Leaf senescence in maize hybrids: plant population, row spacing and kernel set effects

Field Crops Research 82 (2003) 13±26

Leaf senescence in maize hybrids: plant population, row spacing and kernel set effects L. BorraÂs, G.A. Maddonni*, M.E. Otegui Dpto. de ProduccioÂn Vegetal, Facultad de AgronomõÂa, Universidad de Buenos Aires, Av. San MartõÂn 4453 (C1417DSE), Buenos Aires, Argentina Received 25 September 2002; received in revised form 8 December 2002; accepted 15 December 2002

Abstract Maize crop management involves decision making on several cultural practices aimed to maximize grain yield, like plant population and row spacing. These practices affect the light environment perceived by plants and the post-¯owering source±sink ratio, but there is scarce information on the way they in¯uence plant leaf senescence. The objectives of our research were to: (i) characterize the development of leaf area senescence for contrasting canopy architectures (i.e. plant population  row spacing), and (ii) analyze the response of leaf senescence to changes in the light environment and the post-¯owering source±sink ratio. Field experiments were conducted in Argentina between 1997/1998 and 2000/2001. Four hybrids were grown at a wide range of plant populations (3, 9, 10 and 12 plants m 2), row spacings (0.35, 0.7 and 1 m) and pollination treatments (natural and restricted pollination). Senescence development was well described (r 2 ˆ 0:61 0:99; P < 0:05) as a bilinear process, starting always at around 500 8C day (base temperature of 8 8C) before silking. Senescence progressed at a lower rate during the ®rst phase of the process than during the second one (1.4 vs. 5.5 cm2 per plant per 8C per day). The second phase always started between silking and 400 8C day after silking. Increased plant population increased senescence rate during the whole plant cycle, but never affected the ontogenic stage when senescence was initiated or accelerated in all hybrids. Increased plant population promoted: (i) an enhanced light attenuation within the canopy (k coefficient ˆ 0:43, 0.55, 0.53 and 0.65 for 3, 9, 10 and 12 plants m 2, respectively), (ii) an augmented post-¯owering source±sink ratio (11.6 cm2 of green plant leaf area per kernel at 9 and 12 plants m 2 compared to 8.3 cm2 per kernel at 3 plants m 2), and (iii) a decreased grain protein concentration. Senescence was reduced by kernel set restrictions that enhanced post-¯owering assimilate availability, indicating the process was accelerated by assimilate starvation at high plant populations independently of the green leaf area established per growing kernel. Row spacing altered light quality (red:far-red ratio) perceived at the lowermost leaf stratum at the highest plant populations, but had no effect on senescence development. Senescence during grain ®lling was related to the local light quantity perceived by leaves and to N availability for actively growing kernels. Although senesced leaf area was in¯uenced by crop growing conditions, senescence initiation and the onset of increased senescence rate were not. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Leaf senescence; Plant population; Row spacing; Source±sink ratio; Zea mays

1. Introduction *

Corresponding author. Tel.: ‡54-11-4524-8039; fax: ‡54-11-4514-8739. E-mail address: [email protected] (G.A. Maddonni).

A normal process in the life cycle of plants is senescence. It is a terminal phase in the development of every organ, including leaves, stems, ¯owers and

0378-4290/03/$ ± see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-4290(03)00002-9

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fruits (Dangl et al., 2000). Senescence generally occurs without simultaneous growth, following organ maturity. It is in¯uenced by environmental or endogenous (e.g. hormonal) perturbations by initiating or accelerating the different steps of the process: initiation, degeneration, and terminal phase (NoodeÂn et al., 1997). In the present paper, we focus on leaf area senescence at a whole plant level, considering only the terminal phase. This is visualized by the yellowish color of the laminas, and is commonly followed by organ death (i.e. necrosis). In summer crops, such as sun¯ower, maize and sorghum, senescence starts before all leaf area is fully developed (i.e. previous to ¯owering), and progresses at an increased rate during the grain-®lling period (Eik and Hanway, 1965; Muchow and Carberry, 1989; Sadras et al., 2000; Lafarge and Hammer, 2002). Consequently, green leaf area duration has always been shown to depend on the availability of assimilates to sustain grain growth during the post-¯owering period. Thomas (1992) proposed leaf senescence to be triggered by both a shortage or an excess of assimilates. He de®ned an assimilate availability ``window'' between an upper and a lower threshold. As long as a leaf remains within this ``window'' senescence is not initiated. Considering the source of assimilates together with the sink demand established by growing kernels, Rajcan and Tollenaar (1999a) established an optimum post-¯owering source±sink ratio that maximized green leaf area duration at the whole plant level. When the ratio is below the threshold, nitrogen (N) demand by actively growing kernels sets a source de®ciency of this nutrient, which effects leaf senescence due to N remobilization from vegetative to reproductive structures. Contrarily, high source±sink ratios increase senescence rate in response to the accumulation of non-structural carbohydrates, producing a feedback inhibition of photosynthesis (Christensen et al., 1981; Ceppi et al., 1987; Rajcan and Tollenaar, 1999b). Plant population affects the post-¯owering source±sink ratio through its effects on plant leaf area, the amount of light intercepted per plant and kernel number per plant. All these traits decrease in response to increased stand density (Tetio-Kagho and Gardner, 1988a,b; Westgate et al., 1997; Andrade et al., 2000; Maddonni et al., 2001), but there is scarce information on how the relationship between them (i.e. the source±sink ratio) is affected.

