# Guanosine tetraphosphate (ppGpp) signalling promotes high-light tolerance in Arabidopsis thaliana

Shanna Romand, Chen Hu, Aurélie Crepin, Wojciech Nawrocki, Cécile Lecampion, Brigitte Ksas, Sylvie Citerne, Michel Terese, Matteo Sugliani, Stefano Caffarri, et al.

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Shanna Romand, Chen Hu, Aurélie Crepin, Wojciech Nawrocki, Cécile Lecampion, et al. Guanosine tetraphosphate (ppGpp) signalling promotes high-light tolerance in Arabidopsis thaliana. 2025. hal-05356649

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Distributed under a Creative Commons Attribution 4.0 International License
Guanosine tetraphosphate (ppGpp) signalling promotes
high-light tolerance in *Arabidopsis thaliana*.

Shanna Romand<fnref n="1" />, Chen Hu<fnref n="2" />, Aurélie Crepin<fnref n="1" />, Wojciech Nawrocki<fnref n="2" />, Cécile Lecampion<fnref n="1" />, Brigitte Ksas<fnref n="3" />, Sylvie Citerne<fnref n="4" />, Michel Terese<fnref n="5" />, Matteo Sugliani<fnref n="1" />, Stefano Caffarri<fnref n="1" />, Roberta Croce<fnref n="2" />, Michel Havaux<fnref n="3" />, Ben Field<fnref n="1" />

**Abstract**

Guanosine tetraphosphate (ppGpp) is a hyperphosphorylated nucleotide originally discovered in prokaryotes and found in the chloroplasts of plants and algae. In plants, ppGpp signalling plays a role as a regulator of photosynthetic activity, which is important in acclimation to environmental stresses such as nitrogen limitation. However, the full range of stresses involving ppGpp signalling is not yet known. Here, we investigated the role of ppGpp accumulation in the acclimation of plants to high light. We found that the over-accumulation of ppGpp in transgenic lines that overexpress *RSH3* (OX:RSH3) increases tolerance to high light intensity. Although ppGpp leads to higher non-photochemical energy dissipation (NPQ) than in wild-type plants, we show that NPQ is not critical for the enhanced high-light tolerance. Rather, our results show that ppGpp accumulation leads to broad changes in plant physiology that prime plants to resist and subsequently recover from high light exposure. ppGpp levels themselves also increase in response to high light exposure, suggesting that ppGpp signalling may play a physiological role in high light acclimation. Our work highlights the importance of ppGpp signalling in plant stress acclimation.

<footnote n="1">¹ Aix-Marseille University, CEA, CNRS, BIAM, LGBP Team, Marseille, France.</footnote>

<footnote n="2">² Biophysics of Photosynthesis, Department of Physics and Astronomy, Faculty of Science, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.</footnote>

<footnote n="3">³ Aix-Marseille University, CEA, CNRS, BIAM, PEPSS Team, Saint-Paul-lez-Durance, France.</footnote>

<footnote n="4">⁴ Institut Jean-Pierre Bourgin, INRAE, AgroParisTech, CNRS, Université Paris-Saclay, 78000 Versailles, France</footnote>

<footnote n="5">⁵ Current address: Independent researcher, Lyon, France.</footnote>
**Introduction**

Sunlight is essential for driving photosynthesis and growth in plants. However, light quality and intensity can change rapidly, necessitating rapid acclimation mechanisms to optimise growth rates and protect cellular components. Indeed, the absorption of excess energy by the photosynthetic machinery can promote the over-excitation of chlorophyll and the formation of singlet oxygen (¹O₂) and other reactive oxygen species (ROS), sources of oxidative stress that damage lipids and proteins in the thylakoid membranes (Lee and Kim 2024).

Plants possess both constitutive and inducible mechanisms for dissipating the excess energy absorbed by the photosynthetic machinery harmlessly in the form of heat. The main inducible mechanism for energy dissipation is known as non-photochemical quenching (NPQ) (Bassi and Dall'Osto 2021; Ruban and Wilson 2021). NPQ consists of several components, including the well-known qE component. qE is induced within seconds by excess-light triggered acidification of the thylakoid lumen, reaches its maximum after a few minutes, and relaxes rapidly in the dark. During qE, lumen acidification is sensed by the PsbS protein, which is then thought to alter the conformation of the light-harvesting antenna of PSII (LHCII and monomeric Lhcb) to promote quenching. Lumen acidification also activates the lumen-localised violaxanthin de-epoxidase (VDE) to produce the xanthophyll zeaxanthin, which enhances NPQ.

The ppGpp signalling pathway is emerging as another major stress acclimation mechanism in the chloroplast. ppGpp (or guanosine 3',5'-diphosphate) is synthesised from ATP and GDP/GTP in the chloroplast by nuclear-encoded enzymes of the RelA/SpoT (RSH) homolog family (Boniecka et al. 2017; Mehrez et al. 2023). Arabidopsis encodes four RSH proteins, grouped into three subfamilies: RSH1, a ppGpp hydrolase that lacks synthase activity; RSH2 and RSH3, which function redundantly as bifunctional synthases/hydrolases; and the calcium-activated CRSH (Ca²⁺-dependent RSH) that is activated in the dark (Mehrez et al. 2023). We previously showed that RSH3 is directly controlled by the TOR kinase pathway, linking chloroplast ppGpp metabolism to central growth regulation (D'Alessandro et al. 2024).

In prokaryotes, ppGpp (and other 3',5'-phosphorylated guanosine nucleotides) regulate bacterial growth and promote stress acclimation (Irving et al. 2021). In plants, ppGpp is associated with stress acclimation, and is known to be involved in the regulation of plant growth and development (Sugliani et al. 2016; Ono et al. 2021; Turkan et al. 2023), plant immunity (Abdelkefi et al. 2018; Qiu et al. 2023), and responses to nitrogen deprivation (Maekawa et al. 2015; Honoki et al. 2018; Goto et al. 2022; Li et al. 2022a; Romand et al. 2022). Over-accumulation of ppGpp in the chloroplast can down-regulate photosynthetic activity in both land plants (Maekawa et al. 2015; Sugliani et al. 2016; Honoki et al. 2018; Harchouni et al. 2022; Ito et al. 2022; Li et al. 2022a) and algae (Avilan et al. 2021). ppGpp is likely to control these processes via the downregulation of plastid gene expression (Mehrez et al. 2023), nucleotide metabolism (Nomura et al. 2014a, 2014b; Romand et al. 2022) and other processes (Zegarra et al. 2025). Recently, ppGpp was shown to play a physiological role in the acclimation
of Arabidopsis to nitrogen starvation by inducing the remodelling of the photosynthetic electron transport chain to reduce photosynthetic activity and reduce oxidative stress (Romand et al. 2022). Intriguingly, ppGpp over-accumulation in Arabidopsis also promotes a strong NPQ (Honoki et al. 2018) suggesting a potential mechanism for reducing oxidative stress during nitrogen starvation.

The increased NPQ in response to ppGpp over-accumulation, along with its role in protecting plants from oxidative stress during nitrogen starvation, led us to ask whether ppGpp might contribute to high light acclimation in Arabidopsis- a situation in which the photosynthetic machinery absorbs excess energy. In this study, we show that the accumulation of ppGpp in transgenic lines that overexpress RSH3 (OX:RSH3) indeed increases tolerance to high light intensity. However, we show that NPQ is surprisingly not critical for this increased high-light tolerance. Rather, we find that ppGpp accumulation promotes high-light tolerance at multiple levels, effectively priming OX:RSH3 plants for exposure to high light. Finally, we find that ppGpp increases in response to high light in wild type plants, suggesting that ppGpp can play a direct role in the acclimation of plants to high light stress.

## Results

### ppGpp accumulation improves high light tolerance

ppGpp signalling protects plants against photo-oxidative stress during nitrogen limitation (Romand et al. 2022). We therefore tested whether ppGpp over-accumulation alters the resistance of plants to high light induced photooxidative stress. We exposed OX:RSH3 plants, which have around 6 times higher ppGpp levels than the wild type (Bartoli et al. 2020), to sustained high light stress. While the wild type showed extensive photobleaching after 48 hrs of high light treatment, OX:RSH3 plants showed almost none (Fig. 1a). In agreement with this observation, chemiluminescence imaging of peroxidised lipids revealed the presence of significant amounts of peroxidised lipids in the wild type but not in OX:RSH3 (Fig. 1b). Quantification of lipid peroxidation products (hydroxy-octadecatrienoic acids [HOTEs], oxidation products of linolenic acid, the main fatty acid in Arabidopsis leaves) confirmed these results: the wild-type line showed a strong increase in HOTEs after exposure to stress while two independent OX:RSH3 lines did not (Fig. 1c). These results show that high ppGpp levels reduce oxidative damage under high light conditions.

