# Bidirectional regulation between TOR kinase and chloroplasts: emerging connections in photosynthetic organisms

Jose Crespo, Benoît Menand, Ben Field

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Jose Crespo, Benoît Menand, Ben Field. Bidirectional regulation between TOR kinase and chloroplasts: emerging connections in photosynthetic organisms. Journal of Experimental Botany, In press, 10.1093/jxb/eraf338. hal-05226411

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Submitted on 27 Aug 2025

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
1 Bidirectional regulation between TOR kinase and chloroplasts: emerging
2 connections in photosynthetic organisms

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4 Jose L. Crespo¹,*, Benoît Menand²,*, Ben Field²,.*

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6 1. Instituto de Bioquímica Vegetal y Fotosíntesis (CSIC-Universidad de Sevilla), 41092

7 Sevilla, Spain

8 2. Aix Marseille Univ, CEA, CNRS, BIAM, LGBP Team, 13009 Marseille, France

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10 * Correspondence:

11 crespo@ibvf.csic.es

12 benoit.menand@univ-amu.fr

13 ben.field@univ-amu.fr

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**Highlight**

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This review examines the central role of the TOR kinase in linking nutrient availability to chloroplast function, detailing emerging evidence of reciprocal regulation between TOR signalling and photosynthesis in plants and algae.

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**Abstract**

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Eukaryotic growth relies on intricate mechanisms that couple nutrient availability to the cell growth machinery. Central to this regulation is the evolutionarily conserved target of rapamycin (TOR), a large Ser/Thr kinase belonging to the atypical phosphatidylinositol kinase-related kinase (PIKK) family. TOR integrates diverse nutrient, energy, and hormonal signals to stimulate biosynthetic pathways and repress catabolism, thus promoting cell growth across eukaryotes- from yeast and algae to animals and plants. Despite extensive research on TOR signalling, the interplay between TOR activity and chloroplast function, particularly in regulating photosynthesis, has only recently begun to receive attention. Emerging evidence indicates a reciprocal relationship, where TOR influences chloroplast function through transcriptional and post-translational mechanisms, and where chloroplast-derived signals modulate TOR activity. This highlights crucial yet understudied links between nutrient signalling, chloroplast activity, and cellular growth. In this review, we discuss recent advances that emphasise the pivotal role of TOR as a mediator connecting stress and nutrient signals to chloroplast function in plants and algae.

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**Keywords**

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chloroplast, kinase, metabolism, photosynthesis, signalling, stress, TOR

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41 **Introduction**

42 The evolutionarily-conserved TOR (target of rapamycin) kinase operates as a central
43 integrator of nutrient, energy and hormone signals to control growth in eukaryotes
44 (González and Hall, 2017; Liu et al., 2025). TOR signalling is particularly interesting in
45 photosynthetic eukaryotes, where growth and chloroplast function must be closely
46 coordinated. TOR operates primarily through the dimeric TORC1 complex (Yang et al.,
47 2016), comprising TOR itself along with conserved regulatory partners RAPTOR
48 (regulatory associated protein of mTOR) and LST8 (lethal with SEC13 protein 8). Unlike in
49 yeast and animals, which have two distinct TOR complexes, plants and algae appear to
50 possess only TORC1, although gene copy numbers for RAPTOR and LST8 vary across
51 species. For example, the flowering plant Arabidopsis thaliana (Arabidopsis) contains
52 two RAPTOR genes, whereas the green alga Chlamydomonas reinhardtii
53 (Chlamydomonas) has only one (Díaz-Troya et al., 2008; Menand et al., 2002).
54 Consequently, complete loss-of-function tor mutants in Arabidopsis are embryo-lethal,
55 underscoring the essential role of TOR, while viable mutants for individual RAPTOR or
56 LST8 genes have provided valuable insights into the consequences of partial TOR
57 downregulation (Menand et al., 2002; Salem et al., 2018; Forzani et al., 2019). Similarly,
58 in Chlamydomonas, an LST8 knock-down mutant displaying compromised TOR signaling
59 has been characterized (Couso et al., 2020). Alongside these genetic tools, selective
60 chemical inhibitors have significantly advanced the study of TOR signaling in plants and
61 algae (see **Box 1**). TOR activity responds dynamically to nutrient cues such as nitrogen,
62 phosphorus, glucose, amino acids, and light, to modulate the activity of downstream
63 targets including the S6 kinase (S6K) (Liu et al., 2025). Phosphorylation of ribosomal
64 protein S6 (RPS6, recently renamed eS6 according to a new nomenclature for plant
65 ribosomal proteins (Scarpin et al., 2023)) by S6K stimulates protein synthesis.
66 Consequently, phosphorylation of both S6K and RPS6 is widely used as a marker for TOR
67 activity across diverse eukaryotic species (Liu et al., 2025). Nevertheless, despite recent
68 progress, critical questions remain, notably about how chloroplast signals control TOR
69 activity and how TOR specifically regulates photosynthesis and growth, highlighting the
70 need to further explore these unique aspects of TOR signaling in plants and algae.
71 Dissecting this bidirectional TOR-chloroplast dialogue will not only illuminate how
72 photosynthetic eukaryotes assimilated a once-free-living cyanobacterium into their
73 growth-control circuitry- a breakthrough that underpins the evolutionary success of
74 plants and algae- but will also reveal strategies to sustain photosynthetic efficiency,
75 balanced growth, and, ultimately, crop yield as climate stresses intensify.
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77 **1. Regulation of TOR by chloroplast function**

