Jun 8, 1987 - tosynthetic gas exchange in flag leaf blades during grain filling .... were determined after drying to constant weight in a forced air oven at 80°C.
Plant Physiol. (1987) 85, 667-673 0032-0889/87/85/0667/07/$O 1.00/0
Photosynthetic Gas Exchange Characteristics of Wheat Flag Leaf Blades and Sheaths during Grain Filling THE CASE OF A SPRING CROP GROWN UNDER MEDITERRANEAN CLIMATE CONDITIONS Received for publication February 13, 1987 and in revised form June 8, 1987
JosE L. ARAUS* AND Luis TAPIA Departament de Biologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, Barcelona 08028, Spain ABSTRACT The rate of net CO2 assimilation (A), the stomatal (g,) and residual (g,) conductances to C02, the intercellular CO2 concentration, the CO2 compensation points at 21% 02 (r2l) and at 2% 02 (r2) and the amounts of dry matter, nitrogen, and carbohydrates were determined, from anthesis through grain filling, in the flag leaf blade and sheath of spring wheat (Triticum aestirum L. cv Kolibri). The nitrogen content and the rate of net CO2 assimilation declined slowly until the onset of senescence in both orpns, about 3 weeks after anthesis. During senescence the reduction of A in both organs was not primarily caused by a decrease in g;, the main factor is the decrease in g,. From values of r2, and r2 it is suggested that the rate of respiration in the light contributing to the CO2 compensation point is higher in sheaths than in blades irrespective of the 02 level considered. The role of sheaths storing and later transporting assimilates to the developing grains seems to be more important for shoot yield than that of sheaths functioning as photosynthetic organs after the onset of senescence occurs. It is suggested that accumulation of carbohydrates in leaves might somehow trigger senescence in the flag leaf blade and sheath simultaneously.
In wheat, the main source of assimilates for the grains is the flag leaf (8). The flag leaf has two components: the blade and the sheath. Most authors usually refer only to the flag leaf blade as the total flag leaf, thus neglecting the role of the flag leaf sheath. However, morphological characteristics of the flag leaf sheath are highly correlated to shoot yield, even more than characteristics of the flag leaf blade (19). The role of the sheath in the final shoot yield could be of particular importance in spring crops, which develop their grain filling period under warm and dry conditions such as those of a Mediterranean climate. This is based on three considerations. First, throughout the grain filling period the flag leaf sheath is more protected than the flag leaf blade from adverse environmental conditions. This could accelerate the senescence of the blades. Ledent and Moss (19) suggested the high ranking of sheaths in determining final shoot yield may be due to their functioning as photosynthetic organs during the latter part of the grain filling stage, when the flag leaf blade is senescing. There are some experimental results supporting this hypothesis (25). Second, within the canopy it appears that the photosynthetic contribution of the upper parts of the shoot (the ear, peduncle, and flag leaf blade) is relatively less important at lower latitudes, whereas the contribution of the flag
and the blade) and later transport them to the developing grain after the initiation of flag senescence. This may be most important in crops with a short grain filling period such as spring crops under warm conditions (29). Even though the characteristics and the time-course of photosynthetic gas exchange in flag leaf blades during grain filling have been studied, much less information is available about gas exchange characteristics of sheaths (especially under field conditions). Furthermore, sheaths carry out processes, other than photosynthesis, which are accompanied by a significant release of CO2. The structures of blades and sheaths differ considerably. Blade structure is adapted for higher gas exchange rates, while sheaths are partially rolled, holding a segment of shoot and exposing only about one-third of their area of light and gases. Upper leaves are also major contributors of nitrogen to the grain. Higher temperatures increase the rate of nitrogen uptake (24). Therefore, warm temperatures during the grain filling period increase the nitrogen content ofthe grain, but also accelerate the depletion of the nitrogen reserves in the vegetative parts (24), which might accelerate leaf senescence and thus reduce photosynthesis (14). On the other hand, warm temperatures also enhance the rate of grain growth and shorten its duration (29). Thus, in conditions of adequate nitrogen supply, the capacity for utilization of photosynthates by grains may become a significant limiting factor for yield of a spring crop under Mediterranean climate. It is usually observed that nonstructural carbohydrates accumulate in the flag leaf blade and sheath, and also in the stem during the period of linear grain growth (17, 30). These assimilates are either stored or respired (8). There are indications that carbohydrate accumulation may inhibit wheat leaf photosynthesis via feedback effects (13, 23) and that it may also induce, or at least accelerate, leaf senescence (18). The purpose of this study was to investigate further the pattern and physiological nature of ontogenetic changes in gas exchange of flag wheat leaves during the grain filling period. Particular attention has been given to the study of the role of flag leaf sheaths, especially with respect to net CO2 assimilation rates, and nitrogen and carbohydrate remobilization.
