The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H+-pyrophosphatase (V-PPase) and the vacuolar H+-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH.
Article Figures and data Abstract eLife digest Introduction Results Discussion Material and methods References Decision letter Author response Article and author information Metrics Abstract A major feature of embryogenesis is the specification of stem cell systems, but in contrast to the situation in most animals, plant stem cells remain quiescent until the postembryonic phase of development. Here, we dissect how light and metabolic signals are integrated to overcome stem cell dormancy at the shoot apical meristem. We show on the one hand that light is able to activate expression of the stem cell inducer WUSCHEL independently of photosynthesis and that this likely involves inter-regional cytokinin signaling. Metabolic signals, on the other hand, are transduced to the meristem through activation of the TARGET OF RAPAMYCIN (TOR) kinase. Surprisingly, TOR is also required for light signal dependent stem cell activation. Thus, the TOR kinase acts as a central integrator of light and metabolic signals and a key regulator of stem cell activation at the shoot apex. https://doi.org/10.7554/eLife.17023.001 eLife digest Plants are able to grow and develop throughout their lives thanks to groups of stem cells at the tips of their shoots and roots, which can constantly divide to produce new cells. Energy captured from sunlight during a process called photosynthesis is the main source of energy for most plants. Therefore, the amount and quality of light in the environment has a big influence on how plants grow and develop. An enzyme called TOR kinase can sense energy levels in animal cells and regulate many processes including growth and cell division. Plants also have a TOR kinase, but it is less clear if it plays the same role in plants, and whether it can respond to light. Plant stem cells only start to divide after the seed germinates. In shoots, a protein called WUSCHEL is required to maintain stem cells in an active state. Here, Pfeiffer et al. studied how shoot stem cells are activated in response to environmental signals in a plant known as Arabidopsis. The experiments show that light is able to activate the production of WUSCHEL independently of photosynthesis via a signal pathway that depends on TOR kinase. The stem cells do not directly sense light; instead other cells detect the light and relay the information to the stem cells with the help of a hormone called cytokinin. Further experiments show that information about energy levels in cells is relayed via another signal pathway that also involves the TOR kinase. Therefore, Pfeiffer et al.’s findings suggest that the activation of TOR by light allows plant cells to anticipate how much energy will be available and efficiently tune their growth and development to cope with the environmental conditions. Future challenges are to understand how TOR kinase is regulated by light signals and how this enzyme is able to act on WUSCHEL to trigger stem cell division. https://doi.org/10.7554/eLife.17023.002 Introduction Light is the sole energy source of plants and therefore one of the most important environmental factors influencing their development and physiology. Consequently, several of the core developmental decisions during the lifecycle of a plant from germination to seedling development and flowering are strongly influenced by light conditions. After germination, higher plants undergo two distinct developmental programs depending on the availability of light, termed skotomorphogenesis and photomorphogenesis. Skotomorphogenesis, the dark adaptation program, is characterized by an etiolated phenotype, including an elongated hypocotyl, closed cotyledons, the formation of an apical hook and etioplast development. Importantly, stem cells at the shoot and root tip remain dormant and thus growth in etiolated seedlings is mainly dependent on cell elongation rather than cell division. In contrast, photomorphogenesis, the developmental program triggered in light, leads to seedlings with short hypocotyls, unfolded cotyledons and development of chloroplasts. In the light, shoot and root meristems are activated, leading to root growth and development of the first leaves by cell division and expansion (reviewed in Nemhauser and Chory 2002). Based on evolutionary evidence, photomorphogenesis is the default pathway, since gymnosperms for example do not