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USDA-ARS, Crop Genetics and Production Research Unit, P.O. Box 345, Stoneville, MS 38776
* Corresponding author (bpettigrew{at}ars.usda.gov).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSNOTESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: CER, CO2 exchange rate • Chl, chlorophyll • DAP, days after planting • Fv/Fm, dark adapted chlorophyll fluorescence variable to maximal ratio • LAI, leaf area index • qP, photochemical quenching • PPFD, photosynthetic photon flux density • PSI, photosystem I • PSII, photosystem II • qNP, nonphotochemical quenching • SLW, specific leaf weight
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Although there has been considerable research documenting the growth and physiological response of cotton to moisture deficit stress, most of it has been conducted with pots in artificially controlled growth environments of greenhouses (Jordan, 1970; Radin, 1981; Radin and Ackerson, 1981; Loffroy et al., 1983; Ball et al., 1994) and growth chambers (Genty et al., 1987; Nepomuceno et al., 1998), or has been conducted under field conditions in arid climates (Turner et al., 1986; Puech-Suanzes et al., 1989; Ephrath et al., 1990; Ephrath et al., 1993; Leidi et al., 1993; López et al., 1995; Lacape et al., 1998; Leidi et al., 1999) where moisture deficit stresses are more prevalent and extreme. Field studies under temperate humid conditions have been conducted by McMichael and Hesketh (1982) and Faver et al. (1996). From these studies, we know that moisture deficit stress promotes stunted growth in cotton with reduced leaf area expansion (Turner et al., 1986; Ball et al., 1994; Gerik et al., 1996). Lint yield is generally reduced because of reduced boll production, primarily because of fewer flowers but also because of increased boll abortions when the stress is extreme and when it occurs during reproductive growth (Grimes and Yamada, 1982; McMichael and Hesketh, 1982; Turner et al., 1986; Gerik et al., 1996; Pettigrew, 2004). Leaf photosynthesis is also reduced when plants are grown under moisture deficit conditions because of a combination of stomatal and nonstomatal limitations (McMichael and Hesketh, 1982; Marani et al., 1985; Turner et al., 1986; Genty et al., 1987; Ephrath et al., 1990; Faver et al., 1996). As in most plants, leaf water potential (
l) is reduced under drought conditions, but cotton has the ability to osmotically adjust and maintain a higher leaf turgor potential (t) (Turner et al., 1986; Nepomuceno et al., 1998).
Although these controlled growth environment studies have proven insightful, overall cotton growth and yield is reduced when the root zone volume is constrained by a finite container size (Carmi and Shalhevet, 1983). How applicable these controlled-environment studies are to what the plants would experience and respond to under natural field conditions is not clear. Similarly, the arid environment, where the vast majority of field studies have been conducted, would tend to lead to early, rapid, and extreme moisture deficit stress developing in the cotton plants. A later, slower developing, and less severe stress, as would tend to occur in more humid, temperate environments, may modify or delay the physiological alterations in response to moisture deficit stress.
The introduction of transgenic cotton varieties into large-scale production has occurred within the last decade. With the uncertainty of gene insertion position for a given transformation event utilizing current transformation technologies, DNA could be inserted into a chromosomal region containing a gene and thereby disrupt the gene's function. Most of these unfavorable insertions are eliminated through an extensive screening and selection process that culls all lethal mutations and mutations that prove deleterious to one of the major agronomic or quality traits. More subtle alterations could potentially pass through this screen. This possibility, combined with the current cotton yield stagnation and instability problems, has lead to the question of whether transgenic cotton lines are more sensitive to abiotic stresses.
The primary objective of this research was to compare the performance of various physiological traits under irrigated and dryland conditions in a temperate, humid environment for a diverse group of cotton genotypes. A secondary objective was to assess whether transgenic cottons demonstrated enhanced sensitivity to abiotic stress by including two transgenic-recurrent parent pairs among the genotypes grown under the two soil moisture regimes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The experimental design was a randomized complete block with a split-plot arrangement of treatments. Five replicates were used from 1998 through 2000, and four replicates were used in 2001. Two soil moisture treatments (irrigated and dryland) were the main plots and the eight genotypes comprised the subplots. The soil moisture treatment main plots and genotype subplots were randomly assigned each year.
Experimental units or plots consisted of four rows 7.62 m long with a 1-m row spacing and were planted on 23 April 1998, 21 April 1999, and 26 April 2000 and 2001. These plots were initially overseeded and then hand-thinned to the desired population density of approximately 97000 plants ha–1. The experimental area was subsoiled each fall after cotton stalk destruction. Each year, the experimental area received 112 kg N ha–1 in a preplant application. Recommended insect and weed control measures were employed throughout each growing season as needed.
