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Relationships Between C4 Enzyme Activities and Yield in Soybeans (Glycine max (L.) Merr.)
Journal of Integrative Agriculture
March 2013
2013, 12(3): 406-413
RESEARCH ARTICLE
Relationships Between C4 Enzyme Activities and Yield in Soybeans (Glycine max (L.) Merr.) HUANG Shan-shan1*, LI Chang-suo1, 2*, YANG Ming-liang1, LI Wen-bin1 and WANG Ji-an1 Key Laboratory of Soybean Biology, Ministry of Education/Soybean Research Institute, Northeast Agricultural University, Harbin 150030, P.R.China 2 Plant Protection Institute, Heilongjiang Academy of Land Reclamation Sciences, Harbin 150038, P.R.China 1
Abstract To study the relationships between C4 enzyme activities and yield, C4 enzyme activities (phosphoenolpyruvate carboxylase (PEPCase), NADP-malate dehydrogenase (NADP-MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK)) in different organs of ten soybean cultivars with different yields were measured at different growth stages in China. The result showed that four enzyme activities in C4 pathway were obviously different among cultivars, especially PPDK activity was not detected in the leaves of Dongnong 1567 and Dongnong 1068 and the young leaves of Gongjiao 9107-1 and Dongnong 97-172, but there were weak activities in pod coats. The order of C4 enzyme activities is young leaves < old leaves < pod coats. The correlation coefficients between PEPCase activity and yield and between NADP-MDH activity at blooming stage and yield were 0.6979 and 0.6565, respectively, and both reached the significant level (5%), and PEPCase activity kept significant positive correlation with plant photosynthetic rate. There was a negative correlation between NADP-ME activity and yield, and no correlation was found between PPDK activity and yield. Key words: soybean, photosynthetic rate, yield, C4 enzyme
INTRODUCTION Land plants can be divided into three major photosynthetic types: C3, C4, and crassulacean acid metabolism (CAM) plants based on the differences in the mechanism of CO2 assimilation. In C3 plants, CO2 is fixed as the C3 compound, phosphoglycerate. This reaction is inhibited up to 50% when atmospheric O2 competes with CO2 at the active site of RuBPCase, and the resulting fixed O2 wastes energy via the process of photorespiration (Matsuoka et al. 1998). However, as a morphological and biochemical innovation (Hatch and Slack 1966), the C4 photosynthetic pathway is proposed to have been an adaptation to hot, dry environments, and Received 2 May, 2012
CO2 deficiency (Cerling et al. 1997). In the case of lower CO2 concentration, through the enzyme system: PEPCase, NADP-MDH, NADP-ME, and PPDK, CO2 is effectively assimilated, and C4 pathway makes crops maintain higher carbon assimilation efficiency, which helps to improve the photosynthetic efficiency, and ultimately leads to increase crop yield. Therefore, many productive crops in agriculture use the C4 photosynthetic pathway, such as corns. However, some important crops in agriculture are C3 plants exhibiting a lower photosynthetic efficiency, such as soybeans (Makoto et al. 1998). Some scientists found that the division between C3 and C4 plants was not absolute, and there may be C4 pathway existing in C3 plants (Duffus 1973; Nutbeam 1976; Hibberd and Quick 2002; Svensson et al.
Accepted 9 October, 2012
HUANG Shan-shan, E-mail: [email protected]; Correspondence WANG Ji-an, Tel: +86-451-55190692, E-mail: [email protected] * These authors contributed equally to this study. © 2013, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S2095-3119(13)60240-3
Relationships Between C4 Enzyme Activities and Yield in Soybeans (Glycine max (L.) Merr.)
2003). It has been proposed that the key C4 enzyme, such as PEPCase and PPDK, were found at lower levels in C3 plants (Hata and Matsuoka 1987; Aoyagi and Chua 1988; Rosche et al. 1994). Some studies suggested that the evolution of C4 photosynthesis occurred through the recruitment and alteration of pre-existing genes in C3 plants (Rosche et al. 1994; Ku et al. 1996). Due to the high photosynthetic capacity of C4 pathway, many people expected to transfer the high photosynthetic capacity of C4 plants to C3 plants through a cross between C3 and C4 plants many years ago. Then there were many reports with discovering C4 enzyme genes (Sheen 1999; Brown et al. 2005; Nomura et al. 2005; Hibberd et al. 2008). Some attempts have been made to enhance the activities of C4 photosynthesis enzyme in C3 plants through recombinant DNA techniques (Hudspeth et al. 1992; Kogami et al. 1994; Gehlen et al. 1996). Although it has been reported that some key C4 enzyme genes were transferred into C3 plants, trying to improve the photosynthetic efficiency (Makoto et al. 1998), only a few were successful (Gehlen et al. 1996; Ku et al. 1999; Zhang et al. 2009). However, it is possible to screen cultivars with the higher expression of C4 enzyme from C3 plants, and make genetic improvement to enhance photosynthetic efficiency of C3 plants. In the past 50 yr, with improving soybean cultivars and production conditions, soybean production has changed very obviously in China, so that soybean yield was greatly increased. It has been not reported that whether or not C4 enzyme effects on soybean yield and what the relation between them is. Here, ten representative soybean cultivars, growing in a large area in the
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past 50 yr, were studied, from an enzymological perspective, to find out the rule of C4 enzyme in the process of soybean cultivars alternation. Four C4 enzyme activities were determined to further identify whether or not there were C4 enzyme activities in all tissues and organs, whether or not they were different among cultivars, and whether or not there were some relations between C4 enzyme activities and soybean yield, which will be important theory reference to further study on soybean high-yield mechanism and breed soybean cultivars with high photosynthetic efficiency.
RESULTS The relations among PEPCase, yield and photosynthetic rate PEPCase activities of ten cultivars were determined in field and in the pots at seed-filling stage. The result showed that there were significant differences in the cultivars (Table 1). The maximum difference of PEPCase activities in field was 0.034 mol mg-1 pro min-1, and the highest activity was about 2.3 times higher than that of the lowest. The variation range of PEPCase activities in different cultivars in the pots was 0.016-0.065 mol mg-1 pro min-1, and the highest activity was about 4.1 times higher than that of the lowest. The results showed that there were significant differences in different cultivars not only in field, but also in the pots. The correlation coefficients between PEPase activity and yield were 0.6979 in field and 0.6721 in the
Table 1 Relations with PEPCase activity, yield and photosynthetic rate Field Cultivar Gongjiao 9107-1 Dongnong 9339 Dongnong 1567 Heinong 39 Heinong 41 Dongnong 97-172 Dongnong 1068 Gongjiao 9404 Jilin 43 Jilin 47 Correlation coefficient
Photosynthetic rate (mol CO2 m-2 s-1) 12.31±0.452 11.54±0.252 12.25±0.426 13.16±0.211 11.08±0.764 10.36±0.339 8.57±0.320 10.23±0.199 12.38±0.423 13.25±0.339 0.9071 **
PEPCase activity (mol mg-1 pro min-1) 0.051±0.008 0.038±0.005 0.044±0.005 0.061±0.004 0.034±0.002 0.036±0.002 0.027±0.002 0.038±0.003 0.056±0.003 0.058±0.004 1.0000
Pot Yield (kg ha-1)
Photosynthetic rate (mol CO2 m-2 s-1)
PEPCase activity (mol mg-1 pro min-1)
Yield (g/pot)
2 516±7.211 2 347±19.287 2 319±8.888 2 529±8.185 2 421±15.133 2 641±22.113 2 127±11.533 2 346±7.937 2 634±22.517 2 689±10.817 0.6979 *
14.17±0.303 16.25±0.358 15.38±0.161 11.23±0.111 13.49±0.459 10.15±0.530 10.08±0.391 10.12±0.111 13.24±0.062 15.13±0.079 0.7369 *
0.028±0.004 0.065±0.002 0.061±0.006 0.018±0.001 0.041±0.004 0.028±0.006 0.032±0.007 0.016±0.002 0.023±0.003 0.034±0.008 1.0000
60.2±0.953 62.6±3.835 69.8±1.389 61.3±1.345 58.2±1.709 53.6±2.488 56.2±1.308 52.7±2.207 57.9±1.153 63.6±1.136 0.6721 *
** , 0.01 significant level; *, 0.05 significant level. Values are means±SE. The same as below.
© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.
