Hormone-regulated expansins: Expression, localization, and cell wall biomechanics in Arabidopsis root growth.

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at 3-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell-type-specific combination of homogenous vs. 3-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young's modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone (TZ). The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN:XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fourier-transform infrared spectroscopy in the root TZ. The EXPA1-induced CW remodeling is associated with the shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.

Epigenetics and plant hormone dynamics - a functional and methodological perspective.

Plant hormones, pivotal regulators of plant growth, development, and response to environmental cues, have recently emerged as central modulators of epigenetic processes governing gene expression and phenotypic plasticity. This review addresses the complex interplay between plant hormones and epigenetic mechanisms, highlighting the diverse methodologies that have been harnessed to decipher these intricate relationships. We present a comprehensive overview to understand how phytohormones orchestrate epigenetic modifications, shaping plant adaptation and survival strategies. Conversely, we explore how epigenetic regulators ensure hormonal balance and regulate the signalling pathways of key plant hormones. Furthermore, our investigation includes a search for novel genes that are regulated by plant hormones under the control of epigenetic processes. Our review offers a contemporary overview of the epigenetic-plant hormone crosstalk, emphasizing its significance in plant growth, development, and potential agronomical applications.

Mutually opposing activity of PIN7 splicing isoforms is required for auxin-mediated tropic responses in Arabidopsis thaliana.

Advanced transcriptome sequencing has revealed that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Combining approaches of genetics, biochemistry and advanced confocal microscopy, we describe the impact of alternative splicing on the PIN7 gene in the model plant Arabidopsis thaliana. PIN7 encodes a polarly localized transporter for the phytohormone auxin and produces two evolutionarily conserved transcripts, PIN7a and PIN7b. PIN7a and PIN7b, differing in a four amino acid stretch, exhibit almost identical expression patterns and subcellular localization. We reveal that they are closely associated and mutually influence each other's mobility within the plasma membrane. Phenotypic complementation tests indicate that the functional contribution of PIN7b per se is minor, but it markedly reduces the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. Our results establish alternative splicing of the PIN family as a conserved, functionally relevant mechanism, revealing an additional regulatory level of auxin-mediated plant development.

Cytokinin signalling regulates organ identity via the AHK4 receptor in Arabidopsis.

Mutual interactions of the phytohormones, cytokinins and auxin determine root or shoot identity during postembryonic de novo organogenesis in plants. However, our understanding of the role of hormonal metabolism and perception during early stages of cell fate reprogramming is still elusive. Here we show that auxin activates root formation, whereas cytokinins mediate early loss of the root identity, primordia disorganisation and initiation of shoot development. Exogenous and endogenous cytokinins influence the initiation of newly formed organs, as well as the pace of organ development. The process of de novo shoot apical meristem establishment is accompanied by accumulation of endogenous cytokinins, differential regulation of genes for individual cytokinin receptors, strong activation of AHK4-mediated signalling and induction of the shoot-specific homeodomain regulator WUSCHEL. The last is associated with upregulation of isopentenyladenine-type cytokinins, revealing higher shoot-forming potential when compared with trans-zeatin. Moreover, AHK4-controlled cytokinin signalling negatively regulates the root stem cell organiser WUSCHEL RELATED HOMEOBOX 5 in the root quiescent centre. We propose an important role for endogenous cytokinin biosynthesis and AHK4-mediated cytokinin signalling in the control of de novo-induced organ identity.

Cell wall integrity modulates Arabidopsis thaliana cell cycle gene expression in a cytokinin- and nitrate reductase-dependent manner.