Two soil moisture treatments (irrigated and dryland) were used in the study. In 1998, 1999, and 2000, the irrigated plots received four furrow irrigations for a total of 10.16 cm each year. Only three furrow irrigations totaling 7.62 cm were applied in 2001. Tensiometers were used to monitor soil moisture content at a 30-cm depth, with irrigations triggered when readings reached 40 to 50 centibars. However, this irrigation schedule often had to be adjusted (either accelerated or delayed) to accommodate required insecticide spraying and any resulting restricted reentry interval. To enhance the degree of moisture deficit stress occurring in the dryland treatment, rainfall was prevented from entering the soil by covering the soil surface between rows with black polyethylene film from mid June until after harvest (Pettigrew, 2004).
Dry matter harvests were taken at 69 and 96 days after planting (DAP) in 1998, at 63 and 98 DAP in 1999, at 55 and 88 DAP in 2000, and at 55 and 90 DAP in 2001. The early harvest date roughly corresponded to a harvest date during the early blooming period, while the later harvest date corresponded to a cutout harvest date. Cutout refers to a period of slowing vegetative growth and flowering because of a strong demand for assimilates by the existing boll load. One of the inner two plots rows was designated for use in the dry matter harvests. On each harvest date, the aboveground portions of plants from 0.3 m of row were harvested and separated into leaves, stems and petioles, squares, and blooms and bolls. Leaf area was determined with a LI-3100 leaf area meter (LI-COR, Lincoln, NE1), and main-stem nodes were counted. Samples were dried for at least 48 h at 60°C, and dry weights were recorded.
The percentage of photosynthetic photon flux density (PPFD) intercepted by the canopies was determined with a LI 190SB point quantum sensor (LI-COR) positioned above the canopy and a 1-m-long LI 191SB line quantum sensor placed on the ground perpendicular to and centered on the row. Two measurements were taken per plot, and the mean of those two measurements was used for statistical analyses. These measurements were taken under clear skies at 68 and 90 DAP in 1998; 97 DAP in 1999; 68 and 90 DAP in 2000; and 53 and 89 DAP in 2001.
Canopy temperature measurements were taken under clear skies during the afternoon at 75 and 103 DAP in 1998; 70 and 98 DAP in 2000; and 71 and 98 DAP in 2001 by a Telatemp Model AG-42 infrared thermometer (Telatemp Corp., Fullerton, CA). This instrument recorded both canopy surface temperature and the difference between canopy surface temperature and ambient air temperature. Two instantaneous measurements were taken per plot, and the mean of those two measurements were used for statistical analyses.
Water relations data were collected at approximately 1330 h CDT on 96 to 100 DAP in 1999, 89 to 93 DAP in 2000, and 88 to 95 DAP in 2001. Components of leaf water potential (l) for the youngest fully expanded leaf per plant (fourth or fifth leaf from the top on the plant) were determined for leaves from three plants per plot with leaf cutter thermocouple psychrometers (JRD Merrill Specialty Equipment, Logan, UT). After rapidly cutting and inserting the leaf disk into the chamber, the samples were equilibrated for 3 h in a 30°C water bath and then the l was measured. At least four l readings were taken on each leaf disk after the period of equilibration. Stable readings from the three psychrometers per plot were averaged together for subsequent statistical analysis. Following l determinations, the samples were frozen overnight in a –20°C freezer, then allowed to reequilibrate for another 3 h in the 30° water bath; then the leaf osmotic potential (
) was determined. Leaf turgor (t) was estimated as the difference between l and .
Leaf CER and other gas exchange parameters were measured on the youngest fully expanded, disease-free, fully sunlit leaves in each plot with a LI-6200 portable photosynthesis system (LI-COR) with a 1-L chamber. All measurements were taken between 0900 and 1200 CDT with individual leaves oriented perpendicular to the sun. The PPFD reaching the adaxial leaf surfaces were 
1600 µmol m–2 s–1 on all measurements. Measurements were taken on two leaves per plot with the average of the two leaves used for all statistical analysis.
