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The recent COVID-19 pandemic has highlighted the value of technologies that allow a fast setup and production of biopharmaceuticals in emergency situations. The plant factory system can provide a fast response to epidemics/pandemics. Thanks to their scalability and genome plasticity, plants represent advantageous platforms to produce vaccines. Plant systems imply less complicated production processes and quality controls with respect to mammalian and bacterial cells. The expression of vaccines in plants is based on transient or stable transformation systems and the recent progresses in genome editing techniques, based on the CRISPR/Cas method, allow the manipulation of DNA in an efficient, fast, and easy way by introducing specific modifications in specific sites of a genome. Nonetheless, CRISPR/Cas is far away from being fully exploited for vaccine expression in plants. In this review, an overview of the potential conjugation of the renewed vaccine technologies (i.e., virus-like particles—VLPs, and industrialization of the production process) with genome editing to produce vaccines in plants is reported, illustrating the potential advantages in the standardization of the plant platforms, with the overtaking of constancy of large-scale production challenges, facilitating regulatory requirements and expediting the release and commercialization of the vaccine products of genome edited plants.

Breeding for abiotic stress tolerance means employing genetics to increase the capacity of a crop to withstand physical stress factors without penalizing yield. Over 50 years of studies on barley stress tolerance have contributed to the description of the genetic diversity and of the molecular components underlying the stress response as well as to the identification of the main genes and loci involved in stress resistance. This knowledge has been translated into new varieties with improved stress tolerance, although the new challenges imposed by climate change still require extensive work to select barley adapted to the new climatic scenarios and new breeding strategy is needed. This chapter reviews the fundamental knowledge acquired on barley tolerance and adaptation to abiotic stresses and describes how the new tools available for breeding, namely Genomics Selection and Genome Editing, can be applied to increase the rate of genetic progress in breeding for stress-prone environments and to expand genetic diversity introgressing new resistant traits in barley cultivars.

Cereal species can be damaged by frost either during winter or at flowering stage. Frost tolerance per se is only a part of the mechanisms that allow plants to survive during winter, while winter-hardiness also considers other biotic or physical stresses that challenge the plants during the winter season, limiting their survival rate. While frost tolerance can also be tested in controlled environments, winter-hardiness can only be determined with field evaluations. Post-heading frost damage occurs from radiation frost events in spring during the reproductive stages. A reliable evaluation of winter-hardiness or of post heading frost damage should be carried out with field trials replicated across years and locations to overcome the irregular occurrence of natural conditions which satisfactorily differentiate genotypes. The evaluation of post-heading frost damage requires a specific attention to plant phenology. The extent of frost damage is traditionally determined with a visual score at the end of the winter, although, recently an image-based phenotyping coupled with unmanned aerial vehicles (UAVs) has been proposed.

Background In viticulture, rootstock genotype plays a critical role to improve scion physiology, berry quality and to adapt grapevine (Vitis vinifera L.) to different environmental conditions. This study aimed at investigating the effect of two different rootstocks (1103 Paulsen - P - and Mgt 101–14 - M) in comparison with not grafted plants - NGC - on transcriptome (RNA-seq and small RNA-seq) and chemical composition of berry skin in Pinot noir, and exploring the influence of rootstock-scion interaction on grape quality. Berry samples, collected at veraison and maturity, were investigated at transcriptional and biochemical levels to depict the impact of rootstock on berry maturation. Results RNA- and miRNA-seq analyses highlighted that, at veraison, the transcriptomes of the berry skin are extremely similar, while variations associated with the different rootstocks become evident at maturity, suggesting a greater diversification at transcriptional level towards the end of the ripening process. In the experimental design, resembling standard agronomic growth conditions, the vines grafted on the two different rootstocks do not show a high degree of diversity. In general, the few genes differentially expressed at veraison were linked to photosynthesis, putatively because of a ripening delay in not grafted vines, while at maturity the differentially expressed genes were mainly involved in the synthesis and transport of phenylpropanoids (e.g. flavonoids), cell wall loosening, and stress response. These results were supported by some differences in berry phenolic composition detected between grafted and not grafted plants, in particular in resveratrol derivatives accumulation. Conclusions Transcriptomic and biochemical data demonstrate a stronger impact of 1103 Paulsen rootstock than Mgt 101–14 or not grafted plants on ripening processes related to the secondary metabolite accumulations in berry skin tissue. Interestingly, the MYB14 gene, involved in the feedback regulation of resveratrol biosynthesis was up-regulated in 1103 Paulsen thus supporting a putative greater accumulation of stilbenes in mature berries.

