# A Periodic 4-h Extension of the Dark Period Did Not Cause Long-Term Changes in the Circadian Regulation of Photosynthesis and Sugar Levels in Lettuces

Cédric Dresch, Véronique Vidal, Séverine Suchail, Huguette Sallanon,
Florence Charles, Vincent Truffault

<figure><img src="image_1.png" /></figure>

## To cite this version:

Cédric Dresch, Véronique Vidal, Séverine Suchail, Huguette Sallanon, Florence Charles, et al. A Periodic 4-h Extension of the Dark Period Did Not Cause Long-Term Changes in the Circadian Regulation of Photosynthesis and Sugar Levels in Lettuces. Plant Direct, 2025, 9 (4), pp.e70062. 10.1002/pld.3.70062. hal-05075565

HAL Id: hal-05075565

https://amu.hal.science/hal-05075565v1

Submitted on 11 Jul 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH ARTICLE

OPEN ACCESS

# A Periodic 4-h Extension of the Dark Period Did Not Cause Long-Term Changes in the Circadian Regulation of Photosynthesis and Sugar Levels in Lettuces

Cédric Dresch<fnref n="1" />,² ID | Véronique Vidal<fnref n="1" /> ID | Séverine Suchail³ ID | Huguette Sallanon<fnref n="1" /> | Florence Charles<fnref n="1" />,⁴ ID |
Vincent Truffault² ID

**Correspondence:** Cédric Dresch (cedric.dresch@futuragaia.com) | Florence Charles (florence.charles@univ-avignon.fr) | Vincent Truffault (vincent@futuragaia.com)

**Received:** 20 October 2023 | **Revised:** 16 December 2024 | **Accepted:** 4 January 2025

**Funding:** This work was funded by National Association for Research in Technology (ANRT, CIFRE No. 2020/0819).

**Keywords:** carbohydrates | circadian rhythm | controlled environment agriculture | energy savings | indoor farming | photoperiod | photosynthesis | stomatal conductance

## ABSTRACT

The photoperiod in controlled environment agriculture can be adjusted to minimize electricity consumption, even if it differs from the plant's circadian rhythm. Daily modifications of the photoperiod disrupt the plant's circadian resonance state, resulting in altered growth and yield. However, the effects of periodic, rather than daily, photoperiod adjustments remain less understood. This study aims to investigate the effects of a 4-h extension of the dark period every 3 days on the circadian regulation of photosynthetic activity and sugar content, as well as on lettuce yield. Control lettuces were grown under a 16/8 photoperiod, while EPD lettuces ("Exceptionally long Period of Darkness") were grown under a repeated 16/12-16/8-16/8 photoperiod pattern from the beginning to the end of cultivation. The experiment was repeated twice, and the 4-h extension induced a loss of photosynthetic activity of 7% and 11% during the following lighting period in the first and second experiments, respectively. The yields were not affected. The stomatal conductance followed the circadian rhythm of lettuce rather than directly responding to photoperiod modifications. Furthermore, no long-term changes in starch and sucrose content were observed. Taken together, these results show that extending the dark period by 4 h every 3 days did not cause long-term disruption of the circadian regulation of photosynthesis and sugar levels in lettuce. These results provide new insights for optimizing light management in controlled environment agriculture, suggesting that the management of dark periods is crucial for maintaining yields and reducing energy consumption.

## 1 | Introduction

Light management in controlled environment agriculture is in-dependent of natural sunlight. The standard photoperiod applied is 16/8, consisting of 16 h of lighting and 8 h of darkness (Pennisi

et al. 2020). However, the photoperiod can be adjusted to reduce electricity consumption, a major challenge in controlled environment agriculture. Photoperiod has a direct impact on growth. Indeed, when the light period is too short, growth is limited by insufficient light quantity (Kelly et al. 2020). However,

<footnote n="1">¹Avignon Université, UMR95 Qualisud, 84916 Avignon, France. Qualisud, Univ Montpellier, Avignon Université, CIRAD, Institut Agro, Université de La Réunion, Montpellier, France | ²Futura Gaïa Technologies, Mas de Polvelière, Chemin du pont des îles, Rodilhan, France | ³Biomarqueurs Environnement Santé, Institut Méditerranéen de Biodiversité et d'Ecologie marine et continentale (IMBE), UMR 7263, Université d'Avignon et des Pays du Vaucluse Pole Agrosciences 301 rue Baruch de Spinoza, Avignon, France | ⁴UMR Sécurité et Qualité des Produits d'Origine Végétale (SQPOV), Avignon, France</footnote>

<footnote>This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>

<footnote>© 2025 The Author(s). Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.</footnote>
increasing the photoperiod beyond 20/4 to 22/2 or 24/0 (light/dark) also reduces the fresh weight of lettuces (Silva et al. 2022). The plant's circadian rhythm plays a key role in this growth limitation, particularly through the regulation of photosynthetic activity. In fact, the net assimilation rate of CO₂ is 67% higher in the control than in *Arabidopsis thaliana* with a defective circadian clock (CCA1 gene overexpressed) (Dodd et al. 2005). Furthermore, net photosynthesis primarily depends on stomatal conductance, which regulates CO₂ levels in the leaf (Hennessey and Field 1991) and is under circadian control. The stomata of *Vicia* sp., *Arachis*, and *Avena* sp. open and close following a 22- to 26-h cycle under constant lighting conditions (Pallas et al. 1974; Brogårdh and Johnsson 1975; Gorton et al. 1989). Other photosynthetic parameters are also under circadian control, as genes encoding the reaction center of photosystem II cycle (Harmer et al. 2000) and the efficiency of photosystem II exhibit daily variations, which have already been measured in marine algae (*Gonyaulax polyedra*), *Crassulacean acid metabolism* (CAM) plants (*Kalanchoe daigremontiana*), *Brassicaceae* (*Arabidopsis thaliana*), and lettuces (*Lactuca sativa*) (Samuelsson et al. 1983; Hennessey and Field 1991; Rascher et al. 2001; Gould et al. 2009).

