Sugarcane cystatins: From discovery to biotechnological applications.

Phytocystatins are tight-binding cysteine protease inhibitors produced by plants. The first phytocystatin described was isolated from Oryza sativa and, since then, cystatins from several plant species were reported, including from sugarcane. Sugarcane cystatins were unraveled in Sugarcane EST project database, after sequencing of cDNA libraries from various sugarcane tissues at different developmental stages and six sugarcane cystatins were cloned, expressed and characterized (CaneCPI-1 to CaneCPI-6). These recombinant proteins were produced in different expression systems and inhibited several cysteine proteases, including human cathepsins B and L, which can be involved in pathologies, such as cancer. In this review, we summarize a comprehensive history of all sugarcane cystatins, presenting an updated phylogenetic analysis; chromosomal localization, and genomic organization. We also present protein docking of CaneCPI-5 in the active site of human cathepsin B, insights about canecystatins structures; recombinant expression in different systems, comparison of their inhibitory activities against human cysteine cathepsins B, K, L, S, V, falcipains from Plasmodium falciparum and a cathepsin L-like from the sugarcane weevil Sphenophorus levis; and enlighten their potential and current applications in agriculture and health.

Construction of a novel synthetic root-specific promoter and its characterization in transgenic tobacco plants.

Synthetic promoter technology offers a framework for designing expression cassettes that could provide precise control of transgene expression. Such artificially designed promoters enable defined transgene regulation, reduce unwanted background expression, and can overcome homology-dependent gene silencing in transgenic plants. In the present study, a synthetic root-specific module was designed using characterized cis-acting elements, fused with minimal promoter (86 bp) from PortUbi882 promoter, and cloned in pCAMBIA1305.1 by replacing CaMV 35S promoter so as to drive GUS expression. Two constructs were made; one had the synthetic module at the 5' end of the minimal promoter (SynR1), whereas in the other construct, the module was present in both 5' and 3' ends (SynR2). Furthermore, the synthetic promoter constructs were transformed in tobacco wherein SynR1 promoter drove constitutive expression, whereas SynR2 conferred root-specific expression though slight leaky expression was present in stem. GUS assay in the roots of transgenic tobacco plants (T1) indicated that SynR2 promoter expressed significantly higher GUS activity than the CaMV 35S promoter. The real-time quantitative PCR (RT-qPCR) analysis of GUS gene further confirmed that SynR2 promoter conferred 2.1-fold higher root-specific expression when compared to CaMV 35S promoter.

Vacuolar targeting of r-proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice.

Sugarcane is an ideal candidate for biofarming applications because of its large biomass, rapid growth rate, efficient carbon fixation pathway and a well-developed storage tissue system. Vacuoles occupy a large proportion of the storage parenchyma cells in the sugarcane stem, and the stored products can be harvested as juice by crushing the cane. Hence, for the production of any high-value protein, it could be targeted to the lytic vacuoles so as to extract and purify the protein of interest from the juice. There is no consensus vacuolar-targeting sequence so far to target any heterologous proteins to sugarcane vacuole. Hence, in this study, we identified an N-terminal 78-bp-long putative vacuolar-targeting sequence from the N-terminal domain of unknown function (DUF) in Triticum aestivum 6-SFT (sucrose: fructan 6-fructosyl transferase). In this study, we have generated sugarcane transgenics with gene coding for the green fluorescent protein (GFP) fused with the vacuolar-targeting determinants at the N-terminal driven by a strong constitutive promoter (Port ubi882) and demonstrated the targeting of GFP to the vacuoles. In addition, we have also generated transgenics with His-tagged ß-glucuronidase (GUS) and aprotinin targeted to the lytic vacuole, and these two proteins were isolated and purified from the transgenic sugarcane and compared with commercially available protein samples. Our studies have demonstrated that the novel vacuolar-targeting determinant could localize recombinant proteins (r-proteins) to the vacuole in high concentrations and such targeted r-proteins can be purified from the juice with a few simple steps.