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79 **Activation of TOR by photosynthesis**

80 As photosynthetic organisms, plants fix atmospheric CO₂ to produce carbohydrates.

81 Organic carbon is a primary nutrient acting upstream of TOR in evolutionary distant
organisms. In plants, glucose is a key upstream signal for TOR activation, acting through glycolysis and mitochondrial energetics (Xiong et al., 2013) (Fig. 1A). For instance, under stress-conditions low sugar levels lead to SNRK1 activation, which in turn inhibits TOR via the phosphorylation of the Raptor subunit (Nukarinen et al., 2016). TOR also integrates light and hormone signals to activate growth at the shoot apex (Pfeiffer et al., 2016; Li et al., 2017). Glucose alone is sufficient to activate TOR in root apexes, but both glucose and light are required for full TOR activation in photosynthetic tissues (Li et al., 2017). The mechanisms by which these cues are transmitted to TOR are being uncovered. One key discovery is the identification of the plant-specific small GTPase ROP2 (Rho GTPase 2) as a key regulator, which directly binds TOR and transduces light-auxin signals to promote shoot apex growth (Li et al., 2017; Schepetilnikov et al., 2017). In contrast, glucose signalling appears independent of ROP2, suggesting distinct regulatory branches for glucose and light (Li et al., 2017).

In microalgae such as Chlamydomonas, TOR activity is promoted by photosynthetic CO2 assimilation. Addition of bicarbonate stimulates photosynthesis and activates TOR (Mallén-Ponce et al., 2022). This activation depends on the Calvin-Beson-Bassham cycle, as inhibiting phosphoribulokinase with glycolaldehyde suppresses TOR activity, and abolishes bicarbonate-induced activation (Mallén-Ponce et al., 2022). TOR is also strongly light-regulated in Chlamydomonas, and its activity decreases in the dark (Mallén-Ponce et al., 2022, 2025). Notably, inhibition of CO2 fixation with glycolaldehyde fully prevents TOR reactivation upon re-illumination, indicating that light regulates TOR activity via photosynthesis in this alga (Mallén-Ponce et al., 2022). Although these observations may initially suggest that TOR activation by photosynthesis differs fundamentally between plants and algae, it is more likely that the core mechanisms are conserved. For example, growth in energy-starved root meristems of Arabidopsis depends on TOR, and supplementation with bicarbonate alone is sufficient to reactivate growth (Xiong et al., 2013). Nevertheless, the precise molecular mechanism linking photosynthetic activity directly to TOR activation remains to be clearly demonstrated in plants.

Important insights into the molecular mechanisms by which TOR senses photosynthetic activity are beginning to emerge from studies in microalgae. A recent metabolomic study in light/dark synchronized Chlamydomonas cells identified dihydroxyacetone phosphate (DHAP), produced in the chloroplast during photosynthesis, as a key metabolite connecting carbon availability and light signals to TOR (Mallén-Ponce et al., 2025) (Fig. 1B). Addition of dihydroxyacetone (DHA), the triose precursor of DHAP, strongly activates TOR even in the dark. In microalgae, fixed carbon is primarily exported from the chloroplast as DHAP by TPT3 (triose-phosphate translocator 3), located in the inner envelope membrane (Huang et al., 2023). TPT3 null mutants show pleiotropic defects affecting growth, photosynthesis and carbon partitioning, due to the accumulation of
CBB cycle and glycolytic intermediates caused by impaired DHAP export. Interestingly, these mutants fail to properly regulate TOR in response to CO₂ or light, but TOR activity is fully restored by exogenous DHA, confirming that DHAP export is essential for TOR regulation (Mallén-Ponce *et al.*, 2025). The role of DHAP as a regulatory metabolite may be evolutionarily conserved, as glucose signalling to mTORC1 in mammals also appears to be mediated by DHAP rather than glucose itself (Orozco *et al.*, 2020). While the precise molecular mechanism by which DHAP is sensed by TOR remains unknown in mammals and microalgae, whether a similar mechanism operates in plants has yet to be determined.
<figure><img src="image_2.png" /><figcaption>**Figure 1. Regulation of TOR function by chloroplast-derived metabolites. (A)** Photosynthetically derived sugars exported from the chloroplast activate TOR (via phosphorylation, P) in distal plant tissues. Sugar regulation of TOR can occur via SnRK1, or potentially via other routes (dashed arrow). Notably, TOR activation by sugar in roots requires mitochondrial function. (B) DHAP, a triose phosphate product of photosynthesis, positively regulates TOR in the green alga Chlamydomonas. (C) Active TOR directly phosphorylates the ABA receptor PYL, thereby inhibiting it. ABA, whose precursors are made in the chloroplast, binds PYL. ABA-bound PYL binds and inhibits the PP2C phosphatase, activating SNRK2. Active SNRK2 directly phosphorylates the RAPTOR subunit of TOR, inhibiting TOR activity. Lines ending in arrowheads denote positive regulation or metabolic flux; those terminating in a perpendicular “T”-bar indicate inhibition; and a thicker arrow that pierces the double chloroplast envelope represents transport across the plastid membrane. In every case, the line may stand for a multi-step process. The Arabidopsis TOR complex was rendered from an AlphaFold model (Abramson et al., 2024) (RAPTOR, yellow; TOR, light blue; LST8, cyan). A monomer is shown, though the active form is likely a homodimer. TOR, target of rapamycin; RAPTOR, regulatory associated protein of TOR; LST8, lethal with SEC13 protein 8; CBB, Calvin-Benson-Bassham cycle; PSII, photosystem II; PSI, photosystem I; DHAP, dihydroxyacetone phosphate; TPT3, triose phosphate transporter; ABA, abscissic acid; SNRK, SNF1-related protein kinase; PP2C, protein phosphatase 2C; PYL, PYR1-like. The figure was prepared in Inkscape.</figcaption></figure>

