Nitrogen supply has substantial effects on plant growth and development, as it is one of the main constituents of leaf cell components, particularly those associated with the photosynthetic apparatus,    including   carboxylating enzymes and membranes proteins (Pandey et al., 2000). N deficiency inhibits plant growth and development, especially in the older leaves near the base of the plant and ultimately they turn yellow and fall off under severe stress condition. Yield is also severely affected by lower grain number and less grain weight which is caused by fewer fertilized ovum, kernel abortion and other changes at physiological and biochemical level (Uhart and Andrade, 1995 a). On the other hand, problems of eutrophication and ground water contamination occur when high amount of N fertilizers are applied to the soil. MacDonald et al., (1997) reported that unfavorable environmental conditions decreased crop yield, but it was not due to less N content in the soil. Plants could perform considerably better even under limited N condition provided that partitioning of N within the plant and efficient utilization of N at the cellular level (Sattelmacher et al., 1994). This emphasizes the fact that plant breeders need to develop varieties and hybrids with high N use efficiency (NUE). For annual crops (like rice, wheat, maize, sorghum etc.), N uptake is regulated by the crop dry mass accumulation under non-limiting nitrogen supply within species and across environments (Lemaire et al., 2007).  Tollenaar and Wu (1999) indicated that increased leaf longevity, increased water and nutrient uptake, and greater assimilate supply during grain filling were related to increased low N tolerance in Canadian maize hybrids. N stress cause reduction in leaf area, enhances leaf senescence, and decreases radiation use efficiency (RUE) (Uhart and Andrade, 1995 b). Results of Subedi and Ma (2005) indicated that hybrids with greater yield or NUE were accompanied by greater dry matter production and more N uptake during the grain-filling period. Comparative study between early-senescing maize hybrid and late-senescing maize hybrid, both grown under field conditions with a high fertilisation input involving large quantities of fertilizer  showed that leaf senescence occurs independently of the source-to-sink transition at the high level of fertilisation used involving large quantities of fertilizer (Martin et al., 2005). Gastal and Nelson (1994) found that synthesis of Rubisco and other chloroplastic proteins occur largely from recycling of N that was previously incorporated into proteins during cell production and N content was the highest in cell production zone. The relationship between plant photosynthetic capacities, chlorophyll degradation during leaf senescence, and the shift from N assimilation to N remobilization has been investigated in a number of crops by studying the impact of prolonged green leaf area duration on yield of maize (Ma and Dwyer, 1998; Rajcan and Tollenaar, 1999 a, b) and other major crops (Thomas and Smart, 1993; Borrell et al., 2001; Spano et al., 2003). Research is also carried out to identify some of the components responsible for the physiological control of the ‘stay-green’ phenotype particularly in relation to NUE. For example, in both sorghum and maize, delayed leaf senescence allowed photosynthetic activity to be prolonged, which had a positive effect on the N uptake capacity of the plant. In sorghum this enabled the plant to assimilate more carbon and use more N for biomass production (Borrel et al., 2001), whilst in maize yields were higher (Ma and Dwyer, 1998; Rajcan and Tollenaar, 1999 a, b). However, further investigation is required to characterize better the physiological and molecular basis of the stay-green phenotype (Verma et al., 2004) in relation to N supply, root N uptake capacity, root architecture, and leaf structure, and to determine whether such a phenotype can be beneficial when N fertilization is reduced (Borrell et al., 2000).

A list of 260 IBM Recombinant Inbred Lines (RILs) was obtained from the Maize Genome Database (Maize GDB, 2004) (http://maizegdb.org/). The RILs were classified into 2 groups based on the constitution of genotype (abundance of B73 and Mo17) in 5 key regions of the genome that had been previously associated with root traits on Chromosome 1 (Liu et al., 2008) and N use efficiency (NUE). The IBM genotypes which have more of B73 in these 5 target regions are classified as IBM RILs with high B73. The IBM genotypes which have more of Mo17 in these regions are classified as IBM RILs with high Mo17. Five genotypes from each group (high B73 and high Mo17) were selected for the experiment along with parental inbreeds B73 and Mo17 to compare the difference among the genotypes and the parents.

Fine, medium and coarse sand were mixed well and used to fill the 120 black plastic cylindrical pots to equal depth. The 10 IBM RILs and their parents (B73 and Mo17) were planted in the Guterman Laboratory (greenhouse) (total of 120 plants). Two seeds were sown in each pot and seedlings were thinned to one per pot shortly after germination. Day temperature was about 21.1°C (70°F) and night temperature 15.6°C (60°F). Along with normal sunlight, artificial lights (1000 Watt metal halide lamps) were provided to supplement enough light for plant growth.

