Molecular and physiological consequences of transgenic overexpression of Rubisco subunit and assembly chaperones in Zea mays under control and chilling conditions

Abstract

Photosynthesis, the incorporation of atmospheric CO2 into organic compounds, is essential for most life on Earth. Rubisco catalyzes a rate limiting step in this reaction and has long been a key target for improvement, but there has been little success due to the lack of understanding of its complex biogenesis pathway. In maize (Zea mays), three assembly chaperones are known to be required for Rubisco assembly: Bundle Sheath Defective 2 (BSD2), Rubisco Assembly Factor 1 (RAF1) and Rubisco Assembly Factor 2 (RAF2). The purpose of the work described in this thesis was to use the knowledge of these assembly factors to explore whether their overexpression, alone or in combination with Rubisco subunits, would have consequences for plant growth and stress tolerance. BSD2 is required for Rubisco assembly and correct bundle sheath (BS) cell differentiation. To test if BSD2 has additional roles in cell development, the bsd2 mutant was complemented with over/underexpression of BSD2 in the BS. BSD2 expression levels correlated with increased/decreased chloroplast coverage (chloroplast area per cell) in the BS, due to increases in individual chloroplast areas rather than number of chloroplasts per cell. This suggests BSD2 has an ancillary role in regulating and maintaining chloroplast size. Next, I aimed to increase Rubisco abundance as a strategy to increase enzyme activity and carbon assimilation. Since overexpression of Rubisco large (LS) and small (SS) subunits does not result in increased Rubisco content in maize, I overexpressed the Rubisco subunits with the assembly chaperone RAF1. This resulted in a >30% increase in Rubisco content that correlated with increases in CO2 assimilation and above ground fresh weight. Rubisco activation state was negatively correlated with Rubisco content, suggesting Rubisco activase may be limiting the full photosynthetic potential of increased Rubisco content. The final goal of this study was to investigate whether increasing Rubisco content in maize could improve performance during chilling stress, where Rubisco abundance is thought to be limiting. I demonstrate that plants with transgenically-increased Rubisco content had increased photoprotection and reduced damage to PSII after two weeks of chilling. CO2 assimilation rates were increased 17%, and increased leaf area, fresh and dry weight and plant height was also observed. These results demonstrate that increased Rubisco improves the ability of maize to cope with chilling stress.