Plant material and growth conditions

Corn hybrids of Gwangpyeongok (hereafter, GWPOK) and P3394, a popular Korean normal corn F1 hybrid were planted at the experimental field of National Institute of Crop Science in Suwon (37.2N, 126.9E) on 1 May 2015. Plants were grown at a density of 66,666 plants per ha with 60 cm row spacing. The plots were shaded using a black shade net at 10 days after silk emergence when kernels begin to fill on the cob (Tollenaar, 1977). The shade was removed every 7 days that days of CS was 7, 14, 21, and 28 ones. Control plots were not shaded. Shades intercepted 65.7% incident light. Air temperature was measured every hour in the control and the shade plot using a thermistor sensor (TR-71U, T&D Corp., Japan). Daily mean air temperature in the shade was lower by about 1.05°C than that in the control. Interestingly, air temperature during the day (7 a.m. to 6 p.m.) was much lower by about 2.55°C in the shade than in the control but that during the night (7 p.m. to 6 a.m.) was rather higher by about 0.43°C in the shade than in the control (Fig. 1). A poor sunshine day less than 1 hour happened three times intermittently during the 28 days of shade (data not shown), which was neglected. Every shading plots were paired with the control neighboring each other. A plot consisted of four rows of each corn hybrid. Fertilizers were applied at 72.5-30-60 kg ha^-1 of N-P₂O₅-K₂O as a basal fertilizer, respectively and was 72.5kg ha^-1 of nitrogen was additionally applied at V6.

Fig. 1

Diurnal air temperature change in the control and in the shade.

Plant growth measurement at removal of shade

For the uppermost ear leaf, the ratio of leaf length to width and the SPAD value were measured using a chlorophyll meter (SPAD-502 Plus, Konica Minolta, Inc., Japan) at every 7 day removal of shade. Stem length and width, number of leaves were also measured at every 7 day removal of shade. Four plants were sampled from the shade and the control at every 7 day removal of shade, respectively. Aerial parts of the plants were separated into organs such as stem including leaf sheath, leaves, ear, and tassel and oven-dried at 60°C for 4 days. The dry matter of each organ was weighed for every plant. Leaf area was measured with a leaf area meter (LI-3100C, Li-Cor, Inc., Nebraska, USA).

The ratio of ear dry matter to biomass and the ratio of ear dry matter to leaf area were calculated for determining dry matter partitioning between source and sink organs. The relative growth rate (hereafter, RGR), the net assimilation rate (hereafter, NAR), the specific leaf area (hereafter, SLA), and leaf area ratio (LAR) were calculated for plant growth analysis as described by Atwell et al. (1999).

RGR (g g−1d−1)=(lnW2−lnW1)÷(t2−t1)

NAR (g m-2d-1) ={(W2 -W1)÷(t2 -t1)}×{(lnA2 -lnA1)÷(A2 -A1)}LAR (m-2g-1) = A÷WbiomassSLA(m-2g-1) = A÷Wleaf

where, W₁ and W₂ are total plant dry matter at t₁ and t₂ of given time, respectively. A₁ and A₂ are leaf area at t₁ and t₂ of given time, respectively. A, W_biomass and W_leaf are leaf area, total plant dry matter, and leaf dry matter at given time, respectively.

Grain yield components measurement at maturity

Nine or ten of the uppermost ears at maturity were harvested and husked at each plot. The rows per ear and kernels per row were counted to determine the maximum number of kernels per ear. Tip-fill length was measured. All these measurements were performed before oven-dry. Grains were harvested from each oven-dried ear by hand. The filled kernels were counted and weighed for every plant. The 100-grain weight was calculated. The percentage of the filled kernels was calculated by dividing the number of the filled kernels by the maximum kernel number (rows per ear × kernels per row).

Plant yield and harvest index determination

Nine or ten corn plants were sampled at each plot at maturity. Plant yield for biomass (total aerial parts), ear, and grain was determined on the basis of dry weight, respectively. Harvest index on the basis of grains was calculated by dividing plant grain yield by plant biomass yield.

Statistical analysis

All data were expressed mean ± standard error calculated using SAS statistical software package (ver. 9.2, SAS Institute, USA). The ANOVA procedure was used to compare the differences among treatments. If F-test for interaction effect between hybrid and shading treatment was not significant (p>0.05), data were pooled over corn hybrids. Otherwise, data from each corn hybrid were presented separately. Means among treatments were compared by Duncan’s Multiple Range Test (α=0.05). Analysis of simple regression and simple correlation was performed. Grain yield components and plant yield were fitted using Sigma Plot (ver. 10.0, Systat Software, Inc., USA).

