Treatments and growth conditions.

Two experiments were conducted in a greenhouse at the Texas A&M University, Texas Agricultural Expt. Sta. in Uvalde (lat. 29°1′N, long. 99°5′W) during April and Sept. 2005. Maximum PPF within the greenhouse at the canopy level was ≈1500 μmol·m⁻²·s⁻¹ using natural summer photoperiods. Five-week-old pepper seedlings were treated using a spray method with film-forming and chemical antitranspirants, and were then exposed to desiccation by withholding water.

The film-forming (physical) antitranspirants used were AntiStress (AS; Enviroshield Products Co., Houston, TX), Vapor Gard (VG; Miller Chemical and Fertilizer Corp., Hanover, PA), and Transfilm (TF; PBI/Gordon Corp., Kansas City, MO). The chemical antitranspirant was ABA (Valent BioSciences Co., Libertyville, IL), whereas AVG (ReTain; Abbot Laboratories, Abbott Park, IL) was used as a potential antagonist of ethylene activity. Spray solution concentrations were prepared according to manufacturer recommendations: 2.2% for AS and VG, 3.8% for TF, 2 g·L⁻¹ for ABA, and 3.8% for AVG. Foliar applications were done with a hand-held sprayer, and plants were sprayed until leaves were evenly covered but before dripping. Untreated control plants were sprayed with tap water.

The first experiment (small scale) addressed changes in plant height, water status, and survival of pepper seedlings exposed to one desiccation cycle. The second experiment (large scale) concentrated on the physiological behavior of seedlings exposed to two desiccation cycles. In the first experiment (Expt. 1), seeds of poblano pepper (cv. Tiburon; Sakata Seed America, Morgan Hill, CA) were sown in peat-lite mix (Speedling, Sun City, FL) and covered with a thin layer of medium-grade horticultural vermiculite. Seedlings were grown in trays (South Cross Vegetable Transplants, Mission, TX) with 35 mL of cell volume for 5 weeks (ready for transplanting in the field) and then sprayed with tested compounds as described earlier. Two days before the onset of treatments, seedlings were fertilized with a soluble fertilizer 30N–4.4P–8.3K (Scotts Miracle-Gro Products, Marysville, OH) at a N rate of 100 mg·L⁻¹. After spraying, plants were exposed to a desiccation period by withholding water for 11 d. Day/night air temperatures were 21 to 25 °C/16 to 18 °C.

During the second experiment (Expt. 2), pepper seeds were sown as described before except that tray cell volume was 28 mL. Plants of the same age as in Expt. 1 were sprayed and exposed to two 4-d water deficit cycles, the first irrigation being 4 d after spray and water deficit treatment (DAT) and the second, 8 DAT. Rewatering was conducted when leaves from all untreated control plants had wilted. The difference in irrigation timing between the two experiments was the result of higher air temperatures in Expt. 2, with day/night temperatures of 25 to 40 °C/18 to 20 °C. During the course of the experiments, seedling flats were rotated across greenhouse benches to avoid position effects.

Growth measurements and analysis.

Plant height from the substrate level to the shoot apex was measured before treatment application and at 3 and 6 DAT in Expt. 1 and at 4, 8, and 19 DAT in Expt. 2. A total of 60 plants/treatment were used, avoiding plants from edge rows. Thirty additional plants per treatment were harvested at 4 and 8 DAT, and were partitioned into leaves, stems, and roots. Leaf area (LA) was measured using LI-3100 (LI-COR, Lincoln, NE). Roots were carefully washed free of rooting medium, separated into segments, and spread out in a transparent tray containing a thin layer of water. Roots were scanned with a digital image analysis system at a resolution of 300 dots per inch and root length was determined (WinRhizo LA 1600; Régent Instruments, Québec, Canada). All tissues were oven-dried at 70 °C until constant weight and dry weights recorded.

Water potential and relative water content.

