Functional Incorporation of Sorghum Small Subunit Increases the Catalytic Turnover Rate of Rubisco in Transgenic Rice      

Abstract

Rubisco limits photosynthetic CO 2 fixation because of its low catalytic turnover rate (k cat ) and competing oxygenase reaction. Previous attempts to improve the catalytic efficiency of Rubisco by genetic engineering have gained little progress. Here we demonstrate that the introduction of the small subunit (RbcS) of high k cat Rubisco from the C 4 plant sorghum (Sorghum bicolor) significantly enhances k cat of Rubisco in transgenic rice (Oryza sativa). Three independent transgenic lines expressed sorghum RbcS at a high level, accounting for 30%, 44%, and 79% of the total RbcS. Rubisco was likely present as a chimera of sorghum and rice RbcS, and showed 1.32-to 1.50-fold higher k cat than in nontransgenic rice. Rubisco from transgenic lines showed a higher K m for CO 2 and slightly lower specificity for CO 2 than nontransgenic controls. These results suggest that Rubisco in rice transformed with sorghum RbcS partially acquires the catalytic properties of sorghum Rubisco. Rubisco content in transgenic lines was significantly increased over wild-type levels but Rubisco activation was slightly decreased. The expression of sorghum RbcS did not affect CO 2 assimilation rates under a range of CO 2 partial pressures. The J max /V cmax ratio was significantly lower in transgenic line compared to the nontransgenic plants. These observations suggest that the capacity of electron transport is not sufficient to support the increased Rubisco capacity in transgenic rice. Although the photosynthetic rate was not enhanced, the strategy presented here opens the way to engineering Rubisco for improvement of photosynthesis and productivity in the future.

Isoleucine 309 acts as a C 4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria

“…The apparent catalytic neutrality of the tobacco S-subunit when assembled with heterologous L-subunits (Fig. 3) (24,29,30) contrasts with the recent success in shaping rice rubisco toward C 4 -like catalysis using heterologous S-subunits from C 4 sorghum (16). Similarly, structural changes to the S-subunit have improved Chlamydomonas rubisco catalysis (17).…”

“…At 25°C under ambient oxygen levels, the carboxylation efficiency (i.e., V C /K C 21%O2 ) of the C 4 -like 309 Ile-containing rubiscos in tob bid and tob flo-bid (145 mM −1 ·s −1 ) were poorer than the C 3 -like 309 Met-L 8 F S 8 t enzymes in tob pring and tob flo (150 and 161 mM −1 ·s −1 , respectively). Thus, because of the low CO 2 levels within (unstressed) C 3 chloroplasts (<10 μM), the faster C 4 -like enzymes probably provide no advantage to plant growth within a C 3 plant (at least at 25°C), as shown recently in rice (16). As modeled recently, optimal CO 2 concentrations required for C 4 rubisco are substantially higher (∼80 μM) (8), indicating that taking full advantage of a faster rubisco in a C 3 plant will require the combinatorial effect of a suitable CCM, for which a number of strategies are being pursued (2).…”

“…The catalytic core of L 8 S 8 rubisco comprises four 52-kDa large (L)-subunit pairs which are capped by two sets of 15-kDa small (S)-subunit tetramers that provide structural stability and influence catalysis (16,17). Although supplementing rice rubisco with Ssubunits from the C 4 plant sorghum was found to improve V C of the heterologous L 8 S 8 enzyme (16), crosses between C 3 and C 4 Flaveria and Atriplex species showed C 4 catalysis to be maternally inherited (18,19) and hence defined by the chloroplast-encoded L-subunit gene (rbcL). Therefore, changes in both Rubisco Land S-subunits can influence catalysis.…”

Photosynthetic Trichomes Contain a Specific Rubisco with a Modified pH-Dependent Activity

“…For instance, expressing higher plant RbcS subunits in a Chlamydomonas reinhardtii mutant that lacks rbcS genes showed that RbcS makes a significant contribution to the Rubisco activity (Genkov et al, 2010). Expressing an RbcS from sorghum, a C4 plant, in rice, a C3 plant, resulted in increased catalytic turnover of Rubisco (Ishikawa et al, 2011).…”

Synthetic biology for CO2 fixation

“…However, construction of such a cycle was still restricted by uncertainties in the expression, activity, stability, and regulation of all enzymes in this pathway. Recently, relocation of natural CO2-fixation pathways has (Kumar et al, 2009) Replaced the tobacco RuBisCO with cyanobacteria RuBisCO and observed significantly increased growth rate of tobacco under high concentration of CO2 2014 (Lin et al, 2014) Constructed a hybrid RuBisCO from different RuBisCO large and small subunits and studied its enzymatic properties - (Genkov et al, 2010;Ishikawa et al, 2011) Reported that over-expressing the sedoheptulose-1-7 bisphosphatase improves photosynthetic carbon gain and yield 2011 (Rosenthal et al, 2011) received much attention, as engineering natural CO2-fixing autotrophic microbes is usually difficult. In 2013, Mattozzi et al divided the 16 steps of the 3-hydroxypropionate cycle from Chloroflexus aurantiacus into four sub-pathways and expressed each sub-pathway in Escherichia coli (Mattozzi et al, 2013).…”