For many years, research has been carried out with the aim of understanding the mechanism of auxin action, its biosynthesis, catabolism, perception, and transport. One central interest is the auxin-dependent gene expression regulation mechanism involving AUXIN RESPONSE FACTOR (ARF) transcription factors and their repressors, the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins. Numerous studies have been focused on MONOPTEROS (MP)/ARF5, an activator of auxin-dependent gene expression with a crucial impact on plant development. This review summarizes over 30 years of research on MP/ARF5. We indicate the available analytical tools to study MP/ARF5 and point out the known mechanism of MP/ARF5-dependent regulation of gene expression during various developmental processes, namely embryogenesis, leaf formation, vascularization, and shoot and root meristem formation. However, many questions remain about the auxin dose-dependent regulation of gene transcription by MP/ARF5 and its isoforms in plant cells, the composition of the MP/ARF5 protein complex, and, finally, all the genes under its direct control. In addition, information on post-translational modifications of MP/ARF5 protein is marginal, and knowledge about their consequences on MP/ARF5 function is limited. Moreover, the epigenetic factors and other regulators that act upstream of MP/ARF5 are poorly understood. Their identification will be a challenge in the coming years.

• Klíčová slova
• Arabidopsis thaliana, auxin, embryogenesis, flowering, meristem, plant, transcription factor, vascularization,
• MeSH
• Arabidopsis * metabolismus   MeSH
• DNA vazebné proteiny genetika   MeSH
• kyseliny indoloctové metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• kyseliny indoloctové MeSH
• proteiny huseníčku * MeSH
• transkripční faktory MeSH

ClearSee alpha and FAST9 were optimized for imaging Arabidopsis seeds up to the torpedo stages. The methods preserve the fluorescence of reporter proteins and seed shape, allowing phenotyping embryos in intact seeds. Tissue clearing methods eliminate the need for sectioning, thereby helping better understand the 3D organization of tissues and organs. In the past fifteen years, clearing methods have been developed to preserve endogenous fluorescent protein tags. Some of these methods (ClearSee, TDE, PEA-Clarity, etc.) were adapted to clear various plant species, with the focus on roots, leaves, shoot apical meristems, and floral parts. However, these methods have not been used in developing seeds beyond the early globular stage. Tissue clearing is problematic in post-globular seeds due to various apoplastic barriers and secondary metabolites. In this study, we compared six methods for their efficiency in clearing Arabidopsis thaliana seeds at post-globular embryonic stages. Three methods (TDE, ClearSee, and ClearSee alpha) have already been reported in plants, whereas the others (fsDISCO, FAST9, and CHAPS clear) are used in this context for the first time. These methods were assessed for seed morphological changes, clearing capacity, removal of tannins, and spectral properties. We tested each method in seeds from globular to mature stages. The pros and cons of each method are listed herein. ClearSee alpha appears to be the method of choice as it preserves seed morphology and prevents tannin oxidation. However, FAST9 with 60% iohexol as a mounting medium is faster, clears better, and appears suitable for embryonic shape imaging. Our results may guide plant researchers to choose a suitable method for imaging fluorescent protein-labeled embryos in intact Arabidopsis seeds.

• Klíčová slova
• Arabidopsis seed clearing, ClearSee alpha, FAST9, Fluorescence preservation, Iohexol, Microscopy imaging,
• MeSH
• Arabidopsis * metabolismus   MeSH
• rostliny MeSH
• semena rostlinná metabolismus   MeSH
• xylitol metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ClearSee MeSH Prohlížeč
• xylitol MeSH

Plants, in contrast to animals, are unique in their capacity to postembryonically develop new organs due to the activity of stem cell populations, located in specialized tissues called meristems. Above ground, the shoot apical meristem generates aerial organs and tissues throughout plant life. It is well established that auxin plays a central role in the functioning of the shoot apical meristem. Auxin distribution in the meristem is not uniform and depends on the interplay between biosynthesis, transport, and degradation. Auxin maxima and minima are created, and result in transcriptional outputs that drive the development of new organs and contribute to meristem maintenance. To uncover and understand complex signaling networks such as the one regulating auxin responses in the shoot apical meristem remains a challenge. Here, we will discuss our current understanding and point to important research directions for the future.

