Shoot-tip derived callus cultures of Sorghum bicolor were transformed by Agrobacterium tumefaciens as well as by bombardment methods with the mutated pyrroline-5-carboxylate synthetase (P5CSF129A) gene encoding the key enzyme for proline biosynthesis from glutamate. The transgenics were selfed for three generations and T4 plants were examined for 100 mM NaCl stress tolerance in pot conditions. The effect of salt stress on chlorophyll and carotenoid contents, photosynthetic rate, stomatal conductance, internal carbon dioxide concentration, transpiration rates, intrinsic transpiration and water use efficiencies, proline content, MDA levels, and antioxidant enzyme activities were evaluated in 40-day-old transgenic lines and the results were compared with untransformed control plants. The results show that chlorophyll content declines by 65% in untransformed controls compared to 30-38% loss (significant at P < 0.05) in transgenics but not carotenoid levels. Photosynthetic rate (PSII activity) was reduced in untransformed controls almost completely, while it declined by 62-88% in different transgenic lines. Salinity induced ca 100% stomatal closure in untransformed plants, while stomatal conductance was decreased only by 64-81% in transgenics after 4 days. The intercellular CO2 decreased by ca 30% in individual transgenic lines. Malondialdehyde (MDA) content was lower in transgenics compared to untransformed controls. The activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6) and glutathione reductase (GR; EC1.8.1.7) were quantified in leaves exposed to 100 mM NaCl stress and found higher in transgenics. The results suggest that transgenic lines were able to cope better with salt stress than untransformed controls by protecting photosynthetic and antioxidant enzyme activities.

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Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) is an important enzyme that participates in lignin biosynthesis especially in the formation of cell wall ferulic esters of plants. It plays a pivotal role in the methylation of the 3-hydroxyl group of caffeoyl CoA. Two cDNA clones that code CCoAOMT were isolated earlier from subabul and in the present study; 3D models of CCoAOMT1 and CCoAOMT2 enzymes were built using the MODELLER7v7 software to find out the substrate binding sites. These two proteins differed only in two amino acids and may have little or no functional redundancy. Refined models of the proteins were obtained after energy minimization and molecular dynamics in a solvated water layer. The models were further assessed by PROCHECK, WHATCHECK, Verify_3D and ERRAT programs and the results indicated that these models are reliable for further active site and docking analysis. The refined models showed that the two proteins have 9 and 10 alpha-helices, 6 and 7 beta-sheets respectively. The models were used for docking the substrates CoA, SAM, SAH, caffeoyl CoA, feruloyl CoA, 5-hydroxy feruloyl CoA and sinapyl CoA which showed that CoA and caffeoyl CoA are binding with high affinity with the enzymes in the presence and absence of SAM. It appears therefore that caffeoyl CoA is the substrate for both the isoenzymes. The results also indicated that CoA and caffeoyl CoA are binding with higher affinity to CCoAOMT2 than CCoAOMT1. Therefore, CCoAOMT2 conformation is thought to be the active form that exists in subabul. Docking studies indicated that conserved active site residues Met58, Thr60, Val63, Glu82, Gly84, Ser90, Asp160, Asp162, Thr169, Asn191 and Arg203 in CCoAOMT1 and CCoAOMT2 enzymes create the positive charge to balance the negatively charged caffeoyl CoA and play an important role in maintaining a functional conformation and are directly involved in donor-substrate binding.