ox seedlings have larger cotyledons than wild-type and CKX1ox seedlings. The photographs were taken 10 d after germination. C, Adult phenotypes of 46-d-old wild-type, CKX1ox, rock2 CKX1ox, and rock3 CKX1ox plants. D, Flowers of wild-type, CKX1ox, rock2 CKX1ox, and rock3 CKX1ox plants (from left to right). E and F, Flowering time of wild-type and mutant plants grown under long-day (E) or short-day (F) conditions. Crosses indicate that no transition to flowering occurred (n = 20). DAG, Days after germination. G, Relative leaf chlorophyll content of the fourth and fifth leaves of 3-week-old soil-grown plants after 7 d in the dark. Chlorophyll content before the start of dark incubation was set to 100% (n = 3). H, Root elongation of seedlings between day 3 and day 9 after germination (n ≥ 25). Error bars represent sd. The statistical significance of differences was calculated by two-way ANOVA. *, P < 0.01 compared with the wild type; °, P < 0.05 compared with CKX1ox plants.

Cytokinin is known to delay leaf senescence (Richmond and Lang, 1957; Gan and Amasino, 1995; Riefler et al., 2006), and, consistently, leaves of CKX1ox plants showed reduced chlorophyll retention in a detached leaf assay (Fig. 1G). Both rock2 and rock3 retarded the breakdown of chlorophyll of CKX1ox plants during dark-induced senescence compared with wild-type and CKX1ox plants. After 7 d in the dark, the leaf chlorophyll content was reduced by almost 80% in the wild type and by 90% in CKX1ox plants, while it was decreased only by 65% and 35% in rock2 CKX1ox and rock3 CKX1ox mutants, respectively. This indicates that rock3 plays a more prevalent role than rock2 in delaying dark-induced leaf senescence.

To study whether rock2 and rock3 also eventually exert a differential influence on root development, we compared primary root elongation in the mutants and the wild type. Figure 1H shows that the roots of rock2 CKX1ox seedlings displayed a complete reversion, and those of rock3 CKX1ox seedlings showed a partial reversion, of the enhanced primary root elongation caused by the reduced cytokinin content of CKX1ox seedlings (Werner et al., 2003).

rock2 and rock3 Increase Sensitivity toward Exogenous Cytokinin

What could be the cause for the suppression of the cytokinin deficiency phenotype of CKX1ox plants? Analysis of the expression of the CKX1 transgene in both the rock2 and rock3 mutants showed that it was not affected by the mutation (Fig. 2A). In addition, outcrossing the 35S:CKX1 transgene from the rock2 CKX1ox or rock3 CKX1ox background revealed that the transgene was functionally intact (data not shown). Comparison of the cytokinin content of wild type, CKX1ox, rock2, and rock3 seedlings showed that the mutations did not increase the endogenous cytokinin content compared with CKX1ox (Fig. 2B; Supplemental Table S2). This suggested that a mechanism different from interference with 35S:CKX1 gene expression or metabolic compensation must be the cause of the phenotypic reversion.

Figure 2.

Figure 2.

Open in a new tab

rock2 and rock3 mutations alter cytokinin sensitivity. A, Real-time quantitative (q)PCR analysis of CKX1 transcript levels in seedlings grown in vitro for 10 d. Expression values were normalized to PP2AA2, and expression in the wild type (WT) was set to 1. Values are averages of three biological replicates ± se. B, Total cytokinin (CK) contents of 2-week-old seedlings. Contents of individual cytokinin metabolites are shown in Supplemental Table S1. Error bars represent sd. *, P < 0.05 compared with the wild type as calculated by two-way ANOVA. FW, Fresh weight. C, Phenotypes of 14-d-old seedlings grown on medium without (−CK) or with 25 nm benzyladenine (BA; +CK). rock2 CKX1ox and rock3 CKX1ox develop pale yellow leaves and show reduced shoot growth.

Next, we analyzed whether rock2 and rock3 change the plants’ sensitivity to exogenously applied cytokinin. Figure 2C shows that wild-type seedlings grew smaller on medium containing 25 nm BA; however, with the exception of a few yellowing leaves, most leaves stayed green. CKX1ox seedlings were less sensitive and grew on BA-containing medium similar as on medium without BA. In contrast, rock2 CKX1ox and rock3 CKX1ox seedlings showed strong hypersensitive reactions to cytokinin. They stayed smaller than control plants grown on standard medium and formed yellow leaves, which is a typical reaction to high exogenous cytokinin concentrations (Ainley et al., 1993). The altered growth responses indicate that rock2 and rock3 enhance the plants’ cytokinin sensitivity and suggest that the altered sensitivity may be causal for the suppression of the cytokinin-deficient phenotype.

rock2 and rock3 Encode Novel Dominant Gain-of-Function Alleles of AHK2 and AHK3

Intriguingly, genetic mapping of the rock2 and rock3 mutations revealed that both were located in genes coding for different cytokinin receptors. The rock2 mutation turned out to be a C-to-T transition in the seventh exon of the AHK2 (At5g35750) gene, leading to a semiconservative substitution of aliphatic Leu by aromatic Phe at amino acid position 552 (L552F) in the fourth predicted transmembrane domain (Fig. 3, A and B). The rock3 mutation was identified as a single base change from C to T in the second exon of AHK3 (At1g27320). This results in a nonconservative amino acid change from polar Thr to aliphatic Ile at position 179 (T179I) located close to the predicted cytokinin-binding domain of the AHK3 receptor (Fig. 3, A and B). A second independent allele, named rock3-2, was identified during the course of this work and caused the exchange of negatively charged Glu to positively charged Lys at position 182 (E182K) in close proximity to the T179I mutation (Fig. 3A). The first identified allele rock3-1 was used for all analyses described in this article and is called rock3 throughout.

Figure 3.

Figure 3.

Open in a new tab

rock2 and rock3 are novel gain-of-function alleles of the AHK2 and AHK3 genes. A, Two segments of the sequence alignment between the cytokinin receptor proteins AHK2, AHK3, and AHK4. The amino acid residues that are mutated (shown in red) in rock2 and rock3 are conserved in all three receptors. Full-length AHK protein sequences were aligned using ClustalW. B, Schematic representation of the AHK2 and AHK3 protein domains and the positions of amino acid substitutions (red arrows) corresponding to the rock2 and rock3 mutations. TM, Transmembrane. C to J, Genetic complementation of the CKX1ox phenotype by AHK2:rock2 and AHK3:rock3. Two independent transgenic AHK2:rock2 (G and H) and AHK3:rock3 (I and J) lines in the CKX1ox background are shown in comparison with the wild type (C), CKX1ox (D), rock2 CKX1ox (E), and rock3 CKX1ox (F) at 18 d after germination.

To prove that the identified mutant alleles of AHK2 and AHK3 are causal for the suppression of the cytokinin-deficient phenotype, the mutant gene variants were cloned under the control of their native promoters and transformed in the CKX1ox background. Transforming CKX1ox was inherently difficult; nevertheless, two independent transgenic AHK2:rock2 CKX1ox lines and three independent AHK3:rock3 CKX1ox lines were identified. All five transgenic lines showed a suppression of the cytokinin deficiency phenotype similar to or even stronger than the rock2 CKX1ox and rock3 CKX1ox suppressor lines (Fig. 3, C–J). This unequivocally confirmed that the phenotypes were caused by the mutations in the AHK2 and AHK3 genes. Thus, rock2 and rock3 encode two novel dominant gain-of-function variants of the cytokinin receptors AHK2 and AHK3.

rock2 and rock3 Enhance Cytokinin Signaling

The revertant morphology and the enhanced cytokinin sensitivity indicated that the rock2 and rock3 alleles might code for cytokinin receptors with increased signaling activity. To test whether the mutant receptors act in a cytokinin-independent manner or could be activated by lower cytokinin concentrations, we used the yeast (Saccharomyces cerevisiae) complementation assay described by Inoue et al. (2001). In this assay, AHK4 rescues, in a cytokinin-dependent manner, the survival of a yeast mutant that lacks the endogenous His kinase SLN1 (Inoue et al., 2001). It is known that the yeast Δsln1 strain carrying one of the other two cytokinin receptors (AHK2 or AHK3) can grow in the absence of cytokinin, but growth may be accelerated in the presence of cytokinin (Mähönen et al., 2006; Tran et al., 2007). However, under our laboratory conditions, the cytokinin-dependent faster growth rate of strains containing AHK2 or AHK3 receptor genes was not observed; therefore, we were not able to test the original rock2 or rock3 mutation in this system. Instead, we constructed rock2 and rock3 variants of AHK4 (named AHK4rock2 and AHK4rock3), as the positions affected by the rock2 and rock3 mutations are conserved in all three cytokinin receptors of Arabidopsis (Fig. 3A). Both rock variants of AHK4 suppressed the lethality of the Δsln1 mutation even without the addition of cytokinin to the medium, while the mutant carrying the wild-type AHK4 allele showed strictly cytokinin-dependent growth rescue (Fig. 4A). This shows that the His kinase activity of AHK4rock2 and AHK4rock3 is independent of cytokinin and that rock2 and rock3 may be constitutively active receptor proteins.

