Averaged across 10 genotypes there was no genotype-N interaction term or genotypic variation for photosynthetic N use efficiency (PNUE) in agreement with Hikosaka (2004) who concluded that variation in PNUE within a species to be smaller compared with interspecific variation. These results suggest that the variation in leaf-level PNUE is not what is contributing to the genotypic variation observed for NP at whole-plant level. This can be due to relatively small differences that exist among genotypes which are hardly distinguished by statistical methods. That said, if one compares the three genotypes (3G, i.e. Takanari, IR 64 and Milyang 23) that exhibited enhanced NP and faster growth at low N (Chapter three) with the seven genotypes (7G) that did not exhibit
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enhanced NP, one does see differences in PNUE and associated traits. For example, when comparing 3G with 7G, 3G maintained higher Vcmax, a25 at low N compared with 7G, while Vcmax, N25 was greater in 3G than 7G (p < 0.01) (Tables 4.1 and 4.3). Further, these 3G appeared to have a greater (p < 0.001) A on N basis (A400, N) compared with their counterparts at low N (Fig. 4.5C, Tables 4.1 and 4.3). Whilst there was no difference in N partitioning pattern among those 3G with 7G at high N, these 3G allocated relatively a greater fraction of N in photosynthesis (i.e. Rubisco and electron transport components) at the expense on non-photosynthetic components (Fig. 4.10, Tables 4.3 and 4.4). By contrast, the other 7G allocated 3% more N to non-photosynthetic components at the expense of N in pigment protein complexes and Rubisco. Thus, the greater fraction of N invested in Rubisco and electron transport components might explain the enhanced leaf PNUE (as indicated by A400, N and Vcmax, N25) in 3G, possibly leading to greater NP at low N.
Both A400, N [NP at low N = 0.909 + 0.111 * A400, N, r2=0.599, p < 0.005] and Vcmax, N25 [NP at low N = 0.937 + 0.027 * Vcmax, N, r2=0.638, p < 0.003] strongly correlated with whole plant NP at low N (Chapter 3 and Fig. 4.11) providing some indication that these parameters at the leaf level might explain variations in NP at whole plant level. Clearly, more studies are needed with multiple genotypes and more replicates to explore this further, particularly given that when analysing all 10 genotypes collectively, there was no statistical evidence of genotypic or N mediated differences. Further work is also needed to see if variation in PNUE at the whole-shoot level [which depends in part on radiation use efficiency (RUE) at canopy level] differs among the genotypes, particularly under low N supply. Here, factors such as canopy architecture, canopy height, light extinction co-efficient (K) that indicates the leaf spread underpinned by leaf angle and curvature of the leaf blade (Peng, 2000) need to be assessed at the whole plant level.
Relative partitioning of N to Rubisco was 16% on average under high N treatment of present study while past studies reported 27% of N in Rubisco in rice compared with wheat (20%) and Maize (8.5%) (Evans, 1989, Makino et al., 2003). This discrepancy could be due to the fact that carboxylation capacity was
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estimated using Michaelis–Menten kinetic constants for carboxylation (KC) and oxygenation (KO) of tobacco. The fraction of N in Rubisco was reported to be 27% in rice when Rubisco content reaches its maximum (Makino et al., 1984). Thus, a lower fraction of N in Rubisco in my study might indicate that at the early vegetative stage, maximum Rubisco content was not been achieved yet.
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Figure 4.10 Pie charts show the percentage of leaf N in pigment-protein complexes, nP; percentage of leaf N in electron
transport components, nE; percentage of leaf N in Rubisco; nR, for each N treatment (2 and 0.06 mM) when averaged (A)
the three genotypes (Takanari, IR 64 and Milyang 23) that maintained growth and nitrogen productivity (see Chapter 3); (B) the other seven genotypes (Opus, Dular, BG 34-8, Koshihikari, Akihikari, Azucena and Nipponbare). nP estimated from
chlorophyll content, nE estimated from maximum electron transport rate normalised to 25°C i.e. Jmax,a25 and nR was
estimated from maximum carboxylation velocity of Rubisco normalised to 25°C i.e. Vcmax,a25. The percentage of leaf N in all
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4.5.3 Rapid estimation of Rubisco via Western blotting using standards pre-determined with [14C]CPBP Rubisco content
assay
The strong correlation between Rubisco quantified with [14C]CPBP and Western blotting confirmed the ability of the Western blotting procedure to accurately estimate Rubisco. Estimation of Rubisco using the Western blotting procedure would be beneficial because it is quicker, thereby allowing more samples to be quantified for Rubisco. Unfortunately, Rubisco was poorly extracted when the tissue-lyser was used to prepare the extract and there was insufficient time to repeat and extend this approach. Thus, this method needs to be validated for rice and other crop and non-crop species. The maximum catalytic turnover of carboxylase (kcat, mol CO2 mol Rubisco sites-1 s-1) was estimated by the slope of the plot (Fig. 4.9B) based on Western blots performed for the rice genotype ‘Takanari’ grown at high and low N supply. A higher kcat means a faster Rubisco where lower numbers of Rubisco sites are required to achieve a given
Figure 4.11 Nitrogen productivity (NP) at 0.06 mM (LN) is plotted against (A) net assimilation rate (N basis) i.e. A400, N at LN and (B)
carboxylation capacity (N basis) i.e. Vcmax, N25 at LN. The solid lines
indicate the relationships between NP and A400, N at LN [NP at LN =
0.909 + 0.111 * A400, N, r2=0.599, p < 0.005] and NP and Vcmax, N25at LN
[NP at LN = 0.937 + 0.027 * Vcmax, N25, r2=0.638, p < 0.003]. n=4 for A400, N and Vcmax, N25. See Chapter 3 for calculation of NP.
