Jo Putterill

GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains

Flowering of Arabidopsis is promoted by long days and delayed by short days. Mutations in the GIGANTEA (GI) gene delay flowering under long days but have little or no effect under short days. We have now isolated the GI gene and show that it encodes a novel, putative membrane protein. By comparing the sequence of the Arabidopsis gene with that of a likely rice orthologue and by sequencing mutant alleles, we identify regions of the GI protein that are likely to be important for its function. We show that GI expression is regulated by the circadian clock with a peak in transcript levels 8–10 h after dawn. The timing, height and duration of this peak are influenced by daylength. We analysed the interactions between GI and the LHY, CCA1 and ELF3 genes, previously shown to affect daylength responses; we show that the rhythmic pattern of GI expression is altered in the elf3, CCA1‐OX and lhy genotypes, and that CCA1 and LHY expression are reduced by gi mutations. Our results are consistent with the idea that GI plays an important role in regulating the expression of flowering time genes during the promotion of flowering by photoperiod.

Multiple Repeats of a Promoter Segment Causes Transcription Factor Autoregulation in Red Apples

Mutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to color phenotypes. Generally, this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin. Here, we describe a rearrangement in the upstream regulatory region of the gene encoding an apple (Malus × domestica) anthocyanin-regulating transcription factor, MYB10. We show that this modification is responsible for increasing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit flesh. This rearrangement is a series of multiple repeats, forming a minisatellite-like structure that comprises five direct tandem repeats of a 23-bp sequence. This MYB10 rearrangement is present in all the red foliage apple varieties and species tested but in none of the white fleshed varieties. Transient assays demonstrated that the 23-bp sequence motif is a target of the MYB10 protein itself, and the number of repeat units correlates with an increase in transactivation by MYB10 protein. We show that the repeat motif is capable of binding MYB10 protein in electrophoretic mobility shift assays. Taken together, these results indicate that an allelic rearrangement in the promoter of MYB10 has generated an autoregulatory locus, and this autoregulation is sufficient to account for the increase in MYB10 transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant.

The Role of Ethylene and Cold Temperature in the Regulation of the Apple POLYGALACTURONASE1 Gene and Fruit Softening

Fruit softening in apple (Malus × domestica) is associated with an increase in the ripening hormone ethylene. Here, we show that in cv Royal Gala apples that have the ethylene biosynthetic gene ACC OXIDASE1 suppressed, a cold treatment preconditions the apples to soften independently of added ethylene. When a cold treatment is followed by an ethylene treatment, a more rapid softening occurs than in apples that have not had a cold treatment. Apple fruit softening has been associated with the increase in the expression of cell wall hydrolase genes. One such gene, POLYGALACTURONASE1 (PG1), increases in expression both with ethylene and following a cold treatment. Transcriptional regulation of PG1 through the ethylene pathway is likely to be through an ETHYLENE-INSENSITIVE3-like transcription factor, which increases in expression during apple fruit development and transactivates the PG1 promoter in transient assays in the presence of ethylene. A cold-related gene that resembles a COLD BINDING FACTOR (CBF) class of gene also transactivates the PG1 promoter. The transactivation by the CBF-like gene is greatly enhanced by the addition of exogenous ethylene. These observations give a possible molecular mechanism for the cold- and ethylene-regulated control of fruit softening and suggest that either these two pathways act independently and synergistically with each other or cold enhances the ethylene response such that background levels of ethylene in the ethylene-suppressed apples is sufficient to induce fruit softening in apples.

Analysis of genetically modified red‐fleshed apples reveals effects on growth and consumer attributes

Consumers of whole foods, such as fruits, demand consistent high quality and seek varieties with enhanced health properties, convenience or novel taste. We have raised the polyphenolic content of apple by genetic engineering of the anthocyanin pathway using the apple transcription factor MYB10. These apples have very high concentrations of foliar, flower and fruit anthocyanins, especially in the fruit peel. Independent lines were examined for impacts on tree growth, photosynthesis and fruit characteristics. Fruit were analysed for changes in metabolite and transcript levels. Fruit were also used in taste trials to study the consumer perception of such a novel apple. No negative taste attributes were associated with the elevated anthocyanins. Modification with this one gene provides near isogenic material and allows us to examine the effects on an established cultivar, with a view to enhancing consumer appeal independently of other fruit qualities.