Figure 4.

Figure 4.

Open in a new tab

The rock2 and rock3 genes code for constitutively active receptors. A, Suppression of the lethal sln1∆ mutation by His kinase activity of AHK4rock2 and AHK4rock3. The growth of the sln1∆ yeast strain expressing AHK4rock2 or AHK4rock3 was independent of the presence of cytokinin. Error bars represent sd (n = 3). B, Relative expression levels of ARR5 in the rock mutants compared with wild type (WT). qPCR analysis was performed using 5-d-old seedlings grown in vitro. Values are averages of three biological replicates ± se. The statistical significance of differences compared with the wild type was calculated by ANOVA. *, P < 0.01. C to K, Histochemical analysis of ARR5:GUS activity in the wild-type (C–E), rock2 (F–H), and rock3 (I–K) backgrounds. Images show whole seedlings and shoot apices of 5-d-old plants stained overnight and primary root tips of 7-d-old plants after 30 min of staining (from left to right).

To test further the effects of rock2 and rock3 on cytokinin signaling output in planta, we compared the expression level of the well-established cytokinin reporter ARR5 in the wild type and both rock mutants. Figure 4B shows increased steady-state mRNA levels of ARR5 in rock2 and rock3, indicating enhanced cytokinin signaling in planta. Next, we introgressed the mutant alleles (without the 35S:CKX1 transgene) into a line harboring the ARR5:GUS reporter (D’Agostino et al., 2000). Figure 4, B to J, shows strongly increased GUS activity in all analyzed tissues of rock2 ARR5:GUS and rock3 ARR5:GUS seedlings. Seven days after germination increased, GUS activity was detected in the vascular tissue of cotyledons and leaves, the shoot apex, the root vasculature, the vascular procambium, and the root cap of the primary roots. rock2 caused an overall stronger increase in GUS activity than rock3 in all analyzed tissues. For example, rock2 mutants showed high GUS activity along the whole root, whereas in rock3 roots, the GUS signal was increased only weakly in the apical part of the root. This correlates well with the observed stronger capacity of rock2 to suppress the cytokinin deficiency phenotypes in shoot and root. The enhanced expression of the cytokinin response gene ARR5:GUS as an output of the cytokinin signaling system, together with the results of the yeast complementation assay, show that the constitutively active rock2 and rock3 activate more strongly the cytokinin signal transduction pathway.

Previous work has provided indications that cytokinin signaling is intimately linked to the metabolic homeostasis of the hormone (Riefler et al., 2006). The identified more active receptors provide an opportunity to test this link further. Therefore, we analyzed whether the endogenous cytokinin concentration responds to the enhanced cytokinin signaling and determined the cytokinin levels of wild-type, rock2, and rock3 seedlings and seedlings expressing AHK2:rock2. Both rock mutants and the transgenic line showed similar reductions of 45% to 56% of the total cytokinin content (Table I). Stronger differences between rock2 and rock3 were found for the biologically active free bases. Both the rock2 mutation and transgenic expression of AHK2:rock2 caused a decrease of about 40% for iP and cZ and of about 55% for tZ in comparison with the wild type (Table I). In rock3 seedlings, the levels of iP, tZ, and cZ were reduced only by 24%, 11%, and 14%, respectively, in comparison with wild-type seedlings. Taken together, the gain-of-function alleles of AHK2 and AHK3 have an impact on cytokinin homeostasis and lower the cytokinin content, supporting a feedback regulation of cytokinin metabolism by the cytokinin signaling pathway. Moreover, these results corroborate the hypothesis that these receptor variants display a high signaling activity independent of the steady-state cytokinin concentration.

Table I. Cytokinin content of rock2 and rock3.

Genotype iP iPR iPRMP iP9G tZ tZR tZRMP Z9G tZOG tZROG cZ cZR cZRMP cZOG cZROG
Wild type 1.91 ± 0.33 1.28 ± 0.18 2.18 ± 0.55 1.38 ± 0.09 0.71 ± 0.17 0.67 ± 0.19 1.29 ± 0.38 5.74 ± 0.90 5.66 ± 1.00 0.46 ± 0.10 0.23 ± 0.04 0.89 ± 0.20 4.40 ± 0.76 12.26 ± 1.82 3.95 ± 1.27
rock2 1.13 ± 0.26 1.01 ± 0.18 2.23 ± 0.03 0.72 ± 0.06 0.32 ± 0.04 0.24 ± 0.05 0.40 ± 0.12 1.56 ± 0.38 1.83 ± 0.36 0.20 ± 0.03 0.14 ± 0.02 0.75 ± 0.23 3.15 ± 0.12 3.65 ± 0.18 4.46 ± 0.13
AHK2:rock2 1.06 ± 0.29 1.25 ± 0.21 1.99 ± 0.31 0.93 ± 0.11 0.31 ± 0.01 0.23 ± 0.02 0.44 ± 0.09 1.88 ± 0.48 1.67 ± 0.35 0.26 ± 0.03 0.13 ± 0.01 0.93 ± 0.18 3.84 ± 0.73 6.11 ± 1.78 4.33 ± 0.81
rock3 1.45 ± 0.41 1.08 ± 0.21 1.31 ± 0.07 0.84 ± 0.10 0.63 ± 0.11 0.20 ± 0.02 0.23 ± 0.06 1.30 ± 0.10 0.98 ± 0.04 0.21 ± 0.01 0.20 ± 0.05 1.02 ± 0.20 3.63 ± 1.03 3.92 ± 0.82 3.13 ± 0.45
Open in a new tab

Arabidopsis seedlings (14 d after germination) were analyzed. iPR, N6-(Δ2-Isopentenyl)adenosine; iPRMP, N6-(Δ2-isopentenyl)adenosine 5′-monophosphate; iP9G, N6-(Δ2-isopentenyl)adenine 9-glucoside; tZR, trans-zeatin riboside; tZRMP, trans-zeatin riboside 5′-monophosphate; tZ9G, trans-zeatin 9-glucoside; tZOG, trans-zeatin O-glucoside; tZROG, trans-zeatin riboside O-glucoside; cZR, cis-zeatin riboside; cZRMP, cis-zeatin riboside 5′-monophosphate; cZOG, cis-zeatin O-glucoside; cZROG, cis-zeatin riboside O-glucoside. Data shown are pmol g−1 fresh weight ± sd; n = 3.

rock2 and rock3 Increase Shoot Growth and Leaf Size

The functional redundancy within the cytokinin receptor family makes it difficult to dissect the biological functions of individual receptor proteins. In this situation, gain-of-function receptor variants can be useful to obtain information on their individual functions. Therefore, we analyzed the consequences of the rock2 and rock3 mutations on growth and development in more detail using lines containing the original rock mutations in the wild-type background as well as transgenic lines expressing the rock2 and rock3 coding sequences under the control of their native promoters. Results are shown for homozygous progeny of lines AHK2:rock2-10 and AHK3:rock3-1. Two other independent lines, AHK2:rock2-7 and AHK3:rock3-5, showed similar results. qRT-PCR analysis showed that the levels of AHK2 transcripts were similar in the wild type, rock2, and the transgenic AHK2:rock2 line (Supplemental Fig. S1A). Likewise, the wild type, rock3, and the transgenic AHK3:rock3 line had comparable AHK3 transcript levels (Supplemental Fig. S1B).

Figure 5 shows different aspects of the shoot development in these plants. rock2 mutants and transgenic plants grew significantly taller than wild-type plants (Fig. 5, A and B). The inflorescence stems of 50-d-old wild-type and rock3 mutant plants measured 33.2 ± 1.3 cm and 35.4 ± 2.3 cm in height (7% increase). rock2 mutant plants grew 42%, transgenic AHK3:rock3 plants grew 60%, and transgenic AHK2:rock2 plants grew even 85% taller than wild-type plants. All the mutant and transgenic lines also had increased stem diameter (Fig. 5, C–E). Also, in this case, the transgenic lines showed the strongest increase, and the stem diameter was increased up to about 36% in comparison with the wild type. Transverse sections of the primary inflorescence stem showed that stem morphology was normal in all genotypes, but the number of cells in stems of transgenic lines was higher than in wild-type stems. Figure 5E shows as an example of transverse sections of wild-type and AHK3:rock3 inflorescence stems. The increased cell number could be due to a higher cambial activity, which has been shown previously to be regulated by cytokinin (Matsumoto-Kitano et al., 2008; Nieminen et al., 2008; Bartrina et al., 2011). This, however, will require further clarification.

Figure 5.

Figure 5.