### Alterations in the photosynthetic machinery in OX:RSH3 plants

Previously, an RSH3 overexpression line was shown to induce high levels of NPQ (Honoki et al. 2018). Higher NPQ levels could potentially explain the reduced oxidative damage and general high-light tolerance we observe in OX:RSH3 plants. We therefore measured NPQ in an OX:RSH3 line generated in our laboratory. The OX:RSH3 line showed a higher NPQ capacity when grown under control conditions compared to the wild-type line (Fig. 1d). These results
demonstrate that elevated ppGpp levels robustly induce a downstream increase in
photoprotective capacity.

In addition to NPQ, OX:RSH3 plants are known to show changes in photosystem architecture, with notably a reduced quantity of PSII core relative to PSII peripheral antenna, and reduced PSII functionality with a low Fv/Fm ratio due to increased basal fluorescence (F0) (Sugliani et al., 2016). We further investigated these changes to understand whether they could be involved in the high light tolerance. Immunoblotting and protein staining confirmed previous observations that two OX:RSH3 lines have a low PSII core to peripheral antenna ratio (Fig. S1, CP43 versus Lhcb1,2). The effects of ppGpp over-accumulation on the PSI core to peripheral antenna ratio was less clear (PsaA versus Lhca1), as it was lower in OX:RSH3.1 and similar to the wild-type control in OX:RSH3.2. OX:RSH3.1 has a slightly stronger phenotype than OX:RSH3.2, and biochemical data also previously suggested lower amounts of PSI core complex subunits in OX:RSH3.1 (Sugliani et al. 2016). Reduced quantities of subunits for cytochrome b6f (Cyt f) and ATP synthase (AtpC) were also observed in both RSH3 over-expression lines. A marked difference compared to previous studies on RSH3 overexpression lines is that Rubisco large subunit (RBCL) accumulation was similar to that in the wild type control. This is very likely due to differences in protein normalisation, which was performed against total chlorophyll levels here compared to against total protein content previously. Finally, immunoblotting showed that levels of PsbS, a key protein in NPQ, were higher in both RSH3 overexpression lines.

Photosystem parameters were then estimated using electrochromic shift measurements in the presence of inhibitors. We noted an increase in PSII photochemical rate in the high ppGpp lines compared to the wild type, although this was significant only for OX:RSH3.2 (Fig. S2). An increased PSII photochemical rate is consistent with the presence of more peripheral antennae per PSII core complex, as also shown by immunoblotting here (Fig. S1) and previously (Sugliani et al. 2016). The PSI rate was significantly faster in both lines, suggesting likewise that there are more antenna per PSI core complex. The effect of ppGpp accumulation did not cause a striking change in the relative quantity of PSI (the PSI/(PSI+PSII) ratio).

Finally, we measured in vivo chlorophyll fluorescence emission at 77 K in plants in state 1. Analysis of the fluorescence spectra revealed a significant increase in the shoulder on the short-wavelength side of the PSII emission peak in RSH3 overexpressers compared to the wild type (Fig. 1e-g; global test, P < 0.00025). The maximal difference was observed between 677 and 678 nm (95% simultaneous CI). LHCII trimers emit at lower wavelengths than the PSII core, indicating that in the overexpressers, part of the peripheral antenna is less efficiently coupled to the PSII core. Taken together with the electrochromic shift measurements and immunoblotting, these results suggest that RSH3 overexpressers not only have an increased antenna-to-PSII core ratio, but also retain a fraction of peripheral antenna complexes that are more loosely connected to the core.
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<figure><img src="image_7.png" /><figcaption>**Figure 1.** OX:RSH3 lines are tolerant to high light exposure. (A) Phenotypes of wild type and OX:RSH3.1 lines before (0h) and after 48 hours (48h) exposure to high light stress (1500 µmol of photons m⁻²s⁻¹ at 4°C) (B) Bioluminescence of peroxidised lipids after 48h high light exposure. Scale = 3 cm. (C) Quantification of hydroxy-octadecatrienoic acid (HOTE) content in plants exposed to high light intensity for 48h. (D) Measurement of NPQ kinetics in plants exposed to an actinic light of 1500 µmol photons m⁻²s⁻¹ (white bar) followed by a relaxation phase in the dark (black bar). Data shown in panels 1D, 2A, and S6 originate from the same experiment. (E) In vivo chlorophyll fluorescence emission spectra at 77 K. Samples were measured in state I. Peaks for PSII and PSI chlorophyll are indicated. Spectra were normalised to the PSII peak at 685 nm. (F) Enlarged view of the PSII region (660–685 nm). (G) Difference spectra across PSII region relative to wild type. Graphs show mean (C,D) or median (E,F,G) ± 95% CI; n = 4 biological replicates. Statistical differences were tested as described in the materials and methods. Letters indicate statistical groups.</figcaption></figure>
### OX:RSH3 tolerance to sustained high light is not dependent on NPQ

The increased NPQ in OX:RSH3 lines appeared a plausible mechanism for their tolerance to sustained high light. The rapid induction and dark relaxation kinetics suggest that the higher NPQ is due to an upregulation of the qE component of NPQ, which is dependent on PsbS and VDE. We therefore generated OX:RSH3.1 lines lacking PsbS (OX:RSH3 *npq4*) or VDE (OX:RSH3 *npq1*) for testing whether NPQ was required for high light tolerance.

We first confirmed that the *npq1* and *npq4* mutations in the OX:RSH3.1 genetic background effectively suppressed the high NPQ phenotype (Fig. 2a). Interestingly, we also observed that the double mutant lines, and in particular OX:RSH3 *npq1*, were larger and greener than the parental OX:RSH3.1 (Fig. S3a). This morphological difference was more pronounced when the double mutants were grown under short day conditions at the same light intensity (Fig. S3b).

The enhanced growth in OX:RSH3 *npq1* was accompanied by a partial restoration of wild-type PSII functionality (Fig. S4). This suggests that the OX:RSH3 growth and photosynthesis may be limited in part by xanthophylls under growth light conditions, and that altered xanthophyll composition in *npq1* can suppress these effects. We therefore analysed pigment pools in OX:RSH3 plants to determine which xanthophylls were responsible (Fig. S5). We found that growth light-acclimated OX:RSH3.1 plants showed a significantly higher VAZ (violaxanthin, antheraxanthin and zeaxanthin) pool than the wild type, with higher levels of the xanthophyll antheraxanthin (an intermediate in zeaxanthin biosynthesis) (Fig. S5). Interestingly, even zeaxanthin, usually associated with high light exposure, was detected in one of the OX:RSH3.1 replicates. In contrast, *npq1* plants did not accumulate detectable levels of zeaxanthin or antheraxanthin, though they showed similar VAZ content to the wild type thanks to the accumulation of violaxanthin. Surprisingly, in OX:RSH3 *npq1* antheraxanthin accumulation was restored to the higher levels found in OX:RSH3 plants. We note that trace amounts of antheraxanthin sometimes persist in *npq1*, and can vary in the presence of other mutant backgrounds (Niyogi et al. 1998; Gilmore 2001; Li et al. 2009). Furthermore, we observed that the VAZ pool in OX:RSH3 *npq1* was intermediate between OX:RSH3.1 and the wild type. This lower VAZ pool could explain the improved growth of OX:RSH3 *npq1* compared to OX:RSH3.1: the high VAZ levels in OX:RSH3 may induce the constitutive dissipation of useful energy. In summary, these unexpected results show that ppGpp-overaccumulation causes changes in the xanthophyll pool that could act to limit photosynthetic performance and growth.