PubMedID: 30962619 The domestication of wild emmer wheat led to the selection of modern durum wheat, grown mainly for pasta production. We describe the 10.45 gigabase (Gb) assembly of the genome of durum wheat cultivar Svevo. The assembly enabled genome-wide genetic diversity analyses revealing the changes imposed by thousands of years of empirical selection and breeding. Regions exhibiting strong signatures of genetic divergence associated with domestication and breeding were widespread in the genome with several major diversity losses in the pericentromeric regions. A locus on chromosome 5B carries a gene encoding a metal transporter (TdHMA3-B1) with a non-functional variant causing high accumulation of cadmium in grain. The high-cadmium allele, widespread among durum cultivars but undetected in wild emmer accessions, increased in frequency from domesticated emmer to modern durum wheat. The rapid cloning of TdHMA3-B1 rescues a wild beneficial allele and demonstrates the practical use of the Svevo genome for wheat improvement. © 2019, The Author(s), under exclusive licence to Springer Nature America, Inc. Western Grains Research Foundation Ministry of Agriculture - Saskatchewan Israel Science Foundation: 1137/17 031A536 FP7 Food, Agriculture and Fisheries, Biotechnology: DROPS ID244347 Natural Sciences and Engineering Research Council of Canada InterOmics PB05 Genome Prairie United States-Israel Binational Science Foundation: 2015409 Genome Canada Saskatchewan Canola Development Commission 2819103915 PIR01_00017 3060-21000-038-00-D We acknowledge the funding support of: the Italian Ministry of Education and Research with projects CNR Flagship InterOmics PB05 (L.M., A.C., G.S.), PON ELIXIR CNR-BiOmics PIR01_00017 (L.M., M.G., M.Mo.) and PON ISCOCEM (P.D.); CREA project Interomics (L.C.); Fondazione in rete per la ricerca agroalimentare AGER project From Seed to Pasta (R.T.); FP7-KBBE Project DROPS ID244347 (R.T.); Genome Canada (A.G.S., C.P.); the Western Grain Research Foundation (A.G.S., C.P.); the Manitoba Wheat and Barley Commission (A.G.S., C.P.); the Saskatchewan Wheat Development Commission (A.G.S., C.P.); the Alberta Wheat Development Commission (A.G.S., C.P.); the Saskatchewan Ministry of Agriculture (A.G.S., C.P.); the administrative support of Genome Prairie (A.G.S., C.P.); Canadian Triticum Applied Genomics -CTAG2-(A.G.S., C.P.); Binational Science Foundation grant no. 2015409 (I.H., A.D.); Israel Science Foundation grant no. 1137/17 (A.D.); USDA-Agricultural Research Service Current Research Information System project 3060-21000-038-00-D (J.D.F., S.S.X.); German Federal Ministry of Food and Agriculture grant no. 2819103915 (N.S., K.F.X.M.); German Ministry of Education and Research grant no. 031A536 (K.F.X.M.); and Natural Sciences and Engineering Council of Canada grant nos. SPG 336119-06 and RGPIN 92787 (G.J.T., C.P.). The authors are grateful to E. Elias (North Dakota State University) for providing nine DW cultivars, included in the Global Tetraploid Wheat Collection and E. Scarpella (University of Alberta, Edmonton, Canada) for assistance with confocal microscopy.

Plant stress response is a complex molecular process based on transcriptional and posttranscriptional regulation of many stress-related genes. microRNAs are the best-studied class of small RNAs known to play key regulatory roles in plant response to stress, besides being involved in plant development and organogenesis. We analyzed the leaf miRNAome of two durum wheat cultivars (Cappelli and Ofanto) characterized by a contrasting water use efficiency, exposed to heat stress, and mild and severe drought stress. On the whole, we identified 98 miRNA highly similar to previously known miRNAs and grouped in 47 MIR families, as well as 85 novel candidate miRNA, putatively wheat specific. A total of 80 known and novel miRNA precursors were found differentially expressed between the two cultivars or modulated by stress and many of them showed a cultivar-specific expression profile. Interestingly, most in silico predicted targets of the miRNAs coming from the differentially expressed precursors have been experimentally linked in other species to mechanisms controlling stomatal movement, a finding in agreement with previous results showing that Cappelli has a lower stomatal conductance than Ofanto. Selected miRNAs were validated through a standardized and reliable stem-loop qRT-PCR procedure.

In cereals, C-repeat binding factor genes have been defined as key components of the light quality-dependent regulation of frost tolerance by integrating phytochrome-mediated light and temperature signals. This study elucidates the differences in the lipid composition of barley leaves illuminated with white light or white light supplemented with far-red light at 5 or 15 &deg, C. According to LC-MS analysis, far-red light supplementation increased the amount of monogalactosyldiacylglycerol species 36:6, 36:5, and 36:4 after 1 day at 5 &deg, C, and 10 days at 15 &deg, C resulted in a perturbed content of 38:6 species. Changes were observed in the levels of phosphatidylethanolamine, and phosphatidylserine under white light supplemented with far-red light illumination at 15 &deg, C, whereas robust changes were observed in the amount of several phosphatidylserine species at 5 &deg, C. At 15 &deg, C, the amount of some phosphatidylglycerol species increased as a result of white light supplemented with far-red light illumination after 1 day. The ceramide (42:2)-3 content increased regardless of the temperature. The double-bond index of phosphatidylglycerol, phosphatidylserine, phosphatidylcholine ceramide together with total double-bond index changed when the plant was grown at 15 &deg, C as a function of white light supplemented with far-red light. white light supplemented with far-red light increased the monogalactosyldiacylglycerol/diacylglycerol ratio as well. The gene expression changes are well correlated with the alterations in the lipidome.

NLR (NOD-like receptor) genes belong to one of the largest gene families in plants. Their role in plants’ resistance to pathogens has been clearly described for many members of this gene family, and dysregulation or overexpression of some of these genes has been shown to induce an autoimmunity state that strongly affects plant growth and yield. For this reason, these genes have to be tightly regulated in their expression and activity, and several regulatory mechanisms are described here that tune their gene expression and protein levels. This gene family is subjected to rapid evolution, and to maintain diversity at NLRs, a plethora of genetic mechanisms have been identified as sources of variation. Interestingly, regulation of gene expression and evolution of this gene family are two strictly interconnected aspects. Indeed, some examples have been reported in which mechanisms of gene expression regulation have roles in promotion of the evolution of this gene family. Moreover, co-evolution of the NLR gene family and other gene families devoted to their control has been recently demonstrated, as in the case of miRNAs.

The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like durum wheat requires the identification and optimization of factors affecting T-DNA delivery and plant regeneration. The purpose of this research was to compare the behavior of diverse durum wheat genotypes during in vitro culture and Agrobacterium tumefaciens-mediated transformation, using immature embryos as explants. Apart from plant genotype, two of the main influencing factors for a successful genetic transformation have been examined here, i.e., auxin source (Dicamba and 2,4-D) and duration of the pre-culture period (one, seven and 21 days). The addition of Dicamba to the media in combination with seven days pre-cultivation resulted in a general enhancement of T-DNA delivery for most of the analyzed cultivars, as revealed by β-glucuronidase (GUS) histochemical assay. Although all genotypes were able to produce calli, significant differences were detected in regeneration and transformation efficiencies, since only two (Karalis and Neolatino) out of 14 cultivars produced fertile transgenic plants. The estimated transformation efficiencies were 6.25% and 1.66% for Karalis and Neolatino, respectively, and χ2 analysis revealed the stable integration and segregation of the gus transgene in T1 and T2 progenies. This research has demonstrated that, among the influencing factors, genotype and auxin type play the most important role in the success of durum wheat transformation.