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157 Regulation of TOR by diverse chloroplast metabolites

158 In addition to their central role in carbon assimilation, chloroplasts, or rather plastids,
159 are the site of biosynthesis for a wide range of metabolites with the potential to influence
160 TOR activity. Notably, the two hormones most clearly established to act upstream of
161 TOR- auxin, which activates TOR, and abscisic acid (ABA), which represses it- are derived
162 from chloroplastic precursors (Fàbregas and Fernie, 2022) (Fig. 1C). Precursors of the
163 stress-response hormone ABA such as β-carotene, zeaxanthin, and xanthoxin are
164 produced in chloroplasts via the methylerythritol phosphate (MEP) pathway, which also
165 supplies precursors for gibberellins, cytokinins, carotenoids and strigolactones.
166 Biosynthesis of the growth-promoting hormone auxin is more complex: indole-3-acetic
167 acid (IAA), the main auxin, can be synthesized via both a cytosolic tryptophan-dependent
168 pathway and a chloroplastic tryptophan-independent pathway using chorismate and
169 anthranilate. Despite this, the role of chloroplast-localized hormone biosynthesis in TOR
170 regulation remains underexplored. Adding further complexity, TOR also feeds back to
171 regulate hormonal signalling, for instance, through phosphorylation of ABA receptors
172 (PYLs)(Wang et al., 2018) and the auxin efflux facilitator PIN2 (Yuan et al., 2020).
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174 Amino acids are another important class of TOR regulators, and their biosynthesis is
175 closely linked to chloroplast function. Seventeen amino acids are synthesized in
176 chloroplasts, and ten are produced exclusively in this organelle (Rolland et al., 2012). In
177 Arabidopsis, overaccumulation of the branched-chain amino acids (BCAAs) valine,
178 leucine, and isoleucine, all synthesized in chloroplasts, leads to TOR overactivation (Cao
179 et al., 2019; Schaufelberger et al., 2019). This suggests that BCAAs act upstream of TOR
180 in plants, as observed in yeast and mammalian cells (González et al., 2020).
181 Consistently, exogenous addition of 15 amino acids to nitrogen-starved Arabidopsis
182 seedlings, or incubation with individual amino acids such as isoleucine or glutamine,
183 stimulates TOR activity (O'Leary et al., 2020; Liu et al., 2021). In Chlamydomonas, central
184 amino acids involved in carbon metabolism, including alanine, leucine, valine,
185 glutamate, and glutamine, have also been implicated in TOR regulation (Mallén-Ponce et
186 al., 2022). How TOR perceives amino acids in plants and algae is currently unclear
187 although ROP2 has been shown to participate in glutamine sensing in Arabidopsis (Liu et
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190 2. Regulation of chloroplast function by TOR

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192 The green hand of TOR: linking TOR activity to chloroplast function

193 TOR regulates numerous chloroplast processes including photosynthesis, nitrogen
194 fixation, lipid and starch metabolism and amino acid biosynthesis (Liu et al., 2025). Since
195 its discovery in plants and algae, TOR activity has been closely linked to chloroplast
196 function. Early studies frequently noted that attenuating TOR activity in Arabidopsis
197 resulted in reduced leaf chlorophyll content. For instance, chlorosis is often observed
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upon attenuation of TOR, whether by silencing (Deprost et al., 2007; Caldana et al., 2013), in TOR subunit mutants (Moreau et al., 2012), or following treatment with chemical inhibitors (Dong et al., 2015).

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Subsequent research further strengthened the link between TOR and the chloroplast by demonstrating that TOR repression using chemical inhibitors or knockdown is accompanied by reduced expression of chloroplast genes in Arabidopsis (Xiong et al., 2013; Dong et al., 2015; Dobrenel et al., 2016; Scarpin et al., 2020) and in both red and green algae (Imamura et al., 2018; Zhu et al., 2022). However, significant discrepancies emerged among Arabidopsis studies, likely due to differences in TOR attenuation methods and treatment durations. Specifically, while long-term (≥24 h) inhibition of TOR reduced transcript levels of nuclear-encoded chloroplast genes (Xiong et al., 2013; Dong et al., 2015; Dobrenel et al., 2016), short-term (2 h) inhibition did not; however, it did significantly reduce chloroplast translation efficiency (Scarpin et al., 2020). To reconcile these contrasting findings, Scarpin and colleagues proposed that downregulation of nuclear-encoded chloroplast genes represents a secondary response, mediated by chloroplast-to-nucleus retrograde signalling, and triggered by an initial decline in chloroplast translation.

