Multiple loci with cumulative effects on late maturity α-amylase (LMA) in wheat.

MAIN CONCLUSION: The cumulative action of combinations of alleles at several loci on the wheat genome is associated with different levels of resistance to late maturity α-amylase in bread wheat. Resistance to late maturity α-amylase (LMA) in bread wheat (Triticum aestivum L.) involves a complex interaction between the genotype and the environment. Unfortunately, the incidence and severity of LMA expression is difficult to predict and once the trait has been triggered an unacceptably low falling number, high grain α-amylase may be the inevitable consequence. Wheat varieties with different levels of resistance to LMA have been identified but whilst some genetic loci have been reported, the mechanisms involved in resistance and the interaction between resistance loci requires further research. This investigation was focused on mapping resistance loci in populations derived by inter-crossing resistant wheat varieties or crossing resistant lines with a very susceptible line and then mapping quantitative trait loci. In addition to the previously reported locus on chromosome 7B for which a candidate gene has been proposed, loci were mapped on chromosomes 1B, 2A, 2B, 3A, 3B, 4A, 6A and 7D. These loci have limited effects on their own but have a cumulative effect in combination with each other. Further research will be required to determine the nature of the causal genes at these loci, to develop diagnostic markers and determine how the genes fit into the pathway that leads to the induction of α-AMY1 transcription in the aleurone of developing wheat grains. Depending on the target environmental conditions, different combinations of alleles may be required to achieve a low risk of LMA expression.

Gibberellins in developing wheat grains and their relationship to late maturity α-amylase (LMA).

MAIN CONCLUSION: α-Amylase synthesis by wheat aleurone during grain development (late maturity α-amylase) appears to be independent of gibberellin unlike α-amylase synthesis by aleurone during germination or following treatment with exogenous GA. Late-maturity α-amylase (LMA) in wheat (Triticum aestivum L.) involves the synthesis of α-amylase by the aleurone tissue during grain development. Previous research identified a putative ent-copalyl diphosphate synthase gene, coding for an enzyme that controls the first step in gibberellin biosynthesis, that underlies the major genetic locus involved in variation in LMA phenotype. The reported results for gene transcript analysis, preliminary gibberellin analysis and the effects of DELLA mutants on LMA phenotype appeared to be consistent with involvement of gibberellin but did not provide definitive proof of a causal link. Conversely, several observations do not appear to be consistent with this hypothesis. In this current study, LMA phenotype, gibberellin profiles and ABA content were recorded for experiments involving susceptible and resistant genotypes, gibberellin biosynthesis inhibitors, genetic lines containing different LMA quantitative trait loci and treatment of distal halves of developing grains with exogenous gibberellin. The results suggested that gibberellin may not be a prerequisite for LMA expression and further that the mechanism involved in triggering α-amylase synthesis did not correspond to the model proposed for germination and gibberellin challenged aleurone of ripe grain. The results provide new insight into LMA and highlight the need to investigate alternate pathways for the induction of α-amylase gene transcription, the function of novel 1-ß-OH gibberellins and other functions of DELLA proteins in developing grains.

Lipoxygenase in Wheat: Genetic Control and Impact on Stability of Lutein and Lutein Esters.

Preservation of lutein concentrations in wheat-based end-products during processing is important both for product quality and nutritional value. A key constituent involved in lutein degradation is endogenous lipoxygenase. Lutein and lutein ester concentrations were compared at intervals during storage of noodle sheets prepared from flour of wheat varieties representing a range in lipoxygenase activity, as well as in different mill streams and in different grain tissues. Higher lipoxygenase concentration was associated with an increased loss of free lutein and lutein mono-esters whereas lutein diesters appeared to be more resistant to degradation. Lutein degradation was reduced in the presence of a lipoxygenase inhibitor, when noodle sheets were heated to destroy enzyme activity or when pH was increased. In addition, three populations were used to investigate the genetic control of lipoxygenase. A previously reported mutation of Lpx-B1.1 was associated with a reduction in activity from high to intermediate whilst a new locus on chromosome 4D was associated with variation between intermediate and near-zero. The gene underlying the 4D locus is a putative lipoxygenase. Stability of lutein could be improved by deployment of the mutations at the 4B and 4D loci and/or by post-harvest storage of grain under conditions that promote esterification.

A Major Locus on Wheat Chromosome 7B Associated With Late-Maturity α-Amylase Encodes a Putative ent-Copalyl Diphosphate Synthase.

Many wheat varieties have the potential to develop unacceptably high levels of α-amylase in the grains if exposed to a cool temperature shock or simply cool temperature during the early to middle stages of grain filling. This phenomenon is referred to as late maturity α-amylase (LMA). The enzyme persists in the grain until harvest and may result in wheat with a low Falling Number that does not meet receival and export specifications. Resistance to LMA is therefore a valuable target for wheat breeders and wheat industries in general. Genetic evidence implicating a locus on the long arm of chromosome 7B in variation in LMA phenotype was confirmed in this investigation. Through intensive fine-mapping an ent-copalyl diphosphate synthase (CPS), hitherto named LMA-1, was identified as the likely candidate gene associated with variation in LMA phenotype. Single Nucleotide Polymorphisms (SNPs) within the LMA-1 coding sequence of Chinese Spring, Maringa and Halberd result in either prematurely terminated or functionally altered proteins that are associated with useful levels of resistance to LMA. LMA-1 transcripts detected in de-embryonated grain tissue from around 15 days after anthesis, several days before the synthesis of α-amylase, were low in the resistant varieties Chinese Spring and Maringa compared with LMA susceptible genotype Spica. This was associated with a dramatic reduction in the concentrations of intermediates in the gibberellin biosynthesis pathway such as GA19, evidence that LMA-1 was functioning as CPS in the gibberellin biosynthesis pathway. A survey of a large collection of Australian and international wheat varieties distinguished 9 major haplotypes at the LMA-1 locus. Generally, within classes, there was notable variation for LMA phenotype and evidence for genotypes whose resistance is presumed to be due to genetic loci located elsewhere on the wheat genome. Further investigation is required to characterize the sequence of steps between LMA-1 and α-amylase synthesis as well as to gain a better understanding of the role and potential impact of other genetic loci. Diagnostic markers for sources of resistance and SNP variation reported in this study should assist breeders to deploy resistance associated with LMA-1 variants in breeding programs.

Dormancy and dormancy release in white-grained wheat (Triticum aestivum L.).

MAIN CONCLUSION: Dormancy in white-grained wheat is conditioned by the cumulative effects of several QTL that delay the onset of the capacity to germinate during ripening and after-ripening. Grain dormancy at harvest-ripeness is a major component of resistance to preharvest sprouting in wheat (Triticum aestivum L.) and an important trait in regions where rain is common during the harvest period. Breeding lines developed in Australia maintained their dormancy phenotype over multiple seasons and during grain ripening, the time between anthesis and the acquisition of the capacity to germinate, dormancy release, increased in line with the strength of dormancy. Genetic dissection of two dormant lines indicated that dormancy was due to the cumulative action of between one and three major genetic loci and several minor loci. This presents a significant challenge for breeders targeting environments with a high risk of sprouting where strong dormancy is desirable. Only around half of the difference in dormancy between the dormant lines and a non-dormant variety could be attributed to the major genetic loci on chromosomes 4A and 3A. A QTL that was mapped on chromosome 5A may be an orthologue of a minor QTL for dormancy in barley. At each locus, the dormancy allele increased the time to dormancy release during ripening. In combination, these alleles had cumulative effects. Embryo sensitivity to abscisic acid was related to the dormancy phenotype of the whole caryopsis, however, changes in concentrations of abscisic acid and gibberellins in embryo sections and de-embryonated grains during ripening and after-ripening could not be linked to the timing of dormancy release.

Late-maturity α-amylase expression in wheat is influenced by genotype, temperature and stage of grain development.

MAIN CONCLUSION: Late-maturity α-amylase (LMA) expression in wheat grains can be induced by either a cool temperature shock close to physiological maturity or continuous cool maximum temperatures during grain development. Late-maturity α-amylase (LMA) is a genetic trait in wheat (Triticum aestivum L.) involving the production of α-amylase during grain development, which can result in an unacceptably low Falling Number (FN) in mature grain and consequent grain downgrading. Comparison of the FN test, an α-amylase activity assay and a high pI α-amylase-specific ELISA on the same meal samples gave equivalent results; ELISA was used for further experiments because of its isoform specificity. A cool temperature shock during the middle stages of grain development is known to induce LMA and is used for phenotypic screening. It was determined that a cool temperature treatment of seven days was required to reliably induce LMA. Glasshouse studies performed in summer and winter demonstrated that temperature affected the timing of sensitivity to cool-shock by altering the rate and duration of grain development, but that the sensitive grain developmental stage was unchanged at 35-45% moisture content. Wheat varieties with Rht-B1b or Rht-D1b dwarfing genes responded to a cool-shock only from mid grain filling until physiological maturity, whilst genotypes with Rht8c or without a dwarfing gene expressed LMA in response to a cool-shock during a wider developmental range. A continuous cool maximum temperature regimen (23 °C/15 °C day/night) during grain development also resulted in LMA expression and showed a stronger association with field expression than the cool-shock treatment. These results clarify how genotype, temperature and grain developmental stage determine LMA expression, and allow for the improvement of LMA phenotypic screening methods.

A GDSL Esterase/Lipase Catalyzes the Esterification of Lutein in Bread Wheat.

Xanthophylls are a class of carotenoids that are important micronutrients for humans. They are often found esterified with fatty acids in fruits, vegetables, and certain grains, including bread wheat (Triticum aestivum). Esterification promotes the sequestration and accumulation of carotenoids, thereby enhancing stability, particularly in tissues such as in harvested wheat grain. Here, we report on a plant xanthophyll acyltransferase (XAT) that is both necessary and sufficient for xanthophyll esterification in bread wheat grain. XAT contains a canonical Gly-Asp-Ser-Leu (GDSL) motif and is encoded by a member of the GDSL esterase/lipase gene family. Genetic evidence from allelic variants of wheat and transgenic rice (Oryza sativa) calli demonstrated that XAT catalyzes the formation of xanthophyll esters. XAT has broad substrate specificity and can esterify lutein, ß-cryptoxanthin, and zeaxanthin using multiple acyl donors, yet it has a preference for triacylglycerides, indicating that the enzyme acts via transesterification. A conserved amino acid, Ser-37, is required for activity. Despite xanthophylls being synthesized in plastids, XAT accumulated in the apoplast. Based on analysis of substrate preferences and xanthophyll ester formation in vitro and in vivo using xanthophyll-accumulating rice callus, we propose that disintegration of the cellular structure during wheat grain desiccation facilitates access to lutein-promoting transesterification.plantcell;31/12/3092/FX1F1fx1.

Wheat grain preharvest sprouting and late maturity alpha-amylase.

Preharvest sprouting (PHS) and late maturity α-amylase (LMA) are the two major causes of unacceptably high levels of α-amylase in ripe wheat grain. High α-amylase activity in harvested grain results in substantially lower prices for wheat growers and at least in the case of PHS, is associated with adverse effects on the quality of a range of end-products and loss of viability during storage. The high levels of α-amylase are reflected in low falling number, the internationally accepted measure for grain receival and trade. Given the significant losses that can occur, elimination of these defects remains a major focus for wheat breeding programs in many parts of the world. In addition, the genetic, biochemical and molecular mechanisms involved in the control of PHS and LMA as well as the interactions with environmental factors have attracted a sustained research interest. PHS and LMA are independent, genetically controlled traits that are strongly influenced by the environment, where the effects of particular environmental factors vary substantially depending on the stage of grain development and ripening. This review is a summary and an assessment of results of recent research on these important grain quality defects.

Toward an understanding of mechanisms involved in non-polyphenol oxidase (Non-PPO) darkening in yellow alkaline noodles (YAN).

Asian noodles prepared from bread wheat flour darken over time due to a combination of polyphenol oxidase (PPO) activity and non-PPO effects. Although the enzymatic mechanism associated with the PPO reaction is well established, the non-PPO component consists of both physical (e.g., changes in surface properties) and chemical reactions. Variations in pH and solvents were used to gain a quantitative estimate of the contribution of physical and chemical components to non-PPO darkening in yellow alkaline noodles (YAN). In a set of five common high-PPO Australian wheat cultivars it was estimated that on average non-PPO darkening accounted for 69% of total darkening, with approximately two-thirds of this due to physical darkening and one-third had a chemical origin. Data from the chemical portion of non-PPO darkening is consistent with the presence of a PPO-like enzyme that oxidizes tyrosine, has a pH maximum of 8.1, and is inhibited by 50% methanol or ethanol but in the noodle is insensitive to PPO inhibitors such as tropolone. Therefore, with low-PPO and PPO-free wheat varieties becoming available, it may be possible to further reduce darkening in YAN by breeding for wheat varieties with low or zero levels of this PPO-like enzyme.

Analysis of high pI α-Amy-1 gene family members expressed in late maturity α-amylase in wheat (Triticum aestivum L.).

Late maturity α-amylase (LMA) is a genetic defect involving the synthesis of high pI isozymes of α-amylase encoded by α-Amy-1 genes during the later stages of grain development. The aims of this investigation were to determine both the number of expressed α-Amy-1 genes and their relative transcript abundance. Sub-cloning and sequencing of expressed high pI α-amylase genes in developing wheat seeds revealed three insertion/deletion patterns in the 3' untranslated region and numerous single nucleotide polymorphisms at the 3' end of α-Amy-1. The genetic variations defined 36 α-Amy-1 gene sequences that were expressed on the onset of LMA in doubled haploid progenies (SpM25, SpM52 and SpM127) derived from the cross Spica (LMA)/Maringa (non-LMA). Five isoelectric point groups were predicted based on the translated partial coding sequences. The potential application of quantitative real-time RT-PCR in screening wheat genotypes for LMA is discussed.

Genetic, hormonal, and physiological analysis of late maturity α-amylase in wheat.

Late maturity α-amylase (LMA) is a genetic defect that is commonly found in bread wheat (Triticum aestivum) cultivars and can result in commercially unacceptably high levels of α-amylase in harvest-ripe grain in the absence of rain or preharvest sprouting. This defect represents a serious problem for wheat farmers, and apart from the circumstantial evidence that gibberellins are somehow involved in the expression of LMA, the mechanisms or genes underlying LMA are unknown. In this work, we use a doubled haploid population segregating for constitutive LMA to physiologically analyze the appearance of LMA during grain development and to profile the transcriptomic and hormonal changes associated with this phenomenon. Our results show that LMA is a consequence of a very narrow and transitory peak of expression of genes encoding high-isoelectric point α-amylase during grain development and that the LMA phenotype seems to be a partial or incomplete gibberellin response emerging from a strongly altered hormonal environment.

Impact of protein on darkening in yellow alkaline noodles.

Darkening in yellow alkaline noodles (YAN) was examined over a 24 h period in noodles made from 4 wheat varieties, including varieties with different levels of polyphenol oxidase (PPO) activity, selected to cover a range of protein levels. Noodles were made in the presence and absence of the PPO inhibitor, tropolone. The darkening was divided into two time periods: 0-4 h and 4-24 h. The first four hours was described by a composite rate equation, and this period was subdivided into two stages. The rate of darkening in the first stage was independent of both protein concentration and PPO activity. The amount of darkening (c), however, was highly dependent on protein concentration during this stage (-tropolone, r = 0.902; +tropolone, r = 0.905), but independent of PPO activity. The first stage darkening was a zero order reaction where additional protein does not increase the reaction rate, but when the protein supply has been depleted, the reaction stops. The rate of darkening during the first stage (k'(1) = 5.6 +/- 1.0) was similar to the rate of change in the protein structure (k'(1) = 6.5 +/- 1.3) as measured using the amide II band by infrared spectroscopy. This suggested that the first stage of darkening represents changes in light reflectance and absorbance caused by changes in hydrogen bonding rather than changes in covalent bonding. During the second stage of darkening, both the rate (k'(2)) and amount of darkening (DeltaL*(4h-c)) were significantly correlated with protein concentration (-tropolone, r = 0.465; +tropolone, r = 0.813), and in the absence of tropolone the amount of darkening was increased by PPO activity. The amount of darkening (DeltaL*(24h-4h)) during the second time period (4-24 h) (or third stage) was significantly correlated in the presence of tropolone (r = 0.375) and in the absence of tropolone (r = 0.428) with protein concentration. However, compared with earlier stages the response of non-PPO darkening during the third stage to change in protein concentration was smaller. Protein oxidation, or more specifically oxidation of tyrosine groups within the protein, appears to be the main mechanism involved in non-PPO darkening in YAN during the second and third stages with glutenin being the main reactant. Albumin and globulin are important substrates for PPO. No differences in darkening were detected in YAN made from the four varieties in the presence of tropolone; however, differences in YAN darkening were observed for the second and third stages due to site and year variation.

Physical-chemical analysis of non-polyphenol oxidase (non-PPO) darkening in yellow alkaline noodles.

Darkening in yellow alkaline noodles (YAN) was measured over 24 h in a high polyphenol oxidase (PPO) bread wheat ( Triticum aestivum L. cv. Tasman) and a very low PPO durum wheat ( Triticum durum cv. Kamilaroi). Over 24 h non-PPO darkening occurred across a range of pH 3.5-10.5, and in Tasman this was overlaid by darkening from PPO activity. The rate of darkening in YAN was separated into two main time periods, 0-4 and 4-24 h. The first 4 h of darkening was further divided into two stages using a composite first-order rate equation. Several specific inhibitors that partially inhibited non-PPO darkening were identified. These inhibitors, as well as the PPO inhibitors SHAM and tropolone, were used to analyze YAN darkening. The rate of the early stage of darkening was not altered by any inhibitors used; however, the magnitude of darkening was reduced by inhibitors specific for non-PPO darkening. Both the rate and extent of non-PPO darkening of the second stage of darkening were decreased in Tasman and Kamilaroi by inhibitors specific for non-PPO darkening, whereas both PPO inhibitors only decreased darkening in Tasman. The second and third stages of darkening showed similar characteristics. The third stage of darkening was examined in YAN made from Kamilaroi over a temperature range from -4 to 65 degrees C. It followed an Arrhenius relationship indicating non-PPO darkening during this stage was nonenzymatic. The inhibitor data suggested that the reactive component(s) was/were present in a reasonably high concentration(s) and that the soluble protein fraction was involved in the non-PPO darkening process.

Water movement into dormant and non-dormant wheat (Triticum aestivum L.) grains.

The movement of water into harvest-ripe grains of dormant and non-dormant genotypes of wheat (Triticum aestivum L.) was investigated using Magnetic Resonance Micro-Imaging (MRMI). Images of virtual sections, both longitudinal and transverse, throughout the grain were collected at intervals after the start of imbibition and used to reconstruct a picture of water location within the different grain tissues and changes over time. The observations were supplemented by the weighing measurements of water content and imbibition of grains in water containing I(2)/KI which stains starch and lipid, thereby acting as a marker for water. In closely related genotypes, with either a dormant or a non-dormant phenotype, neither the rate of increase in water content nor the pattern of water distribution within the grain was significantly different until 18 h, when germination became apparent in the non-dormant genotype. Water entered the embryo and scutellum during the very early stages of imbibition through the micropyle and by 2 h water was clearly evident in the micropyle channel. After 12 h of imbibition, embryo structures such as the coleoptile and radicle were clearly distinguished. Although water accumulated between the inner (seed coat) and outer (pericarp) layers of the coat surrounding the grain, there was no evidence for movement of water directly across the coat and into the underlying starchy endosperm.

alpha-Amylase and programmed cell death in aleurone of ripening wheat grains.

Late maturity alpha-amylase (LMA) in wheat is a genetic defect that may result in the accumulation of unacceptable levels of high pI alpha-amylase in grain in the absence of germination or weather damage. During germination, gibberellin produced in the embryo triggers expression of alpha-Amy genes, the synthesis of alpha-amylase and, subsequently, cell death in the aleurone. LMA also involves the aleurone and whilst LMA appears to be independent of the embryo there is nevertheless some evidence that gibberellin is involved. The aim of this investigation was to determine whether the increase in alpha-amylase activity in LMA-prone genotypes, like alpha-amylase synthesis by aleurone cells in germinating or GA-challenged grains, is followed by aleurone cell death. Programmed cell death was seen in aleurone layers from developing, ripe and germinated grains using confocal microscopy and fluorescent probes specific for dead or living cells. Small pockets of dying cells were observed distributed at random throughout the aleurone of ripening LMA-affected grains and by harvest-ripeness these cells were clearly dead. The first appearance of dying cells, 35 d post-anthesis, coincided with the later part of the 'window of sensitivity' in grain development in LMA-prone wheat cultivars. No dead or dying cells were present in ripening or fully ripe grains of control cultivars. In germinating grains, dying cells were observed in the aleurone adjacent to the scutellum and, as germination progressed, the number of dead cells increased and the affected area extended further towards the distal end of the grain. Aside from the obvious differences in spatial distribution, dying cells in 20-24 h germinated grains were similar to dying cells in developing LMA-affected grains, consistent with previous measurements of alpha-amylase activity. The increase in high pI alpha-amylase activity in developing grains of LMA-prone cultivars, like alpha-amylase synthesis in germinating grains, is associated with cell death, providing further evidence for the involvement of gibberellin in the LMA response.

Effect of grain colour gene (R) on grain dormancy and sensitivity of the embryo to abscisic acid (ABA) in wheat.

The level of grain dormancy and sensitivity to ABA of the embryo, a key factor in grain dormancy, were examined in developing grains of a white-grained wheat line, Novosibirskaya 67 (NS-67), and its red-grained near-isogenic lines (ANK-1A to -1D); a red-grained line, AUS 1490, and its white-grained mutant line (EMS-AUS). ANK lines showed higher levels of grain dormancy than NS-67 at harvest maturity. AUS 1490 grain also showed higher dormancy than EMS-AUS grain. These results suggest that the R gene for grain colour can enhance grain dormancy. However, the dormancy effect conferred by the R gene was not large, suggesting that it plays a minor role in the development of grain dormancy. Water extracts of AUS 1490 and EMS-AUS bran contained germination inhibitors equivalent to 1-10 microM ABA, although there was no difference in the amount of inhibitors between AUS 1490 and EMS-AUS. Thus, the grain colour gene of AUS 1490 did not appear to enhance the level of grain dormancy by accumulating germination inhibitors in its bran. Sensitivity to ABA of embryos was higher in grains collected around harvest-maturity for ANK lines and AUS 1490, compared with NS-67 and EMS-AUS. The R gene might enhance grain dormancy by increasing the sensitivity of embryos to ABA.