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Photosynthesis and seed production under water-deficit conditions in transgenic tobacco plants that overexpress an Arabidopsis ascorbate peroxidase gene. (Cell Biology & Molecular Genetics).

ENVIRONMENTAL STRESSES such as drought, salinity, heat, chilling, and freezing temperatures are the major limiting factors in crop productivity. Many of these stresses have been associated with the increased production of ROS that include superoxide, [H.sub.2][O.sub.2], and hydroxyl radicals (Allen 1995; Holmberg and Bulow, 1998). Because these ROS are highly reactive and can damage membrane lipids, proteins, chlorophyll, and nucleic acids, their overproduction in cells creates a condition known as oxidative stress, leading to significant agricultural losses (Halliwell, 1982; Scandalios, 1993; Hendry and Crawford, 1994). Plants have developed efficient antioxidant defense systems to cope with ROS (Foyer et al., 1994; Allen 1995). One defense mechanism is the synthesis of antioxidant compounds such as ascorbate, glutathione, [alpha]-tocopherol, or carotenoids to react directly with ROS. A second mechanism is to increase or activate existing antioxidant enzymes, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), and catalase (Foyer et al., 1994; Allen, 1995). Expression of these antioxidant enzymes is induced under oxidative stress conditions, and the elevated activities of these enzymes correlate with increased stress tolerance (Mittler and Zilinskas, 1994; Pinhero et al., 1997). Because some stress-tolerant plants have higher antioxidant enzyme activities and because stress tolerance can result from pretreatment with sublethal stress conditions (Pinhero et al., 1997; Holmberg and Billow, 1998), antioxidant enzymes appear to be critical components in oxidative stress defense mechanisms. Plant stress tolerance may, therefore, be improved by the enhancement of in vivo levels of antioxidant enzymes (Foyer et al., 1994; Allen, 1995).

In the last several years, transgenic plants that overexpress genes for enzymes involved in scavenging ROS have been constructed to test the above hypothesis. The results are inconsistent, which may be due to differences in the ability of the antioxidant gene products to function properly, the physiological target being studied, the severity of the stresses imposed, and/or the plant systems used in those transgenic studies (Rennenberg and Polle, 1994; Allen, 1995). For example, overexpression of SOD genes in plants increased tolerance against herbicide treatment (Allen et al., 1997; Breusegem et al., 1999), water deficit (McKersie et al., 1996), low temperature (McKersie et al., 1993; Sen Gupta et al., 1993a; Breusegem et al., 1999; Payton et al., 2001), and ozone treatment (Van Camp et al., 1994). However, in other studies, SOD overexpression did not provide substantial protection against oxidative stress treatments (Tepperman and Dunsmuir, 1990; Pitcher et al., 1991; Payton et al., 1997). Overexpression of glutathione S-transferase or glutathione peroxidases in tobacco increased tolerance to chilling temperature and salt for germinating tobacco seedlings (Roxas et al., 1997). Overexpression of GR increased antioxidant capacity and resistance to photoinhibition in poplar (Populus alba L.; Foyer et al., 1995) and cotton (Gossypium barbadense L.; Payton et al., 2001). Transgenic tobacco plants that overexpress a cytosolic APX or chloroplast-targeted APX showed increased resistance to methyl viologen and improved protection of photosynthesis under chilling conditions (Pitcher et al., 1994; Allen et al., 1997). However, cytosolic APX targeted to chloroplasts did not provide protection against ozone treatment (Torsethaugen et al., 1997). Nevertheless, it appears that antioxidant genes may provide some degree of protection against environmental stresses under certain conditions.

Recently, we observed increased protection against aminotriazole treatment in tobacco plants that express the Arabidopsis APX3 gene (Wang et al., 1999). Aminotriazole is an herbicide that irreversibly inactivates catalase activity in plants and therefore leads to reduced scavenging of [H.sub.2][O.sub.2] in plant cells. This finding indicates that overexpression of the Arabidopsis APX3 in tobacco may have partially compensated for the loss of [H.sub.2][O.sub.2]--scavenging capacity resulting from catalase inhibition during aminotriazole treatment (Wang et al., 1999). However, protection of these plants from aminotriazole says little about the role of APX3 overexpression in improving tolerance to natural environmental stresses. To further understand the relationship between the overexpression of a foreign antioxidant gene to stress tolerance and crop yield, we explored whether overexpression of APX3 in tobacco could enhance the protection of the photosynthetic apparatus during water deficit and improve seed production. We determined that APX3-overexpressing tobacco plants produced more fruits and a greater seed mass than control plants after repeated cycles of water-deficit imposition. We also found that transgenic plants maintained higher photosynthetic rates than control plants under moderate water-deficit conditions.