Flowering on time: genes that regulate the floral transition: Workshop on the molecular basis of flowering time control

The workshop was held at the John Innes Centre, Norwich, UK, July 11–14, 2001. http://www.jic.bbsrc.ac.uk/events/embo/. Workshop Administrator: Dee Rawsthorne, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, Norfolk, UK (Tel: +44 1603 450527; Fax: +44 1603 450025; E‐mail: dee.rawsthorne@bbsrc.ac.uk). ![][1] Successful sexual reproduction in plants depends on the recognition of favourable environmental conditions and the integration of that information with endogenous developmental cues. Flowering in higher plants involves the transition of a vegetative meristem, producing leaves and stems, into a floral meristem, producing flowers. Most of our understanding of the regulation of the floral transition has been developed in the past 10 years using molecular genetic approaches in the model plant Arabidopsis thaliana , a rosette plant in which the initiation of flowering is followed by the elongation of the main stem. The two main environmental factors that promote flowering in Arabidopsis are long days (Figure 1) and exposure to low temperatures (vernalization). Genetic and physiological analysis of flowering time in Arabidopsis has led to the identification of a large number of flowering‐time genes (>80) that regulate flowering time in response to environmental and endogenous cues (reviewed by Simpson et al ., 1999). Regulation occurs through a complex network of genetic pathways, with two main pathways mediating environmental responses (the long‐day and vernalization pathways) and two pathways that function independently of environmental cues: the autonomous pathway, which promotes flowering under all conditions, and the gibberellin (GA) pathway, which is needed for flowering under non‐inductive short‐day conditions. These pathways converge in the induction of floral meristem identity genes and the floral transition (Figure 2). Many flowering‐time genes have been cloned recently, and the primary aim of the EMBO workshop in Norwich, and this report, was to highlight the most recent advances in our understanding of the molecular mechanisms regulating … [1]: /embed/graphic-1.gif

T-DNA tagging of a flowering-time gene and improved gene transfer by in planta transformation of Arabidopsis

Gene tagging with insertional mutagens greatly facilitates the isolation of novel genes. A new collection of Arabidopsis T-DNA tag insertion lines (n=2165) was generated by in planta transformation. Whole plants were vacuum-infiltrated in a suspension of Agrobacterium carrying the pGKB5 tagging vector. The efficiency of transformation increased with addition of the surfactant Silwet L-77 (0.005% v/v) to the Agrobacterium suspension. Visual screens of the T-DNA lines identified two mutants with floral defects. Allelism tests suggested that a mutation in the GIGANTEA gene was responsible for the late-flowering phenotype of one of the mutants. Linkage analysis indicated that the GIGANTEA gene was tagged in this mutant.

Ethylene Regulates Apple (Malus × domestica) Fruit Softening Through a Dose × Time-Dependent Mechanism and Through Differential Sensitivities and Dependencies of Cell Wall-Modifying Genes

In fleshy fruit species that have a strong requirement for ethylene to ripen, ethylene is synthesized autocatalytically, producing increasing concentrations as the fruits ripen. Apple fruit with the ACC OXIDASE 1 (ACO1) gene suppressed cannot produce ethylene autocatalytically at ripening. Using these apple lines, an ethylene sensitivity dependency model was previously proposed, with traits such as softening showing a high dependency for ethylene as well as low sensitivity. In this study, it is shown that the molecular control of fruit softening is a complex process, with different cell wall-related genes being independently regulated and exhibiting differential sensitivities to and dependencies on ethylene at the transcriptional level. This regulation is controlled through a dose × time mechanism, which results in a temporal transcriptional response that would allow for progressive cell wall disassembly and thus softening. This research builds on the sensitivity dependency model and shows that ethylene-dependent traits can progress over time to the same degree with lower levels of ethylene. This suggests that a developmental clock measuring cumulative ethylene controls the fruit ripening process.

Apple EIN3 BINDING F-box 1 inhibits the activity of three apple EIN3-like transcription factors.

Mining the apple genome identified four EIN3 BINDING F-BOX genes, two developmentally regulated and two constitutively expressed. One (EBF1) was found to reduce the activity of three apple EIN3-like genes

Deconstructing the weed

Abstract Arabidopsis Protocols edited by J.M. Martinez-Zapater and J. Salinas Humana Press, 1998. £60.00/

It's time to flower: the genetic control of flowering time

79.50 hbk (xvi+440 pages) ISBN 0 89603 391 0