To compensate for their sessile lifestyle, plants developed several responses to exogenous changes. One of the previously investigated and not yet fully understood adaptations occurs at the level of early subcellular trafficking, which needs to be rapidly adjusted to maintain cellular homeostasis and membrane integrity under osmotic stress conditions. To form a vesicle, the membrane needs to be deformed, which is ensured by multiple factors, including the activity of specific membrane proteins, such as flippases from the family of P4-ATPases. The membrane pumps actively translocate phospholipids from the exoplasmic/luminal to the cytoplasmic membrane leaflet to generate curvature, which might be coupled with recruitment of proteins involved in vesicle formation at specific sites of the donor membrane. We show that lack of the AMINOPHOSPHOLIPID ATPASE3 (ALA3) flippase activity caused defects at the plasma membrane and trans-Golgi network, resulting in altered endocytosis and secretion, processes relying on vesicle formation and movement. The mentioned cellular defects were translated into decreased intracellular trafficking flexibility failing to adjust the root growth on osmotic stress-eliciting media. In conclusion, we show that ALA3 cooperates with ARF-GEF BIG5/BEN1 and ARF1A1C/BEX1 in a similar regulatory pathway to vesicle formation, and together they are important for plant adaptation to osmotic stress.

• Klíčová slova
• Arabidopsis thaliana, ARF, GEF, endocytosis, flippase, osmotic stress, protein trafficking, secretion,
• MeSH
• Arabidopsis * metabolismus   MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• osmotický tlak MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• proteiny huseníčku * MeSH

Much of plant development depends on cell-to-cell redistribution of the plant hormone auxin, which is facilitated by the plasma membrane (PM) localized PIN FORMED (PIN) proteins. Auxin export activity, developmental roles, subcellular trafficking, and polarity of PINs have been well studied, but their structure remains elusive besides a rough outline that they contain two groups of 5 alpha-helices connected by a large hydrophilic loop (HL). Here, we focus on the PIN1 HL as we could produce it in sufficient quantities for biochemical investigations to provide insights into its secondary structure. Circular dichroism (CD) studies revealed its nature as an intrinsically disordered protein (IDP), manifested by the increase of structure content upon thermal melting. Consistent with IDPs serving as interaction platforms, PIN1 loops homodimerize. PIN1 HL cytoplasmic overexpression in Arabidopsis disrupts early endocytic trafficking of PIN1 and PIN2 and causes defects in the cotyledon vasculature formation. In summary, we demonstrate that PIN1 HL has an intrinsically disordered nature, which must be considered to gain further structural insights. Some secondary structures may form transiently during pairing with known and yet-to-be-discovered interactors.

• Klíčová slova
• PIN1, dimerization, hydrophilic hoop, intrinsic disorder, subcellular trafficking,
• MeSH
• Arabidopsis * metabolismus   MeSH
• biologický transport MeSH
• kořeny rostlin metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• vnitřně neuspořádané proteiny * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• membránové transportní proteiny MeSH
• PIN1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku * MeSH
• vnitřně neuspořádané proteiny * MeSH

Salt and osmotic stress are the main abiotic stress factors affecting plant root growth and architecture. We investigated the effect of salt (100 mM NaCl) and osmotic (200 mM mannitol) stress on the auxin metabolome by UHPLC-MS/MS, auxin distribution by confocal microscopy, and transcript levels of selected genes by qRT-PCR in Arabidopsis thaliana ecotype Columbia-0 (Col-0) and DR5rev::GFP (DR5) line. During long-term stress (13 days), a stability of the auxin metabolome and a tendency to increase indole-3-acetic acid (IAA) were observed, especially during salt stress. Short-term stress (3 h) caused significant changes in the auxin metabolome, especially NaCl treatment resulted in a significant reduction of IAA. The data derived from auxin profiling were consistent with gene expressions showing the most striking changes in the transcripts of YUC, GH3, and UGT transcripts, suggesting disruption of auxin biosynthesis, but especially in the processes of amide and ester conjugation. These data were consistent with the auxin distribution observed in the DR5 line. Moreover, NaCl treatment caused a redistribution of auxin signals from the quiescent center and the inner layers of the root cap to the epidermal and cortical cells of the root elongation zone. The distribution of PIN proteins was also disrupted by salt stress; in particular, PIN2 was suppressed, even after 5 min of treatment. Based on our results, the DR5 line was more sensitive to the applied stresses than Col-0, although both lines showed similar trends in root morphology, as well as transcriptome and metabolome parameters under stress conditions.

