Proteolytic and Structural Changes in Rye and Triticale Roots under Aluminum Stress.

Proteolysis and structural adjustments are significant for defense against heavy metals. The purpose of this study was to evaluate whether the Al3+ stress alters protease activity and the anatomy of cereale roots. Azocaseinolytic and gelatinolytic measurements, transcript-level analysis of phytocystatins, and observations under microscopes were performed on the roots of Al3+-tolerant rye and tolerant and sensitive triticales exposed to Al3+. In rye and triticales, the azocaseinolytic activity was higher in treated roots. The gelatinolytic activity in the roots of rye was enhanced between 12 and 24 h in treated roots, and decreased at 48 h. The gelatinolytic activity in treated roots of tolerant triticale was the highest at 24 h and the lowest at 12 h, whereas in treated roots of sensitive triticale it was lowest at 12 h but was enhanced at 24 and 48 h. These changes were accompanied by increased transcript levels of phytocystatins in rye and triticale-treated roots. Light microscope analysis of rye roots revealed disintegration of rhizodermis in treated roots at 48 h and indicated the involvement of root border cells in rye defense against Al3+. The ultrastructural analysis showed vacuoles containing electron-dense precipitates. We postulate that proteolytic-antiproteolytic balance and structural acclimation reinforce the fine-tuning to Al3+.

Triticale Green Plant Regeneration Is Due to DNA Methylation and Sequence Changes Affecting Distinct Sequence Contexts in the Presence of Copper Ions in Induction Medium.

Metal ions in the induction medium are essential ingredients allowing green plant regeneration. For instance, Cu(II) and Ag(I) ions may affect the mitochondrial electron transport chain, influencing the Yang cycle and synthesis of S-adenosyl-L-methionine, the prominent donor of the methylation group for all cellular compounds, including cytosines. If the ion concentrations are not balanced, they can interfere with the proper flow of electrons in the respiratory chain and ATP production. Under oxidative stress, methylated cytosines might be subjected to mutations impacting green plant regeneration efficiency. Varying Cu(II) and Ag(I) concentrations in the induction medium and time of anther culture, nine trials of anther culture-derived regenerants of triticale were derived. The methylation-sensitive AFLP approach quantitative characteristics of tissue culture-induced variation, including sequence variation, DNA demethylation, and DNA de novo methylation for all symmetric-CG, CHG, and asymmetric-CHH sequence contexts, were evaluated for all trials. In addition, the implementation of mediation analysis allowed evaluating relationships between factors influencing green plant regeneration efficiency. It was demonstrated that Cu(II) ions mediated relationships between: (1) de novo methylation in the CHH context and sequence variation in the CHH, (2) sequence variation in CHH and green plant regeneration efficiency, (3) de novo methylation in CHH sequences and green plant regeneration, (4) between sequence variation in the CHG context, and green plant regeneration efficiency. Cu(II) ions were not a mediator between de novo methylation in the CG context and green plant regeneration. The latter relationship was mediated by sequence variation in the CG context. On the other hand, we failed to identify any mediating action of Ag(I) ions or the moderating role of time. Furthermore, demethylation in any sequence context seems not to participate in any relationships leading to green plant regeneration, sequence variation, and the involvement of Cu(II) or Ag(I) as mediators.

In vitro effects of CaO nanoparticles on Triticale callus exposed to short and long-term salt stress.

KEY MESSAGE: Ca2+ NPs enhanced tolerance of Triticale callus under salt stress by improving biochemical activity and confocal laser scanning analysis, conferring salt tolerance on callus cells. CaO NPs (Ca2+) are significant components that act as transducers in many adaptive and developmental processes in plants. In this study, effect of Ca2+ NPs on the response and regulation of the protective system in Triticale callus under short and long-salt treatments was investigated. The activation of Ca2+ NPs was induced by salt stress in callus of Triticale cultivars. MDA, H2O2, POD, and protein activities were determined in callus tissues. Concerning MDA, H2O2, protein activities, it was found that the Ca2+ NPs treatment was significant, and it demonstrated a high correlation with the tolerance levels of cultivars. Tatlicak cultivar was detected for better MDA activities in the short time with 1.5 ppm Ca2+ NPs concentration of 50 g and 100 g NaCl. Similarly, the same cultivar responded with better H2O2 activity at 1.5 ppm Ca2+ NPs 100 g NaCl in the short time. POD activities exhibited a decreasing trend in response to the increasing concentrations of Ca2+ NPs. The best result was observed at 1.5 ppm Ca2+ NPs 100 g NaCl in the short term. Based on the protein content, treatment of short-term cultured callus cells with 1.5 ppm Ca2+ NPs inhibited stress response and it significantly promoted Ca2+ NPs signals as compared to control callus. Confocal laser scanning analysis proved that the application of Ca2+ NPs could alleviate the adverse effects of salt stress by the inhibition of stress severity in callus cells. This study demonstrated, under in vitro conditions, that the application of Ca2+ NPs can significantly suppress the adverse effects of salt stress on Triticale callus; it was also verified that the concentration of Ca2+ NPs could be important parameter to be considered in adjusting the micronutrient content in the media for this plant.