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pots, respectively, and both reached the significant level (5%) (Fig. 1). PEPCase was the important C4 enzyme. The cultivars containing higher PEPase activity had a higher photosynthetic rate. The correlation coefficients between PEPCase activity and photosynthetic rate were 0.9071 in field and 0.7369 in the pots, and reached extremely significant and significant level, respectively (Fig. 2).
PEPCase activity definition in soybean leaves at different growth stages PEPCase activities of ten cultivars were determined at seedling, blooming, pod-setting, seed-filling, and maturity stages (Table 2). As a whole, the mean of PEPCase activity was increased, however, it was substantially decreased at maturity stage (Fig. 3). PEPCase activity at seed-filling stage was the highest 0.0443 mol mg-1 pro min-1. The more and less differences of PEPCase activity in the cultivars were at seedling, maturity, and podsetting stages, respectively. Those indicated that the more vigorous reproduction, the smaller differences of
Fig. 1 Correlation between PEPCase activity and yield in field (A) and in the pots (B) at seed-filling stage.
HUANG Shan-shan et al.
PEPCase activity. The maximum difference in the cultivars was 0.0354 (173.5%), and the lowest and highest PEPCase activity were in Dongnong 1068 (0.0204) and Heinong 39 (0.0558), respectively (Table 2). In short, PEPCase was districted in soybean leaves at each stage, and with growth stages developing, its activity gradually increased. At early maturity stage, PEPCase activities of some cultivars were increased, but others were decreased. There was no regularity, which would be related with inner physiological of the samples. Because there were significant differences of PEPCase activity in the cultivars, it was possible to select soybean cultivars with high PEPCase activity.
PEPCase activities in pod coats and leaves at seed-filling stage PEPCase activities in young leaves, old leaves, and pod coats at seed-filling stage were determined (Table 3). The highest and lowest PEPCase activities were in pod coats and young leaves, respectively. PEPCase activities among cultivars changed very obviously. PEPCase activities were detected in young leaves of eight cultivars except for Dongnong 9339 and Dongnong 1068, and the differences in young leaves, old leaves, and pod coats were 0.02 (0.006-0.026), 0.034 (0.027-0.061), and 0.036 (0.047-0.083), respectively. PEPCase activity in pod coats was the highest, and the difference in different cultivars was significant. PEPCase activity was not detected in the young leaves of Dongnong 9339 and Dongnong 1068. PEPCase activity at seedling stage was the lowest, which indicated there was higher PEPCase activity in the older green organs at vigorous reproduction stage.
NADP-ME activities in pod coats and leaves at seed-filling stage
Fig. 2 Correlation between PEPCase activity and photosynthetic rate in field (A) and in the pots (B) at seed-filling stage.
The results showed the highest and the lowest NADPME activity were in pod coats and young leaves, respectively. There was the maximum difference of NADP-ME activity in pod coats of cultivars. The highest NADP-ME activity was about 1.5 times higher than the lowest. The cultivars containing the lowest and highest NADP-ME activities were young leaves of Dongnong 97-172 (0.21) and Jinlin 43 (0.93), old leaves of
© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.
Seedling Blooming Podsetting Seedfilling Maturity Average Seedling Blooming Podsetting Seedfilling Maturity Seedling Blooming Podsetting Seedfilling Maturity
PEPCase
1)
Dongnong 1567 0.021±0.002 0.035±0.003 0.034±0.004 0.044±0.003 0.031±0.003 0.0330 0.09±0.021 0.73±0.034 1.18±0.013 1.68±0.057 0.73±0.018 0.02±0.001 0.22±0.038 0.17±0.017 0.06±0.003 0.17±0.025
Dongnong 9339
0.013±0.004 0.018±0.004 0.025±0.002 0.038±0.003 0.054±0.004 0.0296 0.14±0.011 0.82±0.055 1.04±0.011 1.75±0.013 0.42±0.015 0.07±0.006 0.31±0.022 0.21±0.024 0.12±0.011 0.21±0.023
Gongjiao 9107-1
0.025±0.004 0.027±0.004 0.031±0.002 0.051±0.004 0.039±0.003 0.0346 0.26±0.0024 1.26±0.022 1.31±0.042 1.21±0.009 0.86±0.029 0.05±0.007 0.28±0.028 0.26±0.021 0.07±0.009 0.14±0.004
The unit of enzyme activity in this study is mol mg-1 pro min-1.
NADP-MDH
NADP-ME
Growth stage
C4 enzyme
Dongnong 97-172 0.016±0.002 0.023±0.003 0.026±0.002 0.036±0.003 0.031±0.003 0.0264 0.21±0.015 0.68±0.029 1.17±0.026 1.03±0.018 0.87±0.054 0.05±0.005 0.27±0.019 0.14±0.011 0.09±0.011 0.23±0.022
Heinong 41 0.017±0.003 0.027±0.003 0.028±0.002 0.034±0.004 0.054±0.004 0.032 0.17±0.018 0.54±0.044 0.94±0.028 1.47±0.016 0.46±0.026 0.13±0.016 0.31±0.030 0.33±0.014 0.17±0.020 0.18±0.012
Heinong 39 0.058±0.006 0.046±0.004 0.048±0.002 0.061±0.004 0.066±0.002 0.0558 0.28±0.023 0.87±0.035 1.42±0.013 1.31±0.032 1.01±0.027 0.06±0.009 0.34±0.014 0.27±0.028 0.13±0.012 0.26±0.015
Table 2 PEPCase, NADP-ME, and NADP-MDH activities of the leaves of the cultivars at different growth stages1) 0.003±0.0004 0.018±0.001 0.037±0.004 0.027±0.003 0.017±0.002 0.0204 0.07±0.009 0.78±0.018 1.36±0.036 1.22±0.017 0.94±0.035 0.02±0.002 0.23±0.028 0.12±0.022 0.07±0.009 0.14±0.011
Dongnong 1068
Jilin 47 0.014±0.002 0.028±0.002 0.036±0.004 0.058±0.005 0.041±0.002 0.0354 0.27±0.022 1.35±0.015 1.82±0.013 1.07±0.009 1.43±0.005 0.14±0.011 0.32±0.021 0.26±0.013 0.18±0.018 0.27±0.011
Jilin 43 0.026±0.003 0.034±0.002 0.039±0.003 0.056±0.009 0.053±0.008 0.0416 0.18±0.019 1.07±0.051 1.27±0.024 1.34±0.026 0.88±0.028 0.11±0.017 0.35±0.018 0.23±0.007 0.16±0.016 0.36±0.032
Ongjiao 9404 0.015±0.001 0.023±0.002 0.039±0.002 0.038±0.003 0.024±0.002 0.0278 0.26±0.009 1.23±0.033 1.54±0.173 1.46±0.028 1.03±0.018 0.07±0.011 0.27±0.031 0.17±0.016 0.13±0.011 0.31±0.013 0.193 0.933 1.305 1.354 0.863 0.072 0.290 0.216 0.118 0.227
0.208 0.279 0.343 0.443 0.410
Average
Relationships Between C4 Enzyme Activities and Yield in Soybeans (Glycine max (L.) Merr.) 409
Fig. 3 PEPCase activity at different growth stages.
