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CROP PHYSIOLOGY & METABOLISM

Photosynthesis, Carbohydrate Metabolism, and Yield of Phytochrome-B-Overexpressing Potatoes under Different Light Regimes

Inst. of Crop and Grassland Sci., Federal Agric. Res. Centre (FAL), Bundesallee 50, 38116 Braunschweig, Germany

^* Corresponding author (siegfried.schittenhelm{at}fal.de).

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Transgenic potatoes (Solanum tuberosum L.) overexpressing Arabidopsis thaliana (L.) Heynh. phytochrome B (phyB) have been reported to exhibit a substantially modified plant architecture, increased photosynthetic performance, reduced photoinhibition, delayed leaf senescence, and increased tuber yield. A greenhouse and a growth chamber experiment were conducted at Braunschweig, Germany, to elucidate the crop physiological basis for the yield differences between moderately phyB-overexpressing transgenic (Dara-5) and wild-type potato plants. In the greenhouse experiment, Dara-5 leaves showed a 23% greater leaf carbon exchange rate (CER) at light saturation, 32% greater leaf conductance, and 21% longer green leaf area duration (GLAD) than the wild-type plants. The transgenic plants partitioned a considerably greater portion of their biomass to stems and roots, but tuber and total biomass yield did not significantly differ among genotypes. The leaves and stems of the transgenic plants had lower starch and soluble sugar concentrations but consistently higher N concentration than those of the nontransgenic plants. Light response curves showed increasing CER superiority of Dara-5 leaves with increasing photosynthetic photon flux (PPF), suggesting higher productivity of the transgenic plants in high-radiation environments. Therefore, the two genotypes were compared in growth chambers at low, medium, and high light levels of 300, 600, and 900 µmol m^–2 s^–1 PPF. Leaf CER of the transgenic plants reached 123, 115, and 120% of the wild-type plants at low, medium, and high PPF, but only at low PPF did the transgenic plants produce significantly greater (+8%) tuber yield than the nontransgenic plants. It is supposed that enhanced C loss from respiration is responsible for the lack of consistent transgenic yield superiority.

Abbreviations: CER, carbon exchange rate • CER_max, light-saturated carbon exchange rate • Chl, chlorophyll • DAE, days after emergence • GLA, green leaf area • GLAD, green leaf area duration • NIRS, near-infrared reflectance spectroscopy • P_FR, far-red light absorbing form of phytochrome • phyB, phytochrome B • PPF, photosynthetic photon flux • RNA, ribonucleic acid • SLW, specific leaf weight • WSC, water soluble carbohydrates

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
POTATO TUBER YIELD does not appear to have significantly improved during the last few decades. A survey of German federal cultivar tests covering the period from 1952 to 1993 revealed little yield progress for the mid-early and late potato cultivars as compared with other major crop species (Schuster, 1997). Likewise, Douches et al. (1996), in a comparison of U.S. potato cultivars representing four breeding periods from pre-1900 to present found no significant differences in total yield with marketable yield being highest for the 1930 to 1949 breeding period. They explained the lack of yield improvement by earlier maturity of the more modern cultivars, the need of selection for several quality traits, and the narrow genetic base of the cultivated potatoes.

The economic yield of any crop is a function of the amount of light energy absorbed by the green foliage, the efficiency of the foliage to use the energy captured for biomass production, and the partitioning of the crop biomass to the harvested plant part. Because potato has one of the highest harvest indices of major crops and there may be little potential for significant shifts in total biomass accumulation, genotypes with superior net photosynthesis will likely be needed for further yield improvement. Flynn et al. (1998), in a study of a historical series of European potato cultivars, in spite of large genetic variation, found little change in the rate of photosynthesis per unit leaf area. They concluded that breeding for improved yield has not increased the single-leaf photosynthetic rate. Because photosynthesis is one of the most highly integrated and regulated metabolic processes, transgenic approaches to photosynthetic enhancement and yield increase might be more successful than conventional breeding methods. As recently reviewed by Dunwell (2000), the transgenic approaches presently underway include the introduction of genes involved in the C₄ type of photosynthesis into C₃ plants, manipulation of the activity of key photosynthetic enzymes, and the alteration of senescence.