Water stress-induced flag leaf senescence may be accelerated by rehydration.

The aim of the study was to determine molecular, biochemical and physiological responses of non-fully recovered DH line of triticale exposed to water stress during generative stage. The study involved two DH lines of winter triticale that produce different number of shoots with ears during rehydration. We analyzed the content of proteins associated with the photosynthetic apparatus and plant senescence. We also determined the content of hydrogen peroxide and assimilation pigments and assessed stomatal conductance and activity of the photosynthetic apparatus. Water stress-initiated senescence did not slow down during rehydration in the not fully recovered DH line. This line showed an increase in pheophorbide a oxygenase (PaO), a protein associated with chlorophyll degradation, and a decrease in the proteins related to its synthesis (chlorophyll synthase - ChS, protochlorophilide oxidoreductase - POR). Pheophorbide a oxygenase is a marker of accelerated cell death as it catalyzes opening of the porphyrin ring in the chlorophyll degradation pathway. The level of hydrogen peroxide remained high during rehydration with the photosynthetic apparatus being one of its sources. Lower content of Rieske protein reduced the quantum yield of electron transport (ϕRo) from the primary acceptors QA/QB to the final acceptors in PSI. Intensification of metabolic processes during rehydration resulted in overloading the electron transport chain in PSII and transfer of electrons from the primary acceptors to oxygen molecule. Overproduction of hydrogen peroxide accelerated senescence during rehydration and significantly reduced plant yield.

Biomass recalcitrance in barley, wheat and triticale straw: Correlation of biomass quality with classic agronomical traits.

The global production of cereal straw as an agricultural by-product presents a significant source of biomass, which could be used as feedstock for the production of second generation biofuels by fermentation. The production of sugars for fermentation is an important measure of straw quality and in its suitability for biofuel production. In this paper, we present a characterization of straw digestibility from a wide range of cereal. Our main objective is to evaluate the variability of fermentable sugars released from different species including wheat (Triticum durum L., Triticum aestivum L.), barley (Hordeum vulgare L.) and triticale (X Triticosecale Wittmack). To this end, we adapted a saccharification method (IAS Method) capable of detecting significant differences of released sugars between cultivars and species, while using separately another method that would serve as a control and with which we could contrast our results (CNAP method). ANOVA analyses revealed that barley has a higher saccharification potential than wheat and triticale and shows more variation between genotypes. Thus, populations derived from crosses among them such as Steptoe × Morex and OWB Dominant × OWB Recessive hold potential for the identification of genetic basis for saccharification-related traits. The correlation of glucose released between the two methods was moderate (R2 = 0.57). An evaluation of the inter- and intra- specific correlation between a number of chemical and agronomical parameters and saccharification suggests that the cell wall thickness and lignin content in straw could be used in breeding programs for the improvement of the saccharification potential. Finally, the lack of correlation between grain yield and saccharification suggests that it would be possible to make a selection of genotypes for dual purpose, low recalcitrance and grain yield.

Comparison on Photosynthesis and Antioxidant Defense Systems in Wheat with Different Ploidy Levels and Octoploid Triticale.

