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.

Comprehensive LC-MS/MS Analysis of Nitrogen-Related Plant Metabolites.

We have developed and validated a novel LC-MS/MS method for simultaneously analyzing amino acids, biogenic amines, and their acetylated and methylated derivatives in plants. This method involves a one-step extraction of 2-5 mg of lyophilized plant material followed by fractionation of different biogenic amine forms and exploits an efficient combination of hydrophilic interaction chromatography (HILIC), reversed phase (RP) chromatography with pre-column derivatization, and tandem mass spectrometry. This approach enables high-throughput processing of plant samples, significantly reducing the time needed for analysis and its cost. We also present a new synthetic route for deuterium-labelled polyamines. The LC-MS/MS method was rigorously validated by quantifying levels of nitrogen-related metabolites in seedlings of seven plant species including Arabidopsis, maize, and barley, all of which are commonly used model organisms in plant science research. Our results revealed substantial variations in the abundance of these metabolites between species, developmental stages, and growth conditions, particularly for the acetylated and methylated derivatives and the various polyamine fractions. However, the biological relevance of these plant metabolites is currently unclear. Overall, this work contributes significantly to the field of plant science by providing a powerful analytical tool and setting the stage for future investigations into the functions of these nitrogen-related metabolites in plants.

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.

Are Histidine Kinases of Arbuscular Mycorrhizal Fungi Involved in the Response to Ethylene and Cytokinins?

Signals are exchanged at all stages of the arbuscular mycorrhizal (AM) symbiosis between fungi and their host plants. Root-exuded strigolactones are well-known early symbiotic cues, but the role of other phytohormones as interkingdom signals has seldom been investigated. Here we focus on ethylene and cytokinins, for which candidate receptors have been identified in the genome of the AM fungus Rhizophagus irregularis. Ethylene is known from the literature to affect asymbiotic development of AM fungi, and in the present study, we found that three cytokinin forms could stimulate spore germination in R. irregularis. Heterologous complementation of a Saccharomyces cerevisiae mutant strain with the candidate ethylene receptor RiHHK6 suggested that this protein can sense and transduce an ethylene signal. Accordingly, its N-terminal domain expressed in Pichia pastoris displayed saturable binding to radiolabeled ethylene. Thus, RiHHK6 displays the expected characteristics of an ethylene receptor. In contrast, the candidate cytokinin receptor RiHHK7 did not complement the S. cerevisiae mutant strain or Medicago truncatula cytokinin receptor mutants and seemed unable to bind cytokinins, suggesting that another receptor is involved in the perception of these phytohormones. Taken together, our results support the hypothesis that AM fungi respond to a range of phytohormones and that these compounds bear multiple functions in the rhizosphere beyond their known roles as internal plant developmental regulators. Our analysis of two phytohormone receptor candidates also sheds new light on the possible perception mechanisms in AM fungi. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phloem exudate metabolic content reflects the response to water-deficit stress in pea plants (Pisum sativum L.).

Drought stress impacts the quality and yield of Pisum sativum. Here, we show how short periods of limited water availability during the vegetative stage of pea alters phloem sap content and how these changes are connected to strategies used by plants to cope with water deficit. We have investigated the metabolic content of phloem sap exudates and explored how this reflects P. sativum physiological and developmental responses to drought. Our data show that drought is accompanied by phloem-mediated redirection of the components that are necessary for cellular respiration and the proper maintenance of carbon/nitrogen balance during stress. The metabolic content of phloem sap reveals a shift from anabolic to catabolic processes as well as the developmental plasticity of P. sativum plants subjected to drought. Our study underlines the importance of phloem-mediated transport for plant adaptation to unfavourable environmental conditions. We also show that phloem exudate analysis can be used as a useful proxy to study stress responses in plants. We propose that the decrease in oleic acid content within phloem sap could be considered as a potential marker of early signalling events mediating drought response.

Sucrose promotes stem branching through cytokinin.

