INTRODUCTION: Chloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle. METHODS: In the present study we integrated comparative proteomic analysis with biochemical, genetic, cellular, ionomic and hormone analysis in order to gain an insight into how chloroplast calcium channels are integrated into signaling circuits under watered condition and under drought stress. RESULTS: Altogether, our results indicate for the first time a link between chloroplast calcium channels and hormone levels, showing an enhanced ABA level in the cmcu mutant already in well-watered condition. Furthermore, we show that the lack of cMCU results in an upregulation of the calcium sensor CAS and of enzymes of chlorophyll synthesis, which are also involved in retrograde signaling upon drought stress, in two independent KO lines generated in Col-0 and Col-4 ecotypes. CONCLUSIONS: These observations point to chloroplasts as important signaling hubs linked to their calcium dynamics. Our results obtained in the model plant Arabidopsis thaliana are discussed also in light of our limited knowledge regarding organellar calcium signaling in crops and raise the possibility of an involvement of such signaling in response to drought stress also in crops.

• Keywords
• calcium channel, calcium sensor, chloroplast, comparative proteomics, drought stress,
• Publication type
• Journal Article MeSH

C-TERMINALLY ENCODED PEPTIDE (CEP) and cytokinin hormones act over short and long distances to control plant responses to environmental cues. CEP and cytokinin pathway mutants share phenotypes, however, it is not known if these pathways intersect. We show that CEP and cytokinin signalling converge on CEP DOWNSTREAM (CEPD) glutaredoxins to inhibit primary root growth. CEP inhibition of root growth was impaired in mutants defective in trans-zeatin (tZ)-type cytokinin biosynthesis, transport, perception, and output. Concordantly, mutants affected in CEP RECEPTOR 1 showed reduced root growth inhibition in response to tZ, and altered levels of tZ-type cytokinins. Grafting and organ-specific hormone treatments showed that tZ-mediated root growth inhibition involved CEPD activity in roots. By contrast, root growth inhibition by CEP depended on shoot CEPD function. The results demonstrate that CEP and cytokinin pathways intersect, and utilise signalling circuits in separate organs involving common glutaredoxin genes to coordinate root growth.

• MeSH
• Arabidopsis * metabolism   MeSH
• Cytokinins * metabolism   MeSH
• Glutaredoxins genetics   metabolism   MeSH
• Plant Roots metabolism   MeSH
• Peptides metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Shoots metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokinins * MeSH
• Glutaredoxins MeSH
• Peptides MeSH

Cytokinin and auxin are plant hormones that coordinate many aspects of plant development. Their interactions in plant underground growth are well established, occurring at the levels of metabolism, signaling, and transport. Unlike many plant hormone classes, cytokinins are represented by more than one active molecule. Multiple mutant lines, blocking specific parts of cytokinin biosynthetic pathways, have enabled research in plants with deficiencies in specific cytokinin-types. While most of these mutants have confirmed the impeding effect of cytokinin on root growth, the ipt29 double mutant instead surprisingly exhibits reduced primary root length compared to the wild type. This mutant is impaired in cis-zeatin (cZ) production, a cytokinin-type that had been considered inactive in the past. Here we have further investigated the intriguing ipt29 root phenotype, opposite to known cytokinin functions, and the (bio)activity of cZ. Our data suggest that despite the ipt29 short-root phenotype, cZ application has a negative impact on primary root growth and can activate a cytokinin response in the stele. Grafting experiments revealed that the root phenotype of ipt29 depends mainly on local signaling which does not relate directly to cytokinin levels. Notably, ipt29 displayed increased auxin levels in the root tissue. Moreover, analyses of the differential contributions of ipt2 and ipt9 to the ipt29 short-root phenotype demonstrated that, despite its deficiency on cZ levels, ipt2 does not show any root phenotype or auxin homeostasis variation, while ipt9 mutants were indistinguishable from ipt29. We conclude that IPT9 functions may go beyond cZ biosynthesis, directly or indirectly, implicating effects on auxin homeostasis and therefore influencing plant growth.