follow a strict skotomorphogenic development in darkness (Wei, 1994). With the advance of land plants and resulting new environmental challenges, such as growth in dense canopy and germination in soil, the evolution of the dark-adapted skotomorphogenesis program ensued an advantage: It allowed plants to allocate the limited energy sources supplied by the seed to maximally grow by elongation, in order to reach favorable light conditions that will provide energy for further growth and development. To faithfully execute these opposing developmental programs, plants have evolved complex mechanisms to perceive light quality and quantity through a whole range of photoreceptors that are mainly absorbing in the blue, red and far-red range of the spectrum. Activation of the blue absorbing CRYPTOCHROMES (crys) and/or the red and far-red absorbing PHYTOCHROMES (phys) overrides the skotomorphogenic program and plants undergo photomorphogenesis within minutes after perception of a light stimulus (reviewed in Chory, 2010). On the molecular level, activated light receptors inhibit the function of the core repressor of photomorphogenesis, CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1), an E3 ubiquitin ligase that targets positive regulators of photomorphogenesis for degradation in darkness (Yi and Deng, 2005). At the same time, a group of potent transcription factors, the PHYTOCHROME INTERACTING FACTORS (PIFs), which promote skotomorphogenesis in darkness, are degraded upon light perception through the PHYTOCHROMES (Leivar and Quail, 2010). The activities of these pathways converge on the differential regulation of thousands of genes resulting in a massive reprogramming of the transcriptome in response to light (Ma et al., 2002; Tepperman et al., 2004; Peschke and Kretsch, 2011; Pfeiffer et al., 2014). Light not only activates photoreceptors, it also fuels photosynthesis and therefore leads to the production of a number of energy rich metabolites including sugars. Plants are able to monitor their metabolic state with several signaling systems (Lastdrager et al., 2014) and recent studies have focused on the evolutionary conserved TARGET OF RAPAMYCIN (TOR) kinase complex (Dobrenel et al., 2016). In other eukaryotes, TOR functions as a central integrator of nutrient, energy, and stress signaling networks and consistently, TOR regulates cell growth and proliferation, ribosome biogenesis, protein synthesis, cell wall integrity and autophagy (Díaz-Troya et al., 2008; Henriques et al., 2014; Lastdragere et al., 2014; Xiong and Sheen, 2014). While other eukaryotes possess two TOR complexes, so far only a single complex has been identified in plants. It is comprised of TOR, FKBP12, LST8 and RAPTOR (Mahfouz et al., 2006; Moreau et al., 2012) and thus resembles the mammalian TOR complex 1 (mTORC1). AtTOR is expressed in the embryo and endosperm and in meristematic regions of the adult plant (Menand et al., 2002). While tor null mutants show premature arrest of embryo development (Menand et al., 2002), knock down of TOR leads to growth reduction and affects the carbohydrate and amino acid metabolism (Caldana et al., 2013). In contrast, the presence of sugars in general promotes TOR kinase activity (Ren et al., 2012; Dobrenel et al., 2013; Xiong et al., 2013). So far, the only known direct downstream targets of AtTOR kinase are S6 kinase 1 (S6K1) (Schepetilnikov et al., 2011, 2013; Xiong et al., 2013), TAP46 (Ahn et al., 2011, 2014) and E2 promoter binding factor a (E2Fa) (Xiong et al., 2013). S6K1 plays an important role in reinitiating translation (Schepetilnikov et al., 2011) as well as in the regulation of the cell cycle (Henriques et al., 2010; Shin et al., 2012). Similarly, E2Fa is associated with cell cycle control through the expression of S-phase genes (Polyn et al., 2015). Though little is known about how TOR is activated on a molecular level in plants, reports from the past decade suggest that TOR functions as a central regulator of protein synthesis, cell proliferation and metabolism in response to metabolic signals. Several photomorphogenic responses, like the inhibition of hypocotyl elongation, unfolding of the hypocotyl hook and cotyledons, as well as chloroplast development can be triggered by a light signal alone, as displayed in dark-grown cop1 and pif1/pif3/pif4/pif5 quadruple mutants (pifq) (Deng and Quail, 1991; Leivar et al., 2009). However, root growth is not induced in cop1 mutants unless sucrose is supplied with the growth medium. Photosynthetic