Cytokinin oxidase/dehydrogenase inhibitors: progress towards agricultural practice.

Cytokinin oxidase/dehydrogenase (CKX) inhibitors reduce the degradation of cytokinins in plants and thereby may improve the efficiency of agriculture and plant tissue culture-based practices. Here, we report a synthesis and structure-activity relationship study of novel urea derivatives concerning their CKX inhibitory activity. The best compounds showed sub-nanomolar IC50 values with maize ZmCKX1, the lowest value yet documented. Other CKX isoforms of maize (Zea mays) and Arabidopsis were also inhibited very effectively. The binding mode of four compounds was characterized based on high-resolution crystal complex structures. Using the soil nematode Caenorhabditis elegans, and human skin fibroblasts, key CKX inhibitors with low toxicity were identified. These compounds enhanced the shoot regeneration of Lobelia, Drosera, and Plectranthus, as well as the growth of Arabidopsis and Brassica napus. At the same time, a key compound (namely 82), activated a cytokinin primary response gene ARR5:GUS and cytokinin sensor TCSv2:GUS, without activating the Arabidopsis cytokinin receptors AHK3 and AHK4. This strongly implies that the effect of compound 82 is due to the upregulation of cytokinin signalling. Overall, this work presents highly effective and easily prepared CKX inhibitors with a low risk of environmental toxicity for further investigation of their potential in agriculture and biotechnology.

Plant nucleoside N-ribohydrolases: riboside binding and role in nitrogen storage mobilization.

Cells save their energy during nitrogen starvation by selective autophagy of ribosomes and degradation of RNA to ribonucleotides and nucleosides. Nucleosides are hydrolyzed by nucleoside N-ribohydrolases (nucleosidases, NRHs). Subclass I of NRHs preferentially hydrolyzes the purine ribosides while subclass II is more active towards uridine and xanthosine. Here, we performed a crystallographic and kinetic study to shed light on nucleoside preferences among plant NRHs followed by in vivo metabolomic and phenotyping analyses to reveal the consequences of enhanced nucleoside breakdown. We report the crystal structure of Zea mays NRH2b (subclass II) and NRH3 (subclass I) in complexes with the substrate analog forodesine. Purine and pyrimidine catabolism are inseparable because nucleobase binding in the active site of ZmNRH is mediated via a water network and is thus unspecific. Dexamethasone-inducible ZmNRH overexpressor lines of Arabidopsis thaliana, as well as double nrh knockout lines of moss Physcomitrium patents, reveal a fine control of adenosine in contrast to other ribosides. ZmNRH overexpressor lines display an accelerated early vegetative phase including faster root and rosette growth upon nitrogen starvation or osmotic stress. Moreover, the lines enter the bolting and flowering phase much earlier. We observe changes in the pathways related to nitrogen-containing compounds such as ß-alanine and several polyamines, which allow plants to reprogram their metabolism to escape stress. Taken together, crop plant breeding targeting enhanced NRH-mediated nitrogen recycling could therefore be a strategy to enhance plant growth tolerance and productivity under adverse growth conditions.

Cytokinin oxidase/dehydrogenase inhibitors: outlook for selectivity and high efficiency.

Inhibitors of cytokinin oxidase/dehydrogenase (CKX) reduce the degradation of cytokinins in plants, and this effect can be exploited in agriculture and in plant tissue culture. In this study, we examine the structure-activity relationship of two series of CKX inhibitors based on diphenylurea. The compounds of Series I were derived from the recently published CKX inhibitors 3TFM-2HM and 3TFM-2HE, and we identified key substituents with increased selectivity for maize ZmCKX1 and ZmCKX4a over AtCKX2 from Arabidopsis. Series II contained compounds that further exceled in CKX inhibitory activity as well as in the ease of their synthesis. The best inhibitors exhibited half-maximal inhibitory concentration (IC50) values in low nanomolar ranges with ZmCKX1 and especially with ZmCKX4a, which is generally more resistant to inhibition. The activity of the key compounds was verified in tobacco and lobelia leaf-disk assays, where N6-isopentenyladenine was protected from degradation and promoted shoot regeneration. All the prepared compounds were further tested for toxicity against Caenorhabditis elegans, and the assays revealed clear differences in toxicity between compounds with and without a hydroxyalkyl group. In a broader perspective, this work increases our understanding of CKX inhibition and provides a more extensive portfolio of compounds suitable for agricultural and biotechnological research.