Next, we tested the high light tolerance of the double mutants. Wild-type plants as well as the *npq1* and *npq4* mutant lines showed extensive photobleaching after 48 hrs (Fig. 2b). OX:RSH3.1 again showed very little photobleaching. The double mutant line OX:RSH3 *npq1* and OX:RSH3 *npq4* showed more photobleaching than OX:RSH3, but at modest levels compared to the wild type and the *npq1* and *npq4* lines. Therefore, removal of PsbS or VDE has a limited effect on the tolerance of OX:RSH3 to sustained high light.
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<figure><img src="image_10.png" /><figcaption>**Figure 2.** PsbS and VDE are not required for the tolerance of OX:RSH3 to high light. (A) NPQ kinetics were acquired from 4-week-old plants of the indicated lines under an actinic light of 1200 µmol photons m⁻² s⁻¹. Curves represent median values ± 95% confidence interval, n = 8 plants. Data shown in panels 1D, 2A, and S6 originate from the same experiment. Plants exposed to continuous high light (1500 µmol of photons m⁻² s⁻¹ at 4°C for 48h) after 4 weeks of growth in long day conditions were imaged to visualize photobleaching (B) and maximum PSII yield was measured at the indicated timepoints (C). The experiment was repeated more than 3 times in 3 different growth facilities with similar results. Graph shows medians ± 95% confidence intervals for four experimental replicates (circles, triangles, squares, crosses). Differences were tested using ANOVA and post-hoc testing. Letters indicate statistical groups with comparisons between lines at each timepoint. Scale bar, 3 cm.</figcaption></figure>
NPQ is important for attenuating photoinhibition in RSH3 overexpressers

Photobleaching is positively correlated with PSII photoinhibition, which occurs when the rate
of oxidative damage to PsbA (a central protein of the PSII reaction centre) exceeds the rate of
repair (Pinnola and Bassi 2018; Nawrocki et al. 2021). We therefore tested the susceptibility
of RSH3 overexpressers to photoinhibition.

In control conditions, OX:RSH3 plants were small and pale, and showed a lower Fv/Fm ratio
than the control (Fig. 2c), as previously observed (Sugliani et al., 2016). As discussed above,
the low Fv/Fm in OX:RSH3 is a result of a high basal fluorescence (F0) caused by the
disconnection of a fraction of LHCII from the PSII core, and is unlikely to reflect the direct
impairment of PSII core function itself. The OX:RSH3 *npq4* lines showed a similar Fv/Fm ratio,
while OX:RSH3 *npq1* showed a ratio between OX:RSH3 and the wild type.

After 24h of high-light exposure, all lines showed a significant decrease in Fv/Fm as a result of photoinhibition. The OX:RSH3.1 *npq1* and OX:RSH3.1 *npq4* lines showed a similar response to *npq1* and *npq4*, with significantly lower Fv/Fm ratios than OX:RSH3.1 or the wild-type (Fig. 2c, note that statistical groups shown are between lines at individual timepoints). After 48h Fv/Fm ratios were similar to those observed after 24h. Nevertheless, despite higher photoinhibition in OX:RSH3.1 *npq1* and OX:RSH3.1 *npq4* we do not observe as much photobleaching as in the wild type. This suggests that, although OX:RSH3 plants experience photoinhibition, elevated ppGpp levels confer a relatively stronger long-term photoprotective effect, allowing them to better cope with sustained damage to the photosynthetic apparatus.

We also obtained NPQ kinetics after high light stress exposure (Fig. S6). NPQ remained higher in OX:RSH3 than in the wild type even after high light exposure. For *npq1*, *npq4*, OX:RSH3.1 *npq1* and OX:RSH3.1 *npq4* the kinetics were relatively similar to those obtained under control conditions, with a low maximum NPQ. Interestingly, for all lines we found that NPQ capacity decreased after exposure to high light. This phenomenon likely reflects competition from photoinhibition-related ql quenching (Nawrocki et al. 2021), together with reduced PSII activity, which reduces lumen acidification and the triggering of qE.

NPQ is also important for the initial response of OX:RSH3 plants to high light

Photobleaching and photoinhibition after 24-48h is the result of short and long-term
processes, both of which may be influenced by ppGpp. To gain insights into the initial
sensitivity of OX:RSH3 plants to high light we therefore quantified photoinhibition in short
time-course experiments on detached leaves (Fig. 3).

We observed the development of strong photoinhibition in all lines after 2h of exposure to
high light (Fig. 3c). To compare the relative effects between lines we calculated the Fv/Fm
normalized to Fv/Fm before high light treatment (0h). Using this measure, the relative
responses of OX:RSH3.1 and the WT to 2h high light were the same, dropping to 74% and 72%
of starting Fv/Fm respectively (Fig. 3d). *npq1* and *npq4* showed higher sensitivity than the wild type (68% and 64% of starting Fv/Fm). Strikingly however, OX:RSH3 *npq1* and OX:RSH3.1 *npq4* showed significantly higher sensitivity than either of their parental lines (56% and 57% of starting Fv/Fm). The same trend between the lines was also observed during recovery from high light exposure. These results show that OX:RSH3 plants have a similar photoinhibition sensitivity to the wild type. However, the *npq* mutant analyses indicate that the qE component of NPQ plays a more important role in protecting OX:RSH3 plants against short-term photoinhibition than in plants with wild-type ppGpp levels (Fig. 3d). Over longer exposures, however, this dependence on qE appears to diminish, suggesting that additional ppGpp-driven protective mechanisms come into play (Fig. 2c).
<figure><img src="image_11.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 3. Removal of PsbS or VDE increases photoinhibition sensitivity in OX:RSH3 plants.** A short high light exposure protocol (A) was used on detached leaves of the indicated genotypes (B). Maximum PSII yield (Fv/Fm) was measured during short high light exposure and is presented as absolute Fv/Fm (C) or as normalized Fv/Fm, setting the average Fv/Fm before high light exposure to 1 (D). Graphs show medians ± 95% confidence intervals for three experimental replicates (circles, triangles, and squares) on 12 detached leaves. Differences were tested using ANOVA and post-hoc testing. Letters indicate statistical groups with comparisons between lines at each timepoint.</figcaption></figure>
Alteration of PSII architecture restores susceptibility of OX:RSH3 to high light.

Our previous experiments indicated that NPQ is not critical for the tolerance of OX:RSH3 to
high light induced photobleaching. We next asked whether high light tolerance is a general
feature of plants with low chlorophyll content like OX:RSH3, and whether modifying the high
LHCII/PSII RC ratio in OX:RSH3 plants could affect sensitivity.

To explore these two possibilities, we worked with two new genetic backgrounds. We crossed OX:RSH3.1 with the mutant *chlorina 1* (*ch1*). The *ch1* line exhibits a deficiency in chlorophyll b due to the presence of a mutation in the chlorophyllide a oxygenase gene (*CAO*) (Espineda et al., 1999). This leads to a lack of the majority of the LHCII proteins that require chlorophyll b for assembly (Reinsberg et al. 2001), resulting in the accumulation of PSII core without peripheral antenna (Havaux et al. 2007). The OX:RSH3 *ch1* line therefore has a low LHCII/PSII RC ratio due to the lack of LHCII antenna. However, ECS measurements also indicate that the relative PSI fraction is lower in the *ch1* mutant and in OX:RSH3 *ch1*, with about 25% total PSI, rather than the 45% total PSI found in OX:RSH3.1 and the wild type (Fig. S7). We also tested the *GOLDEN-LIKE* (*GLK*) double mutant *glk1,2* that lacks the GLK1 and GL2 transcription factors that regulate the expression of nuclear-encoded genes for photosynthesis and chlorophyll biosynthesis (Waters et al. 2009; Li et al. 2022b). *glk1,2*, has low chlorophyll levels due to reduced expression of nuclear-encoded photosynthetic genes but display wild-type-like photosynthetic parameters (Liu et al. 2018).

*ch1*, OX:RSH3.1 *ch1* and *glk1,2* plants are small, pale and show different developmental rates. We therefore analysed high light tolerance at equivalent developmental stages. Strikingly, even after 24h of exposure to high light *ch1* and OX:RSH3.1 *ch1* plants already showed significant areas of photobleaching on the leaves (Fig. 4a). In contrast, only one leaf showed photobleaching in the wild type and none in the OX:RSH3.1 line. Unexpectedly, *glk1,2* showed a similar sensitivity to high light as *ch1*. After 48h all leaves of the *ch1*, OX:RSH3.1 *ch1* and *glk1,2* plants were heavily photobleached. Before high light exposure, *ch1* and *glk1,2* showed similar Fv/Fm ratios to the wild type (Fig. 4b). After 24h high light exposure, *ch1* and OX:RSH3.1 *ch1* showed a strong decrease in Fv/Fm that was larger than in the other lines.