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carboxylase catalytic capacity thus, this can lower costs associated with Rubisco (Evans, 2013). A greater kcat was found for low N-grown plants [.e. 3.97 mol CO2 (mol Rubisco sites)−1 s−1 at 25 °C] compared with high N grown counterparts [i.e. 2.55 mol CO2 (mol Rubisco sites)−1 s−1 at 25 °C]. Rubisco can act as a N storage compound (Warren et al., 2003, Cheng and Fuchigami, 2000, Warren et al., 2000b) in addition to its role as a catalytic enzyme. This smaller kcat at high N could indicate an increased fraction of inactive Rubisco (Cheng and Fuchigami, 2000, Li et al., 2009). Alternatively, inadequate extraction of Rubisco (Warren et al., 2000a, Harrison et al., 2009) from silicon-rich (Ma, 2004) rice leaves could result in overestimation of kcat at low N. The severity of low N treatment in the present study might have made Rubisco extraction more difficult and less sensitive to the methods used to quantify Rubisco. Any change in mesophyll conductance or diffusional constraints for CO2 could also influence the estimated maximum carboxylation rate and kcat. Different kcat values are available in literature for rice depending on different approaches used by researchers when estimating Rubisco while it is known as 30-40% lower compare with other species (Makino, 2003, Makino, 2005). For instance, 2.69 at 28 °C (Sage, 2002) and 1.69 for rice, 2.50 for wheat at 25 °C (Makino et al., 1988), 2.87 (in vitro) and 3.53 (in vivo) for Tobacco at 25 °C and kcat varied between 2-6 mol CO2 (mol Rubisco sites)−1 s−1 across species (Harrison et al., 2009). Thus, the values observed during present study for rice at low and high N conditions appears reasonable.
4.6 Conclusions
When averaged across genotypes, reduced demand for intercellular CO2 was matched with CO2 supply by partially closing stomata at low N. Across all 10 genotypes, there was no genotypic-N interaction term or genotypic variation for PNUE. Further, genotypic differences were not found for patterns of N partitioning to different components of photosynthesis except the reduction of the fraction of N invested in pigment protein complexes at low N. However, there were strong correlations between whole plant NP and PNUE (as indicated by A400, N and Vcmax, N25) at low N providing some evidence that variation in leaf level photosynthetic N use efficiency at low N could be contributing to a greater
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whole-plant NP at low N. Further, a separate comparison of three genotypes (Takanari, IR 64 and Milyang 23) that exhibited high NP at low N (Chapter three) with the other seven genotypes suggests that there might be enhanced PNUE [as indicated by carboxylation capacity and net assimilation rate (N basis)] in those three genotypes at low N. These 3 genotypes exhibited maintenance of carboxylation capacity at low N along with partitioning more N to photosynthesis (Rubisco and electron transport components), while the other seven genotypes exhibited lower, on average, carboxylation capacity and allocated proportionally more N to non-photosynthetic components at low N. Clearly, further work with more replicates is needed to elucidate any genotypic variation in PNUE contributing to NP in these genotypes at low N conditions. The ratio of dark respiration to carboxylation capacity remained largely constant across N treatments and genotypes. Finally, the ability of Western blotting procedure to accurately estimate the amount of Rubisco of a given sample was confirmed by the strong significant correlation found between the amount of Rubisco estimated via Western blotting approach vs. the amount of Rubisco quantified via [14C]CPBP Rubisco content assay. Thus, performing Western blotting procedure with standards pre-determined with [14C]CPBP Rubisco content assay can be considered as a rapid method of estimating Rubisco from multiple samples. A lower catalytic turnover rate of carboxylase (kcat) observed at high N i.e. 2.55 mol CO2 (mol Rubisco sites)−1 s−1 at 25 °C compared with low N i.e. 3.97 mol CO2 (mol Rubisco sites)−1 s−1 at 25 °C could indicate a lower activation state if Rubisco in leaves from plants supplied with high N.
4.7 Future directions
Clearly, additional work with more replicates is needed to elucidate any genotypic variation for PNUE of rice genotypes at both leaf and shoot level and its ability to explain variation in NP at low N conditions. Moreover, it is important to understand the effect of radiation use efficiency of these genotypes and its contribution to PNUE at whole plant level and its linkage with NP particularly at low N conditions. Due to time constraints light-saturated photosynthesis (A) was measured at two atmospheric CO2 concentrations i.e. at
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400 ppm (A400, assuming A is Rubisco limited) and at 1500 ppm (A1500, assuming A is RuBP regeneration limited). In a future experiment it would be recommended to perform A-Ci curves to confirm these results for the three
genotypes that performed well at low N. Further work is also needed to understand activation state of Rubisco, canopy architecture, differences in light interception patterns of three key performers. It would be useful to assess mesophyll conductance and leaf anatomy to understand diffusional constraints for CO2 within leaves as kcat can vary depending on maximum carboxylation rate which can be influenced by diffusional constraints particularly at high N supply. The rapid method of Rubisco estimation from unknown samples using Western blotting procedure with standards pre-determined with [14C]CPBP Rubisco content assay need to be repeated as Rubisco was not fully extracted during present study particularly from low N grown silicon-rich rice leaves due to the use of tissuelyzer instead of Tenbroeck homogenizer. Further, it would be useful to validate the above method for different other crop and non-crop species.
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