The number of plants per unit land area may in¯uence leaf area senescence through its effects on this relationship. Plant population effects on the post¯owering source±sink ratio are not only related to the amount of light capture by each plant but also to its distribution within the canopy. Maize crops cultivated at high plant populations or in narrow rows exhibit an increased light attenuation within the different leaf strata (FleÂnet et al., 1996; Maddonni et al., 2001), which is known to affect the vertical pro®le of leaf N content of maize crops (Drouet and Bonhomme, 1999). Finally, increased plant population also affects light composition perceived by leaves. As light travels downwards through a canopy, it suffers a reduction in its photosynthetic photon ¯ux density and a signi®cant alteration in its spectral composition. Because absorption by green tissues is more intense in the blue (400±500 nm) and red (600±700 nm) wavebands and re¯ection is more intense in the far-red waveband (700±800 nm), the red:far-red ratio reaching the plant base is greatly reduced at high leaf area indexes (Holmes and Smith, 1977; Smith, 1986; Sattin et al., 1993). Rousseaux et al. (1999) determined that N exportation from the lowermost leaves in sun¯ower is promoted by low red:far-red ratios perceived at the surface of these organs. Moreover, several monocotyledon and dicotyledon species present an enhanced leaf senescence under environments enriched with farred radiation or with low red:far-red ratio (VarletGrancher and Gautier, 1995). Recently, it has been shown that maize leaves perceive the far-red radiation environment at which they grow (Maddonni et al., 2002). Thus, the vertical pro®le of light quantity and quality within a canopy are known to regulate leaf senescence rate. To our knowledge, there is no study showing the relative importance of the light pro®le and the post-¯owering source±sink ratio on maize leaf senescence at the whole plant level. The objectives of our research were to: (i) characterize the development of leaf area senescence under contrasting plant growing conditions, and (ii) analyze the response of leaf senescence to changes in the light environment and the post-¯owering source± sink ratio. For these purposes, two types of experiments were conducted, one with a wide range of leaf area index and light attenuation achieved with plant population density (3, 9 and 12 plants m 2) and row

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spacing (0.35 and 0.70 m) treatments, and another one with different post-¯owering source±sink ratios and light attenuation achieved with pollination control and row spacing (0.35 and 1 m) treatments. 2. Materials and methods 2.1. Experimental design Field experiments were conducted in Argentina during the growing seasons of 1997/1998 and 1998/ 1999 at Salto (348330 S, 608330 W), and during 2000/ 2001 at Pergamino (338560 S, 608340 W), both on silty clay loam soils (Typic Argiudoll). At Salto, sowing took place on 8 October 1997, and 12 November 1998. Treatments at this site were a factorial combination of: (i) three plant populations (3, 9 and 12 plants m 2), (ii) two row spacings (0.35 and 0.70 m), and (iii) two (DK696 and DK757 during 1997/1998) or three (DK696, DK757 and Exp980 during 1998/1999) hybrids. At Pergamino, sowing took place on 10 October 2000, and treatments were a factorial combination of: (i) two row spacings (0.35 and 1 m), (ii) two hybrids (DK696 and DK664), and (iii) two pollination treatments. This experiment was conducted using a constant plant population of 10 plants m 2. In all experiments, treatments were arranged in a split split-plot design with three replicates. At Salto, row spacing was the main factor, plant population the sub-factor and hybrids the sub-sub-factor. Each plot was ®ve (1997/1998) or 12 (1998/1999) rows, 0.7 m apart, and 20 m long. At Pergamino row spacing was the main factor, hybrids the sub-factor and the pollination treatment the sub-sub-factor. At this site, each plot was 12 rows 13 m long. Plots were hand-planted at three seeds per hill, and thinned to the desired plant population at the three-leaf (ligulated leaves) stage (V3). Rows always had an east±west orientation. In order to minimize N restrictions, urea was applied at V4 (200 kg N ha 1). Plots were kept free of weeds, insects, and diseases. Water stress was prevented by means of furrow (at Salto) or sprinkler (at Pergamino) irrigation, with the soil near ®eld capacity throughout the growing season. Mean air temperature and solar radiation were recorded daily at each experimental site. Pollination treatments (restricted and natural pollination) were performed at Pergamino in order to alter

15

the source±sink ratio during the grain-®lling period. At least six plants per each hybrid  row spacing  pollination treatment combination were tagged at random 15 days before silking, and were individually identi®ed. The date of silking (®rst silks visible) of the apical and sub-apical ears was registered for each tagged plant. Only a proportion of all exposed silks was pollinated in the restricted pollination treatment (see BorraÂs and Otegui, 2001, for details). This treatment was performed by bagging apical ears 2 days after they silked in order to decrease the number of pollinated ovaries. When present, all sub-apical ears were bagged prior to their silking to prevent pollination. The open-pollinated plants were never bagged. 2.2. Leaf area senescence dynamic The development of leaf area senescence was characterized for the whole cycle at Salto and during the grain-®lling period at Pergamino. At Salto, ®ve successive plants were tagged in the central row of each plot. Tags were placed at identi®ed positions along the stem (e.g. between leaves 3 and 4), which allowed the identi®cation of individual leaves (ln) and the determination of total leaf number. Tags were ®rst placed at V3 between l3 and l4, and then moved upward at V10 (placed between l8 and l9) and at anthesis (placed exactly below the ear leaf), in order to ease counting. At Pergamino, plants were tagged at anthesis, with tags placed below the ear leaf. The number of senesced leaves was recorded weekly for all tagged plants. A leaf was considered senesced when half or more of its area had yellowed. Lamina length (L) and maximum lamina width (W) were registered, and used to calculate the area of a leaf (A) as in Montgomery (1911; Eq. (1)): A ˆ aLW

