Phenylalanine Ammonia-lyase Activity in Barley After Infection with Bipolaris sorokiniana or Treatment with its Purified Xylanase

Phenylalanine ammonia-lyase (PAL) activity was determined from leaves and roots of two barley (Hordeum vulgare L.) cultivars after infection with a necrotrophic pathogen, Bipolaris sorokiniana (Sacc.) Shoem., and treatment with its purified xylanase. PAL activity increased in leaves of both cultivars 16 h after fungal inoculation but two phases, with activity peaks at 24–32 h and 40 h, were recorded only for the more resistant cultivar, Agneta. Attempts to use a PAL inhibitor, χ-amin, ooxyacetic acid, to increase susceptibility to B. sorokiniana in barley leaves were unsuccessful. Treatments of leaves with purified xylanase resulted in more rapid (4–12 h after injection), although reduced, induction of PAL compared with fungal injection. The higher the concentration of xylanase applied the earlier the activity peaks were detected. Fungal inoculation only slightly increased PAL activity in barley roots while xylanase treatment had no effect. The basal level of PAL was however much higher in roots than in leaves. In wheat, Triticum aestivum L. resistant to B. sorokiniana, the time-course of PAL induction after fungal infection and xylanase treatment resembled that for cv. Agneta, while in oats, Avena sativa L. (non-host) PAL activity did not change after the treatments. The results suggest that the second phase of PAL induction, associated only with responses of barley cv. Agneta and wheat, is linked with their resistance to B. sorokiniana infection. The possible role of xylanase as an elicitor of PAL is discussed.     

增强UV-B辐射和氮对谷子叶光合色素及非酶促保护物质的影响

以谷子(Setaria italica(L)Beauv.)为对象,从拔节期开始持续给予低氮(1.875 mmol/L)和高氮(15 mmol/L)两种氮供应条件并从抽穗期开始进行26 d两种强度(4.29、7.12 kJ·m-2·d-1)的增强UV-B辐射处理,研究了谷子叶中光合色素含量、类黄酮含量和苯丙氨酸解氨酶(PAL)活性的变化.结果表明:与高氮供应条件相比,低氮供应条件明显降低了谷子叶中光合色素含量但提高了类胡萝卜素/叶绿素含量比值;在开花期中段和灌浆期中段,高氮供应条件下谷子叶中光合色素含量对增强UV-B辐射比低氮供应条件下的谷子更敏感.从灌浆期开始到处理结束,两种影响因子对谷子叶中类黄酮含量均有显著影响,增强UV-B辐射导致谷子叶中类黄酮含量逐渐升高,且相同增强UV-B辐射强度下低氮供应条件下的谷子叶中类黄酮含量明显高于高氮供应条件下的谷子.谷子叶中PAL活性对两种影响因子的响应较类黄酮含量更加敏感,低氮供应条件使谷子叶中PAL活性明显提高.结合上述指标的相关性分析结果可知,低氮供应条件加强了处于繁殖期主要阶段的谷子叶中类黄酮的积累,并使谷子叶中的类胡萝卜素/叶绿素含量比值明显提高,进而有助于维持谷子叶中光合色素含量在增强UV-B辐射条件下的相对稳定性,对植株抵抗UV-B辐射伤害有利.     

Increased Levels of Cytokinins in Barley Leaves Having the Systemic Acquired Resistance to Bipolaris sorokiniana (Sacc.) Shoemaker

The role of endogenously induced higher level of cytokinins and exogenously applied kinetin in relation to the development of barley leaf spot caused by Bipolaris sorokiniana (Sacc.) Shoemaker (syn. Helminthosporium sativum Pammel, King and Bakke) was studied. Spraying barley leaves with kinetin suppressed the number and the size of necrotic spots caused by the fungus. Inoculation of the lower leaves of barley by a spore suspension of the fungus B. sorokiniana induced resistance on the upper leaves against a subsequent challenge inoculation by the same pathogen 10 days later. An increase in the level of cytokinins was observed in these resistant leaves. Elevated levels of cytokinins may cause a type of juvenility in leaf tissues. The juvenile state could be in a causal relationship with the suppression of necrotic spots caused by the fungus.     