Dongnong 97-172 (1.03) and Dongnong 9339 (1.75), pod coats of Gongjiao 9107-1 (1.36) and Dongnong 9339 (2.09), respectively (Table 3). Therefore, there may be carbon assimilation in old leaves and pot coats. There was a negative relationship between NADP-ME activity and yield, and correlation coefficients were -0.1015 (in young leaves), -0.5256 (in old leaves), and -0.4986 (in pod coats), respectively. They did not reach a significant level, so NADP-ME was not a necessary factor to increase soybean yield.
NADP-ME activities in cultivars at different growth stages
The highest and the lowest NADP-ME activities appeared at seed-filling and seedling stages, respectively (Table 2). The differences of NADP-ME activity among cultivars were significant at each growth stage: 0.21 at seedling stage, 0.81 at blooming stage, 0.88 at podsetting stage, 0.72 at seed-filling stage, and 1.01 at early mature stage. There was a significant difference between any two stages at whole growth period, except for pod-setting and seed-filling stages.
NADP-MDH activities in pod coats and leaves at seed-filling stage
The highest and the lowest NADP-MDH activities appeared in pod coats and young leaves, respectively. NADP-MDH activity in pod coats was about 1.8 and 3.4 times higher than that in old and young leaves, respectively. It seemed that there was a higher NADPMDH activity in the older tissues. The differences of NADP-MDH activity among cultivars were very different: 0.11 in young leaves, 0.10 in old leaves, and
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HUANG Shan-shan et al.
PPDK
0.32±0.003 0.27±0.002 0.16±0.004 0.38±0.005 0.41±0.005 0.27±0.024 0.11±0.007 0.24±0.009 0.37±0.013 0.41±0.011 0.294
NADP-MDH
0.15±0.022 0.22±0.034 0.26±0.006 0.24±0.029 0.31±0.043 0.22±0.012 0.17±0.002 0.21±0.006 0.18±0.009 0.35±0.008 0.231 0.076±0.004 0.063±0.007 0.052±0.003 0.071±0.003 0.058±0.006 0.047±0.002 0.081±0.003 0.074±0.003 0.083±0.002 0.082±0.004 0.0687 0.04±0.003 0.25±0.006 0.24±0.003 0.17±0.005 0.31±0.003 0.21±0.003 0.24±0.005 0.18±0.003 0.164
NADP-MDH activities in cultivars at different growth stages There was NADP-MDH activity in ten cultivars at each growth stage (Table 2). As a whole, the lowest and highest NADP-MDH activities appeared at seedling and blooming stages, respectively. Then, it was slightly decreased after blooming stage until early maturity stage. From Table 2, the correlation coefficient between NADP-MDH activity at blooming stage and yield could be calculated (0.6565, a significant level). There was also a trend of positive correlation at other stages, but that was not significant.
0.08±0.003 0.12±0.008 0.09±0.002 0.13±0.008 0.17±0.003 0.12±0.005 0.08±0.004 0.13±0.004 0.16±0.008 0.18±0.004 0.126 1.21±0.011 1.75±0.063 1.68±0.101 1.31±0.024 1.47±0.044 1.03±0.031 1.22±0.055 1.46±0.038 1.34±0.019 1.07±0.026 1.354
There was not PPDK activity in issues of some cultivars, such as young and old leaves of Dongnong 1567 and Dongnong 1068 (Table 3), however, their yield was very high (Table 1). PPDK activity was also not detected in the young leaves of Gongjiao 91071 and Dongnong 97-172, which showed that PPDK activity was very low, evens no appearance. Hence, PPDK activity could only exit in older tissues and organs. Therefore, there were important C4 enzyme in soybeans, but not all appeared in the leaves of all cultivars, and they appeared in not all of tissues and organs. Hence, there were some conditions for C4 enzyme activity to appear and express in soybeans. However, it was possible to screen soybean cultivars containing high C4 enzyme activities.
0.051±0.005 0.038±0.002 0.044±0.004 0.061±0.007 0.034±0.002 0.036±0.003 0.027±0.002 0.038±0.001 0.056±0.004 0.058±0.002 0.0443
DISCUSSION
-, not been measured out.
0.017±0.002 0.023±0.001 0.011±0.002 0.026±0.003 0.008±0.0004 0.013±0.001 0.007±0.001 0.0006
0.16±0.027 0.02±0.003 0.04±0.006 0.06±0.004 0.04±0.005 0.03±0.003 0.035 0.04±0.005 0.07±0.005 0.04±0.003 0.06±0.002 0.08±0.004 0.06±0.004 0.02±0.003 0.07±0.004 0.10±0.009 0.13±0.004 0.067 0.35±0.023 0.85±0.034 0.38±0.025 0.76±0.051 0.84±0.032 0.21±0.013 0.33±0.024 0.88±0.058 0.93±0.033 0.24±0.029 0.577 0.006±0.0007 -
Gongjiao 9107-1 Dongnong 9339 Dongnong 1567 Heinong 39 Heinong 41 Dongnong 97-172 Dongnong 1068 Gongjiao 9404 Jilin 43 Jilin 47 Average
Pod coats
1.36±0.028 2.09±0.034 1.78±0.012 1.52±0.029 1.37±0.019 1.66±0.042 1.82±0.025 1.93±0.024 1.76±0.012 1.45±0.022 1.674
PEPCase PPDK NADP-MDH
Old leaves
NADP-ME PEPCase PPDK NADP-MDH
Young leaves
0.20 in pod coats. The correlation coefficient between NADP-MDH activity in pod coats and that in old leaves was 0.6818*, a significant level. Therefore, it was possible to screen cultivars containing high NADP-MDH activity by measuring its content in pod coats (Table 3).
PPDK activity
NADP-ME PEPCase
Cultivar
Table 3 PEPCase, NADP-ME, NADP-MDH, and PPDK activities in young leaves, old leaves, and pod coats at seed-filling stage
NADP-ME
410
In this study, the highest and the lowest PEPCase activities appeared in pod coats and young leaves, respectively. Many reviews summarized certain tissues containing relatively high PEPCase activity, for example immature barley pericarp (Duffus and Rosie 1973), the testa of pea seeds (Hedley et al. 1975) and the inner tissue of tomato fruits (Laval et al. 1977). Latzko and Kelly (1983) showed the photosynthetic tissues surrounding cereal grains and the pericarp of those grains consistently displayed PEPCase activity higher than that in leaves. In the pericarp of wheat, PEPCase activity was about four times higher than that in leaves (Wirth et al. 1977). In this study, PEPCase activity in pod coats was about six times higher
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Relationships Between C4 Enzyme Activities and Yield in Soybeans (Glycine max (L.) Merr.)
