Interactive Effects of Intraspecic Competition and Drought on Stomatal Conductance and Hormone Concentrations in Different Tomato Genotypes

We elucidated the effects of intraspecic competition on plant growth, stomatal opening and hormone concentrations in different tomato genotypes under different water regimes. Intraspecic competition reduced plant leaf area and stomatal conductance (g s ) of wild-type tomato (Ailsa Craig), which was accompanied by abscisic acid (ABA) accumulation and ethylene evolution. Intraspecic competition-induced decrease in g s was absent in acca, an ABA-decient mutant, and in never-ripe, a partially ethylene-insensitive genotype, indicating ABA and ethylene involved in plant response to intraspecic competition. As soil water becomes dry, the competition decreased g s by elevating ABA and ethylene accumulations. Under severe drought, the competition-induced decline in g s was covered by the severe drought-induced decrease in g s , as hydraulic signals most probably dominate. Absence of canopy competition had no signicant inuence on plant stomatal opening of well-watered tomato, due to canopy separation minimized the plant neighbor sensing by ethylene and other signals. Whereas under water decit condition, absence of canopy competition signicantly reduced ABA accumulation in roots and then stomatal conductance, indicating the belowground neighbour detection signals maybe enhanced by soil drought. Absence of root competition increased ethylene evolution, conrming the importance of ethylene in neighbor detection and plant response to environmental stress. stomatal opening was insignicant under well-watered condition, as the g s between the WT tomato with and without root competition was comparable. The belowground competition, i.e., absence of canopy competition, signicantly decreased the foliar ABA concentration of the competing plants, while the aboveground competition did not affect the foliar accumulation of ABA. Whereas, both the root separation and canopy separation had no remarkable effects on the concentration of ABA in the well-watered WT tomato roots. For foliar ethylene evolution of tomato, absence of canopy competition reduced the foliar ethylene evolution distinctly, but root separation had insignicant inuence on foliar ethylene evolution.


Introduction
Both biotic and abiotic stresses affect plants normal growth and development, and signi cantly decrease their productivity [1]. As plants responses to these stresses are com-plex, more attentions are focused on plants responses to particular abiotic or biotic stress [2]. Phytohormones play central roles in sensing biotic and abiotic stresses [3][4][5][6][7][8]. Moreover, physiological effects of signal response to various stresses are characterized by interaction and coupling, while the intrinsic mechanism is still unclear.
Inter-/intra-speci c competition for growing space and limited resources are the important biotic and abiotic factors inhibiting plant growth [7][8].
Plants can detect neighbors by multiple hormones and respond to them in multiple ways [3,[9][10][11]. Over-representation of phytohormoneresponsive genes was observed in competing Arabidopsis plants, con-forming that competing-induced involvement of plant hormones [12].
Ethylene is an important hormone in determining plant responses to neighbors, such as shoot elongation and leaf and stomatal movements [7,[13][14]. Pierik et al. [9] found that the ethylene-insensitive transgenic tobacco could reduce shade avoidance responses to the neighbors. Ethylene generally maintains stomata at sub-maximum apertures despite the relatively non-stressful conditions [7]. Vysotskaya et al. [7] explained that the decline in stomatal conductance induced by the neighborhood was due to the increased ethylene production in competing plants. Abscisic acid (ABA), an essential hormone adjusting stomatal opening, also involved in plant's response to neighborhood [7]. Apart from the effect of ABA on stomatal closure, little attention was paid to the interaction between hormones [15][16]. As ethylene has an opposing effect on the stomata by inhibiting ABA-induced stomatal closure [17], more experiments are necessary to investigate the co-regulation of ABA and ethylene in stomatal responses to the presence of neighbors. Moreover, recent researches indicated that cytokinin and auxin participated in plant adaptation to competitions [3,18]. As response of hormones to stressful environments is a complex signaling network, the mechanism of phytohormone's regulation in plant adaptations to stresses was not well elucidated.
Under the condition of low population density, the inter-/intra-speci c competition was mainly observed belowground [19]. However, limitation in aboveground space or belowground resources in high population environments may favor different suites of plant traits [18]. Despite the interdependence of above-and below-ground competition [20], no studies have addressed the effects of below-and above-ground interaction between different species on plant's response to environmental stresses, when grown in mixture under both abiotic and biotic stresses conditions. Stomatal opening is a fundamental response of plants to environment, regulating carbon gain and water loss [7,[21][22]. Signaling net-work of hormones controlling stomatal movement has been well-established [23][24][25][26], while little information was available on stomatal response to the presence of neighbors, more speci cally, the above-and below-ground completion. Therefore, the objectives of this study were to investigate the interactive effects of intraspeci c competition and drought on plant growth and stomatal response in different tomato genotypes, and to analyze the in uence of aboveground and belowground competition on plant hormones ac-cumulation (ABA and ethylene) and stomatal opening.

