To understand Al tolerance mechanisms in lentil, present study sheds light on differential gene expression among cultivars as well as between cultivars and wild genotypes of lentil via de novo transcriptomic examination. Contigs were sequence aligned to publically available databases and functional characterization of DEGs along with pathway prediction was done. When differential gene expression among cultivars was accomplished, top DEGs which were up-regulated between tolerant and sensitive genotypes under Al stress condition were found to be involved in protein transport, protein degradation, defence, cell growth and development. Specifically, PX domain-containing protein EREX is engaged in vacuolar transport of storage proteins. It controls membrane trafficking to protein storage vacuoles (PSVs) and attaches explicitly to phosphatidylinositol 3-monophosphate . Protein Light-Dependent Short Hypocotyls 1 is a plausible transcription regulator that performs as a developmental regulator by stimulating cell growth in reaction to continuous red, far-red and blue light in a phytochrome-dependent mode . BTB/POZ domain-containing protein NPY1 operates as a substrate-specific adapter of an E3 ubiquitin-protein ligase complex (CUL3-RBX1-BTB) which facilitates the ubiquitination and consequent proteasomal degradation of target proteins. It regulates cotyledon development through control of PIN1 polarity and was found to be involved in root gravitropic responses in Arabidopsis [23–25]. ATP-dependent zinc metalloprotease FTSH 7 chloroplastic is a part of a complex that functions as an ATP-dependent zinc metallopeptidase. It is engaged in thylakoid formation and in exclusion of damaged D1 in the photosystem II, averting cell death under high-intensity light conditions [26, 27]. Significantly down-regulated DEGs also have different functions which were suppressed in tolerant cultivar as compared to sensitive one. Among down-regulated DEGs, Zinc-finger homeodomain protein 4 is a putative transcription factor and is implicated in the regulation of floral induction . Carboxylesterase and Aldehyde dehydrogenase family member have catalytic functions whereas protein kinase SD1-13 represses disease resistance signalling pathways .
Top up-regulated DEGs in tolerant control v/s treated group have catalytic activity (TNT 1–94) and antifungal activity (Defensin-like protein 39). DEMETER is involved in gene imprinting and catalyzes the discharge of 5-methylcytosine from DNA by a glycosylase/lyase process. It allows expression of the maternal copy of the imprinted MEA gene before fertilization and acts by nicking the MEA promoter. This transcriptional activator is entailed for stable reproducible patterns of floral and vegetative development [30–32]. Threonine synthase chloroplastic catalyzes removal of phosphate from L-phosphohomoserine and addition of water to yield L-threonine whereas ABC transporter A family member 2 helps in nucleotide binding. DEGs whose functions were receded have roles in catalysis (TNT 1–94, ATX4), RNA and ubiquitin binding (Protein RAE1), disease resistance (Protein RPP5) and plant defence signalling (protein kinase PBL23).
When sensitive cultivar was compared to its control, topmost DEGs whose functions were elevated have roles in cell expansion, root meristem patterning, auxin-transport (SAUR72 protein). Ty5-1 protein YCL074W has been reported as a truncated part of POL protein in mutated non-functional yeast transposon . Sodium Potassium Root Defective 2 is a key up-regulated protein which is involved in metal binding thereby affecting the growth of sensitive genotype under Al stress. Down-regulated DEGs has function in DNA repair and mitotic recombination (CHROMATIN REMODELING 25) , chloroplast biogenesis and cell division (Amido phosphoribosyl transferase 2 chloroplastic) . Calcium uniporter protein 2 mitochondrial constitutes a pore-forming and calcium-conducting subunit which is involved in calcium uptake into mitochondria whereas UDP-galactose/UDP-glucose transporter 3 is an essential sugar transporter required for pollen development and embryo sac progress .
