# Lack or excess of autophagy leads to premature leaf senescence probably owing to unbalanced nutrient management

Maxence James, Jacques Trouverie, Anne Marmagne, Fabien Chardon, Julie Frémont, Philippe Etienne, Céline Masclaux-Daubresse

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## ► To cite this version:

Maxence James, Jacques Trouverie, Anne Marmagne, Fabien Chardon, Julie Frémont, et al., Lack or excess of autophagy leads to premature leaf senescence probably owing to unbalanced nutrient management. Annals of Botany, 2025, 10.1093/aob/mcaf050. hal-05104668

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PLANT SENESCENCE

ORIGINAL ARTICLE

# Lack or excess of autophagy leads to premature leaf senescence probably owing to unbalanced nutrient management

Maxence James¹,†, Jacques Trouverie¹,†,✉, Anne Marmagne²,✉, Fabien Chardon²,✉, Julie Frémont¹, Philippe Etienne¹,†,✉ and Céline Masclaux-Daubresse²,*,†,✉

¹Université de Caen Normandie, UNICAEN, INRAE, UMR 950 EVA, SFR Normandie Végétal (FED4277), 14000 Caen, France, and ²Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin for Plant Sciences (IJPB), 78000 Versailles, France

†Authors with equal contribution.

*For correspondence: E-mail celine.masclaux-daubresse@inrae.fr

Received: 12 September 2024

Returned for revision: 17 March 2025

Editorial decision: 20 March 2025

Accepted: 16 May 2025

* **Background and Aims** Macroautophagy is essential for the degradation and recycling of various macromolecules in eukaryotic cells. In plants, autophagy is involved in the degradation of damaged chloroplasts in response to stress. Autophagy is a key player in nitrogen management at the whole-plant level, and autophagy mutants display strong defects in nitrogen remobilization and early leaf senescence phenotypes, especially during nitrogen source limitation. It is known that leaf senescence is associated with nutrient remobilization processes and is induced by nitrate limitation. However, it remains to be determined which actors are involved in this interplay and whether nutrient remobilization acts as a signal that enhances leaf senescence or whether senescence-associated chloroplast degradation promotes nutrient remobilization. In this context, our aim was to demine whether the level of autophagic activity controls leaf longevity and influences the progress of leaf senescence, balancing resources and waste management.

* **Methods** In this study, we used *Arabidopsis* autophagy knock-out mutants (*atg*) and plants overexpressing autophagy-associated genes (*ATG8*-OE) to compare their phenotypes with wild-type in both nitrate-sufficient conditions and nitrate starvation.

* **Key Results** The transfer from nitrate-sufficient conditions to nitrate starvation accelerated leaf senescence in all the genotypes. Unexpectedly, in both nitrate-sufficient and nitrate-starved conditions, both *atg* mutants and *ATG8* overexpressors exhibited earlier leaf senescence phenotypes compared with wild-type. Given that autophagy is a longevity factor, the more severe leaf senescence phenotype of overexpressors was puzzling. This study highlights a relationship between autophagy, nitrogen remobilization and leaf senescence and shows how the fine-tuning of nutrient management can influence senescence onset.

* **Conclusions** The fine-tuning of autophagy is necessary to control leaf senescence.

**Key words:** *Arabidopsis thaliana* L., autophagy, leaf longevity, nitrogen remobilization, plant longevity, programmed cell death.

INTRODUCTION

Macroautophagy is a universal mechanism that is essential for the degradation of unwanted cytoplasmic components in eukaryotic cells. Macroautophagy (hereafter referred to as autophagy) involves the formation of cytosolic double-membrane vesicles called autophagosomes (Gomez et al., 2021). Within the cell, autophagosomes act like ‘garbage trucks’ that transport unwanted cytoplasmic material to the lytic vacuoles. They sequester cytoplasmic constituents destined for degradation. The substrates for autophagy are diverse, including damaged or unnecessary proteins, protein aggregates, portions of damaged

organelles and membranes (Xiong et al., 2007). By facilitating cellular cleaning, ensuring organelle quality control and recycling macromolecules into smaller molecules that can be reused for cellular nutrition or exported to other organs, autophagy is a longevity factor that maintains cell homeostasis in all eukaryotic organisms (Masclaux-Daubresse et al., 2017).

Autophagosome formation is governed by 18 AUTOPHAGY (ATG) proteins, first discovered in yeast (Tsukada and Ohsumi, 1993). Homologous genes were subsequently identified in plants and animals (Yang and Bassham, 2015). In *Arabidopsis*, as in yeast, several autophagy genes are unique, whereas some are in multicopies and form families. For instance, there are
nine *ATG8* genes in *Arabidopsis* (*ATG8a-i*). The mutants of single *ATG* genes, such as *atg5*, *atg7* or *atg10*, cannot produce autophagosomes. They display strong phenotypes, including early leaf yellowing, reduced yield and plant biomass, and hypersensitivity to various abiotic stresses (Thompson *et al.*, 2005; Phillips *et al.*, 2008; Yoshimoto *et al.*, 2009; Lenz *et al.*, 2011; Guiboileau *et al.*, 2012, 2013; Masclaux-Daubresse *et al.*, 2014). In contrast, owing to functional redundancies, single mutation of one of the autophagy genes belonging to one of the multigene families (such as *ATG4a-b*, *ATG12a-b* and *ATG8a-i*) does not produce any phenotype. Only the accumulation of mutations in each of the genes in the family seems to allow the appearance of symptoms.