Photosynthesis end-products are regulated by the circadian clock, particularly starch and sucrose metabolism (Graf et al. 2010; Scialdone et al. 2013; Sulpice et al. 2014; Fernandez et al. 2017; Mengin et al. 2017; Flis et al. 2019). Starch is essential for normal plant growth (Stitt and Zeeman 2012; Fernandez et al. 2017). Its accumulation follows a linear pattern during the day, and its nighttime degradation rate is regulated by the circadian clock, which anticipates the expected length of the night (Gibon et al. 2004; Graf et al. 2010; Martins et al. 2013; Scialdone et al. 2013; Sulpice et al. 2014). Starch is degraded into sucrose, which is linked to the circadian rhythm through *PSEUDO-RESPONSE REGULATOR 7* (PRR7), involved in morning perception, and *GIGANTEA* (GI), a major regulator of the circadian rhythm (Dalchau et al. 2011; Haydon et al. 2013). Sucrose is exported and subsequently degraded into glucose and fructose, which contribute to the growth and maintenance of the plant (Webb and Satake 2015).

When a plant's circadian rhythm and the photoperiod coincide, the plant is in a circadian resonance state (Dodd et al. 2005), optimal for growth, as the endogenous regulation of photosynthetic activity and sugar levels matches the external conditions. Thus, continuous photoperiod modifications are deleterious

when too drastic, as they cause constant disruption of the plant's circadian resonance state (Ishii et al. 1995; Kang et al. 2013; Dodd et al. 2014; Zhou et al. 2020). However, less is known about the impact of periodic adjustments of the photoperiod. Therefore, the objective of this study is to investigate how a 4-h extension of the dark period every 3 days affects the photosynthetic activity, sugar content, and yield of lettuce. The goal is to determine whether such adjustments induce a long-term disruption of the circadian regulation of photosynthesis and sugar levels in lettuce, providing new insights for optimizing light management in controlled environment agriculture.

## 2 | Material and Methods

### 2.1 | Plant Material and Growth

Butterhead lettuce (*Lactuca sativa* L. var. fairly, “Enza Zaden,” the Netherlands) seeds were sown in 144 (16×9) holes germination trays filled with potting soil (TBSP, “Florentaise,” France). The photoperiod was 16/8 (16 h of light, 8 h of darkness) with respective temperatures of 24/19°C and relative humidity of 65%. Water was provided until saturation by sub-irrigation on a 3-day basis with a germination solution composed of 3.2, 0.6, 0.9, 1.3, 0.8, and 0.1 mmol L⁻¹ of nitrogen, phosphorus, potassium, calcium, magnesium, and sulfur, respectively. The electroconductivity (EC) of the irrigation solution was equal to 0.5 mS cm⁻¹ with a pH of 5.6. Light intensity was set at 130 µmol m² s⁻¹ with red:blue 3:1 and ≈17% of white light, provided by LED lamps (T10 LED Grow Tube Light, HW-GL-T10-1200-36W-3Y, China). After 14 days, plantlets were transferred to a Gigrow rotative cultivation system from Futura Gaïa Technologies (Figure 1A, France), used for industrial purposes thanks to its technical advantages, notably in terms of light homogeneity. Plants were rotating according to a horizontal rotation axis at a speed of 50 min per revolution, which provides a centrifugal force of 1.33×10⁻⁵ Newton, considered neglectable. Two experiments were conducted in July 2022 and July 2023. In the first one, lettuces were transplanted in five holes stainless steel trays (72 cm×15 cm×4.5 cm; length, width, height) with a density of 30 plants m⁻². In the second experiment, lettuces were transplanted in nine holes stainless steel trays (118 cm×15 cm×4.7 cm; 1×w×h) with a density of 39 plants m⁻². Trays were filled with potting soil (VER4, “Florentaise,” France). The light source of the rotative cultivation system is placed at the center and is at the same distance from all plants.

<figure><img src="image_3.png" /><figcaption>**FIGURE 1** | Illustration of the GiGrow cultivation system used (A) and the photoperiod applied for the control lettuces, (16/8, CT lettuces), and the "Exceptionally long Period of Darkness" modality, (16/12–16/8–16/8, EPD lettuces) (B). The lighting periods were numbered in the same way in the first experiment (Exp 1) and the second experiment (Exp 2). The 4-h extension of the dark period in EPD lettuce, repeated every 3 days, causes a phase shift compared with the control.</figcaption></figure>
Two photoperiod modalities were studied (Figure 1B):

* Control (CT) under a constant 16/8 photoperiod.

* Exceptionally long Period of Darkness (EPD) modality under a 16/12–16/8–16/8 photoperiod pattern, from the beginning to the end of cultivation.

Overall light intensity for both modalities was equal to 350 ± 28 µmol of photons m⁻²s⁻¹ provided by ceramic metal-halide lamps (630 W double-ended, “Lumatek Ltd,” Malta). A light specter with a 4200 K color temperature was provided, with a red:green:blue ratio of 1:0.8:0.4. Temperature and vapor pressure deficit (VPD) were set at 24°C and 1 KPa during the day and 19°C and 0.54 KPa during the night. Ambient CO₂ concentration was not regulated. Cultivation time was set for 31 days. Irrigation solutions were made with Nutrimix of Futura Gaïa Technologies. Water supply was managed to saturate the soil in order to avoid water deficiency effects (2.5 L plant⁻¹ for 31 days). The fertilization solution was composed of 29.3, 1.8, 12.5, 10, and 7.5 mmol L⁻¹ of nitrogen, phosphorus, potassium, calcium, and magnesium, respectively, with an EC of 4 mS cm⁻¹ and a pH of 6.5.

## 2.2 | Photosynthetic Measurement Parameters

Net photosynthesis (µmol CO₂ m⁻²s⁻¹), stomatal conductance (mmol H₂O m⁻²s⁻¹) and the quantum yield of PSII electron transport (ΦPSII) were measured using a portable photosynthetic analyzer (Head Version 1.4.7, LI-COR 6800, Li-Cor, Inc. Lincoln, NE, USA) equipped with a LI-COR chamber type 6800-01. Parameters were set at 400 µmol of photons m⁻²s⁻¹ of light (red and blue light, R:B ratio of 9:1) with 420 ppm CO₂. Temperature and VPD were not controlled and therefore remained the same as those in the cultivation chamber (24/18°C and 1/0.54 kPa for the light/dark periods). Plants were removed from the rotative cultivation system for the entire light period, maintained under similar lighting conditions, and rehydrated every hour to avoid water deficit. In the first experiment, measurements were taken during the lighting period following the 4-h extension of the dark period for EPD lettuces, and again the following day (Lighting Periods 2 and 3 in Figure 1B). In the second experiment, measurements were taken at the same periods, additionally during the lighting period before the 4-h extension of the dark period for EPD lettuces (Lighting Periods 1, 2, and 3 in Figure 1B). Measurements were carried out on 10 plants, with two leaves per plant, so that each leaf was measured every hour. Measurements were recorded manually every 3–5 min, with value stability serving as the indicator for recording the data. Daily photosynthesis was calculated by multiplying each net assimilation (in CO₂ m⁻²s⁻¹) by the time interval between two measurements, resulting in a final value in µmol CO₂ m⁻²d⁻¹.