(1)

where a ˆ 0:75. Leaf area senescence per plant was estimated as the sum of the area of all senesced leaves, and was related to thermal time (TT; base temperature 8 8C) from sowing (Ritchie and NeSmith, 1991). Senescence dynamic was analyzed for: (i) the absolute value of all senesced leaf area, and (ii) values relative to maximum green leaf area (the sum of the areas of all green leaves 15 days after silking) within each treatment combination. In a previous work on modeling of leaf

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senescence in maize (Muchow and Carberry, 1989), an exponential function was ®tted to the data. A bilinear model (Eqs. (2) and (3)) was preferred in the present study, because it gives information on: (i) changes in senescence rate along the cycle, and (ii) the ontogenic stage when these changes take place: Leaf area senescence per plant ˆ a‡b TT for

TT  c (2)

Leaf area senescence per plant ˆ a ‡ bc ‡ d…TT-c† for TT > c

(3)

where a is the intercept, b and d the slopes of the ®rst (Phase 1) and second phase (Phase 2) of leaf senescence, and c the breakpoint (in TT) between both phases. The ®tting of the models was performed using the iterative optimization technique in Table Curve V 3.0 (Jandel, 1992). Differences among treatments for the slopes (b and d) were compared by ANOVA and a t test (Steel and Torrie, 1960). The breakpoints (c) were compared with the con®dence interval of the parameter (P < 0:05). 2.3. Leaf area senescence and source±sink ratio Plant population and pollination treatments were performed to establish functional relationships between the source±sink ratio during the grain-®lling period and leaf senescence. The plant source±sink ratio was estimated as the quotient between green leaf area per plant 15 days after silking and kernel number per plant at physiological maturity. Kernel number per plant was counted for all harvested ears (apical and sub-apical ears) at physiological maturity. Leaf senescence rate in Phase 2 (coef®cient d from Eq. (3)) was correlated to the post-¯owering source±sink ratio. 2.4. Leaf area senescence and protein yield The relationship between N demand by kernels set in each treatment and leaf senescence was also analyzed. Kernel protein concentration was measured at physiological maturity at the second experiment in Salto (1998/1999) and at the experiment in Pergamino. Protein concentration was analyzed by nearinfrared re¯ectance (Infratec 1227, Tecator, Sweden) as described in BorraÂs et al. (2002). Protein yield per

plant was calculated as grain yield multiplied by protein concentration (on a dry weight basis). Protein yield per plant was expressed as the absolute value (g per plant), or as a fraction (%) of the absolute maximum value achieved in each year  hybrid combination. 2.5. Leaf area senescence and light environment The light environment beneath maize canopies was characterized after the maximum green leaf area was attained. Light attenuation was estimated from the incoming and transmitted photosynthetically active radiation (IPAR, TPAR) at different canopy layers. Transmitted PAR was measured at: (i) two leaves below the topmost leaf, (ii) immediately below the ear leaf, (iii) three leaves below the ear leaf, and (iv) below all green leaves but above senesced leaves. Five independent measurements were made at each canopy layer within each plot, between 1100 and 1400 h on clear days, with 1 m of a line quantum-sensor (LI191SA, LI-COR, Lincoln, NE). These measurements were made diagonally across the rows (crops at 0.35 and 0.70 m between rows) or perpendicular to the rows (crops at 1 m between rows), in order to ®t the sensor bar between one (crops at 1 m between rows), two (crops at 0.70 m between rows), or three (crops at 0.35 m between rows) inter-row spaces. Thus, width under measurement included: (i) one row of plants matching the middle of the sensor bar in crops grown at 0.70 and 1 m, or (ii) two rows of plants in those grown at 0.35 m (Maddonni et al., 2001). Light attenuation within the fully developed canopy was related to green leaf area index (GLAI) at different canopy layers and an exponential function (FleÂnet et al., 1996) was ®tted (Eq. (4); Jandel, 1992). The GLAI was estimated as the sum of the green leaf area per unit land area occupied by the plants at each plant population: TPAR ˆe IPAR

k GLAI

(4)

where k is the light attenuation coef®cient. Measurements of the red:far-red ratio within the canopy were also performed 15 days after silking using a two channel radiometer (Model SKR 110, Skye Instruments, Powys, UK) with a cosine-corrected head and narrow band ®lters centered at 660

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(red) and 730 nm (far-red). The sensor was connected to a hand-held meter for direct instantaneous readout. The red:far-red ratio was measured at the apical ear position. The sensor was placed at the mid-distance between plants in the row (west side) and in the middle of the inter-row (south side), with its sensing surface facing upwards. Six measurements per plot were performed between 1200 and 1300 h on clear days. The red:far-red ratio at the apical ear position was related to GLAI at the corresponding canopy layer, and an exponential function was ®tted (Eq. (5); Jandel, 1992): Red : far-red ratio ˆ a e

b GLAI

(5)

where a estimates red:far-red ratio arriving to a horizontal surface when canopy does not exist and b the attenuation coef®cient of red:far-red ratio when light penetrates within a canopy. Differences among treatments for the attenuation coef®cient (b) were compared with the con®dence interval of the parameter (P < 0:05). 3. Results Meteorological conditions differed among years (Fig. 1). The 1997/1998 growing season was characterized by intermediate air temperatures, and the lowest daily irradiance values registered during the last 30 years due to a strong El NinÄo phase of the ENSO phenomenon. Contrarily, maize crops cultivated during 1998/1999 experienced higher (ca. 10%) irradiance levels and lower air temperatures than the previous season. During 2000/2001, irradiance values were intermediate between the other growing seasons, but air temperatures were the highest. 3.1. Plant population and pollination treatment effects on the post-¯owering source±sink ratio At the Salto experiments, row spacing produced no differences in the variables under study, so data were pooled for the two (0.35 and 0.7 m) row spacings within each plant population. Plant populations produced a wide range of values for each tested variable (Table 1). Increased plant population was accompanied by a drastic decrease in leaf area per