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## TOR regulates photosynthesis

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Beyond its effects on chloroplast gene expression, TOR inhibition also impacts photosynthetic activity. So far, this was typically measured using chlorophyll fluorescence from photosystem II (PSII), with TOR inhibition causing a reduction in maximal efficiency of PSII (Fv/Fm) in dark-adapted plants, the PSII quantum yield of linear electron transport (ΦPSII) or the relative electron transport rate (ETR) in illuminated plants. In the photosynthetic green alga Chlamydomonas, chemical inhibition of TOR with AZD-8055 leads to a rapid (<2 h) decrease in photosynthetic activity (Ford et al., 2019; Upadhyaya and Rao, 2019). Notably, a reduction in photosynthesis was observed under both photoheterotrophic (Ford et al., 2019) and photoautotrophic culture conditions (Upadhyaya and Rao, 2019). Roustan and Weckwerth (2018) did not observe a drop in Chlamydomonas photosynthetic activity following treatment with rapamycin, although this is likely an inhibitor-dependent effect (see **Box 1**). TOR has also been proposed to control the phosphorylation state of some proteins from the photosynthetic apparatus (Couso et al., 2021) although it is unclear whether this regulation is direct and the underlying mechanisms are unknown. Recently, rapamycin treatment was also shown to diminish photosynthetic activity within 6 h in a distantly related alga of the red lineage, *Nannochloropsis gaditana* (Zhang et al., 2025).

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Decreased photosynthetic activity in response to TOR inhibition was also reported in plants, including Arabidopsis (D'Alessandro et al., 2024; Perdoux et al., 2024) and tomato (Yang et al., 2024). Interestingly, these studies reported observable responses

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after 24-48 h of TOR inhibition. It remains unclear whether photosynthetic responses to TOR repression can also occur over shorter timescales in plants, or whether this reflects inherent differences between plants and green algae.

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Further support for a link between TOR and photosynthetic performance comes from studies modulating TOR complex subunits. In rice, knockout of *RAPTOR2* reduces TOR activity and results in severe defects in thylakoid membrane development (Sun *et al.*, 2016). Interestingly, in Arabidopsis, however, knockout of *RAPTOR1B* also decreases TOR activity though without detectable changes in photosynthetic activity, possibly due to greater compensation by the RAPTOR1A isoform (Salem *et al.*, 2018). This is consistent with the lethality of double RAPTOR knockouts in both species (Anderson *et al.*, 2005; Sun *et al.*, 2016). Similarly, knockout of *LST8-1*, one of the two *LST8* genes in Arabidopsis, reduces TOR activity without altering photosynthesis under standard growth conditions (Forzani *et al.*, 2019; D'Alessandro *et al.*, 2024). Conversely, overexpression of different TOR subunits in rice, Arabidopsis and the alga *Nannochloropsis gaditana* has been associated with enhanced photosynthetic activity, particularly under stress (Bakshi *et al.*, 2017, 2021; Zhang *et al.*, 2024), although TOR activity was not directly measured in these studies. These findings suggest that substantial changes in TOR - either above or below a certain threshold - are necessary to impact photosynthesis, potentially reflecting buffering and regulatory mechanisms within the TOR signalling pathway.

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Taken together, these studies highlight a clear link between TOR signalling and photosynthetic performance across diverse photosynthetic organisms. However, they also reveal that the effects of TOR modulation on photosynthesis are not always consistent or immediate. Differences in experimental conditions, such as the method and duration of TOR inhibition, as well as environmental factors like light availability and nutrient status, may strongly influence whether a photosynthetic phenotype is observed. This suggests that while TOR is an important regulator of photosynthesis, its effects are context-dependent and likely integrated with other regulatory pathways.

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## A TOR-RSH3-ppGpp axis links TOR activity to photosynthesis

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Mounting evidence for TOR-mediated regulation of chloroplast function, particularly photosynthesis, raises questions about the underlying molecular mechanisms. A recent study provided part of the answer by revealing a signalling axis between TOR and guanosine tetraphosphate (ppGpp), a nucleotide signal that acts as a conserved negative regulator of chloroplast function in plants and algae (Mehrez *et al.*, 2023; D'Alessandro *et al.*, 2024) (Fig. 2A). ppGpp is synthesised from GDP or GTP by RelA SpoT homolog (RSH) enzymes. In bacteria, where ppGpp signalling is best understood, ppGpp and related nucleotides regulate targets such as RNA polymerase, translation-related GTPases, and nucleotide biosynthesis enzymes during stress acclimation (Steinchen *et al.*, 2020). In plants and algae, ppGpp similarly regulates chloroplast transcription and

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nucleotide metabolism in response to stress (Mehrez et al., 2023), and was recently shown to inhibit the activity of the chloroplast signal recognition particle involved in inserting photosynthetic proteins into the thylakoid membrane (Zegarra et al., 2025).