During plant growth and defense, cell cycle activity needs to be coordinated with cell wall integrity. Little is known about how this coordination is achieved. Here, we investigated coordination in Arabidopsis thaliana seedlings by studying the impact of cell wall damage (CWD, caused by cellulose biosynthesis inhibition) on cytokinin homeostasis, cell cycle gene expression and cell shape in root tips. CWD inhibited cell cycle gene expression and increased transition zone cell width in an osmosensitive manner. These results were correlated with CWD-induced, osmosensitive changes in cytokinin homeostasis. Expression of CYTOKININ OXIDASE/DEHYDROGENASE 2 and 3 (CKX2, CKX3), which encode cytokinin-degrading enzymes, was induced by CWD and reduced by osmoticum treatment. In nitrate reductase1 nitrate reductase2 (nia1 nia2) seedlings, CKX2 and CKX3 transcript levels were not increased and cell cycle gene expression was not repressed by CWD. Moreover, established CWD-induced responses, such as jasmonic acid, salicylic acid and lignin production, were also absent, implying a central role of NIA1/2-mediated processes in regulation of CWD responses. These results suggest that CWD enhances cytokinin degradation rates through a NIA1/2-mediated process, leading to attenuation of cell cycle gene expression.

Hormonal orchestration of root apical meristem formation and maintenance in Arabidopsis.

Plant hormones are key regulators of a number of developmental and adaptive responses in plants, integrating the control of intrinsic developmental regulatory circuits with environmental inputs. Here we provide an overview of the molecular mechanisms underlying hormonal regulation of root development. We focus on key events during both embryonic and post-embryonic development, including specification of the hypophysis as a future organizer of the root apical meristem (RAM), hypophysis asymmetric division, specification of the quiescent centre (QC) and the stem cell niche (SCN), RAM maturation and maintenance of QC/SCN activity, and RAM size. We address both well-established and newly proposed concepts, highlight potential ambiguities in recent terminology and classification criteria of longitudinal root zonation, and point to contrasting results and alternative scenarios for recent models. In the concluding remarks, we summarize the common principles of hormonal control during root development and the mechanisms potentially explaining often antagonistic outputs of hormone action, and propose possible future research directions on hormones in the root.

Towards Automated Analysis of Grain Spikes in Greenhouse Images Using Neural Network Approaches: A Comparative Investigation of Six Methods.

Automated analysis of small and optically variable plant organs, such as grain spikes, is highly demanded in quantitative plant science and breeding. Previous works primarily focused on the detection of prominently visible spikes emerging on the top of the grain plants growing in field conditions. However, accurate and automated analysis of all fully and partially visible spikes in greenhouse images renders a more challenging task, which was rarely addressed in the past. A particular difficulty for image analysis is represented by leaf-covered, occluded but also matured spikes of bushy crop cultivars that can hardly be differentiated from the remaining plant biomass. To address the challenge of automated analysis of arbitrary spike phenotypes in different grain crops and optical setups, here, we performed a comparative investigation of six neural network methods for pattern detection and segmentation in RGB images, including five deep and one shallow neural network. Our experimental results demonstrate that advanced deep learning methods show superior performance, achieving over 90% accuracy by detection and segmentation of spikes in wheat, barley and rye images. However, spike detection in new crop phenotypes can be performed more accurately than segmentation. Furthermore, the detection and segmentation of matured, partially visible and occluded spikes, for which phenotypes substantially deviate from the training set of regular spikes, still represent a challenge to neural network models trained on a limited set of a few hundreds of manually labeled ground truth images. Limitations and further potential improvements of the presented algorithmic frameworks for spike image analysis are discussed. Besides theoretical and experimental investigations, we provide a GUI-based tool (SpikeApp), which shows the application of pre-trained neural networks to fully automate spike detection, segmentation and phenotyping in images of greenhouse-grown plants.

Auxins and Cytokinins in Plant Development 2018.

The international symposium "Auxins and Cytokinins in Plant Development" (ACPD), which is held every 4⁻5 years in Prague, Czech Republic, is a meeting of scientists interested in the elucidation of the action of two important plant hormones-auxins and cytokinins. It is organized by a group of researchers from the Laboratory of Hormonal Regulations in Plants at the Institute of Experimental Botany, the Czech Academy of Sciences. The symposia already have a long tradition, having started in 1972. Thanks to the central role of auxins and cytokinins in plant development, the ACPD 2018 symposium was again attended by numerous experts who presented their results in the opening, two plenary lectures, and six regular sessions, including two poster sessions. Due to the open character of the research community, which is traditionally very well displayed during the meeting, a lot of unpublished data were presented and discussed. In this report, we summarize the contributions in individual sessions that attracted our attention.