## 2.3 | Sugars Analysis

Glucose, fructose, sucrose, and starch contents were assessed at the end of lighting and dark periods from 18 to 21 days after transplantation. Results are expressed as hours after the initial sampling.

### 2.3.1 | Glucose, Fructose, and Sucrose Contents

In the first experiment, glucose, fructose, and sucrose content were assayed by high performance anion-exchange chromatography (HPAEC). After removing the core, lettuce leaves were frozen in liquid nitrogen, grounded using IKA A11 basic Analytical mill (IKA, Germany), and then freeze-dried using a Cryotec Cosmos freeze-dryer (Cryotec, France). Between 15 and 20 mg of dried lettuces were sampled in ultra-pure water to obtain a concentration of 10 mg of DW mL⁻¹. Samples were homogenized and centrifuged for 4 min at 15,000g, 4°C (Merck 3-16KL, KGaA, Germany). Supernatant was collected and filtered through a PTFE membrane filter (0.2 µm, IC Millex-LG, Merck KGaA, Germany). Glucose, fructose, and sucrose contents were assayed using HPAEC (Dionex ICS-3000, USA) by injecting 5 µL of each sample. Separation was carried out using an IC Dionex CarboPac PA1 analytical anion-exchange column (10 µm, 4 × 250 mm, Dionex, USA). The mobile phase was a solution of H₂O (eluent A) and 250 mM NaOH with 4 mM sodium acetate (eluent B) with an A/B ratio of 35:65 (v/v). An isocratic elution mode at a constant flow rate of 0.7 mL min⁻¹ was performed. Sugars were monitored by pulsed amperometric detection (PAD), with a detection peak at 254 nm. Curves of glucose, fructose, and sucrose contents were obtained by injecting calibration standards with concentrations of 2.5, 5, 7.5, 10, 12.5, 15, and 20 nmol. CHROMELEON software v.6.7. was used to analyze the concentration of sugars. The limit of quantification was calculated according to Rusch et al. (Rusch et al. 2013). In the second experiment, glucose, fructose, and sucrose content were assayed using a sucrose/D-fructose and D-glucose assay kit (K-SUFRG, Megaenzyme, Ireland). Five milligrams of dried lettuce were solubilized in 2 mL ultra-pure water and centrifuged for 5 min at 12,000 g at 4°C before assay. For sucrose analysis, 20 µL of β-fructosidase was added to 10 µL of the sample solution. After incubating for 5 min at 23°C, 190 µL of ultra-pure water, 10 µL of buffer solution (Solution 1, provided), and 10 µL of NADP+/ATP solution (Solution 2, provided) were added. Absorbance was measured at 340 nm. For glucose and fructose analysis, 210 µL of ultra-pure water, 10 µL of buffer solution (Solution 1, provided), and 10 µL of NADP+/ATP solution (Solution 2, provided) were added to 10 µL of the sample. Absorbance was measured at 340 nm. Respective blanks for the sucrose and glucose/fructose analyses were prepared by replacing the 10 µL of sample solution with ultra-pure water. Then, 2 µL of hexokinase/D-glucose-6-phosphate dehydrogenase solution (Solution 3, provided) were added to all samples. After 5 min, absorbance was measured at 340 nm. For fructose analysis, 2 µL of phosphoglucose isomerase solution (Solution 4, provided) were added, and absorbance was measured at 340 nm after 10 min. The sucrose, glucose, and fructose contents were calculated according to the kit's instructions (K-SUFRG, Megaenzyme, Ireland).

### 2.3.2 | Starch Extraction and Starch Content Assays

Starch extraction was performed on 10 mg of dried lettuce. Samples were washed off any free glucose twice with 1 mL of 90% ethanol. They were then centrifuged for 2 min at 10,000g, 4°C, and 1 mL of ultra-pure water was added. Samples were autoclaved for 60 min at 121°C (ALV1131, “Gettinge,” France)
and weighed to determine the water loss during the autoclaving. Five hundred microliters of ultra-pure water were added, and samples were homogenized. Starch content was determined using a starch fluorometric assay kit (MAK368, Sigma-Aldrich, USA). Starch was hydrolyzed and revealed, and starch content was determined by fluorimetry (Synergy HT, BioTek Instruments, USA).

## 2.4 | Agronomical Parameters

Fresh and dry weights were measured at eight different time points throughout the cultivation period and at harvest (31 days after transplantation). Whole plants were harvested, and fresh weight was measured using a precision balance XT 620M (PRECISA, France). Two leaves were dried in an oven at 80°C for at least 3 days to calculate the dry weight.

## 2.5 | Statistical Analysis

Statistical analyses were carried out using the pairwise Wilcoxon-Mann-Whitney test (α = 0.05) on RStudio Version 4.2.2 (R Core Team 2022) using dplyr, ggpattern, ggplot2, ggpubr, grid, gridExtra, multcompView, rcompanion, sf, and tidyr packages. Details of all *p*-values are presented in Supporting Information.

# 3 | Results

## 3.1 | Agronomic Traits Are Not Altered by Repeated 4-h Extensions of the Dark Period Throughout the Growth Period

Fresh and dry weight data are compiled in Table 1. A similar amount of both fresh and dry weight per tray was produced, regardless of the modality or experiment. In the first experiment (Exp 1), differences in fresh weight between the two modalities were only observed at 5 and 8 days after transplantation. In the second experiment (Exp 2), differences were measured at 11, 14, and 19 days after transplantation. No reproducible differences in fresh and dry weights were observed between the two modalities.