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D'Alessandro and colleagues found that the TOR-complex subunit LST8 interacts with the N-terminal of Arabidopsis RSH3 (D'Alessandro et al., 2024), a nuclear-encoded chloroplast enzyme responsible for ppGpp biosynthesis (Maekawa et al., 2015; Sugliani et al., 2016). RSH3 was further found to be poly-phosphorylated on its chloroplast transit peptide in a TOR-dependent manner. Inhibition of TOR reduces RSH3 phosphorylation and promotes its import into the chloroplast, thereby increasing ppGpp accumulation and leading to the downregulation of photosynthesis. Analysis of ppGpp-deficient mutants further confirmed that ppGpp biosynthesis is physiologically important for TOR-mediated regulation of photosynthesis. This mechanism appears to be conserved across land plants and illustrates how TOR integrates eukaryotic growth regulation with chloroplast-based signalling of prokaryotic origin. Interestingly, a similar TOR-dependent regulation of ppGpp metabolism was reported in the red alga *Cyanidioschyzon merolae*, where TOR inhibition induces transcription of the ppGpp synthase gene *RSH4b*, leading to reduced plastid rRNA levels- presumably through ppGpp-mediated inhibition of plastid transcription (Imamura et al., 2018). While post-translational regulation has been most clearly described in land plants, and transcriptional regulation in red algae, both modes may operate in both systems. Together, these findings highlight a conserved functional link between TOR signalling and chloroplast ppGpp metabolism, achieved through distinct but potentially overlapping regulatory layers. The TOR-RSH-ppGpp axis thus represents a striking example of how TOR may exert multi-level control over photosynthesis by modulating chloroplast activity. As such, it opens an exciting new perspective for understanding how growth and photosynthetic activity are coordinated in response to environmental and metabolic cues.

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<figure><img src="image_3.png" /><figcaption>**Figure 2.** Regulation of chloroplast function by TOR. (A) In Arabidopsis, the LST8 subunit of TOR binds to the chloroplast transit peptide of RSH3, and RSH3 is poly-phosphorylated (P) in a TOR-dependent manner. Upon TOR inactivation, non-phosphorylated RSH3 is more efficiently imported into the chloroplast, and ppGpp levels increase. ppGpp accumulation results in downregulation of photosynthesis. (B) In the red alga *C. merolae*, Floridean starch is synthesised in the cytosol from chloroplast-derived triose phosphates. TOR promotes starch degradation by influencing the activity of two enzymes. The starch synthesis enzyme GLG1 undergoes inhibitory TOR-dependent phosphorylation, while the starch degradation enzyme GWD undergoes an activating TOR-dependent phosphorylation. (C) In Arabidopsis guard cells, active TOR promotes the direct or indirect phosphorylation of BAM1 on a serine residue in the chloroplast transit peptide. BAM1 phosphorylation promotes BAM1 accumulation, and the activation of starch degradation in the chloroplast. Lines ending in arrowheads denote positive regulation or metabolic flux; those terminating in a perpendicular "T"-bar indicate inhibition; and a thicker arrow that pierces the double chloroplast envelope represents transport across the plastid membrane. In every case, the line may stand for a multi-step process. The Arabidopsis TOR complex was rendered from an AlphaFold model (Abramson *et al.*, 2024) (RAPTOR, yellow; TOR, light blue; LST8, cyan). A monomer is shown, though the active form is likely a homodimer. TOR, target of rapamycin; RAPTOR, regulatory associated protein of TOR; LST8, lethal with SEC13 protein 8; CBB, Calvin-Benson-Bassham cycle; PSII, photosystem II; PSI, photosystem I; RSH3, RelA SpoT homolog 3; ppGpp, guanosine tetraphosphate; BAM1, β-amylase 1; GLG1, glycogenin 1; GWD, α-glucan water dikinase. The figure was prepared in Inkscape.</figcaption></figure>
331 **TOR regulates chloroplast carbon storage**

