Rubisco catalyzes a rate-limiting step in photosynthesis and has long been a target for improvement due to its slow turnover rate. An alternative to modifying catalytic properties of Rubisco is to increase its abundance within C 4 plant chloroplasts, which might increase activity and confer a higher carbon assimilation rate. Here, we overexpress the Rubisco large (LS) and small (SS) subunits with the Rubisco assembly chaperone RAF1. While overexpression of LS and/or SS had no discernable impact on Rubisco content, addition of RAF1 overexpression resulted in a >30% increase in Rubisco content. Gas exchange showed a 15% increase in CO 2 assimilation (A SAT ) in UBI-LSSS-RAF1 transgenic plants, which correlated with increased fresh weight and in vitro V cmax calculations. The divergence of Rubisco content and assimilation could be accounted for by the Rubisco activation state, which decreased up to 23%, suggesting that Rubisco activase may be limiting V cmax , and impinging on the realization of photosynthetic potential from increased Rubisco content.

Many C 4 plants, including maize, perform poorly under chilling conditions. This phenomenon has been linked in part to decreased Rubisco abundance at lower temperatures. An exception to this is chilling-tolerant Miscanthus, which is able to maintain Rubisco protein content under such conditions. The goal of this study was to investigate whether increasing Rubisco content in maize could improve performance during or following chilling stress. Here, we demonstrate that transgenic lines overexpressing Rubisco large and small subunits and the Rubisco assembly factor RAF1 (RAF1-LSSS), which have increased Rubisco content and growth under control conditions, maintain increased Rubisco content and growth during chilling stress. RAF1-LSSS plants exhibited 12% higher CO 2 assimilation relative to nontransgenic controls under control growth conditions, and a 17% differential after 2 weeks of chilling stress, although assimilation rates of all genotypes were~50% lower in chilling conditions. Chlorophyll fluorescence measurements showed RAF1-LSSS and WT plants had similar rates of photochemical quenching during chilling, suggesting Rubisco may not be the primary limiting factor that leads to poor performance in maize under chilling conditions. In contrast, RAF1-LSSS had improved photochemical quenching before and after chilling stress, suggesting that increased Rubisco may help plants recover faster from chilling conditions. Relatively increased leaf area, dry weight and plant height observed before chilling in RAF1-LSSS were also maintained during chilling. Together, these results demonstrate that an increase in Rubisco content allows maize plants to better cope with chilling stress and also improves their subsequent recovery, yet additional modifications are required to engineer chilling tolerance in maize.

C4 plants, such as maize, strictly compartmentalize Rubisco to bundle sheath chloroplasts. The molecular basis for the restriction of Rubisco from the more abundant mesophyll chloroplasts is not fully understood. Mesophyll chloroplasts transcribe the Rubisco large subunit gene, and when normally quiescent transcription of the nuclear Rubisco small subunit gene family is overcome by ectopic expression, mesophyll chloroplasts still do not accumulate measurable Rubisco. Here we show that a combination of five ubiquitin promoter-driven nuclear transgenes expressed in maize leads to mesophyll accumulation of assembled Rubisco. These encode the Rubisco large and small subunits, Rubisco Assembly Factors 1 and 2, and the assembly factor Bundle Sheath Defective 2. In these plants Rubisco large subunit accumulates in mesophyll cells, and appears to be assembled into holoenzyme capable of binding the substrate analog CABP. Isotope discrimination assays suggest, however, that mesophyll Rubisco is not participating in carbon assimilation in these plants, most likely due to a lack of the substrate ribulose 1,5-bisphosphate and/or Rubisco activase. Overall, this work defines a minimal set of Rubisco assembly factors in planta and may help lead to methods of regulating the C4 pathway.