Midday (1100–1500 hr) stem and leaf xylem water potential were measured with a Scholander-type pressure bomb (Soil Moisture Equipment Corp., Santa Barbara, CA). Stem xylem water potential (ψ_st) was measured 3, 4, 7, and 8 DAT, whereas leaf water potential (ψ_lf) was measured 2, 3, 4, 6, and 8 DAT. At least six plants per treatment were cut at the base of the transplant and ψ_st was measured immediately. On a separate set of plants, the first fully developed leaf, about the third or fourth leaf from the apex, was excised and used for ψ_lf measurements. Relative water content (RWC) was determined on leaves of the same age as those used for ψ_lf. From each of the six plants, an entire leaf was collected and its fresh weight (FW) was recorded. The leaves were rehydrated in distilled water in the dark for 20 h to obtain the turgid weight (TW). Leaves were then dried in an oven until constant weight and dry weights were determined (Turner, 1981). The RWC expressed as percent was calculated as

Electrolyte leakage.

Leaves of about the same age as those sampled for ψ_lf and RWC measurements were used for the electrolyte leakage (EL) determination by a modified procedure described previously (Lutts et al., 1996). Two leaves per replication were taken for each treatment, and EL was determined for the whole leaf. Leaves were washed with three changes of deionized water and incubated in sealed vials filled with 10 mL deionized water and slowly shaken in a water bath at 25 °C. Electrical conductivity of the solution containing the leaves was measured after 24 h (EC₁), and then autoclaved at 120 °C and 103.1 MPa for 20 min. The second conductivity reading (EC₂) was obtained upon equilibration at 25 °C. The EL expressed as percent was calculated as

Gas exchange measurements.

Net photosynthesis (A_CO2), stomatal conductance (g _s), intercellular CO₂ concentration (C_i), and leaf temperature were determined with a portable photosynthesis system (LI-6200; LI-COR) equipped with a 0.25-L chamber using a constant leaf area (2.5 cm²). Fully developed and sun-exposed leaves from five to seven plants per treatment were selected for measurements during midday (1100–1500 hr) at 1, 4, 5, and 8 DAT. All measurements were made under natural ambient weather conditions on clear sunny days with a PAR of 1200 to 1450 μmol·m⁻²·s⁻¹, air temperature of 35 to 40 °C, relative humidity of 40% to 63%, and an air vapor pressure of 2.4 to 4.1 kPa.

Experimental design and statistical analysis.

The experiments were conducted using a randomized complete block design with three replications. General linear models procedures (SAS Institute, Cary, NC) were used for statistical analyses. Data were tested for normality of distribution and homogeneity of variance, and were transformed when necessary.

Effect of treatment on plant height was analyzed by the analysis of covariance, with initial plant height measured at the day of the treatment as the covariate. Treatment effects on plant growth and biomass allocation were examined by calculating relative growth rates (RGRs) for root, stem, leaves, shoot (stem + leaf), and plant dry weight (root + shoot), as well as for root length and LA growth rate for the period between 4 and 8 DAT (Hunt, 1982). Data for calculated RGR values, A_CO2, g _s, ψ_lf, ψ_st, RWC, EL, leaf abscission, and survival were subjected to analysis of variance using PROC GLM with the SAS software, and mean separation was determined by the lsd test.

Allometric relationships between seedling components were calculated to separate further the treatment effects from size effects as plants grew (Gebauer et al., 1996). For each treatment, the amount of total biomass allocated to a plant component was described by the allometric equation

where b₀ and b₁ are constants. The slope b₁ is the allometric coefficient and it represents the relative change in allocation of dry matter to specific organs. When interactions between treatments and log_e (plant dry weight) were significant, differences among slopes were tested using the Tukey-Kramer test.

Growth and water status (Expt. 1).

Plant height was significantly reduced with ABA and AVG compared with control 3 and 6 DAT, whereas plant height was similar for AS, VG, and control treatments (Table 1). At 6 DAT, untreated control plants were able to maintain a high RWC (98.4%) as other treated plants and higher than AS (89.9%). As the water deficit progressed to 8 d, only ABA-treated plants were able to maintain higher leaf RWC (90.9%) with less EL (7.8%) compared with other treatments (Table 1). Severe water stress caused complete drying and death of control plants, and significant reduction for all other treatments except for ABA, which enabled 100% survival (Table 1).