## 3.2 | The Daily Photosynthesis Trend of EPD Lettuces Is Similar to the Control When Expressed as a Function of the Lighting Hours Anticipated by the Plants' Circadian Rhythm

Figures 2 and 3 represent the net photosynthesis (A) and the stomatic conductance (B) of lettuces from the first and second experiments, respectively. In both figures, the data are presented as a function of zeitgeber time (lighting hours) or anticipated lighting hours, based on the plants' endogenous circadian rhythm.

<table><caption>**TABLE 1** | Mean ± standard error of fresh and dry weight per tray for CT (photoperiod: 16/8) and EPD (photoperiod pattern: 16/12–16/8–16/8) lettuce for both experiments at different days after transplantation (DAT). Each tray had five plants in the first experiment (Exp 1) and nine plants in the second experiment (Exp 2).</caption><thead><tr><th rowspan="2"></th><th colspan="4">Fresh weight (g per tray unit)</th><th colspan="3">Dry weight (g per tray unit)</th></tr><tr><th>DAT</th><th>CT</th><th>EPD</th><th>p-value</th><th>CT</th><th>EPD</th><th>p-value</th></tr></thead><tbody><tr><td rowspan="7">Exp 1 (five plants)</td><td>0</td><td>2 ± 0.3</td><td>2 ± 0.4</td><td>1.0000</td><td>0.1 ± 0.0</td><td>0.1 ± 0.0</td><td>0.3095</td></tr><tr><td>5</td><td>4 ± 1</td><td>6 ± 0.3</td><td>0.0079*</td><td>0.4 ± 0.1</td><td>0.4 ± 0.0</td><td>0.1508</td></tr><tr><td>8</td><td>14 ± 1</td><td>17 ± 2</td><td>0.0317*</td><td>1.1 ± 0.1</td><td>1.2 ± 0.1</td><td>0.0952</td></tr><tr><td>11</td><td>48 ± 7</td><td>50 ± 6</td><td>0.5476</td><td>2.5 ± 0.3</td><td>2.9 ± 0.4</td><td>0.1508</td></tr><tr><td>14</td><td>135 ± 27</td><td>137 ± 17</td><td>0.8413</td><td>6.5 ± 1.2</td><td>6.6 ± 1.1</td><td>1.0000</td></tr><tr><td>19</td><td>393 ± 79</td><td>339 ± 69</td><td>0.2222</td><td>16.8 ± 2.8</td><td>15.3 ± 2.2</td><td>0.4206</td></tr><tr><td>24</td><td>774 ± 153</td><td>763 ± 131</td><td>1.0000</td><td>29.8 ± 6.2</td><td>32.6 ± 5.7</td><td>0.4206</td></tr><tr><td rowspan="7">Exp 2 (nine plants)</td><td>0</td><td>5 ± 1</td><td>5 ± 1</td><td>1.0000</td><td>0.4 ± 0.1</td><td>0.4 ± 0.1</td><td>1.0000</td></tr><tr><td>5</td><td>6 ± 1</td><td>6 ± 1</td><td>0.2032</td><td>0.6 ± 0.1</td><td>0.6 ± 0.1</td><td>0.3406</td></tr><tr><td>8</td><td>15 ± 3</td><td>18 ± 4</td><td>0.1058</td><td>1.2 ± 0.2</td><td>1.3 ± 0.3</td><td>0.4776</td></tr><tr><td>11</td><td>49 ± 14</td><td>37 ± 10</td><td>0.0350*</td><td>3.7 ± 0.7</td><td>2.8 ± 0.5</td><td>0.0011*</td></tr><tr><td>14</td><td>157 ± 26</td><td>131 ± 21</td><td>0.0093*</td><td>9.0 ± 1.4</td><td>8.0 ± 1.2</td><td>0.0387*</td></tr><tr><td>19</td><td>364 ± 51</td><td>274 ± 44</td><td>0.0001*</td><td>20.1 ± 3.7</td><td>16.5 ± 2.7</td><td>0.0121*</td></tr><tr><td>24</td><td>799 ± 134</td><td>700 ± 100</td><td>0.0887</td><td>32.5 ± 6.7</td><td>31.6 ± 5.4</td><td>0.8874</td></tr><tr><td></td><td>28</td><td>1333 ± 193</td><td>1152 ± 221</td><td>0.1782</td><td>64.8 ± 20.1</td><td>61.0 ± 19.7</td><td>0.7728</td></tr><tr><td></td><td>31</td><td>1374 ± 120</td><td>1388 ± 149</td><td>0.8874</td><td>62.4 ± 8.0</td><td>60.3 ± 10.1</td><td>0.8874</td></tr></tbody></table>

Note: *p*-values were calculated using the Wilcoxon-Mann-Whitney test, and significant differences are marked with asterisks (α = 0.05).
In both experiments (Figures 2 and 3), net photosynthesis values of CT lettuces are maximal during the first half of the lighting period (from 0 to 8h) and lowest towards the end, between 12 and 16h. Also, during the second lighting period, EPD values are maximal only during the 4h of lighting and then decrease in both experiments. Net photosynthesis daily trend of both modalities in the first lighting period in the second experiment and

in the first and third lighting periods in both experiments are similar. For the second lighting period, after the 4-h extension of the dark period, when net photosynthesis is expressed as a function of lighting hours, CT and EPD lettuces show different daily trends, especially in the first experiment. However, when net photosynthesis values of EPD lettuces are expressed as a function of the lighting hours anticipated by the plants' circadian

<figure><img src="image_4.png" /><figcaption>**FIGURE 2** | Net photosynthesis (A) and stomatal conductance (B) values of control (photoperiod: 16/8, CT lettuces) and EPD lettuces (photoperiod pattern: 16/12–16/8–16/8) of the first experiment, measured using the LI-6800. Measurements were taken after the 4-h extension of the dark period for EPD lettuces (Lighting Period 2), as well as the following day (Lighting Period 3). Data are expressed as a function of a zeitgeber, the hours of lighting (h), or the anticipated lighting hours by the plant, consistent with their circadian rhythm. Each data point represents the mean ± standard error of all measurements taken during the specified periods (n ≥ 10, on 10 plants, two leaves per plant). Statistically significant differences are indicated by asterisks (pairwise Wilcoxon–Mann–Whitney test, α = 0.01).</figcaption></figure>
A

