Redox homeostasis at SAM: a new role of HINT protein

Abstract

ScHINT1 was identified at sugarcane SAM using subtractive libraries. Here, by bioinformatic tools, two-hybrid approach, and biochemical assays, we proposed that its role might be associated to control redox homeostasis. Such control is important for plant development and flowering transition, and this is ensured with some protein partners such as PAL and SBT that interact with ScHINT1. The shoot apical meristem transition from vegetative to reproductive is a crucial step for plants. In sugarcane (Saccharum spp.), this process is not well known, and it has an important impact on production due to field reduction. In view of this, ScHINT1 (Sugarcane HISTIDINE TRIAD NUCLEOTIDE-BINDING PROTEIN) was identified previously by subtractive cDNA libraries using Shoot Apical Meristem (SAM) by our group. This protein is a member of the HIT superfamily that was composed of hydrolase with an AMP site ligation. To better understand the role of ScHINT1 in sugarcane flowering, here its function in SAM was characterized using different approaches such as bioinformatics, two-hybrid assays, transgenic plants, and biochemical assays. ScHINT1 was conserved in plants, and it was grouped into four clades (HINT1, HINT2, HINT3, and HINT4). The 3D model proposed that ScHINT1 might be active as it was able to ligate to AMP subtract. Moreover, the two-hybrid approach identified two protein interactions: subtilase and phenylalanine ammonia-lyase. The evolutionary tree highlighted the relationships that each sequence has with specific subfamilies and different proteins. The 3D models constructed reveal structure conservation when compared with other PDB-related crystals, which indicates probable functional activity for the sugarcane models assessed. The interactome analysis showed a connection to different proteins that have antioxidative functions in apical meristems. Lastly, the transgenic plants with 35S::ScHINT1_AS (anti-sense orientation) produced more flowers than wild-type or 35S::ScHINT1_S (sense). Alpha-tocopherol and antioxidant enzymes measurement showed that their levels were higher in 35S::ScHINT_S plants than in 35S::ScHINT1_AS or wild-type plants. These results proposed that ScHINT1 might have an important role with other proteins in orchestrating this complex network for plant development and flowering.

Keywords: Antioxidant enzymes; HIT superfamily; Plant development; Protein network; Saccharum spp.; Two-hybrid system.