Karen J. Halliday

Cold and light control seed germination through the bHLH transcription factor SPATULA

BACKGROUND Plants integrate signals from the environment and use these to modify the timing of development according to seasonal cues. Seed germination is a key example of this phenomenon and in Arabidopsis is promoted by the synergistic interaction of light and low temperatures in dormant seeds. This signaling pathway is known to converge on the regulation of the gibberellin (GA) biosynthetic genes GA3 oxidase (GA3ox), whose expression is transcriptionally induced by light and cold in imbibed seeds. However, the molecular basis of this response has until now been unknown. RESULTS Here we show that the bHLH transcription factor SPATULA is a light-stable repressor of seed germination and mediates the germination response to temperature. Furthermore, SPT is required in dormant seeds for maintaining the repression of GA3ox transcription. We also show that the related protein PIL5 represses seed germination and GA3ox expression in the dark. CONCLUSIONS We conclude that SPT and PIL5 form part of a regulatory network coupling seed germination and GA3ox expression to light and temperature signaling in the seed.

The clock gene circuit in Arabidopsis includes a repressilator with additional feedback loops

Circadian clocks synchronise biological processes with the day/night cycle, using molecular mechanisms that include interlocked, transcriptional feedback loops. Recent experiments identified the evening complex (EC) as a repressor that can be essential for gene expression rhythms in plants. Integrating the EC components in this role significantly alters our mechanistic, mathematical model of the clock gene circuit. Negative autoregulation of the EC genes constitutes the clocks evening loop, replacing the hypothetical component Y. The EC explains our earlier conjecture that the morning gene PSEUDO‐RESPONSE REGULATOR 9 was repressed by an evening gene, previously identified with TIMING OF CAB EXPRESSION1 (TOC1). Our computational analysis suggests that TOC1 is a repressor of the morning genes LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK ASSOCIATED1 rather than an activator as first conceived. This removes the necessity for the unknown component X (or TOC1mod) from previous clock models. As well as matching timeseries and phase‐response data, the model provides a new conceptual framework for the plant clock that includes a three‐component repressilator circuit in its complex structure.

Phytochromes B, D, and E Act Redundantly to Control Multiple Physiological Responses in Arabidopsis

Phytochrome-mediated perception of the ratio of red to far-red wavelengths in the ambient light environment is fundamental to plant growth and development. Such monitoring enables plants to detect neighboring vegetation and initiate avoidance responses, thus conferring considerable selective advantage. The shade avoidance syndrome in plants is characterized by elongation growth and early flowering, responses that are fully induced by end-of-day far-red light treatments. Elucidating the roles of individual phytochromes in mediating responses to red to far-red has however always been confounded by synergistic and mutually antagonistic coactions between family members. The creation of triple and quadruple mutants in Arabidopsis, deficient in multiple phytochromes, has revealed functional redundancy between phyB, D, and E in controlling flowering time, leaf development, and regulation of the homeobox gene,ATHB-2. In addition, mutant analysis suggests a possible novel role for phyC in suppressing ATHB-2 transcription in the light.

β-AMYLASE4, a Noncatalytic Protein Required for Starch Breakdown, Acts Upstream of Three Active β-Amylases in Arabidopsis Chloroplasts

This work investigated the roles of β-amylases in the breakdown of leaf starch. Of the nine β-amylase (BAM)–like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable β-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized β-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active β-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total β-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that β-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.

Phytochrome B and at Least One Other Phytochrome Mediate the Accelerated Flowering Response of Arabidopsis thaliana L. to Low Red/Far-Red Ratio.

We have investigated the involvement of phytochrome B in the early-flowering response of Arabidopsis thaliana L. seedlings to low red:far-red (R/FR) ratio light conditions. The phytochrome B-deficient hy3 (phyB) mutant is early flowering, and in this regard it resembles the shade-avoidance phenotype of its isogenic wild type. Seedlings carrying the hy2 mutation, resulting in a deficiency of phytochrome chromophore and hence of active phytochromes, also flower earlier than wild-type plants. Whereas hy3 or hy2 seedlings show only a slight acceleration of flowering in response to low R/FR ratio, seedlings that are doubly homozygous for both mutations flower earlier than seedlings carrying either phytochrome-related mutation alone. This additive effect clearly indicates the involvement of one or more phytochrome species in addition to phytochrome B in the flowering response as well as indicating the presence of some functional phytochrome B in hy2 seedlings. Seedlings that are homozygous for the hy3 mutation and one of the fca, fwa, or co late-flowering mutations display a pronounced early-flowering response to low R/FR ratio. A similar response to low R/FR ratio is displayed by seedlings doubly homozygous for the hy2 mutation and any one of the late-flowering mutations. Thus, placing the hy3 or hy2 mutations into a late-flowering background has the effect of uncovering a flowering response to low R/FR ratio. Seedlings that are triply homozygous for the hy3, hy2 mutations and a late-flowering mutation flower earlier than the double mutants and do not respond to low R/FR ratio. Thus, the observed flowering responses to low R/FR ratio in phytochrome B-deficient mutants can be attributed to the action of at least one other phytochrome species.

The TIME FOR COFFEE gene maintains the amplitude and timing of Arabidopsis circadian clocks

Plants synchronize developmental and metabolic processes with the earths 24-h rotation through the integration of circadian rhythms and responses to light. We characterize the time for coffee (tic) mutant that disrupts circadian gating, photoperiodism, and multiple circadian rhythms, with differential effects among rhythms. TIC is distinct in physiological functions and genetic map position from other rhythm mutants and their homologous loci. Detailed rhythm analysis shows that the chlorophyll a/b-binding protein gene expression rhythm requires TIC function in the mid to late subjective night, when human activity may require coffee, in contrast to the function of EARLY-FLOWERING3 (ELF3) in the late day to early night. tic mutants misexpress genes that are thought to be critical for circadian timing, consistent with our functional analysis. Thus, we identify TIC as a regulator of the clock gene circuit. In contrast to tic and elf3 single mutants, tic elf3 double mutants are completely arrhythmic. Even the robust circadian clock of plants cannot function with defects at two different phases.

