Ultrastructure and biochemical traits of bread and durum wheat grains under heat stress

Características bioquímicas e ultraestruturais de grãos de trigo mole e trigo rijo sob estresse térmico

RESEARCH ARTICLE

Ultrastructure and biochemical traits of bread and durum wheat grains under heat stress

Características bioquímicas e ultraestruturais de grãos de trigo mole e trigo rijo sob estresse térmico

Ana S. DiasI,^*; Ana S. Bagulho^II; Fernando C. Lidon^I

^IGrupo de Disciplinas de Ecologia da Hidrosfera, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal

^IIEstação Nacional de Melhoramento de Plantas, Instituto Nacional dos Recursos Biológicos, Elvas, Portugal

ABSTRACT

The yield and grain quality (as well as technological traits) of two heat-stressed genotypes of bread (Triticum aestivum L.) and durum wheat (Triticum turgidum subsp. durum) having different tolerance to high temperatures after anthesis were investigated. Heat stress, during grain filling, triggered grain shrinkage with a reduced weight and ultrastructural changes in the aleurone layer and in the endosperm cells. Heat stress also decreased the sedimentation index SDS, an effect associated with increased protein content in the grain but with decreased levels of essential amino acids. Although the responses to heat stress were similar among the Triticum genotypes, it is further suggested that during grain filling, high temperatures might affect gluten strength, diminishing the wheat flour quality.

Key Words: grain weight, grain quality, high temperature, Triticum aestivum, Triticum turgidum subsp. durum

RESUMO

A produção e a qualidade do grão (incluindo alguns aspectos da qualidade tecnológica) sob estresse térmico, durante o enchimento do grão, foram avaliadas em dois genótipos de trigo mole (Triticum aestivum L.) e trigo rijo (Triticum turgidum subsp. durum) com diferentes tolerâncias às temperaturas elevadas após a antese. O estresse térmico, após a antese, induziu o desenvolvimento de grãos enrugados e com peso reduzido. Observaram-se ainda modificações ultraestruturais em nível da camada de aleurona e nas células do endosperma. Os grãos submetidos a temperaturas mais elevadas mostraram índices de sedimentação SDS menores, tendo estado este efeito associado a um aumento nos teores em proteína do grão e ainda a um decréscimo nos níveis de aminoácidos essenciais. Apesar das respostas às temperaturas elevadas não terem sido diferenciadas entre os genótipos de trigo, os resultados sugerem que o estresse térmico, durante o período de enchimento do grão, pode afectar a força do gluten, diminuindo a qualidade da farinha de trigo.

Palavras-chaves: Triticum aestivum, Triticum turgidum subsp. Durum, grãos de trigo

Introduction

During the growth cycle of the wheat crops, the optimal mean temperature might vary between 15-18ºC (Chowdhury and Wardlaw, 1978), with 20ºC being the optimum value for grain filling (Dupont and Altenbash, 2003). Several studies conducted in Australia and USA (Wardlaw and Wrigley, 1994) further indicated that, each year, crop production decreases about 10-15%, mostly due to high temperatures during anthesis. It was also pointed out (Wardlaw et al., 1989) that a global reduction in crop production of about 3-4% occurs when the mean temperature increases by 1ºC above the optimum value. In this context, even when water is not a limiting factor, Triticum productions with late sowing in Mediterranean environments (where high temperatures occur at the end of the cycle) have lowered yields, mostly as a result of heat stress during grain filling (McDonald et al., 1983; Maçãs et al., 1999, 2000). Following this pattern Sofield et al. (1977) showed that, after anthesis, high temperatures (between 15/10ºC and 21/16ºC) counterbalance a diminished duration of growth, increasing the filling rate (although also triggering a small variation in grain weight). Yet, with higher temperatures, ranging between 21/16ºC and 30/25ºC, those authors further pointed out that the grain filling rate did not display a compensatory increase when correlated with its duration period (thus, leading to a significant reduction of the grain weight at maturity).

It has long been known (Spiertz, 1974) that with high temperatures after anthesis, increasing leaf senescence is coupled to a significant increase in the respiration rates in the grain. Such a response, depending on its extent, might trigger decreased carbohydrate availability (Thornley, 1971), justifying the decline in grain weight. Jenner (1991), further supported by Nicolas et al. (1984) in their work on sucrose concentrations (in the grain and endosperm) made a similar proposal, concluding that after anthesis heat stress affects wheat grain quality through changes in protein composition. During grain filling, under heat stress, the decreased grain weight (and, therefore, the yield reduction) also diminishes wheat flour production (Guedira et al., 2002). Additionally, gluten strength and flour quality for breadmaking, although highly linked to the genotype (Branlard and Dardevet, 1985), are also affected by environmental conditions (Rharrabti et al., 2001). Environmental conditions affect the protein concentration, a parameter associated with the definition of Triticum grain quality, as well as gluten strength. In this context, breadmaking quality of bread wheat depends on the viscoelasticity properties of dough, which are affected by the composition and quantity of the gluten proteins in the endosperm (Wang et al., 2005). Accordingly, a high correlation has been found for protein content and texture and volume of the cooked bread (Pomeranz, 1988).

From previous studies on heat stress after anthesis, our research group found that Triticum aestivum L. genotype Sever is more tolerant than the Golia genotype (Maçãs et al., 1999) and that tolerance of Triticum turgidum subsp. durum genotype TE 9306 prevailed relative to the genotype Acalou (Maçãs et al., 2000). In this study, these genotypes were submitted to heat stress during anthesis, to characterize the modifications related to grain quality.

MATERIALS AND METHODS

Genotypes and growth conditions: Bread wheat (Triticum aestivum L. genotypes Sever and Golia) and durum wheat (Triticum turgidum subsp. durum genotypes TE 9306 and Acalou) grains were washed in distilled water and sterilized by immersion in mercury dichloride solution (1:1000) for 2 min. The grains were then washed five times in deionized water and placed in an oven at 28ºC for 24 h. Immediately thereafter the seeds were transferred to a greenhouse and grown in 25 x 21 cm pots containing a 1:1 perlite and vermiculite mixture. The experiment was conducted using 136 pots. Half of these pots were placed under heat stress after anthesis. For each genotype 17 replicates were used (with and without heat stress). Ten seeds were grown per pot and two weeks later five plants were selected, and the others discarded. Accordingly, 680 plants were used. During all the experiment the position of the pots was changed weekly to minimize the effects due to irradiance variations. Plants were irrigated weekly, alternating distilled water and a standard nutrient solution (in mL per 100 L, starter/pre-anthesis/post-anthesis, Ca (NO₃)₂ 100/100/50; KNO₃ 50/200/100; KH₂PO₄ 100/100/100; MgSO₄ 200/200/100; K₂SiO₃ 100/100/0; Fe(NO₃)₃ 20/5/5; EDTA 25/5/5; MnCl₂ 5/10/5; ZnSO₄ 20/10/10; H₃BO₃ 10/5/2; CuSO₄ 5/5/3; Na₂MoO₄ 15/5/5). During the vegetative and reproductive growth, plants were kept under natural light, between March and May in Lisbon, Portugal (38°42' N; 9°05' W) with a photoperiod varying between 12 and 14 h and a mean daily temperature of 19ºC. At anthesis, the plants were separated into two groups and each was submitted to a different temperature treatment (control and heat stress) in two different greenhouses.

Temperature treatments: Plants under the control treatment were grown with mean day/night temperatures of 25/14ºC and plants submitted to heat stress with a mean regime of 31/20ºC (Figure 1). The average day/night temperatures were calculated as the mean of readings taken every 2 h, over each 24 h period. Additionally, they were also submitted to short periods with mean daily temperatures above 32ºC (21-24 April and 19 May 2002).

Yield components: Tillers were removed periodically from each plant, leaving just the main stem. At physiological maturity (that is, when the maximum grain weight was reached), the selected plants were harvested and dried at 80ºC, for 48 h. The numbers of spikelets and grains in each spike were recorded, as well as the grain weight. The individual grain weight was calculated dividing the yield per spike by the number of grains per spike.

Grain ultrastruture: Grains were used for observations with a scanning electron microscopy according to Hall and Hawes (1991). Samples were dried with CO₂ using a Balzers union CPD 020 (England), and then metallized with an EM Scope for the gold-paladium metalization. A scanning IST-DS-130 microscope (Akashi Beam Technology, Tokyo, Japan) was used.

Total and reducing sugars: Grain in powder form (100 mg) was mixed with 10 mL deionized water and boiled in a water bath during 5 min. After cooling, the extract was diluted with deionized water and Sumner reagent (Sumner, 1925) was added (1:20:1 v/v/v). Twenty five milliliters of this mixture was boiled for 5 min and, after cooling, the initial volume was restored with deionized water. For the determination of reducing sugars, the absorbance of this mixture was read at 540 nm (Lindsay 1973). For total sugar analysis, 5 mL of the extract were incubated with 12.5 µL of invertase (commercial grade -Sigma I-9253), at 30ºC during 2 h. Thereafter, 2 mL of this extract were mixed with 2 mL of Sumner reagent (Sumner 1925) and boiled for 5 min. After cooling, the volume was brought to 25 mL with deionized water and the absorbance measured at 540 nm (Lindsay 1973).

Grain amino acids: Amino acids were extracted from the grains (ca. 2 g), using 2 mL of a solution containing methanol, chloroform and water (12:5:3 v/v/v). After centrifugation, 0.8 mL of water and 0.53 mL of chloroform were added to the supernatant. Five minutes later, the aqueous phase was removed and transferred to an eppendorf tube and the organic phase was further extracted with 1 mL of water. This second aqueous phase was removed and mixed with the first one. After adding trifluoroacetic acid (TFA) to the aqueous phase (1:2 v/v) and centrifuging for 15 min, the supernatant was removed and frozen at -80ºC, during 5 min, and taken to dryness in a Speed-Vac. For sample hydrolysis, 1 mL of the aqueous phase was mixed with HCl 6M containing phenol (1 mg mL^-1) and the sample was kept at 110ºC for 24 h. Thereafter, the sample was dried. Amino acids were quantified according to Hayakawa and Oizumi (1989) after isocratic separation by reverse phase HPLC (Shimadzu, Japan). A NUCLEOSIL 5C₁₈ 250 x 4.6 mm column and an eluente mixture containing acetonitrile: water (40:60 v/v) with 0.1% TFA (at a flow rate of 1.0 mL min^-1) were used. The effluent was monitored at 269 nm. The amino acids were identified in the chromatogram by comparison with an amino acid standard mixture (Sigma).

Mineral analysis: For the determination of Ca, K, Mg, Na, Cu, Zn, Fe and Mn, 1.0 g of dry material, from each sample, was mineralized by incineration at ~550ºC, followed by nitric acid digestion (Vandecasteele and Block, 1993). A Unicam model 939 atomic absorption unit (Cambridge, UK), equipped with a hollow cathode lamp was used for the metal determinations. For the measurement of P concentrations, hot digestion with HNO₃ and H₂SO₄ (Watts and Halliwell, 1996) was carried out. Phosphates were determined by a molecular absorption Unit (Cecil 9000 series, Cambridge, UK), following the formation of a chromophore with ammonium molybdate, in the presence of ascorbic acid and potassium antimonyl tartrate trihydrate (Watanabe and Olsen, 1965).

Wheat technological quality: Protein content and hardness were evaluated by near-infrared reflectance spectroscopy using an Inframatic 8620 system (Perten, France), according to the AACC methods 39-25 and 39-70A, respectively. The sodium dodecyl sulfate (SDS) sedimentation test was performed on whole flour samples, as described by Dick and Quick (1983). All quality parameters were evaluated using three replications for each genotype/treatment.

Statistical analysis: Statistical analysis was performed with a two-way ANOVA (P ≤ 0.05), using STATISTICA, version 6 (2001), by StatSoft, Inc (Tulsa, OK, USA). In figures, each value represents the mean ± SE of three replicates and different letters indicate significant difference between means.

RESULTS AND DISCUSSION

During grain growth, the high temperature treatment (31/20ºC, as opposed to 25/14ºC) promoted grain shrinkage (Figures 2 and 3) and a decrease in weight (Table 1). The individual grain weight was more affected by high temperatures in the durum wheat genotypes, compared to bread wheat (relative to the control, 17% and 14% reduction, respectively). In the heat-stressed Sever genotype, relative to Golia, grain weight was significantly higher due to a superior potential grain weight (Table 1). Under heat stress, TE 9306 showed a lower grain weight reduction compared to Acalou but, despite displaying a lower potential, it did have a higher final grain weight (Table 1). Accordingly, as previously reported (Sofield et al., 1977; Chowdhury and Wardlaw, 1978; Wardlaw et al., 1989), our data also indicate that grain weight is affected by high temperatures after anthesis, with the temperature x genotype interaction being highly significant (Table 1) suggesting the occurrence of genetic variability (Wardlaw et al., 1989).

In the absence of heat stress, the unique aleurone layer of the wheat kernel presented large cells (Figures 2 and 3), surrounding a starchy endosperm (Bradbury et al., 1956). During grain filling, the shrunken grains of Acalou and Sever showed an aleurone layer with disordered cells, but in Golia and in TE 9306 cell arrangement of this layer remained unchanged (Figures 2 and 3). Under heat stress, the endosperm of the kernels also seemed to be increasingly aggregated, with the starch granules, as previously observed by Pyler (1988), embedded in the protein matrix and a dense cellular structure (Figures 2 and 3). Several authors (Bechtel et al., 1986) showed that different types of starch granules can be found: A-type (lenticular shaped) starch granules, and B-type (spherical shaped). In Golia, the endosperm of the grains showed a higher cohesion compared to Sever (Figure 2). The grains developed under high temperatures also revealed deformed starch granules in the endosperm (Figures 2 and 3), as also found by Shi et al. (1994), with lower protein adherence.

Genetic tolerance to high temperature (associated with drought) in wheat is observed at later phases of plant development, i.e., shooting and heading (Wiśniewski and Zagdańska, 2001). It is well known that soluble sugars play a complex essential role in plant metabolism as products of hydrolytic processes, substrates in biosynthetic processes and energy production as well as in a sugar sensing and signaling systems. Recently, it has been claimed that sugar flux may be a signal for metabolic regulation (Gibson, 2005), with the mobilization of storage reserves in the endosperm of cereal seeds being tightly regulated and having a primary pivotal role in the response to high temperatures associated with drought (Finkelstein and Gibson, 2001). In our study, although the concentrations of total sugars in bread and durum wheat during grain filling were not significantly affected by high temperatures, the levels of reducing sugars increased in Golia, while no significant variation was found in Sever and a decrease was recorded for TE 9306 (Table 2). According to Leon and Sheen (2003), this pattern might be considered a tolerance signal, possibly developmentally regulated, modulated by the sugar pool and implying genetic variability in Triticum species.

Under heat stress, the protein content increased significantly in all genotypes (Table 3). These results agree with the conclusion reached by Correll et al. (1994) that during grain filling high temperatures (> 30ºC) were related to a rise in grain protein levels; our results also agree with those of Wardlaw et al. (1989) and Guedira et al. (2002) even when the observed decrease of the grain weight is considered (Table 1). Moreover, the inverse relation between protein content which increased and the sedimentation index which decreased (Table 3) agree with the results obtained by Novaro et al. (1997) and Rharrabti et al. (2003) under Mediterranean conditions. During grain filling, the modifications in the grain protein content (Table 3), associated with high temperatures (> 30ºC), have been related to reductions in the sedimentation index SDS (Graybosh et al., 1995) and seemed to promote a decrease in gluten strength. Data obtained in several studies carried out in the field and confirmed under controlled environments (Blumenthal et al., 1993; Stone and Nicolas, 1994; Wrigley et al., 1994; Panozzo and Eagles, 2000), indicate that a few days of maximum daily temperatures surpassing 32ºC produce grains with weaker dough. This effect on the dough properties, involving modifications in the protein composition associated with high temperatures during grain growth has also been pointed out by several authors (Blumenthal et al., 1993; Wrigley et al., 1994). The lack of interaction genotype x treatment for protein content in the bread wheat, and also in SDS for the bread and durum wheat (Table 3), might indicate an absence of genetic variability in the response of these traits to high temperatures. In bread wheat, the grain hardness increased significantly with heat stress (Table 3) which, according to Guedira et al. (2002), might be an important factor for the wheat technological value. An increase in grain hardness might modify the milling quality, since greater energy will be required for grain rupture procedures and the production of smaller particles (Finney et al., 1987). Additionally, this effect might also impair conservation and increase damage to starch grains during milling (Pomeranz and Williams, 1990).

The amino acid composition of proteins showed that the stability of Lys was strongly affected (18-30%) under heat stress (Table 4). On the other hand, the levels of Thr, which is the second limiting amino acid (after Lys), increased significantly (5-40%) with high temperatures (Table 4). Concerning non-essential amino acids of bread and durum wheat grains, only the contents of Arg and His vary significantly relative to controls (Table 5). The level of His decreased significantly (except in Acalou), between 10% and 26%, in the durum and bread wheat genotypes. Under heat stress conditions, the contents of Arg also showed a similar trend, decreasing between 4-17%.

The contents of K, Na, Cu, and Mn in the grain were not affected by high temperatures, but the levels of Ca, Zn and Fe in the grains of Sever increased significantly by about 3, 0.5 and 1 fold, respectively (Figures 4 and 5).

In Golia, only the concentrations of P increased (by 19%) (Figure 4). Acalou followed an opposite trend, in comparison with the others genotypes, with high temperatures significantly decreasing the levels of Mg in the grain. Additionally, this genotype also showed a signifficant increase of Zn under heat stress conditions (ca. 100%). In general, under control and heat stress coditions, the nutrient contents of the grais were not significantly different betwen both genotypes of each species (Figures 4 and 5). Yet, although under control conditions the level of Fe in the grain of Sever was significantly lower than in Golia, this difference could not be found under high temperature, suggesting a marked increase of this metal content in heat-stressed Sever (Figures 4 and 5). The nutrient contents of the grain, under heat stress, were similar to those found by Calderini and Ortiz-Monasterio (2003) in two varieties of bread wheat. The only exceptions were Ca and Zn which were higher in our work, probably reflecting specific genomic characteristics.

CONCLUSIONS

At an ultrastructural level heat stress triggered changes in the aleurone layer and in the endosperm cells during grain filling. High temperatures, after anthesis, had negative effects on wheat quality, as indicated by the diminished levels of the essential amino acids in the grain following heat stress. A negative trend for the SDS test was associated with an increase in grain protein content, indicating that heat stress could indirectly reduce gluten strength during grain filling.

Received: 25 June 2008; Returned for revision: 06 August 2008; Accepted: 19 August 2008

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Publication Dates

• Publication in this collection
  14 Dec 2009
• Date of issue
  Dec 2008

History

• Reviewed
  06 Aug 2008
• Received
  25 June 2008
• Accepted
  19 Aug 2008