MicroRNAs are a class of post-transcriptional regulators of plant developmental and physiological processes and responses to environmental stresses. Here, we present the study regarding the annotation and characterization of MIR genes conducted in durum wheat. We characterized the miRNAome of leaf and root tissues at tillering stage under two environmental conditions: irrigated with 100% (control) and 55% of evapotranspiration (early water stress). In total, 90 microRNAs were identified, of which 32 were classified as putative novel and species-specific miRNAs. In addition, seven microRNA homeologous groups were identified in each of the two genomes of the tetraploid durum wheat. Differential expression analysis highlighted a total of 45 microRNAs significantly differentially regulated in the pairwise comparisons leaf versus root. The miRNA families, miR530, miR395, miR393, miR5168, miR396 and miR166, miR171, miR319, and miR167, were the most expressed in leaves in comparison to roots. Putative microRNA targets were predicted for both five and three prime sequences derived from the stem-loop of the MIR gene. Gene ontology analysis showed significant overrepresented gene categories in microRNA targets belonging to transcription factors, phenylpropanoids, oxydases, and lipid binding-protein. This work represents one of the first genome wide characterization of MIR genes in durum wheat, identifying leaf and root tissue-specific microRNAs. This genomic identification of microRNAs together with the analysis of their expression profiles is a well-accepted starting point leading to a better comprehension of the role of MIR genes in the genus Triticum.

The Osmyb4 rice gene, coding for a transcription factor, proved to be efficient against different abiotic stresses as a trans(cis)gene in several plant species, although the effectiveness was dependent on the host genomic background. Eight barley transgenic lines carrying the rice Osmyb4 gene under the control of the Arabidopsis cold inducible promoter cor15a were produced to test the efficiency of this gene in barley. After a preliminary test, the best performing lines were subjected to freezing at -11°C and -12°C. Frost tolerance was assessed measured the F(v)/F(m) parameter widely used to indicate the maximum quantum yield of photosystem II photochemistry in the dark adapted state. Three transgenic lines showed significantly increased tolerance. These selected lines were further studied under a complex stress applying cold and hypoxia at germinating stage. In these conditions the three selected transgenic lines outperformed the wild type barley in terms of germination vigour. The transgenic plants also showed a significant modification of their metabolism under cold/hypoxia conditions as demonstrated through the assessment of the activity of key enzymes involved in anoxic stress response. None of the transgenic lines showed dwarfism, just a slight retarded growth. These results provide evidence that the cold dependent expression of Osmyb4 can efficiently improved frost tolerance and germination vigour at low temperature without deleterious effect on plant growth.

The molecular response of plants to abiotic stresses has been often considered as a complex process mainly based on the modulation of transcriptional activity of stress-related genes. Nevertheless, recent findings have suggested new layers of regulation and complexity. Upstream molecular mechanisms are involved in the plant response to abiotic stress, above all in the regulation of timings and amount of specific stress responses. Post-transcriptional mechanisms based on alternative splicing and RNA processing, as well as RNA silencing define the actual transcriptome supporting the stress response. Beyond protein phosphorylation, other post-translational modifications like ubiquitination and sumoylation regulate the activation of pre-existing molecules to ensure a prompt response to stress. In addition, cross-connections exist among these mechanisms, clearly demonstrating further and superimposed complexity levels in the response to environmental changes. Even if not widely identified, the targets of these mechanisms characterised so far are mainly regulatory elements of the stress response pathways. The network of post-transcriptional and post-translational modifications ensures temporally and spatially appropriate patterns of downstream stress-related gene expression. Future attempts of plant engineering could exploit insights from a deeper comprehension of these emerging sites of regulation of stress responses to develop stress resistant plants.

Sowing date, being determinant for growth stage, may play a decisive role in optimising freezing resistance of winter annual plants. In cereal species, in spite of the abundant literature analysing the factors responsible for the acquisition of frost resistance through the cold hardening process, the involvement of the growth stage per se, has been seldom considered, especially at the earlier vegetative phases. In this work the contribution of growth stage in determining resistance to freezing temperature has been analysed in field and growth chamber experiments using winter and spring barley cultivars exposed to different hardening conditions. Field damage was assessed twice during winter on plants sown at three different dates. In the growth chamber experiments several acclimation treatments at 11/7 and/or 3/1 °C (day/night) were simulated. In both field and laboratory experiments the development of cold acclimation was monitored by means of a COR14b specific antibody, since in previous studies the expression of COR14b was found genetically linked to frost resistance. The lowest resistance, found in the youngest plants and in spring cultivars, however, was not always associated with the lowest level of COR14b accumulation. COR14b accumulation correlated with frost resistance at the earlier field sampling date and in plants grown at 11/7 °C. In a following phase of the hardening process (second sampling in field and 4 weeks at 3/1 °C in growth chamber) the accumulation of COR14b was independent of plant stage and genotype, showing no association with freezing resistance. Results suggest that growth stage is crucial for the achievement of maximal resistance in barley, but not for COR14b expression.

Drought and heat tolerance are complex quantitative traits. Moreover, the adaptive significance of some stress-related traits is more related to plant survival than to agronomic performance. A web of regulatory mechanisms fine-tunes the expression of stress-related traits and integrates both environmental and developmental signals. Both post-transcriptional and post-translational modifications contribute substantially to this network with a pivotal regulatory function of the transcriptional changes related to cellular and plant stress response. Alternative splicing and RNA-mediated silencing control the amount of specific transcripts, while ubiquitin and SUMO modify activity, sub-cellular localization and half-life of proteins. Interactions across these modification mechanisms ensure temporally and spatially appropriate patterns of downstream-gene expression. For key molecular components of these regulatory mechanisms, natural genetic diversity exists among genotypes with different behavior in terms of stress tolerance, with effects upon the expression of adaptive morphological and/or physiological target traits.

