The co-operative interaction of puroindolines in wheat grain texture may involve the hydrophobic domain

The puroindoline genes are causatively associated with wheat grain hardness, a commercially significant property. The proteins puroindoline (PIN) A and B are both required in their wild-type (WT) to impart soft grain texture, and absence of/mutations in either/both PIN(s) results in hard wheat. However, there is no biochemical clarity yet that explains this interdependence. This work critically analyses the roles of the tryptophan-rich domain (TRD), the little-known hydrophobic domain (HD), and certain other residues, in the physical associations of PINs. Site-directed mutagenesis-PCR was used to delete the TRD or HD and introduce an Arg39Gly substitution in PINA. The PINB-D1c mutant (Leu60Pro) was also investigated. The yeast two-hybrid system was used to assess the protein–protein interactions (PPI) of proteins. The TRD deletion or Arg39Gly substitution in PINA did not adversely affect its PPI, while deletion of HD resulted in a significant reduction. No effect on PPI was observed for Leu60Pro PINB. The results of this expression system strongly suggest that the HD is essential (but not sufficient) in higher-order associations of PINs. We propose a two-event model that explains the co-operative action of the PINs and why mutations outside the TRD may alter grain texture.     

Identification of MDP (muramyl dipeptide)-binding key domains in NOD2 (nucleotide-binding and oligomerization domain-2) receptor of Labeo rohita

In lower eukaryotes-like fish, innate immunity contributed by various pattern recognition receptor (PRR) plays an essential role in protection against diseases. Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic PRR that recognizes MDP (muramyl dipeptide) of the Gram positive and Gram negative bacteria as ligand and activates signalling to induce innate immunity. Hypothesizing a similar NOD2 signalling pathway of higher eukaryotes, the peripheral blood leucocytes (PBLs) of rohu (Labeo rohita) was stimulated with MDP. The data of quantitative real-time PCR (qRT-PCR) revealed MDP-mediated inductive expression of NOD2 and its down-stream molecule RICK/RIP2 (receptor-interacting serine-threonine protein kinase-2). This observation suggested the existence of MDP-binding sites in rohu NOD2 (rNOD2). To investigate it, 3D model of ligand-binding leucine-rich repeat (LRR) region of rNOD2 (rNOD2-LRR) was constructed following ab initio and threading approaches in I-TASSER web server. Structural refinement of the model was performed by energy minimization, and MD (molecular dynamics) simulation was performed in GROMACS (Groningen Machine for Chemical Simulations). The refined model of rNOD2-LRR was validated through SAVES, ProSA, ProQ, WHAT IF and MolProbity servers, and molecular docking with MDP was carried out in GOLD 4.1. The result of docking identified LRR3-7 comprising Lys820, Phe821, Asn822, Arg847, Gly849, Trp877, Trp901 and Trp931 as MDP-binding critical amino acids in rNOD2. This is the first study in fish to provide an insight into the 3D structure of NOD2-LRR region and its important motifs that are expected to be engaged in MDP binding and innate immunity.     

Novel role of photoinsensitive alleles in adaptation of soybean [Glycine max (L.) Merr.] to rainfed short growing seasons of lower latitudes

Soybean is a temperate photosensitive crop but has adapted to sub-tropical and tropical countries of lower latitudes also. Photoperiodic and maturity genes confer latitudinal adaptation in this crop. Genotyping of accessions of higher latitudes have shown the role of photoinsensitivity, conferred by recessive photoperiodic alleles (e1/e2/e3/e4), in adaptation of the crop to high latitudes but information is not available for lower latitudinal countries like India. We genotyped and calculated the photosensitivity of 101 cultivated Indian soybean varieties and found that majority of the varieties (86) were photosensitive and had the dominant alleles at these loci. Four genotypic classes (e1-as/E2/E3/E4, E1/e2/e3/E4, E1/e2/E3/E4 and E1/E2/e3/E4) were observed for varieties with recessive alleles. Photoinsensitive alleles at E1 and E2 loci significantly reduced the days to flower, maturity and photosensitivity percentage. Adaptive role of photoperiodic alleles was inferred from breeder seed requirement of these varieties for 35 years. Although the photosensitive class contributed 81% to the total seed requirement the weighted mean contribution of this class (380 Q/year) was far less than that of photoinsensitive class (648 Q/year). Photoinsensitivity is essential for perpetuation of crop in higher latitudes. Present report highlights the novel role of photoinsensitive alleles in adaptation of soybean to rainfed, short growing and sub-tropical conditions of lower latitudes by conferring earliness.

     

Puroindolines,Pin alleles,hordoindolines and grain softness proteins are sources of bactericidal and fungicidal peptides

The puroindoline (PIN) proteins in the wheat grain are considered the primary genetic basis of texture. Their lipid-binding nature that enables an effect on texture is also likely related to their antimicrobial properties. A number of mutations leading to hard texture are reported, but their collateral effects on any antimicrobial activities are unknown. To evaluate whether antimicrobial activity is conserved at the tryptophan rich domain (TRD) and affected by point mutations, we designed a series of peptides based on the TRD of proteins encoded by the alleles Pina-D1a, Pina-D1m, Pinb-D1a, Pinb-D1b, Pinb-D1l and Pinb-D1q, as well as by the grain softness protein-1, hordoindoline-a and hordoindoline-b genes. PuroA, Pina-M (Pro-35 to Ser), Hina and GSP-1 peptides displayed measurable antibacterial activity, Pina-M higher than PuroA. Replacement of Arg-39 with Gly decreased the activity considerably, and the five Trp residues in PuroA could not be substituted with Phe without major loss of activity. Except for Pinb-L (Lys-45 to Glu) and Hinb1a, all displayed antifungal activity and none displayed haemolytic activity. The work demonstrates a potential role for this protein family in pathogen protection, stresses the importance of Trp and basic residues, and suggests potential applications in food and health industries.     

Reconciliation of D-genome puroindoline allele designations with current DNA sequence data

Kernel texture is an important trait in cereals, especially wheat (Triticum spp.). Throughout the Triticeae, the puroindoline genes act to soften kernel endosperm. Absence or mutation of either or both of the two puroindolines, ‘a’ and ‘b’, in Triticum aestivum results in harder grain texture. Apparently only one puroindoline haplotype was contributed by the Aegilops tauschii variety that contributed the D-genome during allopolyploidization. Yet, world collections of Ae. tauschii exhibit a range of puroindoline sequence polymorphisms. Consequently, these genes, through synthetic hexaploids (× Aegilotriticum) can enrich the wheat gene pool. Lastly, the puroindolines represent a useful tool for phylogenetic analyses. Here we review original sequence data published and/or available in public databases to reconcile the known gene sequence polymorphisms with a systematic approach to the designating of puroindoline gene and allele symbols in T. aestivum, Ae. tauschii, and × Aegilotriticum. This system follows the recommendations adopted by the International Wheat Genetics Symposium and described in the Catalogue of Gene Symbols for Wheat. Errors, discrepancies and ambiguities in the puroindolines are reviewed; a reconciliation of all existing data is outlined.