Post-Embryonic Lateral Organ Development and Adaxial-Abaxial Polarity Are Regulated by the Combined Effect of ENHANCER OF SHOOT REGENERATION 1 and WUSCHEL in Arabidopsis Shoots.

The development of above-ground lateral organs is initiated at the peripheral zone of the shoot apical meristem (SAM). The coordination of cell fate determination and the maintenance of stem cells are achieved through a complex regulatory network comprised of transcription factors. Two AP2/ERF transcription factor family genes, ESR1/DRN and ESR2/DRNL/SOB/BOL, regulate cotyledon and flower formation and de novo organogenesis in tissue culture. However, their roles in post-embryonic lateral organ development remain elusive. In this study, we analyzed the genetic interactions among SAM-related genes, WUS and STM, two ESR genes, and one of the HD-ZIP III members, REV, whose protein product interacts with ESR1 in planta. We found that esr1 mutations substantially enhanced the wus and stm phenotypes, which bear a striking resemblance to those of the wus rev and stm rev double mutants, respectively. Aberrant adaxial-abaxial polarity is observed in wus esr1 at relatively low penetrance. On the contrary, the esr2 mutation partially suppressed stm phenotypes in the later vegetative phase. Such complex genetic interactions appear to be attributed to the distinct expression pattern of two ESR genes because the ESR1 promoter-driving ESR2 is capable of rescuing phenotypes caused by the esr1 mutation. Our results pose the unique genetic relevance of ESR1 and the SAM-related gene interactions in the development of rosette leaves.

Interpreting Cytokinin Action as Anterograde Signaling and Beyond.

Among the major phytohormones, the cytokinin exhibits unique features for its ability to positively affect the developmental status of plastids. Even early on in its research, cytokinins were known to promote plastid differentiation and to reduce the loss of chlorophyll in detached leaves. Since the discovery of the components of cytokinin perception and primary signaling, the genes involved in photosynthesis and plastid differentiation have been identified as those directly targeted by type-B response regulators. Furthermore, cytokinins are known to modulate versatile cellular processes such as promoting the division and differentiation of cells and, in concert with auxin, initiating the de novo formation of shoot apical meristem (SAM) in tissue cultures. Yet how cytokinins precisely participate in such diverse cellular phenomena, and how the associated cellular processes are coordinated as a whole, remains unclear. A plausible presumption that would account for the coordinated gene expression is the tight and reciprocal communication between the nucleus and plastid. The fact that cytokinins affect plastid developmental status via gene expression in both the nucleus and plastid is interpreted here to suggest that cytokinin functions as an initiator of anterograde (nucleus-to-plastid) signaling. Based on this viewpoint, we first summarize the physiological relevance of cytokinins to the coordination of plastid differentiation with de novo shoot organogenesis in tissue culture systems. Next, the role of endogenous cytokinins in influencing plastid differentiation within the SAM of intact plants is discussed. Finally, a presumed plastid-derived signal in response to cytokinins for coupled nuclear gene expression is proposed.

Diphenylurea-derived cytokinin oxidase/dehydrogenase inhibitors for biotechnology and agriculture.