MATERIALS AND METHODS

Plant Materials

Two independently-transformed, homozygous tobacco lines B3 and Y3 of T4 generation and control plants (N. tabacum cv. Xanthi) were used in the experiments (Wang et al., 1999). Control plants were segregated from B3 primary transformants at the T2 generation and did not have the APX3 transgene construct. Because there were no discernible differences between these control plants and wild-type plants, the control plants were labeled as Xnn equivalent to wild-type plants (N. tabacum cv. Xanthi). Seeds were sterilized with 10% (by volume) bleach for 10 min, washed a few times with sterile water, and planted on MS medium (Murashige and Skoog, 1962), then cultured under white fluorescent light at 28[degrees]C for 10 d. Young seedlings were transferred to soil mix (Ball Growing on Mix, Ball Seed Co., West Chicago, IL) in quarter-gallon pots with 4 plants per pot and grown under the same conditions. After 10 more days, seedlings were transferred to 1-gallon pots in the greenhouse with one plant per pot. More than 200 plants were transferred to the greenhouse for each line (B3, Y3, and Xnn), and they were watered three times a day and fertilized once a week with Hoagland's solution until plants reached about 40 cm in height. Plants with similar height were chosen for experiments, and the fifth or sixth fully developed leaf of each plant was used for physiological measurements.

Water-Deficit Treatments

All plants, transgenic lines and controls, were divided into two sets: one set was used for the water-deficit treatment, the other set was used as the well-watered control. Growth conditions were the same for both sets of plants except for the water condition. To impose water deficit periodically, 50 d-old plants (of equal size) were subjected to 10 repeated cycles of water-deficit treatments by watering plants with Hoagland's solution once a week until mature seeds were harvested. Fruit number and seed weight per plant were recorded after harvesting. To impose water deficit gradually, replenishment of the daily water lost from each pot was progressively restricted for plants grown in the greenhouse at a temperature of 30 [+ or -] 2[degrees]C. The treatment schedule consisted of 3 d at 100% water replenishment (D1), followed by 3 d during which treated plants received only 75% (D2) of the water needed to fully replenish control plants at the same developmental stage. This stage was followed by 3 d at 50% water replenishment (D3), 3 d at 25% water replenishment (D4), and finally, 3 d with no added water (D5). Physiological parameters, including aboveground biomass, leaf area, relative water content (RWC), leaf water potential (LWP), and gas exchange were analyzed on the third day of each stage of the water-deficit treatment for both water-deficit and control plants. After water was withheld from plants for 5 d (i.e., two more days after measurements were performed for D5 stage), the plants were fully rewatered in the morning. Gas-exchange parameters were then measured after 5 h (R0) and 24 h (R1).

Measurement of Parameters

Eight plants per line were analyzed at each stage of water deficit. Among the eight plants, five were repeatedly used throughout the entire treatment cycle for gas-exchange measurement. The other three plants were replaced at each stage with plants exposed to the same water treatment conditions because they were harvested for measurement of biomass, LWP, RWC, and total leaf area. The LWP values for the fifth or sixth leaf of each plant were measured immediately in the greenhouse by using an Scholander pressure chamber (Soil Moisture Equipment Corp., Santa Barbara, CA). The RWC was calculated as the ratio of (FW-DW)/(TW-DW) x 100, where FW was leaf fresh weight, DW was leaf dry weight, and TW was leaf turgid weight. Total leaf area was measured with a leaf area meter (Model LI-3100, Li-Cor Inc., Lincoln, NE). Leaf gas-exchange parameters were measured using a portable photosynthesis system (Model LI6400, Li-Cor Inc.). For plants recovering from water deficit after watering, only leaf gas exchange parameters and LWP were measured on five plants per treatment.

