Identification and Functional Characterization of Sugarcane Invertase Inhibitor (ShINH1): A Potential Candidate for Reducing Pre- and Post-harvest Loss of Sucrose in Sugarcane

Abstract

In sugarcane, invertase enzymes play a key role in sucrose accumulation and are also involved in futile reactions where sucrose is continuously degraded during the pre- and post-harvest period, thereby reducing sugar yield and recovery. 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 revealed that ShINHI is indeed exported to the apoplast. 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. ShINH1 is expressed at variable levels in flowers with no specific association to floral 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. 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 recovery in sugarcane.

Metabolite Profiles of Sugarcane Culm Reveal the Relationship Among Metabolism and Axillary Bud Outgrowth in Genetically Related Sugarcane Commercial Cultivars

“…It is widely known that there is a gradient concentration of sucrose along the sugarcane stem, with mature internodes having higher sucrose levels that decrease toward the top immature internodes. Strikingly, the bud sprouting performance along the stem follows the opposite gradient of sucrose ( Whittaker and Botha, 1997 ; Zhu et al, 1997 ; Vorster and Botha, 1999 ; Uys et al, 2007 ) and the culm metabolism is apparently essential to determine the dormant status of the axillary meristem and bud outgrowth ( Boussiengui-Boussiengui et al, 2016 ). Although few studies aimed to investigate the mechanisms responsible for successful germination/sprouting in this species ( Verma et al, 2013 ; Singh et al, 2016 ; Boussiengui-Boussiengui et al, 2016 ), very little is known about the biochemical and molecular aspects related to this process, especially concerning sink and source interactions of the bud and culm.…”

Cell Wall Proteome of Wheat Grain Endosperm and Outer Layers at Two Key Stages of Early Development

“…Recently, two invertase inhibitor genes were identified in sugarcane, one of them encoding a protein (ShINH1) localized in the apoplast of the root, flower, stalk, and leaf. Heterologous expression of ShINH1 confirmed its possible function as an invertase inhibitor [48]. In the same way, an apoplastic invertase inhibitor was shown to affect seed mass in soybean by regulating extracellular invertase during seed maturation [49].…”

Overexpression of Sucrose Phosphate Synthase Enhanced Sucrose Content and Biomass Production in Transgenic Sugarcane

“…The overexpression of invertases accelerate sucrose hydrolysis and enhance plant growth in cotton, Arabidopsis, and loquat [16,17]. On the other hand, the downregulation of SAI by foliar chemical treatment or the inhibition of SAI activity increases sucrose content in sugarcane [18,19]. Efforts were made to reduce invertase activity using antisense techniques, but there was no significant increase in the yield of sucrose in sugarcane [20].The knowledge of the role of SuSy in sucrose accumulation and usage is rather limited.…”