Chlorophyll variable fluorescence/maximal fluorescence (Fv/Fm) ratios were taken on the same two leaves per plots as used in the CER measurements. In 1998, a CF-1000 Fluorescence Measurement System (P.K. Morgan, Inc., Andover, MA) was used to make the measurements, while in 1999 through 2001, a Hansatech Fluorescence Monitoring System (Hansatech Instruments Ltd., Norfolk, UK). Leaves were allowed to dark adapt for at least 15 min after the CER measurements and before the Chl fluorescence measurements. Fo was determined with a weak, modulated amber light. Fm was determined after a 0.8-s pulse of strong white light (>4000 µmol m–2 s–1 PPFD). In 1999 through 2001, after the dark adapted Chl Fv/Fm measurement, the leaves were exposed and acclimated to 650 µmol m–2 s–1 PPFD at the leaf surface for 90 sec. After this period of acclimation, light adapted Chl fluorescence yields (Fs, steady state fluorescence yield; and F'm, light adapted fluorescence maximum) were measured at 650 µmol m–2 s–1 PPFD. From these light adapted values, the quantum efficiency of photosystem II (
PSII) and electron transport rate were derived. Coupling these light adapted measurements with the dark adapted Fv/Fm measurements allows for determination of the extent of photochemical (qP) and nonphotochemical (qNP) quenching at this level of light adaptation (Schreiber et al., 1986). The average of the two measurements per plot were used for the statistical analyses.
To document alterations in CER and Chl fluorescence behavior at different times during the day, measurements were taken first before solar noon and then again after solar noon on the same day in 2001. The two leaves measured before solar noon were tagged and then measured again after solar noon on the same day. As with the other CER measurements, the PPFD reaching the adaxial leaf surfaces were 1600 µmol m–2 s–1 on all measurements.
Upon completion of the CER and Chl fluorescence, the leaves were collected for specific leaf weight (SLW) and leaf Chl content determinations. One of the leaves had its leaf area determined and dry weight measured (48 h at 60°C) to calculate SLW. Leaf disks were collected from the second leaf for Chl content assays. Chlorophyll was extracted over a 24-h period in darkness at 30°C from two 0.4 cm2 leaf disks per leaf in 950 mL L–1 ethanol. The Chl concentration of the extract was then spectrophotometrically determined according to the methods of Holden (1976).
Statistical analyses were performed by ANOVA (PROC MIXED, SAS Institute, 1996). For traits where year interacted with treatments, or genotypes and environmental effects associated with year were identified, the results were presented by year. When the treatment or genotype differences for a trait were consistent across years, then treatment or genotype means were averaged across years and the year interactions with treatment or genotype were considered a random source of error. When statistically significant interactions were not detected, treatment means were averaged across genotypes and genotype means were averaged across treatments. Means were separated by a protected LSD at the P 
0.05 level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Soil Moisture Effects
The most obvious soil moisture deficit response across time is a reduction in plant stature. Because soil moisture treatments did not interact with genotypes or years, treatment means were averaged across genotypes and years. By the early bloom dry matter harvest, the moisture deficit stress in the dryland plots had not become severe enough to impact any of the growth parameters measured (Table 1). None of the traits differed significantly among soil moisture treatments during this early bloom harvest period. However, by the late bloom, a severe-enough moisture deficit stress had developed to impact most of the growth parameters. Plants in the dryland plots were 16% shorter than the irrigated plants. The taller irrigated plants were caused by the production of 11% more main stem nodes compared with the dryland plants rather than increased internode lengths, because the height-to-node ratio did not differ between soil moisture treatments. These shorter dryland plants also produced 35% less LAI, and thereby reduced overall vegetative growth by 32% compared with the irrigated plants. Although the dryland plants had reduced LAI, the 12% greater SLW of these plants indicate that the leaves may have been thicker or denser than leaves of the irrigated plants. Reproductive growth had not been altered by soil moisture treatment at this stage of growth. However, the similar reproductive weights coupled with reduced vegetative growth of the dryland plants led to a 30% greater harvest index for the dryland plants at this growth stage.
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Water relations of the leaves as measured during the late bloom period were altered by the soil moisture treatments. Afternoon leaf water potentials were 36% more negative in leaves of the dryland plants compared with leaves of the irrigated plants (Table 2). Because the moisture deficit stress was slow to develop, the leaves of the dryland plants were able to osmotically adjust to the developing moisture deficit stress. This osmotic adjustment meant that while the dryland plants had a 30% more negative leaf osmotic potential, they were able to maintain similar leaf turgor as leaves from the irrigated plants. This reduced osmotic potential caused the reduced leaf water potential of the dryland plants at this time, not a loss of leaf turgor. Similar osmotic adjustment has been reported for cotton by Turner et al. (1986) and Nepomuceno et al. (1998).