The ATG8 proteins are the only proteins associated with the membranes of the autophagosomes. The ATG8-phosphatidylethanolamine (PE) conjugates, which decorate autophagosome membranes, are involved in recruiting and sequestering cytoplasmic material to be degraded (Noda et al., 2010; Masclaux-Daubresse et al., 2017). The conjugation of ATG8 proteins with PE and their anchorage to the membrane are essential for the biogenesis, formation and enclosure of autophagosome vesicle. In *Arabidopsis*, single *atg8* mutants do not display specific phenotypes compared with wild-type (WT) plants owing to redundancies between the different ATG8 proteins. By contrast, plants overexpressing *ATG8A* or *ATG8G* (*ATG8*-OE) display a better performance for nutrient use (Chen et al., 2019) and better resilience to various stresses, such as drought (Xia et al., 2012; Li et al., 2015).

In plants, as in other organisms, autophagy is maintained at a basal level in nutrient-rich conditions. However, during nutrient starvation, autophagy is induced to maintain cellular homeostasis and provide substrates for cell survival. In yeasts and plants, it has been shown that most *ATG* genes are upregulated in response to nitrogen starvation, suggesting that the steady-state levels of their corresponding proteins are insufficient to support autophagy induction (Avila-Ospina et al., 2014; Bernard and Klionsky, 2015; Bernard et al., 2015). In nitrogen-rich conditions, autophagy is kept at a basal level through the post-translational repression exerted by the Target of Rapamycin (TOR) kinase on the ATG1–ATG13 regulatory complex (Bernard and Klionsky, 2015; Bernard et al., 2015; Pu et al., 2017). Although it is relatively straightforward to abolish autophagy flux by mutating one of the *ATG* single genes, genetically stimulating autophagy is more challenging because it is not possible to modify TOR activity without creating strong cellular damage and it is difficult to predict which of the *ATG* genes of the core machinery is limiting for autophagy activity. It has been shown in several organisms, including human cells, that the overexpression of *ATG8* stimulates autophagic activity by increasing the size and/or number of autophagosomes (Jin and Klionsky, 2014; Li et al., 2015; Chen et al., 2019). Indeed, in *Arabidopsis*, Masclaux-Daubresse's group showed that the overexpression of the *ATG8A* and *ATG8G* genomic sequences under the control of ubiquitin promoter increased the number of autophagosomes, thereby stimulating autophagic activity and improving plant performance in nitrogen remobilization to the seeds (Chen et al., 2019).

In all organisms, autophagy is recognized as a longevity factor involved in organelle and protein quality control and in cellular homeostasis. However, the question of whether

excessive autophagic activity contributes to cell death remains debated (Guiboileau et al., 2010; Minina et al., 2014; Denton and Kumar, 2019). Until now, most of the studies addressing the pro-death role of autophagy have been conducted in animals regarding developmental questions or cancer (Denton and Kumar, 2019). Given that autophagy gene expression gradually increases during leaf senescence (Avila-Ospina et al., 2014), it can be hypothesized that autophagic activity could participate in leaf senescence symptoms and in the final stages of cell degradation to death. Still, the fact that autophagy mutants exhibit early leaf yellowing and cell death spots in their leaves suggests that during the early stages of leaf senescence, autophagy functions as a longevity factor (Thompson et al., 2005; Yoshimoto et al., 2009). Studies on autophagy mutants by Guiboileau et al. (2012, 2013) demonstrated that autophagy in leaves contributes to protein degradation, nutrient recycling and the export of nutrients from senescing tissues to new growing tissues. It seems plausible that by clearing the cytoplasmic material damaged by the reactive oxygen species that result from malfunction of the photosynthetic apparatus, autophagy extends cell longevity, at least to a certain extent. However, it remains unclear whether autophagy contributes to cell death during the late stages of senescence owing to excessive cytoplasmic degradation.

Knowing that leaf senescence and nitrogen remobilization are tightly linked, the aim of this study was to decipher the effect of autophagy on leaf senescence in parallel with its role in the management of nitrogen flux from source to sink at the whole-plant level. To determine whether the activity of autophagy can influence the progress of leaf senescence, we compared the phenotypes of autophagy-defective plants (well-characterized *atg5* and *atg7* knock-out mutants; Lai et al., 2011; Guiboileau et al., 2012), autophagy-stimulated plants (*ATG8*-OE previously characterized; Chen et al., 2019) and wild-type.