Fig. 1. Solar radiation and mean air temperature as a function of thermal time from sowing (base temperature 8 8C) of maize crops during 1997/1998 (dotted line), 1998/1999 (heavy solid line) and 2000/2001 (thin solid line). The horizontal lines indicate the silking period of crops.

plant, grain yield per plant and kernel number per plant. The proportion of change, however, varied among variables and depended upon the plant population range examined. Between 3 and 9 plants m 2, each additional plant promoted a larger reduction in grain yield (5.7±6.8% per plant) and kernel number (4.2±5.9% per plant) than in leaf area (2±2.7% per plant). In contrast, a further increase in plant population (from 9 to 12 plants m 2) promoted a larger reduction in plant leaf area (4±6% per plant) than in grain yield (0±5.3% per plant) or kernel number (0±4% per plant). Kernel weight also decreased signi®cantly (P < 0:05) in response to increased plant population, but: (i) the magnitude of the response was smaller (maximum of 3.3% per additional plant) than for the other variables, and (ii) the reduction in kernel weight was not larger between 3 and 9 plants m 2 (0.17±1.8% per plant) than between

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Table 1 Leaf area, grain yield and its components, source±sink ratio 15 days after silking, and kernel protein content (concentration in percent and absolute yield per plant) of maize plants cultivated at three plant populations at Salto (values are the mean of two row spacings)a Experiment

1997/1998

1998/1999

Hybrid

Plant Maximum population green leaf area (plants m 2) (cm2 per plant)

Grain yield (g per plant)

Kernel number per plant

Kernel weight (mg)

Source± sink ratiob (cm2 per grain)

DK757

3 9 12

8767 a 6653 b 5578 c

239 a 118 b 109 b

1063 a 611 b 619 b

226 a 194 b 175 b

8.3 b 10.9 a 9.1 b

DK696

3 9 12

8403 a 6807 b 5536 c

239 a 120 b 121 b

910 a 566 b 541 b

263 a 220 b 222 b

9.2 b 12.1 a 10.2 b

DK757

3 9 12

8668 a 6648 b 5211 c

260 a 119 b 78 c

1258 a 619 b 456 c

204 a 192 b 172 c

DK696

3 9 12

8059 a 6596 b 5161 c

260 a 121 b 88 c

923 a 512 b 408 c

Exp980

3 9 12

9014 a 7278 b 5808 c

282 a 110 b 88 c

1153 a 547 b 446 c

Grain proteinc %

g per plant

7.2 b 10.7 a 11.2 a

9.5 a 10.0 a 6.1 b

24.0 a 12.0 b 4.7 c

264 a 235 b 214 c

9.3 b 13.0 a 12.7 a

11.5 a 10.7 a 6.7 b

29.5 a 12.7 b 5.8 c

223 a 200 b 197 c

7.8 b 13.3 a 13.2 a

7.7 a 8.2 a 6.8 b

21.5 a 8.8 b 5.7 c

a Different letters within a column, an experiment and a hybrid indicate signi®cant (P < 0:05) differences between plant population treatments. b Expressed as the ratio between plant green leaf area 15 days after silking and ®nal kernel number per plant. c Protein content was not determined in the 1997/1998 experiment.

9 and 12 plants m 2 (0±3.3% per plant), like observed for kernel number and grain yield. As a result of the differential effect of increased plant population on plant leaf area and kernel number per plant, the post-¯owering source±sink ratio was always signi®cantly (P < 0:05) smaller at 3 than at 9 plants m 2 (Table 1). Source±sink ratio values were stable between 9 and 12 plants m 2 for the 1998/1999 experiment (i.e. a plateau-type response), but decreased for the 12 plants m 2 treatment in 1997/1998 (i.e. an optimum-type response). Pollination treatments imposed soon after silking modi®ed kernel number per plant in all treatment combinations (P < 0:05; Table 2). The restricted pollination treatment reduced kernel number per plant approximately 50% compared to natural pollination, helped established a twofold increase in the source± sink ratio. Thus, the controlled pollination performed at Pergamino allowed a larger shift in the post-¯owering source±sink ratio than the plant population treatments imposed at Salto.