<figure><img src="image_5.png" /></figure>

B

<figure><img src="image_6.png" /></figure>

**FIGURE 3** | Net photosynthesis (A) and stomatal conductance (B) values of control (photoperiod: 16/8, CT lettuces) and EPD lettuces (photoperiod pattern: 16/12–16/8–16/8) of the second experiment, measured using the LI-6800. Measurements were taken the day before (Lighting Period 1), the day after (Lighting Period 2), and the day following (Lighting Period 3) the 4-h extension of the dark period of EPD lettuces. Data are expressed as a function of a zeitgeber, the hours of lighting (h), or the anticipated lighting hours by the plant, consistent with their circadian rhythm. Each data point represents the mean ± standard error of all measurements from two independent repetitions taken during the specified periods (n ≥ 10, on 2×10 plants, two leaves per plant). Statistically significant differences are indicated by asterisks (pairwise Wilcoxon–Mann–Whitney test, α = 0.01).

rhythm, the daily trends of net photosynthesis of both modalities
are more similar in both experiments.

Stomatal conductance values follow the same patterns as net
photosynthesis in both experiments but show more variability.

3.3 | Starch and Sucrose Contents Varied
According to the Photoperiod and Responded to
the 4-h Extension of the Dark Period

Starch, sucrose, glucose, and fructose contents of CT and EPD
lettuces from both experiments are represented in Figure 4.

Starch (Figure 4A) and sucrose (Figure 4B) contents in both ex-
periments, for both CT and EPD modalities, vary according to the
photoperiod. Values are maximal at the end of the lighting period
and minimal at the end of the dark period. In both experiments,
the extended 4-h dark period for EPD lettuces induces a decrease
in starch and sucrose contents compared with their levels after
the 8-h dark period (dark period before the Lighting Period 2).
In the first experiment, the starch and sucrose contents of EPD
lettuces after a 12-h dark period are three and nine times lower,
respectively, than those of the control after an 8-h dark period. In
the second experiment, the starch and sucrose contents of EPD
lettuces after a 12-h dark period are 1.5 and 1.3 times lower, re-
spectively, than those of the control after an 8-h dark period.
<figure><img src="image_7.png" /></figure>

**FIGURE 4** | Carbohydrates contents of control (photoperiod: 16/8, CT lettuces) and EPD lettuces (photoperiod pattern: 16/12–16/8–16/8) over 3 days. Data from the first experiment are shown in row “Exp 1,” and data from the second experiment are shown in row “Exp 2.” Data are expressed as mgg⁻¹ of dry weight (DW). Time is expressed as hours after the initial sampling time (h). White backgrounds represent lighting periods, gray backgrounds represent dark periods, and striped backgrounds represent dark periods for EPD lettuces only (control lettuces under lighting). A double slash for EPD lettuces marks the omission of 4 h of lighting for clarity in representation. (A) Starch content. (B) Sucrose content. (C) Glucose content. (D) Fructose content. Each symbol represents the mean ± standard error of five samples in the first experiment and the mean ± standard error of 2×3 samples from two independent repetitions in the second experiment. Statistically significant differences are indicated by asterisks (pairwise Wilcoxon–Mann–Whitney test, α = 0.05).

Glucose (Figure 4C) and fructose (Figure 4D) did not vary as significantly as sucrose and starch contents. Moreover, in the first experiment, glucose and fructose contents of EPD lettuces are lower than those of the control, while in the second experiment, glucose and fructose contents of EPD lettuces are similar to those of the control.

4 | Discussion

4.1 | The Circadian Rhythm of Stomatal Conductance Limits Photosynthesis After Periodic 4-h Extensions of the Dark Period, With No Impact on Final Yields

In this experiment, net photosynthesis is maximal at the begin-
ning of the lighting period and decreases throughout the day,
consistent with patterns previously described in *Symbodinium*
sp., *Gonyaulax polyedra*, *Phaseolus vulgaris* (common bean),
*Marchantia polymorpha*, and *Zantedeschia aethiopica* (Hastings
et al. 1961; Fredeen et al. 1991; Sorek et al. 2013; Cuitun-
Coronado et al. 2022; Dias et al. 2022). The same pattern can be
observed for stomatal conductance. Indeed, net photosynthesis
and stomatal conductance are decreasing synchronously. We
can precise that net photosynthesis is related to stomatal con-
ductance by a logarithmic relationship, with a higher correla-
tion coefficient of 0.89 for CT lettuces and 0.88 for EPD lettuces
in the second experiment (Figure S1), consistent with previous
studies (Gorton et al. 1989; Gago et al. 2016). In these exper-
iments, extending the dark period by 4 h every 3 days induced
a 4-h shift in the daily trend of net photosynthesis and stoma-
tal conductance values expressed as a function of the lighting
hours. However, this extension did not affect the daily trend
when the values were expressed as a function of the lighting
hours anticipated by the plant's circadian rhythm. Our results
are consistent with literature that previously described that
stomatal conductance is under circadian regulation (Kreps and
Kay 1997) and showed that this regulation is not disrupted by a
periodic extension of the dark period. Thus, net photosynthesis
is also regulated by the circadian rhythm and not directly by

the photoperiod. It should be noted that other photosynthetic
parameters, such as the efficiency of the photosystem II (ΨPSII,
Figure S2), already known to be regulated by an endogenous
circadian rhythm through specific gene expression (Harmer
et al. 2000; Dodd 2005; Dodd et al. 2014), are also decreasing
following a daily trend. However, in this experiment, stoma-
tal conductance consistently decreased before ΨPSII and other
photosynthetic parameters, highlighting its specific relevance in
studies of photosynthesis under modified photoperiods.