Conformational dynamics are a key factor in signaling mediated by the receiver domain of a sensor histidine kinase from Arabidopsis thaliana.

Multistep phosphorelay (MSP) cascades mediate responses to a wide spectrum of stimuli, including plant hormonal signaling, but several aspects of MSP await elucidation. Here, we provide first insight into the key step of MSP-mediated phosphotransfer in a eukaryotic system, the phosphorylation of the receiver domain of the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1RD) from Arabidopsis thaliana We observed that the crystal structures of free, Mg2+-bound, and beryllofluoridated CKI1RD (a stable analogue of the labile phosphorylated form) were identical and similar to the active state of receiver domains of bacterial response regulators. However, the three CKI1RD variants exhibited different conformational dynamics in solution. NMR studies revealed that Mg2+ binding and beryllofluoridation alter the conformational equilibrium of the ß3-α3 loop close to the phosphorylation site. Mutations that perturbed the conformational behavior of the ß3-α3 loop while keeping the active-site aspartate intact resulted in suppression of CKI1 function. Mechanistically, homology modeling indicated that the ß3-α3 loop directly interacts with the ATP-binding site of the CKI1 histidine kinase domain. The functional relevance of the conformational dynamics observed in the ß3-α3 loop of CKI1RD was supported by a comparison with another A. thaliana histidine kinase, ETR1. In contrast to the highly dynamic ß3-α3 loop of CKI1RD, the corresponding loop of the ETR1 receiver domain (ETR1RD) exhibited little conformational exchange and adopted a different orientation in crystals. Biochemical data indicated that ETR1RD is involved in phosphorylation-independent signaling, implying a direct link between conformational behavior and the ability of eukaryotic receiver domains to participate in MSP.

Light Controls Cytokinin Signaling via Transcriptional Regulation of Constitutively Active Sensor Histidine Kinase CKI1.

In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The cross talk between cytokinin response and light has been known for a long time. However, the molecular mechanism underlying the interaction between light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL (LPH) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT1 (CKI1), encoding the constitutively active sensor His kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE1 (HY1) that encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertible phytochromes. Our analysis confirmed the light-dependent regulation of the CKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors (TF) PHYTOCHROME INTERACTING FACTOR3 and CIRCADIAN CLOCK ASSOCIATED1. Changes in CKI1 expression observed in lph/hy1-7 and phy mutants correlate with misregulation of MSP signaling, changed cytokinin sensitivity, and developmental aberrations that were previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate a novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development.

Antibody-mediated modulation of cytokinins in tobacco: organ-specific changes in cytokinin homeostasis.

Cytokinins comprise a group of phytohormones with an organ-specific mode of action. Although the mechanisms controlling the complex networks of cytokinin metabolism are partially known, the role of individual cytokinin types in the maintenance of cytokinin homeostasis remains unclear. Utilizing the overproduction of single-chain Fv antibodies selected for their ability to bind trans-zeatin riboside and targeted to the endoplasmic reticulum, we post-synthetically modulated cytokinin ribosides, the proposed transport forms of cytokinins. We observed asymmetric activity of cytokinin biosynthetic genes and cytokinin distribution in wild-type tobacco seedlings with higher cytokinin abundance in the root than in the shoot. Antibody-mediated modulation of cytokinin ribosides further enhanced the relative cytokinin abundance in the roots and induced cytokinin-related phenotypes in an organ-specific manner. The activity of cytokinin oxidase/dehydrogenase in the roots was strongly up-regulated in response to antibody-mediated formation of the cytokinin pool in the endoplasmic reticulum. However, we only detected a slight decrease in the root cytokinin levels. In contrast, a significant decrease of cytokinins occurred in the shoot. We suggest the roots as the main site of cytokinin biosynthesis in tobacco seedlings. Conversely, cytokinin levels in the shoot seem to depend largely on long-range transport of cytokinin ribosides from the root and their subsequent metabolic activation.