assimilates dominantly promote growth in the root where they can synergistically interact with photoreceptor-triggered light signaling (Kircher and Schopfer, 2012). Recently, Xiong et al. showed that this photosynthesis-driven growth and proliferation in the root is mediated by the TOR kinase (Xiong et al., 2013). Here, we analyzed the role of light and nutrients for post-germination stem cell activation in the shoot apical meristem (SAM) of young seedlings. Stem cell control in the SAM of Arabidopsis thaliana is based on the activity of the homeodomain transcription factor WUSCHEL (WUS), which is expressed in the organizing centre and necessary and sufficient to non-cell- autonomously induce stem cell fate by protein movement. Stem cells in turn express CLAVATA3 (CLV3), a short secreted peptide, that acts via the CLV / CORYNE receptor system to limit the expression of WUS in the organizing center (Schoof et al., 2000; Daum et al., 2014). The use of a reporter system based on the regulatory regions of WUS and CLV3 allowed us to quantitatively trace behavior of stem cells (pCLV3:mCHERRY-NLS) as well as cells of the underlying organizing center (pWUS:3xVENUS-NLS). With the help of these tools, we were able to genetically dissect the individual contribution of light signaling and photosynthesis-driven nutrient sensing on the stem cell system of the SAM. We show that both pathways ultimately converge at the level of TOR kinase activation, revealing a role for TOR as a central regulator of stem cell activation in response to environmental cues. Results Organogenic development is dependent on light and energy metabolites The SAM of Arabidopsis seedlings remains dormant during skotomorphogenesis and therefore, plants are unable to advance to the organogenic stage in the absence of light. However, since light acts as signal and energy source alike, we first asked which of the two roles is dominant for SAM development. While supplementation of sugar to wild-type seedlings grown in the dark is known to be inefficient to trigger development (Figure 1A), activation of the light pathway alone, either physiologically by low level illumination, or genetically by introduction of the cop1 mutation, was shown to induce photomorphogenic development of the hypocotyl and cotyledons in darkness (Deng and Quail, 1991). Despite this stark developmental transition, SAMs of cop1 mutants were unable to produce organs when grown in the dark. However, the SAM was activated and organogenesis initiated in 100% of the dark-grown cop1 mutants when supplemented with sucrose as external energy source (Figure 1B, see also McNellis et al., 1994; Nakagawa and Komeda, 2004). Conversely, the SAM of light-grown wild-type seedlings remained dormant when photosynthesis was compromised by the carotenoid biosynthesis inhibitor norflurazon. In line with our observation of cop1 mutants, supplementing the growth medium of these plants with sucrose rescued the dormant phenotype in approximately every third seedling (Figure 1C). Thus, neither the availability of energy metabolites, nor light perception alone was sufficient for SAM activation. In contrast, light and energy, likely in the form of photosynthetic products, seemed to be sensed independently, and both factors need to act cooperatively to trigger SAM development. Figure 1 with 1 supplement see all Download asset Open asset SAM development depends on light and sugar. (A–C) Five week old plants grown on media with (+) or without (-) sucrose. (A) Wild-type plants grown in darkness, (B) cop1 mutant plants grown in darkness and (C) wild-type seedlings grown in light in the presence of 0.5 µM photosynthesis inhibitor norflurazon . (D–I) Maximum projections of SAMs of four day old seedlings; scale bar 20 µm. (D–F) pCLV3:mCHERRY-NLS (red) and pWUS:3xVENUS-NLS (green). (G–I) pCLV3:mCHERRY-NLS (red) and pWUS:WUS-linker-GFP (green). Quantification of pWUS:3xVENUS-NLS (J) and pCLV3:mCHERRY-NLS (K) expression by fluorescence intensity under different growth conditions (gray = darkness, red = red light (30 µmol*m−2*s−1), solid box = w/o sucrose, hatched box = 1% sucrose, dag = days after germination). https://doi.org/10.7554/eLife.17023.003 WUS expression is independently regulated by light and sucrose Our phenotypic analysis suggested that light and metabolic signals synergize to activate SAM development, and thus we asked which of the known components underlying stem cell homeostasis might be the relevant cellular and molecular