Morphological,Physiological And Molecular Mechanism Of Nitrogen Use Efficiency In Different Sugarcane(Saccharum Spp.) Genotypes Under Variable Nitrogen Conditions

Low nitrogen use efficiency is the worldwide problem because application of nitrogen in large quantity increases the cost of production,and a lot of applied nitrogen goes into the ecosystem which is hazardous.Sugarcane is the main source of sugar and biodiesel,and it cannot grow without sufficient supply of nitrogen.This study was conducted to understand the mechanism of nitrogen use efficiency in sugarcane through screening of sugarcane germplasm,agronomic,physio-biochemical,morphological and molecular changes under low and normal nitrogen application levels.Fifty eight international,national and wild type sugarcane genotypes were grown in pots from single bud setts in triplicate.Two different ammonium nitrate solutions(0.2 and 2 m M)were applied for 130 days after the start of treatment and seedlings were harvested,separated plant organs,weighed for fresh and dry biomass.The sugarcane genotypes GXASF108-1-11and GT11 showed 5 and 14 times more dry biomass for 2 m M than 0.2m M ammonium nitrate solution whereas within genotypes variation was 28 and 23 times for normal and low nitrogen levels respectively.The genotype Q177 attained 3 and 2 times less nitrogen uptake for normal and low nitrogen levels than FJ1 and LC05-136.Internal nitrogen use efficiency and partial factor productivity had 2 and 2.5 fold decrease while non-significant difference for nitrogen uptake efficiency was observed.Shoot/root ratio increased three fold across the nitrogen levels but had non-significant correlation between the two nitrogen levels.Positive correlation of nitrogen uptake was observed with all the other observed parameters except internal nitrogen use efficiency and shoot/root ratio.According to principal component analysis,the most contributing factors were nitrogen uptake,partial factor productivity,nitrogen use index and internal nitrogen use efficiency which contributed to define nitrogen use efficiency of genotypes.Heat map cluster analysis grouped highly,moderately and low efficient genotypes consequently.The sugarcane genotypes with high and low nitrogen efficiency can be further used to study the mechanism of nitrogen use efficiency.On the other hand,nitrogen efficient(GXASF108-1-11)and inefficient(GT11)sugarcane genotypes were grown under low(0.05 m M)and high(5m M)N levels for growth,physio-biochemical and root morphological analyses at 5 leaves,7 leaves and 9 leaves stages,respectively.Analysis of variance showed that the genetic variability,nitrogen levels and growth stages significantly affected the growth,physiological and root morphological indicators.GXASF108-1-11 attained 16%,20%and 23%more plant dry biomass,52%,46%and 48%more plant height than GT11 with respect to sampling stages.GXASF108-1-11 had21.7 mg g^-1 day^-1 relative growth rate,200.31 g day^-1biomass duration and 1.16 leaf area index while ratio of leaf area to growth rate was 73.62 cm²g^-1,lower than GT11.Correlation of plant dry biomass with leaf area,photosynthetic rate and leaf glutamine synthetase activity was 0.93,0.95 and 0.94,respectively.Linear and polynomial regression models of N uptake with internal nitrogen use efficiency and nitrogen use index were ranged from R²and R²nternal nitrogen use efficiency,R²L0²nitrogen use index,respectively.The principal component analysis explained 91.23%,93.60%and 93.53%of the total variation.These results suggested that the GXASF108-1-11 had the efficient mechanism to tolerate low nitrogen conditions,which can be helpful for sustainable yield and lower nitrogen losses into the environment.High throughput sequencing of m RNA was performed to observe the differentially expressed transcripts and genes in different plant organs of nitrogen efficient and inefficient genotypes under low(F1)and normal(F2)nitrogen levels.In this study,clean reads were filtered and sequenced after removing 1-3.4%raw reads for GXASF108-1-11 and 1.37%low quality raw reads for GT11.More clean reads were obtained in leaf of GT11(G1)at normal nitrogen level but followed by those in leaf of GXASF108-1-11(G2)at low nitrogen levels.Over all 83-90%of clean reads were mapped with S.spontaneum,and Venn diagram comparison of G1F1 vs G2F2 showed 6052,7411 and 6121 genes were up regulated in leaf,stem and root,respectively while 3248 up regulated genes were common across the tissues.Gene ontology(GO)enriched 94682 transcripts along with 38427 differentially expressed genes.Metabolic process term from biological processes class annotated 16677,16968,16838 and 17021 genes,intracellular term of cellular component annotated 11233,11274,19352 and 19480 genes,and catalytic activity annotated 15615,15983,15791 and 15859genes for G1F1,G2F1,G1F2 and G2F2,respectively.Kyoto encyclopaedia of genes and genomes enrichment analysis represented 19799 differentially expressed genes which were annotated for different pathways like genetic information processing.Alternative splicing,SNP and In Del were also analysed for all the treatments and organ levels.Highly abundant differentially expressed genes in high nitrogen use efficiency genotypes at low nitrogen might be good candidates for the development of nitrogen efficient sugarcane genotypes.Similarly,i TRAQ based proteomic profiling was performed for all plant organs at low and normal nitrogen levels.On the whole plant basis 1127992 spectra were detected and matched spectra were 21530 only.Total peptides and identified proteins were 99404 and18273,respectively,and the finally filtered proteins were 18229 after sorting and deleting the proteins with missing values.Stem showed the highest identified proteins(7327)and peptides(43349)compared with leaf and root.Total differentially expressed proteins(DEPs)were 1047 in comparison of both genotypes at low nitrogen conditions(G1F1?G2F1)and distributed in leaf(206),stem(485)and root(356),respectively.Out of total DEPs,85,260and 187 DEPs were up regulated in leaf,stem and root.G1F2?G2F2 comparison showed 868total DEPs,and down regulated DEPs in leaf,stem and root were 153 out of 265,133 out of286,and 182 out of 317,respectively.Gene ontology(GO)annotated overall 3047,4107 and3135 DEPs for leaf,stem and root,and clusters of orthologous group(COG)showed 25groups for DEPs in leaf,stem and root.T-translational545and 605 DEPs respectively.STRING analysis presented the interaction of hub proteins,Sspon.03G0016190-1A,Sspon.03G0001210-1A with co-expression level 0.182 were also inter-connected and showed weak expression level.KEGG functional classification for nitrogen metabolism showed that NADPH dependent nitrate reductase(Sspon.06G0004940-1A)and ferredoxin-nitrite reductase(Sspon.04G0002730-1A)DEPs were up regulated during the assimilatory nitrate reductase reaction in leaf and roots.The expression levels and enrichment of DEPs unzipped the pathways that might be helpful for improvement of nitrogen use efficiency in sugarcane.