<Emphasis Type="Italic">LMI1</Emphasis>-like genes involved in leaf margin development of <Emphasis Type="Italic">Brassica napus</Emphasis>

In rapeseed (Brassica napus L.), leaf margins are variable and can be entire, serrate, or lobed. In our previous study, the lobed-leaf gene (LOBED-LEAF 1, BnLL1) was mapped to a 32.1 kb section of B. napus A10. Two LMI1-like genes, BnaA10g26320D and BnaA10g26330D, were considered the potential genes that controlled the lobed-leaf trait in rapeseed. In the present study, these two genes and another homologous gene (BnaC04g00850D) were transformed into Arabidopsis thaliana (L.) Heynh. plants to identify their functions. All three LMI1-like genes of B. napus produced serrate leaf margins. The expression analysis indicated that the expression level of BnaA10g26320D determined the difference between lobed- and entire-leaved lines in rapeseed. Therefore, it is likely that BnaA10g26320D corresponds to BnLL1.     

Induction of two cyclotide-like genes <Emphasis Type="Italic">Zmcyc1</Emphasis> and <Emphasis Type="Italic">Zmcyc5</Emphasis> by abiotic and biotic stresses in <Emphasis Type="Italic">Zea mays</Emphasis>

Cyclotides are small plant disulfide-rich and cyclic proteins with a diverse range of biological activities. Cyclotide-like genes show key sequence features of cyclotides and are present in the Poaceae. In this study the cDNA of the nine cyclotide-like genes were cloned and sequenced using 3′RACE from Zea mays. The gene expression of two of these genes (Zmcyc1 and Zmcyc5) were analyzed by real-time PCR in response to biotic (Fusarium graminearum, Ustilago maydis and Rhopalosiphum maydis) and abiotic (mechanical wounding, water deficit and salinity) stresses, as well as in response to salicylic acid and methyl jasmonate elicitors to mimic biotic stresses. All isolated genes showed significant similarity to other cyclotide-like genes and were classified in two separate clusters. Both Zmcyc1 and Zmcyc5 were expressed in all studied tissues with the highest expression in leaves and lowest expression in roots. Wounding, methyl jasmonate and salicylic acid significantly induced the expression of Zmcyc1 and Zmcyc5 genes, but the higher expression was observed for Zmcyc1 as compared with Zmcyc5. Expression levels of these two genes were also induced in inoculated leaves with F. graminearum, U. maydis and also in response to insect infestation. In addition, the 1000-base-pairs (bp) upstream of the promoter of Zmcyc1 and Zmcyc5 genes were identified and analyzed using the PlantCARE database and consequently a large number of similar biotic and abiotic cis-regulatory elements were identified for these two genes.     

The <Emphasis Type="Italic">angora</Emphasis> gene weakens the effect of interaction of the mutant genes <Emphasis Type="Italic">wellhaarig</Emphasis> and <Emphasis Type="Italic">waved alopecia</Emphasis> in mice

The interaction of the mutant genes wellhaarig (we) and waved alopecia (wal) in mice was earlier demonstrated in our laboratory. The we gene significantly accelerates the appearance of alopecia in double we/wewal/wal homozygotes as compared to that in single +/+wal/wal homozygotes. It has been found in this work that the mutant gene angora-Y (Fgf5 ^go-Y ) weakens the effect of interaction of the we and wal genes. The first signs of alopecia appear in mice of the we/wewal/wal genotype at the age of 14 days, in triple Fgf5 ^go-Y /Fgf5 ^go-Y we/wewal/wal homozygotes alopecia is observed seven days later, i. e., in 21-day-old animals. The progression of alopecia in triple homozygotes is expressed to a lesser degree than in double +/+we/wewal/wal homozygotes. A single dose of the Fgf5 ^go-Y gene also decreases the effect of interaction of the we and wal genes, but less than a double dose of this gene. The first signs of alopecia in mice of the +/Fgf5 ^go-Y we/wewal/wal genotype appear only three days later than in double +/+we/wewal/wal homozygotes. The data obtained demonstrate that the Fgf5 ^go-Y gene is a powerful modifier of mutant genes determining the process of alopecia.     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

