Plasmodiophora brassicae, a soil-borne biotroph, establishes galls as strong physiological sinks on Brassicaceae plants including Brassica napus and Arabidopsis thaliana. We compare transcriptional profiles of phloem dissected from leaf petioles and hypocotyls of healthy and infected B. napus plants. Our results highlight how pathogenesis accompanies phloem-mediated defence responses whilst exerting a strong influence on carbon-nitrogen (C-N) economy. We observe transcriptional changes indicating decreased aliphatic glucosinolate biosynthesis, fluctuating jasmonic acid responses, altered amino acid (AA) and nitrate transport, carbohydrate metabolism and modified cytokinin responses. Changes observed in phloem-dissected from upper versus lower plant organs point to phloem as a conduit in mediating C-N repartitioning, nutrition-related signalling and cytokinin dynamics over long distances during clubroot disease. To assess changes in physiology, we measured AAs, sugars and cytokinins, in phloem exudates from B. napus plants. Despite the decrease in most AA and sucrose levels, isopentyl-type cytokinins increased within infected plants. Furthermore, we employed Arabidopsis for visualising promoter activities of B. napus AA and N transporter orthologues and tested the impact of disrupted cytokinin transport during P. brassicae-induced gall formation using Atabcg14 mutants. Our physiological and microscopy studies show that the host developmental reaction to P. brassicae relies on cytokinin and is accompanied by intense nitrogen and carbon repartitioning. Overall, our work highlights the systemic aspects of host responses that should be taken into account when studying clubroot disease.

• Keywords
• Brassica napus, Plasmodiophora brassicae, clubroot, laser dissection transcriptomics, oilseed rape, phloem,
• MeSH
• Amino Acids metabolism   MeSH
• Arabidopsis * genetics   physiology   MeSH
• Brassica napus * genetics   metabolism   physiology   parasitology   MeSH
• Cyclopentanes metabolism   MeSH
• Cytokinins metabolism   MeSH
• Nitrogen metabolism   MeSH
• Phloem * metabolism   genetics   MeSH
• Glucosinolates metabolism   MeSH
• Plant Leaves genetics   metabolism   MeSH
• Plant Diseases * parasitology   genetics   MeSH
• Oxylipins metabolism   MeSH
• Plasmodiophorida * physiology   MeSH
• Gene Expression Regulation, Plant * MeSH
• Transcriptome MeSH
• Carbon metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Amino Acids MeSH
• Cyclopentanes MeSH
• Cytokinins MeSH
• Nitrogen MeSH
• Glucosinolates MeSH
• jasmonic acid MeSH Browser
• Oxylipins MeSH
• Carbon MeSH

Mineral nutrition is one of the key environmental factors determining plant development and growth. Nitrate is the major form of macronutrient nitrogen that plants take up from the soil. Fluctuating availability or deficiency of this element severely limits plant growth and negatively affects crop production in the agricultural system. To cope with the heterogeneity of nitrate distribution in soil, plants evolved a complex regulatory mechanism that allows rapid adjustment of physiological and developmental processes to the status of this nutrient. The root, as a major exploitation organ that controls the uptake of nitrate to the plant body, acts as a regulatory hub that, according to nitrate availability, coordinates the growth and development of other plant organs. Here, we identified a regulatory framework, where cytokinin response factors (CRFs) play a central role as a molecular readout of the nitrate status in roots to guide shoot adaptive developmental response. We show that nitrate-driven activation of NLP7, a master regulator of nitrate response in plants, fine tunes biosynthesis of cytokinin in roots and its translocation to shoots where it enhances expression of CRFs. CRFs, through direct transcriptional regulation of PIN auxin transporters, promote the flow of auxin and thereby stimulate the development of shoot organs.