• MeSH
• kyseliny indoloctové * metabolismus   MeSH
• meristém * fyziologie   MeSH
• regulace genové exprese u rostlin MeSH
• signální transdukce fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• kyseliny indoloctové * MeSH

Cell polarity is a fundamental feature of all multicellular organisms. PIN auxin transporters are important cell polarity markers that play crucial roles in a plethora of developmental processes in plants. Here, to identify components involved in cell polarity establishment and maintenance in plants, we performed a forward genetic screening of PIN2:PIN1-HA;pin2 Arabidopsis (Arabidopsis thaliana) plants, which ectopically express predominantly basally localized PIN1 in root epidermal cells, leading to agravitropic root growth. We identified the regulator of PIN polarity 12 (repp12) mutation, which restored gravitropic root growth and caused a switch in PIN1-HA polarity from the basal to apical side of root epidermal cells. Next Generation Sequencing and complementation experiments established the causative mutation of repp12 as a single amino acid exchange in Aminophospholipid ATPase3 (ALA3), a phospholipid flippase predicted to function in vesicle formation. repp12 and ala3 T-DNA mutants show defects in many auxin-regulated processes, asymmetric auxin distribution, and PIN trafficking. Analysis of quintuple and sextuple mutants confirmed the crucial roles of ALA proteins in regulating plant development as well as PIN trafficking and polarity. Genetic and physical interaction studies revealed that ALA3 functions together with the ADP ribosylation factor GTPase exchange factors GNOM and BIG3 in regulating PIN polarity, trafficking, and auxin-mediated development.

• MeSH
• ADP-ribosylační faktory metabolismus   MeSH
• Arabidopsis účinky léků   metabolismus   MeSH
• biologický transport účinky léků   MeSH
• brefeldin A farmakologie   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• genetická epistáze účinky léků   MeSH
• GTP-fosfohydrolasy metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• mutace genetika   MeSH
• proteiny huseníčku metabolismus   MeSH
• proteiny přenášející fosfolipidy metabolismus   MeSH
• tabák metabolismus   MeSH
• trans-Golgiho síť účinky léků   metabolismus   MeSH
• vazba proteinů účinky léků   MeSH
• výměnné faktory guaninnukleotidů metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ADP-ribosylační faktory MeSH
• brefeldin A MeSH
• GTP-fosfohydrolasy MeSH
• kyseliny indoloctové MeSH
• proteiny huseníčku MeSH
• proteiny přenášející fosfolipidy MeSH
• výměnné faktory guaninnukleotidů MeSH

In higher plants, germline differentiation occurs during a relatively short period within developing flowers. Understanding of the mechanisms that govern germline differentiation lags behind other plant developmental processes. This is largely because the germline is restricted to relatively few cells buried deep within floral tissues, which makes them difficult to study. To overcome this limitation, we have developed a methodology for live imaging of the germ cell lineage within floral organs of Arabidopsis using light sheet fluorescence microscopy. We have established reporter lines, cultivation conditions, and imaging protocols for high-resolution microscopy of developing flowers continuously for up to several days. We used multiview imagining to reconstruct a three-dimensional model of a flower at subcellular resolution. We demonstrate the power of this approach by capturing male and female meiosis, asymmetric pollen division, movement of meiotic chromosomes, and unusual restitution mitosis in tapetum cells. This method will enable new avenues of research into plant sexual reproduction.

• Klíčová slova
• A. thaliana, SPIM, cell biology, flower, germline, light sheet microscopy, live cell imaging, meiosis, plant biology,
• MeSH
• Arabidopsis cytologie   růst a vývoj   MeSH
• buněčná diferenciace * MeSH
• cytogenetické vyšetření MeSH
• květy cytologie   růst a vývoj   MeSH
• mikroskopie metody   MeSH
• zárodečné buňky rostlin cytologie   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins.

• Klíčová slova
• Arabidopsis thaliana, Calcium-Dependent Protein Kinase-Related Kinase (CRK), GA3, auxin gradient, embryogenesis, polar auxin transport (PAT) proteins,
• MeSH
• Arabidopsis růst a vývoj   metabolismus   MeSH
• ELISA MeSH
• gibereliny analýza   metabolismus   MeSH
• klíčení * MeSH
• kyseliny indoloctové metabolismus   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• receptory buněčného povrchu metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• semena rostlinná anatomie a histologie   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• CRK5 protein, Arabidopsis MeSH Prohlížeč
• gibberellic acid MeSH Prohlížeč
• gibereliny MeSH
• kyseliny indoloctové MeSH
• membránové transportní proteiny MeSH
• protein-serin-threoninkinasy MeSH
• proteiny huseníčku MeSH
• receptory buněčného povrchu MeSH

