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- Materials and Methods
- Supporting Information
Nitrogen (N) fertilization and the development of new plant breeding strategies, such as the production of hybrids, have been the two most powerful tools for increasing kernel yield (KY), particularly in maize (Moose & Mumm, 2008). Nowadays, a combination of both agricultural and economic constraints means that farmers must optimize the application of N fertilizers to prevent pollution by nitrates and the release of nitric oxides to the atmosphere, whilst, at the same time, preserving their economic margin (Hirel et al., 2007a). Cereal kernels provide 60% of the world’s nutrition, either directly in the human diet or indirectly as animal feed. Worldwide, maize is the most important single crop, comprising 35% of overall cereal production. Recent improvements in maize yield of c. 1% each year from 1955 onwards have been estimated to be a result of improvements in agronomic practices (40%) and genetic gains (60%) (Hallauer & Carena, 2009). Maize is not only recognized as a major crop, but also as a model species that is well adapted for fundamental research into the understanding of the genetic basis of yield performance to improve kernel productivity and quality in terms of nutritional value to feed the world’s population (Hirel et al., 2007b).
Therefore, it has become of major importance to select for maize genotypes that take up and utilize N in the most efficient way for silage and kernel production. To reach such an objective, various complementary approaches, including conventional breeding, molecular genetics, whole-plant physiology and the use of improved or alternative farming techniques, have been developed (Hirel et al., 2007b, 2011; Moose & Below, 2009). Whatever the mode of N fertilization, an increased knowledge of the mechanisms controlling plant N economy is essential to improve nitrogen use efficiency (NUE) and to reduce excessive input of fertilizers, whilst maintaining an acceptable yield. Using plants grown under agronomic conditions at low and high N mineral fertilizer input, whole-plant and physiological studies have been combined with gene, protein and metabolite profiling. This has allowed the development of a comprehensive picture depicting the different steps of N uptake, assimilation and recycling in maize to produce either biomass in vegetative organs or proteins in storage organs (Hirel & Lea, 2011).
Moreover, the development of quantitative genetic studies associated with the use of molecular markers has become a powerful tool to identify putative candidate genes involved in the genetic variation of complex physiological traits, such as NUE (Hirel et al., 2007a). Furthermore, the availability of the maize genome sequence (Schnable et al., 2009) and of more detailed genetic maps allows the precise location of chromosomal regions and, ultimately, the key genes influencing the expression of desired traits. In turn, this strategy will be of great potential for plant breeders to carry out marker-assisted selection for improved NUE in relation to yield, particularly under low fertilization input (Ribaud & Hoisington, 1998; Moose & Below, 2009).
Recently, the main steps of N metabolism in the developing ear of the two maize lines F2 and Io have been characterized (Cañas et al., 2009). During the kernel-filling period, the changes in metabolite concentration, enzyme activities and transcript abundance for marker genes of amino acid synthesis and interconversion in both the cob and kernels are strongly dependent on the genetic background (Cañas et al., 2009). This has given rise to the conclusion that, in maize, there is genetic and environmental control of N metabolism not only in vegetative source organs, but also in reproductive sink organs, which could cooperatively contribute to plant productivity.
This preliminary study prompted us to develop a quantitative genetic approach, similar to that already performed on vegetative organs (Hirel et al., 2001; Gallais & Hirel, 2004) and on germinating kernels (Limami et al., 2002), to obtain more information on the genetic basis of N metabolism in the developing ear and its possible relationship to yield. The aim of such a study was to identify coincidences between QTLs for agronomic traits and QTLs for physiological traits related to N metabolism, in both the cob and developing kernel, during the kernel-filling period. In addition, co-mapping of agronomic and physiological QTLs with genes encoding enzymes involved in N and carbon (C) metabolism, and other metabolic and developmental processes, was also investigated in order to provide a genetic meaning for the QTLs.
To further explore the possible relationship between N metabolism in the developing ear, whole-plant and organ-specific NUE-related traits and KY traits, correlation studies were carried out using the entire available dataset for these traits, measured either in the present studies or gathered from previously published work by Coque et al. (2008). These correlation studies were performed on datasets obtained from different years of experimentation in order to overcome potential environmental effects and to strengthen the significance of the correlations between the different agronomic and physiological traits.
- Top of page
- Materials and Methods
- Supporting Information
Previous studies have demonstrated that N metabolism in maize ears is an important component controlling N allocation during the grain-filling period (Seebauer et al., 2004; Cañas et al., 2009, 2010). Moreover, it is well established that N translocation and, presumably, N assimilation in the kernels facilitate the utilization of carbohydrates, thus being a major component in the determination of yield (Below et al., 2000). However, there is a paucity of data on both the physiological and molecular control of this process, although it has been suggested that a strong relationships exists between source and sink organs during kernel filling (Seebauer et al., 2004; Cañas et al., 2010).
Therefore, the present study focused on the identification of key metabolic reactions and candidate genes involved in the control of N assimilation in maize ears by exploiting the genetic variability in a population of maize RILs. In previous investigations, this RIL population allowed the identification of important components of NUE in both vegetative and reproductive organs in relation to yield (Hirel et al., 2001; Limami et al., 2002; Coque et al., 2008).