To investigate the evolutionary differences of wheat with different ploidy levels and octoploid Triticale, photosynthetic capacity, and antioxidant defenses system were compared within and between diploid, tetraploid and hexaploid wheat, and octoploid Triticale seedlings. The results showed that seed germination rate, chlorophyll content, and photochemical activity of photosystems, and the activities of antioxidative enzymes in hexaploid wheat and octoploid Triticale were significantly higher than in diploid and tetraploid wheat. Compared to other two wheat species and octoploid Triticale, hexaploid wheat presented lower levels of reactive oxygen species (ROS). Furthermore, we found that the levels of photosystem II reaction center protein D1, light-harvesting complex II b4 (CP29), and D subunit of photosystem I (PsaD) in diploid wheat were significantly lower compared with hexaploid wheat and octoploid Triticale. Taken together, we concluded that hexaploid wheat and octoploid Triticale have higher photosynthetic capacities and better antioxidant systems. These findings indicate that different ploidy levels of chromosome probably play an important regulatory role in photosystems and antioxidative systems of plants.

The varied ability of grains to synthesize and catabolize ABA is one of the factors affecting dormancy and its release by after-ripening in imbibed triticale grains of cultivars with different pre-harvest sprouting susceptibilities.

Abscisic acid (ABA) is a phytohormone involved in the acquisition of primary dormancy during seeds maturation as well as dormancy maintenance in imbibed seeds. After imbibition, the ABA content decreased to a much lower level in embryos of freshly harvested triticale grains of the Leontino cultivar, which is more susceptible to pre-harvest sprouting (PHS) than embryos of the Fredro cultivar. Lower ABA content in the Leontino cultivar resulted from increased expression of TsABA8'OH1 and TsABA8'OH2, which encode ABA 8'-hydroxylase and are involved in ABA catabolism. Higher ABA content and maintenance of dormancy in Fredro grains were correlated with intensified ABA biosynthesis, which resulted from higher expression of TsNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase. These results suggest that grains of triticale cultivars with different resistance to PHS vary in their ability to metabolize ABA after imbibition. After-ripening did not affect the ABA content in embryos of dry grains of either triticale cultivar. However, after-ripening caused dormancy release in Fredro grains and significantly affected the ABA content and the rate of its metabolism after imbibition. A more rapid decline in ABA content in imbibed Fredro grains was accompanied by decreased transcript levels of TsNCED1 as well as increased expression of TsABA8'OH1 and TsABA8'OH2. Thus, after-ripening may affect dormancy of grains through reduction of the ABA biosynthesis rate and intensified ABA catabolism. Overexpression of TsNCED1 in tobacco increases ABA content and delays germination, while overexpression of TsABA8'OH2 decreases ABA content, accelerates germination, and reduces the sensitivity to ABA of transgenic seeds compared to seeds of wild-type plants. Therefore, these genes might play an important role in the regulation of triticale grain dormancy, thus affecting susceptibility to PHS.

Chromosomes 1BS and 1RS for control of male fertility in wheats and triticales with cytoplasms of Aegilops kotschyi, Ae. mutica and Ae. uniaristata.

KEY MESSAGE: Engineered chromosomes 1BS and 1RS offer a new alternative in the development of hybrid systems in bread wheat and triticale. In the cytoplasmic male sterility system for hybrid wheat based on the cytoplasm of Triticum timopheevi fertility restoration is difficult, with few good restorer genes available. In the system based on the cytoplasms of Aegilops kotschyi, Ae. uniaristata and Ae. mutica, essentially all chromosomes 1B carry locus Rf multi that restores male fertility; male sterility manifests itself in wheats with the 1RS.1BL translocation where 1BS chromosome arm is missing. To generate male sterile wheats without the 1RS.1BL translocation, the 1BS arm was cytogenetically engineered to replace the segment with Rf multi with two short inserts of rye chromatin. Conversely, to enhance fertility restoration by doubling the number of restorers present for eventual use in wheat and triticale, a region of 1BS with Rf multi was inserted into 1RS. Alloplasmic wheats with Rf multi removed were completely male sterile; alloplasmic wheats with engineered 1RS carrying Rf multi and without normal 1B were male fertile. An exception to the ubiquitous presence of Rf multi is T. spelta var. duhamelianum; four accessions tested in this study gave inconsistent results but some did not restore male fertility. Engineered chromosomes 1BS and 1RS and chromosomes 1B of T. spelta offer a new alternative for practical application of a cytoplasmic male sterility system in the development of hybrid wheat and hexaploid triticale.

Deacclimation may be crucial for winter survival of cereals under warming climate.