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

Changes in Photoperiod or Temperature Alter the Functional Relationships between Phytochromes and Reveal Roles for phyD and phyE

The phytochromes are one of the means via which plants obtain information about their immediate environment and the changing seasons. Phytochromes have important roles in developmental events such as the switch to flowering, the timing of which can be crucial for the reproductive success of the plant. Analysis of phyBmutants has revealed that phyB plays a major role in this process. We have recently shown, however, that the flowering phenotype of thephyB monogenic mutant is temperature dependent. A modest reduction in temperature to 16°C was sufficient to abolish thephyB mutant early-flowering phenotype present at 22°C. Using mutants null for one or more phytochrome species, we have now shown that phyA, phyD, and phyE, play greater roles with respect to phyB in the control of flowering under cooler conditions. This change in the relative contributions of individual phytochromes appears to be important for maintaining control of flowering in response to modest alterations in ambient temperature. We demonstrate that changes in ambient temperature or photoperiod can alter the hierarchy and/or the functional relationships between phytochrome species. These experiments reveal new roles for phyD and phyE and provide valuable insights into how the phytochromes help to maintain development in the natural environment.

Light and plant development

Part 1: Photoreceptors. Chapter 1. Phytochromes. Andreas Hiltbrunner, Ferenc Nagy and Eberhard Schafer. Albert-Ludwigs-Universitat Freiburg, Institute of Biology II/ Botany, Schanzlestr. 1, 79104 Freiburg, Germany, and Biological Research Centre, Institute of Plant Biology, P. O. Box 521, 6701 Szeged, Hungary. Chapter 2. Cryptochromes. Alfred Batschauer, Roopa Banerjee and Richard Pokorny. Philipps-University, Biology-Plant Physiology Karl-von-Frisch-Str. 8 35032 Marburg Germany. Chapter 3. Phototropins and Other Lov-Containing Proteins. John M. Christie. Plant Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. Part 2: Photoreceptor Signal Transduction. Chapter 4. Phytochrome Interacting Factors. Peter H. Quail. UC Berkeley, Plant Gene Expression Center, United States Department of Agriculture (USDA), 800 Buchanan Street, Albany, California 94710, USA. Chapter 5. Phosphorylation/De-phosphorylation in Photoreceptor Signalling. Catherine Lillo(1), Trudie Allen(2) and Simon Geir Moller(1,2,3). (1) Department of Mathematics and Natural Sciences, University of Stavanger, 4036 Stavanger, Norway. (2) Department of Biology, University of Leicester, Leicester LE1 7RH, UK. (3) Laboratory of Plant Molecular Biology, Rockefeller University, New York, New York 10021-3699, USA. Chapter 6. The Role of Ubiquitin/Proteasome-Mediated Proteolysis in Photoreceptor Action. Suhua Feng and Xing Wang Deng. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, 06520-8104, USA. Chapter 7. UV-B Perception and Signal Transduction. Gareth I. Jenkins and Bobby A. Brown. Plant Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Bower Building, University of Glasgow, Glasgow G12 8QQ, UK... Part 3: Physiological Responses. Chapter 8. Photocontrol of Flowering. Dr Paul Devlin. School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK. Chapter 9. Red: Far-red Ratio Perception and Shade Avoidance. Keara A. Franklin and Garry C. Whitelam. Department of Biology, University of Leicester, LE1 7RH, UK. Chapter 10. Photoreceptor Interactions with Other Signals. Eve-Marie Josse and Karen J. Halliday. School of Biological Sciences, The University of Edinburgh, Daniel Rutherford Building, The Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK. Part 4: Applied Aspects of Photomorphogenesis. Chapter 11. Photoreceptor Biotechnology. Matthew Hudson. Department of Crop Sciences, University of Illinois, Urbana, Illinois 61801, USA. Chapter 12. Light Quality Manipulation by Horticulture Industry. Professor Nihal C. Rajapakse and Dr Yosepha Shahak. Department of Horticulture, Clemson University, 168 Poole Agricultural Center, Box 340319, Clemson, SC 29634-0319, USA, and Department of Fruit Tree Sciences, Agricultural Research Organization, The Volcani Center, P.O.Box 6, Bet Dagan 50250, Israel

The HY5-PIF Regulatory Module Coordinates Light and Temperature Control of Photosynthetic Gene Transcription

The ability to interpret daily and seasonal alterations in light and temperature signals is essential for plant survival. This is particularly important during seedling establishment when the phytochrome photoreceptors activate photosynthetic pigment production for photoautotrophic growth. Phytochromes accomplish this partly through the suppression of PHYTOCHROME INTERACTING FACTORS (PIFs), negative regulators of chlorophyll and carotenoid biosynthesis. While the bZIP transcription factor LONG HYPOCOTYL 5 (HY5), a potent PIF antagonist, promotes photosynthetic pigment accumulation in response to light. Here we demonstrate that by directly targeting a common promoter cis-element (G-box), HY5 and PIFs form a dynamic activation-suppression transcriptional module responsive to light and temperature cues. This antagonistic regulatory module provides a simple, direct mechanism through which environmental change can redirect transcriptional control of genes required for photosynthesis and photoprotection. In the regulation of photopigment biosynthesis genes, HY5 and PIFs do not operate alone, but with the circadian clock. However, sudden changes in light or temperature conditions can trigger changes in HY5 and PIFs abundance that adjust the expression of common target genes to optimise photosynthetic performance and growth.