Open in a new tab

Enhanced shoot growth of rock2 and rock3 mutants and transgenic plants. A and B, Height of the main inflorescence stem after the termination of flowering (50 d after germination). C and D, Primary inflorescence stems 3 cm above the rosette (C) and their diameter (D). Bar in C = 2 mm. The statistical significance of differences in B and D compared with the wild type (WT) was calculated by ANOVA. *, P < 0.001. Error bars represent sd. E, Stem sections of wild-type and AHK3:rock3 plants at the base of primary inflorescence stems. Sections were stained with Toluidine Blue.

Further inspection of the shoot phenotype revealed that rock2 and rock3 also positively regulate shoot lateral organ size. rock2 and rock3 seedlings developed strongly enlarged cotyledons (Fig. 6A), a phenotype that was more prominent in transgenic rock2 and rock3 lines. Later during development, rock2 and rock3 mutants formed larger rosette leaves (Fig. 6B), with the AHK2:rock2 and AHK3:rock3 transgenic lines forming the largest leaves. The biomass of rosette leaves was increased in rock2 and rock3 mutants (Fig. 6C). As organ size is influenced by cell number and cell expansion, we analyzed the size of epidermal cells in the sixth fully developed rosette leaf of wild-type and rock2 plants. The epidermal cell size of rock2 plants was slightly but not significantly reduced (Fig. 6D). This, together with the increased leaf surface (Fig. 6E), revealed that rock2 rosette leaves formed about 40% to 50% more epidermal cells compared with the wild type. In conclusion, the rock2-dependent changes in organ size are due to prolonged mitotic activity and/or faster cell proliferation during leaf growth.

Figure 6.

Figure 6.

Open in a new tab

Leaf phenotypes of rock2 and rock3 mutants and transgenic plants. A, Cotyledon size of 5-d-old seedlings. Bar = 1 mm. B, Cotyledons and rosette leaves in the order of appearance (from left to right) at 24 d after germination. Bar = 1 cm. C, Fresh weight of rosette leaves at 32 d after germination. D, Average size of abaxial epidermal cells of the sixth rosette leaf of wild-type (WT) and rock2 plants (n = 14). E, Leaf area of the sixth fully grown rosette leaf. The statistical significance of differences compared with the wild type was calculated by ANOVA. *, P < 0.001.

rock3 Prolongs the Life Span of Leaves

It is known that cytokinin delays leaf senescence (Gan and Amasino, 1995; Kim et al., 2006). We compared the natural senescence as well as the dark-induced senescence of detached leaves of rock2 and rock3 with that of the wild type. Visual examination of the sixth rosette leaves throughout their life spans showed that the onset of natural leaf senescence was delayed particularly in rock3 mutant plants (Fig. 7A). rock3 leaves had an ∼7-d-longer life span compared with wild-type leaves, whereas rock2 leaves showed only a slightly delayed leaf senescence, up to 2 d (Fig. 7A). These results were confirmed by measuring the photosynthetic efficiency of PSII (Fv/Fm), which was maintained about 4 d longer at a high level in leaves of rock2 and about 8 d longer in rock3 plants (Fig. 7B). Figure 7C shows that dark-induced leaf senescence also was strongly retarded in rock3 mutant plants. After 7 d in the dark, the chlorophyll content was reduced to 10% in wild-type and rock2 plants, whereas rock3 leaves were still green, with a remaining chlorophyll content of almost 60%. These results show that a gain-of-function mutation in the AHK3 cytokinin receptor significantly delays different senescence-associated symptoms in Arabidopsis. Enhanced activity of AHK2, on the other hand, has only a minor but still significant impact on delaying leaf senescence.

Figure 7.

Figure 7.

Open in a new tab

Natural and dark-induced leaf senescence. A, Age-dependent senescence phenotypes of the sixth leaf of the wild type (WT) and rock2/rock3 mutants grown under long-day conditions starting at 16 d after leaf emergence (DAE). B, Fv/Fm of the sixth leaf at the time points shown in A. C, Dark-induced senescence in a detached leaf assay. The chlorophyll content of the sixth leaf was examined after 7 d in the dark. The leaf chlorophyll content before the start of dark incubation was set at 100% for each genotype tested (n = 10). Error bars represent sd. The statistical significance of differences compared with the wild type was calculated by ANOVA. *, P < 0.001.

The strong delay of leaf senescence in rock3 mutants prompted us to compare rock3 with another AHK3 allele, ore12, reported to delay leaf senescence (Kim et al., 2006). Both the visual inspection and the Fv/Fm values showed significantly later onset of senescence in rock3 than in ore12 (Supplemental Fig. S2), indicating quantitatively different effects of these mutations on AHK3 receptor activity.

rock2 and rock3 Alter Flowering Time and Increase Flower Size and Seed Yield

Under long-day conditions, rock2 mutants and AHK2:rock2 transgenic lines flowered significantly earlier than the wild type, as indicated by their lower number of rosette leaves at flowering time (Fig. 8A). Likewise, the AHK3:rock3 transgenic plants, but not the rock3 mutant, flowered earlier. Interestingly, all mutant and transgenic lines also showed a markedly increased duration of flowering and increased total life span. Seven-week-old wild-type plants ceased to form new flowers, while rock genotypes continued to flower for at least 1 week and up to 2 weeks in the case of AHK2:rock2 transgenic plants (Fig. 8B).

Figure 8.

Figure 8.

Open in a new tab

rock2 and rock3 positively regulate flowering time and flower size. A, Number of rosette leaves at the start of flowering of plants grown under long-day conditions. B, rock2 and rock3 mutants and transgenic plants flower longer than the wild type (WT). Shown are days until the termination of flowering (n = 10). C, Flowers of rock2 and rock3 mutants and transgenic lines compared with the wild type. Bar length is 500 μm. D, Average size of abaxial epidermal cells of petals at stage 13 (Smyth et al., 1990; n = 5). E, Number of siliques on the main stem. Siliques, including unfilled and partially filled siliques, were counted after the end of flowering (n = 15). F, Seed yield of rock2 and rock3 mutants and transgenic lines compared with the wild type. The seed yield of the wild type was set to 100%. Error bars represent sd. The statistical significance of differences compared with the wild type was calculated by Student’s t test. *, P < 0.005.

The size of flowers was enhanced significantly in rock2 and rock3 mutants (Fig. 8C). To determine whether the increase in petal size was the result of increased cell proliferation, cell expansion, or both, abaxial petal epidermal cell size was analyzed exemplarily in AHK2:rock2 flowers. Figure 8D shows that the cell size was not altered significantly, indicating that an enhanced cell proliferation was the cause for the larger petals, similar to that found for rosette leaf size regulation (Fig. 6, D and E).

rock2, AHK2:rock2, and AHK3:rock3 plants formed more siliques than wild-type plants (Fig. 8E). The transgenic AHK2:rock2 line showed the largest increase, producing almost twice as many siliques as the wild type. Interestingly, despite the activity of cytokinin in regulating shoot meristem activity and size (Bartrina et al., 2011), microscopic analysis did not reveal an increased size of rock2 or rock3 inflorescence meristems (Supplemental Fig. S3). Consistently, the analysis of cytokinin signaling output in the inflorescence meristem of rock2 plants using the TCSn:GFP marker gene (Zürcher et al., 2013) showed no significant change in comparison with the wild type (Supplemental Fig. S3), suggesting a dampening of the increased receptor signaling in the inflorescence meristem. This indicates that the formation of more flowers and, consequently, of more siliques was due to a longer flowering phase. Analysis of seed yield did not show an increase in plants harboring a rock2 allele, which was probably accountable to heterostylous flowers displaying disproportionally elongated gynoecia in comparison with the stamen, which caused reduced self-fertilization (Supplemental Fig. S4). However, AHK3:rock3 plants lacking this morphological defect produced about 40% more seeds than wild-type plants. A similar result was found for a second transgenic rock3 line (AHK3:rock3-5), confirming that seed yield is positively influenced by the rock3 mutation.

rock2 and rock3 Reduce Root Growth

In contrast to its promotional role in shoot organs, cytokinin is a negative regulator of root development (Werner and Schmülling, 2009). As expected from the observed suppressor activity (Fig. 1H), root growth was inhibited in all tested rock seedlings grown under in vitro conditions (Fig. 9A). Primary root elongation was reduced by 37% and 28% in rock2 and rock3 seedlings, respectively. The transgenic rock lines AHK2:rock2-10 and AHK3:rock3-1 showed slightly milder root phenotypes, with reductions in root elongation of 23% and 18%, respectively (Fig. 9A). Consistently, root meristem size was decreased in both the rock2 and rock3 mutants (Fig. 9, B and C). The formation of lateral roots was strongly inhibited as well. On average, the number of lateral roots in wild-type plants was around 1.8 to 2.4 times greater than that of the rock mutants, with rock2 showing the strongest reduction of lateral root formation (Fig. 9D). The similar or even weaker consequences of transgenic rock gene expression compared with their original alleles contrasts with their generally stronger effects on the shoot phenotype.

Figure 9.

Figure 9.

