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. 2019 Dec 20;182(3):1297–1309. doi: 10.1104/pp.19.01053

Subdivision of Light Signaling Networks Contributes to Partitioning of C4 Photosynthesis1,[OPEN]

Ross-W Hendron 1, Steven Kelly 1,2,3
PMCID: PMC7054874  PMID: 31862840

Cell-specific activation of photosynthetic machinery is mediated by differences in light signaling networks between photosynthetic cell types; these differences indicate that the regulatory system that facilitates C4 photosynthesis may have evolved to reinforce differences in within-leaf light availability.

Abstract

Plants coordinate the expression of photosynthesis-related genes in response to growth and environmental changes. In species that conduct two-cell C4 photosynthesis, expression of photosynthesis genes is partitioned such that leaf mesophyll and bundle sheath cells accumulate different components of the photosynthetic pathway. The identities of the regulatory networks that facilitate this partitioning are unknown. Here, we show that differences in light perception between mesophyll and bundle sheath cells facilitate differential regulation and accumulation of photosynthesis gene transcripts in the C4 crop maize (Zea mays). Key components of the photosynthesis gene regulatory network differentially accumulated between mesophyll and bundle sheath cells, indicative of differential network activity across cell types. We further show that blue (but not red) light is necessary and sufficient to activate photosystem II assembly in mesophyll cells in etiolated maize. Finally, we demonstrate that 61% of all light-induced mesophyll and bundle sheath genes were induced only by blue light or only by red light, but not both. These findings provide evidence that subdivision of light signaling networks is a component of cellular partitioning of C4 photosynthesis in maize.

Light is of fundamental importance to photoautotrophs, organisms that harness energy from photons to synthesize sugars. Given the central role that light plays in the growth of plants, it is fitting that they have evolved sophisticated methods to sense this environmental cue and use it to optimize their photosynthetic capabilities. In the model plant Arabidopsis (Arabidopsis thaliana), five families of photoreceptors, of varying spectral sensitivities, allow discrimination between different wavelengths of light. These families comprise the phytochromes, cryptochromes, phototropins, UVRs, and Zeitlupe proteins (Lin, 2000; Smith, 2000; Schepens et al., 2004; Rizzini et al., 2011; Christie et al., 2015). In brief, red light stimulates phytochrome signaling, blue light stimulates cryptochromes, phototropins, and Zeitlupes, and UV-B light stimulates UVR8. Once stimulated, photoreceptors either activate signaling cascades (Petroutsos et al., 2016), bind DNA directly (Yang et al., 2018), and/or bind to and affect the DNA binding properties of many transcription factors that serve as light signaling intermediates, such as PHYTOCHROME INTERACTING FACTOR (PIF) and CRYPTOCHROME INTERACTING BASIC-HELIX-LOOP-HELIX (CIB) genes (Yu et al., 2010; Li et al., 2011). Thus, expression of nucleus-encoded photosynthesis genes is modulated both by photoreceptor activity and by a network of light-responsive transcription factors. Different wavelengths of light also influence posttranscriptional regulation of gene expression, modulating translational efficiency through light-induced alternate start codon usage (Kurihara et al., 2018) and protein degradation through ubiquitination pathways (Xu et al., 2015). By linking light cues to the regulation of photosynthesis genes, plants are able to coordinate development of chloroplasts and optimize photosynthetic rates in mature leaf tissue under changing environmental conditions (Ohashi-Kaneko et al., 2006; Waters and Langdale, 2009; Ort and Melis, 2011).

In most angiosperm species, photosynthesis occurs in the leaf mesophyll. In dicotyledonous species, this cell layer contains at least two distinct cell types: the palisade cells that are located adjacent to the upper leaf epidermis and the spongy mesophyll cells located beneath them. All light that strikes a leaf surface is filtered as it penetrates through these cell layers, such that light availability in the spongy mesophyll is reduced by as much as 90% in comparison to the palisade (Cui et al., 1991; Terashima and Hikosaka, 1995). This biophysical light filtration is responsible for a decreasing gradient in chlorophyll and photosynthetic capacity through the mesophyll, such that upper palisade cells carry out the vast majority of photosynthesis in a dicot leaf (Evans and Vogelmann, 2003; Tholen et al., 2012). In addition, different wavelengths of photosynthetically active light vary in their ability to penetrate leaf tissues, with longer wavelength red light penetrating significantly deeper than shorter wavelength blue light (Vogelmann and Evans, 2002; Slattery et al., 2016). Modeling light penetration through the leaf suggests that just 4% of incoming diffuse blue light reaches the second layer of cells. By contrast, 12% of red light reaches these deeper cells; thus, both the intensity and spectrum of light change as light passes through the leaf (Xiao et al., 2016). Although monocotyledonous leaves contain more uniformly shaped mesophyll cells, analogous light filtration is thought to occur (Takahashi et al., 1994; Kume, 2017).

While most plant species conduct all the reactions required to carry out photosynthesis in a single cell, some species have evolved a way to split the process between two specialized cell types. This spatial division of photosynthesis is known as C4 photosynthesis, and it has evolved independently at least 61 times in both eudicots and monocots (Sage, 2016). The result of this spatial partitioning is a depletion of O2 and an increase in CO2 around Rubisco that together reduce energy loss through photorespiration (von Caemmerer and Furbank, 2016). In all two-cell C4 species, these two specialized cell types are arranged concentrically in an outer layer of photosynthetic carbon assimilation tissue surrounding an inner layer of photosynthetic carbon reduction tissue that, in turn, surround the vascular tissue in a concentric wreath-like arrangement known as Kranz anatomy (Nelson and Langdale, 1989; Muhaidat et al., 2007). Thus, in two-cell C4 species, all light reaching the photosynthetic carbon reduction cell layer must first pass through an outer photosynthetic carbon assimilation cell layer. This means that in two-cell C4 species with Kranz anatomy, light reaching the photosynthetic carbon reduction layer is ∼10-fold dimmer and depleted in blue relative to red wavelengths compared to light that reaches the photosynthetic carbon assimilation layer. This light filtering is thought to be one reason why photosynthetic rates are lower under blue light than red light in C4 species such as maize (Zea mays) and miscanthus (Miscanthus × giganteus; Sun et al., 2012, 2014).