Shoot branching is an important aspect of plant architecture because it substantially affects plant biology and agricultural performance. Sugars play an important role in the induction of shoot branching in several species, including potato (Solanum tuberosum L.). However, the mechanism by which sugars affect shoot branching remains mostly unknown. In the present study, we addressed this question using sugar-mediated induction of bud outgrowth in potato stems under etiolated conditions. Our results indicate that sucrose feeding to detached stems promotes the accumulation of cytokinin (CK), as well as the expression of vacuolar invertase (VInv), an enzyme that contributes to sugar sink strength. These effects of sucrose were suppressed by CK synthesis and perception inhibitors, while CK supplied to detached stems induced bud outgrowth and VInv activity in the absence of sucrose. CK-induced bud outgrowth was suppressed in vinv mutants, which we generated by genome editing. Altogether, our results identify a branching-promoting module, and suggest that sugar-induced lateral bud outgrowth is in part promoted by the induction of CK-mediated VInv activity.

Presence and future of plant phenotyping approaches in biostimulant research and development.

Commercial interest in biostimulants as a tool for sustainable green economics and agriculture concepts is on a steep rise, being followed by increasing demand to employ efficient scientific methods to develop new products and understand their mechanisms of action. Biostimulants represent a highly diverse group of agents derived from various natural sources. Regardless of their nutrition content and composition, they are classified by their ability to improve crop performance through enhanced nutrient use efficiency, abiotic stress tolerance, and quality of crops. Numerous reports have described modern, non-invasive sensor-based phenotyping methods in plant research. This review focuses on applying phenotyping approaches in biostimulant research and development, and maps the evolution of interaction of these two intensively growing domains. How phenotyping served to identify new biostimulants, the description of their biological activity, and the mechanism/mode of action are summarized. Special attention is dedicated to the indoor high-throughput methods using model plants suitable for biostimulant screening and developmental pipelines, and high-precision approaches used to determine biostimulant activity. The need for a complex method of testing biostimulants as multicomponent products through integrating other -omic approaches followed by advanced statistical/mathematical tools is emphasized.

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.

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.

Plastidial Phosphoglucose Isomerase Is an Important Determinant of Seed Yield through Its Involvement in Gibberellin-Mediated Reproductive Development and Storage Reserve Biosynthesis in Arabidopsis.

The plastid-localized phosphoglucose isomerase isoform PGI1 is an important determinant of growth in Arabidopsis thaliana, likely due to its involvement in the biosynthesis of plastidial isoprenoid-derived hormones. Here, we investigated whether PGI1 also influences seed yields. PGI1 is strongly expressed in maturing seed embryos and vascular tissues. PGI1-null pgi1-2 plants had ∼60% lower seed yields than wild-type plants, with reduced numbers of inflorescences and thus fewer siliques and seeds per plant. These traits were associated with low bioactive gibberellin (GA) contents. Accordingly, wild-type phenotypes were restored by exogenous GA application. pgi1-2 seeds were lighter and accumulated ∼50% less fatty acids (FAs) and ∼35% less protein than wild-type seeds. Seeds of cytokinin-deficient plants overexpressing CYTOKININ OXIDASE/DEHYDROGENASE1 (35S:AtCKX1) and GA-deficient ga20ox1 ga20ox2 mutants did not accumulate low levels of FAs, and exogenous application of the cytokinin 6-benzylaminopurine and GAs did not rescue the reduced weight and FA content of pgi1-2 seeds. Seeds from reciprocal crosses between pgi1-2 and wild-type plants accumulated wild-type levels of FAs and proteins. Therefore, PGI1 is an important determinant of Arabidopsis seed yield due to its involvement in two processes: GA-mediated reproductive development and the metabolic conversion of plastidial glucose-6-phosphate to storage reserves in the embryo.

Volatiles from the fungal phytopathogen Penicillium aurantiogriseum modulate root metabolism and architecture through proteome resetting.

