1 Introduction

Pathways that communicate chloroplast to nucleus, called retrograde signaling, has been revealed through gun (g enome un coupled) mutants of Arabidopsis thaliana. Retrograde signaling involves plastids signals that induce nuclear gene expression. There are five genes GUN in Arabidopsis (GUN 1-5), which four of these (GUN2-5) encode proteins related to tetrapyrrole biosynthetic pathway (Susek and Chory 1992; Susek et al. 1993; Mochizuki et al. 2001; Larkin et al. 2003). GUN4 encodes a chloroplast protein that activates Mg-chelatase enzyme (MgChl), responsible by protoporphyrinIX (ProtoIX) to Mg-protoporphyrinIX (Mg-ProtoIX) conversion in the chlorophyll biosynthetic pathway (Fig. 1) (Tanaka and Tanaka 2007). Furthermore, GUN4 is required for posttranslational control of tetrapyrrole biosynthesis for promoting the interaction between H-subunit of MgChl enzyme (MgChl-H) and chloroplast membranes (Adhikari et al. 2009; Peter and Grimm 2009). The phenotype of Arabidopsis gun4 mutant was characterized by reduced levels of Mg-ProtoIX, chlorophyll and carotenoids (Larkin et al. 2003). In the other hand, the overexpression of GUN4 in tobacco increased chlorophyll and Mg-ProtoIX levels as well as increased MgChl enzyme activity (Peter and Grimm 2009). Interestingly, GUN4 mutation in cyanobacteria leads to reduced both Fe-chelatase enzyme activity and Heme levels. It suggests that GUN4 is also involved in the biosynthesis of Heme groups, controlling the flow of substrate for chlorophyll or Heme biosynthetic pathway (Wilde et al. 2004; Masuda and Fujita 2008). Although the function of GUN4 and their precursors- and- products for both retrograde signaling and chlorophyll synthesis has been previously demonstrated in Chlamydomonas and Arabidopsis (Koussevitzky et al. 2007; Czarnecki et al. 2012; Formighieri et al. 2012; Zhou et al. 2012), the mechanisms by which GUN4 are involved in retrograde signaling and the impact of GUN4 mutation on photosynthetic process remains poorly understood (Brzezowski et al. 2014).

Fig. 1
figure 1

Schematic representation of a summarized tetrapyrrole and chlorophyll biosynthetic pathway showing the local of GUN4 mutation. Glu l-glutamate, ALA 5-aminolevulinic acid, ProtoIX nprotoporphyrin IX, MgChl magnesium chelatase, FeChl ferrochelatase, Mg-ProtoIX Mg-protoporphyrin IX, Mg-Proto IX ME Mg-protoporphyrin IX monomethyl ester, Divinyl ProtoChlP divinyl Protochlorophyllide, Mono ChlP monovinyl protohlorophylide; Chl a––Chlorophyll a; Heme––heme groups

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Pathways that communicate organelles (plastids and mitochondria) to nucleus are important once coordinate the development and function of the organelles with the whole plant cell (Pfannschmidt 2010). The communication between chloroplast and nucleus involves the exportation of signaling factors from chloroplast to cytosol where the signaling pathway transduces the signal to the nucleus (Kleine et al. 2009). It has been reported that changes in the chloroplast redox potential, the accumulation of reactive oxygen species, Mg-ProtoIX as well as 5-aminolevulinic acid (ALA) biosynthesis could be involved in the retrograde signaling from chloroplast to nucleus (Nott et al. 2006; Zhang 2007; Larkin and Ruckle 2008; Czarnecki et al. 2012). The accumulation of Mg-ProtoIX was reported as a possible signal from chloroplast that regulate light harvesting chlorophyll binding protein-II (LHCBII) genes in the nucleus (Strand et al. 2003; Larkin et al. 2003; Koussevitzky et al. 2007; Larkin and Ruckle 2008). However, strongly evidences suggest that this retrograde signaling cannot be related to Mg-ProtoIX accumulation, indicating that other unknown signal is involved in the chloroplast to nucleus signaling (Mochizuki et al. 2008; Moulin et al. 2008). The current idea is that there is no only one signal in retrograde signaling but rather the result of continuously sensed metabolic and genetic activities in the organelles (Czarnecki et al. 2012). Given these controversies concerning retrograde signaling a more carefully characterization of gun mutants are needed to understand the function of these genes in retrograde signaling and photosynthetic process, especially under light stress conditions.