An alternative transgenic approach to improving crop yield consists in altering expression of the phytochrome genes PHYA and PHYB. By means of phytochrome photoreceptors, plants can sense reduction in the ratio of red (R) to far-red (FR) light when they become shaded by their neighbors and this allows them to avoid shading by increasing their stem extension rate (Smith and Whitelam, 1997). Disabling of the shade avoidance response by transgenic overexpression of phytochrome genes might improve economic yields because resources committed to stem growth in dense stands may be reallocated to economically usable plant parts (Smith, 1995) and might also represent an alternative to the use of chemical growth regulators (Zheng et al., 2001). Libenson et al. (2002) observed that enforcing the shade-avoidance reaction by artificially reducing the R/FR light ratio reaching the stems in sunflower (Helianthus annuus L.) increased stem dry weight and decreased seed dry weight. They expected that the reverse, that is, reducing photomorphogenic response through increasing the steady state level of photoreceptors in a transgenic crop, might increase the economic yield. Transgenic overproduction of phyA in tobacco (Nicotiana tabacum L.) showed an enhanced allocation of assimilates to leaves with a concomitant increase in leaf harvest index when grown at high densities in the field (Robson et al., 1996). The authors concluded that this approach could provide significant improvements in the productivity of other crop plants where leaves are the ultimate goal of production. In an attempt to transfer this approach to potato, Thiele et al. (1999) observed that transgenic plants exhibiting moderate (line Dara-5) and strong (line Dara-12) overexpression of Arabidopsis phyB showed pleiotropic effects including semidwarfism, increased single-leaf photosynthesis, reduced sensitivity to high-light stress, delayed leaf senescence, and higher tuber yield. They expected that reduced photoinhibition and decelerated chlorophyll (Chl) breakdown could make these transgenic potato plants potentially more productive in high-radiation environments and areas with long growing seasons. However, the higher tuber yield of the transgenic plants in their study might be simply a maturity effect, because late genotypes with longer leaf area duration intercept more light than early genotypes. Higher potato tuber yield is particularly advantageous when attained within a similar growth period.

This study was undertaken to (i) find out whether the higher tuber yield of phyB-overexpressing transgenic potatoes could be confirmed, (ii) elucidate whether the differences in photosynthetic performance, life span, or both are causal for yield differences among transgenic and nontransgenic plants, and (iii) test the hypothesis that reduced photoinhibition of the transformed plants makes them especially suitable for high-radiation environments.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Genetic Materials and Plant Culture
Two potato genotypes, Dara-5 and ‘Désirée’, were used in a greenhouse and a growth chamber experiment at the Federal Agricultural Research Centre (FAL), Braunschweig, Germany (52°17' N lat, elevation 80 m). Dara-5 is a selection from an Agrobacterium-mediated transformation of Désirée that is overexpressing Arabidopsis phyB (Wagner et al., 1991). Because of their previously observed higher tuber yield (Thiele et al., 1999), the moderately phyB-overexpressing Dara-5 plants were preferred to the strongly overexpressing Dara-12 plants. Dara-5 and wild-type plants were jointly propagated vegetatively before each experiment to assure that seed tubers had a similar physiological state. In the greenhouse and the growth chamber experiment, one seed tuber each was placed into a black polyvinyl-chloride pot containing 2.9 kg of 1:1 (v:v) heat-sterilized mixture of silica sand and silt loam topsoil. The pregerminated and uniformly sized seed tubers had average weights of 20 g in the greenhouse experiment and 35 g in the growth chamber experiment. Because only a few plants were taken at each sequential harvest in the greenhouse experiment, single-sprouted seed tubers were used to avoid sampling effects. Each day the plants were watered to excess in the morning. Commercial fertilizer was added at weekly intervals until the onset of leaf senescence as follows: N = 120, P = 120, K = 170, and Mg = 20 mg per plant wk^–1.

Greenhouse Experiment
Growth Analysis
The experiment was conducted from May to September 2000 in a naturally lit greenhouse with air temperatures maintained near 20:15°C day:night. On the basis of hourly-logged PPF at the weather station located on the research site and the percentage transparency of the greenhouse, mean PPF during daytime in the greenhouse was 220 µmol m^–2 s^–1 during the growth period. The experiment was a randomized complete block design with two replications planted at a density of 15 plants m^–2. To minimize border effects, the plots were surrounded by one row of guard plants of the same genotype. Date of emergence and beginning of flowering (one flower open) were recorded for all experimental plants. At each of 10 sequential harvests, the height (cm) of individual plants was measured as the distance between soil surface and the apex of the main stem. At each sampling date, four randomly selected plants per replication and genotype were separated into green leaves, stems (including petioles and rachises), tubers, roots, and dead plant parts. The tuber number per plant was determined by counting the tubers larger than 1 cm in diameter. Following harvest, the experimental plants and the guard plants were rearranged to the target plant density. The green leaf area (GLA, m² per plant) was determined by camera-based image analysis (Delta-T, Cambridge, UK). Roots were washed free of soil with water and dried with tissue paper. Samples were freeze-dried for 12 h, reweighed to determine sample dry weight (g per plant), ground with a type ZM1 Retsch mill (Haan, Germany) with a 1-mm grid, and stored under N atmosphere until spectral and chemical analyses. Total biomass was calculated by summing the dry weights of all plant tissue types. Harvest index was calculated by dividing tuber dry weight by total biomass dry weight and multiplying by 100. The green leaf area duration (GLAD, m² d) was determined by integrating the area under the GLA vs. time curve across the complete growing season according to the formula

[1]

where n is the number of harvest dates, GLA_i is the green leaf area at each harvest date, and t_i – t_i–1 is the time duration. The specific leaf weight (SLW, g m^–2) was calculated as leaf dry weight per leaf area.

