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Calcium- and calmodulin-regulated microtubule-associated proteins as signal-integration hubs at the plasma membrane–cytoskeleton nexus
Abstract
Plant growth and development are a genetically predetermined series of events but can change dramatically in response to environmental stimuli, involving perpetual pattern formation and reprogramming of development. The rate of growth is determined by cell division and subsequent cell expansion, which are restricted and controlled by the cell wall-plasma membrane-cytoskeleton continuum, and are coordinated by intricate networks that facilitate intra- and intercellular communication. An essential role in cellular signaling is played by calcium ions, which act as universal second messengers that transduce, integrate, and multiply incoming signals during numerous plant growth processes, in part by regulation of the microtubule cytoskeleton. In this review, we highlight recent advances in the understanding of calcium-mediated regulation of microtubule-associated proteins, their function at the microtubule cytoskeleton, and their potential role as hubs in crosstalk with other signaling pathways.
... Additional elements such as S and N reduce the concentration of chlorophyll and xanthophyll cycle intermediates, degradation of the structure and protein complexes, and changes in the activity of Rubisco (Longoni et al. 2019;Pavlovic et al. 2021;Hayes et al. 2021). Ca is another essential macronutrient in plants playing an essential role in the cell wall and functions as intracellular messenger in the cytosol and stress signal transduction in plant cells (Kölling et al. 2019;Chen et al. 2021). It is known that chloroplasts are a part of the cellular Ca 2+ network and contribute to cytosolic signalling and membrane and cell cytoskeleton integrity (Hepler and Winship 2010;Kölling et al. 2019;Pavlovic et al. 2021). ...
... Ca is another essential macronutrient in plants playing an essential role in the cell wall and functions as intracellular messenger in the cytosol and stress signal transduction in plant cells (Kölling et al. 2019;Chen et al. 2021). It is known that chloroplasts are a part of the cellular Ca 2+ network and contribute to cytosolic signalling and membrane and cell cytoskeleton integrity (Hepler and Winship 2010;Kölling et al. 2019;Pavlovic et al. 2021). Moreover, Ca 2+ is a structural component of PSII and functions in the photosynthetic oxidation of water (oxygen evolution complex; OEC) (Wang et al. 2019b;Huang et al. 2021). ...
... CO 2 diffusion depends on the cell wall porosity and permeabilities of membranes (Dall'Osto et al. 2019;Longoni et al. 2019;Evans 2021). N (and probably S) deficiency may affect aquaporins, which enable an environment conducive to CO 2 diffusion across the plasma membrane (Terashima and Ono 2002;Kölling et al. 2019). ...
Nutritional deficiency in plants triggers significant economic losses for important crops. As a direct consequence, it broadly affects plant growth, and photosynthetic efficiency is strongly influenced by nutritional imbalance. To deepen our understanding of how nutritional deficiencies affects photosynthesis, we removed the supply of macronutrients (N, P, K, Ca, S) one element at a time at hydroponic tobacco cultivation. Essential macronutrient deficiencies affected both photochemical and carboxylative steps of photosynthesis. In addition, interference on light absorption, energy quenching and the electron transport chain influenced carbon reactions. In particular, deficiencies in N and S depleted potential CO2 assimilation, while Ca deficiency affected CO2 diffusion (mainly gs), and light reactions were affected as well. P deficiency promoted severe damage to the antenna complexes and reaction centres of the photosystems. All deficiencies increased energy dissipation (ɸDo) and nonphotochemical dissipation (Kn). Reduction of ETR and probability of electron transport of QA− for plastoquinone (ΨEo and ɸEo) following distinct levels of damages to ETC were observed.
... Therefore, good candidates for persistence length modulation are proteins involved in microtubule-membrane linkage, such as CMU (cellulose-microtubule uncoupling) proteins [258], and certain IQD proteins [260], of which IQD13 functions in metaxylem development [154]. IQD proteins can also be regulated dynamically, in particular through calcium signalling, as they have calmodulin-binding domains [261,262]. This kind of regulation may dynamically influence microtubule persistence length. ...
... Alternatively, rather than physically blocking all membrane-bound proteins, IQD13 might influence the ROP pattern by selectively interacting with active ROP or associated proteins or lipids in ROP nanodomains. IQD13 is part of a large family of IQD proteins, which are emerging as important players in microtubule organisation with an ability to respond to calcium signalling through a calmodulin-binding domain [261,262,293,294]. These ongoing efforts to characterise the functions of IQDs are likely to yield new insights into their regulation and interactions with active and inactive ROPs. ...
... Cells may tune the microtubule persistence length both by controlling the relative number of these proteins, and by regulating their affinities. IQD proteins have calmodulin-binding domains and can therefore be regulated by calcium signalling [261,262], which is in turn influenced by auxin signalling [302]. As there are many IQDs with the ability to bind microtubules and the membrane [260], it is possible that more of them function in a similar way in other cell types. ...
... The missing link between elevated calcium, suppressed callose deposition and block of the cell cycle might be microtubules, since they can be eliminated through modulation of calcium-and calmodulin-binding associated proteins (Kölling et al. 2019). Elimination of microtubules in response to calcium would culminate in disrupted Content courtesy of Springer Nature, terms of use apply. ...
... The elimination of microtubules might well a consequence of the elevated calcium levels induced by kinetin (Fig. 6). A straightforward hypothesis would imply that calmodulin-related proteins (for review see Kölling et al. 2019) modulate the activity of microtubule-associated proteins. In fact, several members of the IQD clade of calmodulin-binding proteins have been shown to convey calcium-triggered subdomains to microtubules (Bürstenbinder et al. 2017). ...
Plant cells can undergo regulated cell death in response to exogenous factors (often in a stress context), but also as regular element of development (often regulated by phytohormones). The cellular aspects of these death responses differ, which implies that the early signalling must be different. We use cytokinin-induced programmed cell death as paradigm to get insight into the role of the cytoskeleton for the regulation of developmentally induced cell death, using tobacco BY-2 cells as experimental model. We show that this PCD in response to kinetin correlates with an arrest of the cell cycle, a deregulation of DNA replication, a loss of plasma membrane integrity, a subsequent permeabilisation of the nuclear envelope, an increase of cytosolic calcium correlated with calcium depletion in the culture medium, an increase of callose deposition and the loss of microtubule and actin integrity. We discuss these findings in the context of a working model, where kinetin, mediated by calcium, causes the breakdown of the cytoskeleton, which, either by release of executing proteins or by mitotic catastrophe, will result in PCD.
... Guided by cortical microtubules, CSCs generate cellulose microfibrils to the extracellular space (Gutierrez et al. 2009;Paredez et al. 2006). Notably, Ca 2+ and CaM affect the structure and dynamics of both the actin and microtubule cytoskeleton (Hepler 2016;Kölling et al. 2019). Recent findings regarding Ca 2+ -and CaM-interacting microtubuleassociated proteins (MAPs) have revealed further roles for Ca 2+ /CaM signaling in regulating the plasma membrane and cytoskeleton in plant cells (Kölling et al. 2019). ...
... Notably, Ca 2+ and CaM affect the structure and dynamics of both the actin and microtubule cytoskeleton (Hepler 2016;Kölling et al. 2019). Recent findings regarding Ca 2+ -and CaM-interacting microtubuleassociated proteins (MAPs) have revealed further roles for Ca 2+ /CaM signaling in regulating the plasma membrane and cytoskeleton in plant cells (Kölling et al. 2019). In this paper, we first reported the effects of calcium-channel inhibitors on SCW patterning. ...
Secondary cell walls (SCWs) accumulate in specific cell types of vascular plants, notably xylem vessel cells. Previous work has shown that calcium ions (Ca²⁺) participate in xylem vessel cell differentiation, but whether they function in SCW deposition remains unclear. In this study, we examined the role of Ca²⁺ in SCW deposition during xylem vessel cell differentiation using Arabidopsis thaliana suspension-cultured cells carrying the VND7-inducible system, in which VND7 activity can be post-translationally upregulated to induce transdifferentiation into protoxylem-type vessel cells. We observed that extracellular Ca²⁺ concentration was a crucial determinant of differentiation, although it did not have consistent effects on the transcription of VND7-downstream genes as a whole. Increasing the Ca²⁺ concentration reduced differentiation but the cells could generate the spiral patterning of SCWs. Exposure to a calcium-channel inhibitor partly restored differentiation but resulted in abnormal branched and net-like SCW patterning. These data suggest that Ca²⁺ signaling participates in xylem vessel cell differentiation via post-transcriptional regulation of VND7-downstream events, such as patterning of SCW deposition.
... Previous studies proposed that IQD proteins act as scaffolds at microtubules and the plasma membrane 3,18,19 . We thus hypothesized that IQDs may coordinate division-plane set-up by facilitating macromolecular complex assembly during cell division. ...
... To identify structural determinants of POK and PHGAP binding, we generated a set of N-and C-terminally truncated variants of IQD8 and tested the interaction in a Y2H assay (Fig. 3g,h). As a control, we included the calcium sensor calmodulin (CaM2), which previously had been shown to bind to the IQ67 domain of IQD proteins 19,23,24 . As expected, all fragments containing the IQ67 domain interacted with CaM2. ...
Spatiotemporal control of cell division is essential for the growth and development of multicellular organisms. In plant cells, proper cell plate insertion during cytokinesis relies on the premitotic establishment of the division plane at the cell cortex. Two plant-specific cytoskeleton arrays, the preprophase band (PPB) and the phragmoplast, play important roles in division-plane orientation and cell plate formation, respectively1. Microtubule organization and dynamics and their communication with membranes at the cortex and cell plate are coordinated by multiple, mostly distinct microtubule-associated proteins2. How division-plane selection and establishment are linked, however, is still unknown. Here, we report members of the Arabidopsis IQ67 DOMAIN (IQD) family3 as microtubule-targeted proteins that localize to the PPB and phragmoplast and additionally reside at the cell plate and a polarized cortical region including the cortical division zone (CDZ). IQDs physically interact with PHRAGMOPLAST ORIENTING KINESIN (POK) proteins4,5 and PLECKSTRIN HOMOLOGY GTPase ACTIVATING (PHGAP) proteins6, which are core components of the CDZ1. The loss of IQD function impairs PPB formation and affects CDZ recruitment of POKs and PHGAPs, resulting in division-plane positioning defects. We propose that IQDs act as cellular scaffolds that facilitate PPB formation and CDZ set-up during symmetric cell division. The IQ67 DOMAIN (IQD) family are plant-specific calmodulin-binding proteins. Several members are associated with the microtubule cytoskeleton, and now IQD6, IQD7 and IQD8 are characterized as functioning in the set-up of the cell division plane in the root meristem of Arabidopsis.
... Calcium serves as an important second messenger in plants, with established roles in cell division and cell expansion, graviperception, salt stress and cold stress (Hepler, 2016;Lazzaro et al., 2018;Kolling et al., 2019). These effects will involve, at least in part, an impact on microtubule function (Wang & Nick, 2017). ...
