Type-B cytokinin response regulators link hormonal stimuli and molecular responses during the transition from endo- to ecodormancy in apple buds.

KEY MESSAGE: Cytokinin together with MdoBRR1, MdoBRR8 and MdoBRR10 genes participate in the downregulation of MdoDAM1, contributing to the transition from endo- to ecodormancy in apple buds. The final step of cytokinin (CK) signaling pathway culminates in the activation of type-B response regulators (BRRs), important transcriptional factors in the modulation of CK-responsive genes. In this study, we performed a genome-wide analysis aiming to identify apple BRR family members and understand their involvement in bud dormancy control. The investigation identified ten MdoBRR protein-coding genes. A higher expression of three MdoBRR (MdoBRR1, MdoBRR9 and MdoBRR10) was observed in dormant buds in comparison to other developmental stages. Interestingly, in ecodormant buds these three MdoBRR genes were upregulated in a CK-dependent manner. Transcription profiles, determined during dormancy cycle under field and artificially controlled conditions, revealed that MdoBRR1 and MdoBRR8 played important roles in the transition from endo- to ecodormancy, probably mediated by endogenous CK stimuli. The expression of MdoBRR7, MdoBRR9, and MdoBRR10 was induced in ecodormant buds exposed to warm temperatures, indicating a putative role in growth resumption after chilling requirement fulfillment. Contrasting expression patternsin vivo between MdoBRRs and MdoDAM1, an essential dormancy establishment regulator, were observed during dormancy cycle and in CK-treated buds. Thereafter, in vivo transactivation assays showed that CK stimuli combined with transient overexpression of MdoBRR1, MdoBRR8, and MdoBRR10 resulted in downregulation of the reporter gene gusA driven by the MdoDAM1 promoter. These pieces of evidences point to the integration of CK-triggered responses through MdoBRRs that are able to downregulate MdoDAM1, contributing to dormancy release in apple.

Genome organization in Mycoplasma hyopneumoniae: identification of promoter-like sequences.

Information related to open reading frame (ORF) organization, transcription regulation and promoter sequence has been available for the Mycoplasma hyopneumoniae 7448 genome, demonstrating that the ORFs are continuously transcribed (cotranscription) in large clusters. A species-specific position-specific scoring matrix was applied to scan for putative promoters upstream of all coding sequences on a genome scale in M. hyopneumoniae. This study consisted of a detailed in silico promoter localization analysis by scanning the position-specific promoters upstream of predicted ORF clusters (OCs) and mCs (monocistronic genes) in the M. hyopneumoniae whole genome; this was combined with experimental data for the promoterless ORFs. Promoter-like sequences were found in 86% of the OCs (from the OC first gene) and in 85% of the mCs. A transcription analysis of the promoterless ORF was performed by RT-PCR. This strategy allowed the definition of a specific promoter sequence for all OCs and mCs indicating that all the transcriptional units are preceded by putative promoter sequences (matrix and manually located) and providing evidence for functional gene organization in the M. hyopneumoniae genome. These results shown that the species-specific, position-specific scoring matrix for promoter prediction is effective, further increasing the knowledge of gene organization and transcription initiation in mycoplasmas.

Functional characterization of the putative FAD synthase from Mycoplasma hyopneumoniae.

In bacteria, the biosynthesis of the cofactor flavin adenine dinucleotide (FAD), important in many physiological responses, is catalyzed by the bifunctional enzyme FAD synthase (FADSyn) which converts riboflavin into FAD by both kinase and adenylylation activity. The in silico 3D structure of a putative FADSyn from Mycoplasma hyopneumoniae (MhpFADSyn), the etiological agent of enzootic pneumonia was already reported, nevertheless, the in vitro functional characterization was not yet demonstrated. Our phylogenetic analysis revealed that MhpFADSyn is close related to the bifunctional FADSyn from Corynebacterium ammoniagenes. However, only the domain related to adenylylation was assigned by InterPro database. The activity of MhpFADSyn was evaluated through in vitro enzymatic assays using cell extracts from IPTG-inducible heterologous expression of MhpFADSyn in Escherichia coli. The flavoproteins were analyzed by HPLC and results showed that IPTG-induced cell lysate resulted in the formation of twofold increased amounts of FAD if compared to non IPTG-induced cells. Consumption of riboflavin substrate was also threefold greater in IPTG-induced lysate compared to non IPTG-induced cell extract. Thus, the recombinant MhpFADSyn protein could be associated to FAD biosynthesis. These findings contribute to expand the range of potential drug targets in diseases control and unveil metabolic pathways that could be attribute to mycoplasmas.

Spring Is Coming: Genetic Analyses of the Bud Break Date Locus Reveal Candidate Genes From the Cold Perception Pathway to Dormancy Release in Apple (Malus × domestica Borkh.).

Chilling requirement (CR) for bud dormancy completion determines the time of bud break in apple (Malus × domestica Borkh.). The molecular control of bud dormancy is highly heritable, suggesting a strong genetic control of the trait. An available Infinium II SNP platform for genotyping containing 8,788 single nucleotide polymorphic markers was employed, and linkage maps were constructed in a F1 cross from the low CR M13/91 and the moderate CR cv. Fred Hough. These maps were used to identify quantitative trait loci (QTL) for bud break date as a trait related to dormancy release. A major QTL for bud break was detected at the beginning of linkage group 9 (LG9). This QTL remained stable during seven seasons in two different growing sites. To increase mapping efficiency in detecting contributing genes underlying this QTL, 182 additional SNP markers located at the locus for bud break were used. Combining linkage mapping and structural characterization of the region, the high proportion of the phenotypic variance in the trait explained by the QTL is related to the coincident positioning of Arabidopsis orthologs for ICE1, FLC, and PRE1 protein-coding genes. The proximity of these genes from the most explanatory markers of this QTL for bud break suggests potential genetic additive effects, reinforcing the hypothesis of inter-dependent mechanisms controlling dormancy induction and release in apple trees.

Repetitive Elements in Mycoplasma hyopneumoniae Transcriptional Regulation.

Transcriptional regulation, a multiple-step process, is still poorly understood in the important pig pathogen Mycoplasma hyopneumoniae. Basic motifs like promoters and terminators have already been described, but no other cis-regulatory elements have been found. DNA repeat sequences have been shown to be an interesting potential source of cis-regulatory elements. In this work, a genome-wide search for tandem and palindromic repetitive elements was performed in the intergenic regions of all coding sequences from M. hyopneumoniae strain 7448. Computational analysis demonstrated the presence of 144 tandem repeats and 1,171 palindromic elements. The DNA repeat sequences were distributed within the 5' upstream regions of 86% of transcriptional units of M. hyopneumoniae strain 7448. Comparative analysis between distinct repetitive sequences found in related mycoplasma genomes demonstrated different percentages of conservation among pathogenic and nonpathogenic strains. qPCR assays revealed differential expression among genes showing variable numbers of repetitive elements. In addition, repeats found in 206 genes already described to be differentially regulated under different culture conditions of M. hyopneumoniae strain 232 showed almost 80% conservation in relation to M. hyopneumoniae strain 7448 repeats. Altogether, these findings suggest a potential regulatory role of tandem and palindromic DNA repeats in the M. hyopneumoniae transcriptional profile.