In this experiment, the dark period was extended by 4 h, but
all lighting periods stayed similar to the control of 16 h. We
calculated the daily net photosynthesis by multiplying each
net assimilation value (in µmol CO₂ m⁻² s⁻¹) by the time in-
terval between two measurements, resulting in a final value
expressed in µmol CO₂ m⁻² d⁻¹. After the 4-h extension of the
dark period, the daily photosynthesis of EPD lettuces was 7%
lower than the control in the first experiment (approximately
118 and 127 CO₂ m⁻² d⁻¹, respectively) and 11% lower than the
control in the second experiment (approximately 142 and 161
CO₂ m⁻² d⁻¹, respectively). Thus, even though the duration
of the lighting periods was equal in both modalities, the net
photosynthesis of the control was more efficient than that of
the EPD lettuces. This difference can be attributed to the dura-
tion of maximal photosynthetic activity. In control lettuces, net
photosynthesis was maximal during the first 8 h of the lighting
period, while in EPD lettuces, it was only maximal during the
first 4 h. In EPD lettuces, the duration of maximal photosyn-
thesis was shortened because the photoperiod was shifted by
4 h and no longer matched with the timing of maximal stoma-
tal opening. As a result, 4 h of highly efficient photosynthesis
at the beginning of the predicted lighting period were replaced
by 4 h of less efficient photosynthesis at the end of the light-
ing period, leading to a decrease in daily net assimilation (for
16 h of lighting day⁻¹). These observations are consistent with
previous studies showing that the net CO₂ assimilation rate
decreases when the photoperiod does not match the plant's
circadian rhythm (Dodd et al. 2005). In the experiments con-
ducted, the loss of net CO₂ assimilation did not affect fresh and
dry yields. Indeed, the fresh weight and dry weight of EPD
lettuces were similar to those of the control. However, previous studies showed that a photoperiod not tuned with the circadian rhythm induces a reduction of photosynthetic activity. Indeed, Dodd et al. (2005) showed that *Arabidopsis thaliana* fixed more CO₂ with a circadian rhythmicity of 24 h (12/12) than with days of 28 h (14/14) or 20 h (10/10). The same pattern has been observed when *Arabidopsis thaliana* expressing a circadian rhythm of 20 h were placed under long photoperiods (28 h) or when *Arabidopsis thaliana* expressing a circadian rhythm of 28 h were placed under short photoperiods of 20 h (Dodd et al. 2005). Moreover, in *Arabidopsis thaliana*, Graf et al. (2010) have measured a 37% decrease in fresh weight when the length of the day reaches 28 h (14/14) instead of 24 h (12/12). In the literature, the decrease in photosynthetic activity is higher than in our experiment, consistent with the fact that we only modified the photoperiod by 4 h on a 3-day basis. Moreover, other experiments applied daily changes in photoperiod, which may affect plants more strongly than the periodic 4-h extension of the dark period applied in this experiment. Finally, in the literature, extended periods of darkness might have induced a transitory depletion state of carbohydrates at the end of the darkness period, which has been assumed to inhibit growth (Gibon et al. 2004; Graf et al. 2010). In the experiments conducted, we can propose that the yield of lettuces was not altered by the disruption of the circadian rhythm because the pool of carbohydrates was not exhausted at the end of the extended dark periods.

## 4.2 | Periodic 4-h Extensions of the Dark Period Do Not Lead to Long-Term Changes in Starch and Sucrose Regulation or Content

Starch varies according to the photoperiod in both CT and EPD modalities. Starch level increased during the lighting period and decreased during the dark periods. When a dark period of 12 h was applied, the starch level in EPD lettuces continued to decline. It can be noted that the initial amount of starch in CT and EPD lettuces was the same, as no change in lighting hours occurred. After a 12-h dark period, 88% and 94% of the starch in EPD lettuces was consumed in the first and second experiments, respectively, consistent with starch consumption observed in *Arabidopsis thaliana* (Sulpice et al. 2014), and higher than the respective control consumption (63% and 90%, respectively).

The increase in starch consumption can be linked to the mismatch between the photoperiod and the circadian rhythm of lettuce. Indeed, when the photoperiod and the circadian rhythm are tuned, the rate of starch degradation during different night lengths is adjusted to ensure that the same amount of starch remains at the end of each dark period (Gibon et al. 2004; Bläsing et al. 2005; Graf et al. 2010; Scialdone et al. 2013; Sulpice et al. 2014; Fernandez et al. 2017). In this experiment, the starch degradation rate has not been adjusted, suggesting that the circadian rhythm of the lettuces did not anticipate the 3-day basis dark period extension applied.

Despite the lower starch content at the end of the 12-h dark period, the starch content in EPD lettuces during and at the end of the following light period was similar to that of the control. In the second experiment, starch accumulation during this light

period in CT and EPD lettuces followed a linear trend, with correlation coefficients of 0.96 and 0.99, respectively (Figure S3). Moreover, no strong differences in starch content were found the following days. Finally, we observed that sucrose content followed the same trends as starch content. This is consistent with the fact that starch is degraded during the night to provide substrates for sucrose synthesis (Martins et al. 2013). Then, this experiment shows that a 4-h extension of the dark period every 3 days does not lead to long-term changes in the regulation of starch and sucrose content. This also suggests that the 16-h light period allows enough photosynthesis to restore sugar levels to control-like levels following a 4-h dark period extension.

Glucose and fructose levels fluctuate less than those of starch and sucrose across light and dark periods, consistent with the fact that starch and sucrose are storage carbohydrates. In the first experiment, glucose and fructose levels of EPD lettuces were lower than the control. On the second one, glucose and fructose levels of CT and EPD lettuces are similar. Furthermore, glucose and fructose levels in the first experiment were higher than those in the second, a difference that does not seem attributed to starch and sucrose content, as these levels were similar between the two experiments. The observed variation may instead be explained by differences in growth. The second experiment used a higher planting density, which led to lower growth rates for the lettuces compared with the first experiment but resulted in the same production per tray. Additionally, the higher planting density led to partial leaf overlap, which may have also influenced glucose and fructose accumulation.

Sugar levels are under circadian control (Harmer et al. 2000), but also regulate the circadian rhythm of plants, with a key role of starch and sucrose (Webb and Satake 2015). However, in this experiment, no long-term effects of periodic 4-h extensions of the dark period on starch and sucrose levels were observed, suggesting that such extensions do not induce long-term changes in the circadian rhythm of lettuces through sugar levels.