Investigation of the porphyrins accumulation in barley leaves incubated with Mn cations and δ-aminolevulinic acid

In greening etiolated primary leaves of barley (Hordeum vulgare L.), Mn²⁺ ions have been shown to inhibit chlorophyll (Chl) accumulation in a dose dependent manner and to lead to an accumulation of protoporphyrin IX (Proto) and Mg-protoporphyrin IX monomethyl ester (MgPE). The amount of MgPE that accumulated, was 2 times higher than Proto. In the dark, Proto and MgPE were observed to have accumulated to high levels in seven-day old green and etiolated leaves in the presence of 5 mmol/L Mn²⁺, but only if 5 mmol/L δ-aminolevulinic acid (ALA) was present. The 24 hours of irradiation of the green barley leaves treated in this way, resulted in a photodynamic destruction of Proto and MgPE as well as of Chl and carotenoids (Car). The observed porphyrin accumulation caused by the Mn²⁺ ions was reversed in the presence of active iron (Fe²⁺). This effect was observed when the iron concentration in incubation solutions was half the Mn²⁺ concentration, most effective for porphyrin synthesis, i.e. 5 mmol/L. The action of Mn²⁺ on porphyrin accumulation is also discussed.     

A novel effector,CsSp1, from Bipolaris sorokiniana,is essential for colonization in wheat and is also involved in triggering host immunity

The hemibiotrophic pathogen Bipolaris sorokiniana causes root rot, leaf blotching, and black embryos in wheat and barley worldwide, resulting in significant yield and quality reductions. However, the mechanism underlying the host–pathogen interactions between B. sorokiniana and wheat or barley remains unknown. The B. sorokiniana genome encodes a large number of uncharacterized putative effector proteins. In this study, we identified a putative secreted protein, CsSp1, with a classic N-terminal signal peptide, that is induced during early infection. A split-marker approach was used to knock out CsSP1 in the Lankao 9-3 strain. Compared with the wild type, the deletion mutant ∆Cssp1 displayed less radial growth on potato dextrose agar plates and produced fewer spores, and complementary transformation completely restored the phenotype of the deletion mutant to that of the wild type. The pathogenicity of the deletion mutant in wheat was attenuated even though appressoria still penetrated the host. Additionally, the infectious hyphae in the deletion mutant became swollen and exhibited reduced growth in plant cells. The signal peptide of CsSp1 was functionally verified through a yeast YTK12 secretion system. Transient expression of CsSp1 in Nicotiana benthamiana inhibited lesion formation caused by Phytophthora capsici. Moreover, CsSp1 localized in the nucleus and cytoplasm of plant cells. In B. sorokiniana-infected wheat leaves, the salicylic acid-regulated genes TaPAL, TaPR1, and TaPR2 were down-regulated in the ∆Cssp1 strain compared with the wild-type strain under the same conditions. Therefore, CsSp1 is a virulence effector and is involved in triggering host immunity.     

Diverse origins of waxy foxtail millet crops in East and Southeast Asia mediated by multiple transposable element insertions

The naturally occurring waxy and low-amylose variants of foxtail millet and other cereals, like rice and barley, originated in East and Southeast Asia under human selection for sticky foods. Mutations in the GBSS1 gene for granule-bound starch synthase 1 are known to be associated with these traits. We have analyzed the gene in foxtail millet, and found that, in this species, these traits were originated by multiple independent insertions of transposable elements and by subsequent secondary insertions into these elements or deletion of parts of the elements. The structural analysis of transposable elements inserted in the GBSS1 gene revealed that the non-waxy was converted to the low-amylose phenotype once, while shifts from non-waxy to waxy occurred three times, from low amylose to waxy once and from waxy to low amylose once. The present results, and the geographical distribution of different waxy molecular types, strongly suggest that these types originated independently and were dispersed into their current distribution areas. The patterns of GBSS1 variation revealed here suggest that foxtail millet may serve as a key to solving the mystery of the origin of waxy-type cereals in Asia. The GBSS1 gene in foxtail millet provides a new example of the evolution of a gene involved in the processes of domestication and its post-domestication fate under the influence of human selection. Electronic Supplementary Material Supplementary material is available for this article at     

Foxtail Millet CBL4 (SiCBL4) Interacts with SiCIPK24, Modulates Plant Salt Stress Tolerance