Open in a new tab

The rock2 and rock3 mutants have reduced root systems. A, Elongation of primary roots between day 4 and day 12 after germination (n = 20). B, Root meristems of the wild type (WT), rock2, and rock3. Roots were analyzed 5 d after germination. White and black arrowheads indicate, respectively, the quiescent center and the start of the transition zone. Bars = 50 µm. C, Number of cortex cells between the quiescent center and the start of the transition zone (n = 10). D, Number of lateral roots at 12 d after germination (n = 20). Error bars represent sd. The statistical significance of differences compared with the wild type was calculated by Student’s t test. *, P < 0.005.

DISCUSSION

From the analysis of the rock2 and rock3 mutant alleles and transgenic lines carrying these alleles, we obtained valuable information about the cytokinin signaling mechanism and the roles of these redundantly acting receptors in regulating various facets of the plant’s phenotype. Both aspects are discussed in the following sections.

rock2 and rock3 Mutations Provide Insight into Transmembrane Signaling by Cytokinin Receptors

How the cytokinin signal is transmitted across the membrane is an entirely unknown process. The rock2 and rock3 mutations identified several amino acid residues that are relevant for transmembrane signaling. Presumably, they induce changes in the receptor structure resembling those induced by cytokinin binding to the CHASE domain and, thus, cause constitutive cytokinin signaling. The fact that the AHK4 receptors harboring rock2 and rock3 mutations conferred growth rescue of Δsln1 yeast to a great extent similar to that caused by cytokinin-induced signaling of wild-type AHK4 suggests that these mutations cause strong, if not maximal, activation of the receptors.

We isolated two independent rock3 mutations that are located in close proximity to each other in a region of the CHASE domain linking the long α-helical stalk domain with the membrane-distal (ligand-binding) PAS domain (Hothorn et al., 2011). More precisely, they are located in a slightly bent region comprising a 310-helix that connects helix α2 with the first β-strand of the membrane-distal PAS domain (Hothorn et al., 2011; Fig. 10). This region, which is not involved in ligand binding, could participate in the receptor domain movement expected to be triggered by cytokinin binding. The rock3 mutations, which alter evolutionarily highly conserved residues (Heyl et al., 2007; Steklov et al., 2013), may provoke a similar intramolecular movement.

Figure 10.

Figure 10.

Open in a new tab

Model of conformational changes associated with transmembrane signaling following cytokinin perception. The schematic topology of the sensory module of cytokinin receptors is based on the crystal structure of AHK4 (Hothorn et al., 2011). The N-terminal helices (α1, α2, and its neighboring 310-helix) of the CHASE domain are shown in orange, and the two PAS domains are depicted schematically. The model predicts that cytokinin binding causes reversible conformational changes (double-headed arrows), causing a piston-type displacement of the different subdomains and ultimately resulting in the transmission of the signal across the membrane. rock2 and rock3 mutations (arrows) are predicted to mimic those changes locking the receptor in a constitutively active conformation.

There is an intriguing structural similarity between the CHASE domain and the sensing domain of a bacterial methyl-accepting chemotaxis protein (MCP), which is a membrane-bound His kinase recognizing Ile. The sensory domains of both receptor types have a long α-helical stalk important to keep the receptor dimers together and to hold two PAS domains, of which the membrane-distal one binds the ligand (Hothorn et al., 2011; Liu et al., 2015). Analysis of the ligand-induced conformational changes of the PAS-sensing domain of MCP revealed that it likely signals by a piston-displacement mechanism (Liu et al., 2015). The signal-binding PAS domain fluctuates between a closed (ligand-bound) and open form, resulting in a conformational change of the proximal PAS domain and movement toward and away from the membrane. This movement is propagated, causing a displacement of the transmembrane helix toward the cytoplasm, thus generating a transmembrane signal (Liu et al., 2015). Considering the close structural similarity between cytokinin receptors and MCP as well as the fact that piston-type signaling is a common theme among other types of His kinases (Chervitz and Falke, 1996; Cheung and Hendrickson, 2009; Moore and Hendrickson, 2009; Bhate et al., 2015), we hypothesize that the CHASE domain also undergoes, upon ligand binding, structural changes emulating a piston-like domain motion resulting in transmembrane signaling (Fig. 10). Interestingly, the last β-strand of the membrane-proximal PAS domain is linked to the stalk helix by a disulfide bridge (Hothorn et al., 2011), which might limit the degree of structural rearrangements. Hence, the domain displacement will probably be of a subtle nature, as known for other His kinases (Chervitz and Falke, 1996).

The rock2 mutation is located in the transmembrane domain connecting the CHASE domain and the cytosolic His kinase domain. In the proposed signaling mechanism (Fig. 10), this transmembrane domain is essential for transmitting the signal from the lumen of the endoplasmic reticulum to the cytoplasm. Noteworthy, this transmembrane domain shows a high degree of sequence conservation among different cytokinin receptors, in contrast to the transmembrane domain delimiting the CHASE domain N terminally (Steklov et al., 2013). The L552F substitution in the rock2 receptor variant affects the highly conserved Leu residue of the helix core motif Axxx(S/A)x(G/L)x(L/F)VIx(L/F)LxG(Y/H)I (Leu-552 underlined). Furthermore, this residue is located in the immediate vicinity of two gain-of-function mutations of the AHK4 receptor that have been reported to cause constitutive cytokinin signaling in a bacterial assay (Miwa et al., 2007) and that are partially conserved in AHK2. Presumably, the described mutations cause constitutive conformational alterations of the transmembrane domain, which normally occur during signaling. Although no crystal structure for any transmembrane domain of any His kinase has been resolved to date, there is a growing body of knowledge (Bhate et al., 2015) allowing us to predict that the structural changes may involve a lateral or vertical displacement of the neighboring transmembrane helices and that such a displaced conformation is being locked by the rock2 and related mutations.

Constitutively Active Receptors Yield Novel Information About Cytokinin-Regulated Processes

The analysis of mutants and transgenic lines expressing constitutively active variants of two cytokinin receptors has revealed how plants with locally strongly enhanced cytokinin signaling look. Only a single gain-of-function cytokinin receptor mutant, ore12, has been described previously in Arabidopsis (Kim et al., 2006), but the description has been limited to the impact on leaf senescence. In addition, the ore12 mutation achieved a lower activation of the AHK3 receptor compared with the rock3 mutations described here, suggesting that this receptor signals in a gradual rather than an all-or-nothing fashion. Others reported that the production of transgenic plants expressing constitutively active receptors was difficult if not impossible (Miwa et al., 2007). Studying the influence of enhanced cytokinin signaling on the plant phenotype is relevant as, until now, our knowledge of the consequences of an enhanced cytokinin status has been derived largely from plants ectopically expressing a cytokinin-synthesizing ISOPENTENYLTRANSFERASE (Rupp et al., 1999; Sun et al., 2003) or LONELY GUY (Kuroha et al., 2009) gene or carrying mutations in cytokinin-degrading CKX genes (Bartrina et al., 2011). However, the impact of an enhanced production of the hormone may differ from the consequences of increased signaling. The former involves a mobile signal that can induce local but also systemic effects through all cytokinin receptors that are present in a given cell or tissue, while the latter acts in a cell-autonomous fashion involving only a single activated receptor. In accord with this notion, the expression of the ARR5:GUS gene confirmed enhanced cytokinin signaling by rock2 and rock3 and showed that signaling generally stays limited to those tissues known previously to activate the cytokinin reporter by the corresponding wild-type receptor (D’Agostino et al., 2000; Stolz et al., 2011). Interestingly, rock2 and rock3 mutants have reduced levels of all analyzed cytokinin metabolites. This is in agreement with the result that ahk loss-of-function mutants have increased cytokinin contents (Riefler et al., 2006) and, thus, underpins the existence of homeostatic control mechanisms. Part of these control mechanisms is an influence of cytokinin signaling on the transcript level of cytokinin metabolism genes (Miyawaki et al., 2004; Werner et al., 2006; Brenner et al., 2012).

Both rock2 and rock3 mutations affect most morphological aspects of the cytokinin deficiency syndrome but revert them to a different degree. The similar although not identical effects of these mutations on the plant phenotype reflect their high degree of functional redundancy revealed by loss-of-function mutants (Higuchi et al., 2004; Nishimura et al., 2004; Riefler et al., 2006). There are a number of notable quantitative differences between the rock2 and rock3 effects, which also are seen in the differential activation of the ARR5:GUS reporter in different tissues. For example, the rock2 mutation has a stronger impact on primary root elongation and root meristem size, which is surprising in view of the proposed central role that AHK3 (but not AHK2) plays in the root apical meristem (Dello Ioio et al., 2007). Similarly, the late-flowering phenotype of CKX1ox plants was only partially reverted by rock3, whereas rock2 CKX1ox flowered even earlier than the wild type. In contrast, rock3, for example, had a stronger effect in retarding leaf senescence. These mostly gradual differences may partly be due to differences in signaling strength caused by the mutations but also may reflect differences in coupling to downstream signaling processes. Indeed, ectopic expression of the rock2 and rock3 alleles under the transcriptional control of the same promoters in Arabidopsis causes partly different responses (A. Stolz and T. Schmülling, unpublished data). This indicates that, although the cytoplasmic domains of both receptors interact with the same AHP proteins in a yeast two-hybrid assay (Dortay et al., 2006), the affinities between signaling proteins may differ in planta. Their interaction also can be modulated by accessory proteins in a similar fashion to that known from bacterial two-component systems (Jung et al., 2012).