MATERIALS AND METHODS Plant Material and Growth Conditions. Spring wheat (Triticum aestivum L. cv Kolibri) was sown on January 25, and thinned to a final stand of 70 plants m-2. Sowing took place in rectangular field plots (1.5 by 15.8 m) at the Experimental Fields of the Faculty of Biology, University of Barcelona. Each plot consisted of nine rows spaced 0.17 m apart. Soil type was mollic xerofluleaf sheath increases with increasing elevation of the sun (22). vent, with 3 to 4% organic matter and high levels of P and K. Third, the flag sheath could store assimilates (produced by itself Ammonium nitrate (150 kg N ha-') was applied prior to seeding. 667
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Plant Physiol. Vol. 85, 1987 Measurements were made between 9 and 13 h (solar time), about every week, and extended from anthesis to the end of grain filling period. At least five different flag leaves (blade and sheath) from
ARAUS AND TAPIA
crop was flood irrigated at seeding, and thereafter was maintained well irrigated. Daily radiant flux density throughout the period of growth of the flag leaf was measured using an integrating solarimeter (Kipp and Zonen). Air temperature was also continuously registered (Fig. 1). To ensure plant uniformity, only main shoots that presented anthesis on the same date (May 21) were selected for the study. Gas Exchange Measurements. A' was measured at ambient CO2 and levels in attached flag wheat leaf blades and sheaths in the field, using an open gas exchange system with an infrared
The
02
gas analyzer as described in a previous work (1). One intact blade or sheath was inserted in a well ventilated leaf chamber provided with a fan positioned below the leaf (boundary layer conductance to diffusion of water vapor was around 4 mol m-2 s-'). Soft rubber gaskets provided an effective seal when leaf blades or sheaths were enclosed. The leaf chamber was arranged to be normal to the solar beam on cloudless days. Quantum flux density (400-700 nm) incident on the leaf was 1700 to 2000 m-2 s-', measured with a quantum sensor (LICOR, model LI-190 SR). A slightly concave aluminium adapter was placed
'4mol
into the photosynthetic chamber before each measurement of sheath photosynthesis. With this adapter at least 75% of incident irradiance was obtained at the back side of sheaths. Leaf temperature was measured with a copper-constantan thermocouple (0.1 mm diameter) in contact with the lower surface, and was controlled by changing air temperature by circulating cold water through a copper coil surrounding the fan. The leaf temperature was 22.0 ± 1.5°C, and the air RH was between 50 and 60%. 35
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the central 4 rows were used on each day of measurements. Sheaths used in gas exchange measurements included the segment of shoot held. Stomatal conductance to water vapor was measured with a 'steady state" porometer (LICOR, model LI1600) immediately before and after the photosynthesis measurement. Leaf to external air vapor pressure difference was between 1.0 and 4.0 kPa. A small methacrylate adaptor was used to measure sheath stomatal conductance to water vapor. This adaptor enclosed 1.56 cm of the sheath cylinder pressed against the LI- 1600 2 cm2 cuvette aperture. Stomatal conductance was recalculated taking into account the sheath enveloping enclosed area. The gas exchange values refer to the projected area of blades and the total enveloping area of sheaths. Calculations of gs, and C, were made according to von Caemmerer and Farquhar (28) and gr as before (2). The CO2 compensation point was measured by using a closed system (4), allowing the leaf to come to equilibrium with its CO2 atmosphere, and taking the r value after 60 min. At least three excised flag leaf blades or sheaths (cut under water) similar to those used in the photosynthesis measurements were placed, with the cut end in water, into a gas exchange leaf chamber. C02-free air with different 02 concentration (21 and 2%) was obtained by mixing ambient air with N2 from a cylinder, and then by passing the resulting gas through two columns of soda lime. The ratio Rd/Vcma. at 21 and 2% 02 was estimated from the values of r2, and r2, respectively. According to the model of Farquhar et al. (10), the leaf CO2 compensation point in C3 species depends fundamentally on the kinetic properties of RuBP carboxylase and on the ratio Rd/V,,ma Rd being the rate of CO2 efflux in the light by processes other than photorespiration and Vcmax the maximum velocity of carboxylation. From this model it can be deduced that