Statistical Analyses

Plants were randomly arranged in the green house, and each plant was treated as a replication. Effects of water-deficit treatment, plant lines, and their interaction on LWP, RWC, aboveground biomass, total leaf area, and gas-exchange parameters were examined by two-way ANOVA on treatment means using a general linear model (Data Description Inc., Ithaca, NY). A one-way ANOVA was used to test for the effect of plant lines on seed mass and fruit number per plant after repeated cycles of water-deficit treatments. A LSD multiple comparison test was used to determine if means of the dependent variables were significantly different at the 0.05 probability level.

RESULTS

APX3-Expressing Plants Produced Greater Seed Mass Than Control Plants under Cyclic Water Stress

We previously characterized several lines of transgenic tobacco plants that overexpress the Arabidopsis APX3 gene and show increased tolerance against aminotriazole treatment (Wang et al., 1999). In the present study, the characteristics of two highly expressing lines, B3 and Y3, were analyzed under water stress conditions. No significant differences in growth and development or seed yield were observed between APX3-expressing transgenic lines and control plants under well-watered conditions. However, seed production was dramatically reduced in control plants after periodic water-deficit treatment, while the APX3-expressing lines Y3 and B3 produced 72 and 87% more seed mass per plant than control plants, respectively (Fig. 1A). The higher seed production of APX3-expressing plants compared with control plants was primarily due to an increase in fruit number per plant (Fig. 1B), while there were no significant differences in the seed mass per fruit between transgenic plants and control plants (data not shown).

[FIGURE 1 OMITTED]

Comparison of APX3-Expressing and Control Plants during Water Deficit

To better understand the potential physiological parameters responsible for the apparent increase in seed yield in APX3-expressing plants, experiments were performed in which water deficit was imposed gradually by restricting the amount of water given to each plant. Thus, the physiological parameters could be measured at different degrees of water deficit stress. The means of LWP and RWC for control plants and APX3-expressing plants maintained under well-watered conditions were not significantly different and changed very little during the experimental period (data not shown). The LWP and RWC for both genotypes remained relatively high before the D4 stage (25% replenishment of water lost; Table 1). High LWP values found at the end of the D3 stage were most likely due to reductions in [g.sub.s] during the cloudy day when the data were obtained (Table 2). In fact, it was not until the D4 stage that water deficit relative to control water status occurred (Fig. 2A). It was at this stage that substantial negative effects on gas-exchange parameters (Fig. 2B,C) and aboveground biomass and leaf area (Fig. 3) were observed. Also, it was the stage at which wilting first occurred in the afternoon. Although a significant reduction in LWP occurred when water supply was reduced to 25% of that lost (D4), RWC for both genotypes remained close to the values exhibited during the D1 stage, suggesting that osmotic regulation was occurring. Completely withholding water (D5) caused a major decline in both LWP and RWC (Table 1), leading to considerable physiological stress relative to well-watered controls (Fig. 2B,C). However, no significant differences in LWP and RWC were observed between transgenic and control plants throughout the imposition of water-deficit conditions (Table 1).

[FIGURES 2-3 OMITTED]

In contrast to the continuous growth of well-watered plants, the total leaf area of water-deficient plants stopped increasing by the D4 stage, whereas the growth of aboveground biomass did not stop until the D5 stage (Table 1 and Fig. 3). There were no significant differences in total leaf area and aboveground biomass between transgenic and control plants under the water-deficit conditions imposed during this experiment (Tables 1, 3).