During maize leaf development, early transcriptional changes give rise to vasculature and determination of mesophyll and bundle sheath cell identities (Sedelnikova et al., 2018). After this cell determination stage, cells at the base of the leaf blade are primed to respond to light cues to promote photosynthetic maturation in expanding leaf blades (Wang et al., 2013b). The leaf blade emerges vertically, and the leaf is exposed to light on both the adaxial and abaxial surfaces during this greening phase. Since bundle sheath cells are always shrouded in a layer of mesophyll cells, the light stimulus is inherently asymmetrical between these cell types during photosynthetic activation. Given the integration of light signals with the transcriptional control of photosynthesis (Wang et al., 2017a), it was hypothesized that the gene regulatory networks that control photosynthesis gene expression may differentially accumulate between photosynthetic carbon assimilation and photosynthetic carbon reduction cells in a manner that is consistent with biophysical light filtration and may therefore facilitate partitioning of photosynthetic reactions in C4 species. Although the regulators that facilitate this partitioning are unknown, two lines of evidence concerning the light-dependent regulation of cell-specific gene expression support this hypothesis. First, the PSII complex is expressed only in mesophyll cells in maize (Drozak and Romanowska, 2006) and the expression of the gene encoding PSII D2 protein (psbD) is blue light responsive and under the control of sigma factor5 (SIG5), which in maize only accumulates under blue light (Tsunoyama et al., 2002). Second, Rubisco is only found in the bundle sheath of maize, and the gene encoding Rubisco small subunit (rbcS-m3) is expressed in maize bundle sheath cells in response to red light induction and is not expressed in mesophyll cells due to blue light repression (Purcell et al., 1995; Markelz et al., 2003). However, the extent to which blue light and red light facilitate partitioning of photosynthetic gene expression between bundle sheath and mesophyll cells is unknown.

Here, we tested the hypothesis that the gene regulatory networks for photosynthesis are partitioned between bundle sheath and mesophyll cells in the C4 plant maize in a manner that is consistent with biophysical light filtration within the leaf. That is, the expression of genes whose transcripts preferentially accumulate in mesophyll cells will be responsive to both blue and red light stimulation, while expression of genes whose transcripts preferentially accumulate in bundle sheath cells will be responsive only to red light stimulation. We show that transcription factors and photoreceptors that are known to regulate photosynthesis differentially accumulated between mesophyll and bundle sheath cells in maize leaves. Through spectrum-specific deetiolation experiments we show that blue light, but not red light, resulted in rapid accumulation of chlorophyll fluorescence from functional PSII assembly. Furthermore, we demonstrate that transcripts encoding mesophyll-specific genes increased in abundance in response to either red or blue light, while transcripts encoding bundle sheath–specific genes increased in abundance in response to red light. Together, these findings provide evidence that subdivision of light signaling networks contributes to cellular partitioning of C4 photosynthesis in maize.

RESULTS

The Photosynthesis Gene Regulatory Network Shows Extensive Partitioning between Bundle Sheath and Mesophyll Cells in Maize

Much of what is known about the regulation of photosynthesis gene expression has been determined in Arabidopsis (Wang et al., 2017a). To date, few regulators of photosynthesis gene expression have been characterized in other species, and little is known about how these genes are regulated in grasses (Wang et al., 2017a). However, it is likely that maize orthologs of known photosynthesis regulatory genes in Arabidopsis provide some of this regulatory function. To estimate the extent to which the gene regulatory network for photosynthesis was partitioned between bundle sheath and mesophyll cells in maize, the complete set of maize genes that are orthologous to Arabidopsis genes that encode known photoreceptors or light-modulated transcription factors were identified and analyzed (accession numbers for all genes are detailed in Supplemental File S1).

Of the 58 known photosynthesis regulatory genes in Arabidopsis, 49 had at least one ortholog in maize. Multiple gene duplication events have occurred in the lineage leading to maize since Arabidopsis and maize last shared a common ancestor (Lee et al., 2013); as a consequence, most of these regulatory genes in Arabidopsis have more than one ortholog in maize. Specifically, the 49 Arabidopsis genes had 97 orthologs in maize. The differential accumulation of transcripts corresponding to these 97 maize genes was assessed in two transcriptome datasets in which the transcriptomes of mesophyll and bundle cells were separately sequenced, one taken from mature maize leaves (Chang et al., 2012) and one from developing maize leaves (Tausta et al., 2014).

Of the 6,210 genes differentially regulated between bundle sheath and mesophyll cells in the developing leaf dataset (Tausta et al., 2014), 3,741 were also differentially regulated in the same way in the mature dataset (agreement = 60%). Of these genes, 1,370 encoded transcription factors. Transcription factors were therefore more likely to be differentially expressed between cell types than randomly sampled genes (P ≤ 0.001, n = 1,000 Monte Carlo simulations). Indeed, the transcription factors that are orthologous to known photosynthesis-regulating transcription factors in Arabidopsis (n = 67) were also more likely to be differentially expressed between bundle sheath and mesophyll cells than randomly sampled genes (P ≤ 0.002, n = 1,000 Monte Carlo simulations). Thus, transcriptional regulators, including those that can be inferred to regulate photosynthesis in maize, show evidence of partitioning between bundle sheath and mesophyll cells. A complete list of all differentially regulated photosynthesis-related transcription factors between bundle sheath and mesophyll cells of the two relevant datasets is provided in Supplemental File S2.

