The Alleviation of Photosynthetic Damage in Tomato under Drought and Cold Stress by High CO2 and Melatonin

The atmospheric CO2 concentration (a[CO2]) is increasing at an unprecedented pace. Exogenous melatonin plays positive roles in the response of plants to abiotic stresses, including drought and cold. The effect of elevated CO2 concentration (e[CO2]) accompanied by exogenous melatonin on plants under drought and cold stresses remains unknown. Here, tomato plants were grown under a[CO2] and e[CO2], with half of the plants pre-treated with melatonin. The plants were subsequently treated with drought stress followed by cold stress. The results showed that a decreased net photosynthetic rate (PN) was aggravated by a prolonged water deficit. The PN was partially restored after recovery from drought but stayed low under a successive cold stress. Starch content was downregulated by drought but upregulated by cold. The e[CO2] enhanced PN of the plants under non-stressed conditions, and moderate drought and recovery but not severe drought. Stomatal conductance (gs) and the transpiration rate (E) was less inhibited by drought under e[CO2] than under a[CO2]. Tomato grown under e[CO2] had better leaf cooling than under a[CO2] when subjected to drought. Moreover, melatonin enhanced PN during recovery from drought and cold stress, and enhanced biomass accumulation in tomato under e[CO2]. The chlorophyll a content in plants treated with melatonin was higher than in non-treated plants under e[CO2] during cold stress. Our findings will improve the knowledge on plant responses to abiotic stresses in a future [CO2]-rich environment accompanied by exogenous melatonin.


Introduction
Climate change is expected to have a significant impact on agricultural production and food security [1,2]. Plants are subjected to various environmental stresses during their lifecycle, the frequency and intensity of which are increasing due to climate change [2]. For instance, drought and cold are significant factors that limit agricultural crop production [3,4]. Elevated CO 2 concentration (e[CO 2 ]) enhances plant photosynthesis and biomass even under adverse environmental conditions [5,6]. The e[CO 2 ] can increase water-use efficiency and enhance plant growth, leading to higher water use [7]. concentration, respectively. The batch marked with "melatonin" indicates the plants were treated by seven times of 1 mM melatonin. "DS", drought stress, 25/20 °C + no irrigation; "CS", cold stress, 12/12 °C + irrigation; "R1" and "R2", recovery, 25/20 °C + irrigation.

Results
As shown in Figure Figure 2E). The CS significantly decreased the leaf temperature of the plants under the four treatments, with no difference between them ( Figure 2E).  The data represent average values ± SD (n = 3). The ANOVA was conducted within all the treatments. Different small letters showed significant differences (p < 0.05).  Figure 4D).   Figure S1). The F q '/F m ' (quantum yield of PSII) and ETR (electron transport rate) of plants under e[CO 2 ] after R1 significantly decreased compared with controls, which was lower than that under a[CO 2 ] after CS (Supplementary Material Figure S2A Figure 3. The data represent average values ± SD (n = 3). Different small letters showed significant differences (p < 0.05).

Discussion
Due to the increase in the frequency and severity of abiotic stresses [2] in field crops and increased a[CO 2 ], it is urgent to understand how plants respond to complex environmental changes and potential alleviation methods. The effect of melatonin on animals has been widely studied compared with plants and the understanding of melatonin's role in plants is just starting to emerge [24]. Previous studies primarily focused on the effect of melatonin on plants under a[CO 2 ] and abiotic stresses [10,12,13]. The question is how melatonin affects plants under abiotic stresses accompanied by e[CO 2 ].

Effects of e[CO 2 ] on Tomato Photosynthesis
The decreased P N of tomato was aggravated with a prolonged water deficit, which partially recovered when the irrigation restarted and remained low under CS (Figure 7). On the one hand, in accordance with previous studies [25][26][27][28], e[CO 2 ] enhanced the P N and starch accumulation under non-stressed conditions (Figure 7). On the other hand, e[CO 2 ] can ease the damage caused by abiotic stresses, such as DS [29,30]. The e[CO 2 ] reduced the effect of drought on grasses and legumes by decreasing H 2 O 2 production and increasing molecular antioxidants [29]. Furthermore, e[CO 2 ] mitigated the effect of reduced irrigation on tomato fruit yield [30]. We found that the alleviating effect of e[CO 2 ] on the P N only occurred in tomato under a moderate water deficit but not under severe DS (Figure 7).