Transgenic Plants Maintained Higher A, [g.sub.s], and [C.sub.i]/[C.sub.a] than Wild-Type Plants Did at Moderately Low LWP

The photosynthetic rates (A) of both transgenic and control plants were similar ([approximately equal to] 20 [micro]mol [m.sup.-2] [s.sup.-1]) under well-watered conditions. Photosynthetic rates of water-deficient plants were similar to those of watered plants during the 75% (D2) and 50% (D3) water-replenishment treatments (Table 2, Fig. 2B), consistent with the similar LWP for these plants (Fig. 2A). Photosynthetic rates decreased dramatically for both transgenic and wild-type plants three days after watering was reduced to 25% replenishment (D4) (Table 2, Fig. 2B). Interestingly, this dramatic decrease in A occurred when LWP fell but RWC remained high (Table 1). However, the reduction in A is not surprising given the nearly six-fold reduction in [g.sub.s] (Table 2). The reduction in A was significantly less for both lines of transgenic plants compared with wild-type plants (Table 2, Fig. 2B), as transgenic plants maintained about a 30% higher A than wild-type plants. These higher values of A in transgenic plants were associated with greater [g.sub.s] and [C.sub.i]/[C.sub.a] ratios, but lower water use efficiencies (A/[g.sub.s]; Table 2). The interaction between plant lines and water treatment was highly significant for A, A/[g.sub.s], and [C.sub.i]/[C.sub.a], but not for [g.sub.s] (Table 3).

During severe water-deficit stress (0% of water lost replenished, D5), A and [g.sub.s] were decreased to near zero, while [C.sub.i]/[C.sub.a] ratios increased in all plants, with no significant differences observed between transgenic and wild-type plants (Table 2, Fig. 2). Photosynthesis for both transgenic and wild-type plants began to recover within 5 h after rewatering (Table 2), but A of transgenic plants recovered more quickly than that of control plants. Five hours after rewatering (R0), A for transgenic plants was 30% of that of well-watered plants, while A for control plants was <20% of well-watered plants (Table 2, Fig. 2B). Consequently, transgenic plants had values of A that were [approximatley equal to] 40% higher than A for control plants during early recovery from the water-deficit treatment. Associated with the higher A for the transgenic plants was a significantly higher [g.sub.s] and [C.sub.i]/[C.sub.a], but A/[g.sub.s] was less for transgenic plants than for wild-type plants. Photosynthesis of all water-deficient plants recovered to [approximatley equal to] 70 to 80% of that for the well-watered plants the next day (R1), with no significant genotypic differences (Table 2).

DISCUSSION

We previously found that overexpression of the Arabidopsis APX3 gene in tobacco increased protection against aminotrialzole treatment that inhibits [H.sub.2][O.sub.2] scavenging via catalase (Wang et al., 1999). However, this finding did not address the ability of enhanced APX3 activity to protect photosynthesis during natural environmental stress. In the present study, these transgenic tobacco plants produced greater seed mass than wild-type plants following repeated cycles of water deficit. Since there was no genotypic difference in seed mass per fruit, the greater seed mass for transgenic plants was related to the greater number of fruits that were produced by APX3-expressing plants in comparison with control plants. The imposition of a gradually developing water deficit affected leaf water status to the same degree for transgenic and wild-type plants. Thus, one might speculate that it was the maintenance of higher A in transgenic plants than wild-type plants during a portion of the water-deficit period and during recovery that was a factor in regulating seed yield after repeated cycle of water deficit.

The overproduction of chloroplastic SOD, APX, and GR in tobacco, cotton, and poplar has resulted in an improvement in the protection of photosynthesis during chilling stress (Sen Gupta et al., 1993a,b; Foyer et al., 1995; Allen et al., 1997; Payton et al., 2001). At least for cotton, the mechanism that results in a greater initial recovery of A following the stress appears to involve an indirect protection of photosystem II activity (Kornyeyev et al., 2001). It has been shown that chloroplasts are very sensitive to extrachloroplastic [H.sub.2][O.sub.2] that can inhibit C[O.sub.2] fixation by 50% at 10 to 100 [micro]M [H.sub.2][O.sub.2] (Kaiser, 1976; Asada, 1992). These extrachloroplastic [H.sub.2][O.sub.2] concentration levels could be reached through increased photorespiration metabolism under water-deficit conditions when stomatal closure reduces the C[O.sub.2] availability (Boveris et al., 1972; Wingler et al., 1999). Although catalase is abundant in the leaf peroxisomes and glyoxisomes (Scandalios, 1994), its high kM value of >1 M makes it an inefficient scavenger of [H.sub.2][O.sub.2] at low concentrations (Asada, 1992). Ascorbate peroxidase, however, has a much higher affinity for [H.sub.2][O.sub.2], with kM values ranging from 20 to 74 [micro]M (Mittler and Zilinskas 1991; Ishikawa et al., 1998).

