Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.)

The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT–PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain

SHINE Transcription Factors Act Redundantly to Pattern the Archetypal Surface of Arabidopsis Flower Organs

Floral organs display tremendous variation in their exterior that is essential for organogenesis and the interaction with the environment. This diversity in surface characteristics is largely dependent on the composition and structure of their coating cuticular layer. To date, mechanisms of flower organ initiation and identity have been studied extensively, while little is known regarding the regulation of flower organs surface formation, cuticle composition, and its developmental significance. Using a synthetic microRNA approach to simultaneously silence the three SHINE (SHN) clade members, we revealed that these transcription factors act redundantly to shape the surface and morphology of Arabidopsis flowers. It appears that SHNs regulate floral organs' epidermal cell elongation and decoration with nanoridges, particularly in petals. Reduced activity of SHN transcription factors results in floral organs' fusion and earlier abscission that is accompanied by a decrease in cutin load and modified cell wall properties. SHN transcription factors possess target genes within four cutin- and suberin-associated protein families including, CYP86A cytochrome P450s, fatty acyl-CoA reductases, GSDL-motif lipases, and BODYGUARD1-like proteins. The results suggest that alongside controlling cuticular lipids metabolism, SHNs act to modify the epidermis cell wall through altering pectin metabolism and structural proteins. We also provide evidence that surface formation in petals and other floral organs during their growth and elongation or in abscission and dehiscence through SHNs is partially mediated by gibberellin and the DELLA signaling cascade. This study therefore demonstrates the need for a defined composition and structure of the cuticle and cell wall in order to form the archetypal features of floral organs surfaces and control their cell-to-cell separation processes. Furthermore, it will promote future investigation into the relation between the regulation of organ surface patterning and the broader control of flower development and biological functions

Development and Application of Molecular Methods for the Study of Polymyxa betae

A simple method for cloning Polymyxa betae DNA from infected sugar-beet roots has been developed and used to isolate a multi-copy genomic DNA fragment, pPbKES-1, located on at least four different P. betae chromosomes. This fragment proved to be a good probe for detecting the fungus by Southern/dot blot hybridisation, and a good source of PCR primer sequences for the specific amplification of P.betae DNA. PCR is now used routinely for detecting P.betae in sugar-beet roots and in experiments investigating the epidemiology of the fungus. Single copy DNA probes have also been isolated and are beginning to provide useful information on variability at the molecular leve

Specific polyclonal antibodies for the obligate plant parasite Polymyxa - a targeted recombinant DNA approach

Highly specific rabbit polyclonal antibodies for the obligate sugar-beet root parasite, Polymyxa betae, were produced using a novel recombinant DNA approach. Parasite cDNA was selectively isolated from infected roots, expressed in vitro, and the purified protein used to raise antibodies. This produced clean, precisely targeted antibodies, and allowed for rigorous screening of candidate genes and their products at the molecular level prior to animal immunization. This approach selects for genes whose products are highly expressed by the parasite in planta, and five such candidate genes from Polymyxa betae were identified and cloned. Polyclonal antiserum developed using the product of one such gene was found to react specifically with P. betae in sugar-beet roots and with the closely related Polymyxa graminis in barley roots, and to cross-react with Plasmodiophora brassicae in cabbage roots, without the need for further purification. No cross-reaction was detected with protein extracts from potato roots infected by the plasmodiophoromycete Spongospora subterranea. In all cases, there was no interaction with proteins from host plants, or from other microorganisms found in association with uninoculated sugar-beet, barley, cabbage and potato rootsPeer reviewe