Leaf Gas Exchange Measurements
To ensure constant climatic conditions before and during the gas exchange measurements, the plants were moved to a growth chamber (20°C, 570 µmol m^–2 s^–1 PPF at leaf level), allowed to acclimate for 30 min, and immediately returned to the greenhouse after the measurement. Measurements were taken with a HCM-1000 differential infrared gas analyzer (Walz, Effeltrich, Germany) on the terminal leaflet of the fifth leaf below the apex > 5-cm length. The leaf cuvette, covering a 2.5-cm² portion of the leaf, was maintained at 25°C air temperature, a CO₂ partial pressure outside the leaf (C_a) of 35 Pa, and a near-saturating PPF of 1500 µmol m^–2 s^–1. Constant light and CO₂ supply was provided by a Walz 1050-H lighting unit and a Walz 1030-D CO₂ dosing unit. The gas exchange measurements were performed the day before each sequential harvest and on one additional occasion between the eighth and ninth harvest date from 1000 to 1230 h on each genotype and replication by altering among genotypes. Leaf CER was averaged across a 60-s period after a steady state rate was attained which took 5 min. Light-saturated leaf carbon exchange rate (CER_max, µmol m^–2 s^–1) and total leaf conductance to water vapor (g_tw, mmol m^–2 s^–1) was calculated by the HCM-1000 operating software which follows the method of von Caemmerer and Farquhar (1981). Because water vapor diffusion through the leaf is substantially more limited by the stomata than by the boundary layer, differences in g_tw largely reflect differences in stomatal conductance. Automated light response curves (CER vs. PPF) at C_a = 35 Pa were measured at four growth stages on each of four plants per genotype at PPFs of 0, 20, 50, 120, 250, 500, 1000, and 1800 µmol m^–2 s^–1 in descending order, at the same leaf position used for the CER_max measurements. Before initiating a CER measurement with an averaging time of 60 s, the leaf was exposed to the indicated PPF levels for 9 min, allowing the leaf to reach steady state photosynthesis. Because each CER vs. PPF measurement took 1 h 45 min, diurnal variation in leaf CER was minimized by altering measurements between genotypes on two consecutive days such that Dara-5 and wild-type plants each had one measurement in early and late morning as well as early and late afternoon.

Determination of Whole Plant Water Soluble Carbohydrates, Starch, and Nitrogen
In the greenhouse experiment, the amount of tissue dry matter was insufficient for applying wet chemistry procedures on a single-plant basis as it was intended. Therefore, the near-infrared reflectance spectroscopy (NIRS) was used to predict the concentration of water soluble carbohydrates (WSC), starch, and N (all expressed in mg g^–1 dry wt.). The dried and ground samples of the individual plant tissue types were scanned with a NIR-Systems scanning monochromator (Model 6500, Silver Spring, MD) in the range of 400 to 2500 nm, and log l/reflectance (log l/R) was recorded at 2-nm intervals. The four samples of each tissue type belonging to the same genotype, replication, and harvest date were combined and the corresponding near-infrared spectra were taken. Pooled samples were then analyzed in duplicate for WSC, starch, and N as described in the next paragraph. Spectral prediction equations were developed by regressing data from the chemical analysis against first derivative transformation of log l/R with modified partial least squares (Shenk and Westerhaus, 1991) with internal cross-validation and outlier elimination. Samples whose spectra had standardized Mahalanobis distances >3.0 from the average were considered to be spectral outliers.

For analysis of WSC, a 1-g ground sample was transferred to a 500-mL volumetric flask containing 200 mL of cold distilled water, shaken for 60 min, diluted to volume, and filtered. Proteins were precipitated by transferring 50 mL of the filtrate into a 100-mL volumetric flask, adding 2 mL of Carrez solution I (230 g zinc acetate L^–1 water), mixing, adding 2 mL of Carrez solution II (150 g potassium ferrocyanide L^–1 water), mixing, and diluting to volume with water. Two milliliters of the filtrate were homogenized with 10 mL anthrone reagent solution, heated in a boiling water bath for 20 min, cooled under flowing water for 10 min, and A₆₂₅ was read using a Unicam 4 UV-Vis spectrophotometer (Nicolet Instruments, Offenbach, Germany). Starch analysis was performed enzymatically. To hydrolyze starch to glucose, 125 mg of ground samples were suspended in 9.8 mL of acetate buffer (0.1 mol L^–1) with 100 µL of thermostable Termamyl 60L -amylase (Novo Industri, Bagsvaerd, Denmark), boiled for 1 h in a water bath, cooled to 60°C, incubated with 100 µL of amyloglucosidase (Roche, Mannheim, Germany) for 16 h at 60°C, and then centrifuged for 10 min at 1000 x g. The amount of glucose in the hydrolysate was determined colorimetrically by mixing 10 µL of the supernatant with 1 mL phosphate buffered (pH 7.3) glucose enzymatic color liquid (Biotrol Diagnostic, Chennevières-lès-Louvres, France), incubating for 10 min at 37°C, and measuring A₅₁₀ with a Unicam 4 UV-Vis spectrophotometer. Starch concentrations were calculated from a calibration curve based on the absorbance of standard glucose solutions correspondingly subjected to the colorimetric assay. The N concentration of each plant organ was determined by Kjeldahl digestion with a Gerhardt (Bonn, Germany) Vapodest 5 autoanalyzer. Total amounts of WSC, starch, and N in each tissue type (g per plant) were calculated by multiplying tissue concentrations by tissue dry weights.