3.2. Leaf area senescence dynamic Leaf area senescence started around 400± 450 8C day from sowing in all treatment combinations at the Salto experiments (Fig. 2). At this site, both growing seasons exhibited a similar senescence progress in time, which was well described as a two-rate (i.e. bilinear) process. The ®rst phase (Phase 1) of this process proceeded at a very low rate (0.84±2.03 cm2 per plant per 8C per day; Table 3) until ca. 100 8C day after silking, and was followed by a second phase (Phase 2) of high senescence rate (2.24±7.84 cm2 per plant 8C per day; Table 3) until the last measurement at physiological maturity. Crops at 3 plants m 2 usually presented signi®cantly (P < 0:05) lower senescence rates in Phase 2 than crops at the other plant populations, except for no signi®cant effect of plant population for DK757 (Table 3). Row spacing had no effect on senescence rate. Leaf senescence dynamic of the pollination treatments at Pergamino was similar to the above-mentioned

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Table 2 Leaf area, grain yield and its components, source±sink ratio 15 days after silking, and kernel protein content (concentration in percent and absolute yield per plant) of maize plants sown at two row spacings (0.35 and 1 m) and a constant plant population of 10 plants m 2 in the 2000/2001 experiment at Pergaminoa Hybrid

DK664

DK696

a b

Row spacing (m)

Pollination treatment

Maximum green leaf area (cm2 per plant)

Grain yield (g per plant)

Kernel number per plant

Kernel weight (mg)

Source± sink ratiob (cm2 per grain)

Grain protein %

g per plant

0.35

Natural Restricted

5821

105 a 61 b

468 a 197 b

225 b 310 a

12.4 b 29.5 a

6.3 c 11.3 a

6.7 b 6.7 b

1

Natural Restricted

6043

121 a 77 b

515 a 242 b

233 b 320 a

11.7 b 26.5 a

7.2 b 11.8 a

8.8 a 9.1 a

0.35

Natural Restricted

5597 b

94 ab 71 b

469 a 257 b

200 b 283 a

11.9 b 21.8 a

6.2 b 9.8 a

6.0 b 6.9 ab

1

Natural Restricted

6480 a

115 a 89 ab

527 a 305 b

219 b 294 a

12.3 b 21.2 a

7.2 b 10.9 a

8.3 ab 9.6 a

Different letters within a column and a hybrid indicate signi®cant (P < 0:05) differences between row spacing and pollination treatments. Expressed as the ratio between plant green leaf area 15 days after silking and ®nal kernel number per plant.

Table 3 Parameters of the model ®tted to leaf senescence evolution of maize plants cultivated at different plant populations, row spacings, and pollination treatments in different experiments (the process was described by a bilinear model with two slopes (Phase 1 and Phase 2 senescence rates) and a breakpoint (thermal time from sowing) between the slopes) Experiment

Leaf senescence ratea

Breakpoint (8C day)

R2, n

7.8 5.8 5.9

1275 a 998 b 955 b

0.97, 34 0.96, 34 0.96, 34

1.05 b 1.44 ab 2.03 a

2.6 b 7.9 a 6.9 a

1119 1136 1143

0.94, 34 0.97, 34 0.96, 34

Natural

0.84 1.27 1.43

2.2 c 6.0 a 4.3 b

974 919 810

0.97, 16 0.99, 16 0.97, 16

10 10 10 10

Natural Restricted Natural Restricted

1.43 1.44 0.78 0.82

7.7 2.7 10.0 4.1

b c a c

1429 1308 1489 1413

0.99, 0.62, 0.99, 0.95,

7 7 7 7

10 10 10 10

Natural Restricted Natural Restricted

1.38 1.17 0.87 0.78

6.1 6.9 10.3 7.5

b b a ab

1403 1398 1478 1478

0.98, 0.99, 0.99, 0.96,

7 7 7 7

Row spacing (m)

Plant population (plants m 2)

Pollination

DK757

0.35±0.70

3 9 12

Natural

1.28 1.72 1.86

DK696

0.35±0.70

3 9 12

Natural

1998/1999

Exp980

0.35±0.70

3 9 12

2000/2001

DK664

0.35

1997±1999

Hybrid

1 DK696

0.35 1

Phase 1 (cm2 per plant per 8C per day)

Phase 2 (cm2 per plant per 8C per day)

a Different letters within a column and a hybrid indicate signi®cant (P < 0:05) differences between stand densities (1997±1999 and 1998/ 1999 experiments) or row spacing  pollination treatments (2000/2001 experiment).

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Fig. 2. Leaf area senescence progress for three maize hybrids (DK696, DK757 and Exp980) cultivated at three plant populations (circles: 3 plants m 2; triangles: 9 plants m 2; squares: 12 plants m 2) and two row spacings (empty symbols: 0.35 m; full symbols: 0.70 m). Data set for DK696 and DK757 included two growing seasons (1997/1998 and 1998/1999). For Exp980, only one experiment was carried out (1998/1999). The dotted lines represent the bilinear model ®tted to each plant population treatment. Arrows indicate silking date.

pattern described for the hybrid  plant population treatments (Table 3). Restricted pollination, which enhanced assimilate availability per kernel during the whole grain-®lling period, always reduced senescence rate in Phase 2 for DK664. This response of senescence rate to the increased source availability only took place under wide-row cropping for DK696 (Table 3). At