targets. By using transcriptional reporters for stem cells (pCLV3:mCherry-NLS) and niche cells (pWUS:3xVENUS-NLS) we found that stem cell identity was actively maintained independently of growth conditions and was even observed in the dormant state mediated by germination in the dark (Figure 1D). In contrast, expression of the reporter for the stem cell inducing WUS transcription factor was critically dependent on environmental signals and preceded meristem activity and the initiation of organogenic development (Figure 1E). To test if our WUS reporter faithfully recapitulated the behavior of the endogenous gene, we used in situ hybridization and were able to confirm strong light dependent induction of WUS mRNA (Figure 1—figure supplement 1C and D). Since WUS protein exhibits complex movement and a short lifetime (Daum et al., 2014), we furthermore analyzed the behavior of WUS-GFP protein in vivo by recording the GFP signal in our rescue line (pWUS:WUS-linker-GFP in wus mutant background [Daum et al., 2014]). Again, we observed a strong light- and sucrose-dependency of the WUS-GFP signal in line with the observed activation of the WUS promoter and accumulation of the endogenous WUS mRNA under these conditions (Figure 1G–I) confirming that the simple pWUS:3xVenus-NLS reporter represents a faithful and quantitative readout for WUS activity. Taken together, these findings on the one hand suggested that CLV3 expression is at least partially independent of WUS and on the other hand that the environmentally dependent transcriptional activation of WUS is the trigger to overcome stem cell dormancy. Using seedlings carrying both reporters grown under wave-length specific LED illumination and image quantification we found that the WUS reporter (pWUS:3xVENUS-NLS) was below detection level in dark-grown seedlings. In contrast, GFP signals were readily detectable in light-grown plants from three days after germination onwards with the signal steadily increasing over time (Figure 1J). Interestingly, WUS expression was also induced in the absence of light, when plants were grown on sucrose-supplemented medium (Figure 1F,J) and when sucrose was supplied to light-grown seedlings, the effect of light and sucrose on WUS expression was additive (Figure 1J). Light and sucrose also had a similar effect on the regulation of CLV3 expression (Figure 1D–I, K), however, since the CLV3 reporter was already detectable in dark-grown seedlings, the induction of expression by light and sucrose was less pronounced and mainly due to an enlargement of the CLV3 domain rather than an increase of signal intensity in individual cells (Figure 1—figure supplement 1A and B). In sum, development of the SAM required both, light signal transduction and the availability of photosynthetic products, whereas WUS expression was induced also by each signal individually. Thus, tracing WUS expression in the SAM of young seedlings represented a sensitive model to decipher the contribution of upstream signals to stem cell activation in a developmentally and physiologically relevant setting. Mechanisms of light dependent stem cell activation Since the expression of the transcriptional WUS reporter showed an early and dynamic response to environmental stimuli that mimicked both endogenous WUS mRNA, as well as WUS-GFP protein, we used the intensity of the reporter signal in four day-old seedlings as an easily quantifiable proxy for stem cell activation. First, we wanted to elucidate the molecular players involved in stem cell activation by light. To this end, we irradiated seedlings with monochromatic light of low intensities (30 µmol*m−2*s−1) to analyze the effect of light signaling with minimal influence of photosynthesis-derived metabolites. Even at low intensities, blue, as well as red light were sufficient to robustly induce WUS expression (Figure 2A). In line with the well-documented biochemistry of the photoreceptors, red-light-induced WUS activation was specifically reduced in the phyB mutant background, while blue-light-induced reporter activity was impaired in the cry1/cry2 double mutant background (Figure 2A). We thus concluded that light perceived through phyB as well as the crys influences the developmental fate of the SAM. Figure 2 with 1 supplement see all Download asset Open asset Light induced WUS expression depends on photoreceptors and is repressed by COP1. Quantification of pWUS:3xVENUS-NLS expression by fluorescence intensity was measured in four day old wild-type (WT) or mutant