Growth Chamber Experiment
The controlled light experiment was conducted in three walk-in growth chambers (BBC York, Mannheim, Germany) from November 2001 to March 2002. The chambers were each equipped with eight 400-W Philips SON-T AGRO and Osram Powerstar HQI-E lamps mounted on a height-adjustable bank and programmed for a 12-h photoperiod at 20°C and a 12-h dark period at 15°C. During a 1-wk acclimation period, all chambers were set at 200 µmol m^–2 s^–1 PPF. Thereafter and until maturity, average PPFs of 300, 600, and 900 µmol m^–2 s^–1 (corresponding to integrated daily PPF levels of 12.9, 25.9, and 38.9 mol m^–2 d^–1) were maintained at the top of the canopy. The different PPF levels were attained by varying the number of operating lamps and the distance between light bank and plant canopy. The experimental design within each chamber was a randomized complete block with two replications. Each plot consisted of 16 plants covering 1 m² on the bench. Of the total 16 plants, two plants were taken to destructively determine GLA when about the maximum haulm mass was attained at 59 d after emergence (DAE). A further six plants were used for repeated physiological and anatomical leaf analyses, and yet another eight plants served for determining dry matter yield at maturity. Of these eight plants, the same four plants were also used for weekly gas exchange measurements performed between 1000 and 1230 h, with measurements altering between genotypes. Measurements were conducted at ambient light conditions in the respective growth chamber. When the harvest-plants had completely lost their green color, they were separated into haulm, tubers, and roots. The dry weight of each tissue was recorded following drying to constant weight at 105°C.

Determination of Chlorophyll, Carbohydrates, Protein, Rubisco Activity, and Anatomy of Single Leaves
After completion of the gas exchange measurements in the greenhouse and growth chamber experiments, the first pair of subterminal leaflets adjacent to the terminal leaflet was detached, quick-frozen in liquid N, after removal of the major veins powdered at –180°C in a mortar, and stored at –80°C. In addition, two 0.8-cm² leaf discs were taken from the terminal leaflet. One leaf disc was crushed with a minipestle in an Eppendorf vial containing liquid N. Leaf pigments were extracted with 80% (v:v) acetone, absorbance measured with an Uvicon 933 spectrophotometer (Goebel Elektro GmbH, Neufahrn, Germany) at 647 nm (Chl a) and 663 nm (Chl b), and the concentration of leaf pigment (µg cm^–2) calculated using the formula given by Lichtenthaler (1987). The other leaf disc, after fixation with formaldehyde-glutaraldehyde (Karnovsky, 1965), dehydration in an acetone series, critical point drying, and gold sputtering, was photographed through a scanning electron microscope (Model ISI-60, International Scientific Instruments, München, Germany) at 200x magnification. Stomatal density (no. mm^–2; adaxial plus abaxial leaf surface), palisade cell length (µm), and total leaf thickness (µm) were determined from photographic prints. Leaf carbohydrates were extracted from the ground leaf samples with a 4:1 (v:v) methanol-water solution (de Bruijn et al., 1999). Glucose, fructose, and sucrose concentrations (mg g^–1 dry wt.) were determined by HPLC as described in Schittenhelm (1999). After removal of soluble carbohydrates, starch in the methanol-extracted residue was sequentially hydrolyzed to glucose using thermostable bacterial -amylase and fungal amyloglucosidase (both enzymes from Sigma-Aldrich, St. Louis, MO) following the procedure of Holm et al. (1986). The glucose concentration (mg g^–1 dry wt.) was measured colorimetrically with a quantitative glucose test kit (Sigma-Aldrich). The total carboxylase activity of D-ribulose 1,5-bisphosphate carboxylase-oxygenase [Rubisco, µmol NADH (ß-nicotinamide adenine dinucleotide, reduced form) oxidized m^–2 s^–1] was measured spectrophotometrically (Keys and Parry, 1990). Ground leaf samples were extracted with 100 mM Tris-HCl buffer (pH 7.8) containing 20 mM MgCl₂, 10 mM NaHCO₃, 10 mM dithiothreitol, 1 mM EDTA, and 0.05% Triton X-100, and centrifuged for 3 min at 10000 x g and 4°C. The crude extract was used in the assay mixture (Leegood, 1993; modified by replacement of Hepes by Tris) for activity measurement at 25°C. After establishing a steady base rate, the reaction was started by adding D-ribulose1,5-bisphosphate and oxidation of NADH was monitored at A₃₄₀. Soluble protein was extracted with the buffer used in determining Rubisco activity and estimated by a protein dye-binding technique (Bradford, 1976) with a bovine serum albumin standard. Total ribonucleic acid (RNA) extraction was performed with a commercial RNA isolation kit (Promega, San Luis Obispo, CA) and the RNA concentration was quantified photometrically. The area and dry weight of leaf material sampled for the above analyses in the greenhouse experiment were added to the leaf area and dry weight obtained at the respective harvest dates. Leaflet area was determined via image analysis from contour drawings done before sampling. Sample leaf dry weight was calculated as the product of sample leaf area and SLW.