We then quantified the short-term induction of photoinhibition in these lines, using both
absolute values (Fig. 5a) and values after normalisation of the starting Fv/Fm to 1 (Fig. 5b). In
*ch1*, OX:RSH3.1 *ch1* and *glk1,2* the relative Fv/Fm dropped to significantly lower levels (57%,
51% and 61% of starting levels respectively) than in the wild-type control or OX:RSH3 after 2h
of high light (70% of starting levels). We also observed that photoinhibition continued to
develop in *ch1* and OX:RSH3.1 *ch1* even after the first 30 minutes of recovery. In contrast to
OX:RSH3.1 and the wild type, *glk1,2* showed an impaired recovery and considerable variability
between experimental replicates, with one replicate showing similar photoinhibition
sensitivity to *ch1* and OX:RSH3.1 *ch1*. These results indicate that *ch1* and OX:RSH3.1 *ch1* are
more sensitive to PSII photoinhibition compared to the OX:RSH3.1 line and the wild type,
consistent with their greater visible sensitivity to photobleaching.

In summary, loss of *CAO* is sufficient to completely suppress the tolerance of OX:RSH3 to high
light in OX:RSH3 *ch1*. *ch1* plants show enhanced production of singlet oxygen at the exposed
PSII core in response to high light (Triantaphylidès et al. 2008; Dall'Osto et al. 2010). This
excess singlet oxygen production is likely to overwhelm any recovery capacity conferred by
ppGpp over-accumulation, and also suggests that the architecture of the photosynthetic
machinery is an important factor for high light tolerance. Likewise, *glk1,2* plants showed an
unexpected sensitivity to photo-bleaching under high light. Photoinhibition sensitivity was
higher and recovery longer than in the wild type, though not as striking as in *ch1* (Fig. 5).
Therefore, the photobleaching sensitivity of *glk1,2* may also result from impaired long-term
acclimation processes, potentially linked to disrupted coordination between nuclear and
chloroplast gene expression in the absence of the GLK transcription factors.
<figure><img src="image_15.png" /><figcaption>**Figure 4.** Removal of peripheral antenna system of PSII confers high-light sensitivity to OX:RSH3. Plants grown to the same developmental stage were exposed to continuous high light (1500 µmol of photons m⁻²s⁻¹ at 4°C for 48h) were imaged to visualize photobleaching (A) and maximum PSII yield was measured at the indicated timepoints (B). Graph shows medians ± 95% confidence intervals for three experimental replicates (circles, triangles, and squares). Differences were tested using ANOVA and post-hoc testing. Letters indicate statistical groups with comparisons between lines at each timepoint.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**Figure 5. Differential photoinhibition sensitivity in *chlorina1* and *goldenlike* mutants.** Maximum PSII yield (Fv/Fm) was measured during short high light exposure and is presented as absolute Fv/Fm values (A) or as normalized Fv/Fm, setting the average Fv/Fm before high light exposure to 1 (B). Graphs show medians ± 95% confidence intervals for three experimental replicates (circles, triangles, and squares) on 12 detached leaves. Differences were tested using ANOVA and post-hoc testing. Letters indicate statistical groups with comparisons between lines at each timepoint.</figcaption></figure>
ppGpp is involved in priming plants for high light acclimation

To identify additional mechanisms that could explain ppGpp-induced high light tolerance we looked at previous data on differential transcript accumulation in OX:RSH3 plants grown under standard conditions (Abdelkefi et al. 2018). We noticed that OX:RSH3 plants show increased accumulation of several transcripts for enzymes involved in responding to high light or oxidative stress including *EARLY LIGHT INDUCIBLE PROTEIN* (*ELIP1* and *ELIP2*), *HYDROPEROXIDE LYASE 1* (*HPL1*), *COPPER/ZINC SUPEROXIDE DISMUTASE 2* (*CSD2*), *STROMAL ASCORBATE PEROXIDASE* (*SAPX*) and methionine sulfoxide reductases (*MSRB6* and *MSRB7*). This suggests, that even under normal growth conditions, OX:RSH3 plants may be constitutively primed for tolerance to high-light exposure. To confirm the relevance of these elevated transcript levels, we tested ELIP2 protein levels by immunoblotting. We observed significantly higher levels of ELIP2 in OX:RSH3.1 plants after a short exposure to high light (Fig. 6a,b).

Building on this transcriptional priming, we next examined whether antioxidant pools were also affected. Tocopherols and plastochromanol-8 are important lipophilic antioxidants capable of quenching singlet oxygen (Havaux 2020). α-Tocopherol, the most abundant antioxidant, accumulated to significantly higher levels in OX:RSH3.1 compared to the wild type with intermediate levels in OX:RSH3.2 (Fig. 6c). γ-tocopherol and plastochromonal-8 also showed a significant increase in OX:RSH3 lines as compared to WT plants.

We then turned to the role of pigments. As shown above, photoprotective xanthophyll pigments are altered under standard conditions in OX:RSH3 lines (Fig. S5). Notably, the regulation of xanthophyll accumulation in response to high light was also altered. OX:RSH3.1 plants showed significantly higher levels of VAZ after a short high light treatment (Fig. 6d, Fig. S8). These elevated levels persisted during the recovery phase, particularly for zeaxanthin and antheraxanthin, leading to a higher DEPS ratio.

Finally, we asked whether ppGpp might itself behave dynamically under high light. To do this, we measured ppGpp accumulation during exposure, and found that ppGpp increased significantly after 45 minutes HL exposure, but remained unchanged under control conditions (Fig. 6e). The dynamics beyond this point were not determined.

Altogether, our results support the view that ppGpp accumulation is involved in upregulation of oxidative-stress responsive genes and enrichment of the lipophilic antioxidants tocopherol and plastochromanol-8 (Fig. 6), along with photoprotection-boosting changes in the xanthophyll pool (Fig. S5). In addition, ppGpp enhances NPQ, VAZ induction, and ELIP2 protein accumulation during high-light exposure itself (Fig. 1, 6a-c). This broad activation of protective mechanisms contributes to high-light tolerance in ppGpp-overaccumulating plants. Importantly, the accumulation of ppGpp itself in response to high light is consistent with
ppGpp acting as a physiologically relevant signal that promotes high-light acclimation, the
longer-term dynamics of this response remain to be investigated.
<figure><img src="image_17.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>PC-8</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_22.png" /></figure>

**Figure 6.** *ppGpp primes plants for high light acclimation.* (A) Immunoblot showing the abundance of ELIP2 in 20 µg total protein extracted from 4-week old plants exposed to 1500 µmol of photons m⁻²s⁻¹ (4°C) for 2h (HL). Total proteins were revealed using Ponceau red stain (PR), 3-4 biological replicates. (B) Quantification of ELIP2 relative protein abundance from immunoblots. (C) Antioxidant content in 4-week old plants grown under standard conditions. α-TC, α-tocopherol; γ-TC, γ-tocopherol and PC-8, plastochromanol-8. (D) Combined amount of violaxanthin, antheraxanthin and zeaxanthin (VAZ) following 2h HL exposure (HL) and 1h of recovery under standard lighting (REC). Values normalized to 100 total chlorophyll (a+b). (E) ppGpp content in plants grown under standard conditions with growth light (GL) for 4 weeks and then exposed to 1500 µmol of photons m⁻²s⁻¹ at 4°C (HL) for the indicated times. Graphs show mean ± 95% confidence interval, n = 3-4 biological replicates. Differences were tested using ANOVA and post-hoc testing. Letters indicate statistical groups.
Discussion

Here, we show that ppGpp accumulation promotes tolerance to high light in Arabidopsis. We first extended previous work characterizing the photosynthetic phenotype of ppGpp-overaccumulating plants (Sugliani et al. 2016; Honoki et al. 2018), providing additional data on photosynthetic complex composition, and confirming the elevated NPQ capacity of these plants (Fig. 1). We then found that while the qE component of NPQ is particularly important for moderating sensitivity to photoinhibition in ppGpp over-accumulators, it is not essential for resistance to photobleaching (Fig. 2,3). OX:RSH3 plants have reduced chlorophyll content, yet photobleaching resistance was not a general feature of two other low chlorophyll lines (Fig. 4). However, the origins of the chlorophyll deficiency differ: in one case chlorophyll levels are lower while the antenna remains large, whereas in the other the antenna itself is reduced. These differences make direct comparison difficult. Instead, ppGpp over-accumulators showed evidence of constitutive priming for high-light resistance with increased expression of genes responsive to high light or oxidative stress, elevated ELIP expression, and larger antioxidant pools (Fig. 6). ppGpp therefore appears to promote a broad spectrum of responses, including NPQ, that together confer resilience against high light. Interestingly, ppGpp levels themselves also rise in response to high light exposure (Fig. 6), suggesting that ppGpp signalling plays a physiological role in acclimation to high light stress. This adds to the growing list of roles for ppGpp in plant physiology (Mehrez et al. 2023), and underscores its importance in plant stress acclimation.