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and 02, (160 Mbar CO2 and 400 mbar 02, respectively); 0 is the partial pressureof 02 (210 and 20 mbar, respectively, corresponding to 21 and 2% 02 at the atmospheric pressure in Barcelona), andr* is the CO2 compensation point in the absence of Rd (3.3 and 30 ubar CO2 at 20 and 210 mbar 02, respectively). The calculation of this ratio has been made as before (3). Determinations of Leaf Area and Dry Matter. The area (one side) of the blades used in the gas exchange measurements was calculated by means of a gravimetric method; total external sheath area was also calculated by assuming a cylindrical shape. The areas of the flag leaf blades used for measurements ranged between 17cm2 and 27cm2 while those from the flag leaf sheaths ranged between 24cm2 and 36cm2. Blade and sheath dry weights were determined after drying to constant weight in a forced air oven at 80°C. SLDW of blades and sheath (gm-2) was then calculated. SLDW of sheaths was determined as the ratio of dry weight to total cylinder surface. SDW was calculated, from the segment of stem held by the sheath, as stem dry matter per unit of stem length. Grain fresh and dry weights were also registered. Nitrogen Assay. Nitrogen concentration was measured using the micro-Kjeldahl method on the same leaves and grains used above. Two determinations were made for each blade, sheath, and grains. Nonstructural Carbohydrate Assay. Carbohydrates were extracted in boiling water for 15 min. Free glucose plus fructose (hexoses), invertase sugars (mostly sucrose), and starch fractions were determined using an enzymic method (4). At least one determination for each of these carbohydrates was made for each
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FIG. 1. Maximum (-) and minimum
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and daily radiant flux density during the study period. 'Abbreviations: A, net CO2 assimilation rate;gs, stomatal CO2 conductance; g,, residual CO2 conductance; C,, leaf intercellular CO2 concentration;r21, CO2 compensation point at 21% 02; F2, CO2 compensation point at 2% 02; N, nitrogen content; SLDW, specific leaf dry weight; SDW, stem dry weight; TNC, total nonstructural carbohydrates; Rd, rate of respiration in thelight; RuBP, ribulose 1,5-bisphosphate.
GAS EXCHANGE PATTERNS IN WHEAT FLAG LEAF BLADES AND SHEATHS blade and sheath used in the photosynthesis measurements. Values were referred to unit blade or sheath area. RESULTS The rate of net CO2 assimilation per unit leaf area in both flag leafblades and sheaths declined slowly with time, until d 24 after anthesis. On this day blade and sheath senescence started as indicated by the rapid decline of A in these organs (Fig. 2). Values of A in flag leaf blades were, on d 2 after anthesis, nearly 4 times larger than those of sheaths (see "Materials and Methods" for calculations). Thereafter, this ratio decreased to about 3. The stomatal conductance to CO2 in flag leaf blades showed a maximum on d 8 after anthesis (349 ± 13 mmol m-2 s-'), followed by lower values which remained more or less constant (around 250 mmol m-2 s-') until d 30, and decreased strongly after this day (Fig. 3). The g, in flag leaf sheaths also showed its maximum value on d 8 after anthesis (128 ± 8 mmol m-2 s-'), decreasing steadily after this day through ageing and senescence (Fig. 3). Maxima in the A values of blades and sheaths (26.2 ± 1.1 ,mol m-2 s-' and 8.9 ± 0.3 Amol m-2 s-', respectively), were also measured on d 8 after anthesis (Fig. 2). Values of g, in blades were about 3 times higher than those of sheaths until the onset of leaf senescence (Fig. 3). In sheaths, the g, after anthesis remained stable from d 8 until the onset of senescence; whereas in blades it declined slightly (20%) before the onset of senescence. However, gr in both organs decreased sharply after d 24 (Fig. 3). The values of g, in blades were nearly 3 times larger than those of sheaths throughout the study period. The intercellular CO2 concentration in the flag leaf blade remained more or less constant (around 220 Ml L-') until d 24 after anthesis, and increased strongly during senescence. The C, in flag leaf sheaths declined slowly before the onset of senescence (from about 220-240 to 200 Ml L-'), and then also increased sharply (Fig. 3). CO2 compensation points at 21% and 2% 02 in flag leaf blades declined slightly during preanthesis, and remained fairly constant (around 40-45 ul L-' amnd 8-10 Ml L-') during about the 3 weeks after anthesis, and thien showed a transient peak before the senescence started. Afterward, the values rose rapidly (Fig. 4). The F2, and F2 in stheaths showed patterns similar to those observed in blades. Hoiwever, until the onset of senescence values measured in sheaths iwere at least 50% higher than those of blades. In blades the ratio Rd/V,max (Fig. 5) was consistently higher at 21% 02 than at 2% 02; whereas in sheaths it was almost the same at the two 012 levels. However, sheaths showed values 30 *
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DAYS AFTER ANTHESIS FIG. 2. Changes in the rate of net CO2 assimilation (A) at ambient CO2 and 02 levels in the flag leaf blade (0) and sheath (0) from anthesis through senescence. The,arrow indicates the day in which the onset of senescence occurred. Each point represents the mean ± SE of 5 to 6 different main culm flag Ileaves. For experimental details see "Materials and Methods."