Our gas-exchange data did not support the idea that overexpression of APX3 results in enhanced protection of photosynthetic processes. During the imposition of water deficit at Stage D4 and after 5 h into the recovery from water deficit (R0), higher A for transgenic plants was associated with higher [g.sub.s] and [C.sub.i]/[C.sub.a]. In addition, water use efficiency (A/[g.sub.s]) was not greater for the transgenic plants than for wild-type plants. These data were strongly indicative that differences in A were controlled by differences in [g.sub.s]. In fact, during the D4 and R0 stages, A/[g.sub.s] for transgenic plants was actually much less than that for wild-type plants. This suggests that A is more strongly responsive to changes in [g.sub.s] in transgenic plants compared with control plants. There was no indication that A was protected by overexpression of APX3 in tobacco leaf cells. We conclude that in comparison with control plants, the higher values of A observed in transgenic plants at stages D4 and R0 were due to the maintenance of higher [g.sub.s], which increased [C.sub.i] and therefore C[O.sub.2] available for photosynthesis.

We offer one hypothesis for why plants overexpressing APX3 exhibited higher [g.sub.s] than control plants at certain stages in the experiment. Since the overexpression of APX3 was constitutive, enhanced APX3 activity may have altered the water-deficit-sensing mechanism of the guard cells. It was recently reported that [H.sub.2][O.sub.2] plays a role in the abscisic acid (ABA)-initiated signal transduction pathway that leads to stomatal closure (Pei et al., 2000). APX3 may have interfered with a rise in [H.sub.2][O.sub.2] levels that transduce the ABA signal in the guard cells. It would be of value to determine whether the overproduction of other isoforms of APX or catalase in leaf cells alters stomatal response to water deficit.

Abbreviations: A, rate of C[O.sub.2]; APX, ascorbate peroxidase; APX3, ascorbate peroxidase 3; [C.sub.a], atmospheric C[O.sub.2]; [C.sub.i], internal C[O.sub.2]; GR, glutathione reductase; [g.sub.s], stomatal conductance; LWP, leaf water potential; ROS, reactive oxygen species; RWC, relative water content; SOD, superoxide dismutase.
Table 1. Leaf water potential (LWP), relative water content (RWC),
total leaf area, and aboveground biomass of APX3-overexpression
plants (Y3 and B3) and control plants (Xnn) under water-deficit
treatments.

                                  LWP                  RWC
 Drought
treatment
([dagger])   Plant line    Mean [+ or -] SD     Mean [+ or -] SD

                                  MPa                   %

D1           Y3           -0.67 [+ or -] 0.06   83.8 [+ or -] 2.0
             B3           -0.70 [+ or -] 0.00   84.7 [+ or -] 0.4
             Xnn          -0.73 [+ or -] 0.06   83.9 [+ or -] 2.6
D2           Y3           -0.80 [+ or -] 0.10   82.4 [+ or -] 2.2
             B3           -0.77 [+ or -] 0.06   85.4 [+ or -] 0.4
             Xnn          -0.77 [+ or -] 0.06   84.6 [+ or -] 0.9
D3           Y3           -0.57 [+ or -] 0.06   86.3 [+ or -] 1.1
             B3           -0.57 [+ or -] 0.06   88.2 [+ or -] 0.8
             Xnn          -0.53 [+ or -] 0.06   87.7 [+ or -] 0.5
D4           Y3           -1.30 [+ or -] 0.10   79.6 [+ or -] 2.0
             B3           -1.30 [+ or -] 0.10   80.1 [+ or -] 4.5
             Xnn          -1.27 [+ or -] 0.06   81.1 [+ or -] 3.8
D5           Y3           -1.70 [+ or -] 0.10   69.5 [+ or -] 2.5
             B3           -1.73 [+ or -] 0.12   68.3 [+ or -] 1.4
             Xnn          -1.67 [+ or -] 0.06   72.4 [+ or -] 6.9