Polyamine oxidases (PAOs, EC 1.5.3.11) are key enzymes responsible for the terminal catabolism of polyamines in plants, bacteria and protozoa. In barley, two PAO isoforms (HvPAO1 and HvPAO2) have been previously analyzed as regards their tissue expression and subcellular localization. Only the major isoform HvPAO2 has been biochemically characterized up to now. In order to study the ear-specific expression of the HvPAO1 isoform in detail, RT-PCR analysis was performed in barley on the whole ear and on various ear tissues. Moreover, HvPAO1promoter::GUS transient expression was examined in barley developing caryopses at 30-day postfertilization. Results from these analyses have demonstrated that the HvPAO1 gene is specifically expressed in all the ear organs analyzed (i.e. basal lemma, rachis, awn, embryo-deprived caryopsis, embryo and sterile spikelets), at variance with the HvPAO2 gene, which is expressed at high levels in sterile spikelets and at very low levels in embryos. We purified HvPAO1 from barley immature caryopses and characterized its catalytic properties. Furthermore, we carried out in vitro synthesis of HvPAO1 protein in a cell-free translation system. The HvPAO1 enzymes purified from immature caryopses and in vitro synthesized showed the same catalytic properties, in particular, an optimum at pH 7.0 for Spd and Spm oxidation and comparable Km values for both substrates, i.e. 0.89 × 10−5 m and 0.5 × 10−5 m for Spd and Spm, respectively. It has been found that HvPAO1 enzyme activity significantly differs in substrate specificity and pH optimum when compared with the major isoform HvPAO2. As a whole, these data strongly suggest that, in barley, the two PAO genes evolved separately, after a duplication event, to code for two distinct tissue-specific enzymes, and they are likely to play different physiological roles.

Previously, we have shown that barley (Hordeum vulgare) plants carrying a mutation preventing chloroplast development are completely frost susceptible as well as impaired in the expression of several cold-regulated genes. Here we investigated the transcriptome of barley albina and xantha mutants and the corresponding wild type to assess the effect of the chloroplast on expression of cold-regulated genes. First, by comparing control wild type against cold-hardened wild-type plants 2,735 probe sets with statistically significant changes (P = 0.05; ≥2-fold change) were identified. Expression of these wild-type cold-regulated genes was then analyzed in control and cold-hardened mutants. Only about 11% of the genes cold regulated in wild type were regulated to a similar extent in all genotypes (chloroplast-independent cold-regulated genes); this class includes many genes known to be under C-repeat binding factor control. C-repeat binding factor genes were also equally induced in mutants and wild-type plants. About 67% of wild-type cold-regulated genes were not regulated by cold in any mutant (chloroplast-dependent cold-regulated genes). We found that the lack of cold regulation in the mutants is due to the presence of signaling pathway(s) normally cold activated in wild type but constitutively active in the mutants, as well as to the disruption of low-temperature signaling pathway(s) due to the absence of active chloroplasts. We also found that photooxidative stress signaling pathway is constitutively active in the mutants. These results demonstrate the major role of the chloroplast in the control of the molecular adaptation to cold.

WRKY proteins constitute a large family of plant specific transcription factors implicated in many different processes. Here we describe Hv-WRKY38, a barley gene coding for a WRKY protein, whose expression is involved in cold and drought stress response. Hv-WRKY38 was early and transiently expressed during exposure to low non-freezing temperature, in ABA-independent manner. Furthermore, it showed a continuous induction during dehydration and freezing treatments. A WRKY38:YFP fusion protein was found to localise into the nucleus upon introduction into epidermal onion cells. Bacterially expressed Hv-WRKY38 was able to bind in vitro to the W-box element (T)TGAC(C/T) also recognisable by other WRKY proteins. Hv-WRKY38 genomic DNA was sequenced and mapped onto the centromeric region of the barley chromosome 6H. Arabidopsis and rice sequences homologous to Hv-WRKY38 were also identified. Our results indicate that Hv-WRKY38 transcription factor may play a regulatory role in abiotic stress response.

Extensive molecular biological studies have led to the cloning of many cold-regulated genes (cor) whose expression is up-regulated in winter cereals during exposure to cold. Among them is cor14b, a nuclear gene coding for a chloroplast-localized protein whose expression level has been associated with frost resistance in Triticeae. Although frost tolerance is recognised as a complex quantitative character, members of homologous group 5 in Triticeae are known to carry major loci determining frost tolerance. In this work we summarize a number of recent experiments where the cold-regulated gene cor14b was used as a tool (i) to understand the genetic relationship between the expression of cor genes and the frost resistance loci and (ii) to identify barley mutants revealing a chloroplast role in the signal transduction pathway leading to the molecular cold response.Key words: Triticeae, cold hardening, frost resistance, cor genes.

The expression of several barley (Hordeum vulgare) cold-regulated (cor) genes during cold acclimation was blocked in the albino mutanta n, implying a chloroplast control on mRNAs accumulation. By using albino and xanthamutants ordered according to the step in chloroplast biogenesis affected, we show that the cold-dependent accumulation ofcor14b, tmc-ap3, andblt14 mRNAs depends on plastid developmental stage. Plants acquire the ability to fully express cor genes only after the development of primary thylakoid membranes in their chloroplasts. To investigate the chloroplast-dependent mechanism involved in cor gene expression, the activity of a 643-bp cor14b promoter fragment was assayed in wild-type and albino mutant a n leaf explants using transient β-glucuronidase reporter expression assay. Deletion analysis identified a 27-bp region between nucleotides −274 and −247 with respect to the transcription start point, encompassing a boundary of some element that contributes to the cold-induced expression of cor14b. However, cor14bpromoter was equally active in green and in albinoa n leaves, suggesting that chloroplast controls cor14b expression by posttranscriptional mechanisms. Barley mutants lacking either photosystem I or II reaction center complexes were then used to evaluate the effects of redox state of electron transport chain components on COR14b accumulation. In the mutants analyzed, the amount of COR14b protein, but not the steady-state level of the corresponding mRNA, was dependent on the redox state of the electron transport chain. Treatments of thevir-zb63 mutant with electron transport chain inhibitors showed that oxidized plastoquinone promotes COR14b accumulation, thus suggesting a molecular relationship between plastoquinone/plastoquinol pool and COR14b.