• Klíčová slova
• Arabidopsis thaliana, abiotic stress, auxin distribution, auxin metabolome, auxin transcriptome, root growth,
• MeSH
• Arabidopsis růst a vývoj   MeSH
• chlorid sodný farmakologie   MeSH
• kořeny rostlin růst a vývoj   MeSH
• kyseliny indoloctové metabolismus   MeSH
• proteiny huseníčku biosyntéza   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• solný stres účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chlorid sodný MeSH
• kyseliny indoloctové MeSH
• proteiny huseníčku MeSH

The proper distribution of the hormone auxin is essential for plant development. It is channeled by auxin efflux carriers of the PIN family, typically asymmetrically located on the plasma membrane (PM). Several studies demonstrated that some PIN transporters are also located at the endoplasmic reticulum (ER). From the PM-PINs, they differ in a shorter internal hydrophilic loop, which carries the most important structural features required for their subcellular localization, but their biological role is otherwise relatively poorly known. We discuss how ER-PINs take part in maintaining intracellular auxin homeostasis, possibly by modulating the internal levels of IAA; it seems that the exact identity of the metabolites downstream of ER-PINs is not entirely clear as well. We further review the current knowledge about their predicted structure, evolution and localization. Finally, we also summarize their role in plant development.

• Klíčová slova
• ER-PINs, PIN proteins, PIN5, PIN8, auxin metabolism, auxin transport,
• Publikační typ
• časopisecké články MeSH
• přehledy 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 plant-specific proteins named PIN-FORMED (PIN) efflux carriers facilitate the direction of auxin flow and thus play a vital role in the establishment of local auxin maxima within plant tissues that subsequently guide plant ontogenesis. They are membrane integral proteins with two hydrophobic regions consisting of alpha-helices linked with a hydrophilic loop, which is usually longer for the plasma membrane-localized PINs. The hydrophilic loop harbors molecular cues important for the subcellular localization and thus auxin efflux function of those transporters. The three-dimensional structure of PIN has not been solved yet. However, there are scattered but substantial data concerning the functional characterization of amino acid strings that constitute these carriers. These sequences include motifs vital for vesicular trafficking, residues regulating membrane diffusion, cellular polar localization, and activity of PINs. Here, we summarize those bits of information striving to provide a reference to structural motifs that have been investigated experimentally hoping to stimulate the efforts toward unraveling of PIN structure-function connections.

• Klíčová slova
• PIN efflux carriers, auxin transport, protein domains, sequence motifs, subcellular trafficking,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Seedling establishment following germination requires the fine tuning of plant hormone levels including that of auxin. Directional movement of auxin has a central role in the associated processes, among others, in hypocotyl hook development. Regulated auxin transport is ensured by several transporters (PINs, AUX1, ABCB) and their tight cooperation. Here we describe the regulatory role of the Arabidopsis thaliana CRK5 protein kinase during hypocotyl hook formation/opening influencing auxin transport and the auxin-ethylene-GA hormonal crosstalk. It was found that the Atcrk5-1 mutant exhibits an impaired hypocotyl hook establishment phenotype resulting only in limited bending in the dark. The Atcrk5-1 mutant proved to be deficient in the maintenance of local auxin accumulation at the concave side of the hypocotyl hook as demonstrated by decreased fluorescence of the auxin sensor DR5::GFP. Abundance of the polar auxin transport (PAT) proteins PIN3, PIN7, and AUX1 were also decreased in the Atcrk5-1 hypocotyl hook. The AtCRK5 protein kinase was reported to regulate PIN2 protein activity by phosphorylation during the root gravitropic response. Here it is shown that AtCRK5 can also phosphorylate in vitro the hydrophilic loops of PIN3. We propose that AtCRK5 may regulate hypocotyl hook formation in Arabidopsis thaliana through the phosphorylation of polar auxin transport (PAT) proteins, the fine tuning of auxin transport, and consequently the coordination of auxin-ethylene-GA levels.