The maintenance of plants growth under excessive amounts of light has been reported mainly as result of increased photosynthetic capacity of the plant and/or through the activation of process that dissipate the excess of energy absorbed in order to reduce the susceptibility of photo-oxidative damage (Apel and Hirt 2004; Walters 2005; Li et al. 2009). In this context, mutations in GUN genes can induce the overexpression of light harvesting chlorophyll binding (LHCB) proteins especially under stress conditions (Mochizuki et al. 2001; Peter and Grimm 2009; Brzezowski et al. 2014). LHCB are important proteins related to the energy transfer from light harvest complex (LHC) of photosystem II (PSII) to the reaction center of PSII (Teramoto et al. 2001). Given the importance of GUN4 for biosynthesis of photosynthetic pigments and the strong connection between GUN genes and LHCB proteins it’s surprisingly that the effect of GUN4 mutation on light energy transfer in LHCII and plant photochemical performance is still unclear. Therefore, we carried out a photosynthetic characterization of gun4 mutant in order to investigate the role of GUN4 gene for the plant photosynthetic performance under light stress conditions.

2 Materials and methods

2.1 Plant material and growth conditions

All Arabidopsis thaliana plants used in this study were of the columbia ecotype (Col-0). The gun4 knockout mutant (Gene ID: AT3G59400) (SALK_011461) were previously described (Mochizuki et al. 2001; Larkin et al. 2003). Seeds of wild type (WT) and gun4 mutant (homozygous) were surface-decontaminated by shaking 70 % ethanol for 5 min, rinsed with sterile distilled H2O. In sequence treated with 100 % ethanol for 1 min, rinsed three times with sterile distilled H2O. The seeds were then allowed to germinate in Petri dishes containing MS medium (25 cm3) (Murashige and Skoog 1962) and cultivated under photoperiod of 14 h illumination and light intensity of 120 µmol m−2 s−1, with day/night temperatures of 20 ± 3 °C. After 7 days the plants were transferred to recipient (0.1 L) containing commercial soil mixture, keeping in the room with the same conditions described above. We carried out a photosynthetic curve under different light intensities in completely expanded leaves of WT Arabidopsis plants in order to choose the level of light stress would be imposed to the plants. After this, five-week-old plants were subjected to light stress for 0, 14 or 28 h under 800 µmol photons m−2 s−1 using white lamps of 1,000 W in ambient with controlled temperature. It was disposed a water layer among the plants and light source in order to avoid heat excess. Unless otherwise described the fully expanded 9th–12th rosette leaves of six-week-old plants were harvested for subsequent analysis.

2.2 Gas exchange analysis and chlorophyll a fluorescence measurements

The gas exchange analysis were carried out in completely expanded leaves of WT and gun4 mutant using infra-red gas analyzer (IRGA, Licor 6400, Lincoln, USA) according Flexas et al. (2007). It was estimated net photosynthetic rate (A, µmol CO2 m−2 s−1), stomatal conductance (g s, mol H2O m−2 s−1), substomatal CO2 concentration (C i µmol CO2 mol−1), and transpiration rate (E, mmol m−2 s−1) at 0 and 14 h of light stress.