To investigate the evolutionary relationships between these transcription factors, a set of orthogroups were defined at the most recent common ancestor of the dicots and monocots. An orthogroup is the complete set of genes descended from a single gene that was present in the most recent common ancestor of a set of species under consideration. Thus, the orthogroups defined here grouped together all maize genes that were descended from a single copy gene in the most recent common ancestor of the monocots and dicots under consideration (species are detailed in Supplemental File S3). This analysis revealed that many of the differentially accumulating transcription factors identified above were related to each other, having arisen through gene duplication events since the monocot-dicot ancestor. Figure 1 shows the maize genes (within the nine orthogroups that contained more than one differentially expressed maize gene) that were differentially expressed in either dataset. Figure 1A displays orthogroups where the maize paralogs differentially accumulated in opposite cell types. This list includes well-characterized photosynthetic regulators such as the GOLDEN-2 LIKE (GLK) genes, PIFs, and CIBs. This subfunctionalization of paralogous genes extends previous studies that have suggested a role for such subfunctionalization for generating cell-specific dimorphic chloroplasts (Wang et al., 2013a).

Figure 1.

Figure 1.

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Orthology and differential expression of the maize orthologs of regulators of photosynthesis gene expression in Arabidopsis. A, Orthogroups wherein paralogous maize genes were differentially expressed in different cell types. B, Orthogroups wherein paralogous maize genes were preferentially expressed in the same cell types. C, Consistent cell type preferential accumulation of phytochrome paralogs. Evolutionary relationships are indicated by a cladogram relating Arabidopsis photosynthesis gene regulatory network components (bold font) and corresponding differentially expressed maize orthologs (lightface font). Orthogroups were selected in which at least two maize paralogs were differentially expressed in either of the two maize RNA-seq datasets examined (DESeq2 differential expression testing with multiple test corrected P < 0.01 for each of two RNA-seq datasets). Yellow circles indicate significant mesophyll cell preferential accumulation of messenger transcripts, green circles indicate significant bundle sheath cell preferential accumulation.

In addition to subfunctionalization, three orthogroups were detected within which all paralogs were preferentially expressed in the same cell type (i.e. ZIM-LIKE genes [ZML1 and ZML2] and SIG2 expressed in the mesophyll and OBF-BINDING PROTEIN3 [OBP3] expressed in the bundle sheath; Fig. 1B). As these duplication events precede the origin of C4 photosynthesis in maize, it is perhaps more likely that these expression patterns represent the ancestral expression state in the C3 ancestor of maize, rather than independent evolution of cell type specificity. Of particular note was that the maize phytochrome genes were all preferentially expressed in bundle sheath cells (Fig. 1C). Hence, of the photosynthetic regulators whose maize paralogs were preferentially expressed in the same cell types, blue light signaling intermediate ZMLs (Shaikhali et al., 2012) were associated with the mesophyll and red light photoreceptors (i.e. PHYs) were associated with bundle sheath cells. Thus, we hypothesized that the partitioning of photosynthetic regulators between cell types may be due to cellular differences in light perception. Specifically, we hypothesized that photosynthetic machinery in bundle sheath cells would be more sensitive to red light than blue light and that the photosynthetic machinery in mesophyll cells would be more responsive to blue light than red light.

Blue Light, But Not Red Light, Stimulates Assembly of PSII in Etiolated Maize Seedlings

To test our hypothesis, we exploited the fact that many C4 monocots and dicots exhibit preferential accumulation of PSII in mesophyll cells (Schuster et al., 1985; Sheen and Bogorad, 1988; Höfer et al., 1992; Meierhoff and Westhoff, 1993). If the hypothesis is correct, mesophyll cells should be responsive to blue light and bundle sheath cells should be more responsive to red light; thus, it should be possible to trigger mesophyll cell–specific gene regulatory networks using blue light, but not red light. Moreover, given that PSII accumulates only in the mesophyll cells of maize, it should be possible to trigger PSII expression specifically with blue light stimulation, but not with red light.

Maize seedlings were deetiolated by illumination with either blue or red light, and functional PSII assembly was monitored by measuring PSII efficiency (фPSII) after 3 h using fluorescence. Significantly more PSII fluorescence was observed following deetiolation under blue light than under red light, irrespective of leaf number or plant age (Fig. 2A). Time-course analysis of the PSII induction kinetics revealed that significant induction of PSII fluorescence was detectable within 90 min of exposure to blue light (Fig. 2B), with only a minor increase in фPSII detected after 3 h of stimulation with red light (Fig. 2B). Despite differences in experimental setup relating to light administration, environmental conditions, and fluorescence measurement devices that generated data for Figure 2, A and B (detailed in “Materials and Methods”), it is clear that the mesophyll regulatory network governing фPSII development exhibited reduced red light sensitivity but was strongly activated by blue light.

Figure 2.

Figure 2.

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PSII assembly is promoted by blue light stimulation in maize. Boxplots indicating фPSII fluorescence following red (red boxes) or blue (blue boxes) light stimulation. A, Measurements taken on a MultispeQ v1.0 after 3 h of spectrum specific deetiolation in a large chamber (7 ≤ n ≤ 10 for each boxplot). Deetiolation time series of maize leaves (B) and barley leaves (C) in a LICOR LI-6800 (n = 9 for each time point and light treatment for both species). Boxplot tails indicate 95% confidence intervals, and asterisks indicate significant differences between light treatments determined by t tests (P < 0.05). The differences in y axis scale between A and B are attributable to differences in the sensitivity and accuracy of the two different measurement devices.