332 In contrast to other eukaryotes, *de novo* synthesis of fatty acids, the basic components 333 of both major membrane glycerolipids and storage lipids (TAG, triacylglycerol), takes 334 place in the plastids of plants and algae (Li-Beisson *et al.*, 2013). Several studies have 335 shown that chemical inhibition of TOR or nitrogen starvation lead to TAG accumulation 336 in lipid droplets in diverse algae, including green algae, red algae, diatoms and Euglena 337 (Imamura *et al.*, 2015, 2016; Mukaida *et al.*, 2016; Prioretti *et al.*, 2017). This is of interest 338 for biotechnology as TAG can be used for biodiesel production and raises the question of 339 the role of TOR in regulating lipid metabolism. Quantitative measurement of 14C-acetate 340 incorporation into fatty acids indicated that both nitrogen starvation and TOR inhibition 341 reduce *de novo* fatty acid biosynthesis in the unicellular green algae Chlamydomonas 342 and *Chlorella sorokiniana* (Vijayan *et al.*, 2024). This was associated with a reduction in 343 membrane lipids, and is in line with the role of TOR activating anabolism for proliferation. 344 Thus, as with nitrogen starvation, TAG accumulation under TOR inhibition may be due 345 not only to *de novo* synthesis but also to recycling of fatty acids from membrane lipids 346 (Li-Beisson *et al.*, 2019). Similarly, downregulation of TOR in Arabidopsis by inducible 347 gene silencing or Torin-2 treatment leads to inhibition of *de novo* fatty acid synthesis 348 indicating that TOR is also an activator of fatty acid biosynthesis in plants (Liu *et al.*, 349 2025). This was associated with downregulation of genes encoding enzymes involved in 350 fatty acid synthesis, such as plastidic acetyl-CoA carboxylase 2, stearoyl-ACP 351 desaturase, ketoacyl-ACP Synthase I, and acyl carrier protein (ACP). Interestingly, a 352 recent study showed that knocking out a negative regulator of plastidic acetyl-CoA 353 carboxylase in Chlamydomonas boosted TAG content by five-fold without affecting 354 proliferation (Li *et al.*, 2025). Therefore, combining such a strategy with partial TOR 355 inhibition, which was also shown to enhance TAG productivity in the diatom 356 *Phaeodactylum tricornutum* (Prioretti *et al.*, 2017), could further boost TAG 357 accumulation by limiting the negative effects of TOR inhibition on fatty acid synthesis. 358

359 Another major form of carbon storage in plants and algae is starch and related polymers, 360 which are typically produced in the chloroplast from the products of photosynthesis. 361 Downregulation of TOR by rapamycin in green and red algae leads to the accumulation 362 of starch (Jüppner *et al.*, 2018; Pancha *et al.*, 2019). The underlying molecular 363 mechanisms by which TOR regulates the synthesis and degradation of starch have been 364 partially elucidated in the red alga *C. merolae* (Fig. 2C), although starch biosynthesis in 365 this species actually occurs in the cytoplasm rather than in plastids. *C. merolae* 366 glycogenin (GLG1), the enzyme that initiates starch synthesis, is phosphorylated in a 367 TOR-dependent manner, and loss of phosphorylation is required for starch 368 accumulation in response to TOR inhibition (Pancha *et al.*, 2019). Furthermore, active 369 TOR appears to promote starch degradation by the TOR-dependent phosphorylation of 370 α-glucan water dikinase (GWD), a key regulator of starch degradation (Komiya *et al.*, 371 2025).
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Downregulation of TOR also leads to starch accumulation in the plant Arabidopsis (Moreau et al., 2012; Caldana et al., 2013). Additional mechanistic insight came from a recent study showing that TOR promotes starch degradation in guard cells and induces stomatal opening (Han et al., 2022) (Fig. 2B). In this context, BAM1, a nuclear-encoded chloroplastic β-amylase, was identified as a target for TOR-dependent phosphorylation. This phosphorylation is required for BAM1 accumulation and starch degradation. Notably, TOR-dependent phosphorylation occurs on the chloroplast transit peptide of BAM1, as is also the case for the ppGpp synthase RSH3 (D'Alessandro et al., 2024). However, these phosphorylation events have contrasting outcomes- while BAM1 phosphorylation promotes protein accumulation, phosphorylation of RSH3 reduces chloroplast import. TOR-dependent regulation of BAM1 occurs under specific conditions in a particular cell type. Although the phosphorylation site is not conserved in other BAM isoforms, the Arabidopsis genome encodes eight additional BAM proteins, which may participate in TOR-mediated starch degradation in other cell types (Han et al., 2022).

TOR and autophagy: TOR activity keeps chloroplasts off the menu

The turnover of chloroplasts themselves is likely to be regulated by TOR. As in other eukaryotes, TOR is a major repressor of autophagy in plants (Liu and Bassham, 2010; Agbemafle et al., 2023; Liu et al., 2025). When active, TOR suppresses autophagy via multiple mechanisms: for example, it phosphorylates ATG1 and ATG13 (Van Leene et al., 2019), inhibiting autophagy initiation, and it phosphorylates and activates TAP46 (Ahn et al., 2011), a regulatory subunit of protein phosphatase 2A (PP2A), which further inhibits autophagy. Under stress conditions such as nutrient deprivation, salt stress or osmotic stress, TOR activity is reduced, lifting the repression of autophagy and allowing the breakdown and recycling of cellular components (Pu et al., 2017). Notably, certain stresses, including oxidative stress and endoplasmic reticulum stress, can induce autophagy even when TOR remains active, indicating the existence of a TOR-independent autophagy pathway (Pu et al., 2017). Negative regulation of autophagy by TOR has also been shown in Chlamydomonas. Rapamycin-mediated inhibition of TOR in this microalga leads to the activation of ATG8, which is essential for autophagy, and the formation of autophagosomes, a landmark of autophagy induction (Pérez-Pérez et al., 2010). This catabolic process is activated in response to a wide range of stress conditions, including nutrient (nitrogen, carbon, phosphorus) limitation, oxidative stress and chloroplast damage (Pérez-Pérez et al., 2010; Heredia-Martínez et al., 2018). However, whether chloroplast material is degraded via autophagy in microalgae is currently unknown.