Table 1. Effect of abscisic acid (ABA), aminoethoxyvinylglycine (AVG), and film-forming materials on plant height, relative water content (RWC), electrolyte leakage (EL), and survival of pepper seedlings exposed to a single water deficit cycle (Expt. 1).

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Gas exchange (Expt. 2).

During the course of this experiment, g _s was not changed (P > 0.05) by the application of AS, TF, and VG compared with the control (Fig. 1A). Application of ABA sharply decreased g _s from 0.864 mol·m⁻²·s⁻¹ (not shown) measured before treatment to 0.390 μmol·m⁻²·s⁻¹ 1 DAT, and it stayed lower compared with all other treatments (P ≤ 0.05) up to 8 DAT. Conversely, AVG application increased g _s compared with control or ABA treatment 1 DAT and with all other treatments 4 DAT (Fig. 1A).

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Net CO₂ assimilation rate measured before treatment applications was 20.5 μmol·m⁻²·s⁻¹, (not shown) and after 24 h of withholding water it stayed in the range of 17.7 to 21.4 μmol·m⁻²·s⁻¹ for all treatments except for ABA-treated plants (Fig. 1B). Depression of A_CO2 by ABA application in comparison with other treatments (P ≤ 0.05) was present until 5 DAT, and it increased up to the control level after the second rewatering period 8 DAT.

Antitranspirant spray treatments did not influence C_i concentration (data not shown) except at 5 DAT, when the C_i of the AVG-treated plants (193 μmol·mol⁻¹) was significantly lower (P ≤ 0.05) in comparison with control (263 μmol·mol⁻¹), AS (277 μmol·mol⁻¹), TF (259 μmol·mol⁻¹), and VG (246 μmol·mol⁻¹). On the same day, C_i concentration was lower for ABA (217 μmol·mol⁻¹) compared with control and AS-treated plants. Differences between leaf and air temperature were not significant (data not shown).

Water potential and relative water content (Expt. 2).

Leaf water potential measured at the day of application averaged –0.14 MPa across treatments (not shown) and decreased after 2 d of water deficit for all treatments (Fig. 2A). The decrease of ψ_lf 2 DAT was most pronounced for AVG-treated plants (–1.15 MPa) followed by VG (–0.95 MPa) and TF (–0.85 MPa). Untreated control plants maintained ψ_lf at the same level (P > 0.05) as plants treated with AS, TF, and VG, except 3 DAT (Fig. 2A). Leaf water potential of ABA-treated plants remained more than –0.37 MPa during the course of the experiment, and it was higher (P ≤ 0.05) compared with other treatments except at days of rewatering (4 and 8 DAT). Stem water potential was not significantly affected by the treatments, except that ψ_st of ABA-treated plants was higher 3 and 7 DAT compared with other treatments (Fig. 2B).

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Relative water content of ABA-treated plants was significantly higher than that of the other treatments during both drying cycles when measured 3 and 7 DAT (Fig. 3A). Overall leaf RWC measured 7 DAT was ≈25% lower than at 3 DAT.

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Plant growth (Expt. 2).

Plant height was generally unaffected by antitranspirant treatments, although a slight increase was observed in TF- and VG-treated plants compared with untreated, and ABA- and AVG-treated plants 4 DAT and 8 DAT (Fig. 3B), but these differences disappeared 19 DAT. Application of ABA and AVG had the most pronounced reduction in leaf abscission compared with control and film-forming materials 4 and 8 DAT (Fig. 3C).

Root RGR was unaffected by application of film-forming materials, but was reduced for ABA- and AVG-treated plants to 40% and 50%, respectively, compared with control plants (Table 2). Enhanced stem RGR was found after AVG and VG application, whereas ABA and TS reduced it significantly compared with control. Application of ABA and AS reduced leaf RGR compared with control and other treatments, whereas shoot RGR was also reduced for ABA-, AS-, and TF-treated plants, but increased with AVG and VG treatments. Similarly, total RGR was significantly reduced with ABA, TF, and AVG.