                                                Aboveground
                           Total leaf area        biomass
 Drought
treatment
([dagger])   Plant line   Mean [+ or -] SD    Mean [+ or -] SD

                             [cm.sup.2]

D1           Y3            603 [+ or -] 85    1.9 [+ or -] 0.7
             B3            620 [+ or -] 209   1.8 [+ or -] 0.9
             Xnn           607 [+ or -] 47    1.8 [+ or -] 0.3
D2           Y3            782 [+ or -] 61    2.3 [+ or -] 0.3
             B3            978 [+ or -] 62    2.9 [+ or -] 0.3
             Xnn           905 [+ or -] 180   2.9 [+ or -] 0.9
D3           Y3           1020 [+ or -] 69    4.0 [+ or -] 0.4
             B3           1008 [+ or -] 200   3.2 [+ or -] 0.9
             Xnn          1096 [+ or -] 79    4.0 [+ or -] 0.2
D4           Y3           1032 [+ or -] 78    4.8 [+ or -] 0.2
             B3           1154 [+ or -] 18    5.0 [+ or -] 1.0
             Xnn          1092 [+ or -] 84    5.5 [+ or -] 0.7
D5           Y3            955 [+ or -] 51    4.8 [+ or -] 0.4
             B3           1023 [+ or -] 205   5.4 [+ or -] 0.7
             Xnn           953 [+ or -] 94    4.8 [+ or -] 0.6

([dagger]) D1, D2, D3, D4, and D5 indicate plants watered with
100, 75, 50, 25, and 0% of water needed to fully replenish control
plants, respectively. Every treatment lasted for 3 d and all
parameters were measured on the third day. Values are mean
[+ or -] SD (n = 3).

Table 2. Photosynthesis (A), stomatal conductance ([g.sub.s]),
A/[g.sub.s], and [C.sub.i]/[C.sub.a], (internal C[O.sub.2]/
atmospheric C[O.sub.2] of APX3-overexpression plants (Y3 and B3)
and control plants (Xnn) under different water-deficit treatments.
Values are mean [+ or -] SD (n = 8 for drought treatment,
n = 5 for recovery).

                              Photosynthesis     Stomatal conductance
  Drought
 treatment     Plant line    Mean [+ or -] SD      Mean [+ or -] SD

                               [micro]mol
                                [m.sup.-2]         mmol [m.sup.-2]
                                [s.sup.-1]            [s.sup.-1]

D1             Y3           20.1 [+ or -] 2.0    726.3 [+ or -] 118.3
  ([dagger])   B3           20.5 [+ or -] 1.3    778.5 [+ or -] 109.9
               Xnn          19.3 [+ or -] 1.6    753.1 [+ or -] 181.2
D2             Y3           21.7 [+ or -] 0.8    936.2 [+ or -] 125.6
               B3           21.4 [+ or -] 1.3    955.6 [+ or -] 88.5
               Xnn          21.7 [+ or -] 1.3    955.8 [+ or -] 166.2
D3             Y3           19.5 [+ or -] 1.0    595.6 [+ or -] 74.5
               B3           19.2 [+ or -] 2.3    604.9 [+ or -] 169.8
               Xnn          19.8 [+ or -] 2.0    602.2 [+ or -] 174.3
D4             Y3            9.6 [+ or -] 1.6A   112.0 [+ or -] 62.4a
                                 ([double              ([section])
                                 dagger])
               B3            9.9 [+ or -] 1.3A   125.0 [+ or -] 87.1a
               Xnn           7.2 [+ or -] 1.5B    68.4 [+ or -] 42.0b
D5             Y3            0.3 [+ or -] 0.3      7.9 [+ or -] 7.1
               B3            0.8 [+ or -] 0.8      8.4 [+ or -] 3.9
               Xnn           0.7 [+ or -] 0.5     10.1 [+ or -] 4.7
R0             Y3            5.4 [+ or -] 0.5A   43.48 [+ or -] 4.9a
               B3            5.5 [+ or -] 1.1A    39.9 [+ or -] 12.7a
               Xnn           3.8 [+ or -] 0.7B    21.1 [+ or -] 4.5b
RI             Y3           13.2 [+ or -] 1.6    154.7 [+ or -] 34.5
               B3           13.8 [+ or -] 1.3    199.7 [+ or -] 34.0
               Xnn          13.5 [+ or -] 1.0    214.7 [+ or -] 46.7