Chloroplasts, which contain up to 80% of total leaf nitrogen in plants, represent a major reservoir for nutrient recycling during growth and stress acclimation. Both nutrient and salt stress promote chlorophagy via multiple autophagic routes, including the piecemeal
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transfer of chloroplast contents to the vacuole through structures such as Rubisco-
containing bodies, ATG8-interacting protein (ATI) bodies and small starch granule-like
bodies (Otegui, 2018; Izumi et al., 2019; Wan and Ling, 2022; Cadena-Ramos and De-la-
Peña, 2024). High-intensity light, which generates oxidative stress and damages the
chloroplast envelope, can instead trigger microautophagy, involving the direct
engulfment of whole chloroplasts into the lumen of the vacuole (Izumi et al., 2017).
Although a direct mechanistic link between TOR and chlorophagy remains to be
established, a pattern is emerging: TOR-dependent autophagy seems primarily
associated with the selective, piecemeal recycling of the chloroplast contents, while
TOR-independent autophagy, triggered by high light induced oxidative stress, favours
whole chloroplast microautophagy. These contrasting routes may reflect differing
physiological priorities. The former allows orderly nutrient salvage, while the latter
enables rapid damage clearance under crisis scenarios.

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Conclusion

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The TOR pathway and the chloroplast are tightly interconnected in plants and algae. On
one hand, chloroplast-derived metabolites regulate TOR activity; and on the other, TOR
influences chloroplast function by regulating nuclear-encoded chloroplast proteins. In
this regard, the TOR-dependent phosphorylation of chloroplast-targeted proteins before
import represents a recently identified regulatory mechanism that might extend beyond
known examples such as RSH3 (for ppGpp metabolism) and BAM1 (for starch
degradation).

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The TOR-chloroplast association is likely to have emerged early after the primary
endosymbiosis of cyanobacteria, as it is observed in both red and green algae. This
association may have been critical for establishing effective communication between
the endosymbiont and the ancestral eukaryotic host, facilitating coordinated nutrient
allocation and energy production. A notable feature of this interaction is the reciprocal
regulation between TOR and photosynthesis, which enables cells to align energy
production with demand. Metabolite signals from photosynthesis help regulate TOR,
which in turn modulates chloroplast activity, creating a feedback loop that supports
balanced growth.

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Further research is needed to determine to what extent this regulatory architecture is
conserved between plant and algal lineages. For instance, it would be interesting to
determine whether DHAP regulates TOR in plants as it does in Chlamydomonas (Mallén-
Ponce et al., 2025). However, the glycolytic pathway is differently compartmentalized in
plants, suggesting that other metabolites may play a more prominent role in relaying light
and carbon signals to TOR. One notably conserved mechanism is the TOR-dependent
control of chloroplast ppGpp metabolism, with mechanisms described at the
transcriptional and post-translational level in the red and green lineages
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respectively (Imamura et al., 2018; D'Alessandro et al., 2024). Interestingly, the
phosphorylated N-terminal region of RSH3 family proteins is also conserved in the
glaucophyte *Cyanophora paradoxa*. However, in vivo assays in *Nicotiana benthamiana*
failed to detect phosphorylation of the glaucophyte RSH N-terminus, leaving open the
question of whether post-translational regulation of ppGpp metabolism predates the
divergence of green algae and land plants (D'Alessandro et al., 2024).

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The impact of unicellular versus multicellular lifestyles on the TOR-chloroplast
association remains unclear. The algae discussed in this article are unicellular, whereas
plants consist of multiple tissues and cell types in which TOR pathway activity and
plastid function can vary. For example, the TOR-BAM1 regulatory axis appears specific
to guard cells (Han et al., 2022). Furthermore, the TOR-plastid relationship is likely to
differ between photosynthetic and non-photosynthetic tissues in plants or between
autotrophic and heterotrophic growth in algae. In Chlamydomonas, for instance, DHAP
and acetate activate TOR under autotrophic and heterotrophic conditions, respectively,
while in Arabidopsis, glucose alone activates TOR in roots but not in leaves (Li et al.,
2017; Mallén-Ponce et al., 2022, 2025). These observations highlight the importance of
tissue-specific and condition-specific analyses to fully understand the roles of the TOR
pathway in photosynthetic eukaryotes.

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**Author contributions**

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JC, BM and BF contributed equally to this work. The order of authorship was determined
by random draw.

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**Conflict of interest**

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The authors declare no conflict of interest.

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**Funding**

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JLC acknowledges funding by Ministerio de Ciencia, Innovación y Universidades (grants
PID2021-123500NB-I00 and TED2021-130912B-I00). BF and BM acknowledge funding by
research grants from the Agence National de la Recherche (ANR-44222-CE20-0033,
ANR-23-CE15-0017, ANR-24-CE13-2215, ANR-20-CE20-0027).

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**Box 1: TOR inhibitors- not all are the same**

Two types of inhibitors with distinct modes of action are commonly used to study the TOR pathway in plants and algae. Rapamycin is an allosteric inhibitor that selectively inhibits TOR by forming a ternary complex with FKBP12 (FK506-binding protein of 12 kDa) and TOR, thereby restricting access to the active site (Yang *et al.*, 2013). In contrast, ATP-competitive inhibitors, such as AZD-8055 or Torin-2 directly compete with ATP at the TOR kinase domain (Chresta *et al.*, 2010).

