Hormonal Regulation and Crosstalk of Auxin/Cytokinin Signaling Pathways in Potatoes In Vitro and in Relation to Vegetation or Tuberization Stages.

Auxins and cytokinins create versatile regulatory network controlling virtually all aspects of plant growth and development. These hormonal systems act in close contact, synergistically or antagonistically, determining plant phenotype, resistance and productivity. However, the current knowledge about molecular interactions of these systems is still scarce. Our study with potato plants aimed at deciphering potential interactions between auxin and cytokinin signaling pathways at the level of respective gene expression. Potato plants grown on sterile medium with 1.5% (vegetation) or 5% (tuberization) sucrose were treated for 1 h with auxin or cytokinin. Effects of these two hormones on expression profiles of genes belonging to main signaling pathways of auxin and cytokinin were quantified by RT-qPCR. As a result, several signaling genes were found to respond to auxin and/or cytokinin by up- or down-regulation. The observed effects were largely organ-specific and depended on sucrose content. Auxin strongly reduced cytokinin perception apparatus while reciprocal cytokinin effect was ambiguous and sucrose-dependent. In many cases, functional clustering of genes of the same family was observed. Promoters in some clusters are enriched with canonic hormone-response cis-elements supporting their direct sensitivity to hormones. Collectively, our data shed new light on the crosstalk between auxin- and cytokinin signaling pathways.

Cytokinin perception in potato: new features of canonical players.

Potato is the most economically important non-cereal food crop. Tuber formation in potato is regulated by phytohormones, cytokinins (CKs) in particular. The present work studied CK signal perception in potato. The sequenced potato genome of doubled monoploid Phureja was used for bioinformatic analysis and as a tool for identification of putative CK receptors from autotetraploid potato cv. Désirée. All basic elements of multistep phosphorelay required for CK signal transduction were identified in the Phureja genome, including three genes orthologous to three CK receptor genes (AHK 2-4) of Arabidopsis. As distinct from Phureja, autotetraploid potato contains at least two allelic isoforms of each receptor type. Putative receptor genes from Désirée plants were cloned, sequenced and expressed, and the main characteristics of encoded proteins were determined, in particular their consensus motifs, modelled structure, ligand-binding properties, and ability to transmit CK signals. In all studied aspects the predicted sensor histidine kinases met the requirements for genuine CK receptors. Expression of potato CK receptors was found to be organ-specific and sensitive to growth conditions, particularly to sucrose content. Our results provide a solid basis for further in-depth study of CK signaling system and biotechnological improvement of potato.

Auxin synthesis gene tms1 driven by tuber-specific promoter alters hormonal status of transgenic potato plants and their responses to exogenous phytohormones.

KEY MESSAGE: Ectopic auxin overproduction in transgenic potato leads to enhanced productivity accompanied with concerted and occasional changes in hormonal status, and causing altered response of transformants to exogenous auxin or cytokinin. Previously, we generated potato transformants expressing Agrobacterium-derived auxin synthesis gene tms1 driven by tuber-specific patatin gene promoter (B33-promoter). Here, we studied the endogenous hormonal status and the response to exogenous phytohormones in tms1 transformants cultured in vitro. Adding indole-3-acetic acid (IAA) or kinetin to culture medium affected differently tuberization of tms1-transformed and control plants, depending also on sucrose content in the medium. Exogenous phytohormones ceased to stimulate the tuber initiation in transformants at high (5-8%) sucrose concentration, while in control plants the stimulation was observed in all experimental settings. Furthermore, exogenous auxin partly inhibited the tuber initiation, and exogenous cytokinin reduced the average tuber weight in most transformants at high sucrose content. The elevated auxin level in tubers of the transformants was accompanied with a decrease in content of cytokinin bases and their ribosides in tubers and most shoots. No concerted changes in contents of abscisic, jasmonic, salicylic acids and gibberellins in tubers were detected. The data on hormonal status indicated that the enhanced productivity of tms1 transformants was due to auxin and not mediated by other phytohormones. In addition, exogenous cytokinin was shown to upregulate the expression of genes encoding orthologs of auxin receptors. Overall, the results showed that tms1 expression and local increase in IAA level in transformants affect both the balance of endogenous cytokinins and the dynamics of tuberization in response to exogenous hormones (auxin, cytokinin), the latter reaction depending also on the carbohydrate supply. We introduce a basic model for the hormonal network controlling tuberization.

Expression of auxin synthesis gene tms1 under control of tuber-specific promoter enhances potato tuberization in vitro.

Phytohormones, auxins in particular, play an important role in plant development and productivity. Earlier data showed positive impact of exogenous auxin on potato (Solanum tuberosum L.) tuberization. The aim of this study was to generate potato plants with increased auxin level predominantly in tubers. To this end, a pBinB33-tms1 vector was constructed harboring the Agrobacterium auxin biosynthesis gene tms1 fused to tuber-specific promoter of the class I patatin gene (B33-promoter) of potato. Among numerous independently generated B33:tms1 lines, those without visible differences from control were selected for detailed studies. In the majority of transgenic lines, tms1 gene transcription was detected, mostly in tubers rather than in shoots. Indoleacetic acid (IAA) content in tubers and the auxin tuber-to-shoot ratio were increased in tms1-expressing transformants. The organ-specific increase in auxin synthesis in B33:tms1-transformants accelerated and intensified the process of tuber formation, reduced the dose of carbohydrate supply required for in vitro tuberization, and decreased the photoperiodic dependence of tuber initiation. Overall, a positive correlation was observed between tms1 expression, IAA content in tubers, and stimulation of tuber formation. The revealed properties of B33:tms1 transformants imply an important role for auxin in potato tuberization and offer prospects to magnify potato productivity by a moderate organ-specific enhancement of auxin content.

