Heat-induced changes in the abundance of wheat Rubisco activase isoforms

The Triticum aestivum (wheat) genome encodes three isoforms of Rubisco activase (Rca) differing in thermostability, which could be exploited to improve the resilience of this crop to global warming. We hypothesized that elevated temperatures would cause an increase in the relative abundance of heat‐stable Rca1β. Wheat plants were grown at 25° C : 18°C (day : night) and exposed to heat stress (38° C : 22°C) for up to 5 d at pre‐anthesis. Carbon (C) assimilation, Rubisco activity, CA1Pase activity, transcripts of Rca1β, Rca2β, and Rca2α, and the quantities of the corresponding protein products were measured during and after heat stress. The transcript of Rca1β increased 40‐fold in 4 h at elevated temperatures and returned to the original level after 4 h upon return of plants to control temperatures. Rca1β comprised up to 2% of the total Rca protein in unstressed leaves but increased three‐fold in leaves exposed to elevated temperatures for 5 d and remained high at 4 h after heat stress. These results show that elevated temperatures cause rapid changes in Rca gene expression and adaptive changes in Rca isoform abundance. The improved understanding of the regulation of C assimilation under heat stress will inform efforts to improve wheat productivity and climate resilience

Increasing metabolic potential: C-fixation

Due to a growing world population, crop yields must increase to meet rising demand. Crop plants also require adaptation to optimise performance in the changing environments caused by climate change. Improving photosynthetic carbon fixation is a promising, albeit technically challenging, strategy whose potential has only just begun to be considered in breeding programs. Rubisco, a fundamental enzyme of carbon fixation, is extremely inefficient and many strategies to improve photosynthesis focus on overcoming the limitations of this enzyme, either by improving Rubisco activity and regulation or by improving the supply of substrates. Although progress is being made, the need to tailor solutions for each crop and their respective environments has been highlighted. Even so, continuing research will be required to achieve these objectives and to grow crops more sustainably in the future

Exploiting diversity in the regulation of carbon assimilation to improve wheat productivity

Rubisco activase (Rca) is a key regulator of carbon assimilation. It removes inhibitory sugar phosphate derivatives from the active site of Rubisco by using the energy from ATP hydrolysis. However, Rca is thermosensitive and limits photosynthesis at moderately high temperatures and during photosynthetic induction. A better understanding of the properties of Rca isoforms present in wheat, a staple crop for human nutrition, will unlock the potential to improve the efficiency and resilience of carbon assimilation to meet future food demands in the face of climate change. The three wheat Rca isoforms were found to vary in the capacity to activate Rubisco in vitro. Rca1β had lower Rubisco activation activity than Rca2β and Rca2α. Ratios of Rubisco active sites to Rca above 6-11:1 resulted in maximal Rubisco activation. Furthermore, residues that conferred ADP sensitivity of Rca1β were identified using site-directed mutagenesis of the ADP-insensitive Rca2β isoform. These findings can inform future efforts to optimise Rubisco activation during shade to sun transitions in crops. To investigate the thermotolerance of native wheat Rca isoforms, temperature responses of ATP hydrolysis and Rubisco activation were assessed. Rca1β exhibited increased thermotolerance but reduced Rubisco activation activity, in contrast to Rca2β. An isoleucine residue was shown to confer thermostability in the mutant Rca2β-M159I, whilst maintaining high Rubisco activation rates and efficiency. To expand the understanding of thermotolerance and isoform abundance in planta, the effect of heat stress on wheat plants was investigated. The thermotolerant Rca1β increased to 6% of the total Rca protein pool after 5 days of heat stress, but Rubisco activation state remained reduced in heat stressed plants. Furthermore, activity of CA1Pase, which metabolises Rubisco inhibitors, increased in plants in recovery conditions (4 h after relief from heat stress), suggesting a build-up of inhibitors during heat stress and a wider role of this enzyme in Rubisco regulation. Exploiting diversity of wheat wild relatives have been suggested as a strategy for improving modern wheat cultivars. Rca in wild relatives was highly conserved and highly similar to the donor-species of the three wheat sub-genomes. Despite the small differences, the observed variation in Rca expression and protein levels might be associated with the ability of these species to adapt to high temperatures. Overall the results from this thesis suggest that engineering thermotolerant Rca isoforms into wheat to improve carbon assimilation will be the most promising way to make Rubisco regulation in wheat more resilient to heat stress in changing environments