Climate warming can change the winter weather patterns. Warmer temperatures during winter result in a lower risk of extreme freezing events. On the other hand the predicted warm gaps during winter will decrease their freezing tolerance. Both contradict effects will affect winter survival but their resultant effect is unclear. In this paper, we demonstrate that climate warming may result in a decrease in winter survival of plants. A field study of winterhardiness of common wheat and triticale was established at 11 locations and repeated during three subsequent winters. The freezing tolerance of the plants was studied after controlled cold acclimation and de-acclimation using both plant survival analysis and chlorophyll fluorescence measurements. Cold deacclimation resistance was shown to be independent from cold acclimation ability. Further, cold deacclimation resistance appeared to be crucial for overwintering when deacclimation conditions occurred in the field. The shortening of uninterrupted cold acclimation may increase cold deacclimation efficiency, which could threaten plant survival during warmer winters. Measurements of chlorophyll fluorescence transient showed some differences triggered by freezing before and after deacclimation. We conclude that cold deacclimation resistance should be considered in the breeding of winter cereals and in future models of winter damage risk.

Is the OJIP Test a Reliable Indicator of Winter Hardiness and Freezing Tolerance of Common Wheat and Triticale under Variable Winter Environments?

OJIP analysis, which explores changes in photosystem II (PSII) photochemical performance, has been used as a measure of plant susceptibility to stress. However, in the case of freezing tolerance and winter hardiness, which are highly environmentally variable, the use of this method can give ambiguous results depending on the species as well as the sampling year and time. To clarify this issue, we performed chlorophyll fluorescence measurements over three subsequent winters (2010/11, 2011/12 and 2012/13) on 220 accessions of common winter wheat and 139 accessions of winter triticale. After freezing, leaves were collected from cold-acclimated plants in the laboratory and field-grown plants. Observations of field survival in seven locations across Poland and measurements of freezing tolerance of the studied plants were also recorded. Our results confirm that the OJIP test is a reliable indicator of winter hardiness and freezing tolerance of common wheat and triticale under unstable winter environments. Regardless of species, the testing conditions giving the most reliable results were identical, and the reliability of the test could be easily checked by analysis of some relationships between OJIP-test parameters. We also found that triticale is more winter hardy and freezing tolerant than wheat. In addition, the two species were characterized by different patterns of photosynthetic apparatus acclimation to cold.

Photosynthesis-dependent physiological and genetic crosstalk between cold acclimation and cold-induced resistance to fungal pathogens in triticale (Triticosecale Wittm.).

The breeding for resistance against fungal pathogens in winter triticale (Triticosecale Wittm.) continues to be hindered by a complexity of the resistance mechanisms, strong interaction with environmental conditions, and dependence on the plant genotype. We showed, that temperature below 4 °C induced the plant genotype-dependent resistance against the fungal pathogen Microdochium nivale. The mechanism involved, at least, the adjustment of the reactions in the PSII proximity and photoprotection, followed by an improvement of the growth and development. The genotypes capable to develop the cold-induced resistance, showed a higher maximum quantum yield of PSII and a more efficient integration of the primary photochemistry of light reactions with the dark reactions. Moreover, induction of the photoprotective mechanism, involving at least the peroxidases scavenging hydrogen peroxide, was observed for such genotypes. Adjustment of the photosynthesis and stress acclimation has enabled fast plant growth and avoidance of the developmental stages sensitive to fungal infection. The same mechanisms allowed the quick regrow of plants during the post-disease period. In contrast, genotypes that were unable to develop resistance despite cold hardening had less flexible balancing of the photoprotection and photoinhibition processes. Traits related to: photosynthesis-dependent cold-acclimation and cold-induced resistance; biomass accumulation and growth; as well as protection system involving peroxidases; were integrated also at a genetic level. Analysing 95 lines of the mapping population SaKa3006×Modus we determined region on chromosomes 5B and 7R shared within all tested traits. Moreover, similar expression pattern of a set of the genes related to PSII was determined with the metaanalysis of the multiple microarray experiments. Comparable results for peroxidases, involving APXs and GPXs and followed by PRXs, indicated a similar function during cold acclimation and defense responses. These data provide a new insight into the cross talk between cold acclimation and cold-induced resistance in triticale, indicating a key role of photosynthesis-related processes.