• Keywords
• auxin, cytokinin, metabolism, plant hormones, root growth,
• Publication type
• Journal Article 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

The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

• Keywords
• auxin, auxin metabolism, flow cytometry, nucleus, subcellular fractionation,
• MeSH
• Arabidopsis drug effects   metabolism   ultrastructure   MeSH
• Cell Nucleus drug effects   metabolism   ultrastructure   MeSH
• Cell Culture Techniques MeSH
• Centrifugation methods   MeSH
• Cell Fractionation instrumentation   methods   MeSH
• Mass Spectrometry MeSH
• Homeostasis physiology   MeSH
• Indoles metabolism   pharmacology   MeSH
• Indoleacetic Acids metabolism   MeSH
• Protoplasts chemistry   MeSH
• Flow Cytometry MeSH
• Plant Growth Regulators metabolism   MeSH
• Plant Cells drug effects   metabolism   ultrastructure   MeSH
• Nicotiana drug effects   metabolism   ultrastructure   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• indole MeSH Browser
• Indoles MeSH
• Indoleacetic Acids MeSH
• Plant Growth Regulators MeSH

Adventitious rooting is a de novo organogenesis process that enables plants to propagate clonally and cope with environmental stresses. Adventitious root initiation (ARI) is controlled by interconnected transcriptional and hormonal networks, but there is little knowledge of the genetic and molecular programs orchestrating these networks. Thus, we have applied genome-wide transcriptome profiling to elucidate the transcriptional reprogramming events preceding ARI. These reprogramming events are associated with the down-regulation of cytokinin (CK) signaling and response genes, which could be triggers for ARI. Interestingly, we found that CK free base (iP, tZ, cZ, and DHZ) content declined during ARI, due to down-regulation of de novo CK biosynthesis and up-regulation of CK inactivation pathways. We also found that MYC2-dependent jasmonate (JA) signaling inhibits ARI by down-regulating the expression of the CYTOKININ OXIDASE/DEHYDROGENASE1 (CKX1) gene. We also demonstrated that JA and CK synergistically activate expression of the transcription factor RELATED to APETALA2.6 LIKE (RAP2.6L), and constitutive expression of this transcription factor strongly inhibits ARI. Collectively, our findings reveal that previously unknown genetic interactions between JA and CK play key roles in ARI.

• Keywords
• Adventitious roots, Arabidopsis, CKX1, MYC2, RAP2.6L, cytokinins, jasmonate, light, vegetative propagation,
• MeSH
• Arabidopsis * genetics   metabolism   MeSH
• Cyclopentanes MeSH
• Plant Roots genetics   metabolism   MeSH
• Oxylipins MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Transcription Factors genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cyclopentanes MeSH
• jasmonic acid MeSH Browser
• Oxylipins MeSH
• Arabidopsis Proteins * MeSH
• RAP2.6L protein, Arabidopsis MeSH Browser
• Transcription Factors MeSH

The spatial location and timing of plant developmental events are largely regulated by the well balanced effects of auxin and cytokinin phytohormone interplay. Together with transport, localized metabolism regulates the concentration gradients of their bioactive forms, ultimately eliciting growth responses. In order to explore the dynamics of auxin and cytokinin metabolism during early seedling growth in Theobroma cacao (cacao), we have performed auxin and cytokinin metabolite profiling in hypocotyls and root developmental sections at different times by using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS). Our work provides quantitative characterization of auxin and cytokinin metabolites throughout early root and hypocotyl development and identifies common and distinctive features of auxin and cytokinin metabolism during cacao seedling development.