The calcineurin B-like (CBL) protein and the CBL-interacting protein kinase (CIPK) signaling pathway play important roles in plant abiotic stress tolerance. To investigate the molecular mechanism of salt stress tolerance of foxtail millet, SiCBL4 and SiCIPK24 were identified and functionally characterized. Both SiCBL4 and SiCIPK24 were induced by salt, abscisic acid (ABA), methyl viologen (MV), and heat shock stress in foxtail millet seedlings. Yeast two-hybrid and bimolecular fluorescence complementation assay showed that SiCBL4 interacted with SiCIPK24. The mutation of the N-myristoylation site of SiCBL4 changed the sub-cellular localization of SiCBL4 and directed the SiCBL4-SiCIPK24 protein complex from plasma membrane to cytoplasm, and disrupted its function in plant salt stress tolerance. Overexpression of SiCBL4 or SiCIPK24 in Arabidopsis sos3-1 or sos2-1 mutant plants rescued the mutant salt hypersensitivity phenotype. In addition, overexpression of SiCIPK24 also enhanced the salt stress tolerance of Arabidopsis wild-type plants. This work helps to understand the structure and function of the foxtail millet CBL and CIPK genes and confirmed that the foxtail millet CBL-CIPK pathway can be manipulated to enhance the plant salt stress tolerance.     

Influence of photodynamic processes induced by 2,2′-dipyridyl on the enzymatic system of chlorophyll biosynthesis

The influence of 2,2′-dipyridyl (2,2′-DP) on the activity of one of the enzymes at the initial stages of chlorophyll (Chl) biosynthesis, δ-aminolevulinic acid dehydratase (ALAD; δ-aminolevulinate hydro-lyase, EC 4.2.1.24), as well as on δ-aminolevulinic acid (ALA) accumulation was investigated in green barley (Hordeum vulgare L.) leaves. In seven-day-old green leaves treated with 3 mM 2,2′-DP for 17 h in darkness and subsequently irradiated with "white light" (15 W m-2) for 4, 8, and 24 h the ALAD activity was 51 % as compared to that in untreated leaves. At the same time, the ALA forming system was most sensitive to the photodynamic processes caused by 2,2′-DP. After 8 h of irradiation, ALA synthesis was entirely inhibited. After the treatment the leaves accumulated exceptionally high amounts of Chl precursors such as protoporphyrin IX (Proto), Mg-protoporphyrin IX (Mg-Proto), its monomethyl ester, and protochlorophyllide (Pchlide) that are photosensitizers of photodynamic processes in plants. A comparatively low Chl and carotenoid (Car) destruction was registered during the subsequent 4 and 8 h of irradiation. At the same time, the content of Chl precursors was negligible. The low photodestruction of Chl and Car included in pigment-protein complexes, against the background of fast porphyrin disappearance, and fast decrease of enzymatic activities at the initial stages of Chl production could mean that the photodynamic effect induced by porphyrins accumulated in the presence of 2,2′-DP affected first the Chl enzymatic system and did not change the pool of already synthesized photosynthetic pigments.     

Influence of Seed‐borne Cochliobolus sativus (Anamorph Bipolaris sorokiniana) on Crown Rot and Root Rot of Barley and Wheat

The effect of seed‐borne pathogens of wheat and barley on crown and root rot diseases of seven barley cultivars (Jimah‐6, Jimah‐51, Jimah‐54, Jimah‐58, Omani, Beecher and Duraqi) and three wheat cultivars (Cooley, Maissani and Shawarir) was investigated. Bipolaris sorokiniana and Alternaria alternata were detected in seeds of at least eight cultivars, but Fusarium species in seeds of only two barley cultivars (Jimah‐54 and Jimah‐58). Crown rot and root rot symptoms developed on barley and wheat cultivars following germination of infected seeds in sterilized growing media. Bipolaris sorokiniana was the only pathogen consistently isolated from crowns and roots of the emerging seedlings. In addition, crown rot and root rot diseases of non‐inoculated barley cultivars correlated significantly with B. sorokiniana inoculum in seeds (P = 0.0019), but not with Fusarium or Alternaria (P > 0.05). These results indicate the role of seed‐borne inoculum of B. sorokiniana in development of crown rot and root rot diseases. Pathogenicity tests of B. sorokiniana isolates confirmed its role in inducing crown rot and root rot, with two wheat cultivars being more resistant to crown and root rots than most barley cultivars (P < 0.05). Barley cultivars also exhibited significant differences in resistance to crown rot (P < 0.05). In addition, black point disease symptoms were observed on seeds of three barley cultivars and were found to significantly affect seed germination and growth of some of these cultivars. This study confirms the role of seed‐borne inoculum of B. sorokiniana in crown and root rots of wheat and barley and is the first report in Oman of the association of B. sorokiniana with black point disease of barley.     