... However, knock-down plants of MAP18 were reported not to result in helical growth (X. Kolling et al., 2019). Taken together, these results seem to support a role for calcium in plant helical growth (Fig. 4). ...
... Calcium serves as an important second messenger in plants, with established roles in cell division and cell expansion, graviperception, salt stress and cold stress (Hepler, 2016;Kolling et al., 2019;Lazzaro et al., 2018). These effects will involve, at least in part, an impact on microtubule function (Wang and Nick, 2017). ...
... However, knock-down plants of MAP18 were reported not to result in helical growth (Kolling et al., 2019;Wang et al., 2007b). ...
Many plant mutants are known that exhibit some degree of helical growth. This “twisted” phenotype has arisen frequently in mutant screens of model organisms but it is also found in cultivars of ornamental plants including trees. The phenomenon, in many cases, is based on defects in cell expansion symmetry. Any complete model to explain the anisotropy of plant cell growth must ultimately explain how helical cell expansion comes into existence – and how it is normally avoided. While the mutations observed in model plants mainly point to the microtubule system, additional affected components involve cell wall functions, auxin transport and more. Evaluation of published data suggests a two‐way mechanism to underlie the helical growth phenomenon: there is, apparently, a microtubular component that determines handedness, but there is also an influence arising in the cell wall that feeds back into the cytoplasm and affects cellular handedness. This is supported by recent reports demonstrating the involvement of the cell wall integrity pathway. In addition there is mounting evidence that calcium is an important relayer of signals relating to the symmetry of cell expansion. These concepts suggest experimental approaches to untangle the phenomenon of helical cell expansion in plant mutants. This article is protected by copyright. All rights reserved.
... SJ14075 and SJ22156 encode a cytoskeletal adhesion protein and a tubulin protein, respectively. They may play a role in the regulation of microtubule cytoskeleton in growth and development as CaM-binding microtubule-associated proteins [67]. SJ12208 was predicted to be a SAC3/GANP family protein. ...
Calmodulins (CaMs) and Calmodulin-like proteins (CMLs) are vital in plant growth, development, and stress responses. However, CaMs and CMLs have not been fully identified and characterized in brown algae, which has been evolving independently of the well-studied green plant lineage. In this study, whole-genome searches revealed one SjCaM and eight SjCMLs in Saccharina japonica, and one EsCaM and eleven EsCMLs in Ectocarpus sp. SjCaM and EsCaM encoded identical protein products and shared 88.59–89.93% amino acid identities with Arabidopsis thaliana AtCaMs, thereby indicating that brown algae CaMs retained a similar Ca2+ sensors function as in plants. The phylogenetic and gene structure analysis results showed that there was significant divergence in the gene sequences among brown algae CMLs. Furthermore, evolutionary analysis indicated that the function of brown alga CMLs was relatively conserved, which may be related to the fact that brown algae do not need to face complex environments like terrestrial plants. Regulatory elements prediction and the expression analysis revealed the probable functioning of SjCaM/CML genes in gametophyte development and the stress response in S. japonica. In addition, the SjCaM/SjCMLs interacting proteins and chemicals were preliminarily predicted, suggesting that SjCaM/SjCMLs might play putative roles in Ca2+/CaM-mediated growth and development processes and stimulus responses. Therefore, these results will facilitate our understanding of the evolution of brown algae CaMs/CMLs and the functional identification of SjCaM/SjCMLs.
... Thus, it is intriguing to test if ROP GTPases may form a decoder network by integrating PTI/ETI signals and cytoskeleton dynamics with diverse Ca 2+ signatures.Besides MFs, Ca 2+ and CaM regulate MTs by microtubule-associated proteins (MAPs). Several MAPs have emerged as the decoder of Ca 2+ signaling, such as MDP25, MAP18, MAP65, IQDs, KCBP, and TON1[94]. In addition, some MAPs have Ca 2+ -dependent actin-severing activity[95], suggesting crosstalk of MF with MT in Ca 2+ signaling. ...
Calcium ions (Ca2+) are prominent intracellular messengers in all eukaryotic cells. Recent studies have emphasized the crucial roles of Ca2+ in plant immunity. Here, we review the latest progress on the spatiotemporal control of Ca2+ function in plant immunity. We discuss discoveries of how Ca2+ influx is triggered upon the activation of immune receptors, how Ca2+-permeable channels are activated, how Ca2+ signals are decoded inside plant cells, and how these signals are switched off. Despite recent advances, many open questions remain and we highlight the existing toolkit and the new technologies to address the outstanding questions of Ca2+ signaling in plant immunity.
... Por otro lado, Marschner (1995) menciona que la producción de biomasa de un cultivo se determina en gran medida por la fotosíntesis, ya que aproximadamente el 90% del peso seco de una planta se considera derivado de los productos formados durante este proceso. En este trabajo se observó que el efecto de las altas concentraciones de B en la solución nutritiva sobre la fotosíntesis también se vio reflejado en la producción de biomasa, donde la mayor actividad de esta variable fisiológica se relacionó con los tratamientos donde se obtuvo la mayor producción de biomasa seca de raíz, hoja y total, principalmente al adicionar 9 y 11 meq L −1 de Ca, resultado que pudo relacionarse a que, además de su participación como segundo mensajero, el Ca cumple funciones en la biogénesis de la pared celular, división, expansión y organización celular (Kölling et al. 2019), por lo que al aumentar la concentración de Ca se logró generar un mayor desarrollo vegetal lo que se reflejó en una mayor biomasa seca. Relacionado a lo anterior, Turan et al. (2009) y Metwally et al. (2018 reportaron que la aplicación de altos niveles de Ca alivió parcialmente los síntomas de toxicidad de B sobre la producción de biomasa seca de brotes y raíz de plantas de trigo y canola. ...
La toxicidad de B es una restricción ambiental que limita la productividad de los cultivos. El objetivo de esta investigación fue determinar la influencia de Ca en plantas de pimiento irrigadas con altas concentraciones de B. Los tratamientos evaluados fueron cinco concentraciones de B (0.5, 1.5, 3, 4.5 y 6 ppm) y tres concentraciones de Ca (7, 9 y 11 meq L-1). La fotosíntesis, altura de planta, peso seco de raíz y total decreció al aumentar la concentración de B en la solución nutritiva; pero el efecto negativo del B fue menor cuando se incrementó la concentración de Ca en la solución nutritiva. La longitud de raíz fue afectada por concentraciones altas de B, pero no se observó el efecto positivo del incremento de Ca. Asimismo, el contenido total de N, P, K, Ca y Mg en las plantas de pimiento decreció al aumentar la concentración de B; sin embargo, a excepción de Mg, este detrimento fue menor al incrementar la concentración de Ca a 9 y 11 meq L-1. El contenido total de B en las plantas de pimiento decreció al aumentar la concentración de B, pero cuando la concentración de Ca en la solución fue mayor a 7 meq L-1 el contenido total de B presentó una disminución más pronunciado. Por lo tanto, al incrementar la concentración de Ca en la solución nutritiva se puede mejorar el crecimiento y absorción de nutrientes de las plantas de pimiento encondiciones de exceso de B.
... Within these diverse signaling pathways, calcium ions (Ca 2+ ) serve as universal second messengers and several studies implicate roles of Ca 2+ in regulation of the cytoskeleton (Hepler, 2016). Ca 2+ signals may be integrated at microtubules via differential binding of calmodulin (CaM)-Ca 2+ sensors to several MAPs (Kölling et al., 2019). One class of CaM targets are IQDs, which are plant-specific microtubule binding proteins that may recruit CaM to microtubules and other specific subcellular compartments in response to Ca 2+ signals to coordinate plant development and cell shape formation (Bürstenbinder et al., 2013(Bürstenbinder et al., , 2017Zang et al., 2021). ...
... Complex integration of both internal and external stimuli is facilitated through the use of secondary messengers such as calcium signaling (Ca 2+ ). Ca 2+ -signaling regulates the microtubule cytoskeleton [58] likely through binding of Ca 2+ -sensors such as calmodulin (CaM) and then calmodulin binding to microtubule-associated proteins (Figure 2) [4, 59,60,61 ]. The tomato SUN protein is a member of the IQ67 Domain (IQD) family that have been found to localize to membranes and microtubules by binding CaM in a Ca 2+ -dependent manner [61 ,62]. ...
The shape of produce is an important agronomic trait. The knowledge of the cellular regulation of organ shapes can be implemented in the improvement of a variety of crops. The plant-specific Ovate Family Proteins (OFPs) regulate organ shape in Arabidopsis and many crops including rice, tomato, and melon. Although OFPs were previously described as transcriptional repressors, recent data support a role for the family in organ shape regulation through control of subcellular localization of protein complexes. OFPs interact with TONNEAU1 RECRUITMENT MOTIF (TRMs) and together they regulate cell division patterns in tomato fruit development. OFPs also respond to changes in plant hormones and responses to stress. The OFP-TRM interaction may work in conjunction with additional shape regulators such as IQ67 Domain (IQD) proteins to modulate the response to tissue level cues as well as external stimuli and stressors to form reproducible organ shapes by regulating cytoskeleton activities.
... Saeed et al. (2014) also, found that gypsum application greatly improved relative water content and decreased electrolyte leakage in leaves of okra plant grown under salinity. Recently, Kölling et al. (2019) added that calcium ions played an essential role in cellular signaling which acts as second messengers that transduce, integrate, and multiply incoming signals during numerous of plant growth processes. These finding indicated that crop productivity is governed by soil and water characteristics (Dinar et al., 1991). ...
Growth, physiological response and productivity of four wheat varieties, namely Masr 1, Giza 171, Gemmeiza 12, and Sids 13, grown under salt-affected soil conditions irrigated with brackish water, and their feedback to reduce applied mineral nitrogen fertilizers from 215 kg N/ha to 145 kg N/ha coupled with the foliar application of 10g L-1 yeast extract or 10g L-1 K2O or 0.3g L-1 ascorbic acid (AA) were the main objective of this study. Therefore, a field trial was conducted in salt-affected soils of South Port Said Governorate, Egypt, in two successive winter growing seasons 2014/2015 and 2015/2016. The experiments were performed in a split plot design with four replications. The main plots were devoted to the four wheat varieties, and the subplots for other treatments; i.e., 145 kg N with yeast extract, 145 kg N with 1% K2O, 145 kg N with 300 ppm ascorbic acid and control with 215 kg N fertilization. Masr 1 significantly surpassed the rest of varieties for grain productivity and proved the most tolerant variety in the present experiment, followed by Giza 171; Sids 13 seemed to be the lowest tolerant variety under such conditions. Non-significant differences were observed between plants fertilized with 215 kg N/ha and those received 145 kg N/ha plus foliar application of yeast extract for plant height, leaf area index (LAI), crop growth rate (CGR), net assimilation rate (NAR), photosynthetic pigments, soluble sugars, catalase (CAT), polyphenol oxidase (PPO), peroxidase (POD), leaf potassium content, spike length, spike weight, 1000-grain weight, grain and straw yields. Plants treated with 145 kg N/ha plus foliar application of K2O or ascorbic acid (AA) followed the aforementioned treatments in their effects for all studied traits. Relative reduction in soil salinity and sodium absorption ratio (SAR) values during the studied two seasons along with increasing soluble Ca 2+ helped in the enhancement of wheat yield and its attributes.