How is ppGpp able to induce constitutive priming? The principal known mode of action for ppGpp signalling in vivo involves repression of chloroplast transcription and translation (Mehrez et al. 2023), that is, plastid gene expression. Repression of plastid gene expression is also a well-known trigger for retrograde signalling to the nucleus (Liebers et al. 2022). Such signalling provides a plausible route through which ppGpp could activate the transcription of high-light and oxidative-stress responsive genes. In support of this view, the high light sensitivity of *glk1,2*, which is likely due to the disruption of retrograde signalling, underscores the importance of correct coordination between the nuclear and chloroplast genomes. Beyond this, in vitro studies have also implicated nucleotide metabolism and protein trafficking as additional targets of ppGpp (Nomura et al. 2014a, 2014b; Zegarra et al. 2025), suggesting that multiple pathways may influence the priming response.

ppGpp accumulation also induces changes in the architecture of the photosynthetic machinery, such as the relative enlargement of the PSII peripheral antenna system. Counterintuitively, the presence of disconnected antenna would be expected to increase sensitivity by enhancing singlet oxygen production, yet OX:RSH3 plants are more resistant to high light. This suggests that additional protective mechanisms must outweigh this potential risk. In particular, the LHCII antenna- although partially disconnected from PSII in OX:RSH3 and therefore expected to be vulnerable -appears to be well protected, at least in part through mechanisms associated with the VAZ xanthophyll cycle and PsbS, which we show are
enhanced by ppGpp accumulation (Fig. S1, S5, S8). Increased ppGpp levels might also be
expected to disrupt the repair cycle of the PSII RC protein D1, which is critical during
photoinhibition and recovery. Surprisingly, however, OX:RSH3 plants show almost identical
photoinhibition and recovery to wild type, suggesting that ppGpp does not have a major
impact on the repair cycle (Fig. 3D). Nevertheless, removal of VDE or PSBS (in npq1 and npq4)
leads to stronger photoinhibition and slower recovery in OX:RSH3 than in either mutant alone
(Fig. 3D). This indicates an interaction between ppGpp signalling and qE, which we speculate
may relate to the enlarged PSII peripheral antenna system in ppGpp-accumulating plants. In
conclusion, while the qE component of NPQ is important for the initial development of
photoinhibition in OX:RSH3 plants, our results reveal that it is only one of several high-light
acclimation processes triggered by ppGpp accumulation. Together, these processes
contribute to the enhanced high-light resistance observed in ppGpp-accumulating plants.

While these mechanisms enhance high-light resistance in OX:RSH3 plants, they also appear to
come at a cost to growth. Over-accumulation of ppGpp often leads to reduced plant growth,
at least under standard nutrient-replete growth conditions (Sugliani et al. 2016; Goto et al.
2022; Li et al. 2022a). Surprisingly, we observed that crossing OX:RSH3.1 with the npq1 mutant
(and to a certain extent npq4) partially restored a wild-type growth phenotype (Fig. S3).
Pigment content, Fv/Fm and ΦPSII also shifted towards wild-type levels in OX:RSH3.1 npq1
(Fig. 3, S4, S5). These changes are reminiscent of reports showing that increased zeaxanthin
accumulation reduces growth in the npq2 mutant (Dall'Osto et al. 2005), and that plants
deficient for PsbS (npq4 mutant line) accumulate more biomass than wild-type plants under
low-light conditions (Khuong et al. 2019). Together, these findings reinforce the idea that
altered NPQ activity and changes in the xanthophyll pool may contribute to the light-limited
phenotype of OX:RSH3 plants by promoting constitutive dissipation of usable energy.

Acknowledgements

We thank Muriel Reissolet and Patrice Crete for plant care, Chloé Maurin and Theo Mege for
assistance with immunoblotting, and Julia Bartoli and Emmanuelle Bouveret for supplying
isotope labeled ppGpp. Nucleotide measurements were performed on the IJPB Plant
Observatory technological platform with support from Saclay Plant Sciences-SPS (ANR-17-
EUR-0007). Work was funded by the Agence Nationale de la Recherche (ANR-17-CE13-0005,
ANR-22-CE20-0033), and work at VU Amsterdam was supported by the Dutch Organization
for Scientific research via a TOP grant.
# Material and Methods

## Plant material and growing conditions

All *Arabidopsis thaliana* lines used in this study are listed in Table 1. Seeds were sown and germinated on potting soil and the seedlings were transferred to individual pots 7 days after germination. The plants were then grown in a growth chamber with a photoperiod of 16h light (at 22°C) for 8h darkness (at 19.5°C) with LED illumination of 115 µmol photons m⁻² s⁻¹, the plants received two weekly waterings and a weekly application of Coïc-Lesaint fertilizer solution.

<table><caption>Table 1: Lines used in this study.</caption><thead><tr><td>Lines</td><td>Designation</td><td>Source or reference</td><td>Additional information</td></tr></thead><tbody><tr><td>Col-0</td><td>WT1, Columbia, N1093 (NASC)</td><td>Nottingham Arabidopsis</td><td></td></tr><tr><td>OX:RSH3.1</td><td>OX:RSH3.1 (H1)</td><td>(Sugliani et al. 2016)</td><td>Over expressor line, genetic background Col-0</td></tr><tr><td>OX:RSH3.2</td><td>OX:RSH3.2 (G8)</td><td>(Sugliani et al. 2016)</td><td>Over expressor, genetic background Col-0</td></tr><tr><td>npq1</td><td>npq1-2</td><td>(Niyogi et al. 1998)</td><td>Genetic background Col-0</td></tr><tr><td>npq4</td><td>npq4-1</td><td>(Li et al. 2000)</td><td>Genetic background Col-0</td></tr><tr><td>OX:RSH3.1 npq1</td><td>OX:RSH3.1 npq1-2</td><td>This study</td><td></td></tr><tr><td>OX:RSH3.1 npq4</td><td>OX:RSH3.1 npq4-1</td><td>This study</td><td></td></tr><tr><td>ch1</td><td>ch1-1</td><td>(Espineda et al. 1999)</td><td></td></tr><tr><td>glk1,2</td><td>glk1,2</td><td>(Fitter et al. 2002)</td><td>Seeds were provided by Jane Langdale</td></tr><tr><td>OX:RSH3.1 ch1</td><td>OX:RSH3.1 ch1-1</td><td>This study</td><td></td></tr></tbody></table>

## Chlorophyll fluorescence

Plants were placed in the dark for 20 min and chlorophyll fluorescence was measured using the Fluorcam FC 800-O fluorometer (Photon system Instruments). The maximum quantum yield of PSII was calculated according to the formula: (Fm-Fo)/Fm. NPQ was calculated according to the formula NPQ = (Fm-Fm')/Fm'.

## Exposure to high light

Plants or detached leaves were placed in a controlled environment culture chamber under LED lighting set at a continuous light intensity of 1500 µmol photons m⁻² s⁻¹ at 4°C. For detached leaf experiments, the 5th and 6th rosette leaves were selected.
Measurement of the chlorophyll fluorescence emission spectrum at 77K

4-week-old plants were placed under far-red light for 60 min, to induce state I, then harvested and ground in liquid nitrogen. 50 µL of buffer (100% (w/v) glycerol, 10 mM Hepes - KOH, pH 7.5) was added to 30 mg of sample in a Pasteur pipette. Chlorophyll fluorescence spectra were obtained using a CARY Eclipse fluorescence spectrophotometer [Varian]. The excitation light was set to 440 nm (maximum absorption wavelength of chlorophyll). The emission spectrum was measured by scanning between 600 and 800 nm. The spectra were normalised to the peak at 685 nm (max PSII emission).

Quantification of HOTEs

Lipids were extracted from about 300 mg of leaves in methanol/chloroform and analyzed by HPLC–UV as described previously (Montillet et al. 2004; Shumbe et al. 2017).

Immunoblotting

Protein extraction and immunoblotting were performed as described previously (Sugliani et
al. 2016). Proteins were normalized on a total chlorophyll or total protein basis. Primary
antibodies (supplied by Agrisera) were used against AtpC, CP43, Cyt f, ELIP2, Lhca 1, PsaA and
PsbS. ELIP2 was quantified after normalising ELIP2 signal to total protein (PR) for each sample.

