Břetislav Brzobohatý

The Histidine Kinases CYTOKININ-INDEPENDENT1 and ARABIDOPSIS HISTIDINE KINASE2 and 3 Regulate Vascular Tissue Development in Arabidopsis Shoots

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 ARABIOPSIS 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.

The putative sensor histidine kinase CKI1 is involved in female gametophyte development in Arabidopsis

Embryo sac formation is a fundamental step in sexual reproduction in plants. However, the key players involved in the development of the female gametophyte remain elusive. We present data indicating that a two-component sensor histidine kinase, CKI1, originally implicated in cytokinin perception, is required for completion of megagametogenesis in Arabidopsis. We isolated a loss-of-function mutation in CKI1 resulting from an insertion of the En-1 transposon into the CKI1 coding sequence. Genetic analysis revealed that the mutant allele, cki1-i , could not be transmitted through the female germ line. Confocal laser scanning microscopy identified a block in megagametogenesis, characterized by the abortion of the central vacuole in mutant embryo sacs, and degradation of the developing female gametophyte after completion of all mitotic divisions. The recovery of two independent stable alleles and one revertant wild-type allele resulting from En-1 excision confirmed unambiguously the causal link between the cki1-i mutation and the abnormal phenotype. In situ localization of CKI1 mRNA and histochemical analysis of stable transformants harboring the uidA gene under the control of CKI1 promoter revealed that expression of CKI1 starts at the very beginning of female gametophyte development, and continues until fertilization. This suggests that the developing embryo sac may remain sensitive to signals recognized by CKI1 throughout megagametogenesis. Furthermore, expression of the paternally transmitted CKI1 was detected early after fertilization. The results indicate a role for a two-component signaling system during female gametophyte development, and provide the first evidence that gametophytic expression of a sensor-like molecule is essential for specific processes during megagametogenesis.

Cytokinin metabolism: implications for regulation of plant growth and development

This review describes recent advances in the study of cytokinin metabolism. It highlights how plant development is influenced by cytokinin synthesis, conjugation and conjugate hydrolysis, and what has been learned of the enzymes that regulate these processes. Although cytokinin metabolism and physiology are complex issues, some of the key enzymatic players are now being identified. This holds out the prospect of rapid progress in the near future. Just as much of what we know about the control of animal cell proliferation was learned by studying the cellular counterparts of viral oncogenes, so important information about the control of plant development by phytohormones has come from studying the genes of bacterial pathogens that subvert host phytohormone metabolism to their own advantage. We will focus on what has been learned from the use of such genes, and describe progress in identifying their functional counterparts in plants.

Effects of Conditional IPT-Dependent Cytokinin Overproduction on Root Architecture of Arabidopsis Seedlings

Cytokinin (CK) has been known to inhibit primary root elongation and suggested to act as an auxin antagonist in the regulation of lateral root (LR) formation. While the role of auxin in root development has been thoroughly studied, the detailed and overall description of CK effects on root system morphology, particularly that of developing lateral root primordia (LRPs), and hence its role in organogenesis is still in progress. Here we examine the effects of conditional endogenous CK overproduction on root architecture and consider its temporal aspect during the early development of Arabidopsis thaliana. We employed the pOp/LhGR system to induce ectopic ipt overexpression with a glucocorticoid dexamethasone at designated developmental points. The transient CaMV 35S>GR>ipt transactivation greatly enhanced levels of biologically active CKs of zeatin (Z)-type and identified a distinct developmental interval during which primary root elongation is susceptible to increases in endogenous CK production. Long-term CK overproduction inhibited primary root elongation by reducing quantitative parameters of primary root meristem, disturbed a characteristic graded distribution pattern of auxin response in LRPs and impaired their development. Our findings indicate the impact of perturbed endogenous CK on the regulation of asymmetric auxin distribution during LRP development and imply that there is cross-talk between auxin and CK during organogenesis in A. thaliana.

Factors enhancing genetic transformation of intact yeast cells modify cell wall porosity

Genetic transformation of intact cells of Saccharomyces cerevisiae, achieved by incubating the cells with plasmid DNA in the presence of PEG, could be enhanced, not only by pretreatment of the cells with Li+ and 2-mercaptoethanol, as has been reported previously, but also by pretreatment with proteolytic enzymes. The efficiency of transformation with 2-mercaptoethanol rose dramatically when the pretreated cells had been handled in osmotically stabilized media. Following all the pretreatments the cells became leaky for nucleic acids as detected by the presence of endogenous RNAs in the medium. The pretreatments evidently facilitated the passage of transforming DNA across the cell wall.

Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: a proteomic analysis

High concentrations of cytokinins (CKs) in the cultivation medium can induce partial photomorphogenesis in dark-grown Arabidopsis seedlings. However, no significant increases in endogenous CK levels have been found in de-etiolated mutants, suggesting that either parallel pathways are involved in the light and CK responses, or changes in the sensitivity to CKs occur during photomorphogenesis. Here it is shown that even modest increases in endogenous CK levels induced by transgenic expression of the CK biosynthetic gene, ipt, can lead to many typical features of light-induced de-etiolation, including inhibition of hypocotyl elongation and partial cotyledon opening. In addition, significant changes in expression of 37 proteins (mostly related to chloroplast biogenesis, a major element of light-induced photomorphogenesis) were detected by image and mass spectrometric analysis of two-dimensionally separated proteins. The identified chloroplast proteins were all up-regulated in response to increased CKs, and more than half are up-regulated at the transcript level during light-induced photomorphogenesis according to previously published transcriptomic data. Four of the up-regulated chloroplast proteins identified here have also been shown to be up-regulated during light-induced photomorphogenesis in previous proteomic analyses. In contrast, all differentially regulated mitochondrial proteins (the second largest group of differentially expressed proteins) were down-regulated. Changes in the levels of several tubulins are consistent with the observed morphological alterations. Further, 10 out of the 37 differentially expressed proteins detected have not been linked to either photomorphogenesis or CK action in light-grown Arabidopsis seedlings in previously published transcriptomic or proteomic analyses.

Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling

Cytokinins are plant hormones involved in regulation of diverse developmental and physiological processes in plants whose molecular mechanisms of action are being intensely researched. However, most rapid responses to cytokinin signals at the proteomic and phosphoproteomic levels are unknown. Early cytokinin responses were investigated through proteome-wide expression profiling based on image and mass spectrometric analysis of two-dimensionally separated proteins and phosphoproteins. The effects of 15 min treatments of 7-day-old Arabidopsis thaliana seedlings with four main cytokinins representing hydroxyisopentenyl, isopentenyl, aromatic, and urea-derived type cytokinins were compared to help elucidate their common and specific function(s) in regulating plant development. In proteome and phosphoproteome maps, significant differences were reproducibly observed for 53 and 31 protein spots, respectively. In these spots, 96 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), providing a snapshot of early links in cytokinin-regulated signalling circuits and cellular processes, including light signalling and photosynthesis, nitrogen metabolism, the CLAVATA pathway, and protein and gene expression regulation, in accordance with previously described cytokinin functions. Furthermore, they indicate novel links between temperature and cytokinin signalling, and an involvement of calcium ions in cytokinin signalling. Most of the differentially regulated proteins and phosphoproteins are located in chloroplasts, suggesting an as yet uncharacterized direct signalling chain responsible for cytokinin action in chloroplasts. Finally, first insights into the degree of specificity of cytokinin receptors on phosphoproteomic effects were obtained from analyses of cytokinin action in a set of cytokinin receptor double mutants.

5-Azacytidine-induced hypomethylation of tobacco HRS60 tandem DNA repeats in tissue culture

The methylation status and 5-azacytidine-induced hypomethylation of CCGG sites within a family of tandemly organized, highly repeated DNA sequences of the Nicotiana tabacum L. nuclear genome (HRS60 family) were studied. As shown by in-situ hybridization experiments, the HRS60 family is clustered in a few regions of some tobacco chromosomes. The DNAs of leaf-derived calli, leaf-derived calli cultured on media with 5-azacytidine, and leaves were cleaved with restriction endonucleases differing in the sensitivity to the methylation of cytosine. After electrophoresis and Southern blotting they were hybridized with the HRS60 probe. We show that (i) CpG dinucleotides, and partially also CpCpG trinucleotides, of the HRS60 family are methylated in DNAs of the non-treated calli and leaves, and (ii) that these DNA repeats are sensitive to the action of a hypomethylating drug, 5-azacytidine.

Cytokinin modulates proteomic, transcriptomic and growth responses to temperature shocks in Arabidopsis.

As sessile organisms, plants must sense environmental conditions and adjust their growth and development processes accordingly, through adaptive responses regulated by various internal factors, including hormones. A key environmental factor is temperature, but temperature-sensing mechanisms are not fully understood despite intense research. We investigated proteomic responses to temperature shocks (15 min cold or heat treatments) with and without exogenous applications of cytokinin in Arabidopsis. Image and mass spectrometric analysis of the two-dimensionally separated proteins detected 139 differentially regulated spots, in which 148 proteins were identified, most of which have not been previously linked to temperature perception. More than 70% of the temperature-shock response proteins were modulated by cytokinin, mostly in a similar manner as heat shock. Data mining of previous transcriptomic datasets supported extensive interactions between temperature and cytokinin signalling. The biological significance of this finding was tested by assaying an independent growth response of Arabidopsis seedlings to heat stress: hypocotyl elongation. This response was strongly inhibited in mutants with deficiencies in cytokinin signalling or endogenous cytokinin levels. Thus, cytokinins may directly participate in heat signalling in plants. Finally, large proportions of both temperature-shock and cytokinin responsive proteomes co-localize to the chloroplast, which might therefore host a substantial proportion of the temperature response machinery.

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

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