Phenotypic variation in photosynthetic traits in wheat grown under field versus glasshouse conditions.

Recognition of the untapped potential of photosynthesis to improve crop yields has spurred research to identify targets for breeding. The CO2-fixing enzyme Rubisco is characterized by a number of inefficiencies, and frequently limits carbon assimilation at the top of the canopy, representing a clear target for wheat improvement. Two bread wheat lines with similar genetic backgrounds and contrasting in vivo maximum carboxylation activity of Rubisco per unit leaf nitrogen (Vc,max,25/Narea) determined using high-throughput phenotyping methods were selected for detailed study from a panel of 80 spring wheat lines. Detailed phenotyping of photosynthetic traits in the two lines using glasshouse-grown plants showed no difference in Vc,max,25/Narea determined directly via in vivo and in vitro methods. Detailed phenotyping of glasshouse-grown plants of the 80 wheat lines also showed no correlation between photosynthetic traits measured via high-throughput phenotyping of field-grown plants. Our findings suggest that the complex interplay between traits determining crop productivity and the dynamic environments experienced by field-grown plants needs to be considered in designing strategies for effective wheat crop yield improvement when breeding for particular environments.

Improving C4 photosynthesis to increase productivity under optimal and suboptimal conditions.

Although improving photosynthetic efficiency is widely recognized as an underutilized strategy to increase crop yields, research in this area is strongly biased towards species with C3 photosynthesis relative to C4 species. Here, we outline potential strategies for improving C4 photosynthesis to increase yields in crops by reviewing the major bottlenecks limiting the C4 NADP-malic enzyme pathway under optimal and suboptimal conditions. Recent experimental results demonstrate that steady-state C4 photosynthesis under non-stressed conditions can be enhanced by increasing Rubisco content or electron transport capacity, both of which may also stimulate CO2 assimilation at supraoptimal temperatures. Several additional putative bottlenecks for photosynthetic performance under drought, heat, or chilling stress or during photosynthetic induction await further experimental verification. Based on source-sink interactions in maize, sugarcane, and sorghum, alleviating these photosynthetic bottlenecks during establishment and growth of the harvestable parts are likely to improve yield. The expected benefits are also shown to be augmented by the increasing trend in planting density, which increases the impact of photosynthetic source limitation on crop yields.

Measuring Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays.

Rubisco is central to carbon assimilation, and efforts to improve the efficiency and sustainability of crop production have spurred interest in phenotyping Rubisco activity. We tested the hypothesis that microtiter plate-based methods provide comparable results to those obtained with the radiometric assay that measures the incorporation of 14CO2 into 3-phosphoglycerate (3-PGA). Three NADH-linked assays were tested that use alternative coupling enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glycerolphosphate dehydrogenase (GlyPDH); phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH); and pyruvate kinase (PK) and lactate dehydrogenase (LDH). To date there has been no thorough evaluation of their reliability by comparison with the 14C-based method. The three NADH-linked assays were used in parallel to estimate (i) the 3-PGA concentration-response curve of NADH oxidation, (ii) the Michaelis-Menten constant for ribulose-1,5-bisphosphate, (iii) fully active and inhibited Rubisco activities, and (iv) Rubisco initial and total activities in fully illuminated and shaded leaves. All three methods correlated strongly with the 14C-based method, and the PK-LDH method showed a strong correlation and was the cheapest method. PEPC-MDH would be a suitable option for situations in which ADP/ATP might interfere with the assay. GAPDH-GlyPDH proved more laborious than the other methods. Thus, we recommend the PK-LDH method as a reliable, cheaper, and higher throughput method to phenotype Rubisco activity for crop improvement efforts.

Quantification of Photosynthetic Enzymes in Leaf Extracts by Immunoblotting.

In this chapter, we describe a method to extract and quantify photosynthetic enzymes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The method is particularly suitable for characterizing altered protein amounts in leaves of plants produced from genetic engineering or gene-editing approaches. We focus on RuBisCO and RuBisCO activase, a molecular chaperone required to sustain the activity of RuBisCO and CO2 fixation, yet the method can be easily adapted to investigate other leaf proteins of interest.

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.