seedlings (WT) or mutant background under different growth conditions (gray = darkness, red = red light (30 µmol*m−2*s−1), blue = blue light (30 µmol*m−2*s−1), solid box = w/o sucrose, hatched box = 1% sucrose). (A) 0.5 mM lincomycin and 5 µM norflurazon, respectively were applied to the growth media of wild-type seedlings. (B) Impact of cop1−4 mutation on WUS expression. https://doi.org/10.7554/eLife.17023.005 We also tested WUS expression under far-red light, which is sensed by phyA and found that reporter activity was only weakly induced by far-red light. Interestingly, phyA mutants showed similar WUS promoter activity under far-red light and in darkness (Figure 2—figure supplement 1A). However, when we supplemented the growth media with 1% sucrose we observed a clear induction of WUS expression in response to far-red light, which was dependent on a functional copy of PHYA. Still, phyA mutants displayed a basal level of WUS promoter activity already in darkness even when grown on plates containing sucrose, suggesting a complex and so far unknown regulatory role for phyA under these conditions. The fact that plants grown in far-red light are photosynthetically inactive and required an exogenous energy source for WUS activation, while plants under blue and red light did not, raised the question whether minimal levels of photosynthetically derived sugars might contribute to WUS expression in blue and red light, despite the low fluence. Therefore, we tested whether the availability of photosynthetic products is a prerequisite for light-induced WUS expression by chemical interference. However, the inhibition of photosynthesis by either norflurazon or lincomycin, did not affect WUS promoter activity in red or in blue light (Figure 2A). In the presence of lincomycin, WUS expression was even slightly increased under both light conditions. To avoid potential side effects of the pharmacological treatments we also tested the effect of CO2 withdrawal on seedling development and WUS expression. Preventing photosynthetic assimilation in a CO2-deficient atmosphere inhibited development of seedlings even when grown in light. This phenotype could be rescued in one third of the plants by adding 1% sucrose to the media, similar to what we observed using norflurazon treatment (compare Figure 1C and Figure 2—figure supplement 1C). Importantly, WUS induction by red light was unaffected by CO2 reduction in the atmosphere (Figure 2—figure supplement 1B). Thus, photosynthetically derived metabolites produced in a low light environment were not required for activation of stem cells, confirming that light signaling alone was sufficient for WUS expression. We next asked how the light signal perceived by PHYTOCHROMES and CRYPTOCHROMES is relayed to the nucleus by testing the contribution of known downstream signaling components, such as COP1 and HY5. The E3-ubiquitin ligase COP1, which targets HY5 but also other factors for degradation in darkness, showed robust inhibitory effects on WUS expression. Cop1-4 mutants displayed photomorphogenic development in darkness, which was accompanied by WUS expression (Figure 2B). Furthermore, the repressive function of COP1 was prominent under all conditions tested and cop1-4 seedlings displayed strongly elevated WUS promoter activity compared to wild-type when grown in dark with or without sugar, and also under low light conditions (Figure 2B). To confirm that these effects were not caused by second site mutations present in the cop1-4 background or specific to the allele tested, we used qRT-PCR to assay WUS expression in seedlings carrying other cop1 loss-of-function alleles. However, since this approach lacked the spatial resolution provided by microscopic quantification of the WUS reporter, it proofed to be much less sensitive. Still, we were able to detect accumulation of endogenous WUS mRNA in response to light in 7d old wild-type seedlings, as well as in cop1-4 mutants in the dark (Figure 2—figure supplement 1D). Importantly, all three cop1 mutant alleles tested showed robust elevation of WUS mRNA levels when grown in the dark (Figure 2—figure supplement 1E), demonstrating that loss of COP1 function leads to activation of WUS. One of the main functions of COP1 is to target the transcription factor HY5, a positive master regulator of photomorphogenesis, for degradation. Thus, we analyzed the role of HY5 working under the hypothesis that in contrast to cop1 mutants, which had shown elevated WUS reporter expression, hy5 mutants