Fluorescence-activated multi-organelle mapping of subcellular plant hormone distribution

Auxins and cytokinins are two major families of phytohormones that control most aspects of plant growth, development and plasticity. Their distribution in plants has been described, but the importance of cell- and subcellular-type specific phytohormone homeostasis remains undefined. Herein, we revealed auxin and cytokinin distribution maps showing their different organelle-specific allocations within the Arabidopsis plant cell. To do so, we have developed Fluorescence-Activated multi-Organelle Sorting (FAmOS), an innovative subcellular fractionation technique based on flow cytometric principles. FAmOS allows the simultaneous sorting of four differently labelled organelles based on their individual light scatter and fluorescence parameters while ensuring hormone metabolic stability. Our data showed different subcellular distribution of auxin and cytokinins, revealing the formation of phytohormone gradients that have been suggested by the subcellular localization of auxin and cytokinin transporters, receptors and metabolic enzymes. Both hormones showed enrichment in vacuoles, while cytokinins were also accumulated in the endoplasmic reticulum

PSB33 protein sustains photosystem II in plant chloroplasts under UV-A light

Plants can quickly and dynamically respond to spectral and intensity variations of the incident light. These responses include activation of developmental processes, morphological changes, and photosynthetic acclimation that ensure optimal energy conversion and minimal photoinhibition. Plant adaptation and acclimation to environmental changes have been extensively studied, but many details surrounding these processes remain elusive. The photosystem II (PSII)-associated protein PSB33 plays a fundamental role in sustaining PSII as well as in the regulation of the light antenna in fluctuating light. We investigated how PSB33 knock-out Arabidopsis plants perform under different light qualities. psb33 plants displayed a reduction of 88% of total fresh weight compared to wild type plants when cultivated at the boundary of UV-A and blue light. The sensitivity towards UV-A light was associated with a lower abundance of PSII proteins, which reduces psb33 plants\u27 capacity for photosynthesis. The UV-A phenotype was found to be linked to altered phytohormone status and changed thylakoid ultrastructure. Our results collectively show that PSB33 is involved in a UV-A light-mediated mechanism to maintain a functional PSII pool in the chloroplast

IPT9, a cis-zeatin cytokinin biosynthesis gene, promotes root growth

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

Xyloglucan Remodeling Defines Auxin-Dependent Differential Tissue Expansion in Plants

Size control is a fundamental question in biology, showing incremental complexity in plants, whose cells possess a rigid cell wall. The phytohormone auxin is a vital growth regulator with central importance for differential growth control. Our results indicate that auxin-reliant growth programs affect the molecular complexity of xyloglucans, the major type of cell wall hemicellulose in eudicots. Auxin-dependent induction and repression of growth coincide with reduced and enhanced molecular complexity of xyloglucans, respectively. In agreement with a proposed function in growth control, genetic interference with xyloglucan side decorations distinctly modulates auxin-dependent differential growth rates. Our work proposes that auxin-dependent growth programs have a spatially defined effect on xyloglucan's molecular structure, which in turn affects cell wall mechanics and specifies differential, gravitropic hypocotyl growth

Timing-dependent effects of salicylic acid treatment on phytohormonal changes, ROS regulation, and antioxidant defense in salinized barley (Hordeum vulgare L.)

Cross-talk between exogenous salicylic acid (SA) and endogenous phytohormone pathways affects the antioxidant defense system and its response to salt stress. The study presented here investigated the effects of SA treatment before and during salt stress on the levels of endogenous plant growth regulators in three barley cultivars with different salinity tolerances: Hordeum vulgare L. cvs. Akhisar (sensitive), Erginel (moderate), and Kalayc (tolerant). The cultivars' relative leaf water contents, growth parameters, proline contents, chlorophyll a/b ratios, and lipid peroxidation levels were measured, along with the activities of enzymes involved in detoxifying reactive oxygen species (ROS) including superoxide-dismutase, peroxidase, catalase, ascorbate-peroxidase, and glutathione-reductase. In addition, levels of several endogenous phytohormones (indole-3-acetic-acid, cytokinins, abscisic acid, jasmonic acid, and ethylene) were measured. Barley is known to be more salt tolerant than related plant species. Accordingly, none of the studied cultivars exhibited changes in membrane lipid peroxidation under salt stress. However, they responded differently to salt-stress with respect to their accumulation of phytohormones and antioxidant enzyme activity. The strongest and weakest increases in ABA and proline accumulation were observed in Kalayc and Akhisar, respectively, suggesting that salt-stress was more effectively managed in Kalayc. The effects of exogenous SA treatment depended on both the timing of the treatment and the cultivar to which it was applied. In general, however, where SA helped mitigate salt stress, it appeared to do so by increasing ROS scavenging capacity and antioxidant enzyme activity. SA treatment also induced changes in phytohormone levels, presumably as a consequence of SA-phytohormone salt-stress cross-talk.Internal Grant Agency of Palacky University [IGA_PrF_ 2019_ 020]; European Regional Development Fund-Project Plants as a tool for sustainable global development [CZ. 02.1.01/0.0/0.0/16_019/0000827]This work was supported by the Internal Grant Agency of Palacky University N. IGA_PrF_ 2019_ 020 and from European Regional Development Fund-Project Plants as a tool for sustainable global development (No. CZ. 02.1.01/0.0/0.0/16_019/0000827). We would also like to thank to Sees-editing Ltd., U.K. for English editing of the manuscript.WOS:0005635386000092-s2.0-85089529111PubMed: 3280791