Data Analysis
The data obtained from the greenhouse and the growth chamber experiments were subjected to ANOVA performed with the PLABSTAT computer program (Utz, 1991). In these ANOVAs, light regimes and genotypes were considered fixed effects, and individual plants and replications random effects. In the greenhouse experiment, separate ANOVAs on a single-plant basis were performed for each sequential harvest. Data from the growth chamber experiment were analyzed separately for each light regime and across light regimes on an individual plant basis. In the combined ANOVA, light regimes were considered as different environments and replication within light regime mean squares were used to test the light regime effect. Because in these analyses the interaction mean squares for replication x genotype within light regime were not significant (P < 0.05), the interaction and error mean squares were pooled and used as the denominator for F tests of genotype and genotype x light regime mean squares. When F ratios were significant (P < 0.05), LSD values at that level were used to compare treatment means. The four parameters derived from the individual CER vs. PPF dependencies with the Photosyn Assistant software (Parsons and Ogston, 1999) were light compensation point as the abscissa intercept, dark respiration as the ordinate intercept, apparent quantum efficiency determined from the initial slope of the curves, and CER_max as the upper asymptote of the model function. Differences between genotype means for these parameters comprising of four repeated measurements within a given sampling date were tested by two-tailed t tests for homogeneous or heterogeneous variances as needed. Unless stated otherwise, significance levels refer to the 0.05 probability level.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Greenhouse Experiment
Agronomic Traits
Differences in plant height between transgenic and nontransgenic plants gradually decreased during the growing period. The Dara-5 plants reached only 56% of the height of the wild-type plants in the early vegetative phase (28 DAE), but 92% of the wild-type plants close to maturity (94 DAE). Plant height varied considerably depending on the distance from the edge of the greenhouse bench, indicating that one row of border plants could not completely block out irradiance from the side. While growth of the transgenic plants was increasingly inhibited as the number of neighboring plants decreased, the nontransgenic plants were almost uniform in height, except for those plants grown directly beside a border plant (Fig. 1).

View larger version (34K): [in this window] [in a new window]   Fig. 1. Relative height of almost full-grown Dara-5 and wild-type potato plants depending on the minimum number of neighboring plants in the greenhouse experiment. The height of plants with at least four neighbors is set to 100%. Data are means ± 1 SE of six plants within the same distance level from the edge of the bench as determined at 73, 94, and 107 d after emergence.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Agronomic traits for Dara-5 and wild-type potato plants as a function of days after emergence in the greenhouse experiment. Vertical bars represent ±1 SE of the mean (n = 8) where these exceed the size of the symbol. GLA, green leaf area; SLW, specific leaf weight.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Photosynthesis related traits for Dara-5 and wild-type potato plants as a function of days after emergence in the greenhouse experiment. Leaf carbon exchange rate measurements were made at photosynthetic photon flux of 1500 µmol m–2 s–1. Vertical bars represent ±1 SE of the mean (n = 8) where these exceed the size of the symbol. CERmax, light-saturated carbon exchange rate; Chl, chlorophyll; gtw, total leaf conductance to water vapor; RNA, ribonucleic acid.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Response of leaf carbon exchange rate to alteration in photosynthetic photon flux (CER vs. PPF) of young fully expanded leaves of Dara-5 and wild-type potato plants measured (a) during vegetative growth (19 and 20 DAE), (b) at the beginning of flowering (33 and 34 DAE), (c) after attainment of three-quarters of leaf mass (47 and 48 DAE), and (d) at the cessation of leaf growth (75 and 76 DAE) in the greenhouse experiment. Each data point represents the mean of four measurements performed during different time periods within a day at CO2 partial pressure outside the leaf = 35 Pa. Vertical bars represent ±1 SE of the mean where these exceed the size of the symbol.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Water soluble carbohydrate (WSC), starch, and N concentration (left column) and total amounts (right column) in leaves, stems, tubers, and roots of Dara-5 and wild-type potato plants as a function of days after emergence in the greenhouse experiment. The root starch data are not shown for clarity. Vertical bars represent ±1 SE of the mean (n = 8) where these exceed the size of the symbol.

Agronomy
The combined ANOVA showed that the light regime significantly affected the dry weights of haulm and roots, plant height, GLA, and SLW (Table 1). Concomitant with increasing PPF was a reduction in haulm dry weight, plant height, and GLA, but an increase in root dry weight and SLW. The genotype x light regime interactions were significant for tuber dry weight, total biomass dry weight, and SLW, indicating that transgenic and nontransgenic plants for these traits responded differently to the light regimes. The Dara-5 plants at low PPF showed significantly greater tuber and biomass dry weights, but were insignificantly different from the wild-type plants for these traits at medium and high PPFs. Averaged across light regimes, the percentage of biomass allocated to the tubers was almost identical for the two genotypes, being 88.8% for the Dara-5 and 89.1% for the wild-type plants. At high PPF, the transgenic plants produced 25% greater number of tubers than nontransgenic plants. Increasing light quantity greatly reduced the maximal plant height, but within a given light regime, the two genotypes did not show significant height differences. Specific leaf weight measured at 59 DAE was markedly affected by the light level. Elevating the PPF from 300 to 900 µmol m^–2 s^–1 increased the SLW of wild-type plants by 170%, and that of the transgenic plants by 261%.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Leaf carbon exchange rate (CER), leaf conductance to water vapor (gtw), total chlorophyll (Chl) concentration, and total Rubisco carboxylase activity of the fifth leaf below the apex as a function of days after emergence for Dara-5 and wild-type potato plants grown in growth chambers at photosynthetic photon flux (PPF) of 300, 600, and 900 µmol m–2 s–1. The Dara-5 chlorophyll and Rubisco data for the last sampling date in the 900 µmol m–2 s–1 PPF treatment are not shown because of a sampling error. Vertical bars represent ±1 SE of the mean (n = 8) where these exceed the size of the symbol.