Three stock solutions following Engels and Kirby (2001) were prepared: solution A in high and low N versions and solution B. Solution A comprised 10 L of high N and 10 L of low N prepared in 2 different plastic containers (Sen et al., 2015). Iron-EDTA was added in solution A. Solution B contained all other nutrients except Ca (NO₃)₂ 4H₂O and Iron-EDTA and a total of 20 L was prepared in a plastic container. The stock solution was diluted to 10 X solution. Beginning at 8^th day after planting, 30 ml solution was applied to each pot and watering was done every other day. At one month after sowing, 30 ml solution was applied every day and continued for 20 days. The plants were harvested at 50 days after planting.

Measurements of dry matter and chlorophyll content

Dry weight                                                                                                                                                                

Plants were separated into leaves, stems, and roots and dry weights of each part were measured after drying them in the oven for five days at 60°C.

Root – shoot ratio

Root-shoot ratio was derived by taking the ratio of root and shoot dry weight. Shoot dry weight of a particular plant was obtained by adding leaf and stem dry weight for that plant.

Leaf fraction of shoot (LFS)

LFS  was  measured by taking the ratio of leaf dry weight and shoot dry weight.

Chlorophyll

Five leaf disks of 0.25 cm were taken from each plant. Chlorophyll was extracted in 1 ml of 95% (v/v) ethanol/water. The absorbance of chlorophyll at 654 nm wavelength was measured in a spectrophotometer. This wavelength was chosen based on the findings of Wintermanns and de Mots (1965) who reported that chlorophyll has an extinction coefficient of 83.4 L/g at this wavelength.

Experimental design and statistics

A split plot design was used for the experiment with 5 replications. Statistical analysis of various observations was done using the JMP software package.

N stress reduces crop photosynthesis by reducing leaf area development and leaf photosynthesis rate and by accelerating leaf senescence (Bänziger et al., 2000; Graham and Vance, 2000). N uptake is positively related to crop growth rate and to biomass accu­mulation (Gastal and   Bemaire,   2002).   In   this   study,   the   chlorophyll concentration in the IBM RILs was lower than that in the parents at high N (except for IBM337 and IBM055) but fell between Mo17 (lowest chlorophyll) and B73 (highest chlorophyll) at low N. IBM337 was high in chlorophyll at both N levels, which suggests good potential breeding value since delayed senescence is an important criteria for abiotic stress tolerance. Delayed senescence (or stay-green) was proposed as an indirect  selection criterion for low N tolerance (Moll et al., 1994). Uhart and Andrade (1995a) reported that leaf N decrease has a direct effect on canopy photosynthesis, resulting in more kernel abortion and lower grain number. The IBM RILs with high B73 showed higher chlorophyll content and dry root weight under low N  conditions  than  the  IBM  RILs  with high Mo17. “Stay green” concept under stress conditions is important for better photosynthetic activities and supplying carbohydrates for developing sink organs (young leaves, kernels, grains) and hence genotypes that had higher chlorophyll content could be further studied for future  improvement   of   low   N   tolerance.   Chlorophyll content of maize leaf can be used effectively in developing recommendations for soil N replenishment (Ványiné et al., 2012).

Costa et al. (2002) and Chun et al. (2005) reported that low N reduced root biomass in all the tested genotypes, which is in accordance with the obtained results showing reduced root dry weight at low N. Root-shoot ratio was higher in N stressed genotypes, which indicates that under low N conditions the shoot growth of the plant is reduced more than the root growth supported by the findings of Amos and Walters (2006). This increased root-shoot ratio at low N may be an adaptive response that conserves plant N while maximizing the ability to acquire more N from the soil and can result from decreased shoot growth with or without increased root biomass (Chun et al., 2005; Yu et al., 2015). The absolute dry root-mass is usually less for plants grown under low N conditions than under normal soil containing sufficient N (Costa et al., 2002; Bänziger et al., 2000), a pattern that is clearly confirmed by root dry weight data from the present study. IBM337 had higher chlorophyll content and high root dry matter content. This establishes the fact that there is direct relationship between higher photosynthetic activity and increase in dry matter content.

Except for the genotypes IBM189 and IBM346 all other genotypes with high B73 composition had higher chlorophyll content. Genotypes with high Mo17 showed more root-shoot ratio than the genotypes with high B73 except the genotype IBM055. The genotypes with high B73 performed better in root dry weight. All the genotypes with high B73 had higher root dry weight than the genotypes with high Mo17 composition. Overall genotypes with high B73 had better performance than the genotypes with high Mo17. Determining the maize chlorophyll content in leaves and maize dry matter content will provide in near future a way to develop maize hybrids with better NUE which can be grown in both resource poor and resource rich environment.

The authors have not declared any conflict of interest.

The authors would like to thank Guterman Laboratory members for their technical assistance.