than that in young leaves. Because PEPCase is the key enzyme of C4 photosynthetic pathway, the cultivars containing higher PEPase activity should have higher photosynthetic rate (Jiao and Chollet 1991; Lepiniec et al. 1994; Chollet et al. 1996). In our study, the correlation coefficients between PEPCase activity and photosynthetic rate were 0.9071 in field and 0.7369 in the pots, respectively, and reached extremely significant and significant level, respectively (Fig. 2). There was also significant correlation between PEPCase activity and yield. Because there are significant differences of PEPCase activity among cultivars, it is possible that the soybean cultivars containing high PEPCase activity are selected out, which could be a indicator to breed soybeans with high yield and photosynthetic efficiency. There was a negative relationship between NADPME activity and yield, and it did not reach a significant level, so it was not a necessary factor to increase soybean yield. The correlation coefficient between NADP-MDH activity at blooming stage and yield was 0.6565, a significant level. There was also a trend of positive correlation at other stages, but that was not significant. In this study, PPDK activity in young leaves of some cultivars, even old leaves, was not detected, but it did not affect high-yield of the cultivars. In contrast to the high activities of PPDK in the green leaves of C4 and CAM plants, very low activity has been detected in C3 plants (Edwards et al. 1982; Meyer et al. 1982). Maybe, PPDK activity in soybeans was so low that it could not be detected at all in some cultivars. Simultaneously, the results also implied that there could be another photosynthetic process that was similar to C4 pathway in soybeans, but it was not indispensable. Both the highest activities of PEPCase and NADPME appeared at seed-filling stage, however, the highest NADP-MDH activity was at blooming stage. All C4 enzyme activities were the lowest at seedling stage, which indicated there were higher C4 enzyme activities in the older green organs at vigorous reproduction stage. Patel and Berry (2008) showed immature leaves of amaranth conduct C 3 photosynthesis, whereas the C 4 occurrences in mature leaves (Wang et al. 1992, 1993; Patel and Berry 2008), which are consistent with our results. In addition to green leaves,
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chloroplast-containing organs, such as green fruits, soybean pods, and rice panicles, had the potential of photosynthesis (Edwards and Warker 1983; Blanke and Lenz 1989). Hedley et al. (1975) also reported high PEPCase activity as well as RuBPCase activity was detected in soybean pods. In this study, C4 enzyme activities of pod coats were the highest, and the difference was significant, which was important for breeders. However, PPDK activity was very low, even no appearance in young leaves. Maybe, PPDK activity could only exit in older tissues and organs.
MATERIALS AND METHODS Plant materials Ten representative soybean cultivars were used in this study. Young leaves, old leaves, and pod coats were studied in field and in the pots at different growth stages (Marris et al. 1991). All samples were from the field in Xiangfang Experiment Station, Heilongjiang Province, China and the pots in Northeast Agricultural University, China. The seeds were weighed to measure yield, after 100 mature samples of each cultivar, from five sites through Punnett’s square, were artificially threshed. Photosynthetic rates were measured with three replications by CI-301PS photosynthesis system (CID, Inc., USA). At least three samples of a cultivar were measured at each growth stage (intensity of light PAR>1 200 mol m-2 s-1, relative humidity (RH) (70±5)%, CO2 concentration (350±10) ppm). The mean was taken as yield and photosynthetic rate of the cultivars.
Extraction and assay of PEPCase The extraction and assay of PEPCase were as already described (Parvathi et al. 2000; Chinthapalli et al. 2003). The samples were quickly extracted using a chilled mortar and pestle with 1 mL of extraction medium containing 100 mmol L-1 Tris-HCl (pH 7.3), 10 mmol L-1 MgCl2, 2 mmol L-1 K2HPO4, 1 mmol L-1 EDTA, 10% (v/v) glycerol, 10 mmol L-1 bmercaptoethanol, 10 mmol L-1 NaF, 2 mmol L-1 PMSF, 10 g mL-1 chymostatin, and 2% (w/v) insoluble PVP. The homogenate was centrifuged at 15 000×g for 5 min and the supernatant was used as “crude extract”. The procedure used for the purification of PEPCase was as described in detail by Gayathri et al. (2000). PEPCase activity was assayed by coupling to MDH and monitoring NADH oxidation at 340 nm in UV-721 spectrophotometer (Xinmao Co., China) at 30°C. The assay mixture contained 50 mmol L-1 Tris-HCl (pH 7.3), 5 mmol L-1 MgCl2, 0.2 mmol L-1 NADH, 2 U MDH, 2.5 mmol L-1 PEP, 10 mmol L-1 NaHCO3. 1 U of enzyme activity was the
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HUANG Shan-shan et al.
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capacity of the enzyme to catalyze the formation of 1 mol oxaloacetate min-1.
Extraction and assay of NADP-ME The samples were quickly extracted with 1 mL of extraction medium containing 100 mmol L-1 Tris-HCl (pH 7.6), 2 mmol L-1 EDTA, 1 mmol L-1 DTT, 5% (w/v) glycerol, 20% (w/v) insoluble polyvinylpyrrolidone and a set of protease inhibitors, and homogenate was centrifuged at 15 000×g for 5 min. The supernatant solution was collected and desalted using a 3.5 cm×15 cm column of Senhadex G-25 (Sigma, United States). NADP-ME activity was determined in UV-721 spectrophotometer at 30°C by monitoring NADPH production at 340 nm. The reaction mixture contained 5 mmol L-1 Tris-HCl (pH 7.5), 1 mmol L-1 MgCl2, 1 mmol L-1 Mn2+, 1 mmol L-1 EDTA, 0.33 mmol L-1 NADP, and 5 mmol L-1 L-malate. 1 U of enzyme activity was defined as the amount of enzyme that resulted in the production of 1 mol NADPH min-1 (Yuu et al. 2000).
Extraction and assay of NADP-MDH The samples were quickly extracted with 1 mL of extraction medium containing 0.1 mol L-1 Tris-HCl (pH 5.3), 50 mmol L-1 mercaptoethanol, 5 mmol L-1 MgCl2, and 5 mmol L-1 EDTA. The homogenate was centrifuged at 15 000×g for 5 min. The procedure used for the purification of MDH was as described in detail by Gayathri et al. (2000). The assay mixture contained 25 mmol L-1 Tris-HCl (pH 8.9), 1 mmol L-1 EDTA, l mmol L-1 NADP, 50 mmol L-1 malate, 20 mmol L-1 glutamate, 0.6 U mL-1 aminosuccinic acid aminotransferase. MDH activity was assayed in UV-721 spectrophotometer at 30°C at 340 nm.
Extraction and assay of PPDK The samples were quickly extracted using a mortar and pestle (as described above). The supernatant was used for assay of enzyme activity at 30°C in a buffer containing 100 mmol L-1 Tris-HCl (pH 8.0), 10 mmol L-1 MgCl2, 5 mmol L-1 NaHCO3, 0.1 mmol L-1 EDTA, 1.25 mmol L-1 pyruvate, 1.25 mmol L-1 ATP, 2.5 mmol L-1 KH2PO4, 5 mmol L-1 DTT, 2 U malate dehydrogenase (MDH), 0.2 mmol L-1 NADH, 6 mmol L-1 glucose-6-phosphate, and 0.3 U of purified PEPC. 1 U of PPDK activity corresponds to 1 mol of pyruvate converted min-1 at 30°C (Salahas et al. 1990).
Correlation analysis
Where, x, enzyme activity; y, yield/photosynthetic rate.
Acknowledgements This study was supported by the National Natural Science Foundation of China (30471092) and the Genetically Modified Organisms Breeding Major Projects, China (2009ZX08009089B).
