Greater mesophyll conductance and leaf photosynthesis in the field through modified cell wall porosity and thickness via AtCGR3 expression in tobacco.

Mesophyll conductance (gm) describes the ease with which CO2 passes from the sub-stomatal cavities of the leaf to the primary carboxylase of photosynthesis, Rubisco. Increasing gm is suggested as a means to engineer increases in photosynthesis by increasing [CO2] at Rubisco, inhibiting oxygenation and accelerating carboxylation. Here, tobacco was transgenically up-regulated with Arabidopsis Cotton Golgi-related 3 (CGR3), a gene controlling methylesterification of pectin, as a strategy to increase CO2 diffusion across the cell wall and thereby increase gm. Across three independent events in tobacco strongly expressing AtCGR3, mesophyll cell wall thickness was decreased by 7%-13%, wall porosity increased by 75% and gm measured by carbon isotope discrimination increased by 28%. Importantly, field-grown plants showed an average 8% increase in leaf photosynthetic CO2 uptake. Up-regulating CGR3 provides a new strategy for increasing gm in dicotyledonous crops, leading to higher CO2 assimilation and a potential means to sustainable crop yield improvement.

Is chloroplast size optimal for photosynthetic efficiency?

Improving photosynthetic efficiency has recently emerged as a promising way to increase crop production in a sustainable manner. While chloroplast size may affect photosynthetic efficiency in several ways, we aimed to explore whether chloroplast size manipulation can be a viable approach to improving photosynthetic performance. Several tobacco (Nicotiana tabacum) lines with contrasting chloroplast sizes were generated via manipulation of chloroplast division genes to assess photosynthetic performance under steady-state and fluctuating light. A selection of lines was included in a field trial to explore productivity. Lines with enlarged chloroplasts underperformed in most of the measured traits. Lines with smaller and more numerous chloroplasts showed a similar efficiency compared with wild-type (WT) tobacco. Chloroplast size only weakly affected light absorptance and light profiles within the leaf. Increasing chloroplast size decreased mesophyll conductance (gm ) but decreased chloroplast size did not increase gm . Increasing chloroplast size reduced chloroplast movements and enhanced non-photochemical quenching. The chloroplast smaller than WT appeared to be no better than WT for photosynthetic efficiency and productivity under field conditions. The results indicate that chloroplast size manipulations are therefore unlikely to lead to higher photosynthetic efficiency or growth.

Guard-cell-targeted overexpression of Arabidopsis Hexokinase 1 can improve water use efficiency in field-grown tobacco plants.

Water deficit currently acts as one of the largest limiting factors for agricultural productivity worldwide. Additionally, limitation by water scarcity is projected to continue in the future with the further onset of effects of global climate change. As a result, it is critical to develop or breed for crops that have increased water use efficiency and that are more capable of coping with water scarce conditions. However, increased intrinsic water use efficiency (iWUE) typically brings a trade-off with CO2 assimilation as all gas exchange is mediated by stomata, through which CO2 enters the leaf while water vapor exits. Previously, promising results were shown using guard-cell-targeted overexpression of hexokinase to increase iWUE without incurring a penalty in photosynthetic rates or biomass production. Here, two homozygous transgenic tobacco (Nicotiana tabacum) lines expressing Arabidopsis Hexokinase 1 (AtHXK1) constitutively (35SHXK2 and 35SHXK5) and a line that had guard-cell-targeted overexpression of AtHXK1 (GCHXK2) were evaluated relative to wild type for traits related to photosynthesis and yield. In this study, iWUE was significantly higher in GCHXK2 compared with wild type without negatively impacting CO2 assimilation, although results were dependent upon leaf age and proximity of precipitation event to gas exchange measurement.

Rubisco production in maize mesophyll cells through ectopic expression of subunits and chaperones.

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 a holoenzyme capable of binding the substrate analog CABP (carboxyarabinitol bisphosphate). Isotope discrimination assays suggest, however, that mesophyll Rubisco is not participating in carbon assimilation in these plants, most probably 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.

Increased Rubisco content in maize mitigates chilling stress and speeds recovery.

Many C4 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 CO2 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.

Overexpression of Rubisco subunits with RAF1 increases Rubisco content in maize.

Rubisco catalyses 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 C4 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 RUBISCO ASSEMBLY FACTOR 1 (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 CO2 assimilation (ASAT) in UBI-LSSS-RAF1 transgenic plants, which correlated with increased fresh weight and in vitro Vcmax 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 Vcmax, and impinging on the realization of photosynthetic potential from increased Rubisco content.