The chlorophyll a fluorescence measurements were performed in completely expanded leaf of WT and gun4 mutant by modulated light fluorometer system (MINI-PAM, Walz, Effeltrich, Germany). For established fluorescence parameters, initially measured minimal (Fo) and maximal fluorescence (Fm) in dark adapted leaves. It was estimated maximal quantum yield of PSII (Fv/Fm) according to Kitajima and Butler (1975). After, it was measured the parameters of light adapted leaves which was used to estimate effective quantum yield of PSII (ΦPSII) (Genty et al. 1989), photochemical quenching of lake model (qL) (qL = (Fm′ − F) × Fo′/(Fm′ − Fo′) × F) (Kramer et al. 2004), non-photochemical quenching (qN = (Fm − Fm′)/(Fm − Fo′) (Bilger and Schreiber 1986), quantum yield of non-regulated non-photochemical energy loss in PSII (ΦNO = F/Fm), quantum yield of regulated non-photochemical energy loss in PSII (ΦNPQ = (F/Fm′) − (F/Fm)). The parameters ΦPSII, ΦNO, and ΦNPQ were estimated according Hendrickson et al. (2004) in which ΦPSII + ΦNO + ΦNPQ = 1. The chlorophyll a fluorescence parameters were also determined in light responses curves. The parameters were estimated from different light intensities (0, 80, 160, 260, 370, 560, 800, 1,060 e 1,660 μmol of photons m−2 s−1).

2.3 Determination of photosynthetic pigments

Immediately after stress treatment, the rosettes of Arabidopsis were frozen at liquid N2 and used for determination of photosynthetic pigments. Total chlorophylls and carotenoids were extracted in acetone:water 80 % (v/v) and their concentration determined spectrophotometrically following Lichtenthaler (1987).

2.4 14CO2 feeding experiment

The redistribution of the photosynthetic metabolic flux was determined in leaves exposed to 14CO2. The 14CO2 was imposed to leaves using oxygen electrode (Hansatech, Kings Lynn, UK) under 400 μmol photons m−2 s−1, at 25 °C, for 60 min. The preparation of the radioactive solution containing 14C as well as the exposition to leaves was realized according DaMatta et al. (2008). After the exposition, leaves were frozen in liquid N2 and store at −80 °C. The extractions as well as the flux distribution between starch, cell wall, basic amino acids, organic acids, and sugars were realized according DaMatta et al. (2008). The incorporated radioactivity was determined using a liquid scintillation analyzer (Beckman LS 6500; Beckman Instruments, Fullerton, CA, EUA).

2.5 Co-expression analysis

The analyses of the co-expression network were performed using microarray data from on line platform ATTED-II (http://atted.jp/) according Obayashi et al. (2011). The network of GUN4 (At3g59400) co-expressed genes was obtained using CoexViewer tool of ATTED-II 5.2 according Obayashi et al. (2009). The data are available in http://atted.jp/top_draw.shtml#CoexViewer. A complete list of genes co-expressed with GUN4 gene is available in ATTED-II on line platform (http://atted.jp/). Further information’s about the significance of the mutual rank values for the gene co-expression can be found in Obayashi and Kinoshita (2009).

2.6 Statistical analysis

To analyze the effect of light stress in each genotype, the data were statistically compared by ANOVA and Dunnet test at 5 % of significance (P < 0.05). Between genotypes, in the same stress treatment, the data were statistically compared by Student t test (P < 0.05). All statistical analyses were performed using the Saeg system version 9.1. The number of replicates (n) is indicated in each figure.

3 Results

3.1 Photosynthetic pigments content are reduced in gun4 plants

The gun4 mutant showed a phenotype with light green/yellowish leaves (Figure S1) according with lower chlorophyll and carotenoids content in the absence of light stress (Table 1). After 14 h of light stress, no significant differences in pigments content were observed in each genotype. However, the gun4 mutant still showed lower total chlorophyll, chlorophyll b and carotenoids content than WT (Table 1).

Table 1 Mean values and standard error of chlorophyll (Chl) and carotenoids (Carot) content (g kg−1 fresh weight) in wild type (WT) and gun4 mutant of Arabidopsis thaliana under 0, 14, or 28 h of light stress
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Light stress has no effect on carotenoids content in both genotypes. However, the chlorophyll content was significantly reduced in both genotypes after 28 h of light stress, except to chlorophyll b content in gun4 mutant (Table 1).