## 5 | Conclusion

In this work, we studied the effects of modifications of the photoperiod by increasing the darkness period on a 3-day basis while maintaining the same lighting period (16/12–16/8–16/8, repeated). Our measurements showed that stomatal conductance in lettuces was regulated by the plant's endogenous circadian rhythm and did not directly respond to the 4-h extension of the dark period. Because stomatal conductance is a key limiting factor for net photosynthesis, net photosynthesis was also regulated by the plant's circadian rhythm. Because stomatal conductance and net photosynthesis were highest at the start of the light period, the extended dark period reduced daily photosynthetic activity by 7% and 11% in experiments one and two, respectively. However, fresh and dry yields were unaffected. Although starch and sucrose levels initially dropped below control levels following the extended dark period, a single 16-h light period was sufficient to restore starch and sucrose to control-like levels, with no differences observed the following day. This supports the idea that extending the dark period of 4 h every 3 days does not cause long-term changes in starch and sucrose content. When combining data on stomatal conductance and sugar regulation,
we can conclude that a 4-h extension of the dark period every
3 days does not cause long-term changes in the circadian regula-
tion of photosynthesis and sugar levels.

This experiment provides two new insights for optimizing light
management in controlled environment agriculture. First, sugar
levels in lettuces exposed to an extended dark period returned to
control-like levels after a single 16-h light period, suggesting that
16 h of light are sufficient to saturate sugar stores in the studied
lettuce. However, the standard photoperiod in controlled en-
vironment agriculture is typically 16/8, which may need to be
reconsidered to optimize light efficiency. Second, our results in-
dicate that it may be more efficient to turn off lighting at the end
of the light period when photosynthetic activity is at its lowest.
Further experiments are needed to confirm whether shortening
the lighting period impacts the plant's circadian rhythm in the
same way as extending the dark period.

The main perspective of this study is that periodic photoperiod modifications may not disrupt the plant's circadian resonance state, although further research is needed to determine the maximum phase shift that can be applied without causing disruption. This will provide new insights to further optimize light management in controlled environment agriculture.

Author Contributions

V.T., H.S., and F.C. initiated and conceived the project. C.D. created the protocol. V.T. and F.C. revised the protocol. C.D. performed the experiments and collected the materials. C.D. and V.V. performed starch assays. C.D. and S.S. performed glucose, fructose, and sucrose assays. C.D. performed the data analysis. C.D. wrote the article. V.T., F.C., and H.S. revised the manuscript. All the authors read and approved the final manuscript.

Acknowledgments

We thank Doriane Dumont for sharing her experiences and advice on starch assays; Louis Ramade and Manon Petigny for their technical help during the experiments.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

All data presented in the article are available at Zenodo, DOI: https://doi.org/10.5281/zenodo.15058505. Data will be shared upon request from the corresponding author, Cédric Dresch (cedric.dresch@futuragaia.com).

Peer Review

The peer review history for this article is available in the Supporting Information for this article.

References

Bläsing, O. E., Y. Gibon, M. Günther, et al. 2005. "Sugars and Circadian Regulation Make Major Contributions to the Global Regulation of Diurnal Gene Expression in *Arabidopsis*." *Plant Cell* 17: 3257-3281.

Brogårdh, T., and A. Johnsson. 1975. "Regulation of Transpiration in Avena. Responses to White Light Steps." *Physiologia Plantarum* 35: 115-125.

Cuitun-Coronado, D., H. Rees, J. Colmer, A. Hall, L. L. de Barros Dantas, and A. N. Dodd. 2022. "Circadian and Diel Regulation of Photosynthesis in the Bryophyte *Marchantia polymorpha*." *Plant, Cell & Environment* 45: 2381-2394.

Dalchau, N., S. J. Baek, H. M. Briggs, et al. 2011. "The Circadian Oscillator Gene *GIGANTEA* Mediates a Long-Term Response of the *Arabidopsis thaliana* Circadian Clock to Sucrose." *Proceedings of the National Academy of Sciences of the United States of America* 108: 5104-5109.

Dias, M. G., T. I. da Silva, R. R. P. Cruz, L. B. Barbosa, J. C. Zanuncio, and J. A. S. Grossi. 2022. "Daily Photosynthetic Course of Calla Lily Plants." *Revista De Agricultura Neotropical* 9: 6962-6962.

Dodd, A. N. 2005. "Plant Circadian Clocks Increase Photosynthesis, Growth, Survival, and Competitive Advantage." *Science* 309: 630-633.

Dodd, A. N., J. Kusakina, A. Hall, P. D. Gould, and M. Hanaoka. 2014. "The Circadian Regulation of Photosynthesis." *Photosynthesis Research* 119: 181-190.

Dodd, A. N., N. Salathia, A. Hall, et al. 2005. "Plant Circadian Clocks Increase Photosynthesis, Growth, Survival, and Competitive Advantage." *Science* 309: 630-633.

Fernandez, O., H. Ishihara, G. M. George, et al. 2017. "Leaf Starch Turnover Occurs in Long Days and in Falling Light at the End of the Day." *Plant Physiology* 174: 2199-2212.

Flis, A., V. Mengin, A. A. Ivakov, et al. 2019. "Multiple Circadian Clock Outputs Regulate Diel Turnover of Carbon and Nitrogen Reserves." *Plant, Cell & Environment* 42: 549-573.

Fredeen, A. L., T. L. Hennessey, and C. B. Field. 1991. "Biochemical Correlates of the Circadian Rhythm in Photosynthesis in *Phaseolus vulgaris*." *Plant Physiology* 97: 415-419.

Gago, J., D. de Menezes Daloso, C. M. Figueroa, J. Flexas, A. R. Fernie, and Z. Nikoloski. 2016. "Relationships of Leaf Net Photosynthesis, Stomatal Conductance, and Mesophyll Conductance to Primary Metabolism: A Multispecies Meta-Analysis Approach." *Plant Physiology* 171: 265-279.

Gibon, Y., O. E. Bläsing, N. Palacios-Rojas, et al. 2004. "Adjustment of Diurnal Starch Turnover to Short Days: Depletion of Sugar During the Night Leads to a Temporary Inhibition of Carbohydrate Utilization, Accumulation of Sugars and Post-Translational Activation of ADP-Glucose Pyrophosphorylase in the Following Light Period." *Plant Journal* 39: 847-862.

Gorton, H. L., W. E. Williams, M. E. Binns, C. N. Gemmell, E. A. Leheny, and A. C. Shepherd. 1989. "Circadian Stomatal Rhythms in Epidermal Peels from *Vicia faba*." *Plant Physiology* 90: 1329-1334.

Gould, P. D., P. Diaz, C. Hogben, et al. 2009. "Delayed Fluorescence as a Universal Tool for the Measurement of Circadian Rhythms in Higher Plants." *Plant Journal* 58: 893-901.