When wild (ILWL-15) and tolerant cultivar (L-4602) was compared, DEGs which were significantly up-regulated in wild are involved in regulation of transcription in differentiating cells; pre-mRNA splicing; catalysis and protein ubiquitination. For example, Ycf49-like protein is a part of cell membrane protein. Similarly, when expression profiles of BM-4 (sensitive cultivar) and ILWL-15 (wild) were compared, significantly up-regulated DEGs in wild have their functions in catalysis , cellular copper and redox homeostasis , processing of poly-ubiquitin precursors as well as that of ubiquitinated proteins and in resistance to the arginine analog canavanine , cell surface adhesion along with endopeptidase activity. As plants lacks centrosomes, so they have acentrosomal microtubule arrays. Protein TPX2 which is down-regulated in wild genotype under Al stress has microtubule binding capability and regulates pro-spindle assembly during late prophase and at the onset of mitosis [40, 41]. Other down-regulated DEGs involves EXO70B1, which is encompassed in Golgi-independent membrane traffic to the vacuole and is a positive regulator of both abscisic acid (ABA)-promoted and mannitol (drought)-endorsed stomatal closure [42, 43] Similarly, Vacuolar protein sorting-associated protein 54 chloroplastic is involved in pollen tube elongation and other polar growth . Octanoyltransferase is essential for de novo plastidial protein lipoylation during seed development . Protein WEAK Chloroplast Movement Under Blue Light 1 is requisite for chloroplast avoidance response under high intensity blue light. This eschewal response results in the repositioning of chloroplasts on the anticlinal side of exposed cells. It sustains the rate of chloroplast photo relocation movement via cp-actin filaments adjustments 
DEGs which were up-regulated in wild genotype when compared to its control involves U-box domain-containing protein 13 which acts as a E3 ubiquitin ligase and 60S acidic ribosomal protein P1-2 which is involved in protein synthesis. Another up-regulated DEG was Auxin response factor 6 which is a type of transcriptional activator that binds precisely to the DNA sequence 5'-TGTCTC-3' located in the auxin-responsive promoter elements (AuxREs). Configuration of heterodimers with Aux/IAA proteins alters their aptitude to modify initial auxin response genes expression. It synchronizes both stamen and gynoecium maturation and fosters jasmonic acid stimulation [47, 48] Top down-regulated DEGs included TMV resistance protein N which is a disease resistance protein and Cyclin-dependent kinase F-1 that modulates CDKD-2 and CDKD-3 activities by phosphorylation of the T-loop . Thought-provokingly, in all the comparison groups involving cultivars as well as wild genotypes, top DEGs were associated with down-regulation of disease resistance genes.
In present study, result for sequence alignment with databases is way ahead of Kaur et al. (2011), which suggest that advancement in the sequencing chemistry has increased the quantity and quality of acquired data that has improved de novo assembly of non-model crop plants . Further, generation of huge number of molecular markers from acquired data will help in improving the development of lentil reference genomic pool i.e. knowpulse.usask.ca. Top 12 DEGs from different comparison groups when evaluated using qRT-PCR, data were validated precisely. ABR 18 and SAUR-72 was up-regulated in 1C-1T comparison group whereas CSA-UDP and Expansin were down-regulated. Study on precocious germination of cultured immature embryos of Pisum sativum has showed that addition of ABA increases production of ABR-18 protein and is accumulated in testa during early seed development . SAUR-72 has role in regulation of cell expansion, root meristem patterning and auxin transport. In a study conducted on tissue-specific and developmentally regulated expression patterns of SAUR genes, it was found that SAUR72 was highly expressed in the steles of Arabidopsis roots and hypocotyls . In 2C-2T comparison group all the four genes which were selected for qRT-PCR validation, viz. 30S5, Isocitrate Lyase, PCBP3 and Peroxidase-15 were up-regulated. Isocitrate lyase is an enzyme of the glyoxylate cycle which accelerates the cleavage of isocitrate to succinate and glyoxylate. Bradyrhizobium japonicum isocitrate lyase has been reported to have an important functional role in desiccation tolerance . Poly(C)-binding proteins (PCBPs) are commonly acknowledged as RNA-binding proteins that interact in a sequence-specific manner with single-stranded poly(C). PCBP1-4 including PCB-3 proteins are concerned chiefly in innumerable posttranscriptional regulations , have reported that transcriptional regulation of PCBPs might itself be standardized by their localization within the cell and it can work as a signal-dependent and coordinated regulator of transcription in eukaryotic cells. In previous