View larger version (32K): [in this window] [in a new window]   Fig. 7. Sucrose, starch, and soluble protein concentration of the fifth leaf below the apex as a function of days after emergence for Dara-5 and wild-type potato plants grown in growth chambers at photosynthetic photon flux (PPF) of 300, 600, and 900 µmol m–2 s–1. The Dara-5 data for the last sampling date in the 900 µmol m–2 s–1 PPF treatment are not shown because of a sampling error. Vertical bars represent ±1 SE of the mean (n = 8) where these exceed the size of the symbol.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

The plant height of greenhouse-grown transgenic and nontransgenic plants increased with increasing distance from the edge of the bench (Fig. 1). This could result from increasing relative amounts of far-red to red light towards the center of the bench, because gradually increasing amounts of red light are filtered out by the Chl-containing leaves. The transgenic plants, however, were less sensitive to detect shade than the wild-type plants and thus required a stronger reduction in the R/FR light ratio before initiating the shade-avoidance response. Contrary to the greenhouse experiment, Dara-5 and wild-type plants in the growth chambers did not differ in plant height (Table 1). This is most likely attributable to the absence of shading because of the concomitance of the comparatively high amounts of light and low foliage mass which selectively inhibited the growth of the wild-type plants. The light conditions in the growth chambers thus resembled those in sparse stands with no competition for light and no reduction in the R/FR light ratio.

Solanum tuberosum is a long-day plant with regard to flowering, or more precisely, a short-night plant. The observation that a considerably higher proportion of Dara-5 than wild-type plants flowered under the 12 h photoperiod with artificial light in the growth chambers indicates that overexpression of Arabidopsis phyB shortens the critical daylength for flowering in potato. Likewise, in a photoperiod study with an Aster variety, overexpression of the oat (Avena sativa L.) PHYA gene shortened the critical daylength for inflorescence development from 14 to 8 h and overexpression of the Arabidopsis PHYB gene reduced the necessary light break during the night from 2 h to 15 min (Wallerstein et al., 2002).

Leaf Senescence
Because of their delayed onset of senescence, the transgenic plants in the greenhouse experiment showed a 9-d longer growing period and a 21% longer GLAD than the nontransgenic plants. The reason for the delayed foliage senescence of the transgenic plants might be attributable to alterations in the canopy light environment. As enhanced shading triggers the onset of leaf senescence (Ludwig et al., 1965; Marshall and Vos, 1991) less internal shading through reduced plant height might have decelerated senescence of Dara-5 plants. It has been shown in soybean, Glycine max (L.) Merr., that plants containing a gene that decreased plant height by 40% exhibited markedly lower light interception than normal genotypes (Wells et al., 1993).

Plants like Dara-5 that display a tendency to retain green leaves are often described as stay-green. So far, five types of stay-green have been identified (Thomas and Smart, 1993; Thomas and Howarth, 2000). Two functional stay-green types are characterized by differences in the initiation (Type A) and the rate of progress of senescence (Type B). The remaining three types are rather cosmetic than functional because the plants are intensely green but exhibit either normal or no photosynthetic capacity. Under greenhouse conditions, the Dara-5 plants displayed later onset of senescence than the wild-type plants, but comparable subsequent rate of senescence as indicated by the similar decline in leaf Chl and protein concentration, leaf CER, and Rubisco activity (Fig. 3). Dara-5 could thus be classified as a Type A stay-green. Although the aforementioned traits were measured on young leaves only, other senescence-related traits determined on whole plants such as GLA (Fig. 2g) and leaf N content (Fig. 5e) are also in support of this type of classification.

In comparison with the greenhouse experiment, longevity of the plants in the growth chamber experiment was shortened by about 2 wk under low PPF and by about 3 wk under medium and high PPF. Earlier senescence in the growth chambers was probably an indirect effect of photoperiod because short days accelerate tuber induction (Vos, 1999). Senescence was further accelerated by higher light levels as indicated by the more rapid decline of the senescence-indicator Chl under high than under low PPF. It has been shown in A. thaliana that high PPF promotes Chl loss and therefore leaves under high PPF do not live as long as leaves under low PPF (Noodén et al., 1996). The transgenic plants in the growth chambers did not exhibit an extended longevity as observed in the greenhouse. This might be attributable to the absence of differences in internal shading which most likely caused the earlier maturity of the wild-type plants in the greenhouse experiment.