Volatile compounds (VCs) emitted by the fungal phytopathogen Penicillium aurantiogriseum promote root growth and developmental changes in Arabidopsis. Here we characterised the metabolic and molecular responses of roots to fungal volatiles. Proteomic analyses revealed that these compounds reduce the levels of aquaporins, the iron carrier IRT1 and apoplastic peroxidases. Fungal VCs also increased the levels of enzymes involved in the production of mevalonate (MVA)-derived isoprenoids, nitrogen assimilation and conversion of methionine to ethylene and cyanide. Consistently, fungal VC-treated roots accumulated high levels of hydrogen peroxide (H2 O2 ), MVA-derived cytokinins, ethylene, cyanide and long-distance nitrogen transport amino acids. qRT-PCR analyses showed that many proteins differentially expressed by fungal VCs are encoded by VC non-responsive genes. Expression patterns of hormone reporters and developmental characterisation of mutants provided evidence for the involvement of cyanide scavenging and enhanced auxin, ethylene, cytokinin and H2 O2 signalling in the root architecture changes promoted by fungal VCs. Our findings show that VCs from P. aurantiogriseum modify root metabolism and architecture, and improve nutrient and water use efficiencies through transcriptionally and non-transcriptionally regulated proteome resetting mechanisms. Some of these mechanisms are subject to long-distance regulation by photosynthesis and differ from those triggered by VCs emitted by beneficial microorganisms.

New aromatic 6-substituted 2'-deoxy-9-(ß)-d-ribofuranosylpurine derivatives as potential plant growth regulators.

Cytokinins are naturally occurring substances that act as plant growth regulators promoting plant growth and development, including shoot initiation and branching, and also affecting apical dominance and leaf senescence. Aromatic cytokinin 6-benzylaminopurine (BAP) has been widely used in micropropagation systems and biotechnology. However, its 9-glucoside (BAP9G) accumulates in explants, causing root inhibition and growth heterogenity. To overcome BAP disadvantages, a series of ring-substituted 2'-deoxy-9-(ß)-d-ribofuranosylpurine derivatives was prepared and examined in different classical cytokinin bioassays. Amaranthus, senescence and tobacco callus bioassays were employed to provide details of cytokinin activity of 2'-deoxy-9-(ß)-d-ribosides compared to their respective free bases and ribosides. The prepared derivatives were also tested for their recognition by cytokinin receptors of Arabidopsis thaliana AHK3 and CRE1/AHK4. The ability of aromatic N6-substituted adenine-2'-deoxy-9-(ß)-d-ribosides to promote plant growth and delay senescence was increased considerably and, in contrast to BAP, no loss of cytokinin activity at higher concentrations was observed. The presence of a 2'-deoxyribosyl moiety at the N9-position led to an increase in cytokinin activities in comparison to the free bases and ribosides. The antioxidant capacity, cytotoxicity and effect on the MHV-68 gammaherpesvirus strain were also examined.

Plant responses to fungal volatiles involve global posttranslational thiol redox proteome changes that affect photosynthesis.

Microorganisms produce volatile compounds (VCs) that promote plant growth and photosynthesis through complex mechanisms involving cytokinin (CK) and abscisic acid (ABA). We hypothesized that plants' responses to microbial VCs involve posttranslational modifications of the thiol redox proteome through action of plastidial NADPH-dependent thioredoxin reductase C (NTRC), which regulates chloroplast redox status via its functional relationship with 2-Cys peroxiredoxins. To test this hypothesis, we analysed developmental, metabolic, hormonal, genetic, and redox proteomic responses of wild-type (WT) plants and a NTRC knockout mutant (ntrc) to VCs emitted by the phytopathogen Alternaria alternata. Fungal VC-promoted growth, changes in root architecture, shifts in expression of VC-responsive CK- and ABA-regulated genes, and increases in photosynthetic capacity were substantially weaker in ntrc plants than in WT plants. As in WT plants, fungal VCs strongly promoted growth, chlorophyll accumulation, and photosynthesis in ntrc-Δ2cp plants with reduced 2-Cys peroxiredoxin expression. OxiTRAQ-based quantitative and site-specific redox proteomic analyses revealed that VCs promote global reduction of the thiol redox proteome (especially of photosynthesis-related proteins) of WT leaves but its oxidation in ntrc leaves. Our findings show that NTRC is an important mediator of plant responses to microbial VCs through mechanisms involving global thiol redox proteome changes that affect photosynthesis.

Cytokinin-Auxin Crosstalk in the Gynoecial Primordium Ensures Correct Domain Patterning.