One important result derived from the phenotypic analysis is that cytokinin signaling is limiting for the growth of different shoot organs and that rock2/rock3-dependent changes in organ size are due to prolonged cell proliferation. This supports the hypothesis that cytokinin primarily controls the duration of the cell proliferation phase in shoot organ primordia by delaying the onset of cell differentiation (Holst et al., 2011). Consistently, a lower cytokinin activity causes a reduced leaf size, and genetic analysis has revealed that this is redundantly controlled by AHK2 and AHK3 (Werner et al., 2003; Higuchi et al., 2004; Nishimura et al., 2004; Riefler et al., 2006). It has been proposed that plant organ growth displays bell‐shaped dose responses to cytokinin (Ferreira and Kieber, 2005) and that cytokinin limits leaf size in the wild type (Efroni et al., 2013). The enhanced growth of different shoot organs in rock2 and rock3 mutants underpins this view. However, the range of cytokinin activity promoting growth appears to be limited, since cytokinin overproduction may result in the formation of smaller leaves (Hewelt et al., 1994; van der Graaff et al., 2001; Sun et al., 2003). This is likely due to strongly increased cell proliferation and the concomitant inability of proper cell differentiation and expansion. Cell number is apparently sensed in plant leaves (Hisanaga et al., 2015), and an increase in cell number above a certain threshold may interfere with cell expansion, resulting in smaller leaves (Dewitte et al., 2003). The increased cell proliferation in rock2 and rock3 mutants was linked to normal cell expansion, suggesting that this threshold was not reached. Our work here further revealed that flower organ size is very sensitive to enhanced cytokinin signaling too. The particularly strongly enlarged petals and gynoecia suggest that cytokinin is especially involved in regulating the development of these organs.

For a long time, it has been known that exogenously applied cytokinin can promote flowering in Arabidopsis (Michniewicz and Kamienska, 1967; Besnard-Wibaut, 1981; Dennis et al., 1996; D’Aloia et al., 2011). However, it has remained unclear whether endogenous cytokinin also would have the same promotive activity. rock2 suppresses the late-flowering phenotype of CKX1ox plants more strongly than rock3. This effect is most obvious under short-day conditions, where CKX1ox remains in the vegetative state and rock2, but not rock3, reverts this nonflowering phenotype. It seems clear that, in Arabidopsis, a certain cytokinin threshold signal is indispensable for flower induction in short days and that cytokinin has a promotive effect on flowering also in long days. The fact that rock2 CKX1ox mutants start to flower even earlier than the wild type underpins the importance of cytokinin signaling for flowering time control. The mechanistic basis of flowering time control by cytokinin is poorly understood. It has been shown that cytokinin promotes flowering independently of FLOWERING LOCUS T but through the transcriptional activation of its paralogue TWIN SISTER OF FLOWERING LOCUS T (TSF; D’Aloia et al., 2011). However, the question of whether the flowering response to cytokinin is mediated entirely through the transcriptional activation of TSF in leaves or whether cytokinin also might promote flowering by direct action in the shoot apical meristem (Corbesier et al., 2003; D’Aloia et al., 2011) needs to be scrutinized further.

An unexpected phenotype was the increased plant longevity and prolonged reproductive growth phase, which was particularly marked in transgenic plants. The prolonged reproductive growth resulted in a strongly increased number of flowers and siliques. This is a very interesting observation, because the monocarpic plant Arabidopsis generates dependent on the growth conditions a specific number of flowers, which is followed by the cessation of reproductive meristematic activity. This correlative inhibition of maternal growth is caused by the offspring (seeds) and is referred to as global proliferative arrest (GPA; Hensel et al., 1994). The molecular mechanism underlying this phenomenon is largely unknown. A recent study has suggested that the low mitotic activity in meristems linked to GPA represents a form of bud dormancy (Wuest et al., 2016). Given that cytokinin is a major factor determining the proliferative activity in axillary buds (Shimizu-Sato et al., 2009), it is tempting to hypothesize that cytokinin counteracts GPA by maintaining cell division activity. This scenario would predict that a drop in cytokinin activity is required for meristematic arrest during GPA and that the constitutive signaling in rock2 and rock3 transgenic plants delays it. How cytokinin could affect GPA is currently unclear. One possibility is that rock2 and rock3, and thus cytokinin, acts locally in the reproductive meristem by sustaining its activity. This idea is consistent with the previous hypothesis that meristematic sink activity, rather than the leaf source capacity, is decisive for the activity of the meristem and that sink strength is particularly triggered by cytokinin (Werner et al., 2008). A second possibility is that the strong capacity of rock3 to delay leaf senescence prolongs the activity of the reproductive meristems and, in addition, provides sufficient source capacity to support the development of supernumerary seeds. However, it has also been discussed that the activity of the reproductive meristem is uncoupled from rosette leaf senescence in Arabidopsis (Hensel et al., 1993; Noodén and Penney, 2001; Wuest et al., 2016). The two possibilities are not mutually exclusive, and further research is needed to understand the mechanism underlying the action of cytokinin during GPA.

In the above context, it is noteworthy that the size of the inflorescence meristems of rock2 and rock3 mutants was not increased, although these receptors are expressed and active in the meristem (Riefler et al., 2006; Stolz et al., 2011; Gruel et al., 2016). This is surprising considering that plants producing more or less cytokinin develop a larger or smaller inflorescence meristem, respectively (Bartrina et al., 2011). One plausible explanation for these apparently incongruous observations might be that, in tissues where AHK2 and AHK3 expression overlaps that of AHK4, as in the inflorescence meristem (Gruel et al., 2016), the enhanced signaling activity might be dampened by the phosphatase activity of AHK4 (Mähönen et al., 2006). In the presence of low cytokinin (as in the rock2 and rock3 plants), the phosphatase activity of the AHK4 receptor may prevail and alleviate downstream signaling, thus counteracting the enhanced kinase activity of rock2 and rock3 receptors. Consistent with this idea, the TCSn:GFP cytokinin reporter indicated similar signaling output activity in wild-type and rock2 inflorescence meristems. It this respect, it would be interesting to analyze the rock2 and rock3 mutations in an ahk4 null mutant background (Inoue et al., 2001). In contrast to rock2/rock3 plants, the ckx3 ckx5 mutation (Bartrina et al., 2011) presumably increases the signaling output of all three cytokinin receptors and lowers the phosphatase activity of AHK4; together, this might lead to a quantitatively and qualitatively different cytokinin output signal.

Last but not least, transgenic expression of the rock3-1 allele caused an ∼50% higher seed yield, which is similar to that brought about by the ckx3 ckx5 mutation (Bartrina et al., 2011). However, the mechanisms leading to increased seed yield appear to be at least partly different. ckx3 ckx5 mutants have a larger inflorescence meristem forming an increased number of flowers and siliques. In addition, siliques contain more seeds due to an increased placenta activity producing more ovules (Bartrina et al., 2011). In the case of rock3 transgenic plants, enhanced yield was due mainly to delayed GPA (see above), leading to taller plants with more siliques (rock2 transgenic plants formed even more flowers but suffered from reduced fertilization). In sum, our results corroborate the role of cytokinin as a regulator of seed yield (Ashikari et al., 2005; Bartrina et al., 2011) and demonstrate that different developmental mechanisms can be involved. We propose the rock2 and rock3 genes as a novel biotechnological tool to achieve yield enhancement. Because the coupling of the receptors to downstream signaling components is promiscuous, they could be used directly for a gain-of-function approach in crop plants to increase cytokinin signaling in a targeted and cell-autonomous fashion.

MATERIALS AND METHODS

Plant Material and Growth Conditions

The Columbia ecotype of Arabidopsis (Arabidopsis thaliana) was used as the wild type. The following lines were described previously: 35S:CKX1-11 (Werner et al., 2003), ahk2-5 (Riefler et al., 2006), ARR5:GUS (D’Agostino et al., 2000), and ore12-1 (Kim et al., 2006). All plants were grown on soil or in vitro on one-half-strength Murashige and Skoog medium under long-day (16 h of light/8 h of darkness) or short-day (8 h of light/16 h of darkness) conditions at 22°C. For root growth assays, seedlings were grown on vertical plates, and the length of the primary root was measured between day 4 and day 12 after germination from digital images using Scion Image software (http://scion-image.software.informer.com/). For the cytokinin sensitivity assay, BA dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a solvent control was added to the medium.