A barley (Hordeum vulgare L.) full-length clone coding for long chain acyl-CoA oxidase (ACX), key enzyme of β-oxidation, was isolated by cDNA library screening and 5′-rapid amplification of cDNA ends. The cDNA encodes for a polypeptide of 667 amino acids, with a molecular mass of 74.5 kDa. The amino acid sequence, beside an extensive similarity with other plant and mammalian ACXs, showed a PTS1 peroxisomal targeting signal at the C terminus and a conserved FAD-binding domain. The gene was over-expressed in E. coli and the fusion protein was shown to possess long chain acyl-CoA oxidase activity. Polyclonal antibodies were raised against a large fragment of the protein encoded by the barley putative ACX gene. Northern and Western analysis demonstrated that a basal level of long chain ACX is always present along the barley life cycle, while a higher level of expression is typical of actively growing tissues such as germinating embryos, ovary before anthesis, developing embryos, shoots and roots apexes. In vitro germination experiments with glucose and glucose analogues provided evidence about the involvement of a glucose-deriving signal in the positive modulation of ACX expression. This result highlights the role of ACX, not only during oil reserve mobilization, but also in plant growth and metabolism.

Triticum durum Desf. cv. Cappelli and xHaynaldoticum sardoum Meletti et Onnis, a recently formed spontaneous hybrid derived from Cappelli, were studied in order to test their response to hardening. xH. sardoum segregates into two natural lines: Culmo Vuoto (hollow stem-CV), a typical winter wheat, and Culmo Pieno (solid stem-CP), a typical spring wheat. T. durum cv. Cappelli is a typical spring wheat. Freezing test and measurements of electrolytical conductivity have shown that CV, the only winter wheat, is able to acclimate more quickly and to a larger extent than spring wheats. SDS-PAGE with activity staining specific for RNases highlighted an increase in WLB (neutral salt-stimulated RNases) with a higher mobility in the leaves of seedlings which have been hardened (2 °C) for 2, 7 and 14 days, but not in leaves of ABA treated seedlings. This increase is more precocious in hardened CV, and the enzyme is more active in acclimated leaves than in unhardened ones. Unlike RNases, COR14 is equally present in winter and spring acclimated plants.

Drought, low temperature and salinity are the most important abiotic stress factors limiting crop productivity. A genomic map of major loci and QTLs affecting stress tolerance in Triticeae identified the crucial role of the group 5 chromosomes, where the highest concentration of QTLs and major loci controlling plant's adaptation to the environment (heading date, frost and salt tolerance) has been found. In addition, a conserved region with a major role in drought tolerance has been localized to the group 7 chromosomes. Extensive molecular biological studies have led to the cloning of many stress-related genes and responsive elements. The expression of some stress-related genes was shown to be linked to stress-tolerant QTLs, suggesting that these genes may represent the molecular basis of stress tolerance. The development of suitable genetic tools will allow the role of stress-related sequences and their relationship with stress-tolerant loci to be established in the near future.

Winterhardiness is a basic trait for successful winter survival barley (Hordeum vulgare L.) crop. Freezing tolerance, a fundamental component of winterhardiness, is based on an inducible process known as hardening or cold acclimation that occurs when plants are exposed to low non-freezing temperatures. In the recent years, many temperature-dependent genes specifically expressed during hardening have been isolated. Current data on relationship between gene expression and cold tolerance are mostly based on plants grown and hardened under environmentally controlled conditions and, usually, over a short period of time. In order to verify whether variations in the molecular response to cold are likely to be of significant adaptive value under natural environments, we have followed the accumulation of several COR genes ( pt59, pao86 and paf93) and proteins (COR14a and COR14b) during the 1996/97 and 97/98 winter seasons in barley cultivars with contrasting winterhardiness capacity grown under field conditions. In the 1996/97 experiment, a winter cultivar Onice and a spring cultivar Gitane were tested for the accumulation of the cold-regulated genes and proteins. The ability of the plants to promote a strong molecular response to cold was found to be associated with the winterhardiness capacity of the two cultivars. This result was further tested in the winter season 1997/98 using 10 barley varieties. All winter cultivars showed high accumulation of the cold-regulated proteins COR14a and COR14b, while some variations for this character were detected in the spring cultivars suggesting that the selection for winter survival has been effective to fix the high COR14 accumulation capacity. We conclude that a high level of COR14 may be a component the winter survival capacity of barley.

MicroRNAs (miRNAs) are a class of endogenous noncoding small RNAs acting on gene regulation at posttranscriptional level, a phenomenon known in plants as posttranscriptional gene silencing. miRNAs are known to play key regulatory roles in plant response to stress, besides being involved in development and morphogenesis. To extend and to update information on miRNAs and their targets in barley and to identify candidate polymorphisms at miRNA target sites, the features of previously known plant miRNAs have been used to systematically search for barley miRNA homologues and targets in the publicly available EST database. Matching sequences have then been related to UniGene clusters on which most of this study was based. One hundred fifty-six microRNA mature sequences belonging to 50 miRNA families have been found to significantly match at least one EST sequence in barley. The predicted miRNA targets were ascribed to different pathways, among others the response to abiotic stress. To verify experimentally the barley miRNAs and their involvement in stress response, the barley miRNome of plants exposed to low temperatures has been characterized by a deep sequencing approach on Illumina GAIIx. Many of the known miRNAs have been found as different isomeric variants, so-called isomiRs, which might increase the target repertoire of the miRNA gene they derive from.

