Title : Invertase inhibitors A novel target for regulation of sucrose yield and quality in sugarcane
A remarkable feature of sugarcane is its capacity to store sucrose to about 50% of its dry weight. However, at crop maturity when the sucrose concentration in the sink tissues reaches saturation, the action of invertase enzymes affects sucrose stabilization, and reduces sucrose yield. Thus, sucrose breakdown by invertases causes a major economic loss to farmers and sugar processors. Invertases [EC 22.214.171.124; b-fructosidase] are a family of enzymes known to perform diverse functions in plants in addition to hydrolysis of sucrose into glucose and fructose. In sugarcane, invertases are mainly involved in regulation of sucrose accumulation and plant development. Sucrose unloaded from the phloem in the sugarcane stalk is transported to three different cellular compartments: the apoplastic space (cell wall), cytoplasm, and vacuole Each compartment has a specific invertase isoform: an apoplastic space located cell wall invertase (CWI), a vacuolar located acid invertase (VAI) also termed a soluble acid invertase (SAI), and a cytoplasmic located neutral invertase (NI). Invertase inhibitor (INVINH) proteins play a key role in post-translation regulation of plant invertases through which sucrose hydrolysis is controlled. INVINH proteins are small (18 kDa) members of the pectin methylesterase inhibitor superfamily and they are moderately conserved across plants. In the present study, we identified two INVINH genes from sugarcane, ShINH1 and ShINH2. In silico characterization of the encoded proteins revealed 43% sequence identity at the amino acid level, confirming the non-allelic nature of the proteins. The presence of putative signal peptide and subcellular targeting sequences revealed that ShINH1 and ShINH2 likely have apoplasmic and vacuolar localization, respectively. Experimental visualization of ShINH1–GFP and ShINH2-GFP revealed localization to the apoplast and vacuole respectively. Differential tissue-specific and developmental expression of ShINH1 between leaf, stalk, flower and root suggest that it plays a role in controlling source-sink metabolic regulation during sucrose accumulation in sugarcane. ShINH1 is expressed at relatively high levels in leaves and stalk compared to flowers and roots, and expression decreases significantly toward internodal maturity during stalk development. Whereas, expression of ShINH2 was relatively low at early stages of plant growth (up to 150 DAP) and there was a sudden increase in the transcript levels after 210 to 240 DAP. This is clearly evident that both ShINH1 and ShINH2 are likely to be play important role in sucrose accumulation at crop maturity. Production of recombinant ShINH1 enabled experimental validation of protein function under in vitro conditions. Recombinant ShINH1 potently inhibited acid invertase (IC50 22.5 nM), making it a candidate for controlling pre- and post-harvest deterioration of sucrose in sugarcane. Genome wide identification of invertase inhibitor genes revealed the presence 18 gene homologues which are located at different chromosomes in the genome, indicating the diversity of invertase inhibitor gene family in sugarcane. Our results indicate that ShINH1 and ShINH2 are likely to play a regulatory role in sucrose accumulation and contribute to the improvement of sugar yield and quality in sugarcane.