CYTOKININ-INDEPENDENT1 Research Articles

Conserved CKI1-mediated signaling is required for female germline specification in Marchantia polymorpha

In land plants, gametes derive from a small number of dedicated haploid cells.1 In angiosperms, one central cell and one egg cell are differentiated in the embryo sac as female gametes for double fertilization, while in non-flowering plants, only one egg cell is generated in the female sexual organ, called the archegonium.2,3 The central cell specification of Arabidopsis thaliana is controlled by the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1), which is a two-component signaling (TCS) activator sharing downstream regulatory components with the cytokinin signaling pathway.4,5,6,7 Our phylogenetic analysis suggested that CKI1 orthologs broadly exist in land plants. However, the role of CKI1 in non-flowering plants remains unclear. Here, we found that the sole CKI1 ortholog in the liverwort Marchantia polymorpha, MpCKI1, which functions through conserved downstream TCS components, regulates the female germline specification for egg cell development in the archegonium. In M.polymorpha, the archegonium develops three-dimensionally from a single cell accumulating MpBONOBO (MpBNB), a master regulator for germline initiation and differentiation.8 We visualized female germline specification by capturing the distribution pattern of MpBNB in discrete stages of early archegonium development, and found that MpBNB accumulation is restricted to female germline cells. MpCKI1 is required for the proper MpBNB accumulation in the female germline, and is critical for the asymmetric cell divisions that specify the female germline cells. These results suggest that CKI1-mediated TCS originated during early land plant evolution and participates in female germ cell specification in deeply diverged plant lineages.

The mutation of CaCKI1 causes seedless fruits in chili pepper (Capsicum annuum)

The seedless mutant tn-1 in chili pepper is caused by a mutation in CaCKI1 (CA12g21620), which encodes histidine kinase involving female gametophyte development. An amino acid insertion in the receiver domain of CaCKI1 may be the mutation responsible for tn-1. Seedlessness is a desirable trait in fruit crops because the removal of seeds is a troublesome step for consumers and processing industries. However, little knowledge is available to develop seedless chili peppers. In a previous study, a chili pepper mutant tn-1, which stably produces seedless fruits, was isolated. In this study, we report characterization of tn-1 and identification of the causative gene. Although pollen germination was normal, confocal laser microscopy observations revealed deficiency in embryo sac development in tn-1. By marker analysis, the tn-1 locus was narrowed down to a 313kb region on chromosome 12. Further analysis combined with mapping-by-sequencing identified CA12g21620, which encodes histidine kinase as a candidate gene. Phylogenetic analysis revealed CA12g21620 was the homolog of Arabidopsis CKI1 (Cytokinin Independent 1), which plays an important role in female gametophyte development, and CA12g21620 was designated as CaCKI1. Sequence analysis revealed that tn-1 has a 3-bp insertion in the 6th exon resulting in one lysine (K) residue insertion in receiver domain of CaCKI1, and the sequence nearby the insertion is widely conserved among CKI1 orthologs in various plants. This suggested that one K residue insertion may reduce the phosphorylation relay downstream of CaCKI1 and impair normal development of female gametophyte, resulting in seedless fruits production in tn-1. Furthermore, we demonstrated that virus-induced gene silencing of CaCKI1 reducednormally developed female gametophyte in chili pepper. This study describes the significant role of CaCKI1 in seed development in chili pepper and the possibility of developing seedless cultivars using its mutation.

The type-B response regulators ARR10, ARR12, and ARR18 specify the central cell in Arabidopsis.

In Arabidopsis thaliana, the female gametophyte consists of two synergid cells, an egg cell, a diploid central cell, and three antipodal cells. CYTOKININ INDEPENDENT 1 (CKI1), a histidine kinase constitutively activating the cytokinin signaling pathway, specifies the central cell and restricts the egg cell. However, the mechanism regulating CKI1-dependent central cell specification is largely unknown. Here, we showed that the type-B ARABIDOPSIS RESPONSE REGULATORS10, 12, and 18 (ARR10/12/18) localize at the chalazal pole of the female gametophyte. Phenotypic analysis showed that the arr10 12 18 triple mutant is female sterile. We examined the expression patterns of embryo sac marker genes and found that the embryo sac of arr10 12 18 plants had lost central cell identity, a phenotype similar to that of the Arabidopsis cki1 mutant. Genetic analyses demonstrated that ARR10/12/18, CKI1, and ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN2, 3, and 5 (AHP2/3/5) function in a common pathway to regulate female gametophyte development. In addition, constitutively activated ARR10/12/18 in the cki1 embryo sac partially restored the fertility of cki1. Results of transcriptomic analysis supported the conclusion that ARR10/12/18 and CKI1 function together to regulate the identity of the central cell. Our results demonstrated that ARR10/12/18 function downstream of CKI1-AHP2/3/5 as core factors to determine cell fate of the female gametophyte.