MicroRNAs (miRNAs) are short non-coding RNA molecules produced from hairpin structures and involved in gene expression regulation with major roles in plant development and stress response. Although each annotated miRNA in miRBase (www.mirbase.org) is a single defined sequence with no further details on possible variable sequence length, isomiRs - namely the population of variants of miRNAs coming from the same precursors - have been identified in several species and could represent a way of broadening the regulatory network of the cell. Next-gen-based sequencing makes it possible to comprehensively and accurately assess the entire miRNA repertoire including isomiRs. The aim of this work was to survey the complexity of the peach miRNome by carrying out Illumina high-throughput sequencing of miRNAs in three replicates of five biological samples arising from a set of different peach organs and/or phenological stages. Three hundred-ninety-two isomiRs (miRNA and miRNA*-related) corresponding to 26 putative miRNA coding loci, have been highlighted by mirDeep-P and analyzed. The presence of the same isomiRs in different biological replicates of a sample and in different tissues demonstrates that the generation of most of the detected isomiRs is not random. The degree of mature sequence heterogeneity is very different for each individual locus. Results obtained in the present work can thus contribute to a deeper view of the miRNome complexity and to better explore the mechanism of action of these tiny regulators.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20–24 nt, known to play key roles in post-transcriptional gene regulation, that can be coded either by intergenic or intragenic loci. Intragenic (exonic and intronic) miRNAs can exert a role in the transcriptional regulation and RNA processing of their host gene. Moreover, the possibility that the biogenesis of exonic miRNAs could destabilize the corresponding protein-coding transcript and reduce protein synthesis makes their characterization very intriguing and suggests a possible novel mechanism of post-transcriptional regulation of gene expression. This work was designed to carry out the computational identification of putative exonic miRNAs in 30 plant species and the analysis of possible mechanisms involved in their regulation. The results obtained represent a useful starting point for future studies on the complex networks involved in microRNA-mediated gene regulation in plants.

Frost tolerance of 30 barley (Hordeum vulgare L.) cultivars have been field evaluated in North Italy during the 1990/1991 winter season that was characterized by exceptionally low temperatures without snow cover. The results showed a significant correlation between cold injury and grain yield loss (r=0.61**). Five cultivars chosen for their varying degree of frost tolerance were further evaluated using laboratory tests. Measurements of survival rate and membrane damage were used to assess the influence of hardening on frost resistance. The reliability of the tests is shown by the high correlation to the field data. For both the laboratory temperature regimes and field conditions, the tested cultivars showed the same order of classification. The effect of a rise in temperature at the end of the hardening treatment on frost tolerance is also reported. The laboratory tests here proposed can be integrated in a breeding programme for improving frost tolerance in barley.

The hardening-triggered accumulation of a barley cold-regulated protein of 75kDa (COR 75) has been investigated in several genotypes featuring contrasting frost tolerance capacity. COR 75 is accumulated at low temperatures, suggesting its involvement in the development of frost resistance. COR 75 is accumulated faster during the first days at low temperature in frost resistant varieties when compared with extremely sensitive varieties; nevertheless after 7 days of hardening, COR 75 is expressed at the same level in all genotypes tested despite their differences in frost tolerance. Besides low temperature, COR 75 can also be induced by ABA or drought stress indicating that this protein may be a general response to variation in tissue moisture status. Using a polyclonal antibody raised against COR 75, it has been demonstrated that this protein is immunologically related to two wheat cold-regulated proteins of 160 and 200 kDa. The role of COR 75 in the development of hardening is discussed.

Barley (Hordeum vulgare L.) production in many regions is constrained by low temperatures. Numerous screening methods have been proposed for studies of freezing tolerance (FT) and winter hardiness (WH). Here, we compared the response to low temperature of 54 barley genotypes released in Europe. Major components of WH were analyzed in fi eld and growth chamber experiments under different hardening and freezing conditions. Phenotype screening of freezing injury (lethal temperature for 50% of the plants [LT 50 ], plant survival, and chlorophyll fl uorescence) and indirect evaluations (molecular-marker-based analysis of vernalization requirement) were used. The maximum quantum yield of the photosystem II photochemistry measured as the variable (F v ) to maximal (F m ) fl uorescence ratio (F v :F m ) analysis was confi rmed as a reliable method of screening genetic diversity for FT in plants at early growth stages. Variability for FT was also found after shorter acclimation at optimal (3/1°C day/night) or suboptimal (12/7°C day/night) hardening temperatures. High levels of FT and WH were found in both winter and facultative growth habits. Facultative genotypes were more responsive to early hardening than spring or winter types. Some of them coped best with frost in both laboratory and fi eld experiments. The most tolerant winter genotypes coped best with prolonged exposure to low temperature in the fi eld experiments. A conservative estimation of the role of FT in WH implies that at least 50% of WH was associated with the level of FT. A combined analysis using physiological and molecular tools is proposed for phenotyping WH in large populations.

We investigated the pigment composition and the transcriptome of albina (alb-e ( 16 ) and alb-f ( 17 )) and xantha (xan-s ( 46 ) and xan-b ( 12 )) barley mutants to provide an overall transcriptional picture of genes whose expression is interconnected with chloroplast activities and to search for candidate genes associated with the mutations. Beside those encoding plastid-localized proteins, more than 3,000 genes involved in non-chloroplast localized metabolism were up-/down-regulated in the mutants revealing the network of chloroplast-dependent metabolic pathways. The alb-e ( 16 ) mutant was characterized by overaccumulation of protoporphyrin IX upon ALA (5-amino levulinic acid) feeding and down-regulation of the gene encoding one subunit of Mg-chelatase, suggesting a block of the chlorophyll biosynthetic pathway before Mg-protoporphyrin IX biosynthesis, while alb-f ( 17 ) overaccumulated Mg-protoporphyrin IX and repressed PorA expression, without alterations in Mg-chelatase mRNA level. The alb-f ( 17 )mutant also showed overexpression of several genes involved in phytochrome and in phytochrome-dependent pathways. The results indicate that the down-regulation of Lhcb genes in alb-e ( 16 ) cannot be mediated by the accumulation of Mg-protoporphyrin IX. After ALA treatment, xan-s ( 46 ) showed overaccumulation of Mg-protoporphyrin IX, while the relative porphyrin composition of xan-b ( 12 ) was similar to wild type. The transcripts encoding the components of several mitochondrial metabolic pathways were up-regulated in albina/xantha leaves to compensate for the absence of active chloroplasts. The mRNAs encoding gun3, gun4, and gun5 barley homologous genes showed significant expression variations and were used to search for co-expressed genes across all samples. These analyses provide additional evidences on a chloroplast-dependent covariation of large sets of nuclear genes.

