In Arabidopsis thaliana Substrate Recognition and Tissue- as Well as Plastid Type-Specific Expression Define the Roles of Distinct Small Subunits of Isopropylmalate Isomerase.

Kurt Lächler,Barbara Ann Halkier,Janet Imhof,Christoph Crocoll,Stefan Binder,Alexander Schulz,Karen Clauss

doi:10.3389/fpls.2020.00808

Kurt Lächler, Barbara Ann Halkier + Show 5 more

Open Access

https://doi.org/10.3389/fpls.2020.00808

Copy DOI

Abstract

In Arabidopsis thaliana, the heterodimeric isopropylmalate isomerase (IPMI) is composed of a single large (IPMI LSU1) and one of three different small subunits (IPMI SSU1 to 3). The function of IPMI is defined by the small subunits. IPMI SSU1 is required for Leu biosynthesis and has previously also been proposed to be involved in the first cycle of Met chain elongation, the first phase of the synthesis of Met-derived glucosinolates. IPMI SSU2 and IPMI SSU3 participate in the Met chain elongation pathway. Here, we investigate the role of the three IPMI SSUs through the analysis of the role of the substrate recognition region spanning five amino acids on the substrate specificity of IPMI SSU1. Furthermore, we analyze in detail the expression pattern of fluorophore-tagged IPMI SSUs throughout plant development. Our study shows that the substrate recognition region that differs between IPMI SSU1 and the other two IMPI SSUs determines the substrate preference of IPMI. Expression of IPMI SSU1 is spatially separated from the expression of IPMI SSU2 and IPMI SSU3, and IPMI SSU1 is found in small plastids, whereas IMPI SSU2 and SSU3 are found in chloroplasts. Our data show a distinct role for IMPI SSU1 in Leu biosynthesis and for IMPI SSU2 and SSU3 in the Met chain elongation pathway.

Highlights

Amino acids serve as building blocks for proteins and have other important functions
In a previously used construct, which allows the expression of the isopropylmalate isomerase (IPMI) SSU1 reading frame under the IPMI SSU3 promoter (Table 1, ipmi ssu2-1/ipmi ssu3-1 + pSSU3:SSU1), we replaced several amino acids in the substrate recognition region in the IPMI SSU1 reading frame by amino acid residues present in this region in IPMI SSU3 (Figure 2, IPMI pSSU3:SSU1/SSU3srr)
In line with the apparent function of the single small IPMI subunit from rice in Leu biosynthesis, the substrate recognition region (SRR) region from this protein is almost identical to the Arabidopsis IPMI SSU1 that functions in Leu metabolism (Supplementary Figure S1; Imhof et al, 2014)

Summary

Introduction

Amino acids serve as building blocks for proteins and have other important functions. In plants for instance, they are initial substrates for the biosynthesis of specialized metabolites such as glucosinolates in the family of the Brassicaceae. According to the parent amino acid, glucosinolates are grouped into three major categories. Aromatic glucosinolates derive from Phe or Tyr whereas those synthesized from Trp represent the indole glucosinolates. The highly diversified aliphatic glucosinolates originate from Ala, from the branched-chain amino acids Ile) or from Met. The compounds derived from Met represent the most important subgroup of aliphatic glucosinolates in terms of quantity and diversity (Wittstock and Halkier, 2002; Halkier and Gershenzon, 2006)

Methods

Results

Conclusion