The probit analysis calculates maximum likelihood estimates of regression parameters and the natural response rate for quantal response data from biological assays (Bliss, 1934; SAS Institute Inc. 2008). Probit analysis was performed to estimate the effect of the consecutive days of shade on plant yield.

All statistical analyses were done using SAS statistical software package (ver. 9.2, SAS Institute, NC, USA).

Effect of shading treatment on the growth of stem and leaf

Shading treatment at 10 days after tassel emergence, the beginning of kernel filling stages, was imposed for 7, 14, 21, and 28 days, respectively. When each shading treatment was removed, stem length and width, the ratio of length to width and SPAD value of the uppermost ear leaf, and the number of leaves and total leaf area per plant were measured, respectively (Fig. 2 and 3).

Fig. 2

Stem length (A) and width (B) in the control (0) and in plants receiving 7, 14, 21, and 28 days of consecutive shade. Error bar is standard error (n = 5 or 6).

Fig. 3

Number of leaves (A), leaf area (B), and SPAD value and ratio of length to width of the uppermost leaf in the control (0) and in plants receiving 7, 14, 21, and 28 days of consecutive shade. Error bar is standard error (n = 5 or 6).

For both corn hybrids, their stem length and width were not significantly changed by shade, even for consecutive 28 days of shade, compared to the control plants (Fig. 2). Like stem, shading treatment had no impact on the number of leaves and leaf area with variations (Fig. 3A and 3B). The SPAD value of the ear leaf was not also affected by shading treatment (Fig. 3C). Ratio of length to width of the ear leaf was not significantly different among all the shading treatments and the control (Fig. 3D). These results indicate that shading treatment at the beginning of kernel filling stages give rise to little impact on the growth of stem and leaves, the vegetative organs.

Effect of shading treatment on the dry matter accumulation

Dry weight of stem, leaves, ear, and the aboveground parts (hereafter, biomass) were measured when each shading treatment was removed, respectively (Fig. 4). There was no interaction effect between two factors, corn hybrid and shading treatment.

Fig. 4

Dry matter accumulation of leaf (A), stem (B), ear (C), and total plant biomass (D) in the control (0) and in plants receiving 7, 14, 21, and 28 days of consecutive shade. Error bar is standard error (n = 4).

Shading treatment even for 28 days of CS didn't cause any significant change in dry weight of the leaves and the stem of both corn hybrids, Gwangpyeongok and P3394 (Fig. 4A and 4B), indicating that shade at the beginning of kernel filling stages has little effect on dry matter accumulation into stem and leaves.

However, dry matter accumulation in the ear was severely reduced by about 28, 44, and 53% at the 14-, 21-, and 28-day shading treatment, compared with the controls, respectively (Fig. 4C). Dry matter accumulation in the biomass was severely reduced by about 25 and 33% at the 21- and 28-day shading treatment, compared with the controls, respectively (Fig. 4D). These data showed that reduction in dry matter accumulation of the biomass by shade at the beginning of the kernel filling stages was due to reduction in that of the ear.

These results suggested that shade at the beginning of kernel filling stages have a stronger and faster effect on the dry matter accumulation in the ear than the biomass.

Effect of shading treatment on source-sink relations

The ratio of ear to biomass, and ratio of ear to leaf area, relative growth rate (RGR), net assimilation rate (NAR), specific leaf area (SLA), and leaf area ratio (LAR) were calculated (Fig. 5, Fig. 6, and Table 1), respectively.

Fig. 5

Ratio of ear dry matter to total plant biomass (A) and leaf area (B) in the control (0) and in plants receiving 7, 14, 21, and 28 days of consecutive shade. Error bar is standard error (n = 4).

Fig. 6

Leaf area ratio (A) and specific leaf area (B) in the control (0) and in plants receiving 7, 14, 21, and 28 days of consecutive shade. Error bar is standard error (n = 4).

The ratio of ear to biomass represents a portion of total biomass taken by ear (Fig. 5A). It was significantly reduced by shading treatment even for 7 days. Shading treatment for 7, 14, 21, and 28 days caused about 11, 23, 26, and 31% decrease in the ratio of ear to biomass, respectively (Fig. 5A). The ratio of ear to leaf area is the dry matter accumulation in the ear per unit leaf area (Fig. 5B). Like the ratio of ear to biomass, shade led to the reduction in it by about 15% for 7 days, 41% for 14 days, 46% for 21 days, and 53% for 28 days, respectively (Fig. 5B). These results indicated that partitioning of photosynthate into the ear may decrease under shade.

The RGR is a prominent indicator of plant strategy with respect to productivity as related to environmental stress. By separate measurement of leaf, stem and ear mass as well as leaf area, good insight into the components underlying growth variation can be obtained. These underlying parameters are related to allocation of photosynthate into the corresponding organs (Pooter, 2013). The RGR is factored into the LAR and the NAR. The former is the amount of leaf area per unit total plant mass, and the latter is the rate of increase in plant mass per unit leaf area (Lambers et al., 2008).