We analyze the effect of the plastoquinone redox state on the regulation of the light-harvesting antenna size at transcriptional and post-transcriptional levels. This was approached by studying transcription and accumulation of light-harvesting complexes in wild type versus the barley mutant viridis zb63, which is depleted in photosystem I and where plastoquinone is constitutively reduced. We show that the mRNA level of genes encoding antenna proteins is almost unaffected in the mutant; this stability of messenger level is not a peculiarity of antenna-encoding genes, but it extends to all photosynthesis-related genes. In contrast, analysis of protein accumulation by two-dimensional PAGE shows that the mutant undergoes strong reduction of its antenna size, with individual gene products having different levels of accumulation. We conclude that the plastoquinone redox state plays an important role in the long term regulation of chloroplast protein expression. However, its modulation is active at the post-transcriptional rather than transcriptional level.

Polyamine oxidases (PAOs, EC 1.5.3.11) are key enzymes responsible for the terminal catabolism of polyamines in plants, bacteria and protozoa. In barley, two PAO isoforms (HvPAO1 and HvPAO2) have been previously analyzed as regards their tissue expression and subcellular localization. Only the major isoform HvPAO2 has been biochemically characterized up to now. In order to study the ear-specific expression of the HvPAO1 isoform in detail, RT-PCR analysis was performed in barley on the whole ear and on various ear tissues. Moreover, HvPAO1promoter::GUS transient expression was examined in barley developing caryopses at 30-day postfertilization. Results from these analyses have demonstrated that the HvPAO1 gene is specifically expressed in all the ear organs analyzed (i.e. basal lemma, rachis, awn, embryo-deprived caryopsis, embryo and sterile spikelets), at variance with the HvPAO2 gene, which is expressed at high levels in sterile spikelets and at very low levels in embryos. We purified HvPAO1 from barley immature caryopses and characterized its catalytic properties. Furthermore, we carried out in vitro synthesis of HvPAO1 protein in a cell-free translation system. The HvPAO1 enzymes purified from immature caryopses and in vitro synthesized showed the same catalytic properties, in particular, an optimum at pH 7.0 for Spd and Spm oxidation and comparable Km values for both substrates, i.e. 0.89x10(-5) M and 0.5x10(-5) M for Spd and Spm, respectively. It has been found that HvPAO1 enzyme activity significantly differs in substrate specificity and pH optimum when compared with the major isoform HvPAO2. As a whole, these data strongly suggest that, in barley, the two PAO genes evolved separately, after a duplication event, to code for two distinct tissue-specific enzymes, and they are likely to play different physiological roles.

Genetic mapping of major genes and quantitative traits loci (QTLs) for many important agricultural traits is increasing the integration of biotechnology with the conventional breeding process. Exploitation of the information derived from the map position of traits with agronomical importance and of the linked molecular markers, can be achieved through marker assisted selection (MAS) of the traits during the breeding process. However, empirical applications of this procedure have shown that the success of MAS depends upon several factors, including the genetic base of the trait, the degree of the association between the molecular marker and the target gene, the number of individuals that can be analyzed and the genetic background in which the target gene has to be transferred. MAS for simply inherited traits is gaining increasing importance in breeding programs, allowing an acceleration of the breeding process. Traits related to disease resistance to pathogens and to the quality of some crop products are offering some important examples of a possible routinary application of MAS. For more complex traits, like yield and abiotic stress tolerance, a number of constraints have determined severe limitations on an efficient utilization of MAS in plant breeding, even if there are a few successful applications in improving quantitative traits. Recent advances in genotyping technologies together with comparative and functional genomic approaches are providing useful tools for the selection of genotypes with superior agronomical performancies.

Barley is grown either in the northern countries close to the polar circle or on the Himalayan mountains up to 4500 m on the sea level. Such a great diffusion, despite the differences in the climatic conditions, already suggests that the barley gene pool should contain characters for wide environmental adaptability and good stress resistance. The genetic adaptation to cold climate can be achieved either by evolving a powerful frost tolerance ability or by limiting the life cycle to the short summer season (escape strategy). It is a known fact that the winter barley varieties are less hardy than winter wheat, rye and triticale, nevertheless barley is grown till the Polar Circle because spring early maturity cultivars are able to run their life cycle in the short summer season. Plant growth habit and heading date can therefore be considered as the basic traits involved in barley adaptation to environments since they allow to synchronise the plant life cycle with seasonal changes. Nevertheless because winter barley has a higher yielding potential than spring ones, there is a great interest to improve its frost resistance capacity.

Drought, low temperature and salinity are the most important abiotic stress factors limiting crop productivity. A genomic map of major loci and QTLs affecting stress tolerance in Triticeae identified the crucial role of the group 5 chromosomes, where the highest concentration of QTLs and major loci controlling plant's adaptation to the environment (heading date, frost and salt tolerance) has been found. In addition, a conserved region with a major role in drought tolerance has been localized to the group 7 chromosomes. Extensive molecular biological studies have led to the cloning of many stress-related genes and responsive elements. The expression of some stress-related genes was shown to be linked to stress-tolerant QTLs, suggesting that these genes may represent the molecular basis of stress tolerance. The development of suitable genetic tools will allow the role of stress-related sequences and their relationship with stress-tolerant loci to be established in the near future.

We report the expression of the barley (Hordeum vulgare L.) COR (cold-regulated) genecor14b (formerly pt59) and the accumulation of its chloroplast-localized protein product. A polyclonal antibody raised against the cor14b-encoded protein detected two chloroplast COR proteins: COR14a and COR14b. N-terminal sequencing of COR14a and expression of cor14b in Arabidopsis plants showed that COR14a is not encoded by thecor14b sequence, but it shared homology with the wheat (Triticum aestivum L.) WCS19 COR protein. The expression of cor14b was strongly impaired in the barley albino mutant an, suggesting the involvement of a plastidial factor in the control of gene expression. Low-level accumulation of COR14b was induced by cold treatment in etiolated plants, although cor14b expression and protein accumulation were enhanced after a short light pulse. Light quality was a determining factor in regulating gene expression: red or blue but not far-red or green light pulses were able to promote COR14b accumulation in etiolated plants, suggesting that phytochrome and blue light photoreceptors may be involved in the control of cor14bgene expression. Maximum accumulation of COR14b was reached only when plants were grown and/or hardened under the standard photoperiod. The effect of light on the COR14b stability was demonstrated by using transgenic Arabidopsis. These plants constitutively expressedcor14b mRNAs regardless of temperature and light conditions; nevertheless, green plants accumulated about twice as much COR14b protein as etiolated plants.