Increasing crop productivity is our major challenge if we are to meet global needs for food, fodder and fuel. Controlling the content of the plant hormone cytokinin is a method of improving plant productivity. Cytokinin oxidase/dehydrogenase (CKO/CKX) is a major target in this regard because it degrades cytokinins. Here, we describe the synthesis and biological activities of new CKX inhibitors derived mainly from diphenylurea. They were tested on four CKX isoforms from maize and Arabidopsis, where the best compounds showed IC50 values in the 10-8 M concentration range. The binding mode of the most efficient inhibitors was characterized from high-resolution crystal complexed structures. Although these compounds do not possess intrinsic cytokinin activity, we have demonstrated their tremendous potential for use in the plant tissue culture industry as well as in agriculture. We have identified a key substance, compound 19, which not only increases stress resistance and seed yield in Arabidopsis, but also improves the yield of wheat, barley and rapeseed grains under field conditions. Our findings reveal that modulation of cytokinin levels via CKX inhibition can positively affect plant growth, development and yield, and prove that CKX inhibitors can be an attractive target in plant biotechnology and agriculture.

Cytokinin fluoroprobe reveals multiple sites of cytokinin perception at plasma membrane and endoplasmic reticulum.

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.

First Come, First Served: Sui Generis Features of the First Intron.

Most of the transcribed genes in eukaryotic cells are interrupted by intervening sequences called introns that are co-transcriptionally removed from nascent messenger RNA through the process of splicing. In Arabidopsis, 79% of genes contain introns and more than 60% of intron-containing genes undergo alternative splicing (AS), which ostensibly is considered to increase protein diversity as one of the intrinsic mechanisms for fitness to the varying environment or the internal developmental program. In addition, recent findings have prevailed in terms of overlooked intron functions. Here, we review recent progress in the underlying mechanisms of intron function, in particular by focusing on unique features of the first intron that is located in close proximity to the transcription start site. The distinct deposition of epigenetic marks and nucleosome density on the first intronic DNA sequence, the impact of the first intron on determining the transcription start site and elongation of its own expression (called intron-mediated enhancement, IME), translation control in 5'-UTR, and the new mechanism of the trans-acting function of the first intron in regulating gene expression at the post-transcriptional level are summarized.

Occurrence and biosynthesis of cytokinins in poplar.

MAIN CONCLUSION: Isoprenoid and aromatic cytokinins occur in poplar as free compounds and constituents of tRNA, poplar isopentenyltransferases are involved in the production of isoprenoid cytokinins, while biosynthesis of their aromatic counterparts remains unsolved. Cytokinins are phytohormones with a fundamental role in the regulation of plant growth and development. They occur naturally either as isoprenoid or aromatic derivatives, but the latter are quite rare and less studied. Here, the spatial expression of all nine isopentenyl transferase genes of Populus × canadensis cv. Robusta (PcIPTs) as analyzed by RT-qPCR revealed a tissue preference and strong differences in expression levels for the different adenylate and tRNA PcIPTs. Together with their phylogeny, this result suggests a functional diversification for the different PcIPT proteins. Additionally, the majority of PcIPT genes were cloned and expressed in Arabidopsis thaliana under an inducible promoter. The cytokinin levels measured in the Arabidopsis-overexpressing lines as well as their phenotype indicate that the studied adenylate and tRNA PcIPT proteins are functional in vivo and thus will contribute to the cytokinin pool in poplar. We screened the cytokinin content in leaves of 12 Populus species by ultra-high performance-tandem mass spectrometry (UHPLC-MS/MS) and discovered that the capacity to produce not only isoprenoid, but also aromatic cytokinins is widespread amongst the Populus accessions studied. Important for future studies is that the levels of aromatic cytokinins transiently increase after daybreak and are much higher in older plants.

Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase.

