Characterization of a cruciferin deficient mutant of Arabidopsis and its utility for overexpression of foreign proteins in plants.

Yimei Lin,C Daniel Riggs,Frédéric Marsolais,Agnieszka Pajak,Peter Mccourt,Haibing Yang

doi:10.1371/journal.pone.0064980

Yimei Lin, C Daniel Riggs + Show 4 more

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https://doi.org/10.1371/journal.pone.0064980

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Abstract

Plant seeds naturally accumulate storage reserves (proteins, carbohydrates, lipids) that are mobilized during germination to provide energy and raw materials to support early seedling growth. Seeds have been exploited as bioreactors for the production to foreign materials, but stable, high level expression has been elusive, in part due to the intrinsic bias for producing the natural reserves in their typical proportions. To identify mutants governing seed filling, we screened a population of mutagenized Arabidopsis plants for a mutant that failed to fill its seeds. Here we report the identification of ssp1, a recessive, viable mutant that accumulates approximately 15% less protein than wildtype seeds. Molecular analyses revealed that ssp1 is due to the introduction of a premature stop codon in CRU3, one of the major cruciferin genes. Unlike many other reserve mutants or transgenic lines in which seed storage protein levels are reduced by antisense/RNAi technologies, ssp1 exhibits low level compensation by other reserves, and represents a mutant background that might prove useful for high level expression of foreign proteins. To test this hypothesis, we used a bean phytohemagglutinin (PHA) gene as a reporter and compared PHA expression levels in single copy insertion lines in ssp1 vs. wildtype. These near isogenic lines allow reporter protein levels to be compared without the confounding and sometimes unknown influences of transgene copy number and position effects on gene expression. The ssp1 lines consistently accumulated more PHA than the backcrossed counterparts, with increases ranging from 12% to 126%. This proof of principle study suggests that similar strategies in crop plants may improve the yield of foreign proteins of agronomic and economic interest.

Highlights

Bacterial, fungal and animal cell cultures have been exploited to produce chemicals and macromolecules of importance to human health, agriculture, and a variety of industrial processes
To identify mutants defective in seed filling and which might represent lesions in sensor/regulatory genes that could be exploited for overexpression of foreign genes, we generated a population of Arabidopsis EMS-mutagenized lines and screened seed protein extracts by gel electrophoresis
Cruciferins are the major type of seed protein in Arabidopsis, and are encoded by three genes, CRU1, CRU2 and CRU3 [22]

Summary

Introduction

Bacterial, fungal and animal cell cultures have been exploited to produce chemicals and macromolecules of importance to human health, agriculture, and a variety of industrial processes. The advent of recombinant DNA and transgenic technologies has revolutionized biomolecular engineering to facilitate large-scale production of such molecules. These systems employ bioreactors that necessitate sterility and require considerable capital investment and maintenance expenses. Research on the biology of naturally occurring metabolites (e.g. seed storage proteins, lipids, starch) and knowledge of the regulatory control of the expression, modification, maturation, targeting, and catabolism of these macromolecules have informed strategies for plant improvement. Translational research successes have been reported in many areas of agriculture and include yield enhancement [1], food quality [2], pathogen resistance [3], and the targeted expression of specific biomolecules of interest to the fields of medicine, pharmacology and industrial processes. Value-added products such as vitamins [4], vaccines [5], biodegradable plastics [6], and biofuels [7], extend the potential economic and agronomic benefits of transgenic plants

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