These different modes of action result in distinct outcomes. In mammalian systems, ATP-competitive inhibitors can inhibit all TOR targets, while rapamycin only blocks phosphorylation of a subset. Consequently, ATP-competitive TOR inhibitors more effectively suppress growth and proliferation, a trend also observed in *Chlamydomonas* and *Arabidopsis* (Montané and Menand, 2019). Notably, *Chlamydomonas* is highly sensitive to rapamycin, which has therefore been widely used in this alga. However, ATP-competitive inhibitors may produce different effects in *Chlamydomonas*, and direct comparisons should be made with caution.

AZD-8055 is one of the most selective TOR inhibitors available and has been genetically validated as TOR-specific *in vivo* in *Arabidopsis* (Chresta et al 2010; Perdoux et al., 2024). It effectively inhibits growth in diverse plants, including monocots and dicots, as well as in various algae, including green algae and diatoms (Montané and Menand, 2013; Couso et al., 2016; Prioretti et al., 2017; da Silva et al., 2021). However, effective concentrations vary between species and treatments should be standardized to produce equivalent levels of growth inhibition for meaningful comparisons across species.

**Figure legends**

**Figure 1. Regulation of TOR function by chloroplast-derived metabolites. (A)** Photosynthetically derived sugars exported from the chloroplast activate TOR (via phosphorylation, P) in distal plant tissues. Sugar regulation of TOR can occur via SnRK1, or potentially via other routes (dashed arrow). Notably, TOR activation by sugar in roots requires mitochondrial function. (B) DHAP, a triose phosphate product of photosynthesis, positively regulates TOR in the green alga *Chlamydomonas*. (C) Active TOR directly phosphorylates the ABA receptor PYL, thereby inhibiting it. ABA, whose precursors are made in the chloroplast, binds PYL. ABA-bound PYL binds and inhibits the PP2C phosphatase, activating SNRK2. Active SNRK2 directly phosphorylates the RAPTOR subunit of TOR, inhibiting TOR activity. Lines ending in arrowheads denote positive regulation or metabolic flux; those terminating in a perpendicular “T”-bar indicate inhibition; and a thicker arrow that pierces the double chloroplast envelope represents transport across the plastid membrane. In every case, the line may stand for
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a multi-step process. The Arabidopsis TOR complex was rendered from an AlphaFold model (Abramson et al., 2024) (RAPTOR, yellow; TOR, light blue; LST8, cyan). A monomer is shown, though the active form is likely a homodimer. TOR, target of rapamycin; RAPTOR, regulatory associated protein of TOR; LST8, lethal with SEC13 protein 8; CBB, Calvin-Benson-Bassham cycle; PSII, photosystem II; PSI, photosystem I; DHAP, dihydroxyacetone phosphate; TPT3, triose phosphate transporter; ABA, abscissic acid; SNRK, SNF1-related protein kinase; PP2C, protein phosphatase 2C; PYL, PYR1-like. The figure was prepared in Inkscape

**Figure 2. Regulation of chloroplast function by TOR. (A)** In Arabidopsis, the LST8 subunit of TOR binds to the chloroplast transit peptide of RSH3, and RSH3 is phosphorylated (P) in a TOR-dependent manner. Upon TOR inactivation, non-phosphorylated RSH3 is more efficiently imported into the chloroplast, and ppGpp levels increase. ppGpp accumulation results in downregulation of photosynthesis. (B) In the red alga *C. merolae*, Floridean starch is synthesised in the cytosol from chloroplast-derived triose phosphates. TOR promotes starch degradation by influencing the activity of two enzymes. The starch synthesis enzyme GLG1 undergoes inhibitory TOR-dependent phosphorylation, while the starch degradation enzyme GWD undergoes an activating TOR-dependent phosphorylation. (C) In Arabidopsis guard cells, active TOR promotes the direct or indirect phosphorylation of BAM1 on a serine residue in the chloroplast transit peptide. BAM1 phosphorylation promotes BAM1 accumulation, and the activation of starch degradation in the chloroplast. Lines ending in arrowheads denote positive regulation or metabolic flux; those terminating in a perpendicular “T”-bar indicate inhibition; and a thicker arrow that pierces the double chloroplast envelope represents transport across the plastid membrane. In every case, the line may stand for a multi-step process. The Arabidopsis TOR complex was rendered from an AlphaFold model (Abramson *et al.*, 2024) (RAPTOR, yellow; TOR, light blue; LST8, cyan). A monomer is shown, though the active form is likely a homodimer. TOR, target of rapamycin; RAPTOR, regulatory associated protein of TOR; LST8, lethal with SEC13 protein 8; CBB, Calvin-Benson-Bassham cycle; PSII, photosystem II; PSI, photosystem I; RSH3, RelA SpoT homolog 3; ppGpp, guanosine tetraphosphate; BAM1, β-amylase 1; GLG1, glycogenin 1; GWD, α-glucan water dikinase. The figure was prepared in Inkscape.
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