Molecular adaptation to cold and drought involves a series of biochemical and molecular changes leading plants to improve their winter hardiness or drought resistance.

Background Alloplasmic lines provide a unique tool to study nuclear-cytoplasmic interactions. Three alloplasmic lines, with nuclear genomes from Triticum aestivum and harboring cytoplasm from Aegilops uniaristata, Aegilops tauschii and Hordeum chilense, were investigated by transcript and metabolite profiling to identify the effects of cytoplasmic substitution on nuclear-cytoplasmic signaling mechanisms. Results In combining the wheat nuclear genome with a cytoplasm of H. chilense, 540 genes were significantly altered, whereas 11 and 28 genes were significantly changed in the alloplasmic lines carrying the cytoplasm of Ae. uniaristata or Ae. tauschii, respectively. We identified the RNA maturation-related process as one of the most sensitive to a perturbation of the nuclear-cytoplasmic interaction. Several key components of the ROS chloroplast retrograde signaling, together with the up-regulation of the ROS scavenging system, showed that changes in the chloroplast genome have a direct impact on nuclear-cytoplasmic cross-talk. Remarkably, the H. chilense alloplasmic line down-regulated some genes involved in the determination of cytoplasmic male sterility without expressing the male sterility phenotype. Metabolic profiling showed a comparable response of the central metabolism of the alloplasmic and euplasmic lines to light, while exposing larger metabolite alterations in the H. chilense alloplasmic line as compared with the Aegilops lines, in agreement with the transcriptomic data. Several stress-related metabolites, remarkably raffinose, were altered in content in the H. chilense alloplasmic line when exposed to high light, while amino acids, as well as organic acids were significantly decreased. Alterations in the levels of transcript, related to raffinose, and the photorespiration-related metabolisms were associated with changes in the level of related metabolites. Conclusion The replacement of a wheat cytoplasm with the cytoplasm of a related species affects the nuclear-cytoplasmic cross-talk leading to transcript and metabolite alterations. The extent of these modifications was limited in the alloplasmic lines with Aegilops cytoplasm, and more evident in the alloplasmic line with H. chilense cytoplasm. We consider that, this finding might be linked to the phylogenetic distance of the genomes., The authors thank Prof. S.S. Maan (North Dakota State University, USA) for providing the alloplasmic lines T183 and T195 and Dr. Alessio Aprile (University of Salento, Lecce, Italy) for help in data analysis. LQ was partly supported by grant # 2770367, the Chief Scientist Fund, the Ministry of Agriculture, Israel.

Many physiological and molecular changes have been shown to occur at low temperatures, but the role and the contribution of each to frost resistance are still poorly understood. These changes include variations in enzyme activity, accumulation of soluble components, alterations in membrane composition and induction of specific genes.

The cold-regulated (COR14) protein of 14 kDa is a polypeptide accumulated under low-temperature conditions in the chloroplasts of barley leaves. In H. vulgare the COR14 antibody cross-reacts with two proteins, with a slightly different relative molecular weight around the marker of 14.4 kDa, referred to as COR14a and COR14b (high and low relative molecular weight, respectively). In a collection of H. spontaneum genotypes a clear polymorphism was found for the corresponding COR proteins. While some accessions showed the same COR pattern as cultivated barley, in 38 out of 61 accessions examined the COR14 antibody cross-reacted with an additional coldregulated protein with a relative molecular weight of about 24 kDa (COR24). The accumulation of COR24 was often associated with the absence of COR14b; the relationship between the COR14b/COR24 polymorphism and the adaptation of H. spontaneum to different environments is discussed. By studying COR14 accumulation in cultivated barley we have found that the threshold induction-temperature of COR14a is associated with the loci controlling winter hardiness. This association was demonstrated by using either a set of 30 cultivars of different origin, or two sets of frost-tolerant and frost-sensitive F1 doubled-haploid lines derived from the cross Dicktoo (winter type) x Morex (spring type). These results suggest that the threshold induction-temperature of COR14a can be a potential biochemical marker for the identification of superior frostresistant barley genotypes.

The optimum development of barley over its life cycle depends on a number of environmental stress factors that can prevent the plant’s expressing its maximum genetic potential. Severe grain losses are often caused by high or low temperatures, drought, anaerobiosis, and such soil anomalies as excess salt. The responses elicited from the plant by these stresses, when not lethal, include alterations in its processes of photosynthesis, respiration, and hormonal regulation through the development of specific, adaptive defense systems and mechanisms that are molecularly controlled. The duration of the stress and the plant’s growth stage at the former’s onset in turn affect yield. One can also find differing reactions as to plant susceptibility to adverse conditions. Thus, genetic variability plays a primary role in determining positive adaptation to environmental stresses and, hence, in supporting the spread of various barley genotypes to extreme climatic conditions (Stanca et al., 1992).

The protein encoded by cDNA clone pt59 and induced in barley (Hordeum vulgare L.) by cold was overexpressed in coli to produce the matching antibody, which in vivo recognized a cold-induced protein of 14 kDa (COR 14) that was found in the chloroplast stroma. The accumulation of COR14 occurred only at low temperatures after even a brief exposure of the plants to light. Plants grown and fully hardened in the dark accumulated a reduced amount of pt59-corresponding mRNA and only traces of COR14. Light exposure for as short as 5 min was enough to normalize the expression of pt59-corresponding mRNA and increase the accumulation of COR14. These findings indicate that one or more light-dependent factors are involved in transcription of the gene and accumulation of the protein. The COR14 protein was stored in amounts only slightly greater in the resistant barley cultivar Onice than in the susceptible cultivar Gitane, although the former had a higher induction-temperature threshold for COR14 than the latter. This fact is an evolutionary advantage, enabling the resistant varieties in the field to prepare for the cold well ahead of the susceptible ones.