Mutagenesis and Gene Mapping

The rock2 and rock3 mutants were identified in a screen of an M2 population of 35S:CKX1 plants mutagenized with ethyl methanesulfonate (Niemann et al., 2015). Mapping populations for rock2 and rock3 were generated by crossing the rock2 35S:CKX1 and rock3 35S:CKX1 plants with the Landsberg erecta ecotype. F2 progeny resistant to hygromycin (cosegregating with 35S:CKX1) and showing the cytokinin deficiency syndrome were used to map the recessive (wild-type) alleles of rock2 and rock3. By analyzing 535 F2 recombinants, rock2 was mapped to a 1.45-Mb region (∼21.1 cM). To map the rock3 locus, 927 F2 recombinants were analyzed and a 350-kb interval (∼0.4 cM) was identified. The rock2 and rock3 mutations were identified by sequencing candidate genes in these intervals and subsequent complementation of 35S:CKX1 transgenic plants by the mutant alleles of the respective gene.

DNA Cloning

The rock2 and rock3 point mutations were introduced into the AHK2:AHK2 and AHK3:AHK3 genes (Stolz et al., 2011) using the QuickChange site-directed mutagenesis kit (Stratagene). Both constructs were introduced into Agrobacterium tumefaciens strain GV3101, and 35S:CKX1 and wild-type plants were transformed using the floral dip method (Clough and Bent, 1998). Transgenic lines were selected on medium containing 50 mg L−1 kanamycin.

Analysis of Transcript Levels by qPCR

Total RNA was extracted from seedlings with the TRIzol method (Thermo Fisher Scientific). Equal amounts of starting material (1 µg of RNA) were used in a 10-µL SuperScript III Reverse Transcriptase reaction (Thermo Fisher Scientific). First-strand cDNA synthesis was primed with a combination of oligo(dT) primers and random hexamers. Real-time qPCR using FAST SYBR Green I technology was performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The quantitative PCR temperature program consisted of the following steps: 95°C for 15 min; 40 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 15 s; followed by melting curve analysis. The relative transcript abundance of each gene was calculated based on the 2−ΔΔCt method (Livak and Schmittgen, 2001). PP2AA2 (At3g25800) was used for normalization. Primers used for reference genes and genes of interest are listed in Supplemental Table S3.

Yeast Complementation Assay

The yeast complementation assay was performed as described before (Inoue et al., 2001; Mähönen et al., 2006). The rock2 and rock3 mutations were introduced into the AHK4 gene (named AHK4rock2 and AHK4rock3) of plasmid p423TEF-CRE1 (Mähönen et al., 2006) using the QuickChange site-directed mutagenesis kit. After sequence confirmation, plasmids were introduced into yeast strain (TM182) ∆sln1 (Inoue et al., 2001), and the yeast complementation assay was performed with either 2% Gal or 2% Glc (w/v) with 0.1 or 10 µm tZ added to the medium. Optical density at 600 nm was measured after 20 h.

GUS Staining, Microscopy, and Scanning Electron Microscopy

GUS staining was performed as described by Köllmer et al. (2014). For microscopic analysis, tissues were cleared according to Malamy and Benfey (1997). Hand-cut cross sections of stems from 5-week-old plants were stained for 5 min in 0.02% aqueous Toluidine Blue O, rinsed, and mounted in water. All samples were viewed with an Axioskop 2 plus microscope (Zeiss). The inflorescence meristem of the main stem from 4-week-old soil-grown plants was dissected and analyzed by scanning electron microscopy as described before (Bartrina et al., 2011). TCSn:GFP fluorescence was analyzed according to Zürcher et al. (2013) using a Leica SP5 confocal microscope. Root meristem size was determined as described by Dello Ioio et al. (2007).

Determination of Cytokinin Content

Plants were grown in vitro for 14 d. For each sample, 100 mg of seedlings was pooled, and five independent samples were analyzed for each genotype. The cytokinin content was determined by ultra-performance liquid chromatography-electrospray-tandem mass spectrometry (Novák et al., 2008).

Analysis of Leaf Senescence and Photosynthetic Parameters

For the analysis of dark-induced leaf senescence, seedlings were grown in vitro for 18 d. The sixth rosette leaf was detached and floated on distilled water. After 7 d in the dark at room temperature, chlorophyll was extracted as described before (Köllmer et al., 2011). The Fv/Fm ratio of dark-adapted plants was measured with FluorCam (Photon Systems Instruments).

Determination of Flowering Time, Stem Diameter, Plant Height, and Yield Parameters

For flowering time analysis, seeds were stratified for 3 d at 4°C and sown on soil. The onset of flowering was defined as the plant age when the first flower was visible. The termination of flowering was defined as the time point when no new flowers were formed at the main inflorescence. The number of rosette leaves was scored at the onset of flowering. The diameter of the main inflorescence stem was determined when individual stems reached a height of 15 cm. The hand-made transverse sections were taken 1 cm above the rosette. The final plant height and the number of siliques were determined after the termination of flowering. For the analysis of seed yield, the weight of all fully ripened and desiccated seeds was determined.

Petal Surface Area and Cell Size Measurement

The petal surface area was measured from digital images of fully expanded organs with Scion Image. Petals were cleared (Malamy and Benfey, 1997), and average cell sizes were calculated from the number of cells per unit area of digital micrographs.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AHK2 (At5G35750), AHK3 (At1G27320), AHK4 (At2G01830), CKX1 (At2G41510), ARR5 (At3G48100), PP2AA2 (At3G25800).

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_173_3_1783__index.html (1.3KB, html)

Acknowledgments

We thank Ildoo Hwang for seeds of the ore12 mutant and Bruno Müller for the TCSn:GFP line.

Glossary

PAS

Per-Arnt-Sim

iP

isopentenyladenine

tZ

trans-zeatin

cZ

cis-zeatin

BA

benzyladenine

qRT

quantitative reverse transcription

GPA

global proliferative arrest

Footnotes

1

This work was supported by the Deutsche Forschungsgemeinschaft (grant no. CRC 429 to T.S. and T.W. and grant no. SPP 1530 to T.S.) and by the Czech Grant Agency (grant no. 15-22322S to M.S.).