An isoleucine residue acts as a thermal and regulatory switch in wheat Rubisco activase

The regulation of Rubisco, the gatekeeper of carbon fixation into the biosphere, by its molecular chaperone Rubisco activase (Rca) is essential for photosynthesis and plant growth. Using energy from ATP hydrolysis, Rca promotes the release of inhibitors and restores catalytic competence to Rubisco-active sites. Rca is sensitive to moderate heat stress, however, and becomes progressively inhibited as the temperature increases above the optimum for photosynthesis. Here, we identify a single amino acid substitution (M159I) that fundamentally alters the thermal and regulatory properties of Rca in bread wheat (Triticum aestivum L.). Using site-directed mutagenesis, we demonstrate that the M159I substitution extends the temperature optimum of the most abundant Rca isoform by 5°C in vitro, while maintaining the efficiency of Rubisco activation by Rca. The results suggest that this single amino acid substitution acts as a thermal and regulatory switch in wheat Rca that can be exploited to improve the climate resilience and efficiency of carbon assimilation of this cereal crop as temperatures become warmer and more volatile

Rubisco activation by wheat Rubisco activase isoform 2β is insensitive to inhibition by ADP

Rubisco activase (Rca) is a catalytic chaperone that remodels the active site, promotes the release of inhibitors and restores catalytic competence to Rubisco. Rca activity and its consequent effect on Rubisco activation and photosynthesis are modulated by changes to the chloroplast environment induced by fluctuations in light levels that reach the leaf, including redox status and ADP/ATP ratio. The Triticum aestivum (wheat) genome encodes for three Rca protein isoforms: 1β (42.7 kDa), 2β (42.2 kDa) and 2α (46.0 kDa). The regulatory properties of these isoforms were characterised by measuring rates of Rubisco activation and ATP hydrolysis by purified recombinant Rca proteins in presence of physiological ADP/ATP ratios. ATP hydrolysis by all three isoforms was sensitive to inhibition by increasing amounts of ADP in the assay. In contrast, Rubisco activation activity of Rca 2β was insensitive to ADP inhibition, while Rca 1β and 2α were inhibited. Two double and one quadruple site-directed mutants were designed to elucidate if differences in the amino acid sequences between Rca 1β and 2β could explain the differences in ADP sensitivity. Changing two amino acids in Rca 2β to the corresponding residues in 1β (T358K & Q362E) resulted in significant inhibition of Rubisco activation in presence of ADP. The results show that the wheat Rca isoforms differ in their regulatory properties and that amino acid changes in the C domain influence ADP sensitivity. Advances in the understanding of Rubisco regulation will aid efforts to improve the efficiency of photosynthetic CO2 assimilation

Spectrophotometric Determination of Rubisco Activity and Activation State in Leaf Extracts

Rubisco plays a central role in photosynthesis and, due to its catalytic inefficiencies,frequently limits CO2 assimilation in fully illuminated leaves at the top of unstressed crop canopies.The CO2-fixing enzyme is heavily regulated and not all the enzyme present in the leaf is active at any given moment. In this chapter,a spectrophotometric assay is described for measuring Rubisco activity and activation state in leaf extracts.Most of the assay components are available commercially and others can be produced by established protocols, making adoption of the assay achievable by most plant biochemistry laboratories.Its relative high-throughput capacity enables large-scale experiments aimed at screening germplasm for improved Rubisco function