Titanium Dioxide Nanoparticles Affect the Percentage of Free Radical Scavenging,Protein Content and DNA Mismatch Repair Genes in <Emphasis Type="Italic">Zea mays</Emphasis> L. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

This paper identifies the potential molecular markers predicting the impact of nTiO₂ on plants and explores the new statistical correlations between the biomarkers and growth parameters. The quantitative mRNA expression of the three genes involved in DNA mismatch repair (MLH1) and cell division (PCNA1 and PCNA2) in Zea mays and Triticum aestivum seedlings were related to the growth parameters measured in response to five nTiO2 treatments. The results indicated that the higher concentrations were harmless to Z. mays but not to T. aestivum. nTiO₂ treatments increased the total protein levels in both species and significantly inhibited the percentage of DPPH radical scavenging in Z. mays compared with T. aestivum seedlings. The exposure to both 50 μg/ml and 30 μg/ml concentrations of nTiO₂ significantly induced the expression of MLH1 and PCNA1 genes in both species; however, the exposure to 30 μg/ml of nTiO₂ also significantly induced the expression of PCNA2 genes in T. aestivum. The exposure to 50, 70 and 140 μg/ml significantly inhibited the expression of PCNA2 in both species, while 70 and 140 μg/ml repressed the expression of MLH1 and PCNA1 in the seedlings of Z. mays. The induction and repression of the expression of the three genes were correlated with some growth parameters and biological indices in both species. This key finding suggests that the above genes may play a vital role in mediating plant stress response to nTiO₂ and could be used as sensitive molecular biomarkers indicative of the oxidative stress of nTiO₂ exposure.     

Participation of <Emphasis Type="Italic">SRM5/CDC28</Emphasis>, <Emphasis Type="Italic">SRM8/NET1</Emphasis>, and <Emphasis Type="Italic">SRM12/HFI1</Emphasis> genes in checkpoint control in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

About twenty genes participating in checkpoint control are known in yeast Saccharomyces cerevisiae. The involvement of SRM genes in the cell cycle arrest under the action of DNA damaging agents was studied in this work. These genes were earlier defined as genes affecting genetic stability and radiosensitivity. It was shown that mutations srm5/cdc28-srm, srm8/net1-srm, and srm12/hfi1-srm fail the cell cycle arrest in the presence of DNA damage and influence the checkpoint arrest in G0/S (srm5, srm8), G1/S (srm5, srm8, srm12), S (srm5, srm12), and G₂/M (srm5). It seems likely that genes SRM5/CDC28, SRM12/HFI1/ADA1, and SRM8/NET1 are involved in a cell response to DNA damage, and in checkpoint regulation in particular.     

Genome-wide identification and expression analysis of sulphate transporter (<Emphasis Type="Italic">SULTR</Emphasis>) genes under sulfur deficiency in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis>

Sulphur is an important mineral element for plant growth and development. It involves in a number of metabolic processes with crucial functions. This study has performed a genome-wide analysis of sulfate transporter (SULTR) genes in Brachypodium distachyon. Ten putative SULTR genes were identified in Brachypodium genome. BdSULTR genes included 6–17 exons encoding a protein of 647–693 residues with basic nature. BdSULTR proteins included both sulfate_transp (PF00916) and STAS (PF01740) domains. BdSULTRs were classified into 4 groups based on the phylogenetic distribution. Promoter regions of all BdSULTR genes, except for BdSULTR3;3 and 3;5 included the SURECOREATSULTR11 elements. A considerable structural overlap was identified between superimposed SULTR1;3 and 3;1 proteins, indicating that SULTR1 members may also involve in plant stress response/tolerance like SULTR3 members. Microarray and RNA-Seq analyses also revealed the differential expression of SULTR 1 and 3 genes under different biotic/abiotic stresses. Protein–protein interaction partners of BdSULTRs were mainly related with adenylyl-sulfate kinases, 5′-adenylylsulfate reductases, ATP sulfurylases, and acyl carrier proteins. Moreover, expression profiles of identified BdSULTR genes under S-deficiency were analyzed using RT-qPCR. It was revealed that BdSULTR1;1 and 3;1 are highly expressed in plant roots as ~tenfold and ~fivefold, respectively, while BdSULTR2 (~15-fold) and 3;1 (~twofold) are abundantly expressed in leaf tissues.     