Moreover, we tested whether this preferential induction of PSII under blue light was also observed in barley (Hordeum vulgare), a C3 monocot. When deetiolated barley leaves were subjected to the same time-course experimental procedure as maize leaves, either blue or red light was individually sufficient to induce PSII development (Fig. 2C). Thus, the light signaling network governing фPSII development in barley was stimulated by both blue and red light. By contrast, the maize mesophyll regulatory network for PSII activation was induced specifically by blue light, whereas red light was not a sufficient stimulus.

Activation of Blue Light Signaling Networks Promotes the Transcription of PSII Assembly Components

While the rapid increase in PSII activation under blue light was likely predominantly due to posttranslational assembly of PSII monomers already in the etioplast (Forger and Bogorad, 1973; Müller and Eichacker, 1999), it was also expected to be driven by spectrum-specific transcriptional responses. To determine which genes were responsible for the differences in PSII assembly, total RNA was isolated from leaves at the end of the red or blue light induction period described above and subjected to transcriptome sequencing. Samples were also taken from etiolated leaves that received no light treatment and were sampled at the same time of day as deetiolated plants, and mRNA transcript abundance was quantified (Supplemental File S4). Transcripts that preferentially accumulated under blue light or under red light were detected using differential expression analysis. These cohorts of blue light preferential transcripts and red light preferential transcripts were tested for enrichment of functional terms (detailed in Supplemental Files S5 and S6). This analysis revealed 13 significantly enriched categories within the red light preferential transcripts, including sugar transporters, plasma membrane intrinsic proteins, and photosynthesis genes (Fig. 3A). By contrast, blue light preferentially induced the expression of transcripts that were enriched for 25 functional categories. These encompassed regulatory functions (RNA processing, translation proteins, and proteins associated with chloroplast targeting), in addition to nitrogen metabolism, tetrapyrrole synthesis, and amino acid metabolism (Fig. 3B).

Figure 3.

Figure 3.

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Enrichment of functional MapMan categories (y axis) in genes that were differentially expressed during deetiolation under blue or red light. All displayed categories contained significantly more genes that were preferentially expressed under red light (A) or blue light (B) compared to genomic background (hypergeometric testing with multiple test correction, P < 0.01). All genes and categories are listed in Supplemental Files S4–S6. C, Proportion of differentially expressed (DE) mesophyll and bundle sheath genes in each category that were preferentially expressed in response to either blue light or red light. Gray bars indicate that no transcripts associated with the specified category and cell type were differentially expressed in response to different wavelengths of light.

Many of these genes differentially induced by red or blue light were also known transcripts that accumulated differentially between mesophyll and bundle sheath cells (called mesophyll or bundle sheath genes). Figure 3C indicates the proportion of differentially light-induced mesophyll and bundle sheath genes in each category which were also induced preferentially by blue or red light. This analysis revealed that, across the vast majority of categories in which at least one mesophyll or bundle sheath gene was differentially induced by light (23/28), mesophyll genes were more likely than bundle sheath genes to be induced by blue light and bundle sheath genes were more likely induced by red light. This pattern was especially clear for large MapMan categories (e.g. protein, RNA, cell). This result indicated that there may be a more general difference in mesophyll and bundle sheath cells that is mediated by differences in light signaling pathways between cell types.

Given that blue light specifically induced the assembly of PSII in maize, we initially hypothesized that the subunits of the PSII complex and its assembly factors would be up-regulated under blue light. However, expression of a higher number of PS subunits increased following red light stimulation than following blue light stimulation (Fig. 3A; see also Supplemental Files S5 and S6). Hence, PS subunit expression alone could not explain the fluorescence phenotype that we observed. However, there were differences in the induction of RNA and protein regulatory genes in response to light treatments. Specifically, blue light preferentially induced regulators of RNA and proteins that facilitate the transcription, translation, import, and assembly of PSII proteins into the chloroplast. A full breakdown of these genes and their expression profiles is included in Supplemental Files S4–S6.

PSII is a multimeric protein with both nuclear and plastid-encoded subunits. Fittingly, nuclear transcriptional regulators that have been linked to PSII transcription were preferentially induced by blue light, including HYPOCOTYL ELONGATION5 (HY5; Ang and Deng, 1994) and SIG1, SIG2, and SIG5. Notably, 97 different pentatricopeptide repeat (PPR)–containing proteins increased in abundance following blue light illumination, while only three increased following red light (Fig. 3B). The list of blue light–induced PPR proteins contains several regulators of photosynthesis whose transcripts preferentially accumulate in mesophyll cells including MATERNAL EFFECT EMBRYO ARREST40 (MEE40), which prevents delayed thylakoid development in rice (Oryza sativa; Su et al., 2012); PENTATRICOPEPTIDE REPEAT5 (PRR5), which prevents chloroplast ribosome deficiency in maize (Beick et al., 2008); and PROTEINACEOUS RNASE P1 (PRORP1), which prevents a pale green phenotype in Arabidopsis (Zhou et al., 2015). This extensive induction of RNA editing proteins, in addition to the blue light induction of the chloroplast-encoded, mesophyll-accumulating RNA editing protein MATURASE K (MATK), indicates that mesophyll RNA editing machinery was preferentially activated by blue light (Supplemental File S5).