## MATERIALS AND METHODS

### Plant material and growth conditions

The *Arabidopsis thaliana* autophagy T-DNA mutants *atg5-1* (SAIL_129B07) and *atg7-2* (GK-655B06) were described previously by Guiboileau *et al.* (2012) and Lai *et al.* (2011). The ATG8 overexpressors (*ATG8a*-OE and *ATG8g*-OE) were constructed using genomic coding sequences and the pUbiQ10 promoter and characterized for stimulation of autophagy by Chen *et al.* (2019). All four genotypes share the Col-0 background. Seeds were stratified for 48 h in 0.1 % agar at 4 °C in the dark, then sown onto lids cut from 0.5 mL Eppendorf tubes filled with 0.7 % agar. Plants of each genotype were placed in a glasshouse on a tank containing 10 L of 3.5 mM NO₃⁻ nutrient solution [1 mM KNO₃, 1.25 mM Ca(NO₃)₂, 0.2 mM KH₂PO₄, 0.4 mM MgSO₄, 0.1 mM K₂SO₄, 50 µM NaFe-EDTA, 50 µM NaFe-EDDHA, 20 µM H₃BO₃, 6 µM MnSO₄, 3 µM ZnSO₄, 0.7 µM CuSO₄, 0.008 µM (NH₄)₆Mo₇O₂₄, 0.1 µM CoCl₂, 0.15 µM NiCl₂, 0.9 µM Si(OH)₄, 0.5 µM CaCl₂, 0.1 µM KCl, 0.01 µM Na₂SeO₄ and 0.2 µM Na₂SiO₃, buffered to pH 6.8 with 0.36 mM CaCO₃]. Plants were grown in this solution for 44 days. This solution was renewed weekly. At 44 days after sowing (DAS), plants were transferred to two contrasting nitrogen conditions: high nitrate (HN; 3.5 mM nitrate) and no nitrate (ON; 0.048 µM
nitrate). The photosynthetic photon flux density was 110 µmol m⁻² s⁻¹, and day and night temperatures were 21 and 18 °C, respectively. After sowing, plants were cultivated in short days (8 h light–16 h dark) until 64 DAS. At 64 DAS, several plants were harvested to image individual leaves to determine leaf- yellowing ratios using ImageJ. Plant material was frozen in liquid nitrogen before storage at –80 °C for subsequent meas- urement of the expression of flowering genes and senescence marker genes. The rest of the plants were transferred to long days (16 h light–8 h dark photoperiod) to induce flowering and to determine biomass and nitrogen allocations at the end of the reproductive stage (Fig. 1A).

Measurement of plant biomass and leaf area

Rosette areas were measured weekly during the first 44 DAS, and individual leaf areas were measured at 64 DAS. The rosette and individual leaves were imaged using a Canon Powershot G10 camera (Canon, Japan, 14.8 Mpx, JPEG). Leaf areas were determined using a custom script developed for ImageJ

software. Total leaf area was calculated using colour detection based on the hue component of the hue–saturation–lightness system. The yellow leaf area was obtained by subtracting the green leaf area from the total leaf area, with the green area being measured previously by the script.

At seed maturity, plants were harvested and dried in an oven at 60 °C for 4 days to obtain dry matter. Different plant organs were separated, and their dry weights were determined. The harvest index was calculated as the ratio between seed biomass and total plant biomass.

Elemental analysis for nitrogen determination

At seed maturity, nitrogen concentrations (in milligrams per 100 mg dry weight) were determined in different plant com- partments (roots, leaves, stems, pods and seeds) using an elem- ental analyser (EA3000, EuroVector, Milan, Italy). Nitrogen partitioning among the plant organs was calculated as the ratio of nitrogen content in each organ relative to the total nitrogen content of the whole plant.

<figure><img src="image_3.png" /><figcaption>FIG. 1. Leaf areas of WT, autophagy mutants (*atg5* and *atg7*) and overexpressors (*ATG8a*-OE and *ATG8g*-OE) reflect different growth rates in high-nitrogen conditions. (A) Diagram of the experimental set-up for the study of autophagy mutants and overexpressors. The WT (Col), autophagy mutants (*atg5* and *atg7*) and overexpressors (*ATG8a*-OE and *ATG8g*-OE) were cultivated in a hydroponic system in high-nitrate (HN) conditions in short days. Forty-four days after sowing (DAS), half of the plants were transferred to no nitrate (ON). At 64 DAS, all the plants were switched to long days. Harvesting time at seed maturity occurred at seed maturity. (B) Rosette areas were recorded once a week during the first 44 days of plant growth under HN. Values represent means ± s.e. for *n* ≥ 11. Significant differences (*P* ≤ 0.05) between genotypes for each day after sowing are indicated by different letters in the table.</figcaption></figure>
Measurement of pigment concentrations in leaves

Extraction of chlorophylls and carotenoids was performed according a protocol described by Launay et al. (2019). Briefly, 100 mg of leaf fresh weight (FWc) was ground in liquid nitrogen and resuspended in 1 mL of 92 % acetone. After centrifugation (10 000g for 10 min at 4 °C), the supernatant was collected and adjusted to 2 mL (Vc) with 80 % acetone. The absorbance (Aλ) of the acetonic extract was read at λ = 480, 638, 645 and 663 nm using 80 % acetone as a blank. Anthocyanins were extracted by incubating 20 mg of leaf fresh weight (FWa) in 0.5 mL (Va) of methanol/HCl (99/1, v/v) at 4 °C for 24 h. After 10 min of centrifugation at 6000g, the absorbance of supernatant was measured at λ = 530 and 657 nm with methanol/HCl as a blank. The concentrations (in micrograms per milligram fresh weight) of anthocyanin (Panuccio et al., 2016), chlorophyll a, chlorophyll b and carotenoids (Launay et al., 2019) were calculated as follows:

$$
\begin{align*}
[\text{Anthocyanin}] &= [A_{530} - (0.025 \times A_{657})] \times (V_a/FW_a) \\
[\text{Chlorophyll a}] &= [(12.7 \times A_{663}) - (2.6 \times A_{645})] \times (V_c/FW_c) \\
[\text{Chlorophyll b}] &= [(22.9 \times A_{645}) - (4.68 \times A_{663})] \times (V_c/FW_c) \\
[\text{Carotenoids}] &= A_{480} + (0.14 \times A_{663} - 0.638 \times A_{645}) \times (V_c/FW_c)
\end{align*}
$$

Total RNA extraction and RT-digital droplet PCR analysis

Total RNAs were extracted using a protocol previously de-
scribed by James et al. (2018). Briefly, 200 mg of frozen leaf
samples ground in liquid nitrogen were suspended in 750 µL
of extraction buffer [0.1 M TRIS, 0.1 M LiCl, 0.01 M EDTA,
1% SDS (w/v), pH 8] and 750 µL of hot phenol (80 °C,
pH 4.3). The mixture was vortexed for 40 s, and 750 µL of
chloroform:isoamylalcohol (24/1: v/v) was added before cen-
trifugation (15 000g for 5 min at 4 °C). The supernatant was
collected, and 750 µL of 4 M LiCl solution (w/v) was added be-
fore incubation overnight at 4 °C to precipitate total RNA. After
centrifugation (15 000g for 20 min at 4 °C), total RNA was sus-
pended in 100 µL of sterile water before purification using the
RNeasy mini kit (Qiagen, Courtaboeuf, France). Quantification
of total RNA was performed by spectrophotometry at 260 nm
(BioPhotometer, Eppendorf, Le Pecq, France). For RT, 1 µg of
total RNA was converted to complementary DNA (cDNA) with
an iScript cDNA synthesis kit according to the manufacturer's
protocol (Bio-Rad, Marne-la-Coquette, France). Digital droplet
PCR (ddPCR) was performed on cDNAs diluted 1000-fold
using a QX200 Droplet Digital PCR System (Bio-Rad, USA).
Each well of a DG8 cartridge (Biorad, USA) was loaded with
20 µL of reaction mixture [4.4 µL of cDNA, 11 µL of EvaGreen
Mix (Biorad, USA) containing DNA polymerase and dNTPs,
0.2 µL of 100x forward and reverse primer specific to house-
keeping or target genes, qsp H2O (Supplementary Data Table
S1) and 70 µL of QX 200 droplet generation oil]. Droplets were
created using the QX 200 droplet generator (Bio-Rad, USA)
and transferred to a 96-well PCR plate for amplification using
the following programme: 94 °C for 10 min, 35 cycles of 94
°C for 15 s and 60 °C for 60 s, and 98 °C for 10 min. Then,
in each well, positive (fluorescing droplets containing target or

housekeeping genes) and negative (without gene) droplets were
counted using a QX200 droplet reader (Bio-Rad, USA). Data
were analysed using QX Manager Software v.2.2.071 (Bio-
Rad, USA). The expression of each target gene was normalized
by the geometric mean of the expression of the three house-
keeping genes (EF1-α, 18s RNA and SBP-1).

Statistical analysis

Data are presented as the mean ± s.e. (n ≥ 3). Shapiro–Wilk tests and Tukey tests were performed using R software. When data did not follow a normal distribution according to Shapiro–Wilk tests, they were transformed using a logarithmic scale. Statistical significance was considered at *P* ≤ 0.05.

RESULTS

Autophagy controls plant growth

Rosette areas were recorded weekly for 44 DAS on autophagy
mutants (*atg5* and *atg7*), *ATG8* overexpressing lines (*ATG8a*-OE
and *ATG8g*-OE; *Supplementary Data Fig. S1*) and wild-type
(WT) plants cultivated in a hydroponic system with sufficient
nitrate supply (HN; Fig. 1A). We decided to use hydroponics,
which permitted better plant homogeneity in comparison to
other growth conditions and which facilitated the access to the
root compartment. Starting at 28 DAS, rosette areas were sig-
nificantly larger in *ATG8a*-OE and *ATG8g*-OE compared with
WT, whereas the areas in *atg5* and *atg7* mutants were signifi-
cantly smaller than in WT (Fig. 1B). This indicated that defects
in the autophagy machinery impaired plant growth, whereas
stimulation of autophagic activity enhanced plant growth and
biomass production with sufficient nitrate supply. These results
highlight the positive role of enhanced autophagic activity in
promoting plant growth in non-stressed conditions.