... Saeed et al. (2014) also, found that gypsum application greatly improved relative water content and decreased electrolyte leakage in leaves of okra plant grown under salinity. Recently, Kölling et al. (2019) added that calcium ions played an essential role in cellular signaling which acts as second messengers that transduce, integrate, and multiply incoming signals during numerous of plant growth processes. These finding indicated that crop productivity is governed by soil and water characteristics (Dinar et al., 1991). ...
Growth, physiological response and productivity of four wheat varieties, namely Masr 1, Giza 171, Gemmeiza 12, and Sids 13, grown under salt-affected soil conditions irrigated with brackish water, and their feedback to reduce applied mineral nitrogen fertilizers from 215 kg N/ha to 145 kg N/ha coupled with the foliar application of 10g L-1 yeast extract or 10g L-1 K2O or 0.3g L-1 ascorbic acid (AA) were the main objective of this study. Therefore, a field trial was conducted in salt-affected soils of South Port Said Governorate, Egypt, in two successive winter growing seasons 2014/2015 and 2015/2016. The experiments were performed in a split plot design with four replications. The main plots were devoted to the four wheat varieties, and the subplots for other treatments; i.e., 145 kg N with yeast extract, 145 kg N with 1% K2O, 145 kg N with 300 ppm ascorbic acid and control with 215 kg N fertilization. Masr 1 significantly surpassed the rest of varieties for grain productivity and proved the most tolerant variety in the present experiment, followed by Giza 171; Sids 13 seemed to be the lowest tolerant variety under such conditions. Non-significant differences were observed between plants fertilized with 215 kg N/ha and those received 145 kg N/ha plus foliar application of yeast extract for plant height, leaf area index (LAI), crop growth rate (CGR), net assimilation rate (NAR), photosynthetic pigments, soluble sugars, catalase (CAT), polyphenol oxidase (PPO), peroxidase (POD), leaf potassium content, spike length, spike weight, 1000-grain weight, grain and straw yields. Plants treated with 145 kg N/ha plus foliar application of K2O or ascorbic acid (AA) followed the aforementioned treatments in their effects for all studied traits. Relative reduction in soil salinity and sodium absorption ratio (SAR) values during the studied two seasons along with increasing soluble Ca 2+ helped in the enhancement of wheat yield and its attributes.
Prostate cancer (PCa) represents one of the most frequent diagnosed cancer in males worldwide. Due to routine screening tests and the efficiency of available treatments, PCa-related deaths have significantly decreased over the past decades. However, PCa remains a critical threat if detected at a late stage in which, cancer cells would have already detached from the primary tumor to spread and invade other parts of the body. Calcium (Ca2+) channels and their protein regulators are now considered as hallmarks of cancer and some of them have been well examined in PCa. Among these Ca2+ channels, isoform 3 of the ORAI channel family has been shown to regulate the proliferation of PCa cells via the Arachidonic Acid-mediated Ca2+ entry, requiring the involvement of STIM1 (Stromal Interaction Molecule 1). Still, no study has yet demonstrated a role of the "neglected" STIM2 isoform in PCa or if it may interact with ORAI3 to promote an oncogenic behavior. In this study, we demonstrate that ORAI3 and STIM2 are upregulated in human PCa tissues. In old KIMAP (Knock-In Mouse Prostate Adenocarcinoma) mice, ORAI3 and STIM2 mRNA levels were significantly higher than ORAI1 and STIM1. In vitro, we show that ORAI3-STIM2 interact under basal conditions in PC-3 cells. ORAI3 silencing increased Store Operated Ca2+ Entry (SOCE) and induced a significant increase of the cell population in G2/M phase of the cell cycle, consistent with the role of ORAI3 as a negative regulator of SOCE. Higher expression levels of CDK1-Y15/Cyclin B1 were detected and mitotic arrest-related death occurred after ORAI3 silencing, which resulted in activating Bax/Bcl-2-mediated apoptotic pathway and caspase-8 activation and cleavage. STIM2 and ORAI3 expression increased in M phase while STIM1 expression and SOCE amplitude significantly decreased. Taken together, ORAI3 -STIM2 complex allows a successful progression through mitosis of PCa cells by evading mitotic catastrophe.
In response to Ca2+ signals, the evolutionarily-conserved Ca2+ sensor calmodulin (CaM) regulates protein targets via direct interaction. Plants possess many CaM-like (CML) proteins, but their binding partners and functions are mostly unknown. Here, using Arabidopsis CML13 as 'bait' in a yeast two-hybrid screen, we isolated putative targets from three, unrelated protein families, namely, IQD proteins, calmodulin-binding transcriptional activators (CAMTAs) and myosins, all of which possess tandem isoleucine-glutamine (IQ) structural domains. Using the split-luciferase complementation assay in planta and the yeast 2-hybrid system, CML13 and CML14 showed a preference for interaction with tandem over single IQ domains. Relative to CaM, CML13 and CML14 displayed weaker signals when tested with the non-IQ, CaM-binding domain of glutamate decarboxylase or the single IQ domains of CNGC20 (cyclic-nucleotide gated channel-20) or IQM1 (IQ motif protein1). We examined IQD14 as a representative tandem IQ-protein and found that only CaM, CML13 and CML14 interacted with IQD14 among 12 CaM/CMLs tested. CaM, CML13 and CML14 bound in vitro to IQD14 in the presence or absence of Ca2+ . Binding affinities were in the nM range and were higher when two tandem IQ domains from IQD14 were present. Green fluorescent protein-tagged versions of CaM, CML13 and CML14 localized to both the cytosol and nucleus in plant cells but were partially relocalized to the microtubules when co-expressed with IQD14 tagged with mCherry. These and other data are discussed in the context of possible roles for these CMLs in gene regulation via CAMTAs and cytoskeletal activity via myosins and IQD proteins.
The unequal (asymmetric) distribution of cell structures and proteins within a cell is designated as cell polarity. Cell polarity is a crucial prerequisite for morphogenetic processes such as oriented cell division and directed cell expansion. Rho-related GTPase from plants (ROPs) are required for cellular morphogenesis through the reorganization of the cytoskeleton and vesicle transport in various tissues. Here, I review recent advances in ROP-dependent tip growth, vesicle transport, and tip architecture. I report on the regulatory mechanisms of ROP upstream regulators found in different cell types. It appears that these regulators assemble in nanodomains with specific lipid compositions and recruit ROPs for activation in a stimulus-dependent manner. Current models link mechanosensing/mechanotransduction to ROP polarity signaling involved in feedback mechanisms via the cytoskeleton. Finally, I discuss ROP signaling components that are upregulated by tissue-specific transcription factors and exhibit specific localization patterns during cell division, clearly suggesting ROP signaling in division plane alignment.
The preprophase band (PPB) is a transient cytokinetic structure that marks the future division plane at the onset of mitosis. The PPB forms a dense cortical ring of mainly microtubules, actin filaments, endoplasmic reticulum, and associated proteins that encircles the nucleus of mitotic cells. After PPB disassembly, the positional information is preserved by the cortical division zone (CDZ). The formation of the PPB and its contribution to timely CDZ set-up involves activities of functionally distinct microtubule-associated proteins (MAPs) that interact physically and genetically to support robust division plane orientation in plants. Recent studies identified two types of plant-specific MAPs as key regulators of PPB formation, the TON1 RECRUITMENT MOTIF (TRM) and IQ67 DOMAIN (IQD) families. Both families share hallmarks of disordered scaffold proteins. Interactions of IQDs and TRMs with multiple binding partners, including the microtubule severing KATANIN1, may provide a molecular framework to coordinate PPB formation, maturation, and disassembly.
The enhanced growth and productivity of soybeans during the past decades were possible due to the application of agrichemicals such as bio-fertilizers, chemical fertilizers, and the use of high yielding, as well as disease resistant transgenic and non-transgenic varieties. Agrichemicals applied as seed primers, plant protectants, and growth regulators, however, had a diminutive significance on growth and productivity improvements across the globe. The utilization of plant growth regulators (PGRs) for vegetative growth, reproduction and yield quality improvements remains unexplored, particularly, the use of cytokinins such as 6-benzyl adenine (6-BAP) to improve soybean response to abiotic stresses. Therefore, an understanding of the role of 6-BAP in the mediation of an array of adaptive responses that provide plants with the ability to withstand abiotic stresses must be thoroughly investigated. Such mitigative effects will play a critical role in encouraging exogenous application of plant hormones like 6-BAP as a mechanism for overcoming drought stress related effects in soybean. This paper discusses the evolving role of synthetic cytokinin 6-bezyl adenine in horticulture, especially the implications of its exogenous applications in soybean to confer tolerance to drought stress.
The impact of calcium (Ca²⁺) under abiotic stress is manifested by both calmodulin (CaM)-dependent and calmodulin-independent pathways of signaling mechanisms in plants. Present work demonstrates the impact of NaCl (120 mM) stress on Ca²⁺-calmodulin activity and its spatial distribution in roots of 2d old sunflower seedlings (Helianthus annuus). NaCl stress significantly increases [Ca²⁺]cyt in roots (69%) which corresponds with 8% increase in activated calmodulin (Ca²⁺-CaM) levels. Thus, NaCl stress induced Ca²⁺ signaling in roots is likely to majorly operate through CaM-independent routes. Trifluoperazine-mediated fluorescence imaging reveals differential distribution of Ca²⁺-CaM complex in the elongation and differentiation zone of seedling roots which indicate spatial regulation of Ca²⁺-CaM signaling operative in the presence of NaCl stress. NaCl stress-induced CaM-independent Ca²⁺ signaling is further accompanied by differential protein phoshorylation of cytosolic proteins in seedling roots and cotyledons. Phosphoproteins exhibiting qualitative differences in roots in response to salt-stress were of both low and high molecular weight range (76–17.5 kDa) unlike in cotyledons where only high molecular weight (>76 kDa) phosphoproteins exhibited major differential expressions in response to 120 mM NaCl stress. Among the various polypeptides in the fraction of OB membrane proteins two polypeptides of 48 and >76 kDa exhibited increased protein phosphorylation in the presence of NaCl stress. NaCl stress for 48 h induces differences in the expression of phosphoproteins in roots and cotyledons, suggesting the long distance signaling of Na⁺ stress from roots to cotyledons. Roots seem to exhibit much stringent regulation of phosphoproteins in comparison with cotyledons which have lesser qualitative differences. Thus, present work provides evidence on the modulation of Ca²⁺-CaM signaling accompanying differential protein phosphorylation in sunflower seedling roots and cotyledons under NaCl stress.