Graf, A., A. Schlereth, M. Stitt, and A. M. Smith. 2010. "Circadian Control of Carbohydrate Availability for Growth in *Arabidopsis* Plants at Night." *PNAS* 107: 9458-9463.

Harmer, S. L., J. B. Hogenesch, M. Straume, et al. 2000. "Orchestrated Transcription of Key Pathways in *Arabidopsis* by the Circadian Clock." *Science* 290: 2110-2113.

Hastings, J. W., L. Astrachan, and B. M. Sweeney. 1961. "A Persistent Daily Rhythm in Photosynthesis." *Journal of General Physiology* 45: 69-76.

Haydon, M. J., O. Mielczarek, F. C. Robertson, K. E. Hubbard, and A. A. R. Webb. 2013. "Photosynthetic Entrainment of the *Arabidopsis thaliana* Circadian Clock." *Nature* 502: 689-692.

Hennessey, T. L., and C. B. Field. 1991. "Circadian Rhythms in Photosynthesis: Oscillations in Carbon Assimilation and Stomatal Conductance Under Constant Conditions." *Plant Physiology* 96: 831-836. https://doi.org/10.1104/pp.96.3.831
Ishii, M., T. Ito, T. Maruo, K. Suzuki, and K. Matsuo. 1995. "Plant Growth and Physiological Characters of Lettuce Plants Grown Under Artificial Light of Different Irradiating Cycles." *Environmental Control in Biology* 33: 143-149.

Kang, J. H., S. KrishnaKumar, S. L. S. Atulba, B. R. Jeong, and S. J. Hwang. 2013. "Light Intensity and Photoperiod Influence the Growth and Development of Hydroponically Grown Leaf Lettuce in a Closed-Type Plant Factory System." *Horticulture, Environment and Biotechnology* 54: 501-509.

Kelly, N., D. Choe, Q. Meng, and E. S. Runkle. 2020. "Promotion of Lettuce Growth Under an Increasing Daily Light Integral Depends on the Combination of the Photosynthetic Photon Flux Density and Photoperiod." *Scientia Horticulturae* 272: 109565.

Kreps, J., and S. Kay. 1997. "Coordination of Plant Metabolism and Development by the Circadian Clock." *Plant Cell* 9: 1235-1244.

Martins, M. C. M., M. Hejazi, J. Fettke, et al. 2013. "Feedback Inhibition of Starch Degradation in Arabidopsis Leaves Mediated by Trehalose 6-Phosphate." *Plant Physiology* 163: 1142-1163.

Mengin, V., E.-T. Pyl, T. A. Moraes, et al. 2017. "Photosynthate Partitioning to Starch in *Arabidopsis thaliana* is Insensitive to Light Intensity but Sensitive to Photoperiod due to a Restriction on Growth in the Light in Short Photoperiods." *Plant, Cell & Environment* 40: 2608-2627.

Pallas, J. E., Y. B. Samish, and C. M. Willmer. 1974. "Endogenous Rhythmic Activity of Photosynthesis, Transpiration, Dark Respiration, and Carbon Dioxide Compensation Point of Peanut Leaves." *Plant Physiology* 53: 907-911.

Pennisi, G., F. Orsini, M. Landolfo, et al. 2020. "Optimal Photoperiod for Indoor Cultivation of Leafy Vegetables and Herbs." *European Journal of Horticultural Science* 85: 329-338.

R Core Team. 2022. *R: A Language and Environment for Statistical Computing*. R Foundation for Statistical Computing. https://www.R-project.org/.

Rascher, U., M.-T. Hütt, K. Siebke, B. Osmond, F. Beck, and U. Lüttge. 2001. "Spatiotemporal Variation of Metabolism in a Plant Circadian Rhythm: The Biological Clock as an Assembly of Coupled Individual Oscillators." *Proceedings of the National Academy of Sciences of the United States of America* 98: 11801-11805.

Rusch, A., S. Suchail, M. Valentin-Morison, J.-P. Sarthou, and J. Roger-Estrade. 2013. "Nutritional State of the Pollen Beetle Parasitoid *Tersilochus heterocerus* Foraging in the Field." *BioControl* 58: 17-26.

Samuelsson, G., B. M. Sweeney, H. A. Matlick, and B. B. Prézelin. 1983. "Changes in Photosystem II Account for the Circadian Rhythm in Photosynthesis in *Gonyaulax polyedra*." *Plant Physiology* 73: 329-331.

Scialdone, A., S. T. Mugford, D. Feike, et al. 2013. "Arabidopsis Plants Perform Arithmetic Division to Prevent Starvation at Night." *eLife* 2: e00669.

Silva, L. M., L. P. Cruz, V. S. Pacheco, E. C. Machado, L. F. V. Purquerio, and R. V. Ribeiro. 2022. "Energetic Efficiency of Biomass Production is Affected by Photoperiod in Indoor Lettuce Cultivation." *Theoretical and Experimental Plant Physiology* 34: 265-276.

Sorek, M., Y. Z. Yacobi, M. Roopin, I. Berman-Frank, and O. Levy. 2013. "Photosynthetic Circadian Rhythmicity Patterns of *Symbiodinium*, the Coral Endosymbiotic Algae." *Proceedings of the Royal Society B* 280: 20122942.

Stitt, M., and S. C. Zeeman. 2012. "Starch Turnover: Pathways, Regulation and Role in Growth." *Current Opinion in Plant Biology* 15: 282-292.

Sulpice, R., A. Flis, A. A. Ivakov, et al. 2014. "*Arabidopsis* coordinates the diurnal regulation of carbon allocation and growth across a wide range of photoperiods." *Molecular Plant* 7: 137-155.

Webb, A. A. R., and A. Satake. 2015. "Understanding Circadian Regulation of Carbohydrate Metabolism in Arabidopsis Using Mathematical Models." *Plant and Cell Physiology* 56: 586-593.

Zhou, J., J. Z. Wang, T. Hang, and P. P. Li. 2020. "Photosynthetic Characteristics and Growth Performance of Lettuce (*Lactuca sativa* L.) Under Different Light/Dark Cycles in Mini Plant Factories." *Photosynthetica* 58: 740-747.

**Supporting Information**

Additional supporting information can be found online in the Supporting Information section. **Supporting Information**

Additional supporting information can be found online in the Supporting Information section.