ECS measurements

The measurements were performed using a JTS-10 spectrophotometer as described (Hu et al. 2023), based on the method reported (Bailleul et al. 2010).

Pigment quantification

Pigments were extracted by addition of acetone 90% 10 mM Tris HCl pH 7.5 to 6 mm leaf discs, followed by a grinding with glass beads for 10 sec at a speed of 6 m.s⁻¹ in a bead beater (Bead Ruptor Elite, Omni International), and a 10 min centrifugation at maximum speed. The supernatant was passed through HPLC according to a protocol described previously (Campoli et al. 2009). The amount of each pigment was normalized to one hundred total chlorophyll. Deepoxidation state (DEPS) was calculated as (Z+0.5A)/VAZ, where Z = zeaxanthin, A = antheraxanthin and V = violaxanthin.

Quantification of antioxidants

Antioxidants (α- and g-tocopherol, plastochromanol (PC)-8) were extracted from leaf tissue and quantified by reverse-phase HPLC as previously described (Ksas et al. 2015). In brief, leaf samples were grinded in ethyl acetate. After centrifugation, the supernatant was filtered and evaporated on ice under a stream of N₂. The residue was recovered in methanol/hexane (17/1) and analyzed by HPLC with fluorescence detection. Separation of tocopherols and PC was performed on a Phenomenex Kinetex column (2.6 µm, 100 × 4.6 mm, 100 Å) in the isocratic mode with methanol/hexane (17/1) as the solvent system and a flow rate of 0.8 ml min⁻¹. Tocopherols and PC were detected by their fluorescence at 330 nm with
excitation at 290 nm. PC standard was a kind gift from Dr. J. Kruk (Krakow, Poland). Tocopherol standards were purchased from Sigma.

## Guanosine tetraphosphate quantification

Nucleotides were extracted quantified by HPLC-MS/MS using a stable isotope labelled ppGpp standard as described previously (Bartoli et al. 2020).

## Data analysis

Analyses were performed in R (R Development Core Team, 2020) using custom annotated R scripts. Command-line scripts were created to pre-process data obtained from the Fluorcam O-800, extract parameters, and calculate the photosynthetic indices. Graphs were produced using the ggplot2 package (Wickham 2009) with confidence intervals determined for normally distributed data using the Rmisc package (Hope 2013), and non-parametric data with bootstrap confidence interval estimation using the boot package (Davison and Hinkley 1997; Canty and Ripley 2021). Statistical analyses were performed on normally distributed data using analysis of variance (ANOVA) followed by Tukey's post hoc test (Kassambara 2021) and on non-parametric data using aligned-ranks ANOVA (Wobbrock et al. 2011) with post-hoc pairwise comparisons performed using emmeans (Lenth 2024).

77K spectra were analyzed in R using median quantile GAMs with line-specific smooths, and contrasts versus wild type were assessed by difference spectra with simultaneous confidence intervals and global supremum tests (Fasiolo et al. 2021). All statistical tests included adjustments for multiple comparisons, and in figure panels 2C, 3C, 3D, 4B, 5A and 5B, the experimental replicates were included in the aligned-ranks ANOVA model as a random factor. The NPQ data shown in Figures 1D, 2A, and S6 were obtained from the same experiment; wild type and OX:RSH3.1 control data are therefore identical across these panels.

## Data availability

Raw and processed data for all figures are available in the Recherche Data Gouv repository under [temporary](https://doi.org/10.57745%2FYZCG1W&version=DRAFT). All R scripts used for analysis and figure generation are available on GitHub: [https://github.com/benfield99/Romand-et-al.-2025-analysis-scripts/](https://github.com/benfield99/Romand-et-al.-2025-analysis-scripts/)
Bibliography

Abdelkefi H, Sugliani M, Ke H, Harchouni S, Soubigou-Taconnat L, Citerne S, Mouille G, Fakhfakh H, Robaglia C, and Field B. Guanosine tetraphosphate modulates salicylic acid signalling and the resistance of *Arabidopsis thaliana* to *Turnip mosaic virus*. Mol Plant Pathol. 2018:19(3):634–646. https://doi.org/10.1111/mpp.12548

Avilan L, Lebrun R, Puppo C, Citerne S, Cuiné S, Li-Beisson Y, Menand B, Field B, and Gontero B. ppGpp influences protein protection, growth and photosynthesis in *Phaeodactylum tricornutum*. New Phytol. 2021:230(4):1517–1532. https://doi.org/10.1111/nph.17286

Bailleul B, Cardol P, Breyton C, and Finazzi G. Electrophromism: a useful probe to study algal photosynthesis. Photosynth Res. 2010:106(1–2). https://doi.org/10.1007/s11120-010-9579-z

Bartoli J, Citerne S, Mouille G, Bouveret E, and Field B. Quantification of guanosine triphosphate and tetraphosphate in plants and algae using stable isotope-labelled internal standards. Talanta. 2020:219:121261. https://doi.org/10.1016/j.talanta.2020.121261

Bassi R and Dall'Osto L. Dissipation of Light Energy Absorbed in Excess: The Molecular Mechanisms. Annu Rev Plant Biol. 2021:72(1):47–76. https://doi.org/10.1146/annurev-arplant-071720-015522

Boniecka J, Prusinska J, Dabrowska GB, and Goc A. Within and beyond the stringent response- RSH and (p)ppGpp in plants. Planta. 2017:246(5):817–842. https://doi.org/10.1007/s00425-017-2780-y

Campoli C, Caffarri S, Svensson JT, Bassi R, Stanca AM, Cattivelli L, and Crosatti C. Parallel pigment and transcriptomic analysis of four barley albina and xantha mutants reveals the complex network of the chloroplast-dependent metabolism. Plant Mol Biol. 2009:71(1–2):173–191. https://doi.org/10.1007/s11103-009-9516-8

Canty A and Ripley B. boot: Bootstrap R (S-Plus) Functions. Version 1.3-28 https://cran.r-project.org/web/packages/boot/index.html. 2021.

D’Alessandro S, Velay F, Lebrun R, Zafirov D, Mehrez M, Romand S, Saadouni R, Forzani C, Citerne S, Montané M-H, et al. Posttranslational regulation of photosynthetic activity via the TOR kinase in plants. Sci Adv. 2024:10(25):eadj3268. https://doi.org/10.1126/sciadv.adj3268
**Dall'Osto L, Caffarri S, and Bassi R.** A Mechanism of Nonphotochemical Energy Dissipation, Independent from PsbS, Revealed by a Conformational Change in the Antenna Protein CP26. Plant Cell. 2005:17(4):1217-1232. https://doi.org/10.1105/tpc.104.030601

**Dall'Osto L, Cazzaniga S, Havaux M, and Bassi R.** Enhanced photoprotection by protein-bound vs free xanthophyll pools: a comparative analysis of chlorophyll b and xanthophyll biosynthesis mutants. Mol Plant. 2010:3(3):576-593. https://doi.org/10.1093/mp/ssp117

**Davison AC and Hinkley DV.** Bootstrap Methods and their Application 1st edition. (Cambridge University Press).

**Espineda CE, Linford AS, Devine D, and Brusslan JA.** The *AtCAO* gene, encoding chlorophyll *a* oxygenase, is required for chlorophyll *b* synthesis in *Arabidopsis thaliana*. Proc Natl Acad Sci. 1999;96(18):10507-10511. https://doi.org/10.1073/pnas.96.18.10507

**Fasiolo M, Wood SN, Zaffran M, Nedellec R, and Goude Y.** Fast Calibrated Additive Quantile Regression. J Am Stat Assoc. 2021;116(535):1402-1412. https://doi.org/10.1080/01621459.2020.1725521

**Fitter DW, Martin DJ, Copley MJ, Scotland RW, and Langdale JA.** *GLK* gene pairs regulate chloroplast development in diverse plant species. Plant J. 2002;**31**(6):713-727. https://doi.org/10.1046/j.1365-313X.2002.01390.x

**Gilmore AM.** Xanthophyll cycle-dependent nonphotochemical quenching in Photosystem II: Mechanistic insights gained from *Arabidopsis thaliana* L. mutants that lack violaxanthin deepoxidase activity and/or lutein. Photosynth Res. 2001;**67**(1-2):89-101. https://doi.org/10.1023/A:1010657000548

