Role of calcium in regulating key steps in phytochrome-induced signaling pathways

Abstract

A major focus in the field of signal transduction pathways in plants has been the role of calcium ions in mediating diverse sensory responses. Among these responses, those initiated by the red-light activated photoreceptor, phytochrome have received increasing attention in recent years. Although not all phytochrome responses are mediated by calcium, many of them are, and a number of recent publications have clarified just how calcium helps to transduce some of the transcriptomic changes induced by phytochrome. Many of these publications reference Dr. Sopory’s laboratory as an important contributor to the initial data documenting that an early step in the signaling pathways induced by phytochrome was an increased uptake of calcium into cells. This review summarizes the strong evidence that calcium-dependent steps play a major role in transducing phytochrome-initiated responses, and it updates the latest reports on specific steps in some phytochrome responses that are dependent on the mediation of calcium-binding protein kinases and calmodulin.

Introduction

The key role of calcium ions in mediating diverse sensory responses in plants has long been a favored topic for scientists studying signal transduction pathways in plants (Hepler 2005). Among these responses, those initiated by red light, which activates the photoreceptor phytochrome, have received increasing attention in recent years. Several reports published within the last 5 years have provided valuable new information documenting the important role of calcium plays in regulating some of the gene expression changes induced by phytochrome. These publications appropriately reference early reports, including prominently that of Das and Sopory (1985), that provided key initial data revealing that an early step in the signaling pathways induced by phytochrome was an increased uptake of calcium into cells. As pointed out by Neuhaus et al. (1993), not all phytochrome responses are mediated by calcium, but some of them are, and new discoveries have clarified just how calcium helps to transduce some of the transcriptomic changes induced by phytochrome. This review summarizes the strong evidence that calcium-dependent steps play a major role in transducing phytochrome-initiated responses, covering both the pioneering papers in the last century that established the phytochrome-calcium signaling link, and the latest reports that document specific steps in phytochrome responses that are dependent on the mediation of calcium-binding proteins.

Initial evidence that a critical early step in phytochrome responses is an increase in [Ca²⁺]_cyt

Following the initial observation of Weisenseel and Ruppert (1977) that the light-induced depolarization of Nitella cells was mediated by phytochrome and cations, Hale and Roux (1980) used the dye murexide, which undergoes a color change in the presence of calcium, to measure a rapid (< 4 min), red-light induced release of Ca²⁺ from phytochrome-rich coleoptiles of etiolated oat seedlings. This light-induced response was also observed in suspension cultures of protoplasts generated from these coleoptiles, was photoreversible by far-red light, and did not occur in oat-leaf mesophyll protoplasts that had no detectable phytochrome. Das and Sopory (1985) then used ⁴⁵Ca²⁺ to provide critical direct evidence that phytochrome could promote calcium influx into maize protoplasts, with peak uptake occurring in about 2 min.

These initial results were consistent with the hypothesis that phytochrome could induce Ca²⁺ fluxes in plant cells. However, because the changes they measured occurred minutes after the light stimulus, and initial signaling steps typically occur within seconds of the stimulus, they did not reveal whether the light-induced flux changes were key early steps, or downstream of the initial changes. They also did not link these changes to any photomorphogenic responses, or document in what subcellular compartment they occurred (e.g., did they result in any changes in [Ca²⁺]_cyt.?). This critical information was provided by Shacklock et al. (1992), who documented that the R-induced swelling of wheat protoplasts was preceded by a transient increase in [Ca²⁺]_cyt that occurred in less than 15 s after the red light stimulus, and could also be induced by the photolytic release of caged calcium from the cytoplasm of the protoplasts. Fallon et al. (1993) then found that the R-induced rapid increase in [Ca²⁺]_cyt was accompanied by equally rapid phosphorylation of a 70-kD peptide, which increased when extracellular calcium was elevated, but decreased with increasing chelation of extracellular Ca²⁺ by EGTA.

To better discover which downstream photomorphogenic changes induced by phytochrome were mediated by changes in [Ca²⁺]_cyt, Neuhaus et al. (1993) developed single-cell assays to visualize phytochrome responses. They used these assays to examine the effects of microinjecting putative signaling intermediates into phytochrome-deficient tomato cells. They found that phytochrome responses initially involved the activation of one or more G-proteins that were then coupled to one of two different pathways, one of which required calcium and activated calmodulin to stimulate the expression of a red-light activated cab-GUS reporter gene and the synthesis and assembly of some of the photosynthetic complexes.

Because phytochrome-induced increase in [Ca²⁺]_cyt would activate cytoplasmic Ca²⁺- binding proteins like calmodulin and annexin, and because these signal transducers are small enough to passively enter nuclei through nuclear pores (Wang and Brattain 2007), they are known to function both in the cytoplasm and the nucleus (Reddy et al. 2011; Clark et al. 2012). Thus the results of Shacklock et al. (1992) would predict that the phytochrome-induced increase in [Ca²⁺]_cyt could stimulate cellular responses both in the cytoplasm and the nucleus.

