Experiments Used for Heat Stress Studies

Three experiments were carried out at INRA Clermont-Ferrand (45° 46'N, 03° 09'E, 329 m a.s.l.) in the Crop Climate Control and Gas Exchange Measurement (C3-GEM) platform (Triboi et al. 1996) to analyze wheat (Triticum aestivum L.) grain responses to heat treatments. Crops were grown outside in 2-m2 containers 0.5 m deep. Seeds were sown in mid-November at a density of 578 seeds m−2. Crops were watered and fertilized to avoid any growth limitation with an objective of grain protein concentration of 12.5 %. At anthesis the containers were transferred under transparent enclosures under natural light in the C3-GEM platform and exposed to the following conditions:

Experiment 1 Moderate high temperature (MHT) treatment. Cultivars Arche and Tamaro were maintained at 23 °C/11 °C (day/night) for the control and at 28 °C/15

°C for the MHT treatment. Grains were sampled at seven stages from 163 °C days (cumulative degree-days after anthesis) to 781 °C days (physiological maturity) after anthesis.

Experiment 2 Very high temperature (VHT) treatment. Cultivar Thésée was maintained at 18 °C/10 °C for the control treatment and 34 °C/10 °C for the VHT treatment. Results of proteomics analysis of endosperm responses to VHT based on this experiment for total proteins were reported by Majoul et al. (2003) and for albuminsglobulins (AG) by Majoul et al. (2004).

Experiment 3 Heat shock (HS) treatment. Cultivar Récital was grown with a day/ night temperature of 18 °C/10 °C. One container was subjected to 4-h periods at 38 °C for four consecutive days (HS treatment) between 300 and 400 °C days after anthesis. Total proteins and AG were analysed just before and 1, 8, and 26 (ripeness) days after HS were applied. Proteomics analysis of grain responses to HS based on this experiment was reported by Majoul-Haddad et al. (2013).

The Multi-location Field Trial A total of 68 genetically diverse wheat cultivars provided by INRA and by 11 wheat private breeding companies were grown in three locations in France in 2009 and 2010. The cultivars were grown in conventional conditions with full mineral supply and fungicide protection. The aim of the multi-location trial was to better understand the genetic and environmental factors which influence three parameters: dough tenacity (P) and extensibility (L) and bread loaf volume. The grain composition and quality characteristics of 240 samples (40 cultivars × 6 environments) were analyzed.

Main Proteomics Responses of Developing Wheat Grain to High Temperature

Major Impacts of High Temperature on Energy Metabolism and Starch Synthesis

In all experiments average single grain mass was significantly reduced with the temperature treatments. In experiment 3, the HS treatment (38 °C for 4 h on 4 consecutive days) caused a 25 % reduction in single grain dry mass at maturity (26 days after the HS period; Majoul-Haddad et al. 2013). Although here the amount of starch per grain was not determined, previous studies have shown that reduced grain masses in response to elevated temperature results from reduced accumulation of starch (e.g. Altenbach et al. 2003; Hurkman et al. 2009). Most proteomics studies have reported a significant decrease in the amount of the small subunits and/or large subunits of the ADP glucose pyrophosphorylase (AGPase, also named glucose 1 phosphate adenyl transferase), the enzyme that catalyses the first committed step in starch biosynthesis pathway, in response to elevated temperature. In experiment 2, at maturity, the AGPase was reduced by 50 % for VHT compared to control. However, in experiment 3, no significant decrease was observed in response to HS. Surprisingly, the abundance of enzymes involved in amylopectin synthesis (starch synthase SSI and SSII; starch branching enzymes SBE I and SBE II; and starch debranching enzyme) was not reduced by elevated temperatures. The abundance of some of these enzymes was slightly, but not consistently, decreased for HS compared to control. This discrepancy may be associated with the specific procedure required for the extraction of these enzymes, a procedure that is rarely performed in proteomic studies (Bancel et al. 2010). The abundance of granule-bound starch synthase (GBSS I) was increased fivefolds for HT compared to control, in good agreement with the higher amylose content previously observed for grain exposed to elevated temperature (Shi et al. 1994).

Transcriptomic and proteomic analyses of grain exposed to HT revealed an increase of transcripts and proteins involved in glycolysis. Here, the amount of phosphohexose isomerase, aldolase, triose phosphate isomerase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, and enolase increased in response to VHT (experiment 2), or were transiently present in response to HS (experiment 3). Grains exposed to HT require more energy (ATP) as evidenced by the higher abundance of the glycolysis enzymes, reducing the amount of glucose available for starch accumulation. The β-amylase present in the late stage of grain filling (Hurkman et al. 2009) was also reported to have increased threefolds at maturity for VHT compared to control in experiment 2. The over expression of β-amylase, usually found in germinating grain, occurs to provide the endosperm with energy.

The above findings indicate that starch accumulates less due to higher glycolysis and is also partly hydrolysed by β-amylase, probably to provide the endosperm cells with energy. In HT samples, the consequences were a reduced volume of B and C starch granules and correlatively an increased proportion of A granules (Table 28.1).

Table 28.1 Percent volume of large (A-type, diameter > 15 μm), intermediate (B-type, diameter = 5–15 μm) and small (C-type, diameter <5 μm) starch granules determined using laser granulometry for mature grains of cv. Récital grown under normal temperature regime (control) or exposed to 38 °C for 4 h on for 4 consecutive days (heat shock, experiment 3)

Percent volume (%)

Temperature treatment





71.0 ± 0.3

27.8 ± 0.3

1.2 ± 0.01

Heat shock

78.9a ± 0.2

20.1a ± 0.2

1.0a ± 0.01

a Significantly different from control at P < 0.0001 Data are means ± 1 SD for n = 3 independent replicates

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