3.2 GUN4 is co-expressed in a network composed by photosystem II related proteins

We carried out an in silico analysis of GUN4 (At3g59400) gene expression from microarray data available using different web platforms. The Arabidopsis eFP Browser on-line platform (Winter et al. 2007) showed that GUN4 is preferentially expressed in photosynthetic tissues, with high expression in chloroplast of leaf mesophyll cells (data not show). The co-expression network analysis by web platform ATTED-II (Obayashi et al. 2011) showed that there is a high co-expression between GUN4 and genes related to photosynthesis, structure and function of both photosystems I and II, proteins of light harvest complex as well as proteins of chlorophyll biosynthetic pathway (Fig. 2). Among different genes co-expressed with GUN4, highlights to genes of light harvesting chlorophyll binding proteins (LHCB2.3, LHCB3, LHCB4, LHCB4.2, LHCB5, LHCB6, LHCA1, LHCA3), genome uncoupled 5 gene (GUN5), non-photochemical quenching related protein (NPQ4), and subunits of photosystems I and II (PSII protein, PSAD-1, PSAD-2, PSAH1, PSAH2, PSBTN, PSAO, PSAF, PSBO2, PSB28, PSBX, PSBW, PSAL, PASK) (Table S1).

Fig. 2
figure 2

Co-expression analysis of GUN4 gene (AT3G59400) obtained from microarray data using CoexViewer tool of ATTED-II 5.2. The function of some of these genes is described in the table S1

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3.3 gun4 plants has greater photochemical efficiency in photosystem II under non-stress conditions

The gun4 mutant showed reduced minimal (Fo) and maximal (Fm) fluorescence of dark-adapted leaves (Fig. 3a, b) as well as reduced quantum yield of unregulated energy dissipation of photosystem II (ΦNO) (data not shown) under non-stress conditions. However, gun4 plants showed increases in non-photochemical quenching (qN) (Fig. 3c), effective quantum yield of PSII (ΦPSII) (Fig. 3e), as well as in photochemical quenching (qL) (Fig. 3f). Higher values of ΦPSII and lower of ΦNO were also found in gun4 under different light intensities (Fig. 4).

Fig. 3
figure 3

Mean values and standard error of minimal (Fo) (a) and maximal (Fm) (b) fluorescence of dark-adapted leaves, non-photochemical quenching (qN) (c), potential quantum yield of the PSII (Fv/Fm) (d), effective quantum yield of the PSII (ΦPSII) (e), and photochemical quenching (qL) (f) in leaves of wild type (WT) and gun4 mutant under different light stress condition. Values followed by the same letter in each genotype and different light stress conditions are not statistically different by Dunnet test (P < 0.05). Asterisks indicate statistic difference between genotypes in the same stress condition by Student t test (P < 0.05) (n = 5)

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Fig. 4
figure 4

Mean values and standard error of unregulated energy dissipation of photosystem II (ΦNO) and photochemical quantum yield of PSII (ΦPSII) (b) in leaves of wild type (WT) and gun4 mutant under different light intensities. Asterisks indicate statistic difference between genotypes in the same light condition by Student t test (P < 0.05) (n = 6)

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3.4 gun4 has greater non-photochemical dissipation of light excess

It was observed significant differences between WT and gun4 in qN (higher values in gun4) and Fo and Fm (lower values in gun4) independent of the stress level (Fig. 3a–c). Increased qN was observed in both genotypes after 14 h of light stress. However, only WT showed significant increases in Fo and significant reduction in potential quantum yield of the PSII (Fv/Fm) after 14 h of stress (Fig. 3a, d). After 28 h of light stress, all photochemical parameters analyzed were significantly different of the non-stress condition, with exception for qN in WT. However, Fo, Fm, and qN were significantly different between WT and gun4 mutant. After this severe stress, the Fv/Fm values were significantly reduced in both genotypes, but higher decrease was observed in WT (74 %) compared to gun4 mutant (54 %) (Fig. 3 D). The gun4 mutant maintains non-photochemical dissipation of light excess through a significant higher qN under light stress (Fig. 3c). Furthermore, there was no difference in Fo between 14 and 28 h of light stress in gun4 plants (Fig. 3a).