References

  1. Ainley WM, McNeil KJ, Hill JW, Lingle WL, Simpson RB, Brenner ML, Nagao RT, Key JL (1993) Regulatable endogenous production of cytokinins up to ‘toxic’ levels in transgenic plants and plant tissues. Plant Mol Biol 22: 13–23 [DOI] [PubMed] [Google Scholar]
  2. Ashikari M, Sakakibara H, Lin S, Yamamoto T, Takashi T, Nishimura A, Angeles ER, Qian Q, Kitano H, Matsuoka M (2005) Cytokinin oxidase regulates rice grain production. Science 309: 741–745 [DOI] [PubMed] [Google Scholar]
  3. Bartrina I, Otto E, Strnad M, Werner T, Schmülling T (2011) Cytokinin regulates the activity of reproductive meristems, flower organ size, ovule formation, and thus seed yield in Arabidopsis thaliana. Plant Cell 23: 69–80 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Besnard-Wibaut C. (1981) Effectiveness of gibberellins and 6-benzyladenine on flowering of Arabidopsis thaliana. Physiol Plant 53: 205–212 [Google Scholar]
  5. Bhargava A, Clabaugh I, To JP, Maxwell BB, Chiang YH, Schaller GE, Loraine A, Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis. Plant Physiol 162: 272–294 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bhate MP, Molnar KS, Goulian M, DeGrado WF (2015) Signal transduction in histidine kinases: insights from new structures. Structure 23: 981–994 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brenner WG, Ramireddy E, Heyl A, Schmülling T (2012) Gene regulation by cytokinin in Arabidopsis. Front Plant Sci 3: 8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brenner WG, Schmülling T (2015) Summarizing and exploring data of a decade of cytokinin-related transcriptomics. Front Plant Sci 6: 29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Caesar K, Thamm AMK, Witthöft J, Elgass K, Huppenberger P, Grefen C, Horak J, Harter K (2011) Evidence for the localization of the Arabidopsis cytokinin receptors AHK3 and AHK4 in the endoplasmic reticulum. J Exp Bot 62: 5571–5580 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chervitz SA, Falke JJ (1996) Molecular mechanism of transmembrane signaling by the aspartate receptor: a model. Proc Natl Acad Sci USA 93: 2545–2550 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cheung J, Hendrickson WA (2009) Structural analysis of ligand stimulation of the histidine kinase NarX. Structure 17: 190–201 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743 [DOI] [PubMed] [Google Scholar]
  13. Corbesier L, Prinsen E, Jacqmard A, Lejeune P, Van Onckelen H, Périlleux C, Bernier G (2003) Cytokinin levels in leaves, leaf exudate and shoot apical meristem of Arabidopsis thaliana during floral transition. J Exp Bot 54: 2511–2517 [DOI] [PubMed] [Google Scholar]
  14. Cortleven A, Nitschke S, Klaumünzer M, Abdelgawad H, Asard H, Grimm B, Riefler M, Schmülling T (2014) A novel protective function for cytokinin in the light stress response is mediated by the ARABIDOPSIS HISTIDINE KINASE2 and ARABIDOPSIS HISTIDINE KINASE3 receptors. Plant Physiol 164: 1470–1483 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. D’Agostino IB, Deruère J, Kieber JJ (2000) Characterization of the response of the Arabidopsis response regulator gene family to cytokinin. Plant Physiol 124: 1706–1717 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. D’Aloia M, Bonhomme D, Bouché F, Tamseddak K, Ormenese S, Torti S, Coupland G, Périlleux C (2011) Cytokinin promotes flowering of Arabidopsis via transcriptional activation of the FT paralogue TSF. Plant J 65: 972–979 [DOI] [PubMed] [Google Scholar]
  17. Dello Ioio R, Linhares FS, Scacchi E, Casamitjana-Martinez E, Heidstra R, Costantino P, Sabatini S (2007) Cytokinins determine Arabidopsis root-meristem size by controlling cell differentiation. Curr Biol 17: 678–682 [DOI] [PubMed] [Google Scholar]
  18. Dennis ES, Finnegan EJ, Bilodeau P, Chaudhury A, Genger R, Helliwell CA, Sheldon CC, Bagnall DJ, Peacock WJ (1996) Vernalization and the initiation of flowering. Semin Cell Dev Biol 7: 441–448 [Google Scholar]
  19. Dewitte W, Riou-Khamlichi C, Scofield S, Healy JMS, Jacqmard A, Kilby NJ, Murray JAH (2003) Altered cell cycle distribution, hyperplasia, and inhibited differentiation in Arabidopsis caused by the D-type cyclin CYCD3. Plant Cell 15: 79–92 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Dortay H, Mehnert N, Bürkle L, Schmülling T, Heyl A (2006) Analysis of protein interactions within the cytokinin-signaling pathway of Arabidopsis thaliana. FEBS J 273: 4631–4644 [DOI] [PubMed] [Google Scholar]
  21. Efroni I, Han SK, Kim HJ, Wu MF, Steiner E, Birnbaum KD, Hong JC, Eshed Y, Wagner D (2013) Regulation of leaf maturation by chromatin-mediated modulation of cytokinin responses. Dev Cell 24: 438–445 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ferreira FJ, Kieber JJ (2005) Cytokinin signaling. Curr Opin Plant Biol 8: 518–525 [DOI] [PubMed] [Google Scholar]
  23. Franco-Zorrilla JM, Martín AC, Leyva A, Paz-Ares J (2005) Interaction between phosphate-starvation, sugar, and cytokinin signaling in Arabidopsis and the roles of cytokinin receptors CRE1/AHK4 and AHK3. Plant Physiol 138: 847–857 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gan S, Amasino RM (1995) Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270: 1986–1988 [DOI] [PubMed] [Google Scholar]
  25. Gruel J, Landrein B, Tarr P, Schuster C, Refahi Y, Sampathkumar A, Hamant O, Meyerowitz EM, Jönsson H (2016) An epidermis-driven mechanism positions and scales stem cell niches in plants. Sci Adv 2: e1500989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hensel LL, Grbić V, Baumgarten DA, Bleecker AB (1993) Developmental and age-related processes that influence the longevity and senescence of photosynthetic tissues in Arabidopsis. Plant Cell 5: 553–564 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hensel LL, Nelson MA, Richmond TA, Bleecker AB (1994) The fate of inflorescence meristems is controlled by developing fruits in Arabidopsis. Plant Physiol 106: 863–876 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hewelt A, Prinsen E, Schell J, Van Onckelen H, Schmülling T (1994) Promoter tagging with a promoterless ipt gene leads to cytokinin-induced phenotypic variability in transgenic tobacco plants: implications of gene dosage effects. Plant J 6: 879–891 [DOI] [PubMed] [Google Scholar]
  29. Heyl A, Riefler M, Romanov GA, Schmülling T (2012) Properties, functions and evolution of cytokinin receptors. Eur J Cell Biol 91: 246–256 [DOI] [PubMed] [Google Scholar]
  30. Heyl A, Wulfetange K, Pils B, Nielsen N, Romanov GA, Schmülling T (2007) Evolutionary proteomics identifies amino acids essential for ligand-binding of the cytokinin receptor CHASE domain. BMC Evol Biol 7: 62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Higuchi M, Pischke MS, Mähönen AP, Miyawaki K, Hashimoto Y, Seki M, Kobayashi M, Shinozaki K, Kato T, Tabata S, et al. (2004) In planta functions of the Arabidopsis cytokinin receptor family. Proc Natl Acad Sci USA 101: 8821–8826 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hisanaga T, Kawade K, Tsukaya H (2015) Compensation: a key to clarifying the organ-level regulation of lateral organ size in plants. J Exp Bot 66: 1055–1063 [DOI] [PubMed] [Google Scholar]
  33. Holst K, Schmülling T, Werner T (2011) Enhanced cytokinin degradation in leaf primordia of transgenic Arabidopsis plants reduces leaf size and shoot organ primordia formation. J Plant Physiol 168: 1328–1334 [DOI] [PubMed] [Google Scholar]
  34. Hothorn M, Dabi T, Chory J (2011) Structural basis for cytokinin recognition by Arabidopsis thaliana histidine kinase 4. Nat Chem Biol 7: 766–768 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Hwang I, Sheen J (2001) Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413: 383–389 [DOI] [PubMed] [Google Scholar]
  36. Inoue T, Higuchi M, Hashimoto Y, Seki M, Kobayashi M, Kato T, Tabata S, Shinozaki K, Kakimoto T (2001) Identification of CRE1 as a cytokinin receptor from Arabidopsis. Nature 409: 1060–1063 [DOI] [PubMed] [Google Scholar]
  37. Jeon J, Kim NY, Kim S, Kang NY, Novák O, Ku SJ, Cho C, Lee DJ, Lee EJ, Strnad M, et al. (2010) A subset of cytokinin two-component signaling system plays a role in cold temperature stress response in Arabidopsis. J Biol Chem 285: 23371–23386 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Jung K, Fried L, Behr S, Heermann R (2012) Histidine kinases and response regulators in networks. Curr Opin Microbiol 15: 118–124 [DOI] [PubMed] [Google Scholar]
  39. Kieber JJ, Schaller GE (2014) Cytokinins. The Arabidopsis Book 12: e0168, doi/10.1199/tab.0168 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kim HJ, Ryu H, Hong SH, Woo HR, Lim PO, Lee IC, Sheen J, Nam HG, Hwang I (2006) Cytokinin-mediated control of leaf longevity by AHK3 through phosphorylation of ARR2 in Arabidopsis. Proc Natl Acad Sci USA 103: 814–819 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Köllmer I, Novák O, Strnad M, Schmülling T, Werner T (2014) Overexpression of the cytosolic cytokinin oxidase/dehydrogenase (CKX7) from Arabidopsis causes specific changes in root growth and xylem differentiation. Plant J 78: 359–371 [DOI] [PubMed] [Google Scholar]
  42. Köllmer I, Werner T, Schmülling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes of Arabidopsis thaliana alters plant growth and development. J Plant Physiol 168: 1320–1327 [DOI] [PubMed] [Google Scholar]