For the first 2 weeks after shading initiation, the RGR of the ear and the biomass was 0.127 and 0.032 g g^-1 d^-1 at the control and 0.111 and 0.030 g d^-1 at the shading treatment, respectively (Table 1). These results showed that the RGR of the ear was much greater than that of the biomass, particularly for the first 2 weeks after shading initiation (Table 1), indicating that ear growth is stronger than any other plant parts at the beginning of kernel filling stages when the shading initiation. For the first 2 weeks, shading treatment significantly reduced the RGR of the ear by about 13% and did that of the biomass by about 6% with no significance, compared with the control (Table 1). For the second 2 weeks, the RGR of the ear and the biomass was extremely lowered to 0.016 and 0.004 g g^-1 d^-1 by shade, respectively, causing about 65 and 84% decrease in the RGR of the ear and biomass, respectively, compared with the control (Table 1). These data suggested that the effect of shade at the beginning of kernel filling stages on the RGR of the ear and the biomass definitely appears for the second 2 weeks rather than the first 2 weeks.

The response of the NAR to shade was very similar to that of the RGR (Table 1). For the first 2 weeks after shading initiation, the NAR of the ear and the biomass was 9.38 and 9.11 g m^-2 d^-1 at the control and 5.75 and 7.40 g m^-2 d^-1 at the shading treatment. For the first 2 weeks, shading treatment significantly reduced the NAR of the ear by about 39% and did that of the biomass by about 19% with no significance, compared with the control (Table 1). For the second 2 weeks, the NAR of the ear and the biomass dramatically declined to 1.54 and 1.05 g m^-2 d^-1 by shade, respectively, causing about 84 and 90% reduction in the NAR of the ear and biomass, respectively, compared with the control (Table 1). These results showed that shade at the beginning of kernel filling stages similarly had an impact on the NAR of the ear and the biomass and did faster effect on the NAR of the ear than that of the biomass.

The SLA of the shaded plants for 14 days or longer was significantly higher by about 7~11% than that of the control ones (Fig. 6B), saying that leaf thickness decreased. It is in agreement with the previous report (Samarraie et al., 1990). They showed that 15 days shading treatment during flowering stage increased SLA by about 35%. Many authors have demonstrated a negative relationship between SLA and light intensity (Blackman et al., 1955; Cooper, 1966; Gmeling Meyling, 1973; Pears and Lee, 1969; Danalatos et al., 1994). At high light intensities, the thickness of the leaves increases and therefore SLA decreases, because carbon fixation exceeds translocation of photosynthate (Danalatos et al., 1994).

The LAR is the ratio of leaf area to the total biomass. The LAR of the shaded plants for 14 days or longer was significantly higher than the control ones like SLA (Fig. 6A). The shaded plants for 14, 21, and 28 days showed about 27, 40, and 48% higher LAR than the control ones, respectively (Fig. 6A), which is due to the biomass reduction (Fig. 4D) with no change in leaf area (Fig. 3B). Samarraie et al. (1990) also reported the similar results to our study that 15 days shading treatment during flowering stage increased LAR by about 36%. Blackman and Wilson (1951) demonstrated that NAR increased with daily irradiance and LAR was greatly decreased in sunflower. The LAR of shade grown soybean plants increased by over 40% than that of control ones (Wu et al., 2016).

According to these responses of RGR and NAR, shade at the beginning of kernel filling stages causes severe reduction in biomass and ear growth mainly due to decrease in the net gain of carbon assimilate per unit leaf area between photosynthesis and respiration, implying that it has an impact on source activity.

Effect of shading treatment on grain yield components

When the primary corn ear reached the physiological maturity after removal of shading treatment, it was harvested. corn grain yield components such as row number per ear, kernel number per ear row, percent of the filled kernels, the filled kernels per ear, and one hundred grain weight were measured (Fig. 7 and 8). There was no interaction effect between shading treatments and corn hybrids for the all measured variables except for row number per ear and 100-grain weight.

Fig. 7

Ear row number per ear (A), kernel number per ear row (B), percent of filled kernels (C), and number of filled kernels per ear (D) in the control (0) and in plants receiving 7, 14, 21, and 28 days of consecutive shade. Error bar is standard error (n = 20 and n = 38 for the shade and the control, respectively).

Fig. 8

One-hundred-grain weight in the control (0) and in plants receiving 7, 14, 21, and 28 days of consecutive shade. Error bar is standard error (n = 20 and n = 38 for the shade and the control, respectively).