Role of SH3b binding domain in a natural deletion mutant of Kayvirus endolysin LysF1 with a broad range of lytic activity.

The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.

Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.

N6-adenosine methylation (m6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m6 A writer proteins in Arabidopsis thaliana. The components required for m6 A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences.

Dirigent proteins in plants: modulating cell wall metabolism during abiotic and biotic stress exposure.

Dirigent (DIR) proteins were found to mediate regio- and stereoselectivity of bimolecular phenoxy radical coupling during lignan biosynthesis. Here we summarize the current knowledge of the importance of DIR proteins in lignan and lignin biosynthesis and highlight their possible importance in plant development. We focus on the still rather enigmatic Arabidopsis DIR gene family, discussing the few members with known functional importance. We comment on recent discoveries describing the detailed structure of two DIR proteins with implications in the mechanism of DIR-mediated catalysis. Further, we summarize the ample evidence for stress-induced dirigent gene expression, suggesting the role of DIRs in adaptive responses. In the second part of our work, we present a preliminary bioinformatics-based characterization of the AtDIR family. The phylogenetic analysis of AtDIRs complemented by comparison with DIR proteins of mostly known function from other species allowed us to suggest possible roles for several members of this family and identify interesting AtDIR targets for further study. Finally, based on the available metadata and our in silico analysis of AtDIR promoters, we hypothesize about the existence of specific transcriptional controls for individual AtDIR genes and implicate them in various stress responses, hormonal regulations, and developmental processes.

Cytokinins influence root gravitropism via differential regulation of auxin transporter expression and localization in Arabidopsis.

Redirection of intercellular auxin fluxes via relocalization of the PIN-FORMED 3 (PIN3) and PIN7 auxin efflux carriers has been suggested to be necessary for the root gravitropic response. Cytokinins have also been proposed to play a role in controlling root gravitropism, but conclusive evidence is lacking. We present a detailed study of the dynamics of root bending early after gravistimulation, which revealed a delayed gravitropic response in transgenic lines with depleted endogenous cytokinins (Pro35S:AtCKX) and cytokinin signaling mutants. Pro35S:AtCKX lines, as well as a cytokinin receptor mutant ahk3, showed aberrations in the auxin response distribution in columella cells consistent with defects in the auxin transport machinery. Using in vivo real-time imaging of PIN3-GFP and PIN7-GFP in AtCKX3 overexpression and ahk3 backgrounds, we observed wild-type-like relocalization of PIN proteins in the columella early after gravistimulation, with gravity-induced relocalization of PIN7 faster than that of PIN3. Nonetheless, the cellular distribution of PIN3 and PIN7 and expression of PIN7 and the auxin influx carrier AUX1 was affected in AtCKX overexpression lines. Based on the retained cytokinin sensitivity in pin3 pin4 pin7 mutant, we propose the AUX1-mediated auxin transport rather than columella-located PIN proteins as a target of endogenous cytokinins in the control of root gravitropism.

Post-Translational Modifications of Histones in Human Sperm.

We examined the levels and distribution of post-translationally modified histones and protamines in human sperm. Using western blot immunoassay, immunofluorescence, mass spectrometry (MS), and FLIM-FRET approaches, we analyzed the status of histone modifications and the protamine P2. Among individual samples, we observed variability in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1, but the level of acetylated (ac) histones H4 was relatively stable in the sperm head fractions, as demonstrated by western blot analysis. Sperm heads with lower levels of P2 exhibited lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1. A very strong correlation was observed between the levels of P2 and H3K9me2. FLIM-FRET analysis additionally revealed that acetylated histones H4 are not only parts of sperm chromatin but also appear in a non-integrated form. Intriguingly, H4ac and H3K27me3 were detected in sperm tail fractions via western blot analysis. An appearance of specific histone H3 and H4 acetylation and H3 methylation in sperm tail fractions was also confirmed by both LC-MS/MS and MALDI-TOF MS analysis. Taken together, these data indicate that particular post-translational modifications of histones are uniquely distributed in human sperm, and this distribution varies among individuals and among the sperm of a single individual.

Xylem development - from the cradle to the grave.