**Goto M, Oikawa A, and Masuda S.** Metabolic changes contributing to large biomass production in the *Arabidopsis* ppGpp-accumulating mutant under nitrogen deficiency. Planta. 2022;**255**(2):48. https://doi.org/10.1007/s00425-022-03835-0

**Harchouni S, England S, Vieu J, Romand S, Aouane A, Citerne S, Legeret B, Alric J, Li-Beisson Y, Menand B, et al.** Guanosine tetraphosphate (PPGPP) accumulation inhibits chloroplast gene expression and promotes super grana formation in the moss *Physcomitrium* (*Physcomitrella*) *patens*. New Phytol. 2022;**236**(1):86-98. https://doi.org/10.1111/nph.18320

**Havaux M.** Plastoquinone In and Beyond Photosynthesis. Trends Plant Sci. 2020;**25**(12):1252-1265. https://doi.org/10.1016/j.tplants.2020.06.011

**Havaux M, Dall'Osto L, and Bassi R.** Zeaxanthin Has Enhanced Antioxidant Capacity with Respect to All Other Xanthophylls in *Arabidopsis* Leaves and Functions Independent of
Binding to PSII Antennae. Plant Physiol. 2007:145(4):1506-1520.
https://doi.org/10.1104/pp.107.108480

**Honoki R, Ono S, Oikawa A, Saito K, and Masuda S.** Significance of accumulation of the alarmone (p)ppGpp in chloroplasts for controlling photosynthesis and metabolite balance during nitrogen starvation in Arabidopsis. Photosynth Res. 2018:135(1-3):299-308. https://doi.org/10.1007/s11120-017-0402-y

**Hope R.** Rmisc: Ryan Miscellaneous. Version 1.5 https://cran.r-project.org/package=Rmisc.
2013.

**Hu C, Elias E, Nawrocki WJ, and Croce R.** Drought affects both photosystems in Arabidopsis thaliana. New Phytol. 2023:240(2):663-675. https://doi.org/10.1111/nph.19171

**Irving SE, Choudhury NR, and Corrigan RM.** The stringent response and physiological roles of (pp)pGpp in bacteria. Nat Rev Microbiol. 2021:19(4):256-271.
https://doi.org/10.1038/s41579-020-00470-y

**Ito K, Ito D, Goto M, Suzuki S, Masuda S, Iba K, and Kusumi K.** Regulation of ppGpp Synthesis and Its Impact on Chloroplast Biogenesis during Early Leaf Development in Rice. Plant Cell Physiol. 2022:pcac053. https://doi.org/10.1093/pcp/pcac053

**Kassambara A.** Rstatix: Pipe-Friendly Framework for Basic Statistical Tests. 2021.

**Khuong TTH, Robaglia C, and Caffarri S.** Photoprotection and growth under different lights of Arabidopsis single and double mutants for energy dissipation (npq4) and state transitions (pph1). Plant Cell Rep. 2019:38(6):741-753.
https://doi.org/10.1007/s00299-019-02403-3

**Ksas B, Becuwe N, Chevalier A, and Havaux M.** Plant tolerance to excess light energy and photooxidative damage relies on plastoquinone biosynthesis. Sci Rep. 2015:5(1):10919. https://doi.org/10.1038/srep10919

**Lee KP and Kim C.** Photosynthetic ROS and retrograde signaling pathways. New Phytol. 2024:244(4):1183-1198. https://doi.org/10.1111/nph.20134

**Lenth R.** emmeans: Estimated Marginal Means, aka Least-Squares Means. 2024.

**Li H, Nian J, Fang S, Guo M, Huang X, Zhang F, Wang Q, Zhang J, Bai J, Dong G, et al.** Regulation of nitrogen starvation responses by the alarmone (p)ppGpp in rice. J Genet Genomics. 2022a:49(5):469-480. https://doi.org/10.1016/j.jgg.2022.02.006

**Li M, Lee KP, Liu T, Dogra V, Duan J, Li M, Xing W, and Kim C.** Antagonistic modules regulate photosynthesis-associated nuclear genes via GOLDEN2-LIKE transcription factors. Plant Physiol. 2022b:188(4):2308-2324. https://doi.org/10.1093/plphys/kiab600
Li X-P, Björkman O, Shih C, Grossman AR, Rosenquist M, Jansson S, and Niyogi KK. A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature. 2000:403(6768):391–395. https://doi.org/10.1038/35000131

Li Z, Ahn TK, Avenson TJ, Ballottari M, Cruz JA, Kramer DM, Bassi R, Fleming GR, Keasling JD, and Niyogi KK. Lutein accumulation in the absence of zeaxanthin restores nonphotochemical quenching in the Arabidopsis thaliana npq1 mutant. Plant Cell. 2009:21(6):1798–1812. https://doi.org/10.1105/tpc.109.066571

Liebers M, Cozzi C, Uecker F, Chambon L, Blanvillain R, and Pfannschmidt T. Biogenic signals from plastids and their role in chloroplast development. J Exp Bot. 2022:73(21):7105–7125. https://doi.org/10.1093/jxb/erac344

Liu X, Li L, Li M, Su L, Lian S, Zhang B, Li X, Ge K, and Li L. AhGLK1 affects chlorophyll biosynthesis and photosynthesis in peanut leaves during recovery from drought. Sci Rep. 2018:8(1):2250. https://doi.org/10.1038/s41598-018-20542-7

Maekawa M, Honoki R, Ihara Y, Sato R, Oikawa A, Kanno Y, Ohta H, Seo M, Saito K, and Masuda S. Impact of the plastidial stringent response in plant growth and stress responses. Nat Plants. 2015:1(12):15167. https://doi.org/10.1038/nplants.2015.167

Mehrez M, Romand S, and Field B. New perspectives on the molecular mechanisms of stress signalling by the nucleotide guanosine tetraphosphate (ppGpp), an emerging regulator of photosynthesis in plants and algae. New Phytol. 2023:237(4):1086–1099. https://doi.org/10.1111/nph.18604

Montillet J, Cacas J, Garnier L, Montané M, Douki T, Bessoule J, Polkowska-Kowalczyk L, Maciejewska U, Agnel J, Vial A, et al. The upstream oxylipin profile of *Arabidopsis thaliana*: a tool to scan for oxidative stresses. Plant J. 2004:40(3):439–451. https://doi.org/10.1111/j.1365-313X.2004.02223.x

Nawrocki WJ, Liu X, Raber B, Hu C, De Vitry C, Bennett DIG, and Croce R. Molecular origins of induction and loss of photoinhibition-related energy dissipation q₁. Sci Adv. 2021:7(eabj0055). https://doi.org/10.1126/sciadv.abj0055

Niyogi KK, Grossman AR, and Björkman O. Arabidopsis Mutants Define a Central Role for the Xanthophyll Cycle in the Regulation of Photosynthetic Energy Conversion. Plant Cell. 1998:10(7):1121–1134. https://doi.org/10.1105/tpc.10.7.1121

Nomura Y, Izumi A, Fukunaga Y, Kusumi K, Iba K, Watanabe S, Nakahira Y, Weber AP, Nozawa A, and Tozawa Y. Diversity in guanosine 3', 5'-bisdiphosphate (ppGpp) sensitivity among guanylate kinases of bacteria and plants. J Biol Chem. 2014a:289(22):15631–15641.
Nomura Y, Nozawa A, and Tozawa Y. Biochemical analyses of ppGpp effect on adenylosuccinate synthetases, key enzymes in purine biosynthesis in rice. Biosci Biotechnol Biochem. 2014b:78(6):1022-1025.
https://doi.org/10.1080/09168451.2014.910103

Ono S, Suzuki S, Ito D, Tagawa S, Shiina T, and Masuda S. Plastidial (p)ppGpp Synthesis by the Ca2+-Dependent RelA-SpoT Homolog Regulates the Adaptation of Chloroplast Gene Expression to Darkness in Arabidopsis. Plant Cell Physiol. 2021:61(12):2077-2086.
https://doi.org/10.1093/pcp/pcaa124

Pinnola A and Bassi R. Molecular mechanisms involved in plant photoprotection. Biochem Soc Trans. 2018:46(2):467-482. https://doi.org/10.1042/BST20170307

Qiu D, Lange E, Haas TM, Prucker I, Masuda S, Wang YL, Felix G, Schaaf G, and Jessen HJ. Bacterial Pathogen Infection Triggers Magic Spot Nucleotide Signaling in Arabidopsis thaliana Chloroplasts through Specific RelA/SpoT Homologues. J Am Chem Soc. 2023:145(29):16081-16089. https://doi.org/10.1021/jacs.3c04445