The Melatonin Played Positive Roles in Tomato Plants Grown Under Cold and e[CO 2 ]
A positive effect of melatonin application was seen in tomato when irrigation restarted, with better leaf cooling under a[CO 2 ] + M than a[CO 2 ] (Figure 7). By comparison, P N partially recovered, with the highest P N under e[CO 2 ] + M, when irrigation restarted and this trend was kept during the CS period, resulting in the highest plant biomass under e[CO 2 ] + M after recovery (Figure 7). Previous studies have shown that melatonin application can enhance P N of plants, including tomato, under CS and a[CO 2 ] by reducing the damage of the low-temperature conditions on the photosynthetic apparatus and protecting the thylakoid membrane [21,22]. Our study provided proof that melatonin played a positive role in photosythesis protection during DS recovery and CS, and thereby benefits biomass accumulation in tomato under e[CO 2 ]. non-stressed conditions (Figure 7). On the other hand, e[CO2] can ease the damage caused by abiotic stresses, such as DS [29,30]. The e[CO2] reduced the effect of drought on grasses and legumes by decreasing H2O2 production and increasing molecular antioxidants [29]. Furthermore, e[CO2] mitigated the effect of reduced irrigation on tomato fruit yield [30]. We found that the alleviating effect of e[CO2] on the PN only occurred in tomato under a moderate water deficit but not under severe DS (Figure 7).  In accordance with previous studies, e[CO 2 ] decreased g s of the tomatoes under the control condition [31,32]. The trends of E positively corresponded to g s under the control, with lower E under e[CO 2 ]. However, this trend disappeared under the DS and recovery stages. Drought and cold stress reduced g s and E but to a lesser degree under e[CO 2 ] than a[CO 2 ], resulting in a lower leaf temperature and better leaf cooling during DS under e[CO 2 ] than a[CO 2 ] (Figure 7). This indicated the leaf temperature was well-controlled through stomatal regulation in the plants grown under e[CO 2 ] even when there were adverse environmental factors, such as a moderate and severe water deficit. These could partially explain why e[CO 2 ] has a mitigation effect on tomato photosynthesis under DS.
Melatonin played roles in the regulation of chlorophyll loss and synthesis for the plants grown under abiotic stresses [33,34]. Li et al. (2018) found that exogenous application of melatonin can increase the chlorophyll content and delay the leaf senescence of wheat (Chl b-deficient mutant ANK32B) under e[CO 2 ] [17]. In accordance, the chlorophyll a content of the tomatoes treated by melatonin was higher than non-treated plants under e[CO 2 ] during CS.
The effects of melatonin on downregulating chlorophyll breakdown or accelerating its synthesis during abiotic stresses were clear under e[CO 2 ]. Moreover, chlorophyll a/b in the plants treated by melatonin was higher under e[CO 2 ] during DS but lower under a[CO 2 ] during CS compared with controls without melatonin. Li et al. (2018) [17] showed that alteration in chlorophyll a/b induced by melatonin was not seen during non-stressed conditions. Chlorophyll a/b could indicate the degree of leaf damage caused by oxidative stress [35]. An increase in chlorophyll a/b of tomato plants under e[CO 2 ] + M compared with e[CO 2 ] during the water deficit showed the alleviating effects of melatonin on the oxidative damage of tomato plants. Li et al. (2015) suggested that melatonin pre-treatment enhanced the drought tolerance of Malus species under a[CO 2 ] through downregulation of ABA, better leaf water conservation, stable chlorophyll content, and increased P N 11 . Liu et al. (2015) concluded that melatonin pretreatment could improve tomato drought tolerance by increasing the photochemical efficiency and protecting against oxidative damage [19]. Furthermore, Ding et al. (2018) found that melatonin pretreatment could induce thick cutin, increase the cuticular wax level, and enhance wax gene expression in tomato leaves under DS, which explains how melatonin improves tomato drought tolerance through the limitation of leaf water loss [20]. Shi et al. (2015) demonstrated the positive role of melatonin treatment in bermudagrass responding to abiotic stresses, including drought, cold, and salt, through physiological, metabolomics, and transcriptomic regulation [36]. However, the response of tomato at the reproductive stage to abiotic stresses and melatonin needs further study in order to check how plant production was affected. More importantly, the underlying mechanism, especially molecular pathways concerning how melatonin works in plants, need to be further investigated.