Effects of intraspeci c competition on plant growth and stomatal opening of WT tomato
At 4 days after competition, there was no signi cant difference in leaf area between the single and competing plants of WT tomato ( Fig. 1a and   b). Subsequently, compared with the single plant, the competing averagely decreased leaf area by 21% under full irrigation and by 26% under de cit irrigation, respectively. Transpiration was calculated per-unit leaf area basis [5]. During the sampling period, the intraspeci c competition signi cantly decreased transpiration, on average 33% and 28% lower than the single plant under full irrigation and de cit irrigation, respectively Under full irrigation, the leaf water potential (LWP) of single WT plant (-0.58±0.03 MPa) was similar to that of the competing plants (-0.60±0.03 MPa). Furthermore, there was no signi cant difference in LWP between the single plant and competing plants under de cit irrigation (Fig. 2a).
Abaisial stomatal conductance (g s ) of fully expanding leaves are shown in Fig. 2b and c. Under the well-watered condition, the intraspeci c competition signi cantly reduced the g s of WT tomato. As soil moisture depleted gradually, the difference in g s between the single tomato and the competing plants was markedly decreased from 26% at 4 days after competition to 16% at 10 days after competition. Although the LWP was similar between the single plant and competing plants, the difference in g s between the two treatments was measured in our experiment. The result indicated that the non-hydraulic signals contributed for regulating stomatal opening of competing plants. However, under severe drought (at 12 days after competition), the g s of single tomato was not signi cantly higher than that of competing plants, as the LWP in competing plants was lower than that in single plant.