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Relationships Between C4 Enzyme Activities and Yield in Soybeans (Glycine max (L.) Merr.)
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March 2013
2013, 12(3): 406-413
RESEARCH ARTICLE
Relationships Between C4 Enzyme Activities and Yield in Soybeans (Glycine max (L.) Merr.) HUANG Shan-shan1*, LI Chang-suo1, 2*, YANG Ming-liang1, LI Wen-bin1 and WANG Ji-an1 Key Laboratory of Soybean Biology, Ministry of Education/Soybean Research Institute, Northeast Agricultural University, Harbin 150030, P.R.China 2 Plant Protection Institute, Heilongjiang Academy of Land Reclamation Sciences, Harbin 150038, P.R.China 1
Abstract To study the relationships between C4 enzyme activities and yield, C4 enzyme activities (phosphoenolpyruvate carboxylase (PEPCase), NADP-malate dehydrogenase (NADP-MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK)) in different organs of ten soybean cultivars with different yields were measured at different growth stages in China. The result showed that four enzyme activities in C4 pathway were obviously different among cultivars, especially PPDK activity was not detected in the leaves of Dongnong 1567 and Dongnong 1068 and the young leaves of Gongjiao 9107-1 and Dongnong 97-172, but there were weak activities in pod coats. The order of C4 enzyme activities is young leaves < old leaves < pod coats. The correlation coefficients between PEPCase activity and yield and between NADP-MDH activity at blooming stage and yield were 0.6979 and 0.6565, respectively, and both reached the significant level (5%), and PEPCase activity kept significant positive correlation with plant photosynthetic rate. There was a negative correlation between NADP-ME activity and yield, and no correlation was found between PPDK activity and yield. Key words: soybean, photosynthetic rate, yield, C4 enzyme
INTRODUCTION Land plants can be divided into three major photosynthetic types: C3, C4, and crassulacean acid metabolism (CAM) plants based on the differences in the mechanism of CO2 assimilation. In C3 plants, CO2 is fixed as the C3 compound, phosphoglycerate. This reaction is inhibited up to 50% when atmospheric O2 competes with CO2 at the active site of RuBPCase, and the resulting fixed O2 wastes energy via the process of photorespiration (Matsuoka et al. 1998). However, as a morphological and biochemical innovation (Hatch and Slack 1966), the C4 photosynthetic pathway is proposed to have been an adaptation to hot, dry environments, and Received 2 May, 2012
CO2 deficiency (Cerling et al. 1997). In the case of lower CO2 concentration, through the enzyme system: PEPCase, NADP-MDH, NADP-ME, and PPDK, CO2 is effectively assimilated, and C4 pathway makes crops maintain higher carbon assimilation efficiency, which helps to improve the photosynthetic efficiency, and ultimately leads to increase crop yield. Therefore, many productive crops in agriculture use the C4 photosynthetic pathway, such as corns. However, some important crops in agriculture are C3 plants exhibiting a lower photosynthetic efficiency, such as soybeans (Makoto et al. 1998). Some scientists found that the division between C3 and C4 plants was not absolute, and there may be C4 pathway existing in C3 plants (Duffus 1973; Nutbeam 1976; Hibberd and Quick 2002; Svensson et al.
Accepted 9 October, 2012
HUANG Shan-shan, E-mail: [email protected]; Correspondence WANG Ji-an, Tel: +86-451-55190692, E-mail: [email protected] * These authors contributed equally to this study. © 2013, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S2095-3119(13)60240-3
Relationships Between C4 Enzyme Activities and Yield in Soybeans (Glycine max (L.) Merr.)
2003). It has been proposed that the key C4 enzyme, such as PEPCase and PPDK, were found at lower levels in C3 plants (Hata and Matsuoka 1987; Aoyagi and Chua 1988; Rosche et al. 1994). Some studies suggested that the evolution of C4 photosynthesis occurred through the recruitment and alteration of pre-existing genes in C3 plants (Rosche et al. 1994; Ku et al. 1996). Due to the high photosynthetic capacity of C4 pathway, many people expected to transfer the high photosynthetic capacity of C4 plants to C3 plants through a cross between C3 and C4 plants many years ago. Then there were many reports with discovering C4 enzyme genes (Sheen 1999; Brown et al. 2005; Nomura et al. 2005; Hibberd et al. 2008). Some attempts have been made to enhance the activities of C4 photosynthesis enzyme in C3 plants through recombinant DNA techniques (Hudspeth et al. 1992; Kogami et al. 1994; Gehlen et al. 1996). Although it has been reported that some key C4 enzyme genes were transferred into C3 plants, trying to improve the photosynthetic efficiency (Makoto et al. 1998), only a few were successful (Gehlen et al. 1996; Ku et al. 1999; Zhang et al. 2009). However, it is possible to screen cultivars with the higher expression of C4 enzyme from C3 plants, and make genetic improvement to enhance photosynthetic efficiency of C3 plants. In the past 50 yr, with improving soybean cultivars and production conditions, soybean production has changed very obviously in China, so that soybean yield was greatly increased. It has been not reported that whether or not C4 enzyme effects on soybean yield and what the relation between them is. Here, ten representative soybean cultivars, growing in a large area in the
407
past 50 yr, were studied, from an enzymological perspective, to find out the rule of C4 enzyme in the process of soybean cultivars alternation. Four C4 enzyme activities were determined to further identify whether or not there were C4 enzyme activities in all tissues and organs, whether or not they were different among cultivars, and whether or not there were some relations between C4 enzyme activities and soybean yield, which will be important theory reference to further study on soybean high-yield mechanism and breed soybean cultivars with high photosynthetic efficiency.
RESULTS The relations among PEPCase, yield and photosynthetic rate PEPCase activities of ten cultivars were determined in field and in the pots at seed-filling stage. The result showed that there were significant differences in the cultivars (Table 1). The maximum difference of PEPCase activities in field was 0.034 mol mg-1 pro min-1, and the highest activity was about 2.3 times higher than that of the lowest. The variation range of PEPCase activities in different cultivars in the pots was 0.016-0.065 mol mg-1 pro min-1, and the highest activity was about 4.1 times higher than that of the lowest. The results showed that there were significant differences in different cultivars not only in field, but also in the pots. The correlation coefficients between PEPase activity and yield were 0.6979 in field and 0.6721 in the
Table 1 Relations with PEPCase activity, yield and photosynthetic rate Field Cultivar Gongjiao 9107-1 Dongnong 9339 Dongnong 1567 Heinong 39 Heinong 41 Dongnong 97-172 Dongnong 1068 Gongjiao 9404 Jilin 43 Jilin 47 Correlation coefficient
Photosynthetic rate (mol CO2 m-2 s-1) 12.31±0.452 11.54±0.252 12.25±0.426 13.16±0.211 11.08±0.764 10.36±0.339 8.57±0.320 10.23±0.199 12.38±0.423 13.25±0.339 0.9071 **
PEPCase activity (mol mg-1 pro min-1) 0.051±0.008 0.038±0.005 0.044±0.005 0.061±0.004 0.034±0.002 0.036±0.002 0.027±0.002 0.038±0.003 0.056±0.003 0.058±0.004 1.0000
Pot Yield (kg ha-1)
Photosynthetic rate (mol CO2 m-2 s-1)
PEPCase activity (mol mg-1 pro min-1)
Yield (g/pot)
2 516±7.211 2 347±19.287 2 319±8.888 2 529±8.185 2 421±15.133 2 641±22.113 2 127±11.533 2 346±7.937 2 634±22.517 2 689±10.817 0.6979 *
14.17±0.303 16.25±0.358 15.38±0.161 11.23±0.111 13.49±0.459 10.15±0.530 10.08±0.391 10.12±0.111 13.24±0.062 15.13±0.079 0.7369 *
0.028±0.004 0.065±0.002 0.061±0.006 0.018±0.001 0.041±0.004 0.028±0.006 0.032±0.007 0.016±0.002 0.023±0.003 0.034±0.008 1.0000
60.2±0.953 62.6±3.835 69.8±1.389 61.3±1.345 58.2±1.709 53.6±2.488 56.2±1.308 52.7±2.207 57.9±1.153 63.6±1.136 0.6721 *