Even prior to the prediction of Shacklock et al. (1992) that light activation of phytochrome could lead to the stimulation of Ca²⁺-regulated enzymes, Dr. Sopory’s laboratory had already published a report on this. Das et al. (1985) showed that light-activated phytochrome stimulated the activity of NADH-Glutamate Dehydrogenase and that Ca²⁺-activated calmodulin enhanced the activity of partially purified preparations of this enzyme. These results indicated that one of the downstream effects of phytochrome-induced increase in [Ca²⁺]_cyt could be the stimulation of NADH-Glutamate Dehydrogenase activity. Other calcium-mediated responses in plants documented by the Sopory lab are reviewed in Pandey et al. (2000).

Most plants have multiple genes that encode calmodulins or calmodulin-like proteins (CMLs). For example, Arabidopsis has seven genes encoding calmodulins and 50 encoding CMLs (Reddy et al. 2011). There are also other Ca²⁺-binding proteins that participate in signaling, such as annexins and proteins that have a C2-domain, which is a Ca²⁺-dependent membrane-targeting module (Reddy et al. 2011). Thus, it seems likely that the initial increase in [Ca²⁺]_cyt could result in the downstream activation of scores of different cellular activities. For example, in Arabidopsis just one family of Ca^2+_binding proteins, the calmodulins, has been documented to regulate transcription, Ca²⁺ ion transport, protein folding, and protein phosphorylation, among other cellular functions (Reddy et al. 2011). Not surprisingly, then, there are currently scores of published papers that present data linking Ca²⁺ mediation to phytochrome responses. Here we will describe just a few examples of the more recent reports that clarify specifically how Ca²⁺ helps to mediate photomorphogenic responses induced by phytochrome.

Recent reports clarifying the role of calcium in mediating specific phytochrome-stimulated responses

The diversity of different plants in which Ca²⁺ fluxes mediate phytochrome responses was expanded recently by the report of Moysset and colleagues (Moysset et al. 2019). Their study showed how red light triggers the movement of calcium in the motor cells of Robinia pseudoacacia pulvini. They provided evidence that cytoplasmic Ca²⁺ serves as a secondary messenger for phytochrome during the nyctinastic closure process. In accord with earlier studies, they also found that red (R) and far-red (FR) light irradiations have an impact on the subcellular location of loosely bound Ca²⁺ and its properties.

A molecular mechanism linking phytochrome-induced increase in [Ca²⁺]_cyt to phytochrome-induced changes in transcription was reviewed by Gangappa et al. (2013). They summarized reports showing that calmodulin 7 (CaM7) in Arabidopsis functions as a transcription factor. It interacts with the Z-box (ATACGTGT), a Z-DNA forming sequence, and turns on the expression of phytochrome-induced CAB and RBCS genes. It functions synergistically with HY5 to promote light-induced inhibition of hypocotyl elongation during de-etiolation. This signaling pathway, illustrated in Fig. 1, is just one example of how a nuclear calmodulin could help transduce phytochrome photoactivation into changes in transcription, but there are others that have been discovered and, likely, even more that will be discovered since multiple transcription factors are activated by calmodulin (Snedden and Fromm 2001).

Fig. 1.

Example of steps in a phytochrome-induced signaling pathway that include those mediated by Ca²⁺-activated calmodulin (CaM*). 1. Photoactivation of phyB in the cytoplasm. 2. Induction of Ca²⁺ entry into the cytoplasm from the extracellular matrix (ECM) by phyB*. 3. Increased [Ca²⁺]_cyt converts inactive CaM to active CaM (CaM*). 4. Activation of cytoplasmic enzyme NADH-Glutamate Dehydrogenase (NGD*) by CaM*. 5. Both phyB* and some cytoplasmic CaM* enters the nucleus through nuclear pores, and one calmodulin, CaM7*, binds to DNA and, together with the transcription factor HY5, induces the differential expression of genes (DEG) needed for phyB*-promoted de-etiolation of dark-grown seedlings. * indicates activated form of protein

Another nuclear protein activated by Ca²⁺-calmodulin and by phytochrome during the de-etiolation of Arabidopsis seedlings was recently reported to be the apyrase, AtAPY1 (Weeraratne et al. 2022). This enzyme is a calmodulin-activated nucleoside triphosphate-diphosphohydrolase that is chromatin-associated, but also functions in Golgi and in the ECM, (Clark et al. 2021). Its expression in roots and in hook-cotyledon tissue is induced in etiolated seedlings by phytochrome, and this upregulation is required in order for photoactivated phytochrome to stimulate the growth of these tissues during de-etiolation (Weeraratne et al. 2022).

Recently, Zhao et al. (2023) discovered that phytochrome’s red-light activation stimulated the activity of two specific Ca2⁺-binding protein kinases, CPK6 and CPK12 (CPK6/12), within seconds. Protein kinases phosphorylated phyB at Ser80/Ser106, promoting its import from the cytoplasm to the nucleus. A mutated version of phyB (phyBS80A/S106) was unable to enter the nuclei and therefore could not replace phyB mutants. The early expansion of cotyledons is phyB-mediated and requires the activation of CPK6/12. Although the cpk6/cpk12 null mutants have similar levels of phytochrome-interacting factors (PIF) 1 and PIF3 as WT plants in darkness, they fail to undergo photomorphogenic changes induced by Pfr due to the absence of light-induced degradation of PIFs. These results show that CPK6/12 plays a critical role in phyB signal transduction and provide a mechanism linking Pfr-induced increase in [Ca2⁺]cyt to seedling de-etiolation.

