A SNP-based consensus genetic map for synteny-based trait targeting in faba bean (Vicia faba L.).

Faba bean (Vicia faba L.) is a globally important nitrogen-fixing legume, which is widely grown in a diverse range of environments. In this work, we mine and validate a set of 845 SNPs from the aligned transcriptomes of two contrasting inbred lines. Each V. faba SNP is assigned by BLAST analysis to a single Medicago orthologue. This set of syntenically anchored polymorphisms were then validated as individual KASP assays, classified according to their informativeness and performance on a panel of 37 inbred lines, and the best performing 757 markers used to genotype six mapping populations. The six resulting linkage maps were merged into a single consensus map on which 687 SNPs were placed on six linkage groups, each presumed to correspond to one of the six V. faba chromosomes. This sequence-based consensus map was used to explore synteny with the most closely related crop species, lentil and the most closely related fully sequenced genome, Medicago. Large tracts of uninterrupted colinearity were found between faba bean and Medicago, making it relatively straightforward to predict gene content and order in mapped genetic interval. As a demonstration of this, we mapped a flower colour gene to a 2-cM interval of Vf chromosome 2 which was highly colinear with Mt3. The obvious candidate gene from 78 gene models in the collinear Medicago chromosome segment was the previously characterized MtWD40-1 gene controlling anthocyanin production in Medicago and resequencing of the Vf orthologue showed a putative causative deletion of the entire 5' end of the gene.

The Arabidopsis plastid-signalling mutant gun1 (genomes uncoupled1) shows altered sensitivity to sucrose and abscisic acid and alterations in early seedling development.

Developing seedlings of the Arabidopsis gun1 (genomes uncoupled1) mutant, which is defective in retrograde plastid-to-nucleus signalling, show several previously unrecognized mutant phenotypes. gun1 seedlings accumulated less anthocyanin than wild-type seedlings when grown in the presence of 2% (w/v) sucrose, due to lower amounts of transcripts of early anthocyanin biosynthesis genes in gun1. Norflurazon and lincomycin, which induce retrograde signalling, further decreased the anthocyanin content of sucrose-treated seedlings, and altered the temporal pattern of anthocyanin accumulation. Lincomycin treatment altered the spatial pattern of sucrose-induced anthocyanin accumulation, suggesting that plastids provide information for the regulation of anthocyanin biosynthesis in Arabidopsis seedlings. The temporal pattern of accumulation of LHCB1 transcripts differed between wild-type and gun1 seedlings, and gun1 seedlings were more sensitive to sucrose suppression of LHCB1 transcript accumulation than wild-type seedlings. Growth and development of gun1 seedlings was more sensitive to exogenous 2% sucrose than wild-type seedlings and, in the presence of lincomycin, cotyledon expansion was enhanced in gun1 seedlings compared to the wild type. gun1 seedlings were more sensitive than wild-type seedlings to the inhibition of seedling growth and development by abscisic acid. These observations clearly implicate GUN1 and plastid signalling in the regulation of seedling development and anthocyanin biosynthesis, and indicate a complex interplay between sucrose and plastid signalling pathways.

Bioinformatics methods to predict protein structure and function. A practical approach.

Protein structure prediction by using bioinformatics can involve sequence similarity searches, multiple sequence alignments, identification and characterization of domains, secondary structure prediction, solvent accessibility prediction, automatic protein fold recognition, constructing three-dimensional models to atomic detail, and model validation. Not all protein structure prediction projects involve the use of all these techniques. A central part of a typical protein structure prediction is the identification of a suitable structural target from which to extrapolate three-dimensional information for a query sequence. The way in which this is done defines three types of projects. The first involves the use of standard and well-understood techniques. If a structural template remains elusive, a second approach using nontrivial methods is required. If a target fold cannot be reliably identified because inconsistent results have been obtained from nontrivial data analyses, the project falls into the third type of project and will be virtually impossible to complete with any degree of reliability. In this article, a set of protocols to predict protein structure from sequence is presented and distinctions among the three types of project are given. These methods, if used appropriately, can provide valuable indicators of protein structure and function.

Timing the switch to phototrophic growth: a possible role of GUN1.

In young Arabidopsis seedlings, retrograde signalling from plastids regulates the expression of photosynthesis-associated nuclear genes in response to the developmental and functional state of the chloroplasts. The chloroplast-located PPR protein GUN1 is required for signalling following disruption of plastid protein synthesis early in seedling development before full photosynthetic competence has been achieved. Recently we showed that sucrose repression and the correct temporal expression of LHCB1, encoding a light-harvesting chlorophyll protein associated with photosystem II, are perturbed in gun1 mutant seedlings. ( 1) Additionally, we demonstrated that in gun1 seedlings anthocyanin accumulation and the expression of the "early" anthocyanin-biosynthesis genes is perturbed. Early seedling development, predominantly at the stage of hypocotyl elongation and cotyledon expansion, is also affected in gun1 seedlings in response to sucrose, ABA and disruption of plastid protein synthesis by lincomycin. These findings indicate a central role for GUN1 in plastid, sucrose and ABA signalling in early seedling development. 

A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation.