                                A/[g.sub.s]             Ci/Ca
  Drought
 treatment     Plant line     Mean [+ or -] SD      Mean [+ or -] SD

D1             Y3            28.0 [+ or -] 3.2     0.77 [+ or -] 0.01
  ([dagger])   B3            26.8 [+ or -] 3.7     0.77 [+ or -] 0.02
               Xnn           27.1 [+ or -] 5.9     0.77 [+ or -] 0.02
D2             Y3            23.5 [+ or -] 2.6     0.78 [+ or -] 0.01
               B3            22.5 [+ or -] 1.5     0.79 [+ or -] 0.01
               Xnn           23.3 [+ or -] 3.9     0.78 [+ or -] 0.01
D3             Y3            33.2 [+ or -] 4.5     0.75 [+ or -] 0.02
               B3            33.8 [+ or -] 8.9     0.75 [+ or -] 0.04
               Xnn           35.6 [+ or -] 11.8    0.74 [+ or -] 0.05
D4             Y3            95.0 [+ or -] 19.3A   0.52 [+ or -] 0.07A
               B3            95.7 [+ or -] 28.1A   0.52 [+ or -] 0.11A
               Xnn          114.9 [+ or -] 24.1B   0.45 [+ or -] 0.09B
D5             Y3            39.6 [+ or -] 36.9    0.81 [+ or -] 0.19A
               B3            85.1 [+ or -] 52.6    0.59 [+ or -] 0.22B
               Xnn           74.3 [+ or -] 53.4    0.64 [+ or -] 0.23B
R0             Y3           123.6 [+ or -] 6.9A    0.42 [+ or -] 0.03A
               B3           143.2 [+ or -] 25.7A   0.34 [+ or -] 0.11A
               Xnn          184.5 [+ or -] 23.0B   0.17 [+ or -] 0.10B
RI             Y3            87.0 [+ or -] 9.9     0.54 [+ or -] 0.17
               B3            70.3 [+ or -] 9.8     0.61 [+ or -] 0.04
               Xnn           67.1 [+ or -] 17.0    0.59 [+ or -] 0.05

([dagger]) Water-deficit treatments were the same as in Fig. 2.

([double dagger]) Means with capital letters indicate significant
difference at the 0.05 level.

([section]) Means with lower case letters indicate significant
difference at the 0.10 level.

Table 3. Analysis of variance tests for leaf water potential (LWP),
relative water content (RWC), aboveground biomass, total leaf
area, photosynthetic rates (A), stomatal conductance ([g.sub.s]),
A/[g.sub.s], and [C.sub.i]/[C.sub.a] (internal C[O.sub.2]/
atmospheric C[O.sub.2]) of transgenic (Y3 and B3) and control
plants (Xnn) under water-deficit treatments.

                                           P value

                           Water-deficit
Factor                       treatment     Plant line   Interaction

LWP                           <0.001          0.757        0.923
RWC                           <0.001          0.296        0.829
Aboveground biomass           <0.001          0.536        0.470
Total leaf area               <0.001          0.204        0.842
Photosynthetic rates (A)      <0.001          0.033        0.039
Stomatal conductance          <0.001          0.409        0.942
  ([g.sub.s])
A/[g.sub.s]                   <0.001         <0.001       <0.001
[C.sub.i]/[C.sub.a]           <0.001         <0.001       <0.001


ACKNOWLEDGMENTS

This work was supported by grants from the Texas Advanced Technology program (003644-072-1997 and 003644-0127-1999).

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Juqiang Yan, Jing Wang, David Tissue, A. Scott Holaday, Randy Allen, and Hong Zhang *

Dep. of Biological Sci., Texas Tech Univ., Lubbock, TX 79409, USA. Received 24 Jan. 2002. * Corresponding author (brahz@ttu.edu).
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Author:Yan, Juqiang; Wang, Jing; Tissue, David; Holaday, A. Scott; Allen, Randy; Zhang, Hong
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Date:Jul 1, 2003
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