KEY MESSAGE: Two new TDZ derivatives (HETDZ and 3FMTDZ) are very potent inhibitors of CKX and are promising candidates for in vivo studies. Cytokinin hormones regulate a wide range of essential processes in plants. Thidiazuron (N-phenyl-N'-1,2,3-thiadiazol-5-yl urea, TDZ), formerly registered as a cotton defoliant, is a well known inhibitor of cytokinin oxidase/dehydrogenase (CKX), an enzyme catalyzing the degradation of cytokinins. TDZ thus increases the lifetime of cytokinins and their effects in plants. We used in silico modeling to design, synthesize and characterize twenty new TDZ derivatives with improved inhibitory properties. Two compounds, namely 1-[1,2,3]thiadiazol-5-yl-3-(3-trifluoromethoxy-phenyl)urea (3FMTDZ) and 1-[2-(2-hydroxyethyl)phenyl]-3-(1,2,3-thiadiazol-5-yl)urea (HETDZ), displayed up to 15-fold lower IC 50 values compared with TDZ for AtCKX2 from Arabidopsis thaliana and ZmCKX1 and ZmCKX4a from Zea mays. Binding modes of 3FMTDZ and HETDZ were analyzed by X-ray crystallography. Crystal structure complexes, solved at 2.0 Å resolution, revealed that HETDZ and 3FMTDZ bound differently in the active site of ZmCKX4a: the thiadiazolyl ring of 3FMTDZ was positioned over the isoalloxazine ring of FAD, whereas that of HETDZ had the opposite orientation, pointing toward the entrance of the active site. The compounds were further tested for cytokinin activity in several cytokinin bioassays. We suggest that the combination of simple synthesis, lowered cytokinin activity, and enhanced inhibitory effects on CKX isoforms, makes 3FMTDZ and HETDZ suitable candidates for in vivo studies.

Maize cytokinin dehydrogenase isozymes are localized predominantly to the vacuoles.

The maize genome encompasses 13 genes encoding for cytokinin dehydrogenase isozymes (CKXs). These enzymes are responsible for irreversible degradation of cytokinin plant hormones and thus, contribute regulating their levels. Here, we focus on the unique aspect of CKXs: their diverse subcellular distribution, important in regulating cytokinin homeostasis. Maize CKXs were tagged with green fluorescent protein (GFP) and transiently expressed in maize protoplasts. Most of the isoforms, namely ZmCKX1, ZmCKX2, ZmCKX4a, ZmCKX5, ZmCKX6, ZmCKX8, ZmCKX9, and ZmCKX12, were associated with endoplasmic reticulum (ER) several hours after transformation. GFP-fused CKXs were observed to accumulate in putative prevacuolar compartments. To gain more information about the spatiotemporal localization of the above isoforms, we prepared stable expression lines of all ZmCKX-GFP fusions in Arabidopsis thaliana Ler suspension culture. All the ER-associated isoforms except ZmCKX1 and ZmCKX9 were found to be targeted primarily to vacuoles, suggesting that ER-localization is a transition point in the intracellular secretory pathway and vacuoles serve as these isoforms' final destination. ZmCKX9 showed an ER-like localization pattern similar to those observed in the transient maize assay. Apoplastic localization of ZmCKX1 was further confirmed and ZmCKX10 showed cytosolic/nuclear localization due to the absence of the signal peptide sequence as previously reported. Additionally, we prepared GFP-fused N-terminal signal deletion mutants of ZmCKX2 and ZmCKX9 and clearly demonstrated that the localization pattern of these mutant forms was cytosolic/nuclear. This study provides the first complex model for spatiotemporal localization of the key enzymes of the cytokinin degradation/catabolism in monocotyledonous plants.

Cytokinin metabolism in maize: Novel evidence of cytokinin abundance, interconversions and formation of a new trans-zeatin metabolic product with a weak anticytokinin activity.

Cytokinins (CKs) are an important group of phytohormones. Their tightly regulated and balanced levels are essential for proper cell division and plant organ development. Here we report precise quantification of CK metabolites and other phytohormones in maize reproductive organs in the course of pollination and kernel maturation. A novel enzymatic activity dependent on NADP(+) converting trans-zeatin (tZ) to 6-(3-methylpyrrol-1-yl)purine (MPP) was detected. MPP shows weak anticytokinin properties and inhibition of CK dehydrogenases due to their ability to bind to an active site in the opposite orientation than substrates. Although the physiological significance of tZ side-chain cyclization is not anticipated as the MPP occurrence in maize tissue is very low, properties of the novel CK metabolite indicate its potential for utilization in plant in vitro tissue culture. Furthermore, feeding experiments with different isoprenoid CKs revealed distinct preferences in glycosylation of tZ and cis-zeatin (cZ). While tZ is preferentially glucosylated at the N9 position, cZ forms mainly O-glucosides. Since O-glucosides, in contrast to N9-glucosides, are resistant to irreversible cleavage catalyzed by CK dehydrogenases, the observed preference of maize CK glycosyltransferases to O-glycosylate zeatin in the cis-position might be a reason why cZ derivatives are over-accumulated in different maize tissues and organs.