A number of physiological traits representative of C and N assimilation in both the cob and developing kernels (Cañas et al., 2009) were first measured in the RIL population grown over two consecutive years. As the heritability over the 2-yr experiment was high (0.7–0.8) for most of the physiological traits of both cob and developing kernels, it can be concluded that there is a highly significant genotypic effect for all measured physiological traits. This finding strengthens the power of the QTLs detected for these traits, and indicates that they may represent good putative biological markers to be used in breeding programs (Moose & Mumm, 2008).
Among the various QTLs or groups of QTLs detected for these sets of ear physiological traits, some showed interesting co-localization with putative candidate genes. One of the most interesting groups of QTLs identified concerned those controlling the concentrations of glycine and serine in the two parts of the developing ear. On chromosome 1, a QTL for serine concentration and, on chromosome 2, a QTL for glycine concentration were found in both the developing kernels and cob. These results are in agreement with correlation studies showing that there is a strong positive correlation between the serine and glycine concentrations of the cob and ear (Fig. 3, Table S1). It was also found that QTLs for both serine and glycine concentrations co-localized with QTLs for yield. This suggests that there is a genetic mechanism shared by the two parts of the developing ear that controls the synthesis and use of these two amino acids in an interactive manner, and that this control is important for the determination of yield. Moreover, it was shown that the glycine concentration in the developing kernels is highly correlated with several traits related to the plant N metabolic status, plant growth and development, such as NHI and NNI, and dry matter accumulation (Table 5, Fig. 4). In addition, it was observed that the glycine concentration of developing kernels is highly correlated with that of alanine, an amino acid shown to be of major importance in plant NUE (Good et al., 2007; Shrawat et al., 2008; Good & Beatty, 2011). In line with this finding, strong relationships were also found between the alanine concentration of the kernels, NHI, NNI, most of the yield traits (KY, KN) and plant dry matter accumulation (Fig. 4). It is therefore probable that alanine and glycine metabolism are strongly interrelated, as revealed by the high level of correlation obtained between these two traits in both the cob and developing kernels. This interaction between glycine and alanine metabolism may occur through the activity of the enzyme alanine:glyoxyate aminotransferase (AGT) which catalyzes the conversion of alanine to glycine. This enzyme has been shown to be mainly involved in the pathway of photorespiration in the leaves of C3 plants (Igarashi et al., 2006). The importance of glycine and alanine metabolism and accumulation during kernel filling is further strengthened by the presence, on chromosome 3, of a group of QTLs for the concentrations of glycine and alanine in the kernels, for ear number and for yield. In this chromosomal region, a gene encoding serine hydroxymethyltransferase (SHMT3), an enzyme that plays an important role in cellular one-carbon pathways by catalyzing the conversion of serine to glycine (which can be reversible depending on the metabolic pathway involved), was also detected. As the reaction catalyzed by the enzyme SHMT provides the largest part of the one-carbon units available to the cell (Douce et al., 2001; Maurino & Peterhansel, 2010), it is probable that this metabolic pathway is of major importance for the determination of yield during kernel development.
On chromosome 5, a QTL for kernel GS activity co-localized with QTLs for KY and TKW, previously found to be coincident with the Gln1.3 gene locus and a QTL for leaf GS activity (Hirel et al., 2001). Such findings reinforce the validity of previous quantitative genetic approaches, as the same QTL for GS activity was found in developing kernels (present study), in leaves (Hirel et al., 2001) and in germinating kernels (Limami et al., 2002). Surprisingly, the parental line F2 provided the favorable allele for kernel GS activity, whereas, for leaf GS activity, it originated from the parental line Io. In agreement with this finding, a low but significant negative correlation between TKW and GS activity in developing kernels was determined (Table S2), whereas it was found that there was a positive correlation with leaf GS activity (Hirel et al., 2001). It is probable therefore that GS activity at the Gln1.3 locus may be controlled by alleles of opposite effects according to the organ examined, which could represent an example of advantageous additive gene expression relative to one or both parents, which can increase the yield in hybrids (Springer & Stupar, 2007).
In developing kernels, there was a positive coincidence of a QTL for GDH activity and QTLs for yield and its components (TKW and KN) at the end of chromosome 1. Moreover, this group of QTLs also showed a coincidence with the Gdh1 locus, which therefore appears to be a good candidate gene, influencing yield. Previously, two QTLs for leaf GDH activity were found to be positively coincident with two QTLs for KY (Dubois et al., 2003; Gallais & Hirel, 2004), which confirms the hypothesis that GDH activity may be an important factor controlling plant productivity (Ameziane et al., 2000; Dubois et al., 2003; Loulakakis et al., 2009).