Pergamino, Phase 2 always started at around 450 8C day after silking, independently of the post-¯owering source±sink ratio (Table 2). Differences in the breakpoint between control pollination and plant population experiments should be taken with care, because the onset of senescence evaluation differed between them. Since senescence records did not start until anthesis in the control pollination experiment, the few data available to ®t the slope during Phase 1 could force the iterative process to set the beginning of Phase 2 at a delayed TT. The consequence of this mathematical artifact would be a skewed estimate of the actual breakpoint. Differences in relative senescence rate in Phase 2 were signi®cantly explained (r 2 ˆ 0:58; P < 0:05) by the post-¯owering source±sink ratio only for the pollination treatments. Conversely, plant population effects on this ratio (Table 1) had no effect on senescence rate in Phase 2 (Fig. 3). For the plant population treatments, senescence rate in Phase 2 was signi®cantly (P < 0:001) and positively related to senescence rate in Phase 1 (Fig. 4). In order to build relative values for Figs. 3 and 4, data from different plant populations were pooled together after values of leaf area senescence per plant were normalized by the corresponding maximum green leaf area record (Table 1). The relationship between senescence rates was not constant, and a signi®cant (P < 0:01) difference in the response was established between growing seasons (Fig. 4). Crops grown under reduced incident solar radiation during 1997/1998 (Fig. 1) had a larger relative senescence rate in Phase 2 than those cropped during 1998/1999. The relationship between Phase 1 and Phase 2 senescence rates canceled out when the source±sink ratio was drastically augmented by means of kernel number reduction through pollination control (Tables 2 and 3). 3.3. Leaf area senescence and protein yield Increased plant population always promoted a signi®cant (P < 0:05) decrease in protein yield per plant (Table 1). The magnitude of the reduction was very similar for all hybrids, and ranged between 50 and 59% from 3 to 9 plants m 2, with an additional 14±30% decrease when plant population was further raised to 12 plants m 2. Percent protein content, however, did not follow exactly the same trend, and a signi®cant (P < 0:05) reduction in this trait was only

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Fig. 3. Relationship between relative senescence rate in Phase 2 and post-¯owering source±sink ratio (estimated as the ratio between green leaf area per plant on silking ‡ 15 days and ®nal kernel number per plant) for all treatment combinations. Genotypes DK696, DK757 and Exp980 cultivated at three plant populations (circles: 3 plants m 2; triangles: 9 plants m 2; squares: 12 plants m 2) are shown in full symbols, and DK696 and DK664 cultivated at 10 plants m 2 are shown as an empty rhombus. The wide post-¯owering source±sink ratio range of DK696 and DK664 at 10 plants m 2 was obtained by means of pollination control (natural and restricted). The full line shows the relationship between both variables for the pollination treatment data (r2 ˆ 0:57; P < 0:05).

detected at the highest plant population. In contrast to these results, protein yield of plants with a reduced number of kernels from the restricted pollination treatment was similar to that obtained with control

plants (Table 2). Percent protein, on the other hand, was always larger for the former than for the latter. As increased plant population reduced grain protein yield per plant and also increased the relative

Fig. 4. Relationship between relative senescence rates. Relative rates in Phase 1 and Phase 2 were determined by a bilinear model ®tted to the progress of relative senescence (i.e. leaf senescence expressed as a fraction of maximum green leaf area) for each data set (circles: 3 plants m 2; triangles: 9 plants m 2; squares: 12 plants m 2). The dotted line indicates the function ®tted to the 1997/1998 data set (empty symbols): senescence rate in Phase 2 ˆ 0:01 ‡ 6:2 senescence rate in Phase 1 (r2 ˆ 0:92; n ˆ 12; P < 0:01). The solid line indicates the function ®tted to the 1998/1999 data set (full symbols): senescence rate in Phase 2 ˆ 0:03 ‡ 2:4 senescence rate in Phase 1 (r2 ˆ 0:60; n ˆ 18; P < 0:01).

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Fig. 6. Response of light composition (i.e. red:far-red ratio of light reaching the ear leaf layer) to the LAI above the apical ear (circles: 3 plants m 2; triangles: 9 plants m 2; rhombus: 10 plants m 2; squares: 12 plants m 2). Empty symbols: rows 0.35 m apart; full symbols: rows 0.70 and 1 m apart. Lines represent the function described in Eq. (5). For 0.35 m, red:far-red ratio ˆ 1:66 e( 0.44 GLAI) (r2 ˆ 0:87; n ˆ 17; P < 0:001). For 0.70 and 1 m, red:far-red ratio ˆ 1:30 e( 0.27 GLAI) (r 2 ˆ 0:89; n ˆ 17; P < 0:001).

Fig. 5. Response of relative senescence rate in Phase 2 to relative protein yield per plant for three maize hybrids cultivated at different plant populations and row spacings. Circles (3 plants m 2), triangles (9 plants m 2), and squares (12 plants m 2), in empty (rows 0.35 m apart) and full (rows 0.70 m apart) symbols, correspond to the plant population  row spacing experiments. Diamonds (rows 0.35 m apart) and rhombus (rows 1 m apart) in empty (restricted pollination) and full (natural pollination) symbols correspond to the row spacing  pollination experiment. Lines represent the ®tted functions. For DK696, senescence rate in Phase 2 ˆ 0:14 0:12 protein yield (r 2 ˆ 0:81; n ˆ 10; P < 0:001). For DK757, senescence rate in Phase 2 ˆ 0:12 0:09 protein yield (r2 ˆ 0:81; n ˆ 6; P < 0:05). For Exp980, senescence rate in Phase 2 ˆ 0:10 0:08 protein yield (r2 ˆ 0:85; n ˆ 6; P < 0:01).

senescence rate during Phase 2, senescence rates were always negatively (P < 0:01) correlated to grain protein yield per plant (Fig. 5). A similar trend between protein yield and senescence rate in Phase 2 was observed when these variables were modi®ed through

kernel set manipulation. Kernels with increased assimilate availability were always heavier than the control ones (P < 0:05) and also had a higher protein concentration in all treatment combinations (P < 0:05; Table 2). So, plants with the reduced kernel number per plant not only yielded the same amount of protein per plant, but also had a higher green leaf area at physiological maturity due to a reduced senescence rate during Phase 2 (Table 3). 3.4. Leaf area senescence and light environment The light environment perceived by plants was affected by plant population and row spacing. Increased plant population promoted a reduction in light quantity within the canopy, and this effect was re¯ected in estimates obtained for the light attenuation coef®cient (k ˆ 0:43, 0.55, 0.53 and 0.65 for 3, 9, 10 and 12 plants m 2, respectively). Crops cultivated at 0.35 and 0.70 m between rows presented similar light attenuation values, except (P < 0:05) for those cultivated at 3 plants m 2 (k ˆ 0:37 and 0.49 for 0.35 and 0.70 m, respectively). When crops were sown at even more contrasting row spacing (0.35 vs. 1 m), however, signi®cant differences (P < 0:05) were detected in light attenuation within the canopy of