Development of eSSR-Markers in Setaria italica and Their Applicability in Studying Genetic Diversity,Cross-Transferability and Comparative Mapping in Millet and Non-Millet Species

Foxtail millet ( Setaria italica L.) is a tractable experimental model crop for studying functional genomics of millets and bioenergy grasses. But the limited availability of genomic resources, particularly expressed sequence-based genic markers is significantly impeding its genetic improvement. Considering this, we attempted to develop EST-derived-SSR (eSSR) markers and utilize them in germplasm characterization, cross-genera transferability and in silico comparative mapping. From 66,027 foxtail millet EST sequences 24,828 non-redundant ESTs were deduced, representing ~16 Mb, which revealed 534 (~2%) eSSRs in 495 SSR containing ESTs at a frequency of 1/30 kb. A total of 447 pp were successfully designed, of which 327 were mapped physically onto nine chromosomes. About 106 selected primer pairs representing the foxtail millet genome showed high-level of cross-genera amplification at an average of ~88% in eight millets and four non-millet species. Broad range of genetic diversity (0.02–0.65) obtained in constructed phylogenetic tree using 40 eSSR markers demonstrated its utility in germplasm characterizations and phylogenetics. Comparative mapping of physically mapped eSSR markers showed considerable proportion of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (~68%), maize (~61%) and rice (~42%) chromosomes. Synteny analysis of eSSRs of foxtail millet, rice, maize and sorghum suggested the nested chromosome fusion frequently observed in grass genomes. Thus, for the first time we had generated large-scale eSSR markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species.     

Accumulation of glycinebetaine during cold acclimation and freezing tolerance in leaves of winter and spring barley plants

A study was performed to examine whether or not betaine (glycinebetaine), a compatible solute, is accumulated in response to cold stress and is involved in mechanisms that protect plants from freezing injury. For this purpose, we used near-isogenic lines of barley, with each line differing only in a single gene for the spring type of growth habit; the various lines were produced by back-crosses to a recurrent cultivar of the winter type. The winter type of growth habit requires a low temperature for triggering of flower development (vernalization), whereas the spring type does not. Betaine was accumulated to five times the basal level over the course of 3 weeks at low temperature (5 °C) in the winter-type cultivar and in a spring-sh line having the sh gene for the spring-type growth habit, but the level was only doubled in the spring-Sh3 line, which carried the Sh₃ gene for the spring-type growth habit. Among near-isogenic lines of the same cultivar, the levels of betaine accumulated in leaves at low temperature were well correlated with the percentages (on a dry weight basis) of green leaves that survived freezing injury (-5 °C). This observation indicates the possibility, separate from the recognized role of betaine in the response to salinity and/or drought, that betaine accumulates in response to cold stress and that the accumulation of betaine during cold acclimation is associated to some extent with freezing tolerance in leaves of barley plants.     

Foxtail millet: a model crop for genetic and genomic studies in bioenergy grasses

Foxtail millet is one of the oldest domesticated diploid C₄ Panicoid crops having a comparatively small genome size of approximately 515?Mb, short life cycle, and inbreeding nature. Its two species, Setaria italica (domesticated) and Setaria viridis (wild progenitor), have characteristics that classify them as excellent model systems to examine several aspects of architectural, evolutionary, and physiological importance in Panicoid grasses especially the biofuel crops such as switchgrass and napiergrass. Foxtail millet is a staple crop used extensively for food and fodder in parts of Asia and Africa. In its long history of cultivation, it has been adapted to arid and semi-arid areas of Asia, North Africa, South and North America. Foxtail millet has one of the largest collections of cultivated as well as wild-type germplasm rich with phenotypic variations and hence provides prospects for association mapping and allele-mining of elite and novel variants to be incorporated in crop improvement programs. Most of the foxtail millet accessions can be primarily abiotic stress tolerant particularly to drought and salinity, and therefore exploiting these agronomic traits can enhance its efficacy in marker-aided breeding as well as in genetic engineering for abiotic stress tolerance. In addition, the release of draft genome sequence of foxtail millet would be useful to the researchers worldwide in not only discerning the molecular basis of biomass production in biofuel crops and the methods to improve it, but also for the introgression of beneficial agronomically important characteristics in foxtail millet as well as in related Panicoid bioenergy grasses.     