Fine mapping of QTL <Emphasis Type="Italic">qCTB10</Emphasis>-<Emphasis Type="Italic">2</Emphasis> that confers cold tolerance at the booting stage in rice

Key message

The QTL qCTB10 - 2 controlling cold tolerance at the booting stage in rice was delimited to a 132.5 kb region containing 17 candidate genes and 4 genes were cold-inducible.

Abstract

Low temperature at the booting stage is a major abiotic stress-limiting rice production. Although some QTL for cold tolerance in rice have been reported, fine mapping of those QTL effective at the booting stage is few. Here, the near-isogenic line ZL31-2, selected from a BC₇F₂ population derived from a cross between cold-tolerant variety Kunmingxiaobaigu (KMXBG) and the cold-sensitive variety Towada, was used to map a QTL on chromosome 10 for cold tolerance at the booting stage. Using BC₇F₃ and BC₇F₄ populations, we firstly confirmed qCTB10-2 and gained confidence that it could be fine mapped. QTL qCTB10-2 explained 13.9 and 15.9% of the phenotypic variances in those two generations, respectively. Using homozygous recombinants screened from larger BC₇F₄ and BC₇F₅ populations, qCTB10-2 was delimited to a 132.5 kb region between markers RM25121 and MM0568. 17 putative predicted genes were located in the region and only 5 were predicted to encode expressed proteins. Expression patterns of these five genes demonstrated that, except for constant expression of LOC_Os10g11820, LOC_Os10g11730, LOC_Os10g11770, and LOC_Os10g11810 were highly induced by cold stress in ZL31-2 compared to Towada, while LOC_Os10g11750 showed little difference. Our results provide a basis for identifying the genes underlying qCTB10-2 and indicate that markers linked to the qCTB10-2 locus can be used to improve the cold tolerance of rice at the booting stage by marker-assisted selection.

     

Engineering <Emphasis Type="Italic">Escherichia coli</Emphasis> for efficient coproduction of polyhydroxyalkanoates and 5-aminolevulinic acid

Single-cell biorefineries are an interesting strategy for using different components of feedstock to produce multiple high-value biochemicals. In this study, a strategy was applied to refine glucose and fatty acid to produce 5-aminolevulinic acid (ALA) and polyhydroxyalkanoates (PHAs). To express the ALA and PHAs dual-production system efficiently and stably, multiple copies of the poly-β-3-hydroxybutyrate (PHB) synthesis operon were integrated into the chromosome of Escherichia coli DH5αΔpoxB. The above strain harboring the ALA C5 synthesis pathway genes hemA and hemL resulted in coproduction of 38.2% PHB (cell dry weight, CDW) and 3.2 g/L extracellular ALA. To explore coproduction of ALA and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the PHBV synthetic pathway was also integrated into engineered E. coli and coexpressed with hemA and hemL; cells produced 38.9% PHBV (CDW) with 10.3 mol% 3HV fractions and 3.0 g/L ALA. The coproduction of ALA with PHB and PHBV can improve the utilization of carbon sources and maximize the value derived from the feedstock.     