Kinetic and structural investigation of the cytokinin oxidase/dehydrogenase active site.

Cytokinins are hormones that regulate plant development and their environmental responses. Their levels are mainly controlled by the cytokinin oxidase/dehydrogenase (CKO), which oxidatively cleaves cytokinins using redox-active electron acceptors. CKO belongs to the group of flavoproteins with an 8α-N1-histidyl FAD covalent linkage. Here, we investigated the role of seven active site residues, H105, D169, E288, V378, E381, P427 and L492, in substrate binding and catalysis of the CKO1 from maize (Zea mays, ZmCKO1) combining site-directed mutagenesis with kinetics and X-ray crystallography. We identify E381 as a key residue for enzyme specificity that restricts substrate binding as well as quinone electron acceptor binding. We show that D169 is important for catalysis and that H105 covalently linked to FAD maintains the enzyme's structural integrity, stability and high rates with electron acceptors. The L492A mutation significantly modulates the cleavage of aromatic cytokinins and zeatin isomers. The high resolution X-ray structures of ZmCKO1 and the E381S variant in complex with N6-(2-isopentenyl)adenosine reveal the binding mode of cytokinin ribosides. Those of ZmCKO2 and ZmCKO4a contain a mobile domain, which might contribute to binding of the N9 substituted cytokinins.

Biochemical characterization of the maize cytokinin dehydrogenase family and cytokinin profiling in developing maize plantlets in relation to the expression of cytokinin dehydrogenase genes.

The cytokinin dehydrogenases (CKX; EC 1.5.99.12) are a protein family that maintains the endogenous levels of cytokinins in plants by catalyzing their oxidative degradation. The CKX family in maize (Zea mays L.) has thirteen members, only two of which--ZmCKX1 and ZmCKX10--have previously been characterized in detail. In this study, nine further maize CKX isoforms were heterologously expressed in Escherichia coli, purified by affinity and ion-exchange chromatography and biochemically characterized. ZmCKX6 and ZmCKX9 could only be expressed successfully after the removal of putative sequence-specific vacuolar sorting signals (LLPT and LPTS, respectively), suggesting that these proteins are localized to the vacuole. Substrate specificity analyses revealed that the CKX isoforms can be grouped into two subfamilies: members of the first strongly prefer cytokinin free bases while members of the second degrade a broad range of substrates. The most active isoform was found to be ZmCKX1. One of the studied isoforms, ZmCKX6, seemed to encode a nonfunctional enzyme due to a mutation in a conserved HFG protein domain at the C-terminus. Site-directed mutagenesis experiments revealed that this domain is essential for CKX activity. The roles of the maize CKX enzymes in the development of maize seedlings during the two weeks immediately after radicle emergence were also investigated. It appears that ZmCKX1 is a key regulator of active cytokinin levels in developing maize roots. However, the expression of individual CKX isoforms in the shoots varied and none of them seemed to have strong effects on the cytokinin pool.

Genetic engineering of cytokinin metabolism: prospective way to improve agricultural traits of crop plants.