• Klíčová slova
• Arabidopsis thaliana, Ca2+/calmodulin-dependent kinase-related kinases (CRKs), polar auxin transport (PAT) proteins, GA3, auxin gradient, ethylene, skotomorphogenesis,
• MeSH
• Arabidopsis účinky léků   fyziologie   MeSH
• biologické markery MeSH
• fenotyp MeSH
• fosforylace MeSH
• hypokotyl fyziologie   MeSH
• klíčení MeSH
• morfogeneze * účinky léků   genetika   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• receptory buněčného povrchu metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• reportérové geny MeSH
• signální transdukce MeSH
• vývoj rostlin * účinky léků   genetika   MeSH
• xantony farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• CRK5 protein, Arabidopsis MeSH Prohlížeč
• GA3 compound MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• proteiny huseníčku MeSH
• receptory buněčného povrchu MeSH
• xantony MeSH

Plant hormones are master regulators of plant growth and development. Better knowledge of their spatial signaling and homeostasis (transport and metabolism) on the lowest structural levels (cellular and subcellular) is therefore crucial to a better understanding of developmental processes in plants. Recent progress in phytohormone analysis at the cellular and subcellular levels has greatly improved the effectiveness of isolation protocols and the sensitivity of analytical methods. This review is mainly focused on homeostasis of two plant hormone groups, auxins and cytokinins. It will summarize and discuss their tissue- and cell-type specific distributions at the cellular and subcellular levels.

• Klíčová slova
• auxin, cellular level, cytokinin, phytohormone metabolism, phytohormone transport, subcellular level,
• MeSH
• biologický transport MeSH
• cytokininy metabolismus   MeSH
• fyziologie rostlin * MeSH
• homeostáza * MeSH
• intracelulární prostor metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• metabolické sítě a dráhy MeSH
• organely metabolismus   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• rostlinné buňky metabolismus   MeSH
• vývoj rostlin * MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• cytokininy MeSH
• kyseliny indoloctové MeSH
• regulátory růstu rostlin MeSH

Coordination of plant development requires modulation of growth responses that are under control of the phytohormone auxin. PIN-FORMED plasma membrane proteins, involved in intercellular transport of the growth regulator, are key to the transmission of such auxin signals and subject to multilevel surveillance mechanisms, including reversible post-translational modifications. Apart from well-studied PIN protein modifications, namely phosphorylation and ubiquitylation, no further post-translational modifications have been described so far. Here, we focused on root-specific Arabidopsis PIN2 and explored functional implications of two evolutionary conserved cysteines, by a combination of in silico and molecular approaches. PIN2 sequence alignments and modeling predictions indicated that both cysteines are facing the cytoplasm and therefore would be accessible to redox status-controlled modifications. Notably, mutant pin2C-A alleles retained functionality, demonstrated by their ability to almost completely rescue defects of a pin2 null allele, whereas high resolution analysis of pin2C-A localization revealed increased intracellular accumulation, and altered protein distribution within plasma membrane micro-domains. The observed effects of cysteine replacements on root growth and PIN2 localization are consistent with a model in which redox status-dependent cysteine modifications participate in the regulation of PIN2 mobility, thereby fine-tuning polar auxin transport.

• Klíčová slova
• Arabidopsis, Auxin, PIN proteins, SRRF, intracellular distribution, plasma membrane protein sorting, protein mobility, protein modeling, root phenotype,
• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• cystein genetika   MeSH
• konzervovaná sekvence * MeSH
• kořeny rostlin růst a vývoj   metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• membránové mikrodomény metabolismus   MeSH
• proteiny huseníčku chemie   genetika   metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystein MeSH
• kyseliny indoloctové MeSH
• PIN2 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH

This article comments on: Kong Q, Ma W, Yang H, Ma G, Mantyla JJ, Benning C. 2017. The Arabidopsis WRINKLED1 transcription factor affects auxin homeostasis in roots. Journal of Experimental Botany 68, 4627–4634.

• Klíčová slova
• Arabidopsis, GH3, IAA conjugation, PIN expression, endoplasmic reticulum, polar auxin transport, transcriptional regulation,
• MeSH
• Arabidopsis MeSH
• biologický transport MeSH
• kyseliny indoloctové * MeSH
• membránové transportní proteiny genetika   MeSH
• proteiny huseníčku genetika   MeSH
• regulace genové exprese u rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• komentáře MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové * MeSH
• membránové transportní proteiny MeSH
• proteiny huseníčku MeSH