3.5 gun4 plants has intrinsic water use efficiency increased despite strong reduction in photosynthetic rates

The gun4 mutant showed decreased net photosynthetic rate (A) (43 %), stomatal conductance (g s) (61 %), and transpiration rate (E) (46 %) compared to WT plants. These reductions lead to a phenotype with increased intrinsic water use efficiency (WUEi), i.e. higher A/g s relationship (Fig. 5). Due to the mild reductions in A and E in WT after light stress, no differences were observed in these parameters between WT and gun4 plants. However, g s was still lower in gun4 after light stress which leads to a higher A/g s relationship in gun4 (Fig. 5). Given the strong light stress imposed on plants whereas that 28 h of light stress dramatically reduced all photochemical parameters (Fv/Fm, ΦPSII, and qL), no net photosynthetic rate was detected in leaves of both genotypes after 28 h of light stress (data not shown).

Fig. 5
figure 5

Mean values and standard error of gas exchange parameters in leaves of wild type (WT) and gun4 mutant submitted to 0 or 14 h of light stress. A net photosynthetic rate, g s stomatal conductance, E transpiration rate, WUE i intrinsic water use efficiency. Asterisks indicate statistic difference between genotypes in the same stress condition by Student t test (P < 0.05) (n = 5)

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3.6 14CO2 incorporation in WT and gun4 leaves

We carried out a feeding experiment in whole leaves submitted to saturate 14CO2 concentration. Under these conditions, total 14C incorporation was not different between WT and gun4 mutant (Table 2). No differences were found in the percentage of 14C flux redistribution to basic amino acids, sugars, starch, and cell wall. However, mild reduction in the percentage of 14C to organic acids was observed in gun4 mutant (Table 2).

Table 2 Mean values and standard error of total absorption (Bq g−1) and redistribution of 14C (%) in leaves of wild type (WT) and gun4 mutant
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4 Discussion

4.1 gun4 has greater light energy transfer efficiency in PSII despite lower both chlorophyll and carotenoid contents

Given the role of GUN4 for chlorophyll biosynthesis, the mutation in this gene leads to reduced level of photosynthetic pigments in Arabidopsis leaves. In turn it may lead to small LHCII and reduced capacity of light energy absorption in gun4 plants, evidenced by the lower Fo values compared to WT. There are evidences in plants and cyanobacteria that the mutation in genes of chlorophyll biosynthetic pathway, including GUN4, result in smaller LHCII due the less components of light harvesting complex and less subunits of PSII (Havaux et al. 2007; Sobotka et al. 2008; Hansson and Jensen 2009). In this context, gun4 phenotype is similar to other mutants of chlorophyll biosynthetic pathway, such as chl27 and sg1. CHL27 codifies the enzyme Mg-ProtoIX monomethyl ester ciclase (EC 1.14.13.81) (Fig. 1). The chl27 mutant has less chlorophyll content and reduced both LHCI (30 % of reduction) and LHCII (50 % of reduction) content. However, the effective quantum yield of both PSI and PSII increase in chl27 (Hansson and Jensen 2009). The photochemical efficiency of chl27 mutant is greater than WT a despite of minor capacity of light absorption, similar to observed here in gun4 mutant. Interestingly, gun4 and chl27 plants showed a flexibility of PSII to increase the capacity of energy transfer despite of fewer molecules for light absorption.