Cytokinins (CKs) are ubiquitous phytohormones that participate in development, morphogenesis and many physiological processes throughout plant kingdom. In higher plants, mutants and transgenic cells and tissues with altered activity of CK metabolic enzymes or perception machinery, have highlighted their crucial involvement in different agriculturally important traits, such as productivity, increased tolerance to various stresses and overall plant morphology. Furthermore, recent precise metabolomic analyses have elucidated the specific occurrence and distinct functions of different CK types in various plant species. Thus, smooth manipulation of active CK levels in a spatial and temporal way could be a very potent tool for plant biotechnology in the future. This review summarises recent advances in cytokinin research ranging from transgenic alteration of CK biosynthetic, degradation and glucosylation activities and CK perception to detailed elucidation of molecular processes, in which CKs work as a trigger in model plants. The first attempts to improve the quality of crop plants, focused on cereals are discussed, together with proposed mechanism of action of the responses involved.

Effect of blue light on endogenous isopentenyladenine and endoreduplication during photomorphogenesis and de-etiolation of tomato (Solanum lycopersicum L.) seedlings.

Light is one of the most important factor influencing plant growth and development all through their life cycle. One of the well-known light-regulated processes is de-etiolation, i.e. the switch from skotomorphogenesis to photomorphogenesis. The hormones cytokinins (CKs) play an important role during the establishment of photomorphogenesis as exogenous CKs induced photomorphogenesis of dark-grown seedlings. Most of the studies are conducted on the plant model Arabidopsis, but no or few information are available for important crop species, such as tomato (Solanum lycopersicum L.). In our study, we analyzed for the first time the endogenous CKs content in tomato hypocotyls during skotomorphogenesis, photomorphogenesis and de-etiolation. For this purpose, two tomato genotypes were used: cv. Rutgers (wild-type; WT) and its corresponding mutant (7B-1) affected in its responses to blue light (BL). Using physiological and molecular approaches, we identified that the skotomorphogenesis is characterized by an endoreduplication-mediated cell expansion, which is inhibited upon BL exposure as seen by the accumulation of trancripts encoding CycD3, key regulators of the cell cycle. Our study showed for the first time that iP (isopentenyladenine) is the CK accumulated in the tomato hypocotyl upon BL exposure, suggesting its specific role in photomorphogenesis. This result was supported by physiological experiments and gene expression data. We propose a common model to explain the role and the relationship between CKs, namely iP, and endoreduplication during de-etiolation and photomorphogenesis.

Novel cytokinin derivatives do not show negative effects on root growth and proliferation in submicromolar range.

BACKGROUND: When applied to a nutrition solution or agar media, the non-substituted aromatic cytokinins caused thickening and shortening of the primary root, had an inhibitory effect on lateral root branching, and even showed some negative effects on development of the aerial part at as low as a 10 nanomolar concentration. Novel analogues of aromatic cytokinins ranking among topolins substituted on N9-atom of adenine by tetrahydropyranyl or 4-chlorobutyl group have been prepared and tested in standardized cytokinin bioassays [1]. Those showing comparable activities with N(6)-benzylaminopurine were further tested in planta. METHODOLOGY/PRINCIPAL FINDINGS: The main aim of the study was to explain molecular mechanism of function of novel cytokinin derivatives on plant development. Precise quantification of cytokinin content and profiling of genes involved in cytokinin metabolism and perception in treated plants revealed several aspects of different action of m-methoxytopolin base and its substituted derivative on plant development. In contrast to standard cytokinins, N9- tetrahydropyranyl derivative of m-topolin and its methoxy-counterpart showed the negative effects on root development only at three orders of magnitude higher concentrations. Moreover, the methoxy-derivative demonstrates a positive effect on lateral root branching and leaf emerging in a nanomolar range of concentrations, in comparison with untreated plants. CONCLUSIONS/SIGNIFICANCE: Tetrahydropyranyl substitution at N9-position of cytokinin purine ring significantly enhances acropetal transport of a given cytokinins. Together with the methoxy-substitution, impedes accumulation of non-active cytokinin glucoside forms in roots, allows gradual release of the active base, and has a significant effect on the distribution and amount of endogenous isoprenoid cytokinins in different plant tissues. The utilization of novel aromatic cytokinin derivatives can distinctively improve expected hormonal effects in plant propagation techniques in the future.