Arabidopsis seeds release large capsules of mucilaginous polysaccharides, which are shaped by an intricate network of cellulosic microfibrils. Cellulose synthase complexes are guided by the microtubule cytoskeleton, but it is unclear which proteins mediate this process in the seed coat epidermis.
Using reverse genetics, we identified IQ67 DOMAIN 9 ( IQD9 ) and KINESIN LIGHT CHAIN‐RELATED 1 ( KLCR1 ) as two highly expressed genes during seed development and comprehensively characterized their roles in cell wall polysaccharide biosynthesis.
Mutations in IQD9 as well as in KLCR1 lead to compact mucilage capsules with aberrant cellulose distribution, which can be rescued by transgene complementation. IQD9 physically interacts with KLCR1 and localizes to cortical microtubules (MTs) to maintain their organization in seed coat epidermal (SCE) cells. IQD9 as well as a previously identified TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4) protein act to maintain cellulose synthase velocity.
Our results demonstrate that IQD9, KLCR1 and TRM4 are MT‐associated proteins that are required for seed mucilage architecture. This study provides the first direct evidence that members of the IQD, KLCR and TRM families have overlapping roles in cell wall biosynthesis. Therefore, SCE cells provide an attractive system to further decipher the complex genetic regulation of polarized cellulose deposition.
Plant diseases caused by fungi are one of the major threats to global food security and understanding the interactions between fungi and plants is of great significance for plant disease control. The interaction between pathogenic fungi and plants is a complex process. From the perspective of pathogenic fungi, pathogenic fungi are involved in the regulation of pathogenicity by surface signal recognition proteins, MAPK signaling pathways, transcription factors, and pathogenic factors in the process of infecting plants. From the perspective of plant immunity, the signal pathway of immune response, the signal transduction pathway that induces plant immunity, and the function of plant cytoskeleton are the keys to studying plant resistance. In this review, we summarize the current research progress of fungi–plant interactions from multiple aspects and discuss the prospects and challenges of phytopathogenic fungi and their host interactions.
The dynamic responses of microtubules (MTs) to internal and external signals are modulated by a plethora of microtubule associated proteins (MAPs). In higher plants, many plant-specific MAPs have emerged during evolution as advantageous to their sessile lifestyle. Some members of the IQ67 domain (IQD) protein family have been shown to be plant-specific MAPs. However, the mechanisms of interaction between IQD proteins and MTs remain elusive. Here we demonstrate that the Domain of Unknown Function 4005 (DUF4005) of the Arabidopsis IQD family protein ABS6/AtIQD16 is a novel MT-binding domain. Co-sedimentation assays showed that the DUF4005 domain binds directly to MTs in vitro. GFP-labeled DUF4005 also decorates all types of MT arrays tested in vivo. Furthermore, we showed that a conserved stretch of 15 amino acid residues within the DUF4005 domain, which shares sequence similarity with the C-terminal MT-binding domain of human MAP Kif18A, is required for the binding to MTs. Transgenic lines over-expressing the DUF4005 domain displayed a spectrum of developmental defects, including spiral growth and stunted growth at the organismal level. At the cellular level, DUF4005 overexpression caused defects in epidermal pavement cell and trichome morphogenesis, as well as abnormal anisotropic cell elongation in the hypocotyls of dark-grown seedlings. These data establish that the DUF4005 domain of ABS6/AtIQD16 is a new MT-binding domain, overexpression of which perturbs MT homeostasis in plants. Our findings provide new insights into the MT-binding mechanisms of plant IQD proteins.
Polarity cues direct tissue patterning by defining the cell division plane. Proteins containing the IQ67 calmodulin-binding domain govern cell division by establishing and maintaining cell polarity during cytokinesis.
Calcium ions are the simplest agents of signaling, possessing no structural information, and therefore require a very complex system of signal interpretation. With time, Ca²⁺ has been implicated in increasingly greater signaling pathways in all organisms. A large number of these roles lie in signaling processes controlling development, which is essentially a hardwired or genetically determined course of events, modified to various extents by the organism’s interaction with its environment. This assumes greatest importance in higher plants due to the sessile nature of their habit, which places a much greater pressure on plants to adapt than it does on animals. Ca²⁺, cytoskeletal components, especially the microtubular network and their association with plasma membranes, have recently been implicated as hubs of signaling controlling plant development. This chapter is basically an in-depth discussion of this rather nascent area and attempts to integrate various pieces of information and see if a unified theory exists.
The visual deficiency symptoms developing on plants constitute the ultimate manifestation of suboptimal nutrient supply. In classical plant nutrition, these symptoms have been extensively used as a tool to characterize the nutritional status of plants and to optimize fertilization. Here we expand this concept by bridging the typical deficiency symptoms for each of the six essential macronutrients to their molecular and physiological functionalities in higher plants. We focus on the most recent insights obtained during the last decade, which now allow us to better understand the links between symptom and function for each element. A deep understanding of the mechanisms underlying the visual deficiency symptoms enables us to thoroughly understand how plants react to nutrient limitations and how these disturbances may affect the productivity and biodiversity of terrestrial ecosystems. A proper interpretation of visual deficiency symptoms will support the potential for sustainable crop intensification through development of new technologies that facilitate automatized management practices based on imaging technologies, remote sensing and in‐field sensors, thereby providing the basis for timely application of nutrients via smart and more efficient fertilization.
Hypotheses:
The drive to survive is a biological universal. Intelligent behaviour is usually recognised when individual organisms including plants, in the face of fiercely competitive or adverse, real world circumstances, change their behaviour to improve their probability of survival.
Scope:
This article explains the potential relationship of intelligence to adaptability and emphasises the need for recognising individual variation in intelligence showing it to be goal directed and thus being purposeful. Intelligent behaviour in single cells and microbes is frequently reported. Individual variation might be underpinned by a novel learning mechanism described in detail. The requirements for real world circumstances are outlined, the relationship to organic selection indicated together with niche construction as a good example of intentional behaviour that should improve survival. Adaptability is important in crop development but the term may be complex incorporating numerous behavioural traits some of which are indicated.
Conclusion:
There is real biological benefit to regarding plants as intelligent both from a fundamental issue of understanding plant life but also from providing a direction for fundamental future research and in crop breeding.
Microtubule arrays play notable roles in cell division, cell movement, cell morphogenesis and signal transduction. Due to their important regulation of microtubule dynamic instability and array-ordering processes, microtubule-associated proteins have been a cutting-edge issue in research. Here, a new maize microtubule-associated protein, ZmGLR (Zea mays glutamic acid- and lysine-rich), was found. ZmGLR bundles microtubules in vitro and targets the cell membrane through an interaction between 24 conserved N-terminal amino acids and specific phosphatidylinositol phosphates (PtdInsPs). Increased Ca2+ levels in the cytoplasm lead to ZmGLR partially dissociating from the cell membrane and moving into the cytoplasm to associate with microtubule. Overexpression and RNAi of ZmGLR both resulted in misoriented microtubule arrays, which led to dwarf maize plants and curved leaves. In addition, the expression of ZmGLR was regulated by BR and auxin through ZmBES1 and ZmARF9, respectively. This study reveals that the microtubule-associated protein ZmGLR plays a crucial role in cortical microtubule reorientation and maize leaf morphogenesis.
Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced anisotropic expansion. Lastly, we demonstrate IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays and provide first evidence for important roles of calcium in regulation of PC morphogenesis. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate anisotropic growth in PCs.
Main conclusion:
Gene expression and functional analysis of the tomato IQD gene SUN24 revealed that it regulates seed germination through ABA signaling pathway. Ca2+ signaling plays crucial roles in diverse biological processes including ABA-mediated seed germination. The plant-specific IQ67-Domain (IQD) proteins are hypothesized to regulate Ca2+ signaling and plant development through interactions with calmodulins (CaMs). Despite a few IQD genes have been identified to regulate herbivore resistance and plant growth and development, the molecular functions of most members in this gene family are not known. In this study, we characterized the role of the tomato IQD gene SUN24 in seed germination. Using pSUN24::GUS reporter lines and by quantitative reverse transcription PCR analysis, we show that SUN24 is mainly expressed in the roots, flowers, young fruits, seeds, and other young developing tissues, and its expression is repressed by ABA treatments. Functional analysis shows that knockdown of SUN24 expression by RNA interference delays seed germination, whereas overexpression of this IQD gene promotes germination. Further gene expression analysis reveals that SUN24 negatively regulates expression of two key ABA signaling genes Solanum lycopersicum ABA-insensitive 3 (SlABI3) and SlABI5 in germinating seeds. Moreover, SUN24, targeting to microtubule and nuclear bodies, can interact with four tomato CaMs (SlCaM1, 2, 3, and 6) in yeast cells. Our results demonstrate that SUN24 regulates seed germination through ABA signaling pathway, expanding our understanding of the roles of the IQD protein family members in plant physiological processes.
The universality of calcium as an intracellular messenger depends on the dynamics of its spatial and temporal release from calcium stores. Accumulating evidence over the past two decades supports an essential role for nuclear calcium signalling in the transduction of specific stimuli into cellular responses. This review focusses on mechanisms underpinning changes in nuclear calcium concentrations and discusses what is known so far, about the origin of the nuclear calcium signals identified, primarily in the context of microbial symbioses and abiotic stresses.
Calcium sensors are indispensable tools to study the role of Ca²⁺ and visualize Ca²⁺ dynamics during biological processes. Over the past years, the field of Ca²⁺ imaging has strongly expanded by the development of a wide palette of sensors and optimization of sample handling. Here, we provide guidelines for imaging of the Ca²⁺ sensor R-GECO1 in Arabidopsis thaliana roots which can be interpolated to other intensiometric Ca²⁺ sensors. Furthermore, we demonstrate a procedure for image analysis of the acquired time-lapse recordings.