Autophagy controls biomass and nitrogen partitioning at harvest

To verify that our hydroponic culture conditions aligned with previous studies from our group, we separated plants into two batches and grew them until seed maturity with either sufficient nitrate supply or nitrate starvation, to verify that phenotypes already described for autophagy mutants (Guiboileau et al., 2012) and ATG8 overexpressing lines (Chen et al., 2019) were reproduced. Regardless of the nitrate conditions, the plant biomass of *atg5* and *atg7* autophagy mutants was lower (only one-fifth) than that of WT at seed maturity (Fig. 2A, B). Biomass partitioning revealed that the primary developmental defect in autophagy mutants was related to the production of reproductive organs (stems, pods and seeds) (Fig. 2C, D). The harvest index (HI), calculated as the ratio of seed yield to total plant biomass, was significantly reduced in autophagy mutants compared with WT in both HN and ON conditions. The nitrogen harvest index (ratio of seed N to total plant N) was sharply lower in autophagy mutants owing to their inability to remobilize nitrogen efficiently from rosette leaves (Fig. 3A, B).

In contrast, the total biomass and organ weights of *ATG8a*-OE
and *ATG8g*-OE were not significantly different from WT,
<figure><img src="image_4.png" /><figcaption>FIG. 2. Dry weights and dry matter partitioning are different in both autophagy mutants and overexpressors compared with wild-type. The dry weights of the different plant organs were recorded on plants harvested at seed maturity (A, B). The partitioning of dry matter in the different organs was calculated from dry weights and expressed as a percentage of the whole plant (C, D). WT, autophagy mutant (*atg5* and *atg7*) and overexpressor (*ATG8a*-OE and *ATG8g*-OE) plants were cultivated in high-nitrate (HN; A, C) or no-nitrate (ON; B, D) conditions. Values represent means ± s.e. for *n* = 5. Significant differences (*P* ≤ 0.05) between genotypes for each nitrogen condition are indicated by different letters.</figcaption></figure>

irrespective of the HN or ON conditions (Fig. 2A, B). However, in HN, the dry weights of the rosettes in *ATG8a*-OE and *ATG8g*-OE were lower than that of WT, whereas pod dry weights were higher (Fig. 2A). Under HN, seed yield was significantly higher in *ATG8g*-OE and tended to be higher in *ATG8a*-OE compared with WT. Consequently, biomass allocation between leaves and seeds in HN differed in *ATG8a*-OE and *ATG8g*-OE compared with WT, with both *ATG8*-OE lines displaying a higher HI than WT in HN (Fig. 2C). These results show that in HN conditions,

*ATG8* overexpression improved plant performance in our hydroponic conditions. However, no differences between WT and the two *ATG8*-OE lines were observed under ON in terms of dry weights, biomass or nitrogen partitioning, as previously shown by *Chen et al.* (2019) (Figs 2D and 3B). The absence of differences in root development, total biomass and N concentrations in the different organs (not shown) between *ATG8*-OE plants and WT in HN suggests that there was no difference in nitrate uptake between these genotypes.
<figure><img src="image_5.png" /><figcaption>FIG. 3. Autophagic activity is correlated with N remobilization from leaves to seeds in *Arabidopsis* under HN conditions. Nitrogen partitioning in the different organs of WT, autophagy mutants (*atg5* and *atg7*) and overexpressors (*ATG8a*-OE and *ATG8g*-OE) was calculated from nitrogen concentrations and dry weights, and expressed as a percentage of the whole plant. The nitrogen concentrations of the different compartments (roots, leaves, stems, pods and seeds) were measured on plants cultivated in high-nitrate (HN; A) or no-nitrate (ON; B) conditions, and harvested at seed maturity. Values represent means ± s.e. for *n* = 5. For each nitrogen condition, different letters indicate significant differences (*P* ≤ 0.05) between genotypes.</figcaption></figure>

These results show that the two *ATG8*-OE lines outperformed WT for rosette to seed nitrogen remobilization in the HN growth conditions, which provide enough nitrate to avoid natural autophagy induction in WT (Supplementary Data Fig. S2), whereas the absence of autophagic activity severely impaired plant biomass and nitrogen remobilization. All these phenotypes were in line with previous results found by Guiboileau *et al.* (2012) and Chen *et al.* (2019).

Both autophagy mutants and *ATG8* overexpressors exhibit more severe leaf senescence phenotypes than wild-type irrespective of growth conditions

To assess leaf senescence in different genotypes cultivated in HN or ON, we separated the first 15 leaves from the rosettes of *atg5*, *atg7*, *ATG8a*-OE, *ATG8g*-OE and WT plants at 64 DAS. The dissected leaves were imaged immediately after separation (Fig. 4A, B). In both HN and ON conditions, more yellow leaves were observed in *atg* mutants and *ATG8* overexpressors than in WT. The yellow and green areas were quantified using imaging tools (Fig. 4C, D). In HN, the proportion of yellow area was three and two times higher in autophagy mutants and *ATG8* overexpressors, respectively, compared with WT (Fig. 4C). In ON, the differences between WT, *atg* mutants and *ATG8*-OE were smaller. However, even during ON, the percentage of yellow area remained significantly higher in *atg* mutants and *ATG8*-OE than in WT (Fig. 4D).