The Influence of Protochlorophyllide, Formed from Exogenous δ-Aminolevulinic Acid, on Chlorophyll Synthesis in Leaves during Flash Illumination

In the presence of large accumulations of protochlorophyllide, derived from exogenous δ-aminolevulinic acid, chlorophyll synthesis in excised leaves of two varieties of barley was less than in untreated leaves. In oat leaves the accumulated protochlorophyllide, from exogenous δ-laminolevulinic acid, stimulated chlorophyll synthesis to above the control level. — These relationships could only be demonstrated when phtodestruction of pigments was minimised by the use of flash illumination (2 milliseconds every 3 minutes). — These was no evidence from in vivo absorption spectra that the pigments in the barley leaves were different to those in leaves studied by other workers. However, the presence of the accumulated protochlorophyllide appeared to prevent the shift of the chlorophyll absorption maximum from 673 nm to 677 nm. — Possible mechanisms of inhibition are discussed.     

Isolation and characterization of a novel peroxisomal choline monooxygenase in barley

Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline → betaine aldehyde → GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [¹⁴C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [¹⁴C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.     

Comparative genetic maps of foxtail millet (Setaria italica) and rice (Oryza sativa)

 A foxtail millet-rice comparative genetic map was constructed using mapped rice RFLP markers and wheat genomic and cDNA clones with known map position in rice. About 74% and 37% of the cDNA and genomic clones, respectively, were transferable to foxtail millet, confirming that conservation at the DNA level is greatest in genic regions. A high degree of conserved colinearity was observed between the two genomes. Five entire foxtail millet chromosomes appear to be colinear with five entire rice chromosomes. The remaining four foxtail millet linkage groups each show colinearity with segments of two rice chromosomes. The rearrangements of rice chromosomes 3 and 10 to form foxtail millet chromosome IX, and 7 and 9 to form chromosome II are very similar to those required to form maize chromosomes 1 and 7 and sorghum linkage groups C and B, indicating Setaria’s clear taxonomic position within the subfamily of the Panicoideae. Received: 18 December 1996 / Accepted: 4 August 1997     

Abscisic Acid accumulation is not required for proline accumulation in wilted leaves

Leaves from dark-grown barley (Hordeum vulgare L. var Larker) seedlings grown in the presence and absence of fluridone were used to determine whether or not abscisic acid (ABA) accumulation was necessary for proline to accumulate in wilted tissue. Wilted tissue (polyethylene glycol-treated) leaves from fluridone-grown seedlings did not accumulate ABA but did accumulate proline at a rate that was not different from the non-fluridone-treated leaves. Thus ABA accumulation is not required for wilting-induced proline accumulation in barley leaves. Proline accumulation in wilted leaves from the wilty tomato (Lycopersicon esculentum) mutant, flacca, was compared to that in the wild type, Rheinlands Ruhm. Proline accumulated in wilted leaves from flacca. The rate of accumulation was faster in flacca compared to the rate in the wild type because the wilty mutant wilted faster. ABA accumulated in wilted leaves from the wild type but not in the wilty mutant. This result is a further confirmation that ABA accumulation is not required for wilting-induced proline accumulation. These results are significant in that proline accumulation in barley leaves can be induced independently by any one of three treatments: wilting, ABA, or salt.     

Accumulation of Free Proline at Low Temperatures

The accumulation of free proline in the first leaves of barley, Hordeum distichum L., and wheat, Triticum aestivum L., in response to a range of low temperatures was examined with 10-day-old plants. In barley (cv. Prior) no proline accumulated at 8°C or above, but in wheat (cv. Gabo) proline accumulated at 12°C and lower temperatures. In barley, the first leaf survived for 29 days following transfer to 5°C and continued to accumulate proline throughout this period. In contrast, the first leaves of plants maintained at 20°C survived for 13 days only and accumulated no proline. Proline accumulation at low temperature was shown to be light-dependent, both in intact plants and excised leaf sections, and the light requirement could not be replaced by supplying leaf segments with precursors of proline. Proline accumulation in response to water stress was not light-dependent at 20°C but was at 5°C. Inter-specific and intra-specific variation in the extent of accumulation in response to low temperature was also examined. Considerable variation was encountered but there was no clear relationship with geographical distribution or chilling sensitivity for the species and no correlation with accumulation in response to water stress in the cultivars of barley examined.