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Averaged across years, morning CER measurements were 6% greater for leaves from the dryland plants compared with irrigated leaves (Table 3). This result is in contrast to the many reports of lower leaf photosynthesis under moisture deficit conditions (McMichael and Hesketh, 1982; Marani et al., 1985; Turner et al., 1986; Genty et al., 1987; Ephrath et al., 1990; Faver et al., 1996). This higher photosynthesis seen in dryland leaves was not accompanied by higher stomatal conductance, and therefore produced a tendency for lower internal CO2 concentrations and for the water use efficiency to be higher with dryland plants. While the maximum quantum efficiency of PSII photochemistry, as measured by the dark adapted Fv/Fm, did not vary between soil moisture treatments, the light adapted PSII quantum efficiency (adapted at 650 µmol m–2 s–1 PPFD) was 9% greater in the dryland leaves compared with the irrigated leaves and allowed for a 9% greater electron transport rate in the dryland leaves (Table 4). While qP was similar between soil moisture treatments, the nonphotochemical quenching was 6% lower in leaves from the dryland plants. Photochemical quenching is caused by the oxidation of the primary electron acceptor (QA) of PSII; in the light this is caused by electron transport through photosystem I (PSI). Nonphotochemical quenching can be caused by (i) the intrathylakoid acidification during light-driven proton translocation across the membrane; (ii) increased distribution of excitation energy to weakly fluorescent PSI at the expense of PSII excitation, regulated by phosphorylation of LHC II; and (iii) photoinhibition of photosynthesis (Krause and Weis, 1991). These fluorescent quenching results contrast with the findings of Genty et al. (1987), who reported that qP was depressed by drought stress but that nonphotochemical quenching was relatively unaffected for pot-grown cotton plants.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Although many previous studies have documented reduced photosynthesis associated with moisture deficit stress (McMichael and Hesketh, 1982; Marani et al., 1985; Turner et al., 1986; Genty et al., 1987; Ephrath et al., 1990; Faver et al., 1996) and the further depression of photosynthesis as the day progressed into the afternoon (Turner et al., 1986; Puech-Suanzes et al., 1989; Ephrath et al., 1990; Ephrath et al., 1993), this study conducted under humid environmental conditions is the first to document a moisture-deficit stress-induced elevated photosynthesis in the morning before plummeting to lower photosynthetic rates in the afternoon compared with irrigated plants (Table 5). This study also documented that while the dark adapted Chl, fluorescence Fv/Fm (maximum PSII quantum efficiency) was not altered by the soil moisture treatments, the light adapted PSII quantum efficiency and photosynthetic electron transport of the dryland leaves were greater during the morning before falling below levels exhibited by the irrigated leaves in the afternoon. Data from this research indicate that both stomatal and nonstomatal factors contributed to this afternoon photosynthetic decline in the dryland leaves.
Smaller leaves with occasionally greater SLW for the dryland plants (Table 6) leads to speculation of a higher concentration of photosynthetic apparatus per unit leaf area for the dryland plants. This speculation is reinforced by the 19% greater Chl content of the dryland plant leaves. These deep Mississippi Delta soils allowed the moisture deficit stress to be slow-developing for the dryland plants. The capacity of these soils to recharge the plant's hydraulic network overnight also allowed the dryland plants to take advantage of this potentially higher photosynthetic apparatus density per unit leaf area, and photosynthesize at a higher rate during the morning before the decreasing leaf water potential became the dominant influence and began shutting down components of the photosynthetic process. Soils under more severe moisture deficit conditions, as in arid environments, or without as large a recharge capacity, may not sufficiently recharge the plant to allow for the higher morning photosynthetic potential to express itself.
The primary genotypic variation seen in the physiological traits measurements was the elevated CER observed with the two okra leaftype lines used in this study compared with the normal leaftype lines. These results were similar to the previously reported higher okra leaftype CER rates in a different genetic background (Pettigrew et al., 1993). The 14% greater light adapted PSII quantum efficiency and electron transport rate, coupled with the 11% reduction in nonphotochemical quenching is new information regarding genotypic variation in photosynthetic components and further explains the higher photosynthetic rates per unit leaf area observed with okra leaftype genotypes. Few, if any, differences were detected for any of the physiological traits measured between the transgenic lines and their conventional recurrent parent lines. The transformation events that introduced the Bt and glyphosate resistance genes did not disrupt the photosynthetic response to developing soil moisture deficits. It appears that the transgenic genotypes did not respond any differently to moisture stress than did their conventional recurrent parent lines.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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NOTES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication November 10, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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l), leaf osmotic potential (