To confirm earlier senescence in the leaves of autophagy mutants and of the *ATG8* overexpressing lines, we measured pigment concentrations (Fig. 5). In HN conditions, the concentrations of total chlorophyll and of chlorophyll *a* (chl-a) in aged leaves were significantly higher in WT than in *atg5*, *atg7*, *ATG8a*-OE and *ATG8g*-OE (Fig. 5A, D). These concentrations were also significantly higher in the entire WT rosette than in the rosettes of *atg5*, *atg7* and *ATG8g*-OE in HN (Fig. 5B, E) and ON (Fig. 5C, F). For chlorophyll *b* (chl-b), differences between WT and the other genotypes are observable and generally follow the same trend as for chl-a, but with a lower degree of significance (Fig. 5G–I). Carotenoid concentrations were less variable between genotypes. Carotenoid concentrations in the old leaves of HN plants were significantly lower in autophagy mutants and *ATG8* overexpressing lines than in WT (Fig. 5J, K). Changes in anthocyanin contents do not allow WT to be distinguished from other genotypes except in ON conditions (Supplementary Data Fig. S3). In addition to pigments, the expressions of genes known to be related to senescence, such as the Senescence Associated Gene 12 (*SAG12*), the NAC transcription factor *ORESARA1* (*ORE1*) and the WRKY DNA-BINDING PROTEIN 70 (*WRKY70*), in the rosettes of plants grown in HN and LN confirmed that both autophagy mutants and *ATG8* overexpressing lines were more senescent than WT (Fig. 6; Besseau *et al.*, 2012; Rauf *et al.*, 2013; James *et al.*, 2018; Rankenberg *et al.*, 2024).

Altogether, the results show that both autophagy deficiency and autophagy over-stimulation modify the onset and progress of leaf senescence.
<figure><img src="image_6.png" /><figcaption>FIG. 4. Autophagy mutants and overexpressors display higher leaf senescence symptoms than wild-type. Leaves were dissected from WT, autophagy mutants (*atg5* and *atg7*) and overexpressors (*ATG8a*-OE and *ATG8g*-OE) 20 days after their transfer to high-nitrate (HN; A, C) or no-nitrate (ON; B, D) conditions [64 days after sowing (DAS)]. Representative pictures of whole rosette and leaf distribution from the base to the apex are presented (A, B). Histograms represent leaf areas split into yellow and green areas (C, D). The red squares in the pictures (A, B) correspond to the 15 oldest leaves used to measure the percentages of yellow (senescent) and green (non-senescent) leaf areas. White scale bars: 20 mm. The measures were performed on the 15 oldest leaves dissected from four plant replicates for each genotype. All analyses were performed at 64 DAS. Values represent means ± s.e. for *n* = 4. Significant differences (*P* ≤ 0.05) between genotypes for each nitrogen condition are indicated by different letters.</figcaption></figure>

## Both defect in and overexpression of autophagic activity modifies bolting time

As shown in Fig. 1A, plants were first grown under short days, then shift to long days at 64 DAS. The expression of the *FT* flowering gene, determined at 64 DAS, was very low and not significantly different from WT in any of the autophagy mutants and *ATG8* overexpressing lines. After transfer to long day, the first flowering buds (0.5 cm over the rosette) were observed at 70 DAS, i.e. 6 days after transfer. Discrepancies in flowering bud emergence were observed between the different genotypes, with globally a delay of bud emergence in *atg5* and *atg7* compared with WT under both HN and ON, whereas buds appeared slightly earlier in both *ATG8a*-OE and *ATG8g*-OE than in WT, at least in ON (Supplementary Data Fig. S4).

## DISCUSSION

Leaf senescence is associated with autophagy, as demonstrated by the gene networks published by Penfold and Buchanan-Wollaston (2014). Autophagy is induced in parallel to leaf ageing, and the senescence-associated transcription factor ATAF1 has been reported to control the expression of autophagy genes in *Arabidopsis* (Garapati et al., 2015; Wang et al., 2020). More recently, Lee et al. (2024) reported that Pif4 and Pif5 (phytochrome interacting factor 4 and 5, respectively) directly regulate autophagy during leaf senescence. Nevertheless, the numerous physiological studies and transcriptional analyses of leaf senescence have not yet clarified the cause-and-effect relationships, and whether the enhancement of catabolic processes involved in nutrient recycling drives the degradation of the photosynthetic apparatus and leads to leaf yellowing or
<figure><img src="image_7.png" /><figcaption>FIG. 5. Chlorophyll concentrations are lower in autophagy mutants and in the *ATG8* overexpressing lines compared with wild-type. Concentrations of total chlorophyll (*a* + *b*; A–C), chlorophyll *a* (D–F), chlorophyll *b* (G–I) and carotenoid (J–L) were measured in WT, autophagy mutants (*atg5* and *atg7*) and *ATG8* overexpressing lines (*ATG8a*-OE and *ATG8g*-OE) grown in high-nitrate (A, B, D, E, G, H, J, K) or low-nitrate (C, F, I, L) conditions. The measures were performed on the 15 oldest leaves (A, D, G, J) and entire rosettes (B, E, H, K) of plants grown in HN conditions and on the entire rosette (C, F, I, L) for plants grown in ON at 64 days after sowing (DAS). Values represent means ± s.e. for *n* = 4 and *n* = 3 for HN and ON conditions, respectively. Significant differences (*P* ≤ 0.05) between genotypes are indicated by different letters.</figcaption></figure>

whether the senescence programme inducing the decay of the photosynthetic apparatus and yellowing is a prerequisite for N remobilization through induction of autophagy.