Key message:
A 159 bp deletion in ClFS1 gene encoding IQD protein is responsible for fruit shape in watermelon. Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is known for its rich diversity in fruit size and shape. Fruit shape has been one of the major objectives of watermelon breeding. However, the candidate genes and the underlying genetic mechanism for such an important trait in watermelon are unknown. In this study, we identified a locus on chromosome 3 of watermelon genome controlling fruit shape. Segregation analysis in F2 and BC1 populations derived from a cross between two inbred lines "Duan125" (elongate fruit) and "Zhengzhouzigua" (spherical fruit) suggests that fruit shape of watermelon is controlled by a single locus and elongate fruit (OO) is incompletely dominant to spherical fruit (oo) with the heterozygote (Oo) being oval fruit. GWAS profiles among 315 accessions identified a major locus designated on watermelon chromosome 3, which was confirmed by BSA-seq mapping in the F2 population. The candidate gene was mapped to a region 46 kb on chromosome 3. There were only four genes present in the corresponding region in the reference genome. Four candidate genes were sequenced in this region, revealing that the CDS of Cla011257 had a 159 bp deletion which resulted in the omission of 53 amino acids in elongate watermelon. An indel marker was derived from the 159 bp deletion to test the F2 population and 105 watermelon accessions. The results showed that Cla011257 cosegregated with watermelon fruit shape. In addition, the Cla011257 expression was the highest at ovary formation stage. The predicted protein of the Cla011257 gene fitted in IQD protein family which was reported to have association with cell arrays and Ca2+-CaM signaling modules. Clear understanding of the genes facilitating the fruit shape along with marker association selection will be an effective way to develop new cultivars.
The cortical microtubule arrays of higher plants are organized without centrosomes and feature treadmilling polymers that are dynamic at both ends. The control of polymer end stability is fundamental for the assembly and organization of cytoskeletal arrays, yet relatively little is understood about how microtubule minus ends are controlled in acentrosomal microtubule arrays, and no factors have been identified that act at the treadmilling minus ends in higher plants. Here, we identify Arabidopsis thaliana SPIRAL2 (SPR2) as a protein that tracks minus ends and protects them against subunit loss. SPR2 function is required to facilitate the rapid reorientation of plant cortical arrays as stimulated by light perception, a process that is driven by microtubule severing to create a new population of microtubules. Quantitative live-cell imaging and computer simulations reveal that minus protection by SPR2 acts by an unexpected mechanism to promote the lifetime of potential SPR2 severing sites, increasing the likelihood of severing and thus the rapid amplification of the new microtubule array.
Brassinosteroids (BRs), a group of plant steroid hormones, play important roles in regulating plant development. The cytoskeleton also affects key developmental processes and a deficiency in BR biosynthesis or signaling leads to abnormal phenotypes similar to those of microtubule-defective mutants. However, how BRs regulate microtubule and cell morphology remains unknown. Here, using liquid chromatography-tandem mass spectrometry, we identified tubulin proteins that interact with Arabidopsis BRASSINOSTEROID INSENSITIVE2 (BIN2), a negative regulator of BR responses in plants. In vitro and in vivo pull-down assays confirmed that BIN2 interacts with tubulin proteins. High-speed co-sedimentation assays demonstrated that BIN2 also binds microtubules. The Arabidopsis genome also encodes two BIN2 homologs, BIN2-LIKE 1 (BIL1) and BIL2, which function redundantly with BIN2. In the bin2-3 bil1 bil2 triple mutant, cortical microtubules were more sensitive to treatment with the microtubule-disrupting drug oryzalin than in wild-type, whereas in the BIN2 gain-of-function mutant bin2-1, cortical microtubules were insensitive to oryzalin treatment. These results provide important insight into how BR regulates plant pavement cell and leaf growth by mediating the stabilization of microtubules by BIN2.
Centrosomes are microtubule-nucleating organelles that facilitate chromosome segregation and cell division in metazoans. Centrosomes comprise centrioles that organize a micron-scale mass of protein called pericentriolar material (PCM) from which microtubules nucleate. During each cell cycle, PCM accumulates around centrioles through phosphorylation-mediated assembly of PCM scaffold proteins. During mitotic exit, PCM swiftly disassembles by an unknown mechanism. Here, we used Caenorhabditis elegans embryos to determine the mechanism and importance of PCM disassembly in dividing cells. We found that the phosphatase PP2A and its regulatory subunit SUR-6 (PP2ASUR-6), together with cortically directed microtubule pulling forces, actively disassemble PCM. In embryos depleted of these activities, ∼25% of PCM persisted from one cell cycle into the next. Purified PP2ASUR-6 could dephosphorylate the major PCM scaffold protein SPD-5 in vitro Our data suggest that PCM disassembly occurs through a combination of dephosphorylation of PCM components and force-driven fragmentation of the PCM scaffold.
Ca(2+) ions play pivotal roles as second messengers in intracellular signal transduction, and coordinate many biological processes. Changes in intracellular Ca(2+) levels are perceived by Ca(2+) sensors such as CaM/CML proteins, which transduce Ca(2+) signals into cellular responses by regulation of diverse target proteins. Insights into molecular functions of CaM targets are thus essential to understand the molecular and cellular basis of Ca(2+) signaling. During the last decade, IQD proteins emerged as the largest class of CaM targets in plants with mostly unknown functions. In the March issue of Plant Physiology, we presented the first comprehensive characterization of the 33-membered IQD family in Arabidopsis thaliana. We showed, by analysis of the subcellular localization of translational GFP fusion proteins, that most IQD members label MTs, and additionally often localize to the cell nucleus or to membranes, where the recruit CaM Ca(2+) sensors. Important functions at MTs are supported by altered MT organization and plant growth in IQD gain-of-function lines. Because IQD proteins share structural hallmarks of scaffold proteins, we propose roles of IQDs in the assembly of macromolecular complexes to orchestrate Ca(2+) CaM signaling from membranes to the nucleus.
Minus end–directed cargo transport along microtubules (MTs) is exclusively driven by the molecular motor dynein in a wide variety of cell types. Interestingly, during evolution, plants have lost the genes encoding dynein; the MT motors that compensate for dynein function are unknown. Here, we show that two members of the kinesin-14 family drive minus end–directed transport in plants. Gene knockout analyses of the moss Physcomitrella patens revealed that the plant-specific class VI kinesin-14, KCBP, is required for minus end–directed transport of the nucleus and chloroplasts. Purified KCBP directly bound to acidic phospholipids and unidirectionally transported phospholipid liposomes along MTs in vitro. Thus, minus end–directed transport of membranous cargoes might be driven by their direct interaction with this motor protein. Newly nucleated cytoplasmic MTs represent another known cargo exhibiting minus end–directed motility, and we identified the conserved class I kinesin-14 (ATK) as the motor involved. These results suggest that kinesin-14 motors were duplicated and developed as alternative MT-based minus end–directed transporters in land plants.
Grain size is a major determinant of grain yield in cereal crops. qSW5/GW5, which exerts the greatest effect on rice grain width and weight, was fine-mapped to a 2,263-bp/21-kb genomic region containing a 1,212-bp deletion, respectively. Here, we show that a gene encoding a calmodulin binding protein, located ∼5 kb downstream of the 1,212-bp deletion, corresponds to qSW5/GW5. GW5 is expressed in various rice organs, with highest expression level detected in young panicles. We provide evidence that the 1,212-bp deletion affects grain width most likely through influencing the expression levels of GW5. GW5 protein is localized to the plasma membrane and can physically interact with and repress the kinase activity of rice GSK2 (glycogen synthase kinase 2), a homologue of Arabidopsis BIN2 (BRASSINOSTEROID INSENSITIVE2) kinase, resulting in accumulation of unphosphorylated OsBZR1 (Oryza sativa BRASSINAZOLE RESISTANT1) and DLT (DWARF AND LOW-TILLERING) proteins in the nucleus to mediate brassinosteroid (BR)-responsive gene expression and growth responses (including grain width and weight). Our results suggest that GW5 is a novel positive regulator of BR signalling and a viable target for genetic manipulation to improve grain yield in rice and perhaps in other cereal crops as well.
The utilization of natural genetic variation greatly contributes to improvement of important agronomic traits in crops. Understanding the genetic basis for natural variation of grain size can help breeders develop high-yield rice varieties. In this study, we identify a previously unrecognized gene, named GSE5, in the qSW5/GW5 locus controlling rice grain size by combining the genome-wide association study (GWAS) with functional analyses. GSE5 encodes a plasma membrane-associated protein with IQ domains (IQD), which interacts with the rice calmodulin protein, OsCaM1-1. We found that loss of GSE5 function caused wide and heavy grains, while overexpression of GSE5 resulted in narrow grains. We showed that GSE5 regulates grain size predominantly by influencing cell proliferation in spikelet hulls. Three major haplotypes of GSE5 (GSE5, GSE5(DEL1+IN1) and GSE5(DEL2)) in cultivated rice were identified based on the deletion/insertion type in its promoter region. We demonstrated that a 950-bp deletion (DEL1) in indica varieties carrying the GSE5(DEL1+IN1) haplotype and a 1212-bp deletion (DEL2) in japonica varieties carrying the GSE5(DEL2) haplotype associated with decreased expression of GSE5, resulting in wide grains. Further analyses indicate that wild rice accessions contain all the three haplotypes of GSE5, suggesting that the GSE5 haplotypes present in cultivated rice are likely to have originated from different wild rice accessions during rice domestication. Taken together, our results indicate that the previously unrecognized GSE5 gene in the qSW5/GW5 locus that is widely utilized by rice breeders controls grain size and reveal that natural variation in the promoter region of GSE5 contributes to grain size diversity in rice.
Key message:
QTL analysis revealed two interacting loci, FS1.2 and FS2.1, underlying round fruit shape in WI7239 cucumber; CsSUN , a homolog of tomato fruit shape gene SUN , was a candidate for FS1.2. Fruit size is an important quality and yield trait in cucumber, but its genetic basis remains poorly understood. Here we reported QTL mapping results on fruit size with segregating populations derived from the cross between WI7238 (long fruit) and WI7239 (round fruit) inbred cucumber lines. Phenotypic data of fruit length and diameter were collected at anthesis, immature and mature fruit stages in four environments. Ten major-effect QTL were detected for six traits; synthesis of information from these QTL supported two genes, FS1.2 and FS2.1, underlying fruit size variation in the examined populations. Under the two-gene model, deviation from expected segregation ratio in fruit length and diameter among segregating populations was observed, which could be explained mainly by the interactions between FS1.2 and FS2.1, and segregation distortion in the FS2.1 region. Genome-wide candidate gene search identified CsSUN, a homolog of the tomato fruit shape gene SUN, as the candidate for FS1.2. The round-fruited WI7239 had a 161-bp deletion in the first exon of CsSUN, and its expression in WI7239 was significantly lower than that in WI7238. A marker derived from this deletion was mapped at the peak location of FS1.2 in QTL analysis. Comparative analysis suggested the melon gene CmSUN-14, a homolog of CsSUN as a candidate of the fl2/fd2/fw2 QTL in melon. This study revealed the unique genetic architecture of round fruit shape in WI7239 cucumber. It also highlights the power of QTL analysis for traits with a simple genetic basis but their expression is complicated by other factors.