The angiosperm seed is composed of three genetically distinct tissues: the diploid embryo that originates from the fertilized egg cell, the triploid endosperm that is produced from the fertilized central cell, and the maternal sporophytic integuments that develop into the seed coat1. At the onset of embryo development in Arabidopsis thaliana, the zygote divides asymmetrically, producing a small apical embryonic cell and a larger basal cell that connects the embryo to the maternal tissue2. The coordinated and synchronous development of the embryo and the surrounding integuments, and the alignment of their growth axes, suggest communication between maternal tissues and the embryo. In contrast to animals, however, where a network of maternal factors that direct embryo patterning have been identified3,4, only a few maternal mutations have been described to affect embryo development in plants5-7. Early embryo patterning in Arabidopsis requires accumulation of the phytohormone auxin in the apical cell by directed transport from the suspensor8-10. However, the origin of this auxin has remained obscure. Here we investigate the source of auxin for early embryogenesis and provide evidence that the mother plant coordinates seed development by supplying auxin to the early embryo from the integuments of the ovule. We show that auxin response increases in ovules after fertilization, due to upregulated auxin biosynthesis in the integuments, and this maternally produced auxin is required for correct embryo development.

• MeSH
• Arabidopsis růst a vývoj   metabolismus   MeSH
• kyseliny indoloctové farmakologie   MeSH
• semena rostlinná účinky léků   růst a vývoj   MeSH
• vývoj rostlin účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH

Auxin is unique among plant hormones due to its directional transport that is mediated by the polarly distributed PIN auxin transporters at the plasma membrane. The canalization hypothesis proposes that the auxin feedback on its polar flow is a crucial, plant-specific mechanism mediating multiple self-organizing developmental processes. Here, we used the auxin effect on the PIN polar localization in Arabidopsis thaliana roots as a proxy for the auxin feedback on the PIN polarity during canalization. We performed microarray experiments to find regulators of this process that act downstream of auxin. We identified genes that were transcriptionally regulated by auxin in an AXR3/IAA17- and ARF7/ARF19-dependent manner. Besides the known components of the PIN polarity, such as PID and PIP5K kinases, a number of potential new regulators were detected, among which the WRKY23 transcription factor, which was characterized in more detail. Gain- and loss-of-function mutants confirmed a role for WRKY23 in mediating the auxin effect on the PIN polarity. Accordingly, processes requiring auxin-mediated PIN polarity rearrangements, such as vascular tissue development during leaf venation, showed a higher WRKY23 expression and required the WRKY23 activity. Our results provide initial insights into the auxin transcriptional network acting upstream of PIN polarization and, potentially, canalization-mediated plant development.

• MeSH
• Arabidopsis genetika   růst a vývoj   MeSH
• geneticky modifikované rostliny MeSH
• genové regulační sítě * účinky léků   MeSH
• kořeny rostlin účinky léků   genetika   růst a vývoj   metabolismus   MeSH
• kyseliny indoloctové metabolismus   farmakologie   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• mikročipová analýza MeSH
• polarita buněk * genetika   MeSH
• proteiny huseníčku genetika   metabolismus   fyziologie   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• stanovení celkové genové exprese MeSH
• transkripční faktory fyziologie   MeSH
• zpětná vazba fyziologická účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• membránové transportní proteiny MeSH
• PIN1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• transkripční faktory MeSH
• WRKY23 protein, Arabidopsis MeSH Prohlížeč

Auxins mediate various processes that are involved in plant growth and development in response to specific environmental conditions. Its proper spatio-temporal distribution that is driven by polar auxin transport machinery plays a crucial role in the wide range of auxins physiological effects. Numbers of approaches have been developed to either directly or indirectly monitor auxin distribution in vivo in order to elucidate the basis of its precise regulation. Herein, we provide an updated list of valuable techniques used for monitoring auxins in plants, with their utilities and limitations. Because the spatial and temporal resolutions of the presented approaches are different, their combination may provide a comprehensive outcome of auxin distribution in diverse developmental processes.

• Klíčová slova
• auxin, auxin distribution, auxin signalling, auxin transport, direct visualization, indirect visualization, receptor, sensor,
• MeSH
• Arabidopsis metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• vývoj rostlin fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• kyseliny indoloctové MeSH