In addition to RNA transcription and editing machinery, translation, chloroplast targeting, and import mechanisms were also preferentially activated by blue light. Fifteen chloroplast ribosome components (detailed in Supplemental File S4 under the MapMan category ‘protein.synthesis.ribosomal protein.prokaryotic’) were preferentially up-regulated under blue light. Blue light also induced TRANSLOCONS OF OUTER CHLOROPLAST (TOC) protein importers, in addition to proteins that function as molecular chaperones such as HEAT SHOCK PROTEIN90 (HSP90) and CHAPERONIN60 (CPN60A). Preferential accumulation under blue light was also apparent for PSII assembly proteins (whose transcripts also accumulate preferentially in mesophyll cells): SEQUENCE RECOGNITION PARTICLE54 (SRP54) and ALBINO3 (ALB3), which insert the PSII light-harvesting complex into thylakoid membranes (Schuenemann et al., 1998), and PSII turnover proteins (FTSH proteases FTSH2 and FTSH5; Kato et al., 2009) were all preferentially activated by blue light stimulation (Supplemental File S5; Supplemental Fig. S1).

Thus, while the transcripts encoding components of the PSII complex were induced by both red and blue light, light-responsive transcripts encoding mesophyll-localized PSII assembly factors were preferentially induced by blue light. This suggests that the mechanism of PSII accumulation in the mesophyll of mature leaves is implemented at two levels: by blue light signaling networks acting on the posttranscriptional assembly of functional PSII complexes and by blue light signaling networks promoting the transcription of the requisite assembly factors in these cells. This result also suggests that the lack of PSII induction response under red light was because red light did not induce expression of mesophyll-localized regulatory genes, whose proteins are important for correct expression, localization, and assembly of functional PSII complexes.

Subdivision of Blue and Red Light Signaling Networks between Mesophyll and Bundle Sheath Cells Is a Component of Cell Type Specification in Maize

Across multiple categories of genes that promote photosynthetic development, mesophyll and bundle sheath genes were differentially up-regulated in response to blue and red light, respectively (Fig. 3). Given this observation, we hypothesized that there would be a transcriptome-wide association between the responsivity of a transcript to light wavelengths and the cell type preferential expression of that transcript in mesophyll and bundle sheath cells. In keeping with the idea that bundle sheath cells experience spectral shading compared to mesophyll cells, we hypothesized that transcripts that accumulated in bundle sheath cells would be preferentially induced by red light and less by blue light. The majority of light-induced genes (3,153 of the total 4,316) were up-regulated uniquely under blue light, with relatively few (119) being up-regulated only under red light (Fig. 4A). Hence, we quantified how many of these uniquely blue light– or red light–induced genes were also mesophyll or bundle sheath genes.

Figure 4.

Figure 4.

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Transcripts encoding genes that preferentially accumulate in bundle sheath and mesophyll cells are differentially expressed depending on light wavelengths. A, Total number of genes whose transcripts significantly increased in abundance following spectrum specific deetiolation. B, C4 genes grouped by cell type specificity and light responses. Parentheses indicate paralogs that have disparate light responses. All gene names are provided in Supplemental File S7. C to E, Observed (obs.) and expected (exp.) counts of transcripts encoding genes that preferentially accumulated in bundle sheath or mesophyll cells following deetiolation by either color light (C), only blue light (D), or only red light (E). Light responses were observed using n = 3 to 4 RNA-seq replicates per light treatment and DESeq2. Error bars indicate 99% confidence intervals for the mean expected counts generated by Monte Carlo resampling (n = 100), and asterisks indicate significantly different observations compared to null expectations at P < 0.01.

Lists of mesophyll and bundle sheath preferentially expressed genes in developing maize leaves were combined with a curated list of C4 cycle enzymes and transporters (Tausta et al., 2014). These were then compared to the lists of differentially expressed genes in response to light (Supplemental File S7). Consistent with previous analysis (Burgess et al., 2016), transcripts encoding C4 cycle enzymes and transporters in maize generally increased in abundance in response to any light (Fig. 4B) except for NADP-MALIC ENZYME (ME), the primary decarboxylase in the maize C4 cycle (Hatch and Kagawa, 1976). Additionally, PHOSPHOENOLPYRUVATE CARBOXYKINASE (PCK) was induced only by blue light, which is consistent with previous observations that PCK activity is higher under high light and reduced by more than 75% in shade (where the blue:red light ratio is further decreased; Finnegan et al., 1999; Sharwood et al., 2014).

Generally, C4 proteins were induced by either blue or red light and did not show strong preference for either blue or red wavelengths. For example, bundle sheath Rubisco subunits were not specifically induced by red light. However, the Rubisco chaperone Rubisco ACTIVASE (RCA), whose expression is correlated with grain yield in maize (Zhang et al., 2019), was induced only by red light. Additionally, key sugar metabolism genes were uniquely induced by red light, including Calvin cycle enzymes FRU BISPHOSPHATASE (FBP), ALDOLASE (ALDOA), and TRANSKETOLASE (TKL), and sugar transporter MALTOSE EXPORTER1 (MEX1). Thus, the expression of C4 photosynthesis sugar metabolism genes was generally consistent with the hypothesis that the bundle sheath was relatively more sensitive to red light signaling.

Overall, ∼35% of the 3,187 transcripts that preferentially accumulate in mesophyll cells in maize (Tausta et al., 2014) increased in abundance in response to any light treatment (Fig. 4C). This is significantly more than would be expected by chance alone (Fig. 4C). By contrast, only ∼11% of the 3,023 transcripts that preferentially accumulate in bundle sheath cells (Tausta et al., 2014) were induced by any light (Fig. 4C). This is significantly fewer than would be expected by chance alone (Fig. 4C).

Of the set of genes that were induced only by blue light (3,153), 20.7% were mesophyll genes, 5.6% bundle sheath genes, and the remainder were genes that were not differentially expressed between these cell types. By contrast, the set of red-only-induced genes (119) was made up of 26.1% mesophyll genes, 26.1% bundle sheath genes, and the remainder were not differentially expressed between cell types. Since mesophyll and bundle sheath genes each account for ∼8.7% of all transcripts that were expressed in the leaf, these light response distributions indicate significant associations between cell type preferential expression and light wavelength preferential induction (Fig. 4, D and E). Hence, while transcripts that preferentially accumulate in mesophyll cells were responsive to both red and blue stimulation (Fig. 4, D and E), transcripts that accumulate in bundle sheath cells were, on average, more responsive to red light than expected and also less responsive to blue light (Fig. 4, D and E).

