Transgenic sugarcane with higher levels of BRK1 showed improved drought tolerance.

KEY MESSAGE: Transgenic sugarcane overexpressing BRK1 showed improved tolerance to drought stress through modulation of actin polymerization and formation of interlocking marginal lobes in epidermal leaf cells, a typical feature associated with BRK1 expression under drought stress. BRICK1 (BRK1) genes promote leaf epidermal cell morphogenesis and division in plants that involves local actin polymerization. Although the changes in actin filament organization during drought have been reported, the role of BRK in stress tolerance remains unknown. In our previous work, the drought-tolerant Erianthus arundinaceus exhibited high levels of the BRK gene expression under drought stress. Therefore, in the present study, the drought-responsive gene, BRK1 from Saccharum spontaneum, was transformed into sugarcane to test if it conferred drought tolerance in the commercial sugarcane cultivar Co 86032. The transgenic lines were subjected to drought stress, and analyzed using physiological parameters for drought stress. The drought-induced BRK1-overexpressing lines of sugarcane exhibited significantly higher transgene expression compared with the wild-type control and also showed improved physiological parameters. In addition, the formation of interlocking marginal lobes in the epidermal leaf cells, a typical feature associated with BRK1 expression, was observed in all transgenic BRK1 lines during drought stress. This is the first report to suggest that BRK1 plays a role in sugarcane acclimation to drought stress and may prove to be a potential candidate in genetic engineering of plants for enhanced biomass production under drought stress conditions.

Isolation and characterization of water-deficit stress-responsive α-expansin 1 (EXPA1) gene from Saccharum complex.

In this study, full-length (1282-1330 bp) α-expansin 1 (EXPA1) gene from three different accessions belonging to Saccharum complex (Saccharum officinarum-SoEXPA1, Erianthus arundinaceus-EaEXPA1, and Saccharum spp. hybrid-ShEXPA1) was isolated using RAGE technique and characterized. The intronic and coding regions of isolated expansin genes ranged between 526-568 and 756-762 bp, respectively. An open reading frame encoding a polypeptide of 252 amino acids was obtained from S. officinarum and commercial sugarcane hybrid, whereas 254 amino acids were obtained in E. arundinaceus, a wild relative of Saccharum. Bioinformatics analysis of deduced protein revealed the presence of specific signature sequences and conserved amino acid residues crucial for the functioning of the protein. The predicted physicochemical characterization showed that the protein is stable in nature with instability index (II) value less than 40 and also clearly shown the dominance of random coil in the protein structure. Phylogenetic analysis revealed high conservation of EXPA1 among Saccharum complex and related crop species, Sorghum bicolor and Zea mays. The docking study of EXPA1 protein showed the interaction with xylose, which is present in xyloglucan of plant cell wall, elucidated the role of the expansin proteins in plant cell wall modification. This was further supported by the subcellular localization experiment in which it is clearly seen that the expansin protein localizes in the cell wall. Relative expression analysis of EXPA1 gene in Saccharum complex during drought stress showed high expression of the EaEXPA1 in comparison with SoEXPA1 and ShEXPA1 indicating possible role of EaEXPA1 in increased water-deficit stress tolerance in E. arundinaceus. These results suggest the potential use of EXPA1 for increasing the water-deficient stress tolerance levels in crop plants.

Genetic transformation and pyramiding of aprotinin-expressing sugarcane with cry1Ab for shoot borer (Chilo infuscatellus) resistance.

We evaluated the insecticidal toxicity of Cry1Aa, Cry1Ab and Cry1Ac toxins against neonate larvae of sugarcane shoot borer Chilo infuscatellus Snellen (Lepidoptera: Crambidae) in vitro on diet surface. With the lowest LC(50) value, Cry1Ab emerged as the most effective among the three toxins. Sugarcane cultivars Co 86032 and CoJ 64 were transformed with cry1Ab gene driven by maize ubiquitin promoter through particle bombardment and Agrobacterium-mediated transformation systems. Gene pyramiding was also attempted by retransforming sugarcane plants carrying bovine pancreatic trypsin inhibitor (aprotinin) gene, with cry1Ab. Southern analysis confirmed multiple integration of the transgene in case of particle bombardment and single site integration in Agrobacterium-mediated transformants. The expression of cry1Ab was demonstrated through Western analysis and the toxin was quantified using ELISA. The amount of Cry1Ab protein in different events varied from 0.007 to 1.73% of the total soluble leaf protein; the events transformed by Agrobacterium method showed significantly higher values. In in vivo bioassay with neonate larvae of shoot borer, transgenics produced considerably lower percentage of deadhearts despite suffering feeding damage by the borer compared with the untransformed control plants. Expressed Cry1Ab content was negatively related to deadheart damage. Aprotinin-expressing sugarcane pyramided with cry1Ab also showed reduction in damage. The potential of producing sugarcane transgenics with cry1Ab and aprotinin genes resistant to early shoot borer was discussed in the light of the results obtained.