On chromosome 9, it was found that a QTL for asparagine concentration in the cob co-localized with a gene encoding asparagine synthetase (AS4). Interestingly, it has been shown previously that this gene is strongly induced during the process of N remobilization and recycling within the developing ear (Cañas et al., 2010). Moreover, the finding that the asparagine concentration of the cob is highly correlated with that of the kernels (Fig. 3c, Table S1) and, within the cob, with the other physiological traits related to amino acid interconversion (Fig. 3b, Table S1), further supports the hypothesis that asparagine is of major importance for C and N translocation between the cob and the developing kernels (Seebauer et al., 2004; Lea et al., 2007; Cañas et al., 2010). By contrast, asparagine may not be directly involved in grain yield if the negative correlations observed between yield components and the asparagine concentration of the developing kernels are considered. These negative correlations are in line with a previous investigation, in which a large accumulation of asparagine was observed during kernel abortion, thus being, in turn, detrimental to the final yield (Cañas et al., 2010). By contrast, the importance of asparagine during the process of ear elongation is an attractive hypothesis, as co-localization between the ear length and asparagine concentration was found on the same region of chromosome 9.
Although a QTL for the DW : FW ratio was only found for the cob on chromosome 6, with no co-localization with other QTLs or any particular candidate gene, this trait may be a good predictor of the water status of the whole plant. This hypothesis is supported by the fact that a significant negative correlation was found between kernel moisture (GMoist) and DW : FW of the kernels (Table S2). Furthermore, it was shown that a high DW : FW in the cob negatively affects KY (Table S2). A negative relationship was also found between DW : FW of both the cob and the kernels and most of the physiological traits measured in the cob (Fig. 3, Table S1). This finding indicates that, when there is a deficit of water in the developing ear, independent of dry matter production, most of the metabolites are not actively synthesized and transported, which may lead to kernel abortion (Zinsemeier et al., 1995; Ribaud et al., 2009). In line with these conclusions, DW : FW ratios of both the cob and developing kernels were highly correlated (Table S1, Fig. 3d), indicating that water deficit occurs in both parts of the developing ear.
In addition to studying QTL detection and the interpretation of their physiological meaning in terms of plant performance, interesting correlations between several physiological and agronomic traits were identified. If we consider that the genotypic effect predominates for most of the traits, in comparison with the genotype × year interaction, these correlation studies can be very informative for identifying important relationships between physiological and agronomic traits. Similarly, in the agronomic study of Coque et al. (2008), the genotype × year interaction was much smaller than the genotypic effect alone. Therefore, when measuring different sets of traits, the presence of genotype × year interaction experiments can only reduce the value of the correlation coefficients, which, in turn, does not bias the interpretation of the results based on the highest correlation coefficients. These correlations were calculated using a large dataset of agronomic and physiological traits obtained over several years of experimentation in order to circumvent potential environmental effects caused by climatic changes and variable N nutrition under field growth conditions. Moreover, stable relationships between traits will be essential if used by breeders to improve plant performance, both in terms of yield and N use. Together, they were consistent with well-known relationships existing between these traits, thus reinforcing the validity of this quantitative genetic approach and correlation studies performed with maize. For example, there is a strong negative correlation between the percentage of sterile plants and the total number of ears per plant (Tables 5, S2), simply because sterile plants have less ears or empty ears. By contrast, the number of ears is positively correlated with yield and its components (Table S2), as a plant with several ears produces generally more kernels than a plant with only one ear (Pan et al., 1986). The total or individual amino acid concentration of the developing ear and visual leaf senescence at 14 DAS were positively correlated, whereas this correlation was negative with visual leaf senescence at 45 DAS and at maturity. Thus, if the level of leaf senescence is high between 10 and 14 DAS, there will be an accumulation of amino acids in the ear because N remobilization to this organ is already occurring. By contrast, if there is a shortage of amino acids at the end of the grain-filling period, premature leaf senescence will occur to provide more amino acids to the developing ear through the N remobilization process.
It is also worth noting that the majority of the physiological traits of the cob are positively correlated with each other (Fig. 3d), suggesting that the C and N metabolic pathways in this organ are interconnected and that, when there is active metabolism, all metabolites are rapidly synthesized and transported.
Conclusions and perspectives
From both the study of correlations among traits and the detection of QTLs for various agronomic and physiological traits, one can conclude that genetic variability for N metabolism may be an important determinant for the yield and its components, not only in vegetative organs, but also in the developing ear of maize. This genetic variability mainly concerns amino acid metabolism and interconversion, mostly in developing kernels and, to a lesser extent, in the cob. One of the major breakthroughs from these studies concerns the metabolism of glycine and serine, and, presumably, the interconversion of glycine to alanine in the kernels, and the putative role of the cognate genes encoding the enzymes involved in these pathways. It is well established that glycine and serine play a major role during photorespiration (Maurino & Peterhansel, 2010), although, in C4 plants, this process is limited, but necessary, for proper functioning of photosynthesis (Lacuesta et al., 1997; Zelitch et al., 2009). Further work is thus needed to investigate the regulation of this metabolic pathway in the kernels, an organ in which photorespiration is normally absent.
Finally, this work has confirmed that both the GS enzyme (Martin et al., 2006) and, possibly, GDH are important in the determination of yield. Experiments are now in progress to overexpress these genes encoding the two enzymes, either constitutively or in an organ-specific manner in both source and sink organs, in order to verify whether grain filling and grain yield are improved.