Involvement of ABA and ethylene in plant response to intraspeci c competition
As a mutant of ABA-de cient, in the WT/FL competing pot, the g s of well-watered FL plant (686±24 mmol m −2 s −1 ) was similar to the value (681±18 mmol m −2 s −1 ) of water-stressed FL tomato (Fig. 3). Like the WT/WT competing pot, the intraspeci c competition from the WT/FL competing pot signi cantly reduced the g s of well-watered WT. When soil moisture reduced from 36-26%, the difference in g s between the single WT tomato and the competing WT in the WT/FL competing pot was averagely 16% (P<0.01); this difference was not signi cant as soil moisture depleted below 20%. The difference response of stomatal opening to intraspeci c competing between WT and FL indicated that ABA involved in tomato respond to competition. ABA concentration in the single and competing WT tomatoes under full and de cit irrigations is shown in Table 1.
Whether under well-watered condition or water de cit condition, the intraspeci c competition markedly increased the foliar ABA accumulation in WT tomato (Table 1). Under full irrigation, the intraspeci c competition signi cantly decreased the root ABA concentration, whereas increased ABA accumulation in roots under soil drought. When growing WT and NR tomato in a pot, whether under full or de cit irrigation, the intraspeci c competition did not reduce leaf area, transpiration and abaxial g s of the NR plant ( Fig. 4). In the WT/NR competing pot, the intraspeci c competition had more signi cant in uence on the g s of WT tomato (Fig. 5a). Under full irrigation, the g s of WT plant was averaging lower than that of NR plant by 22%. The difference in g s between the WT and NR tomato gradually decreased from 25-7% with decreasing in soil moisture. Moreover, this difference was not signi cant as soil moisture was lower than 28%. Overall, the distinct response to intraspeci c competition between WT and NR tomato indicated that ethylene contributed to tomato response to intraspeci c competition.
In the WT/NR competing pot under full irrigation, the foliar accumulation of ABA in competing WT plants (WT-C) was signi cantly greater than that in single WT plant by 39% and 44%, respectively. In contrast, the single WT plant had higher ABA concentration in roots, 35% and 23% greater than that in the competing plants (WT-C), respectively (Fig. 5b). Under water de cit condition, the foliar accumulation of ABA in the competing plants was signi cantly greater than that in the single WT plant, and the value in the competing NR tomato was signi cantly higher (35%) than that in the competing WT plant. However, there was no signi cant difference in root ABA accumulation between the competing plants and the single plant under water de cit condition. The effects of intraspeci c competition on foliar ethylene evolution between the single WT and competing WT and NR tomato are shown in Fig. 5c. Under the well-watered condition, the foliar ethylene evolution in the competing NR and WT tomato was 73% and 28% higher than that in the single WT plant, and the value in the competing WT tomato was signi cantly greater than the competing NR plant by 26%. No signi cant difference in foliar ethylene was detected between the competing plants and the single WT plant under de cit irrigation.

Effects of above-and below-ground competition on plant response to competition
With su cient water supply, the pot size had insigni cant in uences on plant growth, stomatal opening, and hormone synthesis of the competing tomato (data not shown). From the rst day to 10 days after de cit irrigation, soil moisture in the small pot with a volume of 0.94 L was slightly lower than that in the big pot with a volume of 1.86 L, the difference was not signi cant. While at 12 days after de cit irrigation, the value in the big pot was 17.3%, signi cantly greater than 13.0% in the small pot. At 10 days after de cit irrigation, there were insigni cant differences in leaf area, transpiration and foliar ABA concentration of tomato between the small pot and big pot, whereas the difference in foliar ethylene evolution was signi cant (Table 2). At 12 days after de cit irrigation, the signi cant difference in plant growth and hormone synthesis was detected between the small and big pot with severe drought. Effects of absence of canopy competition or root competition on g s , ABA content and ethylene evolution in the WT tomato under different treatments are presented in Table 3. The value of g s in the well-watered WT plants without canopy competition was signi cantly higher than that in the WT plant with above-and below-ground competition by 25%. The in uence of belowground competition on stomatal opening was insigni cant under well-watered condition, as the g s between the WT tomato with and without root competition was comparable.  Table 3 Abaxial stomatal conductance (mmol m -2 s -1 ), ABA content (ng g -1 DW) and ethylene evolution (nl g -1 FW) in competing WT plants under full irrigation and de cit irrigation. NC-without canopy competition, NR-without root competition.