• Keywords
• Theobroma cacao, auxin, cytokinin, phytohormone metabolism, root development,
• Publication type
• Journal Article MeSH

Reaction wood (RW) formation is an innate physiological response of woody plants to counteract mechanical constraints in nature, reinforce structure and redirect growth toward the vertical direction. Differences and/or similarities between stem and root response to mechanical constraints remain almost unknown especially in relation to phytohormones distribution and RW characteristics. Thus, Populus nigra stem and root subjected to static non-destructive mid-term bending treatment were analyzed. The distribution of tension and compression forces was firstly modeled along the main bent stem and root axis; then, anatomical features, chemical composition, and a complete auxin and cytokinin metabolite profiles of the stretched convex and compressed concave side of three different bent stem and root sectors were analyzed. The results showed that in bent stems RW was produced on the upper stretched convex side whereas in bent roots it was produced on the lower compressed concave side. Anatomical features and chemical analysis showed that bent stem RW was characterized by a low number of vessel, poor lignification, and high carbohydrate, and thus gelatinous layer in fiber cell wall. Conversely, in bent root, RW was characterized by high vessel number and area, without any significant variation in carbohydrate and lignin content. An antagonistic interaction of auxins and different cytokinin forms/conjugates seems to regulate critical aspects of RW formation/development in stem and root to facilitate upward/downward organ bending. The observed differences between the response stem and root to bending highlight how hormonal signaling is highly organ-dependent.

• Keywords
• UHPLC-MS/MS, auxins, bending stress, cytokinins, metabolite profiling,
• Publication type
• Journal Article MeSH

In the primary root and young hypocotyl of Arabidopsis, ACAULIS5 promotes translation of SUPPRESSOR OF ACAULIS51 (SAC51) and thereby inhibits cytokinin biosynthesis and vascular cell division. In this study, the relationships between ACAULIS5, SAC51 and cytokinin biosynthesis were investigated during secondary growth of Populus stems. Overexpression of ACAULIS5 from the constitutive 35S promoter in hybrid aspen (Populus tremula × Populus tremuloides) trees suppressed the expression level of ACAULIS5, which resulted in low levels of the physiologically active cytokinin bases as well as their direct riboside precursors in the transgenic lines. Low ACAULIS5 expression and low cytokinin levels of the transgenic trees coincided with low cambial activity of the stem. ACAULIS5 therefore, contrary to its function in young seedlings in Arabidopsis, stimulates cytokinin accumulation and cambial activity during secondary growth of the stem. This function is not derived from maturing secondary xylem tissues as transgenic suppression of ACAULIS5 levels in these tissues did not influence secondary growth. Interestingly, evidence was obtained for increased activity of the anticlinal division of the cambial initials under conditions of low ACAULIS5 expression and low cytokinin accumulation. We propose that ACAULIS5 integrates auxin and cytokinin signaling to promote extensive secondary growth of tree stems.

• Keywords
• ACAULIS5, POPACAULIS5, Populus tremula × Populus tremuloides, cytokinin, polyamine, thermospermine, wood development, xylem,
• Publication type
• Journal Article MeSH

Vegetative propagation relies on the capacity of plants to regenerate de novo adventitious roots (ARs), a quantitative trait controlled by the interaction of endogenous factors, such as hormones and environmental cues among which light plays a central role. However, the physiological and molecular components mediating light cues during AR initiation (ARI) remain largely elusive. Here, we explored the role of red light (RL) on ARI in de-rooted Norway spruce seedlings. We combined investigation of hormone metabolism and gene expression analysis to identify potential signaling pathways. We also performed extensive anatomical characterization to investigate ARI at the cellular level. We showed that in contrast to white light, red light promoted ARI likely by reducing jasmonate (JA) and JA-isoleucine biosynthesis and repressing the accumulation of isopentyl-adenine-type cytokinins. We demonstrated that exogenously applied JA and/or CK inhibit ARI in a dose-dependent manner and found that they possibly act in the same pathway. The negative effect of JA on ARI was confirmed at the histological level. We showed that JA represses the early events of ARI. In conclusion, RL promotes ARI by repressing the accumulation of the wound-induced phytohormones JA and CK.

• Keywords
• Picea abies, adventitious roots, auxin, conifers, cytokinins, jasmonate, red light, root development,
• Publication type
• Journal Article MeSH