should suffer from a much reduced meristem activity due to the absence of an important photomorphogenesis stimulating activity. However, hy5 mutants were unaffected in activation of WUS expression (Figure 2—figure supplement 1F), suggesting that SAM stem cell activation is dependent on another COP1-targeted transcriptional transducer, such as HY5 HOMOLOG (HYH), or a so far unknown regulator. Since the SAM is shielded from the environment especially in etiolated seedlings, where it is buried between the closed cotyledons and protected by the apical hook of the hypocotyl, it seemed questionable that the meristem itself is the site of light perception. We therefore tested the competence of different tissues to perceive light signals and translate them into a stem cell activating output. To this end, we expressed a constitutive active form of phyB (Su and Lagarias, 2007) under different tissue specific promoters (Figure 2—figure supplement 1C,D). Expression of phyB Y276H under an ubiquitous promoter (pUBI10) caused strong cop1-like phenotypes and a substantial activation of the WUS promoter in the SAM showing that transgenic activation of light signaling is sufficient to trigger stem cell activation in darkness (Figure 2—figure supplement 1G,H,J). In line with our hypothesis that light is likely perceived by cells outside the SAM, vascular specific expression of phyB Y276H by the pSUC2, or mesophyll specific expression by pCAB3 promoters (Ranjan et al., 2011) initiated constitutive photomorphogenic phenotypes and WUS expression in dark-grown seedlings. Similar results were also obtained for the epidermal pML1 promoter, in lines showing high expression levels of phyB Y276H (Figure 2—figure supplement 1G–J). These results suggested that the stimulus downstream of light perception can be transmitted between tissues by a mobile signal and raised the question whether the SAM itself even has the ability to respond to light. To explore this, we expressed phyB Y276H specifically in the SAM under the promoter of At3g59270 (Yadav et al., 2009), but in contrast to expression outside of the SAM, we observed fully etiolated seedlings without detectable WUS expression when these plants were grown in the dark. Even when we drove PHYB Y276H expression in cells surrounding the organizing center by the promoter of At1g26680 (Yadav et al., 2009), we only observed a minor reduction in hypocotyl elongation in darkness compared to wild-type and marginal WUS expression (Figure 2—figure supplement 1G,H,J). We therefore concluded that light is perceived by cells outside of the SAM, likely in the cotyledons or the hypocotyl and that this stimulus is transmitted to the SAM by a so far unidentified mobile signal. Amazingly, the SAM does not possess the competence to perceive and/or translate the light stimulus into stem cell activation, but rather is limited to responding to the signals that are transmitted from distant plant organs. Mechanisms of hormonal stem cell activation Since we had shown that light is perceived at a distance from the SAM, which also for energy rich metabolites is not a source, but a sink tissue, we next asked how the information for both environmental inputs is relayed to the stem cell system. Obvious candidates for inter-regional signaling components are plant hormones and there are a number of studies demonstrating their importance in regulating the shoot stem cell niche, especially for cytokinin (CK) and auxin (reviewed in Murray et al., 2012). A previous study had analyzed the environmental influence on organ initiation at the SAM using transfer of light grown tomato plants to darkness as a model and found that light is required for CK signaling and polarized membrane localization of the auxin export carrier PIN1 (Yoshida et al.,2011). However, these studies could not distinguish whether light was perceived as informational cue or energy source. We therefore analyzed CK signaling activity using the pTCSn:GUS cytokinin output sensor (Zürcher et al., 2013) as well as auxin flux directionality using polarization of pPIN1:PIN1-GFP (Benková et al., 2003) as a proxy (Figure 3A–K). In line with the findings of Yoshida et al., CK signaling was strongly activated by light when compared to etiolated seedlings (compare Figure 3H,G). Furthermore, we also found that PIN1 polarly localized to the plasma membrane in a light dependent manner (Figure 3A and B). Interestingly, sucrose treatment of etiolated seedlings did not affect the localization of PIN1 (Figure 