Growth Environmental Condition and Treatments
Seeds of tomato cultivar "Qianxi" (Known-you seed co. LTD, Taiwai, China) were sown in plastic pots with a 9-cm height and 11-cm diameter. This cultivar was chosen since it has been popular in the Chinese market for more than 10 years, which has good market prospects. Pots were filled by Pindstrup 2 (Pindstrup Mosebrug A/S, Ryomgaard, Denmark). Relative humidity was 43-55% and 49-63% and air temperature was 23 and 16 • C for day and night, respectively, in two rooms of a greenhouse. . The environmental parameters of two chambers were set to 25/20 • C (15 h day/9 h night), 60% relative humidity, and 300 µmol m −2 s −1 PPFD during daytime. The parameter settings of the two chambers were the same except the CO 2 concentration. Each chamber had 36 seedlings and half of the seedlings were sprayed by melatonin. All seedlings were irrigated by the same nutrition solution twice a day by a flooding bench for 10 min at 8:00 and 16:00. Half of the 26-day-old plants were continuously sprayed (15 mL per plant) and irrigated (50 mL per plant) by 1 mM melatonin for the fourth time at 16:00 before stress. Then, the 27-day-old plants were subjected to DS by withdrawing irrigation from 8:00 for 32 h. The 28-day-old plants were irrigated at 16:00 as the recover stage 1 (R1) from DS for 28 h. Half of the 28-day-old and 29-day-old plants were continuously sprayed (15 mL per plant) and irrigated (100 mL per plant) by 1 mM melatonin at 16:00. Afterwards, the 29-day-old plants were subjected to CS (12/12 • C, day/night) from 20:00 for 68 h. The 32-day-old plants were subjected to a normal temperature (25/20 • C, day/night) from 16:00 as the recover stage 2 (R2) from CS. Half of the 32-day-old plants were continuously sprayed (15 mL per plant) and irrigated (100 mL per plant) by 1 mM melatonin at 16:00 during R2. In total, melatonin was applied for seven times. Control plants were sprayed and irrigated by the same amount of ddH 2 O (double-distilled water) as melatonin solution throughout the experiment. The first fully expanded leaf from the top was chosen for measurements.
Photosynthesis parameters: P N , g s , E, C i , and leaf temperature were measured using a portable photosynthesis system (CIRAS-2, PP Systems, Amesbury, USA). Measurements with three replicates were taken for the plants under control (before stress), DS for 24 h and 30 h, R1 for 16 h, CS for 60 h, and R2 for 16 h. We started to take records until five parameters were stable. The mean of the last six values were averaged and considered as the final results.
Measurements of the chlorophyll and carbohydrate content were taken from the plants under the control before stress, under DS for 30 h, and under CS for 60 h with three replicates. Leaf disks were punched using a cork borer and then the samples were immersed in 95% 4 • C ethanol for chlorophyll content measurements. Meanwhile, the samples were freeze-dried, ground, and weighed for carbohydrate content measurements [37].
Leaves was dark-adapted using a leaf clip for 25 min. Leaf F v /F m was detected using Handy PEA (Hansatech Instrument, King's Lynn, England). Measurements with four replicates were taken under the control (before stress), DS for 30 h, R1 for 16 h, CS for 60 h, and R2 for 16 h.
Plants were dark-adapted in a dark room for 20 min before quenching analysis. The F q '/F m ', q L , NPQ, and ETR of the plants were detected using MINI-PAM (Walz, Effeltrich, Germany) operated with WinControl 3 software (Walz, Effeltrich, Germany). A PPFD of 300 µmol m −2 s −1 during measurements was provided with an external light source (Schott KL 1500, Göttingen, Germany) through fiber optics. Measurements with three replicates were taken under the control (before stress), R1 for 16 h, CS for 60 h, and R2 for 16 h.
Plant growth parameters with three replicates were measured when the plants were grown under the control before stress, DS for 32 h, and CS for 68 h. Plant height from the cotyledonary node to growth point was recorded using a ruler. Leaf area was detected using a leaf area meter (3100, LI-COR, Lincoln, Nebraska, USA). Finally, FW of the leaf and stem was measured. After 48 h of drying at 80 • C, DW of the leaf and stem was measured.
The data were analyzed using analysis of variance (ANOVA) by SPSS 16.0 (SPSS Inc. Chicago, IL, USA). There was no role in work plan, data analysis and manuscript submission for the funder.