• Keywords
• macronutrient, nitrate, plant development,
• MeSH
• Cytokinins metabolism   MeSH
• Nitrates * metabolism   MeSH
• Plant Roots metabolism   MeSH
• Indoleacetic Acids * metabolism   MeSH
• Soil MeSH
• Gene Expression Regulation, Plant MeSH
• Signal Transduction MeSH
• Plant Shoots MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokinins MeSH
• Nitrates * MeSH
• Indoleacetic Acids * MeSH
• Soil MeSH

Directional organ growth allows the plant root system to strategically cover its surroundings. Intercellular auxin transport is aligned with the gravity vector in the primary root tips, facilitating downward organ bending at the lower root flank. Here we show that cytokinin signaling functions as a lateral root specific anti-gravitropic component, promoting the radial distribution of the root system. We performed a genome-wide association study and reveal that signal peptide processing of Cytokinin Oxidase 2 (CKX2) affects its enzymatic activity and, thereby, determines the degradation of cytokinins in natural Arabidopsis thaliana accessions. Cytokinin signaling interferes with growth at the upper lateral root flank and thereby prevents downward bending. Our interdisciplinary approach proposes that two phytohormonal cues at opposite organ flanks counterbalance each other's negative impact on growth, suppressing organ growth towards gravity and allow for radial expansion of the root system.

• MeSH
• Arabidopsis physiology   MeSH
• Genome-Wide Association Study MeSH
• Cytokinins metabolism   MeSH
• Plants, Genetically Modified physiology   MeSH
• Genome, Plant genetics   MeSH
• Gravitropism MeSH
• Plant Roots metabolism   MeSH
• Oxidoreductases genetics   metabolism   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Proteolysis MeSH
• Plant Growth Regulators metabolism   MeSH
• Systems Biology MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• cytokinin oxidase MeSH Browser
• Cytokinins MeSH
• Oxidoreductases MeSH
• Arabidopsis Proteins MeSH
• Plant Growth Regulators MeSH

An affinity-based chemical proteomic technique enabled direct identification of BAP-interacting proteins in wheat, including the well-known cytokinin-binder, cytokinin-binding protein 1. In this work, we show the development of a chemical proteomic technique for the identification of proteins binding to natural aromatic cytokinins (CKs). 6-benzylaminopurine (BAP) and documented CK-binder, wheat germ-allocated cytokinin-binding protein 1 (CBP-1), were suggested as an ideal proof-of concept affinity pair. Therefore, wheat grains were chosen as a model plant material. The BAP affinity beads were prepared by the immobilization of synthesized BAP-derived ligand to a commercial, pre-activated resin and used to isolate target proteins. The proteomic analysis of complex plant extracts is often complicated by the presence of highly abundant background proteins; in this case, the omnipresent alpha-amylase inhibitors (AAIs). To cope with this problem, we included SDS-PAGE, in-gel trypsin digestion and fraction pooling prior to shotgun analysis, which brought about an obvious drop in the signals belonging to the obstructing proteins. This was accompanied by a sharp increase in the number of identified BAP targets in comparison to a conventional in-solution digestion approach. To distinguish specific CK-binding proteins from those having a general affinity for nucleotide-like compounds, competitive pull-downs with natural nucleotides and free BAP were included in every affinity experiment. By this approach, we were able to identify a group of BAP-interacting proteins, which were subsequently found to be related to biological processes affected by CKs. Moreover, the selected affinity enrichment strategy was verified by the detection of the aforementioned CK-interacting protein, CBP-1. We propose that the developed method represents a promising tool for appealing research of as yet unknown CK molecular partners in plants.

• Keywords
• Affinity purification, Chemical proteomics, Cytokinin, Molecular target identification, Plant proteomics, Wheat,
• MeSH
• Benzyl Compounds metabolism   MeSH
• Chromatography, Liquid MeSH
• Cytokinins metabolism   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Edible Grain metabolism   MeSH
• Proteome metabolism   MeSH
• Proteomics methods   MeSH
• Triticum metabolism   MeSH
• Purines metabolism   MeSH
• Plant Proteins metabolism   MeSH
• Tandem Mass Spectrometry MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• benzylaminopurine MeSH Browser
• Benzyl Compounds MeSH
• Cytokinins MeSH
• Proteome MeSH
• Purines MeSH
• Plant Proteins MeSH