** , 0.01 significant level; *, 0.05 significant level. Values are means±SE. The same as below.
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pots, respectively, and both reached the significant level (5%) (Fig. 1). PEPCase was the important C4 enzyme. The cultivars containing higher PEPase activity had a higher photosynthetic rate. The correlation coefficients between PEPCase activity and photosynthetic rate were 0.9071 in field and 0.7369 in the pots, and reached extremely significant and significant level, respectively (Fig. 2).
PEPCase activity definition in soybean leaves at different growth stages PEPCase activities of ten cultivars were determined at seedling, blooming, pod-setting, seed-filling, and maturity stages (Table 2). As a whole, the mean of PEPCase activity was increased, however, it was substantially decreased at maturity stage (Fig. 3). PEPCase activity at seed-filling stage was the highest 0.0443 mol mg-1 pro min-1. The more and less differences of PEPCase activity in the cultivars were at seedling, maturity, and podsetting stages, respectively. Those indicated that the more vigorous reproduction, the smaller differences of
Fig. 1 Correlation between PEPCase activity and yield in field (A) and in the pots (B) at seed-filling stage.
HUANG Shan-shan et al.
PEPCase activity. The maximum difference in the cultivars was 0.0354 (173.5%), and the lowest and highest PEPCase activity were in Dongnong 1068 (0.0204) and Heinong 39 (0.0558), respectively (Table 2). In short, PEPCase was districted in soybean leaves at each stage, and with growth stages developing, its activity gradually increased. At early maturity stage, PEPCase activities of some cultivars were increased, but others were decreased. There was no regularity, which would be related with inner physiological of the samples. Because there were significant differences of PEPCase activity in the cultivars, it was possible to select soybean cultivars with high PEPCase activity.
PEPCase activities in pod coats and leaves at seed-filling stage PEPCase activities in young leaves, old leaves, and pod coats at seed-filling stage were determined (Table 3). The highest and lowest PEPCase activities were in pod coats and young leaves, respectively. PEPCase activities among cultivars changed very obviously. PEPCase activities were detected in young leaves of eight cultivars except for Dongnong 9339 and Dongnong 1068, and the differences in young leaves, old leaves, and pod coats were 0.02 (0.006-0.026), 0.034 (0.027-0.061), and 0.036 (0.047-0.083), respectively. PEPCase activity in pod coats was the highest, and the difference in different cultivars was significant. PEPCase activity was not detected in the young leaves of Dongnong 9339 and Dongnong 1068. PEPCase activity at seedling stage was the lowest, which indicated there was higher PEPCase activity in the older green organs at vigorous reproduction stage.
NADP-ME activities in pod coats and leaves at seed-filling stage
Fig. 2 Correlation between PEPCase activity and photosynthetic rate in field (A) and in the pots (B) at seed-filling stage.
The results showed the highest and the lowest NADPME activity were in pod coats and young leaves, respectively. There was the maximum difference of NADP-ME activity in pod coats of cultivars. The highest NADP-ME activity was about 1.5 times higher than the lowest. The cultivars containing the lowest and highest NADP-ME activities were young leaves of Dongnong 97-172 (0.21) and Jinlin 43 (0.93), old leaves of
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Seedling Blooming Podsetting Seedfilling Maturity Average Seedling Blooming Podsetting Seedfilling Maturity Seedling Blooming Podsetting Seedfilling Maturity
PEPCase
1)
Dongnong 1567 0.021±0.002 0.035±0.003 0.034±0.004 0.044±0.003 0.031±0.003 0.0330 0.09±0.021 0.73±0.034 1.18±0.013 1.68±0.057 0.73±0.018 0.02±0.001 0.22±0.038 0.17±0.017 0.06±0.003 0.17±0.025
Dongnong 9339
0.013±0.004 0.018±0.004 0.025±0.002 0.038±0.003 0.054±0.004 0.0296 0.14±0.011 0.82±0.055 1.04±0.011 1.75±0.013 0.42±0.015 0.07±0.006 0.31±0.022 0.21±0.024 0.12±0.011 0.21±0.023
Gongjiao 9107-1
0.025±0.004 0.027±0.004 0.031±0.002 0.051±0.004 0.039±0.003 0.0346 0.26±0.0024 1.26±0.022 1.31±0.042 1.21±0.009 0.86±0.029 0.05±0.007 0.28±0.028 0.26±0.021 0.07±0.009 0.14±0.004
The unit of enzyme activity in this study is mol mg-1 pro min-1.
NADP-MDH
NADP-ME
Growth stage
C4 enzyme
Dongnong 97-172 0.016±0.002 0.023±0.003 0.026±0.002 0.036±0.003 0.031±0.003 0.0264 0.21±0.015 0.68±0.029 1.17±0.026 1.03±0.018 0.87±0.054 0.05±0.005 0.27±0.019 0.14±0.011 0.09±0.011 0.23±0.022
Heinong 41 0.017±0.003 0.027±0.003 0.028±0.002 0.034±0.004 0.054±0.004 0.032 0.17±0.018 0.54±0.044 0.94±0.028 1.47±0.016 0.46±0.026 0.13±0.016 0.31±0.030 0.33±0.014 0.17±0.020 0.18±0.012
Heinong 39 0.058±0.006 0.046±0.004 0.048±0.002 0.061±0.004 0.066±0.002 0.0558 0.28±0.023 0.87±0.035 1.42±0.013 1.31±0.032 1.01±0.027 0.06±0.009 0.34±0.014 0.27±0.028 0.13±0.012 0.26±0.015
Table 2 PEPCase, NADP-ME, and NADP-MDH activities of the leaves of the cultivars at different growth stages1) 0.003±0.0004 0.018±0.001 0.037±0.004 0.027±0.003 0.017±0.002 0.0204 0.07±0.009 0.78±0.018 1.36±0.036 1.22±0.017 0.94±0.035 0.02±0.002 0.23±0.028 0.12±0.022 0.07±0.009 0.14±0.011
Dongnong 1068
Jilin 47 0.014±0.002 0.028±0.002 0.036±0.004 0.058±0.005 0.041±0.002 0.0354 0.27±0.022 1.35±0.015 1.82±0.013 1.07±0.009 1.43±0.005 0.14±0.011 0.32±0.021 0.26±0.013 0.18±0.018 0.27±0.011
Jilin 43 0.026±0.003 0.034±0.002 0.039±0.003 0.056±0.009 0.053±0.008 0.0416 0.18±0.019 1.07±0.051 1.27±0.024 1.34±0.026 0.88±0.028 0.11±0.017 0.35±0.018 0.23±0.007 0.16±0.016 0.36±0.032
Ongjiao 9404 0.015±0.001 0.023±0.002 0.039±0.002 0.038±0.003 0.024±0.002 0.0278 0.26±0.009 1.23±0.033 1.54±0.173 1.46±0.028 1.03±0.018 0.07±0.011 0.27±0.031 0.17±0.016 0.13±0.011 0.31±0.013 0.193 0.933 1.305 1.354 0.863 0.072 0.290 0.216 0.118 0.227
0.208 0.279 0.343 0.443 0.410
Average
Relationships Between C4 Enzyme Activities and Yield in Soybeans (Glycine max (L.) Merr.) 409
Fig. 3 PEPCase activity at different growth stages.
