Redirecting the Cyanobacterial Bicarbonate Transporters BicA and SbtA to the Chloroplast Envelope: Soluble and Membrane Cargos Need Different Chloroplast Targeting Signals in Plants.

Vivien Rolland,G Dean Price,Murray R Badger

doi:10.3389/fpls.2016.00185

Abstract

Most major crops used for human consumption are C3 plants, which yields are limited by photosynthetic inefficiency. To circumvent this, it has been proposed to implement the cyanobacterial CO2-concentrating mechanism (CCM), principally consisting of bicarbonate transporters and carboxysomes, into plant chloroplasts. As it is currently not possible to recover homoplasmic transplastomic monocots, foreign genes must be introduced in these plants via nuclear transformation. Consequently, it is paramount to ensure that resulting proteins reach the appropriate sub-cellular compartment, which for cyanobacterial transporters BicA and SbtA, is the chloroplast inner-envelope membrane (IEM). At present, targeting signals to redirect large transmembrane proteins from non-chloroplastic organisms to plant chloroplast envelopes are unknown. The goal of this study was to identify such signals, using agrobacteria-mediated transient expression and confocal microscopy to determine the sub-cellular localization of ∼37 GFP-tagged chimeras. Initially, fragments of chloroplast proteins known to target soluble cargos to the stroma were tested for their ability to redirect BicA, but they proved ineffective. Next, different N-terminal regions from Arabidopsis IEM transporters were tested. We demonstrated that the N-terminus of AtHP59, AtPLGG1 or AtNTT1 (92–115 amino acids), containing a cleavable chloroplast transit peptide (cTP) and a membrane protein leader (MPL), was sufficient to redirect BicA or SbtA to the chloroplast envelope. This constitutes the first evidence that nuclear-encoded transmembrane proteins from non-chloroplastic organisms can be targeted to the envelope of plant chloroplasts; a finding which represents an important advance in chloroplast engineering by opening up the door to further manipulation of the chloroplastic envelope.

Highlights

In the last decade, improving plant traits through genetic engineering has become an area of increased focus (Mittler and Blumwald, 2010; Ort et al, 2015)
It was suggested that the first step toward implementing the cyanobacterial concentrating mechanism (CCM) in the chloroplast of key C3 crop plants be the addition of bicarbonate transporters into the innerenvelope membrane (IEM) (Price et al, 2013; McGrath and Long, 2014)
To carry out a similar function in higher plants, the cyanobacterial bicarbonate transporters BicA and SbtA need to be localized in the chloroplast IEM

Summary

Introduction

In the last decade, improving plant traits through genetic engineering has become an area of increased focus (Mittler and Blumwald, 2010; Ort et al, 2015). In the context of global food security, improving crop yield has emerged as a critical issue, and maintaining current population growth is predicted to require a doubling of the total food production by 2050 (Zhu et al, 2010; Price et al, 2013; Long et al, 2015) To tackle this problem, several avenues have been proposed; one of them being the implementation of components of the CO2-concentrating mechanism (CCM) from cyanobacteria into crop plants such as rice and wheat to improve their photosynthetic capacity (Price et al, 2011a, 2013; McGrath and Long, 2014). Because BicA and SbtA are coded by single genes, they are the more ideal candidates for transfer into higher plants

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