3C) but lead to a mild activation of the TCS reporter also in the absence of light (Figure 3I), suggesting that there is specificity in the hormonal response. Figure 3 with 1 supplement see all Download asset Open asset Hormonal control of the SAM. (A–F) Confocal images of four day old seedlings expressing pPIN1:PIN1-GFP in WT (A–D) or cop1-4 (E,F) background under diverse growth conditions. The lower row shows a magnification of the meristem shown in the picture above. (G–K) GUS staining of four day old plants expressing pTCSn:GUS (light = white light (150 µmol*m−2*s−1), + suc = 1% sucrose, nor = 5 µM norflurazon, scale bar = 20 µm). (L) Wild-type seedlings after 20 days on plates containing CK (75 µM benzyladenine) supplemented with (+) or without (-) sucrose. https://doi.org/10.7554/eLife.17023.007 To test the light signaling response independently from impeding effects of photosynthesis, we treated plants grown in light with the photosynthesis inhibitor norflurazon. While PIN1 localization was still light responsive, no activity of the TCS reporter was detectable under these conditions (Figure 3D,J). We also observed a reduction of TCS signal when plants were grown in a CO2-deficient environment (Figure 3—figure supplement 1C). Since in both cases TCS reporter activity could be restored by sucrose supplementation (Figure 3K and Figure 3—figure supplement 1D), we concluded that CK signaling output is dependent on the availability of energy metabolites. However, light signaling and photosynthesis together had a much stronger effect on CK output than nutrient availability alone, suggesting that both signals synergize to stimulate CK signaling at the SAM. In contrast, PIN1 localization to the plasma membrane was fully dependent on light perception and could not even be restored by the cop1 mutation (Figure 3E,F). If CK signaling indeed integrates energy status and light perception, it may be sufficient to activate SAM development. In line with this idea, the importance of CK in light dependent SAM activation had already been demonstrated (Chory et al., 1994; Skylar et al., 2010) and Yoshida et al. had shown that the application of CK to tomato apices can induce organogenesis in the dark (Yoshida et al.,2011). Consistently, etiolated Arabidopsis seedlings treated with CK produced leaf like structures even in darkness (Figure 3L and Chory et al. 1994). However, this developmental transition was strictly dependent on the presence of an external energy source, similar to the behavior of cop1 mutants and in the absence of sucrose, CK treated seedlings failed to develop leaves in the dark (Figure 3L). Thus, our experiments were consistent with CK being an important
A major feature of embryogenesis is the specification of stem cell systems, but in contrast to the situation in most animals, plant stem cells remain quiescent until the postembryonic phase of development. Here, we dissect how light and metabolic signals are integrated to overcome stem cell dormancy at the shoot apical meristem. We show on the one hand that light is able to activate expression of the stem cell inducer WUSCHEL independently of photosynthesis and that this likely involves inter-regional cytokinin signaling. Metabolic signals, on the other hand, are transduced to the meristem through activation of the TARGET OF RAPAMYCIN (TOR) kinase. Surprisingly, TOR is also required for light signal dependent stem cell activation. Thus, the TOR kinase acts as a central integrator of light and metabolic signals and a key regulator of stem cell activation at the shoot apex.
Significance Cell–cell communication is a prerequisite of multicellular development and noncell autonomous stem cell induction has been conserved during evolution. Cytoplasmic bridges, called plasmodesmata, which facilitate the exchange of molecules between neighboring cells, are a striking innovation for cell–cell signaling in plants. Here, we show that plasmodesmata function is required for the activity of shoot apical stem cells in Arabidopsis and provide evidence that the stem cell inducing transcription factor WUSCHEL moves from the niche into the stem cells via this route. WUSCHEL movement is functionally relevant and mediated by multiple protein domains. Because parts of the protein that restrict movement are required for homodimerization, the formation of WUSCHEL dimers might contribute to the regulation of stem cell activity in Arabidopsis .
ABSTRACT To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.