Shade at the beginning of kernel filling stages had little or a little impact on the row number per ear (Fig. 7A). The response of the row number per ear to the CS was a little different between two corn hybrids, P3394 and GWPOK. In GWPOK, it was slightly but consistently decreased by about 6 to 11% with statistical significance at the all shading treatments (Fig. 7A). In P3394, however, it was not significantly reduced even for 28 days of shade, compared with the control (Fig. 7A).

On the other hand, kernel number per ear row and the percent of the filled kernels logistically declined with the length of CS increased (Fig. 7B and 7C). The kernel number per ear row significantly decreased by about 14%, compared with that of the control for 7 days of shade. This decrease did not get any more with the length of CS increased (Fig. 7B). The percent of the filled kernels, however, was reduced by about 19 and 25% for 14 and 28 days of shade, respectively.

The number of the filled kernels is the function of the row number per ear, kernel number per ear row and the percent of the filled kernels. Thus it also declined logistically with the length of CS increased (Fig. 7D). For consecutive 7 days of shade, it was significantly reduced by about 31%, compared with that of the control. This reduction did not significantly get any more with the length of CS increased (Fig. 7D).

The response of 100-grain weight to the CS was definitely different between two corn hybrids (Fig. 8). In both hybrids, it was not affected by shade for the first 2 weeks after shading initiation, but for the second 2 weeks was reduced by about 30% only in P3394 (Fig. 8). Interestingly, the 100-grain weight of Gwangpyeongok was a little affected with no statistical significance even at the longest shading treatment. It is to be investigated further in the future study. Several authors (Reed et al., 1988; Uhart and Andrade, 1991) also demonstrated the similar results that shade during kernel filling stages reduced 100-grain weight. The shades intercepted 50% incident light throughout the kernel filling stages (onset of linear kernel dry matter accumulation to physiological maturity) reduced the 100-grain weight by about 12.8% (Reed et al., 1988).

In summary, the 7 or 14 days of shade did a substantial and irreparable damage to kernel number per ear row, percent of the filled kernels and number of filled kernels per ear but did a restorable effect on the 100-grain weight after removal of shading treatment. The row number per ear was not significantly affected by shade.

Effect of shading treatment on plant yield

Plant yield was measured based on biomass, grain, and ear, respectively. There was no interaction effect between shading treatment and corn hybrids. Plant yield data were pooled over corn hybrids. Plant yield of biomass, grain, and ear logistically declined with the length of the CS increased (Fig. 9A, 9B and 9C). Harvest index acted on the length of CS in similar manner to plant ear and grain yield (Fig. 9D).

Fig. 9

Plant yield of biomass (A), ear (B), and grain (C), and harvest index (D) in the control (0) and in plants receiving 7, 14, 21, and 28 days of consecutive shade. Error bar is standard error (n = 20 and n = 38 for the shade and the control, respectively).

The corn plants shaded at the beginning of kernel filling stages got an irreversible damage on these all kinds of plant yield even for consecutive 7 days of shade (Fig. 9). Biomass yield gradually decreased with the CS increased, which was reduced by about 14 and 28% at the 14- and 28-day CS, respectively (Fig. 9A). The yield of ear and grain dramatically declined by about 32 and 34% at the 7-day CS, respectively (Fig. 9B and 9C). The ear and the grain yield at the 28-day CS were reduced by about 52 and 50%, respectively (Fig. 9B and 9C). Harvest index abruptly decreased by about 24 and 35% at the 7- and 28-day CS (Fig. 9D), respectively, indicating that dry matter partitioning into kernels is not to be recovered even for 7-day shade at the beginning of kernel filling stages.

Probit analysis was carried out to estimate how the length of consecutive days of shade has an impact on these three kinds of plant yield. The days of CS to cause 25 and 50% reduction in plant yield (hereafter, RD₂₅ and RD₅₀) was obtained from probit analysis, respectively. The results are summarized in Table 2.

The RD₂₅ for the plant yield of biomass, ear, and grain was 22.5, 4.0, 3.7 days of CS, respectively (Table 2). During these corresponding periods, every single day of the shade at the beginning of kernel filling stages caused about 1.1, 5.9, and 6.3% loss in plant yield of biomass, ear, and grain, respectively (Table 2). The RD₅₀ for the plant yield of biomass was not to be estimated because it escaped from the given sample period of 28 days of CS (Table 2). The RD₅₀ for the plant yield of ear and grain was 21.6 and 23.1 days of CS, respectively (Table 2). During these corresponding periods, every single day of the shade caused about 1.4 and 1.3% loss in plant yield of ear and grain, respectively (Table 2). These results suggested that shade during kernel filling stages have a severer impact on ear and grain yield than biomass and the plant yield loss may be achieved mainly for the first week of CS.