How cell shape is controlled is a fundamental question in developmental biology, but the genetic and molecular mechanisms that determine cell shape are largely unknown. Arabidopsis trichomes have been used as a good model system to investigate cell shape at the single-cell level. Here we describe the trichome cell shape 1 (tcs1) mutants with the reduced trichome branch number in Arabidopsis. TCS1 encodes a coiled-coil domain-containing protein. Pharmacological analyses and observations of microtubule dynamics show that TCS1 influences the stability of microtubules. Biochemical analyses and live-cell imaging indicate that TCS1 binds to microtubules and promotes the assembly of microtubules. Further results reveal that TCS1 physically associates with KCBP/ZWICHEL, a microtubule motor involved in the regulation of trichome branch number. Genetic analyses indicate that kcbp/zwi is epistatic to tcs1 with respect to trichome branch number. Thus, our findings define a novel genetic and molecular mechanism by which TCS1 interacts with KCBP to regulate trichome cell shape by influencing the stability of microtubules.
Calcineurin B-like protein (CBL)-CBL-interacting protein kinase (CIPK) network is one of the vital regulatory mechanisms which decode calcium signals triggered by environmental stresses. Although the complicated regulation mechanisms and some novel functions of CBL-CIPK signaling network in plants need to be further elucidated, numerous advances have been made in its roles involved in the abiotic stresses. This review chiefly introduces the progresses about protein interaction, classification and expression pattern of different CBLs and CIPKs in Arabidopsis thaliana, summarizes the physiological roles of CBL-CIPK pathway while pointing out some new research ideas in the future, and finally presents some unique perspectives for the further study. The review might provide new insights into the functional characterization of CBL-CIPK pathway in Arabidopsis, and contribute to a deeper understanding of CBL-CIPK network in other plants or stresses.
The preprophase band of microtubules performs the critical function of marking the plane of cell division. Although the preprophase band depolymerises at the onset of mitosis, the division plane is 'memorized' by a cortical division zone to which the phragmoplast is attracted during cytokinesis. Proteins have been discovered that are part of the molecular memory but little is known about how they contribute to phragmoplast guidance. Previously, we found that the microtubule-associated protein AIR9 is found in the cortical division zone at preprophase and returns during cell plate insertion but is absent from the cortex during the intervening mitosis. To identify new components of the preprophase memory we searched for protein interactors of AIR9. We detected the kinesin-like calmodulin binding protein, KCBP, which can be visualised at the predicted cortical site throughout division. A truncation study of KCBP indicates that its MyTH4-FERM domain is required for linking the motor domain to the cortex. These results suggest a mechanism by which minus end-directed KCBP helps guide the centrifugally expanding phragmoplast to the cortical division site.
© 2015. Published by The Company of Biologists Ltd.
Members of the plant-specific IQ67-domain (IQD) protein family are involved in plant development and the basal defense response. Although systematic characterization of this family has been carried out in Arabidopsis, tomato (Solanum lycopersicum), Brachypodium distachyon and rice (Oryza sativa), systematic analysis and expression profiling of this gene family in soybean (Glycine max) have not previously been reported. In this study, we identified and structurally characterized IQD genes in the soybean genome. A complete set of 67 soybean IQD genes (GmIQD1-67) was identified using Blast search tools, and the genes were clustered into four subfamilies (IQD I-IV) based on phylogeny. These soybean IQD genes are distributed unevenly across all 20 chromosomes, with 30 segmental duplication events, suggesting that segmental duplication has played a major role in the expansion of the soybean IQD gene family. Analysis of the Ka/Ks ratios showed that the duplicated genes of the GmIQD family primarily underwent purifying selection. Microsynteny was detected in most pairs: genes in clade 1-3 might be present in genome regions that were inverted, expanded or contracted after the divergence; most gene pairs in clade 4 showed high conservation with little rearrangement among these gene-residing regions. Of the soybean IQD genes examined, six were most highly expressed in young leaves, six in flowers, one in roots and two in nodules. Our qRT-PCR analysis of 24 soybean IQD III genes confirmed that these genes are regulated by MeJA stress. Our findings present a comprehensive overview of the soybean IQD gene family and provide insights into the evolution of this family. In addition, this work lays a solid foundation for further experiments aimed at determining the biological functions of soybean IQD genes in growth and development.
The Arabidopsis proline-rich, extensin-like receptor-like kinases (PERKs) are a small group of receptor-like kinases that are thought to act as sensors at the cell wall through their predicted proline-rich extracellular domains. In this study, we focused on the characterization of a subclade of three Arabidopsis predicted PERK genes, PERK8, -9, and -10, for which no functions were known. Yeast two-hybrid interaction studies were conducted with the PERK8,- 9, and -10 cytosolic kinase domains, and two members of the Arabidopsis AGC VIII kinase family were identified as interacting proteins: AGC1-9 and the closely related kinesin-like calmodulin-binding protein (KCBP)-interacting protein kinase (KIPK). As KIPK has been identified previously as an interactor of KCBP, these interactions were also examined further and confirmed in this study. Finally, T-DNA mutants for each gene were screened for altered phenotypes under different conditions, and from these screens, a role for the PERK, KIPK, and KCBP genes in negatively regulating root growth was uncovered.
Domestication of fruit and vegetables resulted in a huge diversity of shapes and sizes of the produce. Selections that took place over thousands of years of alleles that increased fruit weight and altered shape for specific culinary uses provide a wealth of resources to study the molecular bases of this diversity. Tomato (Solanum lycopersicum) evolved from a wild ancestor (S. pimpinellifolium) bearing small and round edible fruit. Molecular genetic studies led to the identification of two genes selected for fruit weight: FW2.2 encoding a member of the Cell Number Regulator family; and FW3.2 encoding a P450 enzyme and the ortholog of KLUH. Four genes were identified that were selected for fruit shape: SUN encoding a member of the IQD family of calmodulin-binding proteins leading to fruit elongation; OVATE encoding a member of the OVATE family proteins involved in transcriptional repression leading to fruit elongation; LC encoding most likely the ortholog of WUSCHEL controlling meristem size and locule number; FAS encoding a member in the YABBY family controlling locule number leading to flat or oxheart shape. For this article, we will provide an overview of the putative function of the known genes, when during floral and fruit development they are hypothesized to act and their potential importance in regulating morphological diversity in other fruit and vegetable crops.
The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament-severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca(2+), in vitro. Analysis of a mutant that bears a point mutation at the Ca(2+) binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca(2+) level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament-severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth.
The dynamic arrangement of cortical microtubules (MTs) plays a pivotal role in controlling cell growth and shape formation in plants, but the mechanisms by which cortical MTs are organized to regulate these processes are not well characterized. In particular, the dynamic behavior of cortical MTs is critical for their spatial organization, yet the molecular mechanisms controlling MT dynamics remain poorly understood. In this study, we used the puzzle piece-shaped pavement cells of Arabidopsis thaliana leaves as a model system to study cortical MT organization. We isolated an ethyl methanesulfonate mutant with reduced interdigitation of pavement cells in cotyledons. This line carried a mutation in IQ67 DOMAIN5 (IQD5), which encodes a member of the plant-specific IQ motif protein family. Live-cell imaging and biochemical analyses demonstrated that IQD5 binds to MTs and promotes MT assembly. MT depolymerizing drug treatment and in vivo MT dynamics assays suggested that IQD5 functions to stabilize MTs. Hence, our findings provide genetic, cell biological, and biochemical evidence that IQD5 regulates MT dynamics that affect MT organization and subsequent cell shape formation.
Calcium (Ca2+) is among the most important intracellular messengers in living organisms. Understanding of the players and dynamics of Ca2+ signalling pathways in plants may help to unravel the molecular basis of their exceptional flexibility to respond and to adapt to different stimuli. In the present review we focus on new tools that have recently revolutionized our view of organellar Ca2+ signalling as well as on the current knowledge regarding the pathways mediating Ca2+ fluxes across intracellular membranes. The contribution of organelles and cellular subcompartments to the orchestrated response via Ca2+ signalling within a cell is also discussed, underlining the fact that one of the greatest challenges in the field is the elucidation of how influx and efflux Ca2+ transporters/channels are regulated in a concerted manner to translate specific information into a Ca2+ signature.
Genetically encoded biosensors that directly interact with a molecule of interest were first introduced more than 20 years ago with fusion proteins that served as fluorescent indicators for calcium ions. Since then, the technology has matured into a diverse array of biosensors that have been deployed to improve our spatiotemporal understanding of molecules whose dynamics have profound influence on plant physiology and development. In this review, we address several types of biosensors with a focus on genetically encoded calcium indicators, which are now the most diverse and advanced group of biosensors. We then consider the discoveries in plant biology made by using biosensors for calcium, pH, reactive oxygen species, redox conditions, primary metabolites, phytohormones, and nutrients. These discoveries were dependent on the engineering, characterization, and optimization required to develop a successful biosensor; they were also dependent on the methodological developments required to express, detect, and analyze the readout of such biosensors.
Temporally and spatially defined changes in Ca2+ concentration in distinct compartments of cells represent a universal information code in plants. Recently, it has become evident that Ca2+ signals not only govern intracellular regulation but also appear to contribute to long distance or even organismic signal propagation and physiological response regulation. Ca2+ signals are shaped by an intimate interplay of channels and transporters, and during past years important contributing individual components have been identified and characterized. Ca2+ signals are translated by an elaborate toolkit of Ca2+-binding proteins, many of which function as Ca2+ sensors, into defined downstream responses. Intriguing progress has been achieved in identifying specific modules that interconnect Ca2+ decoding proteins and protein kinases with downstream target effectors, and in characterizing molecular details of these processes. In this review, we reflect on recent major advances in our understanding of Ca2+ signaling and cover emerging concepts and existing open questions that should be informative also for scientists that are currently entering this field of ever-increasing breath and impact.
Plant cortical microtubules have crucial roles in cell wall development. Cortical microtubules are tightly anchored to the plasma membrane in a highly ordered array, which directs the deposition of cellulose microfibrils by guiding the movement of the cellulose synthase complex. Cortical microtubules also interact with several endomembrane systems to regulate cell wall development and other cellular events. Recent studies have identified new factors that mediate interactions between cortical microtubules and endomembrane systems including the plasma membrane, endosome, exocytic vesicles, and endoplasmic reticulum. These studies revealed that cortical microtubule-membrane interactions are highly dynamic, with specialized roles in developmental and environmental signaling pathways. A recent reconstructive study identified a novel function of the cortical microtubule-plasma membrane interaction, which acts as a lateral fence that defines plasma membrane domains. This review summarizes recent advances in our understanding of the mechanisms and functions of cortical microtubule-membrane interactions.
Classical microtubule associated proteins (MAPs) were originally identified based on their co-purification with microtubules assembled from mammalian brain lysate. They have since been found to perform a range of functions involved in regulating the dynamics of the microtubule cytoskeleton. Most of these MAPs play integral roles in microtubule organization during neuronal development, microtubule remodeling during neuronal activity, and microtubule stabilization during neuronal maintenance. As a result, mutations in MAPs contribute to neurodevelopmental disorders, psychiatric conditions, and neurodegenerative diseases. MAPs are post-translationally regulated by phosphorylation depending on developmental time point and cellular context. Phosphorylation can affect the microtubule affinity, cellular localization, or overall function of a particular MAP and can thus have profound implications for neuronal health. Here we review MAP1, MAP2, MAP4, MAP6, MAP7, MAP9, Tau and DCX, and how each is regulated by phosphorylation in neuronal physiology and disease. This article is protected by copyright. All rights reserved.