Predators influence the behaviour of prey and by doing so they potentially reduce pathogen transmission by a vector. Arthropod predators have been shown to reduce the consumption of plant biomass by pest herbivores, but their cascading non-consumptive effect on vector insects' feeding behaviour and subsequent pathogen transmission has not been investigated experimentally before. Here we experimentally examined predator-mediated pathogen transmission mechanisms using the plant pathogen Wheat Dwarf Virus that is transmitted by the leafhopper, Psammotettix alienus. We applied in situ hybridization to localize which leaf tissues were infected with transmitted virus DNA in barley host plants, proving that virus occurrence is restricted to phloem tissues. In the presence of the spider predator, Tibellus oblongus, we recorded the within leaf feeding behaviour of the herbivore using electrical penetration graph. The leafhopper altered its feeding behaviour in response to predation risk. Phloem ingestion, the feeding phase when virus acquisition occurs, was delayed and was less frequent. The phase when pathogen inoculation takes place, via the secretion of virus infected vector saliva, was shorter when predator was present. Our study thus provides experimental evidence that predators can potentially limit the spread of plant pathogens solely through influencing the feeding behaviour of vector organisms.
Plant development is continually fine-tuned based on environmental factors. How environmental perturbations are integrated into the developmental programs and how poststress adaptation is regulated remains an important topic to dissect. Vegetative to reproductive phase change is a very important developmental transition that is complexly regulated based on endogenous and exogenous cues. Proper timing of flowering is vital for reproductive success. It has been shown previously that AGAMOUS LIKE 16 (AGL16), a MADS-box transcription factor negatively regulates flowering time transition through FLOWERING LOCUS T (FT), a central downstream floral integrator. AGL16 itself is negatively regulated by the microRNA miR824. Here we present a comprehensive molecular analysis of miR824/AGL16 module changes in response to mild and recurring heat stress. We show that miR824 accumulates gradually in response to heat due to the combination of transient transcriptional induction and posttranscriptional stability. miR824 induction requires heat shock cis-elements and activity of the HSFA1 family and HSFA2 transcription factors. Parallel to miR824 induction, its target AGL16 is decreased, implying direct causality. AGL16 posttranscriptional repression during heat stress, however, is more complex, comprising of a miRNA-independent, and a miR824-dependent pathway. We also show that AGL16 expression is leaf vein-specific and overlaps with miR824 (and FT) expression. AGL16 downregulation in response to heat leads to a mild derepression of FT. Finally, we present evidence showing that heat stress regulation of miR824/AGL16 is conserved within Brassicaceae. In conclusion, due to the enhanced post-transcriptional stability of miR824, stable repression of AGL16 is achieved following heat stress. This may serve to fine-tune FT levels and alter flowering time transition. Stress-induced miR824, therefore, can act as a "posttranscriptional memory factor" to extend the acute impact of environmental fluctuations in the poststress period.
Summary In some plant–virus interactions plants show a sign of healing from virus infection, a phenomenon called symptom recovery. It is assumed that the meristem exclusion of the virus is essential to this process. The discovery of RNA silencing provided a possible mechanism to explain meristem exclusion and recovery. Here we show evidence that silencing is not the reason for meristem exclusion in Nicotiana benthamiana plants infected with Cymbidium ringspot virus (CymRSV). Transcriptome analysis followed by in situ hybridization shed light on the changes in gene expression in the shoot apical meristem (SAM) on virus infection. We observed the down‐regulation of meristem‐specific genes, including WUSCHEL ( WUS ). However, WUS was not down‐regulated in the SAM of plants infected with meristem‐invading viruses such as turnip vein‐clearing virus (TVCV) and cucumber mosaic virus (CMV). Moreover, there is no connection between loss of meristem function and fast shoot necrosis since TVCV necrotized the shoot while CMV did not. Our findings suggest that the observed transcriptional changes on virus infection in the shoot are key factors in tip necrosis and symptom recovery. We observed a lack of GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE ( GAPDH ) expression in tissues around the meristem, which likely stops virus replication and spread into the meristem.