The Arabidopsis (Arabidopsis thaliana) gynoecium consists of two congenitally fused carpels made up of two lateral valve domains and two medial domains, which retain meristematic properties and later fuse to produce the female reproductive structures vital for fertilization. Polar auxin transport (PAT) is important for setting up distinct apical auxin signaling domains in the early floral meristem remnants allowing for lateral domain identity and outgrowth. Crosstalk between auxin and cytokinin plays an important role in the development of other meristematic tissues, but hormone interaction studies to date have focused on more accessible later-stage gynoecia and the spatiotemporal interactions pivotal for patterning of early gynoecium primordia remain unknown. Focusing on the earliest stages, we propose a cytokinin-auxin feedback model during early gynoecium patterning and hormone homeostasis. Our results suggest that cytokinin positively regulates auxin signaling in the incipient gynoecial primordium and strengthen the concept that cytokinin regulates auxin homeostasis during gynoecium development. Specifically, medial cytokinin promotes auxin biosynthesis components [YUCCA1/4 (YUC1/4)] in, and PINFORMED7 (PIN7)-mediated auxin efflux from, the medial domain. The resulting laterally focused auxin signaling triggers ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN6 (AHP6), which then represses cytokinin signaling in a PAT-dependent feedback. Cytokinin also down-regulates PIN3, promoting auxin accumulation in the apex. The yuc1, yuc4, and ahp6 mutants are hypersensitive to exogenous cytokinin and 1-napthylphthalamic acid (NPA), highlighting their role in mediolateral gynoecium patterning. In summary, these mechanisms self-regulate cytokinin and auxin signaling domains, ensuring correct domain specification and gynoecium development.

Resorcinol-Type Strigolactone Mimics as Potent Germinators of the Parasitic Plants Striga hermonthica and Phelipanche ramosa.

Strigolactones are a particular class of plant metabolites with diverse biological functions starting from the stimulation of parasitic seed germination to phytohormonal activity. The expansion of parasitic weeds in the fields of developing countries is threatening the food supply and calls for simple procedures to combat these weeds. Strigolactone analogues represent a promising approach for such control through suicidal germination, i.e., parasitic seed germination without the presence of the host causing parasite death. In the present work, the synthesis of resorcinol-type strigolactone mimics related to debranones is reported. These compounds were highly stable even at alkaline pH levels and able to induce seed germination of parasitic plants Striga hermonthica and Phelipanche ramosa at low concentrations, EC50 ≈ 2 × 10-7 M ( Striga) and EC50 ≈ 2 × 10-9 M ( Phelipanche). On the other hand, the mimics had no significant effect on root architecture of Arabidopsis plants, suggesting a selective activity for parasitic seed germination, making them a primary target as suicidal germinators.

Arabidopsis ROCK1 transports UDP-GlcNAc/UDP-GalNAc and regulates ER protein quality control and cytokinin activity.

The formation of glycoconjugates depends on nucleotide sugars, which serve as donor substrates for glycosyltransferases in the lumen of Golgi vesicles and the endoplasmic reticulum (ER). Import of nucleotide sugars from the cytosol is an important prerequisite for these reactions and is mediated by nucleotide sugar transporters. Here, we report the identification of REPRESSOR OF CYTOKININ DEFICIENCY 1 (ROCK1, At5g65000) as an ER-localized facilitator of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) transport in Arabidopsis thaliana. Mutant alleles of ROCK1 suppress phenotypes inferred by a reduced concentration of the plant hormone cytokinin. This suppression is caused by the loss of activity of cytokinin-degrading enzymes, cytokinin oxidases/dehydrogenases (CKXs). Cytokinin plays an essential role in regulating shoot apical meristem (SAM) activity and shoot architecture. We show that rock1 enhances SAM activity and organ formation rate, demonstrating an important role of ROCK1 in regulating the cytokinin signal in the meristematic cells through modulating activity of CKX proteins. Intriguingly, genetic and molecular analysis indicated that N-glycosylation of CKX1 was not affected by the lack of ROCK1-mediated supply of UDP-GlcNAc. In contrast, we show that CKX1 stability is regulated in a proteasome-dependent manner and that ROCK1 regulates the CKX1 level. The increased unfolded protein response in rock1 plants and suppression of phenotypes caused by the defective brassinosteroid receptor bri1-9 strongly suggest that the ROCK1 activity is an important part of the ER quality control system, which determines the fate of aberrant proteins in the secretory pathway.