Two types of leaf senescence are described in plants. Sequential senescence is characteristic of the vegetative stage and occurs after the leaves have reached optimal growth and maturity. First, sequential senescence is associated with the

metabolic shift that promotes transfer of nutrients from ageing leaves to developing leaves that represent new sinks for the plant (Avice and Etienne, 2014). The second type of senescence is enhanced after flowering; it is called monocarpic senescence. It plays a central role in the transfer of nutrients from vegetative tissues to the reproductive organs and was especially investigated in crops. In *Arabidopsis*, both sequential and
<figure><img src="image_8.png" /><figcaption>FIG. 6. Senescence-associated genes are overexpressed in autophagy mutants (*atg5* and *atg7*) and *ATG8* overexpressors (*ATG8a*-OE and *ATG8g*-OE). The expression levels of *SAG12*, *ORE-1* and *WRKY70* in the rosette leaves of WT, autophagy mutant (*atg5* and *atg7*) and *ATG8* overexpressor (*ATG8a*-OE and *ATG8g*-OE) plants grown in HN or 0N, were measured by RT-digital droplet PCR. Values were normalized with the geometric mean of three housekeeping genes (*EF1-α*, 18S RNA and *SBP-1*). Means of gene expressions (*n* = 3) were calculated and expressed relative to WT as log₂ fold change (log₂FC). Scale from blue to red represent the values of log₂FC.</figcaption></figure>

monocarpic senescence have been observed (Diaz et al., 2006), and both are involved in nutrient remobilization, although towards different sinks (Diaz et al., 2008). In our study, the early leaf-yellowing phenotypes of both the autophagy mutants and the *ATAG8* overexpressing lines were observed as early as 60 DAS, several days before the transfer to long-day conditions and before the appearance of a flowering bud. At 64 DAS, the expression of the flowering gene was absent across all the genotypes (data not shown). Thus, it can be concluded that the early senescence phenotypes of autophagy mutant and *ATAG8* overexpressing lines are unrelated to flowering and are typical of sequential senescence that appears on leaves at the vegetative stage. In addition, we noticed that after the transfer to long days, flowering occurred earlier in the autophagy mutants and was delayed in the lines overexpressing *ATAG8* compared with the WT. This pattern underscores the lack of correlation between leaf yellowing and flowering time.

The earlier leaf senescence phenotypes of autophagy mutants and *ATAG8* overexpressing lines could, however, be related to discrepancies in nitrogen management. Indeed, it is well known that leaf senescence is closely associated with protein degradation, nitrogen (N) remobilization and CO₂ fixation. In crops, the ‘stay-green’ phenotypes often lead to reduced seed protein concentrations owing to prolonged carbon fixation and delayed N remobilization (Gregersen et al., 2013). In *Brassica napus*, defective remobilization during the sequential senescence of the rosette leaves leads to poor performance in nitrogen remobilization (Avice and Etienne, 2014). The noticeable effect of defective autophagy on the plant nitrogen budget observed under 0N in *atg5* and *atg7* confirms that functional autophagy is crucial for N remobilization (Fig. 3) and seed production (Fig. 2; Guiboileau et al., 2012). The overexpression of *ATG8a* and *ATG8g*, known to increase autophagic activity (Supplementary Data Fig. S1; Chen et al., 2019), significantly improves nitrogen remobilization to the seeds, which globally improves nitrogen use efficiency (NUE) relative to WT in the nitrate-sufficient conditions (Chen et al., 2019). The Fig. 3A

nicely illustrates the opposite effects of autophagy defect and autophagy stimulation on nitrogen allocation to the seeds.

The earlier leaf senescence observed in both *atg* mutants and *ATG8*-OE compared with WT shows that timely and fine-tuned autophagy is important not only for nitrogen remobilization but also for leaf senescence and thus photosynthetic capacity. The fact that both autophagy mutants and *ATG8* overexpressing lines are altered for nutrient remobilization in opposite ways while they all exhibit a phenotype of early leaf senescence suggests that their early yellowing is associated with distinct physiological events, although potentially related to resource management (Fig. 7; Guiboileau et al., 2013; Chen et al., 2019). It is well established that the lack of cellular clearance in autophagy mutants limits nutrient remobilization (Guiboileau et al., 2012; Lornac et al., 2020) and results in the accumulation of toxic compounds in source leaves (Fig. 7A, B). Although early senescence phenotype of *atg* mutants is attributable, in part, to the overproduction of salicylic acid in their leaves (Yoshimoto et al., 2009; Guiboileau et al., 2012), studies combining autophagy mutations with mutations in salicylic acid biosynthesis (*sid2*) or with the overexpression of *NahG* demonstrated that salicylic acid is not the only cause of early yellowing. Indeed, the early senescence phenotype persists in salicylic acid-deficient *atg* plants (Yoshimoto et al., 2009; Guiboileau et al., 2012). The inability of *atg* mutants to remove damaged cytoplasmic components might be a cause of the early yellowing phenotype. Several reports show that autophagy is essential to endomembrane recycling, and especially important for endoplasmic reticulum (ER) quality control (McLoughlin et al., 2018; Fan et al., 2019; Havé et al., 2019). ER stress is known to be a checkpoint in programmed cell death. Thus, the lack of control of ER stress damages, and defects in cellular waste management might explain the early senescence phenotype of *atg* mutants (Liu et al., 2012; Yang et al., 2016).