Significance
Plants have the ability to live and grow for many thousands of years due to the activity of groups of cells called meristems. Meristems contain stem cells that can survive the entire life of the plant and ensure the continuous supply of new cells. Stem cells are thought to be qualitatively different compared with their neighboring daughter cells. Here we show that in the case of the proximal root meristem, there does not seem to be such an on-off type of organization. We show that the majority of transcripts, together with other cellular properties, gradually transition from stem cell activity to differentiation, by opposing gradients. This impacts our understanding of meristem organization and will determine the direction of future research.
Plant cytokinesis is orchestrated by a specialized structure, the phragmoplast. The phragmoplast first occurred in representatives of Charophyte algae and then became the main division apparatus in land plants. Major cellular activities, including cytoskeletal dynamics, vesicle trafficking, membrane assembly, and cell wall biosynthesis, cooperate in the phragmoplast under the guidance of a complex signaling network. Furthermore, the phragmoplast combines plant-specific features with the conserved cytokinetic processes of animals, fungi, and protists. As such, the phragmoplast represents a useful system for understanding both plant cell dynamics and the evolution of cytokinesis. We recognize that future research and knowledge transfer into other fields would benefit from standardized terminology. Here, we propose such a lexicon of terminology for specific structures and processes associated with plant cytokinesis.
Calcium signals occur in specific spatio‐temporal patterns in response to various stimuli and are coordinated with, for example, hormonal signals, for physiological and developmental adaptations. Quantification of calcium together with other signalling molecules is required for correlative analyses and to decipher downstream calcium‐decoding mechanisms.
Simultaneous in vivo imaging of calcium and abscisic acid has been performed here to investigate the interdependence of the respective signalling processes in Arabidopsis thaliana roots. Advanced ratiometric genetically encoded calcium indicators have been generated and in vivo calcium calibration protocols were established to determine absolute calcium concentration changes in response to auxin and ATP .
In roots, abscisic acid induced long‐term basal calcium concentration increases, while auxin triggered rapid signals in the elongation zone. The advanced ratiometric calcium indicator R‐ GECO 1‐ mT urquoise exhibited an increased calcium signal resolution compared to commonly used Förster resonance energy transfer‐based indicators. Quantitative calcium measurements in Arabidopsis root tips using R‐ GECO 1‐ mT urquoise revealed detailed maps of absolute calcium concentration changes in response to auxin and ATP.
Calcium calibration protocols using R‐ GECO 1‐ mT urquoise enabled high‐resolution quantitative imaging of resting cytosolic calcium concentrations and their dynamic changes that revealed distinct hormonal and ATP responses in roots.
Spatial control of cell-wall deposition is essential for determining plant cell shape [1]. Rho-type GTPases, together with the cortical cytoskeleton, play central roles in regulating cell-wall patterning [2]. In metaxylem vessel cells, which are the major components of xylem tissues, active ROP11 Rho GTPases form oval plasma membrane domains that locally disrupt cortical microtubules, thereby directing the formation of oval pits in secondary cell walls [3-5]. However, the regulatory mechanism that determines the planar shape of active Rho of Plants (ROP) domains is still unknown. Here we show that IQD13 associates with cortical microtubules and the plasma membrane to laterally restrict the localization of ROP GTPase domains, thereby directing the formation of oval secondary cell-wall pits. Loss and overexpression of IQD13 led to the formation of abnormally round and narrow secondary cell-wall pits, respectively. Ectopically expressed IQD13 increased the presence of parallel cortical microtubules by promoting microtubule rescue. A reconstructive approach revealed that IQD13 confines the area of active ROP domains within the lattice of the cortical microtubules, causing narrow ROP domains to form. This activity required the interaction of IQD13 with the plasma membrane. These findings suggest that IQD13 positively regulates microtubule dynamics as well as their linkage to the plasma membrane, which synergistically confines the area of active ROP domains, leading to the formation of oval secondary cell-wall pits. This finding sheds light on the role of microtubule-plasma membrane linkage as a lateral fence that determines the planar shape of Rho GTPase domains.
Refined understanding of the preprophase band
Because plant cells do not move, plant tissues are constructed according to how they place the divisions of their constituent cells. Schaefer et al. found a mutation in the model plant Arabidopsis that abolishes a visible precursor of cell division, the preprophase band. Despite loss of the band—previously thought essential to define the division plane—the general orientations of cell division planes in the roots of these plants were normal. However, individual division orientations showed more variance than normal. Thus, the preprophase band serves to focus and refine the final orientation of the nascent cell division plane.
Science , this issue p. 186
Establishment and maintenance of the polar site are important for root hair tip growth. We previously reported that Arabidopsis (Arabidopsis thaliana) MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18) functions in controlling the direction of pollen tube growth and root hair elongation. Additionally, the Rop GTPase ROP2 was reported as a positive regulator of both root hair initiation and tip growth in Arabidopsis. Both loss-of-function of ROP2 or knock-down of MAP18 leads to a decrease in root hair length, whereas overexpression of either MAP18 or ROP2 causes multiple tips or a branching hair phenotype. However, it is unclear whether MAP18 and ROP2 coordinately regulate root hair growth. In the present study, we demonstrate that MAP18 and ROP2 interact genetically and functionally. MAP18 physically interacts with ROP2 in vitro and in vivo and preferentially binds to the inactive form of the ROP2 protein. MAP18 promotes ROP2 activity during root hair tip growth. Further investigation revealed that MAP18 competes with RhoGTPase GDP dissociation inhibitor 1 (AtRhoGDI1)/SUPERCENTIPEDE1 (SCN1) for binding to ROP2, in turn affecting localization of active ROP2 in the plasma membrane of the root hair tip. These results reveal a novel function of MAP18 in the regulation of ROP2 activation during root hair growth.
Significance
Higher plants are built from three major tissue types: epidermis, ground tissue, and vascular tissue. Each of these differentiates into several functionally distinct cell types. Although identity switches for the different cell types within the major three tissues have been identified, mechanisms that trigger the initiation of the three tissues themselves have remained obscure. Auxin response, in particular the auxin-dependent transcription factor MONOPTEROS (MP), plays a critical role in Arabidopsis embryonic root initiation. In our study, we identify a set of embryonic MP target genes and show that MP acts as a very first regulator of ground tissue initiation. Moreover, our data provide a framework for the simultaneous formation of multiple cell types by the same transcriptional regulator.
Calcium (Ca2+) signaling and dynamic reorganization of the cytoskeleton are essential processes for the coordination and control of plant cell shape and cell growth. Calmodulin (CaM) and closely related CaM-like polypeptides (CML) are principal sensors of Ca2+ signals. CaM/CMLs decode and relay information encrypted by the second messenger via differential interactions with a wide spectrum of targets to modulate their diverse biochemical activities. The plant-specific IQ67-DOMAIN (IQD) family emerged as the possibly largest class of CaM interacting proteins with undefined molecular functions and biological roles. Here, we show that the 33 members of the IQD family in Arabidopsis thaliana differentially localize, using GFP-tagged proteins, to multiple and distinct subcellular sites, including microtubule (MT) arrays, plasma membrane microdomains, and nuclear compartments. Intriguingly, the various IQD-specific localization patterns coincide with the subcellular patterns of IQD-dependent recruitment of CaM, suggesting that the diverse IQD members sequester Ca2+-CaM signaling modules to specific subcellular sites for precise regulation of Ca2+-dependent processes. Because MT localization is a hallmark of most IQD family members, we quantitatively analyzed GFP-labeled MT arrays in tobacco cells transiently expressing GFP-IQD fusions and observed IQD-specific MT patterns, which point to a role of IQDs in MT organization and dynamics. Indeed, stable overexpression of select IQD proteins in Arabidopsis altered cellular MT orientation, cell shape, and organ morphology. Because IQDs share biochemical properties with scaffold proteins, we propose that IQD families provide an assortment of platform proteins for integrating CaM-dependent Ca2+ signaling at multiple cellular sites to regulate cell function, shape, and growth.
The microtubule, F-actin and neurofilament networks play a critical role in neuronal cell morphogenesis, polarity and synaptic plasticity. Significantly, the assembly/disassembly and stability of these cytoskeletal networks is crucially modulated by protein phosphorylation and dephosphorylation events. Herein, we aim to more closely examine the role played by three major neuronal Ser/Thr protein phosphatases, PP2A, PP1 and calcineurin, in the homeostasis of the neuronal cytoskeleton. There is strong evidence that these enzymes interact with and dephosphorylate a variety of cytoskeletal proteins, resulting in major regulation of neuronal cytoskeletal dynamics. Conversely, we also discuss how multi-protein cytoskeletal scaffolds can also influence the regulation of these phosphatases, with important implications for neuronal signalling and homeostasis. Not surprisingly, deregulation of these cytoskeletal scaffolds and phosphatase dysfunction are associated with many neurological diseases.
The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells.
Cellulose is the most abundant biopolymer on Earth and is the major contributor to plant morphogenesis. Cellulose is synthesized by plasma membrane-localized cellulose synthase complexes (CSCs). Nascent cellulose microfibrils become entangled in the cell wall, and further catalysis therefore drives the CSC forward through the membrane: a process guided by cortical microtubules via the protein CSI1/POM2. Still, it is unclear how the microtubules can withstand the forces generated by the motile CSCs to effectively direct CSC movement. Here, we identified a family of microtubule-associated proteins, the cellulose synthase-microtubule uncouplings (CMUs), that located as static puncta along cortical microtubules. Functional disruption of the CMUs caused lateral microtubule displacement and compromised microtubule-based guidance of CSC movement. CSCs that traversed the microtubules interacted with the microtubules via CSI1/POM2, which prompted the lateral microtubule displacement. Hence, we have revealed how microtubules can withstand the propulsion of the CSCs during cellulose biosynthesis and thus sustain anisotropic plant cell growth.
The membranes of eukaryotic cells create hydrophobic barriers that control substance and information exchange between the inside and outside of cells and between cellular compartments. Besides their roles as membrane building blocks, some membrane lipids, such as phosphoinositides (PIs), also exert regulatory effects. Indeed, emerging evidence indicates that PIs play crucial roles in controlling polarity and growth in plants. Here, I highlight the key roles of PIs as important regulatory membrane lipids in plant development and function.