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hybrid DK696. For this genotype, rows at 0.35 m had a larger light attenuation than rows at 1 m (k values of 0.61 and 0.35, respectively). Plant population affected the light quality perceived by plants at the lowest leaf stratum. There was a consistent reduction in the red:far-red ratio at the ear level (Fig. 6) whenever plant population was increased. The reduction in red:far-red ratio through the canopy was less pronounced in wide-rows compared to narrow rows (P < 0:05) at plant populations 9 plants m 2 (Fig. 6). When genotypes DK664 and DK696 were sown at 1 m between rows, the red:farred attenuation was reduced (36 and 62% for DK664 and DK696, respectively) compared to the 0.35 m row spacing. 4. Discussion The development of leaf area senescence registered from early measurements along the crop cycle revealed that the initiation of the process is highly conservative. Senescence was always initiated at around 400±450 8C day from sowing, independently of the growing environment perceived by plants. Highly contrasting plant populations, row spacings and irradiance values did not modify the early ontogenic stage (ca. V6±V9) at which senescence of the lowermost leaves started. This stage took place well before signi®cant light interception (Otegui et al., 1996) and shading among plants (Maddonni et al., 2001) usually occur. Our results are in agreement with those obtained for other maize genotypes cultivated under diverse sowing dates (Muchow and Carberry, 1989) or phosphorus availabilities (Colomb et al., 2000), which promoted a wide range in maximum leaf area. These evidences, therefore, indicate that leaf senescence is not only a programmed process on a cell or an individual leaf basis (Dangl et al., 2000), but also at a whole plant level, where the initiation of the process is genetically controlled (NoodeÂn et al., 1997). Like for senescence initiation, treatments had no effect on the TT for the start of active senescence rate (i.e. Phase 2). Most tested conditions had the onset of Phase 2 between silking and 400 8C day after silking, independent of plant population, row spacing or assimilate availability per kernel. Only one hybrid (DK757) exhibited a delay of the breakpoint when

23

sown at 3 plants m 2, which was the response expected a priori for all hybrids at this growing condition (i.e. almost isolated plants with direct sunlight on all leaves). Lack of response of the breakpoint to plant population in the other hybrids suggests that this trait is strongly regulated by the shift in assimilate partitioning that takes place around silking (Uhart and Andrade, 1991), and not only by the amount of light that reaches a leaf. Estimates of the breakpoint between both senescence phases obtained in this work matched the onset of active ear growth at silking (Otegui and Bonhomme, 1998) or of the effective grain-®lling period soon after silking (Maddonni et al., 1998; BorraÂs and Otegui, 2001). The stable estimate of the breakpoint obtained in the pollination experiment, on the other hand, suggests that the source±sink ratio had no effect on the shift from slow to active leaf senescence. The onset of an active assimilate demand from a large reproductive sink (i.e. small source±sink ratio) soon after pollination did not accelerate the start of active leaf senescence. Since senescence presented a preferential acropetal progression (data not shown), as usually described in literature (Ceppi et al., 1987; Pearson and Jacobs, 1987), differences in senescence rates between Phase 1 and Phase 2 re¯ected the areas of the individual leaves comprised in each phase. The vertical pro®le of the area of mature leaves in maize plants is normally described by a bell-shaped function (Dwyer and Stewart, 1986), with small leaves at the extremes of the plant and large leaves at the middle of the stalk. The amplitude of the bell is determined by the area of the largest leaf, which is always located 1 or 2 leaves immediately below the one containing the apical ear (Dwyer et al., 1992; Maddonni and Otegui, 1996). Due to this leaf area distribution along the plant, senescence during Phase 1 progressed at a low rate because it involved the eight lowermost leaves from a total of at least 20 in the hybrids under study. Contrarily, senescence during Phase 2 involved leaves with larger areas and shorter longevity (Colomb et al., 2000) than those included in Phase 1, determining greater senescence rates. The most stressful conditions for plant growth (e.g. high stand densities, low irradiance) determine a decreased leaf longevity (Colomb et al., 2000), which promotes increased senescence rate like observed during the second phase of this process in our experiments