Arabidopsis Responds to Alternaria alternata Volatiles by Triggering Plastid Phosphoglucose Isomerase-Independent Mechanisms.

Volatile compounds (VCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote photosynthesis, growth, and the accumulation of high levels of starch in leaves through cytokinin (CK)-regulated processes. In Arabidopsis (Arabidopsis thaliana) plants not exposed to VCs, plastidic phosphoglucose isomerase (pPGI) acts as an important determinant of photosynthesis and growth, likely as a consequence of its involvement in the synthesis of plastidic CKs in roots. Moreover, this enzyme plays an important role in connecting the Calvin-Benson cycle with the starch biosynthetic pathway in leaves. To elucidate the mechanisms involved in the responses of plants to microbial VCs and to investigate the extent of pPGI involvement, we characterized pPGI-null pgi1-2 Arabidopsis plants cultured in the presence or absence of VCs emitted by Alternaria alternata We found that volatile emissions from this fungal phytopathogen promote growth, photosynthesis, and the accumulation of plastidic CKs in pgi1-2 leaves. Notably, the mesophyll cells of pgi1-2 leaves accumulated exceptionally high levels of starch following VC exposure. Proteomic analyses revealed that VCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism, and growth that can account for the observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants can respond to VCs emitted by phytopathogenic microorganisms by triggering pPGI-independent mechanisms.

Haustorium initiation in the obligate parasitic plant Phelipanche ramosa involves a host-exudated cytokinin signal.

The heterotrophic lifestyle of parasitic plants relies on the development of the haustorium, a specific infectious organ required for attachment to host roots. While haustorium development is initiated upon chemodetection of host-derived molecules in hemiparasitic plants, the induction of haustorium formation remains largely unknown in holoparasitic species such as Phelipanche ramosa. This work demonstrates that the root exudates of the host plant Brassica napus contain allelochemicals displaying haustorium-inducing activity on P. ramosa germinating seeds, which increases the parasite aggressiveness. A de novo assembled transcriptome and microarray approach with P. ramosa during early haustorium formation upon treatment with B. napus root exudates allowed the identification of differentially expressed genes involved in hormone signaling. Bioassays using exogenous cytokinins and the specific cytokinin receptor inhibitor PI-55 showed that cytokinins induced haustorium formation and increased parasite aggressiveness. Root exudates triggered the expression of cytokinin-responsive genes during early haustorium development in germinated seeds, and bio-guided UPLC-ESI(+)-/MS/MS analysis showed that these exudates contain a cytokinin with dihydrozeatin characteristics. These results suggest that cytokinins constitutively exudated from host roots play a major role in haustorium formation and aggressiveness in P. ramosa.

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.

Cytokinins influence root gravitropism via differential regulation of auxin transporter expression and localization in Arabidopsis.

Redirection of intercellular auxin fluxes via relocalization of the PIN-FORMED 3 (PIN3) and PIN7 auxin efflux carriers has been suggested to be necessary for the root gravitropic response. Cytokinins have also been proposed to play a role in controlling root gravitropism, but conclusive evidence is lacking. We present a detailed study of the dynamics of root bending early after gravistimulation, which revealed a delayed gravitropic response in transgenic lines with depleted endogenous cytokinins (Pro35S:AtCKX) and cytokinin signaling mutants. Pro35S:AtCKX lines, as well as a cytokinin receptor mutant ahk3, showed aberrations in the auxin response distribution in columella cells consistent with defects in the auxin transport machinery. Using in vivo real-time imaging of PIN3-GFP and PIN7-GFP in AtCKX3 overexpression and ahk3 backgrounds, we observed wild-type-like relocalization of PIN proteins in the columella early after gravistimulation, with gravity-induced relocalization of PIN7 faster than that of PIN3. Nonetheless, the cellular distribution of PIN3 and PIN7 and expression of PIN7 and the auxin influx carrier AUX1 was affected in AtCKX overexpression lines. Based on the retained cytokinin sensitivity in pin3 pin4 pin7 mutant, we propose the AUX1-mediated auxin transport rather than columella-located PIN proteins as a target of endogenous cytokinins in the control of root gravitropism.