Calcium-dependent protein kinases (CDPKs) are at the forefront of decoding transient Ca2+ signals into physiological responses. They play a pivotal role in many aspects of plant life starting from pollen tube growth,
during plant development, and in stress response to senescence and cell death. At the cellular level, Ca2+ signals have a distinct, narrow distribution, thus requiring a conjoined localization of the decoders. Accordingly, most
CDPKs have a distinct subcellular distribution which enables them to ‘sense’ the local Ca2+ concentration and to interact specifically with their targets. Here we present a comprehensive overview of identified CDPK
targets and discuss them in the context of kinase–substrate specificity and subcellular distribution of the CDPKs. This is
particularly relevant for calcium-mediated phosphorylation where different CDPKs, as well as other kinases, were frequently
reported to be involved in the regulation of the same target. However, often these studies were not performed in an in situ context. Thus, considering the specific expression patterns, distinct subcellular distribution, and different Ca2+ affinities of CDPKs will narrow down the number of potential CDPKs for one given target. A number of aspects still remain
unresolved, giving rise to pending questions for future research.
The centrosome, known as the cell’s major microtubule organizing center, is a multifunctional organelle of ca. 1 μm in diameter without membrane boundaries; it relies on precise regulation to nucleate microtubules for specific functions throughout the cell cycle. The centrosome also serves as hub for signal transduction molecules and orchestrates signal transduction through its microtubule network. It holds key roles in cell cycle regulation and directly or indirectly affects cell cycle progression and cellular metabolism.
Centrosome dysfunctions have been implicated in numerous diseases and disorders including neurodegenerative diseases and cancer. Centrosome functions are also affected in aging cells in which aneuploidy is associated with loss of centrosome and microtubule integrity leading to chromosome mis-segregation, as seen in senescing somatic cells and in mammalian reproductive cells in which meiotic spindles become dysfunctional resulting in fertility problems and developmental disorders.
This chapter is centered on centrosome–microtubule interactions and their dysfunctions in disease and disorders with focus on (1) Centrosome–microtubule dynamics; (2) Centrosome dysfunctions in aging cells; (3) Centrosome dysfunctions in cancer cells. Several avenues are discussed to understand centrosome abnormalities and restore function in affected reproductive and somatic cells.
Calmodulin (CaM) is a well-studied calcium sensor that is ubiquitous in all eukaryotes and contributes to signaling during developmental processes and adaptation to environmental stimuli. Among eukaryotes, plants have a remarkable variety of CaM-like proteins (CMLs). The expansion of genomic data sets offers the opportunity to explore CaM and CML evolution among the green lineage from algae to land plants. Database analysis indicates that a striking diversity of CaM and CMLs evolved in angiosperms during terrestrial colonization and reveals the emergence of new CML classes throughout the green lineage that correlate with the acquisition of novel biological traits. Here, we speculate that expansion of the CML family was driven by selective pressures to process environmental signals efficiently as plants adapted to land environments.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Rho-type small GTP-binding plant proteins function as two-state molecular switches in cellular signalling. There is accumulating evidence that Rho-of-plants (ROP) signalling is positively controlled by plant receptor kinases, through the ROP guanine nucleotide exchange factor proteins. These signalling modules regulate cell polarity, cell shape, hormone responses, and pathogen defence, among other things. Other ROP-regulatory proteins might also be subjected to protein phosphorylation by cellular kinases (e.g., mitogen-activated protein kinases or calcium-dependent protein kinases), in order to integrate various cellular signalling pathways with ROP GTPase-dependent processes. In contrast to the role of kinases in upstream ROP regulation, much less is known about the potential link between ROP GTPases and downstream kinase signalling. In other eukaryotes, Rho-type G-protein-activated kinases are widespread and have a key role in many cellular processes. Recent data indicate the existence of structurally different ROP-activated kinases in plants, but their ROP-dependent biological functions still need to be validated. In addition to these direct interactions, ROPs may also indirectly control the activity of mitogen-activated protein kinases or calcium-dependent protein kinases. These kinases may therefore function as upstream as well as downstream kinases in ROP-mediated signalling pathways, such as the phosphatidylinositol monophosphate kinases involved in cell polarity establishment.
Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
The microinjection of calcium-saturated calmodulin into living fibroblasts causes the rapid disruption of microtubules and stress fibers in a sharply delimited region concentric with the injection site. This effect is specific to the calcium-bearing form of calmodulin; neither calcium-free calmodulin nor calcium ion at similar levels affects the cytoskeleton. If cells have previously been microinjected with calcium-free calmodulin, elevation of their intracellular calcium levels to 25 mM potentiates the disruption of microtubules throughout the cytoplasm. Approximately 400 mM free calcium is required to cause an equivalent disruption in uninjected cells. The level of calmodulin necessary to disrupt the full complement of cellular microtubules is found to be approximately in 2:1 molar ratio to tubulin dimer. These results indicate that calmodulin can be localized within the cytoplasm in a calcium-dependent manner and that it can act to regulate the calcium lability of microtubules at molar ratios that could be achieved locally within the cell. Our results are consistent with the hypothesis that calmodulin may be controlling microtubule polymerization equilibria in areas of high local concentration such as the mitotic spindle.
The kinesin-like calmodulin (CaM) binding protein (KCBP), a minus end–directed microtubule motor protein unique to plants, has been implicated in cell division. KCBP is negatively regulated by Ca²⁺ and CaM, and antibodies raised against the CaM binding region inhibit CaM binding to KCBP in vitro; therefore, these antibodies can be used to activate KCBP constitutively. Injection of these antibodies into Tradescantia virginiana stamen hair cells during late prophase induces breakdown of the nuclear envelope within 2 to 10 min and leads the cell into prometaphase. However, mitosis is arrested, and the cell does not progress into anaphase. Injection of antibodies later during cell division has no effect on anaphase transition but causes aberrant phragmoplast formation and delays the completion of cytokinesis by ∼15 min. These effects are achieved without any apparent degradation of the microtubule cytoskeleton. We propose that during nuclear envelope breakdown and anaphase, activated KCBP promotes the formation of a converging bipolar spindle by sliding and bundling microtubules. During metaphase and telophase, we suggest that its activity is downregulated.
Key message
PCaP2 plays a key role in maintaining the nucleus at a relatively fixed distance from the apex during root hair growth by modulating actin filaments.
Abstract
During root hair growth, the nucleus localizes at a relatively fixed distance from the apex. In Arabidopsis thaliana, the position of the nucleus is mainly dependent on the configuration of microfilaments (filamentous actin). However, the mechanisms underlying the regulation of actin dynamics and organization for nuclear positioning are largely unknown. In the present study, we demonstrated that plasma membrane-associated Ca2+ binding protein 2 (PCaP2) influences the position of the nucleus during root hair growth. Abnormal expression of PCaP2 in pcap2 and PCaP2 over-expression plants led to the disorganization of actin filaments, rather than microtubules, in the apex and sub-apical regions of root hairs, which resulted in aberrant root hair growth patterns and misplaced nuclei. Analyses using a PCaP2 mutant protein revealed that actin-severing activity is essential for the function of PCaP2 in root hairs. We demonstrated that PCaP2 plays a key role in maintaining nuclear position in growing root hairs by modulating actin filaments.
Stimuli-induced fluctuations in intracellular free calcium (Ca2+) serve as secondary messenger signals that regulate diverse biochemical processes in eukaryotic cells, such as developmental transitions and responses to biotic and abiotic stresses. Stimuli-specific Ca2+ signals are manifested as spatially and temporally defined differential Ca2+ signatures that are sensed, decoded, and transduced to elicit distal responses via an array of Ca2+ binding proteins (CBPs) that function as intracellular Ca2+ sensors. Calmodulin (CaM), the most important eukaryotic CBP, senses and responds to fluctuations in intracellular Ca2+ levels by binding to this ubiquitous second messenger, and transducing given Ca2+ signatures that differentially activate distal effector (target) proteins regulating a broad range of biochemical responses. Ca2+/CaM targets include an increasing number of proteins whose functions continue to be elucidated. Hundreds of reports have highlighted the importance of CaM, and other CBPs, in the transduction of Ca2+-mediated signals involved in transcriptional regulation, protein phosphorylation/dephosphorylation, and metabolic shifts. Other Ca2+-binding proteins are known to play significant functional roles in plant cells as well. This review is primarily focused on the role of CaM in some key plant processes, and discusses recent advances in understanding the pivotal role of CaM in an ever-increasing number of plant cell functions and biochemical responses. We also discuss recent work highlighting the emerging importance of CaM in nuclear and organellar signaling.
Background
Plant cell walls are complex matrices of carbohydrates and proteins that control cell morphology and provide protection and rigidity for the plant body. The construction and maintenance of this intricate system involves the delivery and recycling of its components through a precise balance of endomembrane trafficking, which is controlled by a plethora of cell signalling factors. Phosphoinositides (PIs) are one class of signalling molecules with diverse roles in vesicle trafficking and cytoskeleton structure across different kingdoms. Therefore, PIs may also play an important role in the assembly of plant cell walls.ScopeThe eukaryotic PI pathway is an intricate network of different lipids, which appear to be divided in different pools that can partake in vesicle trafficking or signalling. Most of our current understanding of how PIs function in cell metabolism comes from yeast and mammalian systems; however, in recent years significant progress has been made towards a better understanding of the plant PI system. This review examines the current state of knowledge of how PIs regulate vesicle trafficking and their potential influence on plant cell-wall architecture. It considers first how PIs are formed in plants and then examines their role in the control of vesicle trafficking. Interactions between PIs and the actin cytoskeleton and small GTPases are also discussed. Future challenges for research are suggested.
The cell growth-promoting peptide phytosulfokine (PSK) is perceived by LRR receptor kinases. To elucidate PSK receptor function we analyzed PSKR1 kinase activity and binding to Ca(2+) sensors and evaluated the contribution of these activities to growth control in planta. Ectopically expressed PSKR1 was capable of auto- and transphosphorylation. Replacement of a conserved lysine within the ATP binding region by a glutamate resulted in the inhibition of auto- and transphosphorylation kinase activities. Expression of the kinase-inactive PSKR1(K762E) receptor in the pskr null background did not restore root or shoot growth. Instead, the mutant phenotype was enhanced suggesting that the inactive receptor protein exerts growth inhibitory activity. Bioinformatic analysis predicted a putative calmodulin (CaM) binding site within PSKR1 kinase subdomain VIa. Bimolecular fluorescence complementation analysis demonstrated that PSKR1 binds to all isoforms of CaM, more weakly to the CaM-like protein CML8 but apparently not to CML9. Mutation of a conserved tryptophan (W831S) within the predicted CaM binding site strongly reduced CaM binding. Expression of PSKR1(W831S) in the pskr null background resulted in growth inhibition that was similar to that of the kinase-inactive receptor. We conclude that PSK signaling requires Ca(2+) /CaM binding and kinase activity of PSKR1 in planta. We further propose that the inactivated kinase interferes with other growth-promoting signaling pathway(s). This article is protected by copyright. All rights reserved.