studies, impediment of peroxidase was linked with Al tolerance as it helps in maintaining H2O2 levels desirable for non-enzymatic wall loosening [55, 56]. But similar to our study, peroxidases were up-regulated during transcriptomic profiling of wheat near isogenic lines under Al stress and were reported to be connected to decreased growth rate . Since, in our study this has been up-regulated in sensitive genotype under Al stress, we cannot associate it with Al stress tolerance. Instead similar to study conducted in wheat NILs, here also it might be due to reduced growth rate under Al stress. In 3C-3T comparison group, CYP45081E8, HSP 17.1, were up-regulated and IRX9, UPBEAT1 were downregulated. CYP45081E8 is a probable monooxygenase which exhibit activity with isoflavones such as formononetin, biochanin, pseudobaptigenin, daidzein, genistein, isoformononetin and prunetin, or with flavonoids including naringenin, liquiritigenin, apigenin, luteolin, or kaempferol . Up-regulated HSP 17.1 is a part of small heat shock protein (HSP20) family. These genes signify the highly profuse class amid the HSPs in plants. Hsp20 genes have been reported to be connected with stress triggered by HS and other abiotic influences in soybean . Down-regulated IRX9 gene encrypts a putative family 43 glycosyl transferase. It was reported to be co-ordinately articulated with the cellulose synthase subunits in the course of secondary cell wall formation . Cell wall examination exposed a reduction in the richness of xylan in the irx9 mutant, demonstrating that IRX9 is essential for xylan production [60, 61]. IRX9 was also identified as MUCI65 in a reverse genetic screen for Mucilage-Related genes. Despite producing only a few seeds, the irx9-1 mutant displays normal mucilage properties [62, 63]. UPBEAT1 is a transcription factor which regulates the equilibrium between cellular propagation and differentiation in root growth. It does not act via cytokinin and auxin signaling, but by curbing peroxidase expression in the elongation zone .
During overall pathway analysis, more number of prominent BINs pertaining to cell wall was found in tolerant sample when compared to sensitive and wild genotypes under Al stress. Aluminium ion binds to the cell wall causing increase in cell wall rigidity . Thereby, it hampers the cell wall growth and cell elongation . Many systems such as efflux of organic acids, signalling due to Al ion involves the role of cell wall . Enhanced activity of cell wall related genes in tolerant genotype when compared to sensitive and wild ones under Al stress affirms its involvement in Al tolerance. Also, release of secondary metabolites from the root apex is well established mechanism for Al tolerance . Increase in number of BINs for secondary metabolites activity in wild indicates that the Al tolerance is rendered through production of different secondary metabolites. Further, pathway analysis of top 135 DEGs in all the comparison groups have disclosed many unique and uncharacterized Al stress related proteins which are involved in organic acid synthesis and exudation, phytohormone response, Al induced ROS detoxification, callose synthesis along with genes of alternate pathway. Plants protects against Al damage by exudation of organic acids from root apexes. This exudation is accompanied by release /uptake of ions to balance membrane potential. Organic anions chelate Al3+ to prevent it from binding to cell membrane. PDK is involved in organic acid synthesis and plays a crucial role in intermediary metabolism by acting as a negative regulator of mitochondrial dehydrogenase complex . In soybean, Al triggered organic acid secretion from roots was found to be more in tolerant genotypes than the sensitive ones, although their biosynthesis was not the rate restricting step for exudation of organic acids . Transporters acts as channels for organic acid exudation. These include members of Al activated Malate transporter (ALMT) and multidrug export protein AcrE (MATE) families which encrypt membrane proteins that expedite organic anion efflux crosswise cell membrane. ALMT genes are involved in organic acid transportation which were significantly up-regulated in tolerant genotype when compared to its control. These genes are classified in the Fusaric acid resistance protein like superfamily, CL0307 and it was the first Al tolerance gene to be recognized in case of plants represented by wheat . ALMT 9 is a chloride channel which is activated by physiological concentration of cytosolic malate and mediates vascular malate uptake . VvALMT 9 has facilitated the accumulation of malate and tartrate in the vacuole of grape berries . ALMT 12 functions as quickly activating anion channel mainly for chloride and nitrate ions . The action of AtALMT12 is controlled by phosphorylation through kinase Open Stomata 1, which in turn is activated by ABA under stress conditions to regulate stomata closure .