Carbon and Nitrogen Metabolism
Under greenhouse and low PPF conditions in the growth chamber, the transgenic plants had lower carbohydrate but higher N levels in their leaf and stem tissues than the wild-type plants (Fig. 5e, 7). Because phyB-overexpressing plants are able to form more physiologically active far-red light absorbing phytochrome (P_FR) (Schopfer and Brennicke, 1999), and P_FR stimulates the transcription of genes for nitrate reductase (Gatz et al., 1998), the greater N concentrations in stems and leaves of transgenic plants might be attributable to their greater potential to assimilate N. A stronger accumulation of N compounds must be accompanied by a more intense supply of C skeletons acting as N acceptors. This regulatory interaction of C and N metabolism may explain the observation of reduced carbohydrate and increased N concentration in the aboveground tissues of the transgenic plants. In addition, protein biosynthesis is energy costly and occurs at the expense of the carbohydrate pool. Toward the end of the growing period, Dara-5 plants in the greenhouse showed considerably higher WSC and starch levels in stems and leaves than the wild-type plants. This increase occurred in concomitance with a moderate decrease of N and Chl concentration (compare Mascleaux et al., 2000) as well as photosynthesis rate and Rubisco activity. During senescence, the production of C skeletons obviously continues but cannot be directed to the formation of amino acids and proteins. An alternative explanation for the accumulation of carbohydrates in leaves and stems, particularly in the transgenic plants, is the growing evidence that the lipid to sugar pathway of gluconeogenesis is activated during leaf senescence. The C salvaged from lipids of the Chl membranes, which possibly occurred to a greater extent in Dara-5 leaves with their higher Chl contents, can either be used as sources of respirable energy to meet metabolic and transport demands or can be converted into transportable sucrose by the reversible steps of glycolysis and be exported to sinks elsewhere in the plant (Dangl et al., 2000). Presumably, in Dara-5 plants, respiration dominated over the export to sinks because the two genotypes did not significantly differ in final tuber yield.

Leaf Activity Traits
In both experiments, Dara-5 plants exhibited substantially greater leaf CER and leaf conductance than the wild-type plants for most of the growing season (Fig. 3, 6). Moreover, leaf conductance was significantly (P < 0.001) correlated with leaf CER, both in the greenhouse (r = 0.98 for Dara-5 and r = 0.96 for wild-type plants) and across light levels in the growth chambers (r = 0.71 for Dara-5 and r = 0.78 for wild-type plants). The greater stomatal density of the transgenic plants (Fig. 3e) suggests that their increased leaf CER may have an anatomical basis. The intercellular leaf CO₂ partial pressure (C_i) is determined by the balance between CO₂ supply into the leaf via the stomata and the CO₂ demand in photosynthesis. The C_i of Dara-5 plants averaged 99.7% of the wild-type in the greenhouse and 101.8% of the wild-type in the growth chambers. Unlike C_i, the transgenic plants had considerably greater CER:C_i ratios, averaging 117 and 115% of the nontransgenic plants in the greenhouse and growth chambers, respectively. It is therefore assumed that nonstomatal rather than stomatal factors were responsible for the differences in the photosynthetic activity between the two potato genotypes. This assumption is consistent with the recent observation that stomatal density does not represent a bottleneck for leaf gas exchange in potatoes because leaf conductance is effectively regulated through changes in stomatal aperture (Lawson et al., 2002). This would also explain the observation that Dara-5 and wild-type plants, despite lower leaf conductance, had higher leaf CER under high than under medium PPF in the growth chamber experiment (Fig. 6). Because the greater leaf CER of Dara-5 was generally associated with greater Chl concentration and total Rubisco activity, these seem to be the responsible nonstomatal factors.

Thiele et al. (1999) assumed that phyB-overexpressing transgenic plants might be more productive in high-radiation environments because of reduced photoinhibition. Contrary to this expectation, the Dara-5 plants in the growth chamber experiment exhibited almost the same leaf CER superiority at low as well as high light level. Obviously, results from studies with short-term exposure of single leaves to high PPF are not simply transferable to systems where whole plants are exposed to high PPF during the entire growth period.

Tuber and Biomass Yield
Despite the greater light-saturated leaf CER and the longer GLAD, tuber and total biomass yield of Dara-5 and wild-type plants did not significantly differ in the greenhouse experiment. Likewise, considerably greater leaf CERs of the transgenic plants in the growth chamber experiment only resulted in slightly higher biomass and tuber dry matter yield in the low-PPF treatment. The results observed are thus in contrast to those of Thiele et al. (1999), who reported 56% greater tuber yield for Dara-5 compared with wild-type plants under greenhouse conditions. It is noticeable that the tuber fresh matter yields per plant of 345 g for Dara-5 and 380 g for the wild-type plants in the greenhouse experiment, despite of a shorter growth period, were two- to threefold greater than those in the Thiele et al. (1999) study. This discrepancy may be attributable to differences in the type of planting material. Thiele et al. (1999) used in vitro plantlets whereas tubers were applied in the present study. In vitro plantlets, because of their slower development and reduced vigor, are probably less productive than plants grown from tubers. Nevertheless, this does not explain the large yield superiority of the transgenic plants in their study. Braun et al. (2002), in a 2-yr field experiment in Germany, likewise could not confirm the yield superiority of Dara-5 and Dara-12 plants. However, because of their comparatively slow early growth, the phyB-overexpressing plants took longer to complete soil coverage. The population density of 3.8 plants m^–2 applied in their study (A. Braun, 2002, personal communication) might thus have been suboptimal for the transgenic plants.