Screening and identification of CKI1 upstream transcription regulators in Arabidopsis

Arabidopsis CKI1 (cytokinin independent 1) is a histidine kinase protein involved in the two-component system, which can activate two-component signaling via the downstream histidine phospho-transfer proteins, playing the essential roles in central cell fate determination and development regulation in embryo sacs. However, studies on CKI1 upstream transcription regulators are still limited. In the present study, promoter activities with varying fragments were investigated, and CKI1 upstream transcription regulators were screened and identified by the yeast-one hybrid technique. Results indicated F5/R2 fragments located in the intron region showed promoter activities in embryo sacs, which is consistent with CKI1 full-length promoters. Then three tandem repeats of F5/R2 fragments were used to construct the bait expression vector, and Arabidopsis pistils were collected for cDNA library construction. Totally, 226 positive clones were screened by the yeast-one hybrid technique, 66 readable sequences were retrieved after removing sequences with low quality and redundant repeats, among which eight proteins could act as DNA-binding proteins. These results provided some important clues to study the molecular function of CKI1 in the transcription regulation network.

The gymnosperm ortholog of the angiosperm central cell‐specification gene CKI1 provides an essential clue to endosperm origin

A defining feature of angiosperms is double fertilization involving the female gametophyte central cell and formation of a nutrient-storing tissue called endosperm. The route for the evolutionary origin of endosperm from a gymnosperm ancestor, particularly the molecular steps involved, has remained elusive. Recently, the histidine kinase gene Cytokinin-Independent 1 (CKI1), an activator of cytokinin signaling, was described as a key to specification of the endosperm precursor central cell in Arabidopsis. Here, we have investigated the function and expression of a putative ortholog of CKI1 in the gymnosperm Ginkgo biloba. We demonstrate that Ginkgo CKI1 can partially rescue an Arabidopsis cki1 mutant and promote weak activation of the cytokinin signaling pathway in the Arabidopsis embryo sac, but does not confer central cell specification. Ginkgo CKI1 is expressed in both male and female gametophytes of Ginkgo. In the latter, it is expressed in the ventral canal cell, which is sister to the egg cell in the archegonium. As in Arabidopsis, Ginkgo CKI1 is not expressed in the egg cell. The similarities in expression patterns of CKI1 in Ginkgo and Arabidopsis female gametophytes suggest that extant gymnosperms possess an essential component of the molecular machinery required for angiosperm endosperm development, and provide new insights into endosperm origin from a gymnospermous ancestor.

AHP2, AHP3, and AHP5 act downstream of CKI1 in Arabidopsis female gametophyte development.

Histidine phosphotransfer proteins (HPs) are key elements of the two-component signaling system, which act as a shuttle to transfer phosphorylation signals from histidine kinases (HKs) to response regulators (RRs). CYTOKININ INDEPENDENT 1 (CKI1), a key regulator of central cell specification in the Arabidopsis female gametophyte, activates the cytokinin signaling pathway through the Arabidopsis histidine phosphotransfer proteins (AHPs). There are five HP genes in Arabidopsis, AHP1-AHP5, but it remains unknown which AHP genes act downstream of CKI1 in Arabidopsis female gametophyte development. Promoter activity analysis of AHP1-AHP5 in embryo sacs revealed AHP1, AHP2, AHP3, and AHP5 expression in the central cell. Phenotypic studies of various combinations of ahp mutants showed that triple mutations in AHP2, AHP3, and AHP5 resulted in defective embryo sac development. Using cell-specific single and double markers in the female gametophyte, the ahp2-2 ahp3 ahp5-2/+ triple mutant ovules showed loss of central cell and antipodal cell fates and gain of egg cell or synergid cell attributes, resembling the cki1 mutant phenotypes. These data suggest that AHP2, AHP3, and AHP5 are the major factors acting downstream of CKI1 in the two-component cytokinin signaling pathway to promote Arabidopsis female gametophyte development.

Light Controls Cytokinin Signaling via Transcriptional Regulation of Constitutively Active Sensor Histidine Kinase CKI1.