BACKGROUND: The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7-10 d and high seedling density and fungal contamination may result in failure to recover transformants. RESULTS: A method for identifying transformed seedlings in as little as 3.25 d has been developed. Arabidopsis T1 seeds obtained after floral dip transformation are plated on 1% agar containing MS medium and kanamycin, phosphinothricin or hygromycin B, as appropriate. After a 2-d stratification period, seeds are subjected to a regime of 4-6 h light, 48 h dark and 24 h light (3.25 d). Kanamycin-resistant and phosphinothricin-resistant seedlings are easily distinguished from non-resistant seedlings by green expanded cotyledons whereas non-resistant seedlings have pale unexpanded cotyledons. Seedlings grown on hygromycin B differ from those grown on kanamycin and phosphinothricin as both resistant and non-resistant seedlings are green. However, hygromycin B-resistant seedlings are easily identified as they have long hypocotyls (0.8-1.0 cm) whereas non-resistant seedlings have short hypocotyls (0.2-0.4 cm). CONCLUSION: The method presented here is an improvement on current selection methods as it allows quicker identification of transformed seedlings: transformed seedlings are easily discernable from non-transformants in as little as 3.25 d in comparison to the 7-10 d required for selection using current protocols.

Characterisation and expression of the pathway from UDP-glucose to UDP-xylose in differentiating tobacco tissue.

The pathway from UDP-glucose to UDP-xylose has been characterised in differentiating tobacco tissue. A xylogenic suspension cell culture of tobacco has been used as a source for the purification of the enzymes responsible for the oxidation of UDP-glucose to UDP-glucuronic acid and its subsequent decarboxylation to UDP-xylose. Protein purification and transcriptional studies show that two possible candidates can contribute to the first reaction. Most of the enzyme activity in the cultured cells could be accounted for by a protein with an Mr of 43 kDa which had dual specificity for UDP-glucose and ethanol. The cognate cDNA, with similarity to alcohol dehydrogenases (NtADH2) was expressed in E. coli to confirm the dual specificity. A second UDP-glucose dehydrogenase, corresponding to the monospecific form, ubiquitous amongst plants and animals, could not be purified from the tobacco cell cultures. However, two cDNAs were cloned with high similarity to the family of UDP-glucose dehydrogenases. Transcripts of both types of dehydrogenase showed highest expression in tissues undergoing secondary wall synthesis. The UDP-glucuronate decarboxylase was purified as polypeptides of Mr 87 and 40 kDa. Peptide fingerprinting of the latter polypeptide identified it as a form of UDP-glucuronate decarboxylase and functionality was established by expressing the cognate cDNA in E. coli. Expression of 40 kDa polypeptide and its corresponding mRNA was also found to be highest in tissues associated with secondary wall formation.

AP1 genes in Fugu indicate a divergent transcriptional control to that of mammals.

The draft genomic sequence of the Japanese puffer fish, Fugu rubripes, has now been announced. This is the first complete sequence of a teleost fish and the second available vertebrate sequence, the first being that of human. For the first time, whole-genome comparisons between two vertebrates can be undertaken. Early analysis has suggested that there may be surprising differences in gene regulation between human and fish. In mammals, a gene commonly has several functions, and this may not always be the case in fish. Many gene families comprise more members in fish than they do in mammals, possibly because each fish gene has evolved an individual function. Complexities of gene regulation in mammals has hampered studies of all biological processes from cell proliferation to cell death. Determining the activities of the AP1 transcription factor proteins has been non-trivial. The AP1 complex typically comprises two proteins, a Jun (c-Jun, JunB, and JunD) and a Fos (c-Fos, FosB, Fra1, and Fra2). These proteins can form both homodimers and heterodimers among-themselves and can interact with additional proteins; thus, dissecting their individual roles has been difficult. We have determined that Fugu has more Jun and Fos genes than mammals, and if each proves to have a separate function, then addressing the roles of the individual AP1 proteins in Fugu may be simpler than in human.

Molecular characterisation of the SAND protein family: a study based on comparative genomics, structural bioinformatics and phylogeny.

The activities of vertebrate lysosomes are critical to many essential cellular processes. The yeast vacuole is analogous to the mammalian lysosome and is used as a tool to gain insights into vesicle mediated vacuolar/lysosome transport. The protein SAND, which does not contain a SAND domain (PFAM accession number PF01342), has recently been shown to function at the tethering/docking stage of vacuole fusion as a critical component of the vacuole SNARE complex. In this publication we have identified SAND in diverse eukaryotes, from single celled organisms such as the yeasts to complex multi-cellular chordates such as mammals. We have demonstrated subfamily divisions in the SAND proteins and show that in vertebrates, a duplication event gave rise to two SAND sequences. This duplication appears to have occurred during early vertebrate evolution and conceivably with the evolution of lysosomes. Using bioinformatics we predict a secondary structure, solvent accessibility profile and protein fold for the SAND proteins and determine conserved sequence motifs, present in all SAND proteins and those that are specific to subsets. A comprehensive evaluation of yeast and human functional studies in conjunction with our in silico analysis has identified potential roles for some of these motifs.