Flowering Time

Adaptation genes have a major role to play in the response of plants to environmental changes. Flowering time is a key adaptive trait, responding to environmental and endogenous signals that ensure reproductive growth and devel- opment occurs under favorable environmental conditions. Under a climate change scenario, temperature and water conditions are forecast to change and/or fluctuate, while photoperiods will remain constant at any given latitude. By assessing the current knowledge of the flowering-time pathways in both model (Arabidopsis thaliana) and key cereal (rice, barley, wheat, maize), temperate forage and biofuel grasses (perennial ryegrass, Miscanthus, sugarcane), root (sugar beet), and tree (poplar) crop species, it is possible to define key breeding targets for promoting adaptation and yield stability under future climatic conditions. In Arabidopsis, there are four pathways controlling flowering time, and the genetic and/or epigenetic control of many of the steps in these pathways has been well characterized. Despite A.R. Bentley • I.J. Mackay • E. Mutasa-Go ¨ttgens • J. Cockram (*) The John Bingham Laboratory, NIAB, Huntingdon Road, Cambridge CB3 0LE, UK e-mail: [email protected] E.F. Jensen • I.P. Armstead • C. Hayes • D. Thorogood • A. Lovatt Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth SY23 3EB, UK H. Ho ¨nicka • M. Fladung Johann Heinrich von Thu ¨nen Institute, Institute of Forest Genetics, Sieker Landstr. 2, 22927 Grosshansdorf, Germany K. Hori • M. Yano National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan J.E. Mullet Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA R. Morris • N. Pullen Computational and Systems Biology Department, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK C. Kole (ed.), Genomics and Breeding for Climate-Resilient Crops, Vol. 2, DOI 10.1007/978-3-642-37048-9_1, © Springer-Verlag Berlin Heidelberg 2013 1 this, even in this model species, there is little published information on the molecu- lar basis of adaptation to the environment. In contrast, in crop and tree species, flowering time has been continually selected, either directly or indirectly as breeders and growers have selected the material that best suits a particular location. Understanding the genetic basis of this adaptive selection is now being facilitated via cloning of major genes, the mapping of QTL, and the use of marker-assisted breeding for specific flowering targets. In crop species where the genetic basis of flowering is not well understood (i.e., in the emerging biofuel grass, Miscanthus), such work is in its infancy. In cases where the genetic basis is well established, however, there are still grounds for important discovery, via new and emerging methods for mapping and selecting for flowering-time traits (i.e., QTL mapping in MAGIC populations, RABID selection), as well as methods for creating new genetic combinations with potentially novel flowering-time phenotypes (i.e., via targeted mutagenesis). In the future it is likely that computational modeling approaches which incorporate gene networks and the range of phenological response to measurable environmental conditions will play a central role in predicting the resilience of crop and tree species under climate change scenarios.Peer reviewe

Gibberellin as a factor in floral regulatory networks

Gibberellins (GAs) function not only to promote the growth of plant organs, but also to induce phase transitions during development. Their involvement in flower initiation in long-day (LD) and biennial plants is well established and there is growing insight into the mechanisms by which floral induction is achieved. The extent to which GAs mediate the photoperiodic stimulus to flowering in LD plants is, with a few exceptions, less clear. Despite evidence for photoperiod-enhanced GA biosynthesis in leaves of many LD plants, through up-regulation of GA 20-oxidase gene expression, a function for GAs as transmitted signals from leaves to apices in response to LD has been demonstrated only in Lolium species. In Arabidopsis thaliana, as one of four quantitative floral pathways, GA signalling has a relatively minor influence on flowering time in LD, while in SD, in the absence of the photoperiod flowering pathway, the GA pathway assumes a major role and becomes obligatory. Gibberellins promote flowering in Arabidopsis through the activation of genes encoding the floral integrators SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), LEAFY (LFY), and FLOWERING LOCUS T (FT) in the inflorescence and floral meristems, and in leaves, respectively. Although GA signalling is not required for floral organ specification, it is essential for the normal growth and development of these organs. The sites of GA production and action within flowers, and the signalling pathways involved are beginning to be revealed.Peer reviewe

Modification of gibberellin signalling (metabolism & signal transduction) in sugar beet: analysis of potential targets for crop improvement

Sugar beet, Beta vulgaris spp. vulgaris is a biennial long day plant with an obligate requirement for vernalization (prolonged exposure to low temperature). As a spring crop in temperate European climates, it is vulnerable to vernalization-induced premature bolting and flowering, resulting in reduced crop yield and quality. Gibberellins (GAs) play important roles in key physiological processes including stem elongation (bolting) and flowering and are, therefore, potential targets for controlling reproductive growth in sugar beet. We show that the BvGA20ox gene, which encodes an enzyme necessary for GA biosynthesis, was transcriptionally activated in apices of sugar beet plants after vernalization and that GA metabolism can be manipulated to delay bolting in vernalized plants. We demonstrate that down-regulation of GA responses by transformation with the Arabidopsis thaliana gai gene (which represses GA signalling), under its own promoter (pgai::gai) or deactivation of GA by over-expression of the Phaseolus coccineus (bean) GA2ox1 gene, which inactivates GA, increased the required post vernalization thermal time (an accurate and stable measure of physiological time), to bolt by similar to 300A degrees Cd. This resulted in agronomically significant bolting time delays of similar to 2 weeks and 3 weeks in the pgai::gai and 35S::PcGA2ox1 plants, respectively. Our data represent the first transgenic sugar beet model to (1) show that GA signalling can be used to improve crops by manipulation of the transition to reproductive growth; and (2) provide evidence that GA is required for seed set in sugar beet.Peer reviewe