In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The cross talk between cytokinin response and light has been known for a long time. However, the molecular mechanism underlying the interaction between light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL (LPH) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT1 (CKI1), encoding the constitutively active sensor His kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE1 (HY1) that encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertible phytochromes. Our analysis confirmed the light-dependent regulation of the CKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors (TF) PHYTOCHROME INTERACTING FACTOR3 and CIRCADIAN CLOCK ASSOCIATED1. Changes in CKI1 expression observed in lph/hy1-7 and phy mutants correlate with misregulation of MSP signaling, changed cytokinin sensitivity, and developmental aberrations that were previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate a novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development.

The CKI1 Histidine Kinase Specifies the Female Gametic Precursor of the Endosperm

Since the discovery of double fertilization, it has been recognized that flowering plants produce two highly dimorphic female gametes, the egg cell and central cell. These give rise, respectively, to the embryo and the endosperm, a nourishing tissue unique to flowering plants. Here we show that in Arabidopsis, endosperm formation requires the CYTOKININ INDEPENDENT 1 (CKI1) histidine kinase, an activator of the cytokinin signaling pathway, which specifies central cells and restricts egg cell fate. Dimorphism of the two adjacent gametes is mechanistically established in the syncytial embryo sac by spatially restricted CKI1 expression, followed by translocation of ER-localized CKI1 protein via nuclear migration. Cell specification by CKI1 likely involves activation of the cytokinin signaling pathway mediated by histidine phosphotransferases. Ectopic CKI1 expression generates non-propagating seeds with dual fertilized endosperms and no embryos. We conclude that CKI1-directed specification of the endosperm precursor central cell results in seeds containing an embryo and an endosperm.

Antibodies against CKI1RD, a receiver domain of the sensor histidine kinase in Arabidopsis thaliana: From antigen preparation to in planta immunolocalization

Immunodetection is a powerful tool in functional studies of all organisms. In plants, the gene redundancy and presence of gene families composed of highly homologous members often impedes the unambiguous identification of individual gene products. A family of eight sensor histidine kinases (HKs) mediates the transduction of diverse signals into Arabidopsis thaliana cells, thereby ensuring the initiation of appropriate adaptive responses. Antibodies recognizing specific members of the HK family would be valuable for studying their functions in Arabidopsis and other plant species including important crops. We have focused on developing and applying antibodies against CYTOKININ-INDEPENDENT 1 (CKI1), which encodes a constitutively active membrane-bound sensor HK that regulates the development of female gametophytes and vascular tissue in Arabidopsis. A coding sequence delimiting the C-terminal receiver domain of CKI1 (CKI1RD) was expressed in Escherichia coli using the IPTG-inducible expression system and purified to give a highly pure target protein. The purified CKI1RD protein was then used as an antigen for anti-CKI1RD antibody production. The resulting polyclonal antibodies had a detection limit of 10ng of target protein at 1:20,000 dilution and were able to specifically distinguish CKI1, both in vitro and in situ, even in a direct comparison with highly homologous members of the same HK family AHK4, CKI2 and ETR1. Finally, anti-CKI1RD antibodies were able to selectively bind CKI1–GFP fusion protein in a pull-down assay using crude lysate from an Arabidopsis cell suspension culture. Our results suggest that the receiver domain is a useful target for the functional characterization of sensor HKs in immunological and biochemical studies.

ArabidopsisHistidine Kinase CKI1 Acts Upstream of HISTIDINE PHOSPHOTRANSFER PROTEINS to Regulate Female Gametophyte Development and Vegetative Growth

Cytokinin signaling is mediated by a multiple-step phosphorelay. Key components of the phosphorelay consist of the histidine kinase (HK)-type receptors, histidine phosphotransfer proteins (HP), and response regulators (RRs). Whereas overexpression of a nonreceptor-type HK gene CYTOKININ-INDEPENDENT1 (CKI1) activates cytokinin signaling by an unknown mechanism, mutations in CKI1 cause female gametophytic lethality. However, the function of CKI1 in cytokinin signaling remains unclear. Here, we characterize a mutant allele, cki1-8, that can be transmitted through female gametophytes with low frequency (approximately 0.17%). We have recovered viable homozygous cki1-8 mutant plants that grow larger than wild-type plants, show defective megagametogenesis and rarely set enlarged seeds. We found that CKI1 acts upstream of AHP (Arabidopsis HP) genes, independently of cytokinin receptor genes. Consistently, an ahp1,2-2,3,4,5 quintuple mutant, which contains an ahp2-2 null mutant allele, exhibits severe defects in megagametogenesis, with a transmission efficiency of <3.45% through female gametophytes. Rarely recovered ahp1,2-2,3,4,5 quintuple mutants are seedling lethal. Finally, the female gametophytic lethal phenotype of cki1-5 (a null mutant) can be partially rescued by IPT8 or ARR1 (a type-B Arabidopsis RR) driven by a CKI1 promoter. These results define a genetic pathway consisting of CKI1, AHPs, and type-B ARRs in the regulation of female gametophyte development and vegetative growth.