Table 2. Effect of abscisic acid (ABA), aminoethoxyvinylglycine (AVG), and film-forming materials on relative growth rate (RGR) of root, stem, leaf, shoot and total plant weight, root length, and leaf area of pepper seedlings exposed to two water deficit cycles (Expt. 2).

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Root length and leaf area RGR were significantly reduced for all treatments, except for AVG, compared with the control (Table 2). Aminoethoxyvinylglycine-treated plants showed higher stem and shoot RGR than control; but, as a result of reduced root RGR, they had lower total plant RGR.

Allometric analyses revealed treatment differences in dry matter allocation to below- and aboveground plant parts (Table 3). Plants treated with AS allocated more biomass to stems (b₁ = 0.489) at the expense of leaves (b₁ = 0.298). Aminoethoxyvinylglycine-treated plants allocated more biomass to leaves (b₁ = 0.662) at the expense of root production (b₁ = 0.107). Plants treated with ABA, VG, and control had similar allocation patterns to each other, whereas TF had more allocation to roots and less to stems compared with control.

Table 3. Allometric coefficients (b₁) for biomass distribution among plant tissues of pepper seedlings as affected by antitranspirant treatment and water deficit exposure (Expt. 2).

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Film-forming materials.

The film-forming antitranspirants used in the current study (AS, TF, and VG) did not significantly influenced leaf gas exchange (A_CO2, g _s, or C_i) and water relations of pepper seedlings exposed to water deficit stress when compared with control untreated seedlings. Leaf and stem xylem water potential of plants treated with all film-forming materials decreased at the same rate as those of untreated plants (Fig. 2). In both experiments, no improvement in RWC or reduction of cell membrane injury was found with the application of film-forming materials.

Plaut et al. (2004) found that acrylic film application decreased g _s and A_CO2 of greenhouse-grown pepper plants. Differences in leaf and plant age, as well as the imposition of water deficit stress, might explain the responses of their study and ours. Seedlings typically have much smaller leaves and may not yet be as physiologically competent and responsive to treatments as fully developed leaves from mature plants. Developing leaves from seedlings may also be more susceptible to injury, as suggested by the decrease in leaf area RGR for all film-forming materials. This was most pronounced for AS (Tables 2 and 3), although leaf abscission was similar to other film-forming treatments and control. Irmak et al. (1999) reported that VG reduced transpiration and increased the vegetative growth of tomato plants. This is in agreement with our findings, because VG enhanced stem growth rate and positively affected shoot growth rate compared with control. It is highly possible that variability in species/cultivar responses to film-forming materials, as reported for ornamentals, woody, desert or horticultural crops (Hummel, 1990, Laurie et al., 1994; Plaut et al., 2004), could explain the differences between studies. Film-forming antitranspirants act as a physical barrier to water lost from leaves and thus they are primarily active in the adaxial leaf surface. If spray methods could ensure uniform coverage on both the adaxial/abaxial epidermal leaf surfaces, the potential for reducing water loss through stomata could be greater to what was observed.

Aminoethoxyvinylglycine and abscisic acid.