Relationship between Leaf Activity and Yield
It was surprising that the transgenic plants in spite of consistently greater photosynthetic rates did not produce overall higher tuber and total biomass yield than the wild-type plants. Because the routine CER measurements were all performed around noon, diurnal fluctuation was not taken into account. Nevertheless, the light response curves taken in the greenhouse experiment at four different times of the day indicated either similar or greater CER of Dara-5 than wild-type plants (data not shown). The lack of positive association between photosynthesis and yield in the greenhouse experiment might arise from the fact that CER measurements were done at light saturation whereas the mean daily PPF amounted only to 220 µmol m^–2 s^–1. Under these conditions, a large portion of crop photosynthesis occurred at nonsaturating PPF, that is, under conditions where transgenic and nontransgenic plants exhibit little leaf CER difference (Fig. 4). Nevertheless, Dara-5 plants under medium and high PPF in the growth chambers also failed to produce superior tuber and biomass yields. When measured on a leaf area basis, there is often a poor correlation between photosynthesis and dry matter production or yield because genotypes with increased leaf CER sometimes have lower total leaf areas and thus lower total canopy photosynthesis (Evans, 1993). However, this explanation is not applicable to the present study because the higher leaf CER of Dara-5 relative to wild-type plants was associated with similar or longer GLAD and thus greater photosynthetic capacity.

We therefore assume that the reason why the greater leaf CER of Dara-5 plants did not result in increased yields is explained by poor C-use efficiency, low sink strength of the tubers, or both. The transgenic plants exhibited relatively higher investment of biomass into photosynthetic leaf area, stems, and roots and less to tubers than the nontransgenic plants. It has been shown that dark respiration rate differs significantly among plant tissue types (Winkler, 1971; McCutchan and Monson, 2001; Vose and Ryan, 2002). Because tubers have lower respiration rates than other potato plant parts, especially leaves (Winkler, 1971), the transgenic plants most likely had higher respiration losses than the nontransgenic plants. In addition to the development of more intensively respiring plant tissue by the transgenic plants, these tissues also had greater N and hence protein concentration. More than 50% of the total leaf N in C₃–plants is bound in photosynthetically effective protein (Evans and Seemann, 1989). It has been estimated that respiratory costs for the cyclic degradation and resynthesis of leaf proteins (protein turnover) accounts for a considerable portion of total respiration (de Visser et al., 1992; Bouma et al., 1994). Thus, the benefit of providing and maintaining an increased photosynthetic machinery in the form of extra Chl and other photosynthesis components might have been a waste of resources. Although in this study respiration during the night was not measured, the assumption of a poor C-use efficiency of the transgenic plants is supported by two observations. First of all, Dara-5 leaves exhibited significantly greater leaf dark respiration than the wild-type plants at one of four light-response measurement dates. Second, at the only occasion when Dara-5 plants had significantly higher tuber and biomass yield than the wild-type plants (at low PPF in the growth chamber), the two genotypes had similar total Chl concentration and Rubisco activity (Fig. 6).

A different explanation for the lack of Dara-5 yield superiority is the yield structure of Désirée, which produces relatively large but few tubers. The transgenic plants had significantly more tubers than wild-type plants at the final harvest in the greenhouse experiment (+41%) and under conditions of high PPF in the growth chambers (+25%). The tuber number of the transgenic plants in the greenhouse experiment sharply increased toward the end of the growing period (Fig. 2f). This might be interpreted as an attempt of the transgenic plants to increase an insufficient sink capacity to cope with the continued carbohydrate production. The simultaneous sharp rise of Dara-5 leaf starch concentration (Fig. 5c) corroborates this hypothesis. Overexpressing phyB in potato cultivars with a greater genetically fixed sink number may enable the formation of higher tuber yield.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
The findings from this study do not confirm the reported greater yield of phyB-overexpressing transgenic potato plants. It is assumed that enhanced C loss from respiration was the reason why the transgenic plants were unable to translate their superior leaf activity and partially greater GLAD into consistently greater tuber and total biomass yield. There was no evidence of special suitability of the transgenic plants for areas with high radiation. On the contrary, the results suggest that the greater physiological potential of the transgenic plants owing to an enhanced allocation of N to the leaves might be more advantageous under low rather than high PPF conditions. The findings from this study affirm earlier reports that phyB overexpression represents a tool for manipulating senescence. However, transgenic approaches to delaying senescence are hardly required in potato because substantial genetic variation is available for breeding cultivars that fit into the growing season of almost any specific area. In the present study, the plants were well supplied with N, other nutrients, and water. Additional research appears worthwhile in elucidating whether phyB-overexpressing potato plants benefit from a greater root system under conditions of water and N shortage.

	ACKNOWLEDGMENTS

 
The authors thank Sabine Peickert, Birgit Pohl, Claudia Lüders, and Dirk Hillegeist for the excellent technical assistance, Dr. Christian Paul and Merle Alex for their help with the NIRS data analysis, and Professor Christiane Gatz (Albrecht-von-Haller-Institute, University of Göttingen, Germany) for providing the transgenic tuber material.

Received for publication January 29, 2003.

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