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at high plant populations. These results are in agreement with previous ®ndings obtained in different grain species, which determined enhanced leaf senescence for crops grown under N (Eik and Hanway, 1965; Pearson and Jacobs, 1987) or phosphorus (Colomb et al., 2000) de®ciencies, water de®cit (Muchow and Carberry, 1989; Sadras et al., 1991), high plant populations (Eik and Hanway, 1965), or increased air temperature (Slafer and Miralles, 1992). Intra-speci®c competition promoted by increased plant population sets a gradual type of stress that intensi®es along the cycle due to progressive interplant interference related to plant growth. This condition was re¯ected in the high correlation established between senescence rates of both phases in this study. The relationship between leaf senesce rates cancelled out when plants grown under similar pre-¯owering conditions were subjected to a sudden shift in the post-¯owering source±sink ratio (i.e. natural and restricted pollination treatments). Reduced senescence rate in Phase 2, registered on maize plants with restricted kernel set, indicated that leaf senescence of maize plants cultivated at a high stand density (10 plants m 2) was accelerated by assimilate starvation (Tollenaar and Daynard, 1982). Restricted pollination that reduced kernel set decreased the grain N demand coming from leaf remobilization (Reed et al., 1988; Uhart and Andrade, 1995), and increased assimilate availability for sinks other than grains. It is known that increased carbon supply to the roots increase N uptake during grain ®lling (Pan et al., 1995; Uhart and Andrade, 1995), which has an important contribution in maize total N uptake (Pearson and Jacobs, 1987). Plants grown at a high plant population of 10 plants m 2 that were subjected to a 50% reduction in kernel number per plant con®rmed this hypothesis. They exported to the grains the same amount of N per plant than those under natural pollination, but presented a lower senescence rate during grain ®lling than the latter, suggesting a higher N uptake during this period. Plants growing at the lowest plant population (3 plants m 2) con®rmed this conceptual frame, because they presented the highest protein yields and highest protein concentration and had the lowest senescence rate during Phase 2. These trends at 3 plants m 2 were independent of the post-¯owering source±sink ratio (i.e. green leaf area per growing kernel), suggesting that N availability was not a limiting factor in this treatment.

Our results are in agreement with previous ®ndings (BorraÂs et al., 2002) showing that only when grain protein concentration is maximized there is an accumulation of N in organs other than kernels at physiological maturity. Although plant population permitted a wide range in maximum plant leaf area and kernel number per plant, it slightly modi®ed the post-¯owering source±sink ratio expressed as plant green leaf area per kernel. Similar results were obtained when the source±sink ratio was quanti®ed as plant weight gain per kernel during the effective grain-®lling period of crops cultivated at 3 and 9 plants m 2 (BorraÂs and Otegui, 2001). Consequently, acclimation of maize plants to the environment imposed by increased plant population did not produce a wide range of post-¯owering source±sink ratio, as expected a priori for this treatment. A 300% plant population increase (3±9 plants m 2) not only did not reduce the post-¯owering source±sink ratio but increased it (11±36%). Leaf senescence rate, however, was increased (ca. 100%) whenever plant population was augmented. Thus, differences in senescence rates between plant populations were not correlated to the corresponding post-¯owering source±sink ratios. During the grain-®lling period, less irradiance penetrated within the different leaf strata at plant populations 9 plants m 2. Light quality at the lowermost leaf stratum was also enriched in far-red radiation, a light signal which modulates leaf senescence in several species (Varlet-Grancher and Gautier, 1995; Rousseaux et al., 1999). Results of the plant population experiments suggested that the senescence process during the effective grain-®lling period was more related to the local light environment perceived by leaves than to the photosynthetically active leaf area per kernel (i.e. post-¯owering source±sink ratio) established at the onset of this period. This is based on the fact that senescence was increased when light quantity and quality at the lowermost leaf strata were reduced as plant population increased. We could isolate the effect of both light signals on leaf senescence only for plants cultivated at 9 and 12 plants m 2. For these plant populations, plants cultivated in wide rows (0.70 m) perceived a higher red:far-red ratio (ca. 0.40) at the ear leaf stratum than those cultivated in narrow rows (ca. 0.20), despite the similar light attenuations. Senescence rate during Phase 2, however, was similar for plants cultivated at wide or narrow rows within the

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same plant population. These results suggest that at plant populations 9 plants m 2 light quality did not affect the senescence process. In summary, senescence of maize leaves at a plant level is a complex process regulated by both internal and external factors. Our results gave further evidence of the modulating effect of the environment perceived by plants on leaf senescence, which on the other hand did not control the temporal frame of the process. The onset of this process seems to be genetically controlled. Among the cultural practices analyzed in our work, plant population had the largest effect on senescence progress, especially on senescence rate increase during Phase 2. But again, growing conditions had no effect on the onset of this second phase. Increased plant population imposed an assimilate starvation condition and an impoverished light environment (i.e. low light quantity) perceived by plants along the whole growing period. Plants less limited by the light source, as those at a low plant population, probably partitioned more carbohydrates to roots enhancing post-¯owering nitrogen uptake and diminishing senescence progression during this period. Acknowledgements This work was partially supported by Dekalb-Monsanto Argentina and the Agencia Nacional de PromocioÂn Cientõ®ca y TecnoloÂgica (PICT-99 No. 0806608). L. BorraÂs held a grant from, and G.A. Maddonni and M.E. Otegui are members of CONICET, the Research Council of Argentina. References Andrade, F.H., Otegui, M.E., Vega, C.R.C., 2000. Intercepted radiation at ¯owering and kernel number in maize. Agron. J. 92, 92±97. BorraÂs, L., Otegui, M.E., 2001. Maize kernel weight response to post¯owering source±sink ratio. Crop Sci. 41, 1816±1822. BorraÂs, L., CuraÂ, J.A., Otegui, M.E., 2002. Maize kernel composition and post-¯owering source±sink ratio. Crop Sci. 42, 781±790. Ceppi, D., Sala, M., Gentinetta, E., Verderio, A., Motto, M., 1987. Genotype-dependent leaf senescence in maize. Plant Physiol. 85, 720±725. Christensen, L.E., Below, F.E., Hageman, R.H., 1981. The effects of ear removal on senescence and metabolism of maize. Plant Physiol. 68, 1180±1185.

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