In contrast to film-forming materials, ABA and AVG were probably translocated within plant tissues, and both compounds had more influence on the physiology and growth of pepper seedlings exposed to desiccation stress. An enhancement of g _s by AVG compared with untreated plants was measured in the absence of visible water stress 1 DAT, and immediately after rewatering 4 DAT (Fig. 1). This suggests that by blocking ethylene production, AVG induced stomatal opening, as previously reported (Tissera and Ayres, 1986). In addition, a significant decline in ψ_lf for AVG-treated plants measured 2 and 6 DAT suggests that AVG may have interfered with regulation of stomatal function during water deficit stress, whereas other treated plants were only under moderate water deficit stress. The lower g _s of AVG-treated plants compared with control and film-forming materials 5 DAT is probably a result of “overcompensation” for excessive water loss (Gu et al., 1996). In contrast to the g _s responses to AVG, A_CO2 did not changed significantly throughout the drying cycles (Fig. 1). However, A_CO2 levels of AVG-treated plants were higher than those of controls (2% to 33%), depending on day of measurement. The enhanced leaf dry matter allocation and leaf area RGR at the expense of root mass allocation and root growth (Tables 2 and 3) raises the question of AVG allocation within plants. Root-applied AVG can modify processes at the shoot level (Islam et al., 2003; Leskovar et al., 2006; Locke et al., 2000); however, whether shoot-applied AVG influences root growth is unknown. The reduction in whole-plant RGR of AVG-treated plants (almost 30% compared with control) was probably a consequence of excessive water loss that resulted from the lower capacity of those plants to regulate stomata closure during water stress. Maintenance of a favorable plant water status is crucial for expansive growth and overall dry matter accumulation.

The reduced leaf abscission by both AVG and ABA treatments (Fig. 3C) may have occurred possibly through interactive mechanisms. It is known that AVG reduces ethylene production and thus prevents leaf abscission (Abeles et al., 1992). On the other hand, ABA-induced stomatal closure can prevent leaf dehydration, senescence, and abscission. In addition, ABA possibly limited the extent of ethylene synthesis, as was found for jack pine (Pinus banksana Lamb.) seedlings, and citrus (Citrus sinensis L.) plants (Gómez-Cadenas et al., 2002; Rajasekaran and Blake, 1999). The decrease of EL observed in ABA- but not in AVG-treated plants (Table 1) also suggests that ethylene evolution is not the only factor contributing to membrane damage under desiccation stress (Rajasekaran and Blake, 1999). Enhanced osmotic adjustment and higher turgor pressure and hence leaf ψ_lf and RWC in ABA-treated leaves may also be important in membrane protection under drought stress (Wang et al., 2003).

Stomatal conductance for ABA-treated plants compared with untreated plants decreased significantly from 47% 1 DAT to 60% 5 DAT; however, the magnitude of this change diminished after the second rewatering 8 DAT. A significant but less pronounced trend was also measured for A_CO2. Because Ci concentration of ABA-treated plants was not changed in comparison with control, except at 5 DAT, it seems that stomatal functioning was not the only limiting factor for A_CO2 (Jifon and Syvertsen, 2003). Abscisic acid might have had a nonstomatal influence on biochemical changes during the photosynthetic reaction (Popova et al., 1996). A decrease in g _s and A_CO2 after ABA application was observed as soon as 1 h after treating french bean (Phaseolus vulgaris L.), sugar beet (Beta vulgaris L.), and maize (Zea mays L.) (Pospisilova and Batkova, 2004). The same effect was found in well-watered or drought-stressed barley and poplar (Populus L.) plants (Popova et al., 1996; Yin et al., 2004).

Reduced RGR of root and all shoot growth components in ABA-treated plants led to 80% reduction of total RGR compared with control plants (Table 2). However, patterns of root and shoot biomass accumulation were similar to those of control plants (Table 3). Because the ABA-induced reduction in RGR is transient, this further confirms the potential use of ABA to control transplant growth in nurseries (Leskovar and Cantliffe, 1992).

In summary, application of film-forming antitranspirants during water deficit stress did not significantly affect physiological or growth parameters of pepper seedlings. Treatment with AVG resulted in increased g _s and reduced ψ_lf and total plant growth despite a reduction in leaf abscission. The potential of AVG as an antitranspirant seems limited because the excessive water loss resulting from increased g _s would predispose the newly transplanted seedlings to dehydration, retarded growth, and poor establishment. The ABA-induced transient reduction in g _s (and presumably transpirational water loss) could potentially protect seedlings from desiccation injury associated with transplant shock by allowing plants to conserve water. Further research across species aiming at the potential application of natural ABA, or ABA analogs having similar physiological effects, would make this approach economically feasible for large-scale industry use.