Soluble Carbohydrates Regulate Auxin Biosynthesis via PIF Proteins in Arabidopsis

Plants are necessarily highly competitive and have finely tuned mechanisms to adjust growth and development in accordance with opportunities and limitations in their environment. Sugars from photosynthesis form an integral part of this growth control process, acting as both an energy source and as signaling molecules in areas targeted for growth. The plant hormone auxin similarly functions as a signaling molecule and a driver of growth and developmental processes. Here, we show that not only do the two act in concert but that auxin metabolism is itself regulated by the availability of free sugars. The regulation of the biosynthesis and degradation of the main auxin, indole-3-acetic acid (IAA), by sugars requires changes in the expression of multiple genes and metabolites linked to several IAA biosynthetic pathways. The induction also involves members of the recently described central regulator PHYTOCHROME-INTERACTING FACTOR transcription factor family. Linking these three known regulators of growth provides a model for the dynamic coordination of responses to a changing environment

Endogenous Hypoxia in Lateral Root Primordia Controls Root Architecture by Antagonizing Auxin Signaling in Arabidopsis

As non-photosynthesizing organs, roots are dependent on diffusion of oxygen from the external environment and, in some instances, from the shoot for their aerobic metabolism. Establishment of hypoxic niches in the developing tissues of plants has been postulated as a consequence of insufficient diffusion of oxygen to satisfy the demands throughout development. Here, we report that such niches are established at specific stages of lateral root primordia development in Arabidopsis thaliana grown under aerobic conditions. Using gain- and loss-of-function mutants, we show that ERF-VII transcription factors, which mediate hypoxic responses, control root architecture by acting in cells with a high level of auxin signaling. ERF-VIIs repress the expression of the auxin-induced genes LBD16, LBD18, and PUCHI, which are essential for lateral root development, by binding to their promoters. Our results support a model in which the establishment of hypoxic niches in the developing lateral root primordia contributes to the shutting down of key auxin-induced genes and regulates the production of lateral roots. This study shows that reduced oxygen availability marks lateral root primordia at late developmental stages in Arabidopsis. Exploiting transgenic plants with altered hypoxia responsiveness, it was established that the local activation of ERF-VII transcription factors by low oxygen antagonizes auxin responses and contributes to terminating the primordium development, thus achieving optimal root system architecture

Jasmonate Signalling Contributes to Primary Root Inhibition Upon Oxygen Deficiency in Arabidopsis thaliana

Plants, including most crops, are intolerant to waterlogging, a stressful condition that limits the oxygen available for roots, thereby inhibiting their growth and functionality. Whether root growth inhibition represents a preventive measure to save energy or is rather a consequence of reduced metabolic rates has yet to be elucidated. In the present study, we gathered evidence for hypoxic repression of root meristem regulators that leads to root growth inhibition. We also explored the contribution of the hormone jasmonic acid (JA) to this process in Arabidopsis thaliana. Analysis of transcriptomic profiles, visualisation of fluorescent reporters and direct hormone quantification confirmed the activation of JA signalling under hypoxia in the roots. Further, root growth assessment in JA-related mutants in aerobic and anaerobic conditions indicated that JA signalling components contribute to active root inhibition under hypoxia. Finally, we show that the oxygen-sensing transcription factor (TF) RAP2.12 can directly induce Jasmonate Zinc-finger proteins (JAZs), repressors of JA signalling, to establish feedback inhibition. In summary, our study sheds new light on active root growth restriction under hypoxic conditions and on the involvement of the JA hormone in this process and its cross talk with the oxygen sensing machinery of higher plants

Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis

Auxin (indole-3-acetic acid, IAA) plays fundamental roles as a signalling molecule during numerous plant growth and development processes. The formation of local auxin gradients and auxin maxima/minima, which is very important for these processes, is regulated by auxin metabolism (biosynthesis, degradation, and conjugation) as well as transport. When studying auxin metabolism pathways it is crucial to combine data obtained from genetic investigations with the identification and quantification of individual metabolites. Thus, to facilitate efforts to elucidate auxin metabolism and its roles in plants, we have developed a high-throughput method for simultaneously quantifying IAA and its key metabolites in minute samples (<10 mg FW) of Arabidopsis thaliana tissues by in-tip micro solid-phase extraction and fast LC-tandem MS. As a proof of concept, we applied the method to a collection of Arabidopsis mutant lines and identified lines with altered IAA metabolite profiles using multivariate data analysis. Finally, we explored the correlation between IAA metabolite profiles and IAA-related phenotypes. The developed rapid analysis of large numbers of samples (>100 samples d(-1)) is a valuable tool to screen for novel regulators of auxin metabolism and homeostasis among large collections of genotypes