Discussion
Intra-/inter-speci c competition is the key external factor in uencing plant growth, physiologies and functions [3,27]. The few studies that have explored links plant growth traits and competition have shown the relationships were complex, as a few of plant hormones involved in plant response to competition [3,7,9,11,14,18]. Growing space for shoot and roots plays an important role in interception of radiation and absorption of water and nutrients, respectively. These authors usually planted the single plant and competing plants in pots with same volume, the difference in growing space was overlooked. Unlike to the competition literatures, the effect of pot size on plant response was explicitly accounted for in our experiment. Our results shown that the effects of pot size on plant growth and hormone synthesis were dependent on soil water availability. Soil drying stimulates ABA formation in roots, translocation to leaves, then reduction in stomatal opening and plant growth [24]. Moreover, soil drying promotes soil compaction and in-crease in plant ethylene production [28]. Our results also demonstrated that soil drying-induced compaction increased foliar ethylene evolution in both the big and small pot. The severe drought (12 days after de cit irrigation) signi cantly decreased ABA and ethylene synthesis in the small pot compared with the big pot. Moreover, as the physical properties were different between the compost we used in this experiment and soil, soil drying-induced compaction in soil maybe more signi cant. Therefore, to compare soil drying-induced in uences on plant physiology and phenotype in different volumes, soil compaction and soil water availability need to be considered.
Plant hormone ABA and ethylene are known to regulate stomatal opening in response to competition from a neighbor [3,7,18]. With growing WT tomato and NR or FL tomato in one pot, we also concluded that ABA and ethylene involved in plant response to intraspeci c competition. The decreased in stomatal conductance, leaf area and transpiration, which was induced by intraspeci c competition, was accompanied by an increased in plant hormone concentrations. Vysotskaya et al. [7] found no signi cant difference in ABA concentration in xylem sap between single tomato and competing tomato. Whereas the elevated foliar ABA concentration in competing plants was measured in our experiment, which was in accord with earlier conclusions [18,29]. Vysotskaya et al. [18] indicated that the competing from neighbors increased ABA concentration in lettuce shoot. Kurepin et al. [29] attributed the increased ABA concentration in Helianthus annuus leaves to shade light-reduced R/FR ratio. Intraspeci c competition increased ABA concentration in tomato in our experiment, which contrasted with the lettuce data of Vysotskaya et al. [18]. This may be explained by different species, soil and environments.
Both ABA and ethylene are known to regulate stomatal opening in response to reduced water availability [28, [30][31]. As soil dries, the competition decreased g s by elevating ABA and ethylene accumulations, whereas, under severe drought, the competition cannot reduce g s even increasing in these plant hormones accumulations. During the early stages of soil drought before hydraulic signals were produced, plant hormones dominated plant in response to stress [32]. Under severe drought, chemical signals became less important when LWP declines and leaves wilt [30]. The results indicated that the competition-induced decline in g s may be covered by the severe soil-drying-induced decrease in g s , as hydraulic signals most probably dominate (Fig. 2a).
Except for severe, competition regulated plant stomatal closure mainly through non-hydraulic signals [11][12]. Ethylene is an important phytohormone of sensing competing neighbors and determining plant responses to neighbors [7,[13][14]. Under well-watered condition, the absence of canopy competition cannot signi cantly reduce the stomatal opening (Table 3). Ethylene and the red: far-red light ratio (R:FR) are most important above-ground signals of plant neighbor detection [13,33]. Canopy separation minimized the plant neighbor sensing by ethylene and R: FR. Although root exudates can serve as a belowground neighbor detection signal [34], belowground neighbor detection most prob-ably occur through reduction of local soil water and nutrients [35]. As presence of above-ground sensing signals in the competing pot with root separation, the competition-induced in uences on plant growth occurred observably.
Neighbors can be detected by several sensing signals in aboveground and below-ground through neighbor-induced changes in resource availability. The absence of canopy competition signi cantly decreased ABA accumulation in roots and then stomatal conductance, which contrasted with the result under full irrigation, indicating the below-ground neighbor detection signals were enhanced by soil drought. Although ethylene evolution was increased by drought, elevated ethylene synthesis did not signi cantly affect stomatal opening. Under some circumstances, ethylene can modulate stomatal responses to a given ABA concentration [17,[36][37].
Vysotskaya et al. [7,18] suggested that several plant hormones, such as ABA, ethylene, auxin and cytokinins, involved in plant growth response to competition from neighbors, while the interactive mechanism of multi-hormones regulating plant response to competition still was unclear. Further experiments are necessary to learn more about interaction between competition and defense responses. Moreover, only a few of competition experiments were conducted under natural eld conditions. Therefore, better understanding the multi-hormones mediated plant-plant interactions could help to optimize plant density and to understand plant behaviors in natural environment.