  43. Kuroha T, Tokunaga H, Kojima M, Ueda N, Ishida T, Nagawa S, Fukuda H, Sugimoto K, Sakakibara H (2009) Functional analyses of LONELY GUY cytokinin-activating enzymes reveal the importance of the direct activation pathway in Arabidopsis. Plant Cell 21: 3152–3169 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Liu YC, Machuca MA, Beckham SA, Gunzburg MJ, Roujeinikova A (2015) Structural basis for amino-acid recognition and transmembrane signalling by tandem Per-Arnt-Sim (tandem PAS) chemoreceptor sensory domains. Acta Crystallogr D Biol Crystallogr 71: 2127–2136 [DOI] [PubMed] [Google Scholar]
  45. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25: 402–408 [DOI] [PubMed] [Google Scholar]
  46. Lomin SN, Krivosheev DM, Steklov MY, Arkhipov DV, Osolodkin DI, Schmülling T, Romanov GA (2015) Plant membrane assays with cytokinin receptors underpin the unique role of free cytokinin bases as biologically active ligands. J Exp Bot 66: 1851–1863 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Mähönen AP, Bonke M, Kauppinen L, Riikonen M, Benfey PN, Helariutta Y (2000) A novel two-component hybrid molecule regulates vascular morphogenesis of the Arabidopsis root. Genes Dev 14: 2938–2943 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Mähönen AP, Higuchi M, Törmäkangas K, Miyawaki K, Pischke MS, Sussman MR, Helariutta Y, Kakimoto T (2006) Cytokinins regulate a bidirectional phosphorelay network in Arabidopsis. Curr Biol 16: 1116–1122 [DOI] [PubMed] [Google Scholar]
  49. Malamy JE, Benfey PN (1997) Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124: 33–44 [DOI] [PubMed] [Google Scholar]
  50. Maruyama-Nakashita A, Nakamura Y, Yamaya T, Takahashi H (2004) A novel regulatory pathway of sulfate uptake in Arabidopsis roots: implication of CRE1/WOL/AHK4-mediated cytokinin-dependent regulation. Plant J 38: 779–789 [DOI] [PubMed] [Google Scholar]
  51. Matsumoto-Kitano M, Kusumoto T, Tarkowski P, Kinoshita-Tsujimura K, Václavíková K, Miyawaki K, Kakimoto T (2008) Cytokinins are central regulators of cambial activity. Proc Natl Acad Sci USA 105: 20027–20031 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Michniewicz M, Kamienska A (1967) Effect of kinetin on the content of endogenous gibberellins in germinating seeds of some plant species. Naturwissenschaften 54: 372. [DOI] [PubMed] [Google Scholar]
  53. Miwa K, Ishikawa K, Terada K, Yamada H, Suzuki T, Yamashino T, Mizuno T (2007) Identification of amino acid substitutions that render the Arabidopsis cytokinin receptor histidine kinase AHK4 constitutively active. Plant Cell Physiol 48: 1809–1814 [DOI] [PubMed] [Google Scholar]
  54. Miyawaki K, Matsumoto-Kitano M, Kakimoto T (2004) Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: tissue specificity and regulation by auxin, cytokinin, and nitrate. Plant J 37: 128–138 [DOI] [PubMed] [Google Scholar]
  55. Moore JO, Hendrickson WA (2009) Structural analysis of sensor domains from the TMAO-responsive histidine kinase receptor TorS. Structure 17: 1195–1204 [DOI] [PubMed] [Google Scholar]
  56. Müller B, Sheen J (2007) Advances in cytokinin signaling. Science 318: 68–69 [DOI] [PubMed] [Google Scholar]
  57. Niemann MCE, Bartrina I, Ashikov A, Weber H, Novák O, Spíchal L, Strnad M, Strasser R, Bakker H, Schmülling T, et al. (2015) Arabidopsis ROCK1 transports UDP-GlcNAc/UDP-GalNAc and regulates ER protein quality control and cytokinin activity. Proc Natl Acad Sci USA 112: 291–296 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Nieminen K, Immanen J, Laxell M, Kauppinen L, Tarkowski P, Dolezal K, Tähtiharju S, Elo A, Decourteix M, Ljung K, et al. (2008) Cytokinin signaling regulates cambial development in poplar. Proc Natl Acad Sci USA 105: 20032–20037 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Nishimura C, Ohashi Y, Sato S, Kato T, Tabata S, Ueguchi C (2004) Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis. Plant Cell 16: 1365–1377 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Noodén LD, Penney JP (2001) Correlative controls of senescence and plant death in Arabidopsis thaliana (Brassicaceae). J Exp Bot 52: 2151–2159 [DOI] [PubMed] [Google Scholar]
  61. Novák O, Hauserová E, Amakorová P, Dolezal K, Strnad M (2008) Cytokinin profiling in plant tissues using ultra-performance liquid chromatography-electrospray tandem mass spectrometry. Phytochemistry 69: 2214–2224 [DOI] [PubMed] [Google Scholar]
  62. Richmond AE, Lang A (1957) Effect of kinetin on protein content and survival of detached Xanthium leaves. Science 125: 650–65113421662 [Google Scholar]
  63. Riefler M, Novak O, Strnad M, Schmülling T (2006) Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism. Plant Cell 18: 40–54 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Romanov GA, Lomin SN, Schmülling T (2006) Biochemical characteristics and ligand-binding properties of Arabidopsis cytokinin receptor AHK3 compared to CRE1/AHK4 as revealed by a direct binding assay. J Exp Bot 57: 4051–4058 [DOI] [PubMed] [Google Scholar]
  65. Rupp HM, Frank M, Werner T, Strnad M, Schmülling T (1999) Increased steady state mRNA levels of the STM and KNAT1 homeobox genes in cytokinin overproducing Arabidopsis thaliana indicate a role for cytokinins in the shoot apical meristem. Plant J 18: 557–563 [DOI] [PubMed] [Google Scholar]
  66. Shimizu-Sato S, Tanaka M, Mori H (2009) Auxin-cytokinin interactions in the control of shoot branching. Plant Mol Biol 69: 429–435 [DOI] [PubMed] [Google Scholar]
  67. Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development in Arabidopsis. Plant Cell 2: 755–767 [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Spíchal L, Rakova NY, Riefler M, Mizuno T, Romanov GA, Strnad M, Schmülling T (2004) Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay. Plant Cell Physiol 45: 1299–1305 [DOI] [PubMed] [Google Scholar]
  69. Steklov MY, Lomin SN, Osolodkin DI, Romanov GA (2013) Structural basis for cytokinin receptor signaling: an evolutionary approach. Plant Cell Rep 32: 781–793 [DOI] [PubMed] [Google Scholar]
  70. Stolz A, Riefler M, Lomin SN, Achazi K, Romanov GA, Schmülling T (2011) The specificity of cytokinin signalling in Arabidopsis thaliana is mediated by differing ligand affinities and expression profiles of the receptors. Plant J 67: 157–168 [DOI] [PubMed] [Google Scholar]
  71. Sun J, Niu QW, Tarkowski P, Zheng B, Tarkowska D, Sandberg G, Chua NH, Zuo J (2003) The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis. Plant Physiol 131: 167–176 [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Suzuki T, Miwa K, Ishikawa K, Yamada H, Aiba H, Mizuno T (2001) The Arabidopsis sensor His-kinase, AHk4, can respond to cytokinins. Plant Cell Physiol 42: 107–113 [DOI] [PubMed] [Google Scholar]
  73. Tran LSP, Urao T, Qin F, Maruyama K, Kakimoto T, Shinozaki K, Yamaguchi-Shinozaki K (2007) Functional analysis of AHK1/ATHK1 and cytokinin receptor histidine kinases in response to abscisic acid, drought, and salt stress in Arabidopsis. Proc Natl Acad Sci USA 104: 20623–20628 [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Ueguchi C, Sato S, Kato T, Tabata S (2001) The AHK4 gene involved in the cytokinin-signaling pathway as a direct receptor molecule in Arabidopsis thaliana. Plant Cell Physiol 42: 751–755 [DOI] [PubMed] [Google Scholar]
  75. van der Graaff EE, Hooykaas PJJ, Auer CA (2001) Altered development of Arabidopsis thaliana carrying the Agrobacterium tumefaciens ipt gene is partially due to ethylene effects. Plant Growth Regul 34: 305–315 [Google Scholar]
  76. Werner T, Holst K, Pörs Y, Guivarc’h A, Mustroph A, Chriqui D, Grimm B, Schmülling T (2008) Cytokinin deficiency causes distinct changes of sink and source parameters in tobacco shoots and roots. J Exp Bot 59: 2659–2672 [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Werner T, Köllmer I, Bartrina I, Holst K, Schmülling T (2006) New insights into the biology of cytokinin degradation. Plant Biol (Stuttg) 8: 371–381 [DOI] [PubMed] [Google Scholar]
  78. Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, Schmülling T (2003) Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15: 2532–2550 [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Werner T, Schmülling T (2009) Cytokinin action in plant development. Curr Opin Plant Biol 12: 527–538 [DOI] [PubMed] [Google Scholar]
  80. Wuest SE, Philipp MA, Guthörl D, Schmid B, Grossniklaus U (2016) Seed production affects maternal growth and senescence in Arabidopsis. Plant Physiol 171: 392–404 [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Wulfetange K, Lomin SN, Romanov GA, Stolz A, Heyl A, Schmülling T (2011) The cytokinin receptors of Arabidopsis are located mainly to the endoplasmic reticulum. Plant Physiol 156: 1808–1818 [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Yamada H, Suzuki T, Terada K, Takei K, Ishikawa K, Miwa K, Yamashino T, Mizuno T (2001) The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane. Plant Cell Physiol 42: 1017–1023 [DOI] [PubMed] [Google Scholar]
  83. Zürcher E, Müller B (2016) Cytokinin synthesis, signaling, and function: advances and new insights. Int Rev Cell Mol Biol 324: 1–38 [DOI] [PubMed] [Google Scholar]
  84. Zürcher E, Tavor-Deslex D, Lituiev D, Enkerli K, Tarr PT, Müller B (2013) A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network in planta. Plant Physiol 161: 1066–1075 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Data
supp_173_3_1783__index.html (1.3KB, html)
supp_pp.16.01903_PP2016-01141D_Supplemental_Materialfigs.pdf (1.3MB, pdf)
supp_pp.16.01903_PP2016-01141D_Supplemental_Materialtbls.pdf (124.6KB, pdf)

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES