Effect of secondary plant substances on winter wheat resistance to grain aphid

The association between the concentrations of hydroxamic acids, total phenols, and indole alkoloids in winter wheat cultivars with their antibiotic resistance to the grain aphid Sitobion avenae (F.) (Homoptera: Aphididae) was studied. The antibiosis was measured under field conditions using the intrinsic rate of natural increase (r_m). The content of the studied allelochemicals was estimated in flag leaf at the anthesis growth stage. Highly significant negative correlations were found between the r_m values and the concentrations of hydroxamic acids (r=–0.905) and total phenols (r=–0.946), but not with indole alkaloids (r=–0.380). The contribution of these allelochemicals to the resistance of winter wheat to S. avenae is discussed.

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Résumé Les effets combinés de la concentration en substances allélochimiques des feuilles flétries et de la résistance antibiotique aux pucerons des grains ont été étudiés sur 4 cultivars de blé d'hiver, dont 2 modérément résistants: Saga et Grana et 2 sensibles: Emika et Liwilla. L'antibiose a été évaluée dans les conditions de la nature, aux stades d'épiaison et d'anthèse, en utilisant le taux intrinsèque de croissance r_m. La quantification des substances allélochimiques a été effectuée par colorimétrie.Des corrélations négatives hautement significatives ont été trouvées entre les valeurs de R_m et la concentration en acides hydroxamiques (r=–0,905 pour 10 dl, P<0,01) et la teneur totale en phénols (r=–0,946, pour 10 dl, P<0,01). La teneur en alcaloïdes indols était elle aussi corrélée négativement, mais pas significativement avec r_m (r=–0,380, pour 10 dl, P>0,1). La discussion porte sur la contribution de ces substances allélochimiques à la résistance du blé d'hiver à Sitobion avenae.

     

QTL mapping and comparative genome analysis of agronomic traits including grain yield in winter rye

Key message

Genetic diversity in elite rye germplasm as well as F _2:3 testcross design enables fast QTL mapping to approach genes controlling grain yield, grain weight, tiller number and heading date in rye hybrids.

Abstract

Winter rye (Secale cereale L.) is a multipurpose cereal crop closely related to wheat, which offers the opportunity for a sustainable production of food and feed and which continues to emerge as a renewable energy source for the production of bioethanol and biomethane. Rye contributes to increase agricultural crop species diversity particularly in Central and Eastern Europe. In contrast to other small grain cereals, knowledge on the genetic architecture of complex inherited, agronomic important traits is yet limited for the outbreeding rye. We have performed a QTL analysis based on a F_2:3 design and testcross performance of 258 experimental hybrids in multi-environmental field trials. A genetic linkage map covering 964.9 cM based on SSR, conserved-orthologous set (COS), and mixed-phase dominant DArT markers allowed to describe 22 QTL with significant effects for grain yield, heading date, tiller number, and thousand grain weight across seven environments. Using rye COS markers, orthologous segments for these traits have been identified in the rice genome, which carry cloned and functionally characterized rice genes. The initial genome scan described here together with the existing knowledge on candidate genes provides the basis for subsequent analyses of the genetic and molecular mechanisms underlying agronomic important traits in rye.

     

Antifreeze protein produced endogenously in winter rye leaves

After cold acclimation, winter rye (Secale cereale L.) is able to withstand the formation of extracellular ice at freezing temperatures. We now show, for the first time, that cold-acclimated winter rye plants contain endogenously produced antifreeze protein. The protein was extracted from the apoplast of winter rye leaves, where ice forms during freezing. After partial purification, the protein was identified as antifreeze protein because it modified the normal growth pattern of ice crystals and depressed the freezing temperature of water noncolligatively.     

Coleoptile removal-induced ethylene production in winter rye seedlings

Coleoptile removal-induced ethylene production was investigated in light-grown winter rye seedlings. Removal of the coleoptile induced 1-aminocyclopropane-l-carboxylic acid (ACC) synthesis and ethylene production by primary leaves and caused an inhibition of elongation growth of the leaves. The activity of ethylene-forming enzyme (EFE) was associated with the increase in ethylene evolution. Both rise in ethylene and ACC production, as well as EFE activity were inhibited by cycloheximide. Wounding the tissue 40 min after the initial treatment resulted in the second increase in ethylene evolution. Derooting of the seedlings without coleoptile removal did not induce ethylene production. It is suggested that the coleoptile represents a barrier for wound-induced ethylene production from actively growing leaf tissue.     

Cold-active winter rye glucanases with ice-binding capacity

Extracellular pathogenesis-related proteins, including glucanases, are expressed at cold temperatures in winter rye (Secale cereale) and display antifreeze activity. We have characterized recombinant cold-induced glucanases from winter rye to further examine their roles and contributions to cold tolerance. Both basic beta-1,3-glucanases and an acidic beta-1,3;1,4-glucanase were expressed in Escherichia coli, purified, and assayed for their hydrolytic and antifreeze activities in vitro. All were found to be cold active and to retain partial hydrolytic activity at subzero temperatures (e.g. 14%-35% at -4 degrees C). The two types of glucanases had antifreeze activity as measured by their ability to modify the growth of ice crystals. Structural models for the winter rye beta-1,3-glucanases were developed on which putative ice-binding surfaces (IBSs) were identified. Residues on the putative IBSs were charge conserved for each of the expressed glucanases, with the exception of one beta-1,3-glucanase recovered from nonacclimated winter rye in which a charged amino acid was present on the putative IBS. This protein also had a reduced antifreeze activity relative to the other expressed glucanases. These results support the hypothesis that winter rye glucanases have evolved to inhibit the formation of large, potentially fatal ice crystals, in addition to having enzymatic activity with a potential role in resisting infection by psychrophilic pathogens. Glucanases of winter rye provide an interesting example of protein evolution and adaptation aimed to combat cold and freezing conditions.     

Mapping QTLs for alpha-amylase activity in rye grain

Genetic control of alpha-amylase activity in rye grain was investigated by QTL mapping based on DS2 x RXL10 intercross consisting of 99 F5-6 families propagated at one location during four vegetation seasons. A wide range of variation in alpha-amylase activity and transgression effects were found among families and parental lines. This variation was shown to be determined in 40.1% by 7 significant (LOD score not less than 2.5) and 2 putative QTLs (2 < LOD < 2.5) distributed on all rye chromosomes except 4R. Two significant QTLs located on 3RL and 5RL chromosome arms were expressed each year. The third significant QTL was detected in three years (1RL). The other four significant QTLs (2RL, 5RS, 6RL, 7RL) were found in one year of study. The number and composition of QTLs were specific for a given year varying from three to six. QTLs were not correlated with isoenzyme polymorphisms at the structural alpha-Amy1 loci. A QTL associated with a region containing the alpha-Amy3 locus was detected on chromosome 5RL. Both high- and low-activity QTL alleles were found in each parental line, which explains the appearance of transgressive recombinants in the segregating population.     

Permeability of the suberized mestome sheath in winter rye

Mestome sheath cells of winter rye (Secale cereale L. cv Puma) deposit suberized lamellae in their secondary cell walls. Histochemical tests including acid digestion and staining with Sudan IV and Chelidonium majus root extract were used to detect the presence of suberin in the primary cell wall. There was no evidence of a Casparian band between adjacent mestome sheath cells. Fluorescent dye techniques were used to trace solute movement through the rye leaf apoplast. Calcofluor white M2R, a fluorescent dye which binds to cell walls as it moves apoplastically, proved to be too limited in its mobility in leaves to test mestome sheath permeability. Trisodium 3-hydroxy-5,8,10 pyrene trisulfonate, a fluorescent dye which is mobile in the apoplast, moved easily up the vascular bundles in the transpiration stream, and diffused outward from the veins to the epidermal cell walls within minutes of reaching a particular level in the leaf. We conclude that the suberized mestome sheath of rye leaves is freely permeable to solutes moving apoplastically through radial primary cell walls.     

Exploring new alleles for frost tolerance in winter rye

Key message

Rye genetic resources provide a valuable source of new alleles for the improvement of frost tolerance in rye breeding programs.

Abstract

Frost tolerance is a must-have trait for winter cereal production in northern and continental cropping areas. Genetic resources should harbor promising alleles for the improvement of frost tolerance of winter rye elite lines. For frost tolerance breeding, the identification of quantitative trait loci (QTL) and the choice of optimum genome-based selection methods are essential. We identified genomic regions involved in frost tolerance of winter rye by QTL mapping in a biparental population derived from a highly frost tolerant selection from the Canadian cultivar Puma and the European elite line Lo157. Lines per se and their testcrosses were phenotyped in a controlled freeze test and in multi-location field trials in Russia and Canada. Three QTL on chromosomes 4R, 5R, and 7R were consistently detected across environments. The QTL on 5R is congruent with the genomic region harboring the Frost resistance locus 2 (Fr–2) in Triticeae. The Puma allele at the Fr–R2 locus was found to significantly increase frost tolerance. A comparison of predictive ability obtained from the QTL-based model with different whole-genome prediction models revealed that besides a few large, also small QTL effects contribute to the genomic variance of frost tolerance in rye. Genomic prediction models assigning a high weight to the Fr–R2 locus allow increasing the selection intensity for frost tolerance by genome-based pre-selection of promising candidates.

     

Proteomic analysis of developing rye grain with contrasting resistance to preharvest sprouting

Significant differences in the two-dimensional electrophoresis patterns of proteins from developing rye grain were found to be associated with resistance and susceptibility to preharvest sprouting (PHS). Mass spectrometry of individual spots showing different abundance in PHS-resistant and PHS-susceptible lines identified proteins involved in: reaction to biotic and abiotic stresses, including oxidative stress, energy metabolism and regulation of gene expression. Highly differentiated abundance of proteins found in developing grain suggest that the diversification of processes leading to developing PHS resistance or PHS susceptibility starts from an early stage of grain development. A part of the identified proteins in rye grain were also reported to be associated with PHS in wheat and rice, which suggests that some mechanisms affecting precocious germination might be common for different cereal species.     

Identification of rye chromosomes in triticales by giemsa staining in relation to grain shrivelling

In hexaploid triticale, cultivar UPT 72142 having highly shrivelled grains, there were only five pairs of rye chromosomes, whereas cultivars UPT 78268 and UPT 79245 having medium grains had six and five pairs, respectively. Plump grained cultivars UPT 79347 and UPT 79339 had 2 pairs of rye chromosomes. A decrease in the number of rye chromosomes in triticales is con-elated with grain improvement.     

The interrelationship of growth and frost tolerance in winter rye

The reduction in growth of winter cereals that occurs in the fall is thought to be required for the development of frost resistance. In the present study, the interrelationship of freezing tolerance and growth was examined by raising winter rye ( Secale cereale cv. Puma) plants at 20/16°C (day/night) and at 5/3°C under 8-, 16- and 24-h daylengths to vary growth rates and frost tolerance. Temperature and irradiance were quantified as thermal time, photothermal time and photosynthetic photon flux and examined by multiple linear regression in order to determine their effects on growth and frost tolerance of rye shoots. At low temperature, both growth and frost tolerance were markedly influenced by daylength and irradiance. Plants grown at 5/3°C with a short daylength accumulated shoot dry weight and increased frost tolerance at a greater rate per unit photothermal time or photon flux than plants grown at longer daylengths. Moreover, 5/3°C plants grown with a 16-h day grew more slowly and were less frost tolerant than plants grown with a 24-h day. We conclude that the interrelationship between growth and frost tolerance is a quantitative one. Frost tolerance is induced only by low temperature, but the development of forst tolerance is dependent upon both irradiance, which affects the amount of photoassimilate available, and daylength, which may affect the partitioning of photoassimilates between growth and frost tolerance.     

The effect of gametocides on microsporogenesis of winter rye

The effect of gametocides FW-450, maleic hydrazide and Dalapone on the course of microsporogenesis, gametogenesis and starch synthesis in pollen grains of winter rye ev. ‘Esto’ was investigated. Solutions containing various concentrations of tested substances were applied on leaves in amount of 2.5 ml and 5.0 ml per plant at the beginning, at the full and at the end of shooting. The effect of gametocides was detectable already in the phase of microspore formation when a decrease of anther weight, pollen grain size and starch content in grains and an increase of the number of microspores with nonfinished development were observed. The manner and the extent of injuries were dependent on the applied substance, its concentration and on period of its application.     

A single grain screening technique for breeding alkylresorcinol-poor rye

Summary A quick and simple semi-quantitative method of selecting single seeds of rye for their content of 5-alkyl-resorcinols is presented. The resorcinols are extracted from whole, intact caryopses with acetone. Under the conditions described the germination rate is hardly decreased. The single grain method offers especially striking advantages when selffertile inbred lines are used.     

Immunogold localization of glucanase-like antifreeze protein in cold acclimated winter rye

Summary Apoplastic antifreeze proteins (AFPs) accumulate in winter rye (Secale cereale L. cv. Musketeer) leaves during cold acclimation. Two of the rye AFPs with molecular masses of 32 and 35 kDa are similar in their amino acid sequences and epitopes to -1, 3-endoglucanase. Localization of these AFPs, which we refer to as glucanase-like proteins (GLPs), was carried out with antiserum raised against the 32 kDa AFP. Specimens from leaves and roots of non-acclimated (NA) plants and cold acclimated (CA) plants were prepared by freeze-substitution for high resolution immunoelectron microscopy. In CA leaves, high levels of GLPs were observed in cell walls of mesophyll cells adjacent to intercellular spaces and in secondary thickenings of xylem vessels. Taken together with the absence of GLPs in vacuoles, these results confirm the apoplastic accumulation of AFPs in CA winter rye. Within the cells of CA leaves, GLPs were localized in cisternae of the rough endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which indicates that GLPs are secreted via an exocytic bulk-flow pathway. The occurrence of high levels of GLPs in CA leaves, their low presence in NA leaves and the lack of GLPs in roots all suggest that there is a correlation between increased accumulation of GLPs and increased freezing tolerance of these plant materials. Furthermore, the localization of GLPs in the immediate vicinity of pathways for free water within the tissues supports the view that these proteins have an important role in the crystallization and/or recrystallization of water when the leaves of CA winter rye are exposed to freezing temperatures.Abbreviations AFP antifreeze protein - BSA bovine serum albumin - CA cold acclimated - GAR goat antirabbit antiserum conjugated with colloidal gold - GLP glucanase-like protein - NA non-acclimated - PBS phosphate buffered saline - PR pathogenesis related     

Leucine transport in cells isolated from cold-hardened and nonhardened winter rye

The properties of the leucine transport systems of cells isolated from dark-grown cold-hardened and nonhardened winter rye (Secale cereale L. cv. Puma) epicotyls were remarkably similar. After 1 hour of incubation, leucine was accumulated in the cells 80- to 100-fold above that of the external medium, but the transported leucine was not metabolized. Approximately one-third of the accumulated leucine was present in the vacuole after 40 minutes of incubation. At 25°C, efflux of leucine from the vacuole was 6 to 10 times slower than it was from the cytoplasm, while at 5°C efflux from the cells was inhibited.     

Reduced sensitivity to photoinhibition following frost-hardening of winter rye is due to increased phosphate availability

The possibility of a role for phosphate metabolism in the photosynthetic regulation that occurs during frost hardening was investigated in winter rye (Secale cereale L. cv. Musketeer). Leaves of frost-hardened and non-hardened winter rye were studied during photosynthetic induction, and at steady state after being allowed to take up 20 mM orthophosphate through the transpiration stream for 3 h. At the growth irradiance (350 mol·m^-2·s^-1) frost-hardening increased the stationary rate of CO₂-dependent O₂ evolution by 57% and 25% when measured at 5 and 20° C, respectively. Frosthardening also reduced the lag phase to stationary photosynthesis by 40% at 5° C and decreased the susceptibility of leaves to oscillations during induction and after interruption of the actinic beam during steady-state photosynthesis. These responses are all indicative of increased phosphate availability in frost-hardened leaves. As reported previously by Öquist and Huner (1993, Planta 189, 150–156), frost-hardening also decreased the reduction state of Q_A, the primary, stable quinone acceptor of PSII, and decreased the sensitivity of winter rye to photoinhibition of photosynthesis. Non-hardened rye leaves fed orthophosphate also showed an increased photosynthetic capacity (25% at 20° C and light saturation), lower reduction state of Q_A, a reduced sensitivity to photoinhibition and lower susceptibility to oscillations resulting from a brief interruption of the actinic light. Thus, the data indicate that phosphate metabolism plays a key role in photosynthetic acclimation of winter rye to low temperatures.Abbreviations F_o and F_o minimal fluorescence when all PSII reaction centres are open in dark-and light-acclimated leaves, respectively - F_m and F_m maximal fluorescence when all PSII reaction centres are closed in dark-and light-acclimated leaves, respectively - F_v variable fluoresence (F_m -F_o) in dark-acclimated leaves - F_v variable fluorescence (F_m-F_o) in light-acclimated leaves - PCR photosynthetic carbon reduction - PPFD photosynthetic photon flux density - Q_A the primary, stable quinone acceptor of PSII - q_P photochemical quenching of fluorescence - q_N non-photochemical quenching of fluorescence This work was supported by the Swedish Natural Sciences Research Council. The authors are indebted to Dr. N. Huner, Department of Plant Sciences, UWO, London, Canada, for helpful discussions during the initiation of this work and for the gift of rye seeds.     

Characteristics of resistance to the grain aphid Sitobion avenue in winter wheat

Five stocks of winter wheat were resistant to S. avenae in glasshouse screening tests, where resistance was more evident in older than younger plants but different types of resistance were not separated. Antibiosis was measured from the growth and reproduction of caged individual aphids and antixenosis from the settling of adult aphids on detached leaf portions. Cvs Bounty, Rapier and Virtue were resistant due to antibiosis, whilst cv. Kador and line A4501-4E showed antixenosis. These and other differences indicated that these five wheat stocks probably include at least four distinct sources of resistance to S. avenae. The existence of different types of resistance could account for variability in the results of screening tests.     

Genomic architecture of alpha-amylase activity in mature rye grain relative to that of preharvest sprouting

Bi-directional selective genotyping (BSG) carried out on two opposite groups of F₉(541 × Ot1-3) recombinant inbred lines (RILs) with extremely low and extremely high alpha-amylase activities in mature (dry) grain of rye, followed by molecular mapping, revealed a complex system of selection-responsive loci. Three classes of loci controlling alpha-amylase activity were discerned, including four major AAD loci on chromosomes 3R (three loci) and 6RL (one locus) responding to both directions of the disruptive selection, 20 AAR loci on chromosomes 2RL (three loci), 3R (three loci), 4RS (two loci), 5RL (three loci), 6R (two loci) and 7R (seven loci) responding to selection for low alpha-amylase activity and 17 AAE loci on chromosomes 1RL (seven loci), 2RS (two loci), 3R (two loci), 5R (two loci) and 6RL (four loci) affected by selection for high alpha-amylase activity. The majority of the discerned AA loci also showed responsiveness to selection for preharvest sprouting (PHS). Two AAD loci on chromosome arm 3RL coincided with PHSD loci. The AAD locus on chromosome arm 3RS was independent from PHS, whereas that on chromosome 6RL belonged to the PHSR class. AAR-PHSR loci were found on chromosomes 4RS (one locus) and 5R (two loci) and AAE-PHSE loci were identified on chromosomes 1RL (one locus) and 5RL (one locus). Some PHSD loci represented the AAE (chromosomes 1RL, 3RS and 3RL) or AAR classes (chromosome 5RL). AAR and AAE loci not related to PHS were found on chromosomes 1RL, 2R, 3RS, 4R, 6RL and 7RL. On the other hand, several PHS loci (1RL, 3RS, 5RL, 6RS and 7RS) had no effect on alpha-amylase activity. Allele originating from the parental line 541 mapped in six AA loci on chromosomes 2R (two loci), 5R (three loci) and 7R (one locus) exerted opposite effects on PHS and alpha-amylase activity. Differences between the AA and PHS systems of loci may explain the weak correlation between these two traits observed among recombinant inbred lines. Strategies for the breeding of sprouting-resistant varieties with low alpha-amylase and high PHS resistance are discussed.     

Structural studies on water-soluble arabinoxylans in rye grain using enzymatic hydrolysis

The major water-soluble arabinoxylan fraction from rye grain, containing 4-linked β- -xylopyranosyl residues of which about 43% were substituted solely at O-3 and 7% at both O-2 and O-3 with terminal - -arabinofuranosyl units, was hydrolysed to different extents using semi-purified xylanase from Trichoderma reesei. Products were fractionated on Biogel P-2 and structurally elucidated by sugar, methylation and high-field ¹H-NMR analysis. Moderate hydrolysis released arabinose, xylose, xylobiose, xylotriose and xylotetraose together with xylo-oligosaccharides (DP ≥ 4) in which one or more of the residues were substituted at O-3 with a terminal arabinose unit. The xylose residues substituted with arabinose units at both O-2 and O-3 became enriched in the remaining polymeric fraction. Extensive hydrolysis with the enzyme released arabinose, xylose and xylobiose as major products together with small amounts of two oligosaccharides and a polymeric fraction. One of the oligosaccharides was identified as xylotriose in which the non-reducing end was substituted at O-2 and O-3 with terminal arabinose units and the other as xylotetraose in which one of the interjacent residues was substituted with arabinose units in the same way. The polymeric fraction contained a main chain of 4-linked xylose residues in which 60–70% of the residues were substituted at both O-2 and O-3 with arabinose units.

The semi-purified enzyme contained xylanase and arabinosidase activities which rapidly degraded un- and mono-substituted xylose residues while the degradation of double-substituted xylose residues was much slower. The results show that the mono- and double-substituted xylose residues were present in different polymers or different regions of the same polymer.