Thus, the transcriptional networks that promote the accumulation of transcripts for mesophyll genes and bundle sheath genes differ in terms of both general light sensitivity and responses to specific light spectra. Of all transcripts that differentially accumulated between bundle sheath and mesophyll cells in mature maize leaves, 23% were detectably induced by light. Of these light-inducible genes, the majority (61%) were uniquely induced by either blue or red light. Thus, subdivision of light signaling networks between specialized C4 cell types is a component of C4 photosynthesis partitioning in maize.

Wavelength Specificity of Maize Blue Light and Red Light Transcriptome Responses Is Not Conserved in Arabidopsis

Given the association between light spectrum and cell-specific gene expression observed in maize, we examined to what extent these associations were conserved or divergent in orthologous genes in Arabidopsis. To facilitate this analysis, an analogous transcriptomic dataset of gene expression following spectrum-specific deetiolation in Arabidopsis was interrogated (Hartmann et al., 2016). Here, the complete set of orthogroups described above was used for analysis. If a maize gene and an Arabidopsis gene from the same orthogroup behaved the same way in response to light stimulation, this was recorded as analogous behavior and the light response was said to be conserved for that orthogroup. Otherwise, the light response was deemed to be divergent, indicating a lack of conservation in light response behavior in orthologous genes of these two species.

Consistent with the larger quantity of genes induced by blue light in maize, these genes comprised a larger number of orthogroups whose genes were responsive only to blue light. Overall, 1,914 maize orthogroups were induced only by blue light, 680 orthogroups by either blue or red light, and 68 by red light alone. By contrast, in Arabidopsis 493 orthogroups were induced by blue light only, 1,516 by either type of light, and 380 by red light only. Thus, although there were more orthogroups induced by blue light than by red overall, the number of orthogroups in the two categories was much more similar in Arabidopsis than in maize (Supplemental Fig. S2).

As shown in Table 1, of the 680 orthogroups in which maize genes were responsive to light (irrespective of wavelength), 523 contained Arabidopsis genes with consistent light responses. Hence, ∼77% of orthogroups contained genes that in both species responded in the same way to light. However, when the wavelength of light was taken into consideration the results were dramatically different. Here, only 7–9% of orthogroups that contained a maize gene whose expression was altered by spectrum-specific light treatment also contained Arabidopsis genes with consistent behavior. Thus, while the genes that are responsive to light are highly similar in the two species (i.e. 77% of orthologous genes), the spectrum of light to which they respond is now different.

Table 1. Conservation and divergence in light responses of orthologous Arabidopsis and maize genes.

Light responses were observed in an Arabidopsis deetiolation experiment and a maize deetiolation experiment. Orthogroups were said to be induced by blue light only, red light only, or either type of light, based on the light responses of orthologs within those orthogroups. Orthogroups containing maize genes that were induced by either blue or red light were likely to have Arabidopsis orthologs that were induced by either wavelength as well. Orthogroups with maize genes that responded to only one type of light rarely had Arabidopsis orthologs that responded to the same type of light. These differences were statistically significant by hypergeometric testing. Orthogroups with divergent light-induction responses were enriched for maize MapMan functions. Functions are labeled according to the largest enriched hierarchical MapMan term. CHO, sugar; NA, not applicable.

Type of Light Induction Orthogroups in Which an Arabidopsis Gene Showed a Given Light Response Orthogroups in Which a Maize Gene Showed a Given Light Response Orthogroups Where Maize Light Response Is Conserved in Arabidopsis Proportion of Conserved Maize Orthogroups Enrichment P Value for Conserved Light Responses Functional Groups Enriched within Divergent Maize Orthogroups
Either blue or red light 1,516 680 523 0.77 Overrepresented None
P = 3.38E−271
Blue light only 493 1,914 137 0.07 Underrepresented Protein synthesis
P = 1.23E−241 Tricarboxylic acid cycle
Transcription regulation
Red light only 380 68 6 0.09 Underrepresented Photosynthesis
P = 5.71E−4 Major CHO metabolism
Total 2,389 2,662 666 0.25 NA NA
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Given this substantial difference in behavioral response to light wavelengths, we tested whether the orthogroups that exhibited divergent light responses between Arabidopsis and maize were enriched for genes that perform specific functions. This analysis revealed that genes that were induced by blue light in maize, but not in Arabidopsis, were enriched for regulatory genes, including transcription factors and translational regulators (Table 1; also detailed in Supplemental File S8). Similarly, the genes that were induced only by red light in maize, but not in Arabidopsis, were enriched for photosynthesis genes. Specifically, these red light–induced photosynthesis genes comprised PSI subunits and Calvin cycle genes, in addition to sugar and starch metabolism components (Table 1; also detailed in Supplemental File S9), all of which are defining characteristics of bundle sheath cells in maize.