Light stress has been reported as signal to reduce the expression of nuclear genes that codify light-harvesting chlorophyll a/b-binding proteins (LHCB) via a pathway that involve GUN4 and ABI4 transcription factor (abscisic acid-insensitive 4) (Larkin et al. 2003; Larkin and Ruckle 2008). Probably this is a photo-protection response in order to reduce light excess absorption in PSII and minimize oxidative damages caused by excess of light in WT plants. Furthermore, gun mutants exhibited high-level expression of LHCB genes under stress (Susek et al. 1993; Mochizuki et al. 2001; Eberhard et al. 2008; Peter and Grimm 2009; Brzezowski et al. 2014). It suggests that the expression of GUN and LHCB genes are highly coordinated. Indeed, the analysis of GUN4 co-expression network showed that many genes related to photosynthesis and structure of photosystems are co-expressed with GUN4, including many genes of LHCB family (Table S1). Similarly, the single sg1 mutant and the double sg1 gun4 mutant presented low levels of chlorophyll but high levels of genes related to photosystem II (psbA, psbB) and carbon fixation (RbcS, RbcL) (Hu et al. 2014). It suggests that GUN4, like SG1, is important not only for chlorophyll biosynthesis but also for expression of genes related to the photosynthetic process; which was indeed showed in this work. The up-regulation of LHCB genes in gun4 mutant under stress may optimize the light energy transfer from LHCII to reaction center of PSII, contributing to the greater light energy transfer efficiency in LHCII/PSII despite low chlorophyll and carotenoids content. Furthermore, in the absence of light stress, gun4 plants showed lower values of ΦNO than WT. This reduction in constitutive loss of energy can be a flexibility of gun4 LHCII to maximize the energy transfer to reaction center of PSII, contributing for the greater photochemical capacity of gun4 plants.

4.2 GUN4 mutation minimize the effects of light stress

The decrease in Fv/Fm and the increase in Fo in both genotypes after 28 h of light stress suggest the occurrence of photoinhibition on photochemical apparatus (Maxwell and Johnson 2000; Müller et al. 2001). The increases in Fo can be related to damages in the reaction center of PSII and/or in the LHCII (Demmig-Adams and Adams 1992; Bolhàr-Nordenkampf and Öquist 1993). Damages on PSII light stress-induced can also be associated with increases in unregulated energy dissipation of PSII (ΦNO) (Klughammer and Schreiber 2008). Indeed it was observed increases in ΦNO in both genotypes after 14 h of light stress (data not shown). It is important to highlight that although light stress was sufficient to induce damages on PSII of both WT and gun4 mutant, the decreases observed in Fv/Fm and the increases in Fo after severe light stress were minor in gun4 plants. Furthermore, WT plants were unable to keep a high qN value after 28 h of light stress as observed in gun4 plants. Thus, considering that gun4 mutant absorbs less light energy (lower Fo values) and has greater capacity to dissipate energy (higher qN values), it suggests that the oxidative damage on PSII light stress-induced is minor in gun4 plants. However, the mechanism by which gun4 plants dissipate more efficiently the excess of energy absorbed is still unknown.

4.3 Total 14CO2 leaf absorption suggests a diffusive limitation for photosynthesis in gun4 mutant

Although gun4 plants showed lower values of stomatal conductance (g s) and net photosynthetic rate (A) compared to WT in the absence of stress, no differences were detected between the genotypes in the total 14CO2 fixation as well as in starch fraction after 14CO2 fixation, a photosynthetic product accumulated in the light (Zeeman et al. 2007). It suggests that the lower capacity for CO2 fixation observed in gun4 plants in absence of stress can be related to the diffusional limitation of CO2 rather than a biochemical limitation, once the feeding experiment was carried out under saturating levels of CO2 where stomata present no barrier to CO2 assimilation.

5 Conclusions

Taken together, the results of this work showed that GUN4 mutation have high impact on gas exchange through the leaves, photosynthetic pigment contents, and on light energy transfer capacity of the PSII. It suggests that this gene is important not only for the chlorophyll biosynthesis but also for process that regulates the transfer of energy from LHCII to reaction center of PSII. Furthermore, because of the phenotype displayed by gun4 mutant differs from other gun mutants, it indicates that gun4 phenotype cannot be explained solely by the reduction in chlorophyll content. The flexibility of gun4 PSII to maximize light energy transfer reflects an acclimation of LHCII in order to compensate the reduced ability of light absorption. It highlights the importance of gun4 mutant to investigate the mechanisms that control LHCII plasticity, an important aspect to understand plant light stress tolerance.