Dongnong 97-172 (1.03) and Dongnong 9339 (1.75), pod coats of Gongjiao 9107-1 (1.36) and Dongnong 9339 (2.09), respectively (Table 3). Therefore, there may be carbon assimilation in old leaves and pot coats. There was a negative relationship between NADP-ME activity and yield, and correlation coefficients were -0.1015 (in young leaves), -0.5256 (in old leaves), and -0.4986 (in pod coats), respectively. They did not reach a significant level, so NADP-ME was not a necessary factor to increase soybean yield.
NADP-ME activities in cultivars at different growth stages
The highest and the lowest NADP-ME activities appeared at seed-filling and seedling stages, respectively (Table 2). The differences of NADP-ME activity among cultivars were significant at each growth stage: 0.21 at seedling stage, 0.81 at blooming stage, 0.88 at podsetting stage, 0.72 at seed-filling stage, and 1.01 at early mature stage. There was a significant difference between any two stages at whole growth period, except for pod-setting and seed-filling stages.
NADP-MDH activities in pod coats and leaves at seed-filling stage
The highest and the lowest NADP-MDH activities appeared in pod coats and young leaves, respectively. NADP-MDH activity in pod coats was about 1.8 and 3.4 times higher than that in old and young leaves, respectively. It seemed that there was a higher NADPMDH activity in the older tissues. The differences of NADP-MDH activity among cultivars were very different: 0.11 in young leaves, 0.10 in old leaves, and
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HUANG Shan-shan et al.
PPDK
0.32±0.003 0.27±0.002 0.16±0.004 0.38±0.005 0.41±0.005 0.27±0.024 0.11±0.007 0.24±0.009 0.37±0.013 0.41±0.011 0.294
NADP-MDH
0.15±0.022 0.22±0.034 0.26±0.006 0.24±0.029 0.31±0.043 0.22±0.012 0.17±0.002 0.21±0.006 0.18±0.009 0.35±0.008 0.231 0.076±0.004 0.063±0.007 0.052±0.003 0.071±0.003 0.058±0.006 0.047±0.002 0.081±0.003 0.074±0.003 0.083±0.002 0.082±0.004 0.0687 0.04±0.003 0.25±0.006 0.24±0.003 0.17±0.005 0.31±0.003 0.21±0.003 0.24±0.005 0.18±0.003 0.164
NADP-MDH activities in cultivars at different growth stages There was NADP-MDH activity in ten cultivars at each growth stage (Table 2). As a whole, the lowest and highest NADP-MDH activities appeared at seedling and blooming stages, respectively. Then, it was slightly decreased after blooming stage until early maturity stage. From Table 2, the correlation coefficient between NADP-MDH activity at blooming stage and yield could be calculated (0.6565, a significant level). There was also a trend of positive correlation at other stages, but that was not significant.
0.08±0.003 0.12±0.008 0.09±0.002 0.13±0.008 0.17±0.003 0.12±0.005 0.08±0.004 0.13±0.004 0.16±0.008 0.18±0.004 0.126 1.21±0.011 1.75±0.063 1.68±0.101 1.31±0.024 1.47±0.044 1.03±0.031 1.22±0.055 1.46±0.038 1.34±0.019 1.07±0.026 1.354
There was not PPDK activity in issues of some cultivars, such as young and old leaves of Dongnong 1567 and Dongnong 1068 (Table 3), however, their yield was very high (Table 1). PPDK activity was also not detected in the young leaves of Gongjiao 91071 and Dongnong 97-172, which showed that PPDK activity was very low, evens no appearance. Hence, PPDK activity could only exit in older tissues and organs. Therefore, there were important C4 enzyme in soybeans, but not all appeared in the leaves of all cultivars, and they appeared in not all of tissues and organs. Hence, there were some conditions for C4 enzyme activity to appear and express in soybeans. However, it was possible to screen soybean cultivars containing high C4 enzyme activities.
0.051±0.005 0.038±0.002 0.044±0.004 0.061±0.007 0.034±0.002 0.036±0.003 0.027±0.002 0.038±0.001 0.056±0.004 0.058±0.002 0.0443
DISCUSSION
-, not been measured out.
0.017±0.002 0.023±0.001 0.011±0.002 0.026±0.003 0.008±0.0004 0.013±0.001 0.007±0.001 0.0006
0.16±0.027 0.02±0.003 0.04±0.006 0.06±0.004 0.04±0.005 0.03±0.003 0.035 0.04±0.005 0.07±0.005 0.04±0.003 0.06±0.002 0.08±0.004 0.06±0.004 0.02±0.003 0.07±0.004 0.10±0.009 0.13±0.004 0.067 0.35±0.023 0.85±0.034 0.38±0.025 0.76±0.051 0.84±0.032 0.21±0.013 0.33±0.024 0.88±0.058 0.93±0.033 0.24±0.029 0.577 0.006±0.0007 -
Gongjiao 9107-1 Dongnong 9339 Dongnong 1567 Heinong 39 Heinong 41 Dongnong 97-172 Dongnong 1068 Gongjiao 9404 Jilin 43 Jilin 47 Average
Pod coats
1.36±0.028 2.09±0.034 1.78±0.012 1.52±0.029 1.37±0.019 1.66±0.042 1.82±0.025 1.93±0.024 1.76±0.012 1.45±0.022 1.674
PEPCase PPDK NADP-MDH
Old leaves
NADP-ME PEPCase PPDK NADP-MDH
Young leaves
0.20 in pod coats. The correlation coefficient between NADP-MDH activity in pod coats and that in old leaves was 0.6818*, a significant level. Therefore, it was possible to screen cultivars containing high NADP-MDH activity by measuring its content in pod coats (Table 3).
PPDK activity
NADP-ME PEPCase
Cultivar
Table 3 PEPCase, NADP-ME, NADP-MDH, and PPDK activities in young leaves, old leaves, and pod coats at seed-filling stage
NADP-ME
410
In this study, the highest and the lowest PEPCase activities appeared in pod coats and young leaves, respectively. Many reviews summarized certain tissues containing relatively high PEPCase activity, for example immature barley pericarp (Duffus and Rosie 1973), the testa of pea seeds (Hedley et al. 1975) and the inner tissue of tomato fruits (Laval et al. 1977). Latzko and Kelly (1983) showed the photosynthetic tissues surrounding cereal grains and the pericarp of those grains consistently displayed PEPCase activity higher than that in leaves. In the pericarp of wheat, PEPCase activity was about four times higher than that in leaves (Wirth et al. 1977). In this study, PEPCase activity in pod coats was about six times higher
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Relationships Between C4 Enzyme Activities and Yield in Soybeans (Glycine max (L.) Merr.)