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FIG. 3. Changes in the stomatal (ge, 0- -0) and residual (g, O-O) conductances to C02, and in the leaf intercellular CO2 concentration (C,, *-*) in the flag leaf blade and sheath from anthesis through senescence. The arrow indicates the day in which the onset of senescence occurred. Values are means ± SE of 5 to 6 replicates.
of this ratio consistently higher than those of blades, even with values several times higher at 2% 02 N in the flag leaf blade declined slowly during the 24 d after anthesis; then N declined very rapidly. The N in the flag leaf sheath followed a similar pattern (Fig. 6). The values of N in blades were around 50% higher than those of sheaths throughout the studied period. In both organs A rates increased with increasing N, following saturation pattern when N in the blade and in the sheath exceeded 125 mmol m-2 (Fig. 7: in sheaths, values are referred to unit of half-enveloping area). SLDW of flag leaf blades increased to a maximum value (68.7 2.4 g m2) on d 24; after this day the SLDW rapidly decreased (Fig. 8). Sheaths showed about 30% higher SLDW values than blades, and the maximum SLDW value (87.2 ± 2.0 g m-2) was also observed on d 24, followed by a sharp decrease (Fig. 8). The SDW also showed a consistent maximum (1.70 ± 0.05 g m-') on d 24, afterward decreasing dramatically. The highest values of daily temperature occurred on d 20 to 23 (Fig. 1); that is, just before the attainment of maximum SLDW and SDW values. Dry weight and nitrogen accumulation in the grain increased in a linear manner until about 5 weeks after anthesis, then grain growth
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the grain began to ripen; the water per kernel on d 43
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Total nonstructural carbohydrates (starch plus sucrose plus hexoses) in flag leaf blades and sheaths (Fig. 10) followed patterns similar to those observed in the respective SLDW; however, only
670
ARAUS AND TAPIA
Plant Physiol. Vol. 85, 1987
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FIG. 4. Changes in the CO2 compensation point (r) at 21% 02 (0) and 2%02 (0) of the flag leaf blade and sheath from about I week prior to anthesis through senescence.
blades attained their maximum value of TNC on d 24 after anthesis (32.7 ± 2.5 g m-2). Thus, during the grain filling period changes in SLDW values, at least in blades, were mostly accounted for by changes in TNC levels. However, fructosans, a storage carbohydrate in sheaths (17), were not measured in our work. The starch fraction represented in both organs almost 80% of TNC (Fig. 10). The starch fraction in both organs declined slightly during about the first week after anthesis, coinciding with the beginning of the grain growth (Figs. 9 and 10), increasing somewhat on d 14 and then remaining fairly constant until the end of senescence. The relative constancy in the starch fraction suggested that variations in other carbohydrate fractions could be more important for determining the maximum SLDW values. In this respect, maximum SLDW in blades was basically because of a peak (9.5 ± 1.5 g m-2) in the content of soluble carbohydrates (sucrose plus hexose). The hexose (glucose plus fructose) fraction in both organs represented less than 20% ofsoluble carbohydrates during the grain filling.
The patterns shown by the rate of net CO2 assimilation, the stomatal and residual C02 conductances, and the intercellular CO2 concentration were basically similar in both organs irrespective of differences in their absolute values (Figs. 2 and 3). Throughout the period studied, differences in A and g, between flag leaf blades and sheaths are in agreement with reports on RuBP carboxylase activity of these organs (21): throughout the grain filling period RuBP carboxylase activity of blades was, at least, 3 times larger than that of sheaths. The time-course of changes in gas exchange parameters in blades as well as the changes in nitrogen and dry matter content (in both organs) agreed well with our results obtained the year before under very similar experimental conditions (1, 2).
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DAYS AFTER ANTHESIS
FIG. 5. Changes in the ratio between the rate of dark respiration in the light and maximal RuBP carboxylase activity (Rd/V,cma) of the flag leaf blade and sheath. Values have been calculated from F at 21% 02 (0) and 2% 02 (0). For other experimental details, see "Materials and Methods." 2.4
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