DOI: 10.25911/bwj0-ak03
title: Improving wheat yield through increases in heat tolerance of leaf carbon exchange GRDC ANU2304-001RTX

description: We will screen diverse populations of wheat germplasm - known to differ in heat tolerance as measured by yields during hot seasons - for variability in the heat responses of key biochemical steps in the photosynthetic and respiratory pathways. By coupling hyperspectral measurements to trait screens, we will deliver new high throughput, low-cost tools that enable improved selection accuracy for heat tolerance in breeding programs. We will combine the results of our screens with population genetic structure to identify loci, markers and candidate genes to enable breeding of heat tolerant germplasm that can: maintain photosynthetic CO2 uptake on hot days; and, reduce respiratory CO2 release during hot nights. Our integrated strategy will determine which underlying metabolic traits best define and determine yield in hot climates, and can and should be used in genomic selection approaches to achieve complementary trait stacking.
description: We will screen diverse populations of wheat germplasm - known to differ in heat tolerance as measured by yields during hot seasons - for variability in the heat responses of key biochemical steps in the photosynthetic and respiratory pathways. By coupling hyperspectral measurements to trait screens, we will deliver new high throughput, low-cost tools that enable improved selection accuracy for heat tolerance in breeding programs. We will combine the results of our screens with population genetic structure to identify loci, markers and candidate genes to enable breeding of heat tolerant germplasm that can: maintain photosynthetic CO2 uptake on hot days; and, reduce respiratory CO2 release during hot nights. Our integrated strategy will determine which underlying metabolic traits best define and determine yield in hot climates, and can and should be used in genomic selection approaches to achieve complementary trait stacking.

Our research has led to the development of the high-throughput tools needed to screen for variability in photosynthetic and respiratory metabolic pathways.

Hyperspectral screening: we developed a machine learning (ML) frameworks, based on Partial Least Squares Regression, deep learning and hyperspectral reflectance, that enable prediction of Rubisco capacity (Vcmax) (Furbank et al., 2021, Silva-Perez et al., 2018) and respiratory O2 uptake (Coast et al., 2019b) at 25°C in wheat leaves with high accuracy and speed - 30 s to 1 min per leaf. There is now an opportunity to extend these ML frameworks to predict leaf level photosynthetic and respiratory performance at heat wave temperatures (e.g. 35-40°C), and to scale up leaf-level models to enable canopy level, higher-throughput screening of thousands of lines at multiple growth stages. Ultimately, we envisage development of canopy-level heat tolerance phenomarkers that enable screening at scale in breeding programs.

Heat tolerance of PSII: with the support of GRDC US00080 - 2016.02.01G (Bramley, 2016) and aligned Fellowship US1904-003RTX - 9177346, we developed a high-throughput tool to screen for variability in Tcrit, the temperature at which photosystem II (PSII) loses functionality (Posch et al., 2022a, Coast et al., 2022). PSII is key step in the light reactions, controlling delivery of energy needed to drive photosynthetic CO2 fixation. Using a custom-made fluorescence-based assay, we can screen 200-300 leaf samples per day. Application of the method has shown that there is heritable variation in Tcrit in wheat (Coast et al., 2022) and that Tcrit increases (i.e. acclimates) when wheat is exposed to sustained heat (Posch et al., 2022a). The next step will be to quantify variation in Tcrit among a wide range of wheat lines differing in heat tolerance (Trethowan et al., 2022, Thistlethwaite et al., 2021) – at multiple field sites and seasons – and then establish the genetic basis for variation in intrinsic Tcrit and/or the capacity to thermally-acclimate Tcrit. By tying high throughput measurements of Tcrit to hyperspectral scans, a ML framework could also be developed that would enable even more rapid measurements of this trait in pre-breeding trials.

Photosynthetic O2 evolution capacity: in addition to being able to assay for variability in Tcrit, we are now able to quantify heat tolerance of the actual rate of O2 evolution by PSII. As part of our recently funded HeDWIC-FFAR grant (Doody, 2022, Atkin et al., 2022), we have re-engineered our high throughput O2 fluorophore Astec-Global Q2 system (Scafaro et al., 2017a) with a saturating light source so that maximal rates of PSII-dependent O2-evolution (PSII-O2) – measured in the presence of saturating CO2 and thus unconfounded by stomatal limitations – can be measured in tandem with Rdark. The ability to screen rates of PSII-O2 over a range of supra-optimal temperatures (e.g. 30-40°C) will enable genotypic and environmental variation in the high-temperature optimum of PSII-O2 to be quantified in Australian wheat germplasm.

Stomatal conductance and quantum yield: breeding for high yield over many decades has unintentionally included selection for increased stomatal conductance (gs) (Fischer et al., 1998, Faralli and Lawson, 2020, Lu et al., 1998, De Vita et al., 2007, Rebetzke et al., 2012). The positive effect of gs on yield arise due to: increased availability of CO2 for photosynthesis; reduced photorespiration, resulting in increased Rubisco efficiency; and, increased evaporative cooling to maintain optimal temperature for A (Faralli and Lawson, 2020, Lawson and Blatt, 2014). Maintenance of gs thus has implications for photosynthetic performance at high temperatures (McAusland et al., 2020). Developments in porometer and fluorometer technology mean that is now possible to combine rapid measurements of gs (5-15 seconds per leaf) along with quantification of the quantum yield of fluorescence (FPSII), the latter being a measure of the efficiency that light absorbed by PSII is used in biochemistry.