In contrast, the stimulation of autophagic activity in the *ATG8a*-OE and *ATG8g*-OE is believed to improve cellular quality control and to participate in better metabolism and resource management. In HN conditions, where autophagy is naturally repressed in WT, stimulation of autophagy in the *ATG8* overexpressing lines enhances nitrogen remobilization (Figs 3A and 7A), which is in good agreement with Chen et al. (2019). However, more intensive nutrient remobilization in *ATG8*-OE might deplete cellular resources more rapidly than in WT, ultimately leading to nutrient exhaustion, which compromises cell survival. Also, autophagy machinery, which requires the formation of new membranes for autophagosome biogenesis, is an energy-demanding process for the cell. Nutrient depletion occurring earlier (Fig. 3A) and extra energy consumption in the leaves of *ATG8*-OE could explain the earlier onset of leaf senescence compared with WT in favourable nitrogen conditions.

During 0N, autophagy is naturally enhanced in WT plants in response to nitrate starvation, which abolishes the differences observed in HN with *ATG8*-OE for nitrogen allocation at the whole-plant level (Chen et al., 2019; present study). Nevertheless, although there is no difference between WT and *ATG8*-OE for nitrogen partitioning under 0N, it is noticeable that the earlier leaf yellowing phenotype remains visible in the *ATG8*-OE plants in 0N. This suggests that autophagy-dependent N remobilization is not the only cause of the
<figure><img src="image_9.png" /><figcaption>FIG. 7. Schematic representation of the role of autophagy in nitrogen remobilization to the seeds and of potential impacts on leaf senescence. Relationships between autophagy, nitrogen remobilization and leaf senescence are represented for plants grown in high-nitrate (A) or low-nitrate (B) conditions. Pink motifs illustrate autophagic activity in *atg* mutants (no autophagy) wild-type (natural autophagy) and *ATG8*-OE (constitutively stimulated autophagy). Histograms represent the nitrogen repartition in the different organs (from bottom to top: black, roots; light green, rosette; dark green, stems; orange, pods; and yellow, seeds). Proportions match data presented in Fig. 3. The yellow–green triangles represent senescence progression with ageing for the different genotypes and growth conditions. Black arrows indicate nitrogen fluxes. Red dashed arrows indicate causal effects.</figcaption></figure>
discrepancy observed for leaf yellowing between *ATG8*-OE and WT. Additional roles of autophagy in pro-death might thus be possible.

Based on leaf yellowing, pigment concentrations and senescence markers, our study demonstrates that both the excess and the deficiency of autophagy-related nitrogen remobilization lead to early leaf yellowing. The fine-tuning of autophagy is thus essential for the regulation of leaf senescence. Investigating leaf senescence regulators in the framework of autophagy deserves further attention (Lee *et al.*, 2024). Several studies suggest that stimulation of autophagy can significantly improve plant performance (Slavikova *et al.*, 2008; Xia *et al.*, 2012; Li *et al.*, 2015; Chen *et al.*, 2019). Our study stresses that stimulation of autophagy can also have adverse outcomes by inducing precocious senescence in favourable environmental conditions.

## SUPPLEMENTARY DATA

Supplementary data are available at *Annals of Botany* online and consist of the following.

Table S1: primers used for ddPCR analysis. Figure S1: expression of *ATG8A* and *ATG8G* genes in the leaves of *ATG8* overexpressing (*ATG8*-OE) lines. Figure S2: *ATG8a*-OE and *ATG8g*-OE outperform wild-type for seed production and nitrogen remobilization to the seeds under HN. Figure S3: histograms of the anthocyanin concentration of WT, autophagy mutants (*atg5* and *atg7*) and overexpressors (*ATG8a*-OE and *ATG8g*-OE) in high-nitrogen (A, B) or low-nitrogen (C) conditions. Figure S4: flower bud emergence after switching to long day.

## ACKNOWLEDGEMENTS

We are most grateful to PLATIN' (Plateau d'Isotopie de Normandie) core facility for all element and isotope analysis used in this study, and the technical staff of UMR 950 EVA (Magalie Bodereau, Josiane Pichon, Elise Nexer and Kenza Boulouiz) for their help in harvesting and dissecting the plants.

## AUTHOR CONTRIBUTIONS

Designed research: P.E., J.T., A.M., F.C. and C.M.-D.; conducted experiments: M.J. and J.F.; supervision of experiments: P.E. and J.T.; analysed data: M.J. and C.M.-D.; discussed results: M.J., J.F., P.E., J.T., A.M., F.C. and C.M.-D.; wrote paper: M.J., C.M.-D. and P.E.

## CONFLICT OF INTEREST

None declared.

## FUNDING

This work was funded by the French National Research Agency (ANR-19-CE14-0009-02 hAPPEN: Autophagy, Proteases and plant Performances). The IJPB benefits from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007).

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