The Histidine Kinases CYTOKININ-INDEPENDENT1 and ARABIDOPSIS HISTIDINE KINASE2 and 3 Regulate Vascular Tissue Development inArabidopsisShoots

The development and activity of the procambium and cambium, which ensure vascular tissue formation, is critical for overall plant architecture and growth. However, little is known about the molecular factors affecting the activity of vascular meristems and vascular tissue formation. Here, we show that the His kinase CYTOKININ-INDEPENDENT1 (CKI1) and the cytokinin receptors ARABIDOPSIS HISTIDINE KINASE2 (AHK2) and AHK3 are important regulators of vascular tissue development in Arabidopsis thaliana shoots. Genetic modifications of CKI1 activity in Arabidopsis cause dysfunction of the two-component signaling pathway and defects in procambial cell maintenance. CKI1 overexpression in protoplasts leads to cytokinin-independent activation of the two-component phosphorelay, and intracellular domains are responsible for the cytokinin-independent activity of CKI1. CKI1 expression is observed in vascular tissues of inflorescence stems, and CKI1 forms homodimers both in vitro and in planta. Loss-of-function ahk2 and ahk3 mutants and plants with reduced levels of endogenous cytokinins show defects in procambium proliferation and an absence of secondary growth. CKI1 overexpression partially rescues ahk2 ahk3 phenotypes in vascular tissue, while the negative mutation CKI1H405Q further accentuates mutant phenotypes. These results indicate that the cytokinin-independent activity of CKI1 and cytokinin-induced AHK2 and AHK3 are important for vascular bundle formation in Arabidopsis.

CYTOKININ INDEPENDENT-1 regulates levels of different forms of cytokinin in Arabidopsis and mediates response to nutrient stress

The gene CYTOKININ INDEPENDENT-1 (CKI-1), previously isolated by enhancer trap screening, has been hypothesised to play a role in cytokinin perception. Alternative hypotheses suggest that it is required for the production of cytokinins or that it has no direct role in cytokinin signalling but simply interferes with the pathway when overexpressed. These hypotheses were investigated by producing transgenic Arabidopsis plants expressing CKI-1 cDNA in antisense orientation. In standard conditions, the phenotype of the plants was similar to wild type. Significantly higher amounts of the free base and riboside forms of cytokinin and lower amounts of membrane-impermeable cytokinins were found in the antisense lines. This supports the hypothesis that CKI-1 is involved in cytokinin perception and demonstrates the existence of a feedback loop altering cytokinin metabolism in response to the level of receptor abundance. An elevation in the content of free bases and ribosides of zeatin and isopentenyladenine, along with a reduction in the content of ribotide forms, suggests that a cytokinin ribotide 5'-ribonucleotidase may be a site at which CKI-1 exerts feedback control. When seed homozygous for the transgene was germinated on medium with reduced total mineral nutrient levels, the cotyledons of seedlings with reduced levels of CKI-1 failed to expand and green, and vegetative growth was inhibited. A similar phenotype was observed on low-phosphate media, suggesting that this failure resulted from an interaction between phosphate and cytokinins.

An Arabidopsis histidine kinase is essential for megagametogenesis.

Cytokinin-Independent 1 (CKI1) belongs to a group of putative plant histidine kinases whose members do not appear to act as ethylene receptors. The deduced protein structure, combined with the observation that Arabidopsis callus cultures overexpressing CKI1 exhibit a "cytokinin-independent" cell division and greening phenotype, led to the hypothesis that CKI1 is involved in cytokinin signaling, perhaps acting as a cytokinin receptor. To test the function of CKI1, we used a reverse-genetic approach to identify plants carrying T-DNA insertions in CKI1. Two independent alleles were identified, which produce the same developmental phenotype. Analyses of populations segregating for the cki1-5 or cki1-6 T-DNA insertion alleles failed to reveal any homozygous cki1 plants, indicating that the homozygous mutant condition was lethal. Based on segregation distortion, transmission studies, a microscopy-based examination of developing female gametophytes, and mRNA expression data, we suggest that CKI1 function is required for megagametophyte development. Our work with CKI1 mutants indicates that signal transduction by means of a HisAsp phosphorelay system may play an important and previously unsuspected role in female gametophyte development in Arabidopsis.