Plant materials
Tomato (the wild-type, Never-ripe mutant, and acca mutant) was used as a model species. The wild-type (WT) of tomato was Ailsa Craig. The Never-ripe mutant (NR) was the partially ethylene-insensitive genotype, and the acca mutant (FL) was the abscisic ac-id (ABA) -de cient tomato mutant. Seeds from the three genotypes, which were obtained from the Tomato Genetics Resource Center (University of California, Davis, USA), were germinated in compost (John Innes No.2) and covered with black plastic. After 6-7 days, the plastic was removed to prevent etiolation of the seedlings. After a further 8-days, seedlings were transferred to pots, which were lled with the same substrate, and grown in a walk-in controlled environment room with a day/night temperature of 32/16℃ and a 12h photo-period (06:00-18:00). Light intensity at plant height was between 400 and 600 µ mol m −2 s −1 PPFD (Photosynthetic Photon Flux Density).

Experimental design
Two irrigation levels were designed as full irrigation (irrigation amount=daily transpiration) and de cit irrigation (60% of irrigation amount in full irrigation). Three kinds of competition were designed, i.e., with root and canopy competition, without root competition and without canopy competition, respectively. There were three sub-treatments (two plants in one pot) in each competition, i.e., WT/WT, WT/NR and WT/FL, respectively. CK was single plant of the three species in one pot. Two different size pots were used for the treatment of single plant, i.e., 1.86 L and 0.94 L, respectively. The pot size for the competing plants was 1.86 L. Therefore, 30 treatments with 10 replications were carried out in this experiment (Table 4). Each treatment was replicated twice. The pot in the treatment with-out root competition was completely separated into two equal parts using an acrylic divider, which was glued to the inner wall and bottom of pot. For the treatment without canopy competition, a transparent glass barrier was placed between the aerial portions of two plants to totally separate the shoot components. Samples were then placed on a shaker in a cold room (4℃) overnight to extract ABA. A standard curve was determined with standards in a serial dilution of synthetic unlabeled (±)-cis, trans-ABA (Sigma Let., Dorset, UK). ABA concentration was calculated by reference to the standard curve after linearization using the 'logit' transformation.
For the determination of ethylene evolution rates, lea ets and roots on the same plant sampled for ABA determination were weighed and placed in 28 ml glass vials containing saturated lter paper, which was then sealed with a rubber puncture cap. The lea ets and roots samples were incubated for 60 min under a lamp (200 µ mol m −2 s −1 ) and in a dark chamber, respectively. A 1 ml headspace sample was withdrawn using a gastight syringe, then manually injected into a gas chromatograph (6890N, Agilent Technologies UK Ltd, Wokingham, UK; Networked GC system, method: Ethylenesplit. M, software: Enhanced Chemstation Online GC) equipped with a J&W HP-AL/S (50 m×0.537 mm×15.0 mm) column (HiChrom Ltd, Reading, UK). This was maintained for the rst 5 min at 100℃ to resolve ethylene, and then ramped at 15℃ min −1 to 150℃ and held for 1.5 min to drive off any water vapor introduced onto the column by sample injection. The carrier gas was helium at a ow rate of 5.7 mL min −1 , and detection was by ame ionization. The rate of ethylene evolution was determined with reference to peak areas of known ethylene standards (99.995% minimum purity, BOC Special Gases, Manchester, UK), and corrected for tissue FW and time in incubation.

Statistical analysis
All data were analyzed by one-way analysis of variance (ANOVA). Less signi cant difference (LSD) and Student's t-test were carried out with SPSS21.

Declarations
Author Contributions: Data Availability Statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Figure 1
Variations of leaf area and transpiration in CK and the treatment of competition under full irrigation and de cit irrigation. * indicates signi cant difference between treatments (*P<0.05, **P<0.01).

Figure 2
Page 10/11 Variation of leaf water potential (a-de cit irrigation), abaxial stomatal conductance (b-full irrigation, c-de cit irrigation) in the single plant and competing plants of wild type tomato (*P<0.05, **P<0.01).