Plant responses to calcium and light signals impact more than just photomorphogenesis. In Arabidopsis thaliana, the Ca²⁺-activated protein kinase SALT OVERLY SENSITIVE2 (SOS2) positively regulates salt tolerance. A recent study by Ma et al. (2023) revealed that phytochromes enhance the activity of SOS2 when plants are exposed to both light and salt stress. In the presence of light, photoactivated phyA and phyB interact with SOS2 in both the cytosol and nucleus, boosting the kinase activity of salt-activated SOS2. Although phytochrome activation and salt stress both increase [Ca²⁺]_cyt, it is unclear whether the increase in [Ca²⁺]_cyt caused by light is involved in the phytochrome-mediated enhancement of SOS2 activity. Further investigation is needed in this regard (Han et al. 2023).

A recent report by Jiang et al. (2019) found that the abundance of phyB in cells can be affected by calcium and the plastidial metabolite MEcPP (methylerythritol cyclodiphosphate). The authors found that the MEcPP-activated, calcium-dependent transcriptional regulator CAMTA3 plays a critical role in maintaining phyB abundance. Their biochemical analyses showed that high levels of phyB in MEcPP-accumulating plants resulted from reduced expression of phyB antagonists and decreased levels of the hormone that negatively regulates phyB abundance, auxin. Overall, these findings show that a single plastidial metabolite can influence various regulatory networks that control the abundance of phyB, including specifically Ca²⁺ signaling (Jiang, et al. 2019).

An unanswered question is what types of Ca²⁺ channels are activated by phytochrome. Among the better-studied channels in plants are glutamate receptor-like (GLR) channels, which when activated, promote the uptake of Ca²⁺ into plant cells (Ahmed et al. 2023). They have been reported to play a role in transducing different developmental and environmental stimuli, and recently Krzeszowiec et al. (2023) studied the role of two types of GLR channels, NMDA-like and AMPA-like, in photomorphogenic responses to different light treatments. They found that both types of GLR channels help regulate plant responses to blue and red light. They observed changes in cellobiose-induced calcium wave after GLR inhibitors were applied, and their results suggested that both types of channels likely work together in shaping the light-induced growth and development changes of Arabidopsis seedlings. These results are the first experimental evidence that two types of GLR channels-NMDA-like and AMPA-like-function in plants. They showed that the involvement of these channels in seedling growth and development was at least partially through modulation of calcium signaling, but because antagonists of GLR channels affected dark growth more than light growth, the authors concluded that the channels may not play a major role in photomorphogenesis.

Conclusion

This review covered only a fraction of the scores of publications that document a role for Ca²⁺ in mediating phytochrome responses, with a focus only on the key initial reports and those published within the last 5 years, Nonetheless, they provided both support for hypotheses that proposed this role several decades ago, and new insights on the mechanisms by which the mediation occurred. They can serve as valuable guides for the future research that will be needed to further evaluate the Ca²⁺-phytochrome signaling connection and clarify the mechanisms by which this connection is made.

Major unanswered questions

Although the results reviewed here provide further support for the hypothesis that the early phytochrome-induced increase in [Ca²⁺]_cyt plays a major role in promoting downstream photomorphogenic changes, the mechanisms by which these changes occur remain largely unresolved. Here we note 4 of the more obvious unresolved questions:

1. How does photoactivated phyB (phyB*) promote an increase in the concentration of cytoplasmic Ca²⁺? If phyB* induces a Ca²⁺ channel to open, does it do so by interacting with channels directly, or indirectly through some intermediate?

2. Is the phytochrome-induced increase in [Ca²⁺]_cyt due to Ca²⁺ entry only through the plasma membrane from the extracellular matrix (cell wall), or does the red-light stimulus also induces the release of Ca²⁺ from internal organellar stores, such as the chloroplast (Navazio et al. 2020) and the vacuole and endoplasmic reticulum (Stael et al. 2012), all of which are now recognized to help control [Ca²⁺]_cyt.

3. Since nuclei regulate their internal [Ca²⁺] ([Ca²⁺]_nuc) independent of the [Ca²⁺]_cyt (Charpentier 2018), does the phyB-induced increase in [Ca²⁺]_cyt induces a similar change in [Ca²⁺]_nuc? Is a change in [Ca²⁺]_nuc needed for Ca²⁺ to mediate any of the transcriptional changes induced by phytochrome?

4. There are multiple CaM-regulated transcription factors in plant nuclei (Reddy et al. 2011), so what mechanism specifies which ones are activated by CaM to help mediate phytochrome-induced transcription changes?

Funding

Some of the authors’ research was funded by the U.S. National Science Foundation [Grant Number IOS-1027514], Texas Crop Science [Grant Number UTA13-000682].