DISCUSSION

C4 photosynthesis is one of the most remarkable examples of anatomical, physiological, and biochemical convergence in eukaryotic biology (Sage, 2016). The relative frequency with which it has evolved suggests that such large differences must be attributable to a small number of key regulatory changes (Wang et al., 2017b). Here, we provide insight into this regulation by showing that light perception is unevenly distributed between mesophyll and bundle sheath cells in maize. We demonstrate that mesophyll cells preferentially accumulate transcripts that respond to both red light and blue light, while bundle sheath cells preferentially accumulate transcripts that respond only to red light. Consistent with this partitioning, we show that blue light, but not red light, is able to induce development of PSII fluorescence, a functional readout of a protein complex that is specific to maize mesophyll cells. In contrast to C4 maize, in C3 barley leaves, both blue and red light are individually sufficient to induce PSII activation. We further show that the wavelength-specific responses observed in maize were not conserved in Arabidopsis, indicating that the gene regulatory networks have substantially diverged. Finally, we reveal that 61% of the light-induced mesophyll and bundle sheath genes were induced by stimulation with specifically blue light or red light, but not by stimulation with both wavelengths of light. Taken together, these data reveal that subdivision of light signaling networks is a component of the regulatory mechanism that facilitates photosynthetic partitioning between bundle sheath and mesophyll cells in maize.

Although we have shown that there is extensive partitioning of the light-dependent gene regulatory network between bundle sheath and mesophyll cells and that the light responses of bundle sheath and mesophyll cells differ dramatically, the precise molecular links connecting these two phenomena were not determined. Our analysis of gene expression data has revealed multiple correlations between wavelength sensitivity and the expression of protein-binding regulatory genes that corroborates and expands on previous observations that have indicated that posttranslational mechanisms provide an important role in determining asymmetric protein accumulation in C4 species (Meierhoff and Westhoff, 1993). For example, although both PSII subunits and Rubisco subunits were not consistently differentially sensitive to either wavelength of light, assembly factors that bind to these proteins were significantly differentially expressed such that PSII assembly factors were preferentially up-regulated by blue light and Rubisco-associated genes by red light. Experimental interrogation of light signaling networks, such as the systematic deletion of all maize photoreceptor genes, will undoubtedly yield further insights. For example, which (if any) of the four bundle sheath–associated maize phytochrome genes is responsible for the red light–dependent transcriptional activation of bundle sheath–specific genes in maize. It will take a considerable amount of time and experimentation to determine the full details of light signaling network partitioning between mesophyll and bundle sheath cells.

While the work described here has focused on a single C4 species, maize, it will be interesting to determine the extent to which differences in light perception between bundle sheath and mesophyll cells is conserved in different C4 species. In this context, we have shown that the genes that respond to light are predominantly conserved between Arabidopsis and maize; thus, it is likely that these genes will also respond to light in other plant species. However, the spectrum of light to which these genes respond has changed such that the genes’ illumination with different wavelengths of light activates and represses almost completely different sets of genes in the two species. Thus, it may be that different sets of genes respond to spectrum-specific light treatments in different C4 species and that bundle sheath and mesophyll cells have greater or lesser extents of spectral partitioning in other C4 lineages.

Finally, we propose that the asymmetries we have shown are, in part, a natural consequence of the differential light availability in mesophyll and bundle sheath cells in maize. Specifically, all light that strikes a leaf surface is filtered as it penetrates through cell layers, such that light availability after passing through a single photosynthetic cell layer is reduced by as much as 90% (Cui et al., 1991; Terashima et al., 2009) and depleted in blue wavelengths relative to deeper penetrating red wavelengths (Vogelmann and Evans, 2002; Slattery et al., 2016). As bundle sheath cells are always positioned inwards of a layer of mesophyll cells, they always receive light that is dimmer and depleted in shorter wavelengths than the mesophyll. It is therefore tempting to speculate that this purely biophysical phenomenon could represent a simple difference that became exploited and exaggerated during the evolutionary transition from C3 to C4 and that helped facilitate biased cell type expression of genes in C4 species (Fig. 5). Thus, the difference between mesophyll and bundle sheath cells in C4 species may be in part just a trick of the light.

Figure 5.

Figure 5.

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A model for C4 photosynthesis evolution from an ancestral C3 network. A, An ancestral C3 network of blue light– and red light–induced gene regulatory networks (depicted as blue or red circles) regulate gene expression (represented by pink transcription factor on a double helix) and chloroplast development within the same cell in response to blue and red light beams. Mature photosynthetic chloroplasts (green) contain thylakoids (stacked dark green bars) that synthesize starch (white dots). B, A derived C4 gene regulatory network wherein differential accumulation and activity of blue light– and red light–sensitive regulatory components between cell types (indicated by differences in blue and red circle sizes), consistent with different penetration of light beams into the two cell types, help to partition the expression of cell type–specific genes.

MATERIALS AND METHODS

Identification of Orthogroups and Phylogenetic Tree Inference

The 58 light signaling Arabidopsis (Arabidopsis thaliana) genes (detailed in Supplemental File S1) comprised all experimentally validated photoreceptors and light signaling transcription factors as recently reviewed (Wang et al., 2017a); circadian clock genes were removed to focus the list on purely photosynthetic functions. Maize (Zea mays) orthologs were identified for 49 of these Arabidopsis genes by running OrthoFinder v2.2.7 (Emms and Kelly, 2015) with setting ‘-S diamond’ on a set of 16 representative plant species, including both maize and Arabidopsis, as well as suitable relatives and outgroups (Supplemental File S10). OrthoFinder identified orthologs, and these were manually checked by aligning the protein sequences for the complete orthogroups using mafft-linsi (Katoh and Standley, 2013) and constructing maximum likelihood gene trees using FastTree 2 (Price et al., 2010).

Deetiolation of Seedling Leaves in Light Chambers

B73 maize seed was sterilized using 70% (v/v) ethanol and 25% (v/v) bleach. Seeds were planted on vermiculite, watered, and grown with the light off in a growth cabinet set to 28°C. Six-, 9-, and 12-d-old etiolated B73 maize seedlings were put in a chamber where a light-emitting diode (LED) panel administered 100 µmol m−2 s−1 of blue or red light (at leaf level) over whole plants from above. For each of the blue light and red light treatments, a minimum of seven replicates were measured, each from different plants. The emission spectra for the LED lights in the light chamber were measured using an i-phos spectrophotometer and Theremino software (Supplemental Fig. S3) and were consistent with the manufacturer’s specifications. After 3 h, a MultispeQ v1.0 device (Kuhlgert et al., 2016) was used to measure фPSII 2 cm in from leaf tips of leaves 1 and 2 using a custom-designed protocol and macro tailored to the experimental settings. The protocol code and analysis macro are provided in Supplemental File S10.