Reinsberg D, Ottmann K, Booth PJ, and Paulsen H. Effects of chlorophyll a, chlorophyll b, and xanthophylls on the *in vitro* assembly kinetics of the major light-harvesting chlorophyll a/b complex, LHCIIb1. J Mol Biol. 2001:308(1):59-67.
https://doi.org/10.1006/jmbi.2001.4573

Romand S, Abdelkefi H, Lecampion C, Belaroussi M, Dussenne M, Ksas B, Citerne S, Caius J, D'Alessandro S, Fakhfakh H, et al. A guanosine tetraphosphate (ppGpp) mediated brake on photosynthesis is required for acclimation to nitrogen limitation in Arabidopsis. eLife. 2022:11:e75041. https://doi.org/10.7554/eLife.75041

Ruban AV and Wilson S. The Mechanism of Non-Photochemical Quenching in Plants: Localization and Driving Forces. Plant Cell Physiol. 2021:62(7):1063-1072.
https://doi.org/10.1093/pcp/pcaa155

Shumbe L, D'Alessandro S, Shao N, Chevalier A, Ksas B, Bock R, and Havaux M. METHYLENE BLUE SENSITIVITY 1 (MBS1) is required for acclimation of Arabidopsis to singlet oxygen and acts downstream of β-cycocitral. Plant Cell Environ. 2017:40(2):216-226.
https://doi.org/10.1111/pce.12856

Sugliani M, Abdelkefi H, Ke H, Bouveret E, Robaglia C, Caffarri S, and Field B. An Ancient Bacterial Signaling Pathway Regulates Chloroplast Function to Influence Growth and Development in Arabidopsis. Plant Cell. 2016:28(3):661-679.
https://doi.org/10.1105/tpc.16.00045

Triantaphylidès C, Krischke M, Hoeberichts FA, Ksas B, Gresser G, Havaux M, Van Breusegem F, and Mueller MJ. Singlet Oxygen Is the Major Reactive Oxygen Species Involved in
Photooxidative Damage to Plants. Plant Physiol. 2008:148(2):960-968.
https://doi.org/10.1104/pp.108.125690

Turkan S, Kulasek M, Zienkiewicz A, Mierek-Adamska A, Skrzypek E, Warchoł M, Szydłowska-Czerniak A, Bartoli J, Field B, and Dąbrowska GB. Guanosine tetraphosphate (ppGpp) is a new player in Brassica napus L. Seed development. Food Chem. 2023:137648. https://doi.org/10.1016/j.foodchem.2023.137648

Waters MT, Wang P, Korkaric M, Capper RG, Saunders NJ, and Langdale JA. GLK Transcription Factors Coordinate Expression of the Photosynthetic Apparatus in *Arabidopsis*. Plant Cell. 2009:21(4):1109-1128. https://doi.org/10.1105/tpc.108.065250

Wickham H. Ggplot2: elegant graphics for data analysis (Springer: New York).

Wobbrock JO, Findlater L, Gergle D, and Higgins JJ. The aligned rank transform for nonparametric factorial analyses using only anova procedures. In: Proceedings of the SIGCHI Conference on Human Factors in Computing Systems. (ACM: Vancouver BC Canada), pp. 143-146. https://doi.org/10.1145/1978942.1978963

Zegarra V, Weiland P, Plitzko PA, Thiery J, Czech L, Willmund F, Bedrunka P, and Bange G. Structural and mechanistic basis for the regulation of the chloroplast signal recognition particle by (p)ppGpp. FEBS Lett. 2025:599(10):1373-1385. https://doi.org/10.1002/1873-3468.70008
Supplemental data

<figure><img src="image_23.png" /><figcaption>**Figure S1. Photosynthetic protein levels in OX:RSH3 plants.** (A) Immunoblots showing the abundance of the indicated proteins extracted from 4-week old plants. Total proteins were revealed using Ponceau stain (Ponceau S). (B) Lhcb1,2 abundance from Coomassie staining of thylakoid extracts in the indicated lines. (C) Quantification of relative protein amounts in OX:RSH3.1 and OX:RSH3.2 compared to the wild-type. Samples were normalised to total chlorophyll (Chl) content. Results are shown for two independent OX:RSH3 lines derived from a single experiment. PSII RC, PSII reaction centre; PSI RC, PSI reaction centre.</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>**Figure S2.** PS rates in OX:RSH3 lines. The photochemical rate of PSI and PSII *in vivo* and the PSI/(PSI+PSII) ratio were determined *in vivo* using electrochromic shift measurements. Means ± 95% confidence interval, n = 3 biological replicates). Differences were tested using ANOVA and post-hoc testing. Letters indicate statistical groups.</figcaption></figure>
<figure><img src="image_25.png" /><figcaption>A</figcaption></figure>

WT

OX:RSH3.1

npq1

npq4

OX:RSH3.1

npq1

OX:RSH3.1

npq4

<figure><img src="image_26.png" /><figcaption>B</figcaption></figure>

WT

OX:RSH3.1

npq1

npq4

OX:RSH3.1

npq1

OX:RSH3.1

npq4

**Figure S3: NPQ mutations suppress the growth defects of OX:RSH3.** (A) Phenotypes of the different lines after 4 weeks of growth in long day conditions. (B) Phenotypes of the different lines after 4 weeks of growth in short day conditions. Scale bar, 3 cm.
<figure><img src="image_27.png" /><figcaption>**Fig. S4. PSII operating efficiency under growth light.** Dark adapted plants were subjected to a saturating pulse to obtain maximal PSII efficiency (Fv/Fm), before exposure to an actinic light at growth light intensity (120 µmol m⁻² s⁻¹ PAR) with saturating pulses at the indicated time points to obtain PSII operating efficiency, ΦPSII. The actinic light was turned off after one hour (3600 s) to monitor ΦPSII recovery. Points represent median ± 95% CI, n=4.</figcaption></figure>
<figure><img src="image_28.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_29.png" /></figure>

**Figure S5. Pigment levels in growth-light acclimated plants.** Pigments were extracted from 4-week old plants and analysed by HPLC. (a) Individual pigments. (b) Combined amount of violaxanthin, antheraxanthin and zeaxanthin (VAZ), and the de-epoxidation state (DEPS, (zeaxanthin+0.5*antheraxanthin)/VAZ)) parameter. Mean values, ±95% CI, normalized for 100 total chlorophyll (a+b), n=4 biological replicates. Differences were tested using ANOVA and post-hoc testing. Letters indicate statistical groups. *npq1* was excluded from tests for antheraxanthin and zeaxanthin due to the absence of these pigments.
<figure><img src="image_30.png" /><figcaption>**Figure S6** The effects of high light exposure on NPQ. 4-week-old plants were exposed to 1500 µmol of photons m⁻²s⁻¹ (4°C) and NPQ was measured by chlorophyll fluorescence imaging of individual plants after 24h (T24) and 48h (T48). NPQ kinetics under an actinic light of 1200 µmol photons m⁻²s⁻¹. Curves represent median values ± 95% confidence interval, n = 4 biological replicates. Data shown in panels 1D, 2A, and S6 originate from the same experiment.</figcaption></figure>
<figure><img src="image_31.png" /><figcaption>**Figure S7.** ECS measurements in *ch1* and *OX:RSH3 ch1*. The photochemical rate of PSI and PSII and the PSI/(PSI+PSII) ratio were determined *in vivo* using electrochromic shift measurements. Means ± 95% confidence interval, n = 3 biological replicates. Differences were tested using ANOVA and post-hoc testing. Letters indicate statistical groups.</figcaption></figure>
<figure><img src="image_32.png" /><figcaption>**Figure S8. Pigment levels in growth-light acclimated plants.** 4-week-old plants were exposed to 1500 µmol of photons m⁻²s⁻¹ (4°C) (HL) and allowed to recover for 1h under standard conditions (REC). Pigments were extracted analysed by HPLC after HL and REC treatments. (a) Individual pigments. (b) Combined amount of violaxanthin, antheraxanthin and zeaxanthin (VAZ), and the de-epoxidation state (DEPS, (zeaxanthin+0.5*antheraxanthin)/VAZ)) parameter. Mean values, ±95% CI, normalized for 100 total chlorophyll (a+b), n=4 biological replicates. Differences were tested using ANOVA and post-hoc testing. Letters indicate statistical groups.</figcaption></figure>