Dependence of Rubisco activity on Rubisco Activase: Our collaborative work with BASF Agricultural Solutions and the Carmo-Silva lab (Partner Investigator for this GRDC Tender application) has discovered the importance of Rubisco activase (Rca) as a target for improving photosynthetic heat tolerance in wheat. Rca is a heat-labile chaperone that removes tightly-bound inhibitors from the active site of Rubisco (Carmo-Silva et al., 2015). In wheat, photosynthesis is impaired at temperatures exceeding 35°C which can be attributed to loss of Rca function (Perdomo et al., 2017). However, we have discovered a heat stable isoform of wheat Rca (Rca1-b) that is preferentially expressed during heat stress, and can maintain activity at temperatures exceeding 40°C, several °C above the temperature at which the standard wheat Rca isoform denatures (Scafaro et al., 2019, Degen et al., 2021). In wheat, increased Rca1-b expression leads to improved stability of the heterooligomeric Rca holoenzyme extracted from leaves. The extent to which Rca1-b abundance in leaves varies among wheat genotypes is not known. Noting this, there is now an opportunity to determine whether genotypic differences in heat tolerance are linked to levels of Rca1-b gene expression and protein abundance, and in turn, maintenance of Rubisco activity at 40°C. Such knowledge would provide breeders with powerful selection markers to identify genotypes with superior genetics relating to heat tolerance.

Dark respiration and its relationship with photosynthesis: Another key factor determining the impact of heat on yield is the response of leaf dark respiration (Rdark) to high temperatures (Scafaro et al., 2021). Rdark is a temperature sensitive process, with mitochondrial CO2 release and O2 uptake increasing sharply with rising temperature to meet heat-induced demands for respiratory ATP (e.g. by increased protein turnover). Hot conditions can lead to excessive carbon loss by Rdark, with negative effects for daily carbon gain, biomass accumulation and yield (Posch et al., 2022b, Coast et al., 2021, Ferguson et al., 2020). Using our recently developed, high-throughput technique for measuring oxygen-based Rdark (Scafaro et al., 2017a, Coast et al., 2019b), we have shown that there is a two-fold variation in leaf Rdark among 160 wheat lines (Scafaro et al., 2017a), and a significant heritability (30%) in our recent survey of CIMMYT and Australia wheat panels at Obregon, Mexico and Ginninderra, Australia (Gaju et al., 2021). The method has also been applied as part of our GRDC project (US00080 - 2016.02.01G) where we showed that while Rdark increases when wheat leaves are heated, sustained warming leads to reduced Rdark at given leaf temperature (i.e. Rdark thermally acclimates). We have also shown that Rdark at 25°C can be predicted from hyperspectral signatures (Coast et al., 2019b). There is now an opportunity to apply this methodology to determine whether the GRDC-identified heat tolerant lines exhibit intrinsically lower rates of Rdark at high leaf temperatures (i.e. 35-40°C) and/or an improved capacity to thermally acclimate Rdark when challenged with heat.

Importantly, by quantifying both PSIIcap and oxygen-based Rdark using the Astec-Global Q2 system, we can establish the Rdark to photosynthetic assimilation capacity (Rdark/Acap) ratio for individual plants, in replication across trial plots, measured on the same leaf from the same plant. The Rdark/Acap ratio is a powerful indicator of energy efficiency as it can distinguish crops that can achieve the greatest amount of photosynthesis for the least amount of respiratory loss (i.e. the germplasm with the lowest Rdark/Acap ratio).

Equipment

· High-throughput O2 exchange screening robot (ANU and UWA): high-throughput fluorescence-based system (Astec-Global Q2 system) for measuring O2 exchange that we developed (Fan et al., 2022b, Posch et al., 2022b, Scafaro et al., 2017a). The system allows hundreds of measurements of O2 exchange to be assessed simultaneously; it has four Peltier-controlled compartments, enabling rates to be measured over the 20-40°C range, and allowing us to quantify the temperature sensitivity of O2-based leaf Rdark and photosynthesis. Three of these units are available for the project.

· High-throughput fluorescence-based measurements of PSII heat tolerance: A custom-built system that combines an imaging fluorometer (FluorCam 800MF, Photon Systems Instrument, Brno, Czech Republic), a thermoregulatory attached to the fluorometer (TR2000, Photon Systems Instrument, Brno, Czech Republic) and a 48-well Peltier block. The Peltier block measured 8 × 12 cm, and each well on the block was 1 cm in diameter. This set-up was capable of temperature regulation within the 10–70°C range. Two of these set ups are available for the project. Used for measuring the Tcrit of PSII.

· High-throughput Sample Preparation Robotics Facility aka FROSTY (ANU): enables high-throughput sample preparation for plant genomics, metabolomics and proteomics research

· Licor 6400XT infra-red gas analyzers (ANU, USyd and UNE) – for measuring net CO2 and H2O exchange

· Licor 600 porometer/fluorometers (ANU and USyd): high-throughput device for measuring stomatal conductance and photosynthetic quantum yield

· ASD FieldSpec Spectroradiometers – high-throughput devices to measure hyperspectral signatures on all leaves used for trait analyses