than that in young leaves. Because PEPCase is the key enzyme of C4 photosynthetic pathway, the cultivars containing higher PEPase activity should have higher photosynthetic rate (Jiao and Chollet 1991; Lepiniec et al. 1994; Chollet et al. 1996). In our study, the correlation coefficients between PEPCase activity and photosynthetic rate were 0.9071 in field and 0.7369 in the pots, respectively, and reached extremely significant and significant level, respectively (Fig. 2). There was also significant correlation between PEPCase activity and yield. Because there are significant differences of PEPCase activity among cultivars, it is possible that the soybean cultivars containing high PEPCase activity are selected out, which could be a indicator to breed soybeans with high yield and photosynthetic efficiency. There was a negative relationship between NADPME activity and yield, and it did not reach a significant level, so it was not a necessary factor to increase soybean yield. The correlation coefficient between NADP-MDH activity at blooming stage and yield was 0.6565, a significant level. There was also a trend of positive correlation at other stages, but that was not significant. In this study, PPDK activity in young leaves of some cultivars, even old leaves, was not detected, but it did not affect high-yield of the cultivars. In contrast to the high activities of PPDK in the green leaves of C4 and CAM plants, very low activity has been detected in C3 plants (Edwards et al. 1982; Meyer et al. 1982). Maybe, PPDK activity in soybeans was so low that it could not be detected at all in some cultivars. Simultaneously, the results also implied that there could be another photosynthetic process that was similar to C4 pathway in soybeans, but it was not indispensable. Both the highest activities of PEPCase and NADPME appeared at seed-filling stage, however, the highest NADP-MDH activity was at blooming stage. All C4 enzyme activities were the lowest at seedling stage, which indicated there were higher C4 enzyme activities in the older green organs at vigorous reproduction stage. Patel and Berry (2008) showed immature leaves of amaranth conduct C 3 photosynthesis, whereas the C 4 occurrences in mature leaves (Wang et al. 1992, 1993; Patel and Berry 2008), which are consistent with our results. In addition to green leaves,
411
chloroplast-containing organs, such as green fruits, soybean pods, and rice panicles, had the potential of photosynthesis (Edwards and Warker 1983; Blanke and Lenz 1989). Hedley et al. (1975) also reported high PEPCase activity as well as RuBPCase activity was detected in soybean pods. In this study, C4 enzyme activities of pod coats were the highest, and the difference was significant, which was important for breeders. However, PPDK activity was very low, even no appearance in young leaves. Maybe, PPDK activity could only exit in older tissues and organs.
MATERIALS AND METHODS Plant materials Ten representative soybean cultivars were used in this study. Young leaves, old leaves, and pod coats were studied in field and in the pots at different growth stages (Marris et al. 1991). All samples were from the field in Xiangfang Experiment Station, Heilongjiang Province, China and the pots in Northeast Agricultural University, China. The seeds were weighed to measure yield, after 100 mature samples of each cultivar, from five sites through Punnett’s square, were artificially threshed. Photosynthetic rates were measured with three replications by CI-301PS photosynthesis system (CID, Inc., USA). At least three samples of a cultivar were measured at each growth stage (intensity of light PAR>1 200 mol m-2 s-1, relative humidity (RH) (70±5)%, CO2 concentration (350±10) ppm). The mean was taken as yield and photosynthetic rate of the cultivars.
Extraction and assay of PEPCase The extraction and assay of PEPCase were as already described (Parvathi et al. 2000; Chinthapalli et al. 2003). The samples were quickly extracted using a chilled mortar and pestle with 1 mL of extraction medium containing 100 mmol L-1 Tris-HCl (pH 7.3), 10 mmol L-1 MgCl2, 2 mmol L-1 K2HPO4, 1 mmol L-1 EDTA, 10% (v/v) glycerol, 10 mmol L-1 bmercaptoethanol, 10 mmol L-1 NaF, 2 mmol L-1 PMSF, 10 g mL-1 chymostatin, and 2% (w/v) insoluble PVP. The homogenate was centrifuged at 15 000×g for 5 min and the supernatant was used as “crude extract”. The procedure used for the purification of PEPCase was as described in detail by Gayathri et al. (2000). PEPCase activity was assayed by coupling to MDH and monitoring NADH oxidation at 340 nm in UV-721 spectrophotometer (Xinmao Co., China) at 30°C. The assay mixture contained 50 mmol L-1 Tris-HCl (pH 7.3), 5 mmol L-1 MgCl2, 0.2 mmol L-1 NADH, 2 U MDH, 2.5 mmol L-1 PEP, 10 mmol L-1 NaHCO3. 1 U of enzyme activity was the
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HUANG Shan-shan et al.
412
capacity of the enzyme to catalyze the formation of 1 mol oxaloacetate min-1.
Extraction and assay of NADP-ME The samples were quickly extracted with 1 mL of extraction medium containing 100 mmol L-1 Tris-HCl (pH 7.6), 2 mmol L-1 EDTA, 1 mmol L-1 DTT, 5% (w/v) glycerol, 20% (w/v) insoluble polyvinylpyrrolidone and a set of protease inhibitors, and homogenate was centrifuged at 15 000×g for 5 min. The supernatant solution was collected and desalted using a 3.5 cm×15 cm column of Senhadex G-25 (Sigma, United States). NADP-ME activity was determined in UV-721 spectrophotometer at 30°C by monitoring NADPH production at 340 nm. The reaction mixture contained 5 mmol L-1 Tris-HCl (pH 7.5), 1 mmol L-1 MgCl2, 1 mmol L-1 Mn2+, 1 mmol L-1 EDTA, 0.33 mmol L-1 NADP, and 5 mmol L-1 L-malate. 1 U of enzyme activity was defined as the amount of enzyme that resulted in the production of 1 mol NADPH min-1 (Yuu et al. 2000).
Extraction and assay of NADP-MDH The samples were quickly extracted with 1 mL of extraction medium containing 0.1 mol L-1 Tris-HCl (pH 5.3), 50 mmol L-1 mercaptoethanol, 5 mmol L-1 MgCl2, and 5 mmol L-1 EDTA. The homogenate was centrifuged at 15 000×g for 5 min. The procedure used for the purification of MDH was as described in detail by Gayathri et al. (2000). The assay mixture contained 25 mmol L-1 Tris-HCl (pH 8.9), 1 mmol L-1 EDTA, l mmol L-1 NADP, 50 mmol L-1 malate, 20 mmol L-1 glutamate, 0.6 U mL-1 aminosuccinic acid aminotransferase. MDH activity was assayed in UV-721 spectrophotometer at 30°C at 340 nm.
Extraction and assay of PPDK The samples were quickly extracted using a mortar and pestle (as described above). The supernatant was used for assay of enzyme activity at 30°C in a buffer containing 100 mmol L-1 Tris-HCl (pH 8.0), 10 mmol L-1 MgCl2, 5 mmol L-1 NaHCO3, 0.1 mmol L-1 EDTA, 1.25 mmol L-1 pyruvate, 1.25 mmol L-1 ATP, 2.5 mmol L-1 KH2PO4, 5 mmol L-1 DTT, 2 U malate dehydrogenase (MDH), 0.2 mmol L-1 NADH, 6 mmol L-1 glucose-6-phosphate, and 0.3 U of purified PEPC. 1 U of PPDK activity corresponds to 1 mol of pyruvate converted min-1 at 30°C (Salahas et al. 1990).
Correlation analysis
Where, x, enzyme activity; y, yield/photosynthetic rate.
Acknowledgements This study was supported by the National Natural Science Foundation of China (30471092) and the Genetically Modified Organisms Breeding Major Projects, China (2009ZX08009089B).
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