Structural Polymorphism of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> Genes Related to Virulence and Salt Tolerance

Analysis of the structural polymorphism of eight genes in Sinorhizobium meliloti (nodA, nodB, nodC, and nodH, as well as betA, betB, betC, and betB2) involved in virulence control and salt tolerance, respectively, was carried out in native populations from two geographically distant areas of alfalfa diversity. These areas are located in the North Caucasian gene center of cultivated plants (NCG) and in the modern center of introgressive hybridization of alfalfa located next to the Aral Sea area (PAG) subjected to salinization. RFLP types (alleles) of the nod and bet genes, similar to those in the reference strain Rm1021 (A-type) and different from them (divergent, or D-type alleles) were revealed. The combinations for A- and D-type alleles of the aforementioned genes (analysis of the linkage disequilibrium, LD) were studied in both populations. It was shown that D-type alleles of the nod genes were two times more frequent in the NCG population, while D-type alleles of the bet genes were predominantly identified in the PAG population. At the same time, different combinations of D-type alleles of both the nod and bet genes prevailed in populations. For instance, in the case of the glycine betaine metabolism pathway, these were the betC and betB2 genes in NCG population and betB and betA genes in PAG population. The state of linkage disequilibrium was shown for 60.7% of combinations of alleles of the nod and bet genes in the S. meliloti strains from NCG and more than twice less in strains from the PAG population. It was concluded that clonal lines prevailed in NCG, while the PAG population of S. meliloti had a panmictic structure with revealed single clonal lines.     

Towards improvement of marker assisted selection of apple scab resistant cultivars: <Emphasis Type="Italic">Venturia inaequalis</Emphasis> virulence surveys and standardization of molecular marker alleles associated with resistance genes

Molecular breeding for pathogen resistance faces two major problems that delay its widespread adoption, resistance breakdown and difficulties in unambiguously identifying the alleles of the markers associated with specific resistance genes. Since the breakdown of the Rvi6 (Vf) gene in the Northern part of Europe breeders have intensified the search for new resistance sources to be introduced into their breeding programs. Alternative major genes to Rvi6 are available (e.g. Rvi2, Rvi4, Rvi5, Rvi10; Rvi11, Rvi12, Rvi13, and Rvi15, respectively Vh2, Vh4, Vm, Va, Vbj, Vb, Vd, Vr2 according to the old apple scab resistance gene nomenclature) but, with few exceptions (i.e., Rvi4, Rvi5 and, Rvi13), they have so far not been incorporated in commercial varieties. Pyramiding, i.e., combining several of these major resistance genes (R-genes) in individual plants, is one of the most promising strategies currently available to develop apple cultivars with durable apple scab resistance. But, which genes are the best suited to produce such new cultivars? Although the most interesting genes are surely those whose resistance so far has not been broken by the pathogen, genes with resistance that has been overcome coupled with only limited spread of the virulence may also be used in the pyramiding process. However, obtaining information on whether an R-gene is overcome and if so, the extent of the spread of the virulence is difficult and time consuming. Furthermore, often such reports are not up-to-date and the correctness of the data is difficult to verify. To solve these problems, the initiative “Monitoring of Venturia inaequalis virulences” has been proposed. The monitoring is based on a network of orchards of selected differential hosts. Incidence and severity of scab on these genotypes will be collected yearly; and after validation, the data will be published through the homepage of the project (www.vinquest.ch). Here, we present an outline of this initiative. A second major obstacle for broad adoption of marker assisted selection is the lack of tools to align marker analyzes performed in different laboratories to unambiguously identify the alleles linked to specific resistances. The identification of the alleles of the markers in coupling with the resistance genes is often very difficult, if the same genotype used to develop the markers is not simultaneously analyzed. In this paper we present an approach to standardize the size of the alleles in coupling with the resistance genes, using easily accessible cultivars. The proposed procedure has been applied to selected markers for the apple scab resistance genes Rvi2, Rvi4, Rvi5, Rvi6, Rvi11, Rvi12, Rvi13, Rvi14 and Rvi15 (respectively Vh2, Vh4, Vm, Vf, Vbj, Vb, Vd, Rvi14 and Vr2 according to the old nomenclature).