Deetiolation of Seedling Leaves in a LI-6800 Chamber

To obtain a time series over the 3-h induction period, etiolated leaves were clamped in a LICOR LI-6800 Portable Photosynthesis System (LICOR) equipped with a multiphase flash fluorimeter head. For maize, 9-d-old maize second leaves were sampled. For barley (Hordeum vulgare), the first leaf of ‘Golden Promise’ 7-d-old seedlings was sampled (having been grown in the same conditions as the maize seedlings). The emission spectra LICOR LEDs were measured using an i-phos spectrophotometer (Supplemental Fig. S3) and were consistent with the manufacturer-quoted spectra. For deetiolation, 100 µmol m−2 s−1 of either 100% red or 100% blue light was used. The LICOR was configured with a flow rate of 500 µmol s−1, 400 µmol mol−1 CO2, leaf temperature 28°C, and 60% humidity, to match the environmental conditions of the growth chamber. Fluorescence was measured every 15 min for 3 h.

To rule out whether the observed results were influenced by the fluorescence measuring beam, the same experimental design was repeated, but only a single fluorescence measurement was made after 2 h. The values obtained for фPSII at 2 h were identical to those observed for plants subjected to regular measurement over the same time window (Supplemental Fig. S4). Thus, we concluded that the fluorescence measuring pulses provided no additional developmental stimulus. Negative фPSII values were assumed to be measurement artifacts of fully saturated reaction centers and therefore set to 0.

It has been shown that фPSII measurements can be erroneously inflated under blue light sources compared to red light sources, resulting from overestimation of the maximal fluorescence value (Evans et al., 2017). It should be noted that this effect cannot explain the lack of PSII induction by red light in maize, nor that blue light also preferentially induced PSII fluorescence when the MultispeQ device was used, because the MultispeQ uses different measuring LED spectra that administer orange light rather than red light.

RNA Collection and Sequencing

Immediately following 3 h of deetiolation in the LICOR chamber under either 100 µmol m−2 s−1 of either 100% red or 100% blue light (as described above), 3 cm of maize leaf tissue (starting at 2 cm from the tip) that had been exposed to blue light, red light, or no light treatment were cut and frozen in liquid nitrogen. Samples were collected in a dark room at the same time of day for each replicate. Frozen tissue was ground, and total RNA was extracted and purified using the TRIzol method (Rio et al., 2010) in conjunction with a TurboDNAse treatment. Ten samples were sequenced using the BGIseq-500 RNA-sequencing (RNA-seq) platform: four replicates of blue light, three of red light, and three of no light treatment.

Analysis of Gene Expression Data

Following transcriptome sequencing of deetiolated maize samples, raw read files were trimmed using Trimmomatic v0.38 (Bolger et al., 2014), with settings ‘LEADING: 10, TRAILING: 10, SLIDINGWINDOW:5:15, MINLEN:25.’ Transcript counts and effective lengths were then quantified using Salmon v0.10.0 (Patro et al., 2017) with settings ‘-l A –seqBias–gcBias.’ Transcript counts from all gene models corresponding to the same gene were summed to generate abundance estimates at the gene locus level. Previously published datasets were processed using the same RNA-seq analysis pipeline. Raw counts for each locus were analyzed using DESeq2 (Love et al., 2014), from which a set of maize mesophyll and bundle sheath genes were defined from the DESeq2 analysis using an adjusted P-value cutoff (q < 0.01; Supplemental File S2).

Functional Term Enrichment Analysis

MapMan categories for maize genes were downloaded (Usadel et al., 2009), and a standard hypergeometric test was carried out to identify functional groups that were enriched within the sets of differentially expressed genes during deetiolation. In all cases, the P values obtained were subjected to Bonferroni multiple test correction. When functions are mentioned in the text, the largest hierarchical grouping was used, but full detail of subgroups is available Supplemental Files S5 and S6.

Monte Carlo Resampling

The total population of maize genes was subjected to Monte Carlo resampling to generate expected numbers of differentially expressed transcription factors and photosynthesis regulatory genes. The list of maize transcription factors was downloaded from the Plant Transcription Factor Database (Jin et al., 2017). P values were calculated as (1 + no. of simulations where the simulated value was greater than observed value)/(1 + no. of simulations).

The total population of genes whose expression could be detected in either mesophyll or bundle sheath cells in maize were subjected to Monte Carlo resampling to generate expected numbers of light-induced mesophyll and bundle sheath genes. The 99% confidence intervals were used to confirm that randomly simulated data were significantly different from observed data.

Accession Numbers

All referenced gene names and accessions are detailed in Supplemental Files S1, S2, and S4–S9. Additional RNA-seq data for transcriptomes used in this study were previously published and are available in the National Center for Biotechnology Information Sequence Read Archive under the following accession numbers: SRP009063 (Z. mays) and SRP060410 (Arabidopsis). The RNA-seq data generated in this study have been deposited to ArrayExpress under accession E-MTAB-7200.

Supplemental Data

The following supplemental materials are available.

Footnotes

1

This work was supported by the European Union’s Horizon 2020 research and innovation program (grant no. 637765) and by the Biotechnology and Biological Sciences Research Council (BB/P003117/1). R.-W.H. is supported by a Biotechnology and Biological Sciences Research Council studentship (BB/J014427/1). S.K. is a Royal Society University Research Fellow.

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