When tolerant and sensitive genotypes were compared, MATE was found to be significantly up-regulated in tolerant genotype. MATEs are big lineage of proteins which functions as subordinate active carriers using electrochemical gradient of other ions . Furukava et al. (2007) have identified HvAACT1 gene in barley which belongs to MATE family and is responsible for citrate exudation under Al stress . Al stress alters the function of plasma membrane by interfering with membrane lipids which result in increase in highly toxic ROS. To mitigate the effects of increased ROS, Al toxicity tolerance pathway involving activation of detoxifying enzymes comes into action. Many genes for Al induced ROS detoxifying enzymes e.g. SOD, Peroxidases etc. were witnessed to be up-regulated in tolerant genotype. Similar to our findings, in roots of aspen, Fe SOD genes were also found to be up-regulated under Al stress which were identified via transcriptomics . Wu et al. (2017) have identified that Arabidopsis peroxidase 64 gene (AtPrx64) was up-regulated in tobacco under Al stress and it was associated with the formation of plant secondary cell wall . With affirmation to our study, they also revealed that AtPrx64 is always up-regulated under Al stress suggesting that this gene plays protective role under this type of stress. AtPrx 42 and 64 were identified by microarrays to be involved in xylem secondary wall formation . AtPrx53 was expressed in vascular bundles by merging of regulatory sequence with β glucouronidase and the corresponding proteins were found to be involved in lignification . Also, Metacaspases were found to be induced in tolerant genotype which structures the alternative pathway for Al stress tolerance. Metacaspases are cysteine reliant proteases present in protozoa, fungi and plants that are involved in proteolytic pathway. Metacaspase-1 is a positive regulator of cell death and needs conserved caspase like- putative catalytic residues while Metacaspase-4 is positive regulator of biotic and abiotic induced programme cell death and it does not cleave caspase specific substrate . According to microarray study in poplar metacaspase 9 plays a role in xylem programmed cell death . Another signalling network for Al stress tolerance involves phytohormones which activates various Al tolerance genes or suppresses root growth under Al stress conditions . Some phytohormone signalling pathway genes were also up-regulated in tolerant genotype. For e.g. Auxin efflux carrier components which are required for optimal auxin distribution in root tips that is vital for modulating root growth under stress conditions . The impediment of root elongation by deposition of auxin is accompanied by increased production of ethylene, which regulates auxin biosynthesis and basipetal auxin shipping in root apex . Yang et al. (2017) have demonstrated that up-regulation of ethylene synthesis under Al stress suppresses root growth via jasmonic acid and cytokinin responsive pathways . Al stress also induces production of callose, which is a cell wall associated polysaccharide whose synthesis is not only an early sign of Al stress in plants, but it also occupies a central position in toxicity pathway that leads to hampering of root growth . Al induces callose sedimentation adjacent to plasmodesmata that could obstruct cell to cell transportation and communication . Callose synthesis was also increased in tolerant genotype. Callose synthase is multi subunit membrane associated enzyme complex whose activity is primarily under post-translational control but transcriptional regulation can also ensue . Most of the Al tolerance pathways described above is under control of one or the other regulation system, one being sensitive to Proton rhizotoxicity 1 (Stop 1) regulation system, which in this study has significantly contributed to Al stress response in tolerant genotype by substantial upregulation of its protein. STOP 1 is zinc finger transcription factor which plays significant functions in regulating expression of downstream genes. STOP 1 like proteins are conserved among land plant species . AtSTOP1 was encompassed in the positive regulation of 3 important Al resistance genes viz. AtALMT1, AtMATE, and AtALS3 in Arabidopsis. It also controls a series of potential H+ tolerance genes . Fan et al. (2015) have isolated and characterized VuSTOP 1 gene from rice bean and found that it was mainly involved in H+ tolerance .
When wild genotype was compared to tolerant cultivar, ROS mediated antioxidants signalling pathway gene were significantly upregulated. APX and GPX are key antioxidant enzymes of scavenging systems which influences maximally to hydrogen peroxide detoxification. APX is one of the highly regulated enzymes . In our previous studies also, we have identified significant increase in GPX activity in wild lentil genotypes . For organic acids synthesis pathway, several genes were significantly up-regulated in wild genotype, one being that of Aconitatehydratase (Aco). Aco is an enzyme highly sensitive to irreversible oxidative inactivation by H2O2. This enzyme catalyzes isomerization of citrate to isocitrate via cis-aconitate in the TCA cycle. Aco have been shown to be involved in regulating tolerance to oxidative stress and cell death in Arabidopsis and Nicotiana benthamiana .