The development and growth of plants, as well as their successful adaptation to a variety of environments, is highly dependent on the conduction of water, nutrients and other important molecules throughout the plant body. Xylem is a specialized vascular tissue that serves as a conduit of water and minerals and provides mechanical support for upright growth. Wood, also known as secondary xylem, constitutes the major part of mature woody stems and roots. In the past two decades, a number of key factors including hormones, signal transducers and (post)transcriptional regulators have been shown to control xylem formation. We outline the main mechanisms shown to be essential for xylem development in various plant species, with an emphasis on Arabidopsis thaliana, as well as several tree species where xylem has a long history of investigation. We also summarize the processes which have been shown to be instrumental during xylem maturation. This includes mechanisms of cell wall formation and cell death which collectively complete xylem cell fate.

Hormonal regulation of secondary cell wall formation.

Secondary cell walls (SCWs) have critical functional importance but also constitute a high proportion of the plant biomass and have high application potential. This is true mainly for the lignocellulosic constituents of the SCWs in xylem vessels and fibres, which form a structured layer between the plasma membrane and the primary cell wall (PCW). Specific patterning of the SCW thickenings contributes to the mechanical properties of the different xylem cell types, providing the plant with mechanical support and facilitating the transport of solutes via vessels. In the last decade, our knowledge of the basic molecular mechanisms controlling SCW formation has increased substantially. Several members of the multi-layered regulatory cascade participating in the initiation and transcriptional regulation of SCW formation have been described, and the first cellular components determining the pattern of SCW at the subcellular resolution are being uncovered. The essential regulatory role of phytohormones in xylem development is well known and the molecular mechanisms that link hormonal signals to SCW formation are emerging. Here, we review recent knowledge about the role of individual plant hormones and hormonal crosstalk in the control over the regulatory cascades guiding SCW formation and patterning. Based on the analogy between many of the mechanisms operating during PCW and SCW formation, recently identified mechanisms underlying the hormonal control of PCW remodelling are discussed as potentially novel mechanisms mediating hormonal regulatory inputs in SCW formation.

RNA processing in auxin and cytokinin pathways.

Auxin and cytokinin belong to the 'magnificent seven' plant hormones, having tightly interconnected pathways leading to common as well as opposing effects on plant morphogenesis. Tremendous progress in the past years has yielded a broad understanding of their signalling, metabolism, regulatory pathways, transcriptional networks, and signalling cross-talk. One of the rapidly expanding areas of auxin and cytokinin research concerns their RNA regulatory networks. This review summarizes current knowledge about post-transcriptional gene silencing, the role of non-coding RNAs, the regulation of translation, and alternative splicing of auxin- and cytokinin-related genes. In addition, the role of tRNA-bound cytokinins is also discussed. We highlight the most recent publications dealing with this topic and underline the role of RNA processing in auxin- and cytokinin-mediated growth and development.

Illuminating light, cytokinin, and ethylene signalling crosstalk in plant development.

Integrating important environmental signals with intrinsic developmental programmes is a crucial adaptive requirement for plant growth, survival, and reproduction. Key environmental cues include changes in several light variables, while important intrinsic (and highly interactive) regulators of many developmental processes include the phytohormones cytokinins (CKs) and ethylene. Here, we discuss the latest discoveries regarding the molecular mechanisms mediating CK/ethylene crosstalk at diverse levels of biosynthetic and metabolic pathways and their complex interactions with light. Furthermore, we summarize evidence indicating that multiple hormonal and light signals are integrated in the multistep phosphorelay (MSP) pathway, a backbone signalling pathway in plants. Inter alia, there are strong overlaps in subcellular localizations and functional similarities in components of these pathways, including receptors and various downstream agents. We highlight recent research demonstrating the importance of CK/ethylene/light crosstalk in selected aspects of plant development, particularly seed germination and early seedling development. The findings clearly demonstrate the crucial integration of plant responses to phytohormones and adaptive responses to environmental cues. Finally, we tentatively identify key future challenges to refine our understanding of the molecular mechanisms mediating crosstalk between light and hormonal signals, and their integration during plant life cycles.