Rapid activation of specific phospholipase(s) D by cytokinin in Amaranthus assay system.

The suggested link between intracellular cytokinin signaling and phospholipase D (PLD, EC 3.1.4.4.) activity (Romanov et al. 2000, 2002) was investigated. The activity of PLD in the early period of cytokinin action was studied in vivo in derooted Amaranthus caudatus seedlings, using the level of phosphatidylbutanol production as a measure of PLD activity. Rapid activation of phosphatidylbutanol synthesis was demonstrated as early as within 5 min of cytokinin administration. Neomycin, a known phosphatidylinositol-4,5-bisphosphate (PIP(2)) antagonist, strongly repressed both physiological cytokinin effect and cytokinin-dependent PLD activation. N-acylethanolamine (NAE 12), an inhibitor of alpha-class PLD, did not influence significantly cytokinin effect on Amaranthus seedlings. Together, results suggest the involvement of PIP(2)-dependent non-class alpha-PLD in the molecular mechanism of cytokinin action.

Global View on the Cytokinin Regulatory System in Potato.

Cytokinins (CKs) were earlier shown to promote potato tuberization. Our study aimed to identify and characterize CK-related genes which constitute CK regulatory system in the core potato (Solanum tuberosum) genome. For that, CK-related genes were retrieved from the sequenced genome of the S. tuberosum doubled monoploid (DM) Phureja group, classified and compared with Arabidopsis orthologs. Analysis of selected gene expression was performed with a transcriptome database for the S. tuberosum heterozygous diploid line RH89-039-16. Genes responsible for CK signaling, biosynthesis, transport, and metabolism were categorized in an organ-specific fashion. According to this database, CK receptors StHK2/3 predominate in leaves and flowers, StHK4 in roots. Among phosphotransmitters, StHP1a expression largely predominates. Surprisingly, two pseudo-phosphotransmitters intended to suppress CK effects are hardly expressed in studied organs. Among B-type RR genes, StRR1b, StRR11, and StRR18a are actively expressed, with StRR1b expressing most uniformly in all organs and StRR11 exhibiting the highest expression in roots. By cluster analysis four types of prevailing CK-signaling chains were identified in (1) leaves and flowers, StHK2/3→S t H P1a→StRR1b/+; (2) shoot apical meristems, stolons, and mature tubers, StHK2/4→S t H P1a→StRR1b/+; (3) stems and young tubers, StHK2/4→S t H P1a→StRR1b/11/18a; and (4) roots and tuber sprouts, StHK4→S t H P1a→StRR11/18a. CK synthesis genes StIPT3/5 and StCYP735A are expressed mainly in roots followed by tuber sprouts, but rather weakly in stolons and tubers. By contrast, CK-activation genes StLOGs are active in stolons, and StLOG3b expression is even stolon-confined. Apparently, the main CK effects on tuber initiation are realized via activity of StLOG1/3a/3b/7c/8a genes in stolons. Current advances and future directions in potato research are discussed.

Cytokinin activity of N6-benzyladenine derivatives assayed by interaction with the receptors in planta, in vitro, and in silico.

Biological effects of hormones in both plants and animals are based on high-affinity interaction with cognate receptors resulting in their activation. The signal of cytokinins, classical plant hormones, is perceived in Arabidopsis by three homologous membrane receptors: AHK2, AHK3, and CRE1/AHK4. To study the cytokinin-receptor interaction, we used 25 derivatives of potent cytokinin N6-benzyladenine (BA) with substituents in the purine heterocycle and/or in the side chain. The study was focused primarily on individual cytokinin receptors from Arabidopsis. The main in planta assay system was based on Arabidopsis double mutants retaining only one isoform of cytokinin receptors and harboring cytokinin-sensitive reporter gene. Classical cytokinin biotest with Amaranthus seedlings was used as an additional biotest. In parallel, the binding of ligands to individual cytokinin receptors was assessed in the in vitro test system. Quantitative comparison of results of different assays confirmed the partial similarity of ligand-binding properties of receptor isoforms. Substituents at positions 8 and 9 of adenine moiety, elongated linker up to 4 methylene units, and replacement of N6 by sulfur or oxygen have resulted in the suppression of cytokinin activity of the derivative toward all receptors. Introduction of a halogen into position 2 of adenine moiety, on the contrary, often increased the ligand activity, especially toward AHK3. Features both common and distinctive of cytokinin receptors in Arabidopsis and Amaranthus were revealed, highlighting species specificity of the cytokinin perception apparatus. Correlations between the extent to which a compound binds to a receptor in vitro and its ability to activate the same receptor in planta were evaluated for each AHK protein. Interaction patterns between individual receptors and ligands were rationalized by structure analysis and molecular docking in sensory modules of AHK receptors. The best correlation between docking scores and specific binding was observed for AHK3. In addition, receptor-specific ligands have been discovered with unique properties to predominantly activate or block distinct cytokinin receptors. These ligands are promising for practical application and as molecular tools in the study of the cytokinin perception by plant cells.