Physiological Plasticity Is Important for Maintaining Sugarcane Growth under Water Deficit.

The water availability at early phenological stages is critical for crop establishment and sugarcane varieties show differential performance under drought. Herein, we evaluated the relative importance of morphological and physiological plasticity of young sugarcane plants grown under water deficit, testing the hypothesis that high phenotypic plasticity is associated with drought tolerance. IACSP95-5000 is a high yielding genotype and IACSP94-2094 has good performance under water limiting environments. Plants were grown in rhizotrons for 35 days under three water availabilities: high (soil water matric potential [Ψm] higher than -20 kPa); intermediate (Ψm reached -65 and -90 kPa at the end of experimental period) and low (Ψm reached values lower than -150 kPa). Our data revealed that morphological and physiological responses of sugarcane to drought are dependent on genotype and intensity of water deficit. In general, IACSP95-5000 showed higher physiological plasticity given by leaf gas exchange and photochemical traits, whereas IACSP94-2094 showed higher morphological plasticity determined by changes in leaf area (LA) and specific LA. As IACSP94-2094 accumulated less biomass than IACSP95-5000 under varying water availability, it is suggested that high morphological plasticity does not always represent an effective advantage to maintain plant growth under water deficit. In addition, our results revealed that sugarcane varieties face water deficit using distinct strategies based on physiological or morphological changes. When the effectiveness of those changes in maintaining plant growth under low water availability is taken into account, our results indicate that the physiological plasticity is more important than the morphological one in young sugarcane plants.

Short-term physiological changes in roots and leaves of sugarcane varieties exposed to H2O2 in root medium.

The aim of this study was to evaluate the differential sensitivity of sugarcane genotypes to H2O2 in root medium. As a hypothesis, the drought tolerant genotype would be able to minimize the oxidative damage and maintain the water transport from roots to shoots, reducing the negative effects on photosynthesis. The sugarcane genotypes IACSP94-2094 (drought tolerant) and IACSP94-2101 (drought sensitive) were grown in a growth chamber and exposed to three levels of H2O2 in nutrient solution: control; 3 mmol L(-1) and 80 mmol L(-1). Leaf gas exchange, photochemical activity, root hydraulic conductance (Lr) and antioxidant metabolism in both roots and leaves were evaluated after 15 min of treatment with H2O2. Although, root hydraulic conductance, stomatal aperture, apparent electron transport rate and instantaneous carboxylation efficiency have been reduced by H2O2 in both genotypes, IACSP94-2094 presented higher values of those variables as compared to IACSP94-2101. There was a significant genotypic variation in relation to the physiological responses of sugarcane to increasing H2O2 in root tissues, being root changes associated with modifications in plant shoots. IACSP94-2094 presented a root antioxidant system more effective against H2O2 in root medium, regardless H2O2 concentration. Under low H2O2 concentration, water transport and leaf gas exchange of IACSP94-2094 were less affected as compared to IACSP94-2101. Under high H2O2 concentration, the lower sensitivity of IACSP94-2094 was associated with increases in superoxide dismutase activity in roots and leaves and increases in catalase activity in roots. In conclusion, we propose a general model of sugarcane reaction to H2O2, linking root and shoot physiological responses.

Superoxide dismutase and ascorbate peroxidase improve the recovery of photosynthesis in sugarcane plants subjected to water deficit and low substrate temperature.

The physiological responses of C4 species to simultaneous water deficit and low substrate temperature are poorly understood, as well as the recovery capacity. This study investigated whether the effect of these abiotic stressors is cultivar-dependent. The differential responses of drought-resistant (IACSP94-2094) and drought-sensitive (IACSP97-7065) sugarcane cultivars were characterized to assess the relationship between photosynthesis and antioxidant protection by APX and SOD isoforms under stress conditions. Our results show that drought alone or combined with low root temperature led to excessive energetic pressure at the PSII level. Heat dissipation was increased in both genotypes, but the high antioxidant capacity due to higher SOD and APX activities was genotype-dependent and it operated better in the drought-resistant genotype. High SOD and APX activities were associated with a rapid recovery of photosynthesis in IACSP94-2094 plants after drought and low substrate temperature alone or simultaneously.