Robert A. Sharrock

Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation

Two genomic clones (λUbi-1 and λUbi-2) encoding the highly conserved 76 amino acid protein ubiquitin have been isolated from maize. Sequence analysis shows that both genes contain seven contiguous direct repeats of the protein coding region in a polyprotein conformation. The deduced amino acid sequence of all 14 repeats is identical and is the same as for other plant ubiquitins. The use of transcript-specific oligonucleotide probes shows that Ubi-1 and Ubi-2 are expressed constitutively at 25°C but are inducible to higher levels at elevated temperatures in maize seedlings. Both genes contain an intron in the 5′ untranslated region which is inefficiently processed following a brief, severe heat shock. The transcription start site of Ubi-1 has been determined and a transcriptional fusion of 0.9 kb of the 5′ flanking region and the entire 5′ untranslated sequence of Ubi-1 with the coding sequence of the gene encoding the reporter molecule chloramphenicol acetyl transferase (CAT) has been constructed (pUBI-CAT). CAT assays of extracts of protoplasts electroporated with this construct show that the ubiquitin gene fragment confers a high level of CAT expression in maize and other monocot protoplasts but not in protoplasts of the dicot tobacco. Expression from the Ubi-1 promoter of pUBI-CAT yields more than a 10-fold higher level of CAT activity in maize protoplasts than expression from the widely used cauliflower mosaic virus 35S promoter of a 35S-CAT construct. Conversely, in tobacco protoplasts CAT activity from transcription of pUBI-CAT is less than one tenth of the level from p35S-CAT.

The phytochrome apoprotein family inArabidopsis is encoded by five genes: the sequences and expression ofPHYD andPHYE

Two novelArabidopsis phytochrome genes,PHYD andPHYE, are described and evidence is presented that, together with the previously describedPHYA, PHYB andPHYC genes, the primary structures of the complete phytochrome family of this plant are now known. ThePHYD- andPHYE-encoded proteins are of similar size to the other phytochrome apoproteins and show sequence similarity along their entire lengths. Hence, red/far-red light sensing in higher plants is mediated by a diverse but structurally conserved group of soluble photoreceptors. The proteins encoded by thePHYD andPHYE genes are more closely related to phytochrome B than to phytochromes A or C, indicating that the evolution of thePHY gene family inArabidopsis includes an expansion of a PHYB-related subgroup. The PHYB and PHYD phytochromes show greater than 80% amino acid sequence identity but the phenotypes ofphyB null mutants demonstrate that these receptor forms are not functionally redundant. The fivePHY mRNAs are, in general, expressed constitutively under varying light conditions, in different plant organs, and over the life cycle of the plant. These observations provide the first description of the structure and expression of a complete phytochrome family in a higher plant.

Patterns of expression and normalized levels of the five Arabidopsis phytochromes.

Using monoclonal antibodies specific for each apoprotein and full-length purified apoprotein standards, the levels of the five Arabidopsis phytochromes and their patterns of expression in seedlings and mature plants and under different light conditions have been characterized. Phytochrome levels are normalized to the DNA content of the various tissue extracts to approximate normalization to the number of cells in the tissue. One phytochrome, phytochrome A, is highly light labile. The other four phytochromes are much more light stable, although among these, phytochromes B and C are reduced 4- to 5-fold in red- or white-light-grown seedlings compared with dark-grown seedlings. The total amount of extractable phytochrome is 23-fold lower in light-grown than dark-grown tissues, and the percent ratios of the five phytochromes, A:B:C:D:E, are measured as 85:10:2:1.5:1.5 in etiolated seedlings and 5:40:15:15:25 in seedlings grown in continuous white light. The four light-stable phytochromes are present at nearly unchanging levels throughout the course of development of mature rosette and reproductive-stage plants and are present in leaves, stems, roots, and flowers. Phytochrome protein expression patterns over the course of seed germination and under diurnal and circadian light cycles are also characterized. Little cycling in response to photoperiod is observed, and this very low amplitude cycling of some phytochrome proteins is out of phase with previously reported cycling ofPHY mRNA levels. These studies indicate that, with the exception of phytochrome A, the family of phytochrome photoreceptors in Arabidopsis constitutes a quite stable and very broadly distributed array of sensory molecules.

Differential Patterns of Expression of the Arabidopsis PHYB, PHYD, and PHYE Phytochrome Genes

The Arabidopsis thaliana phyB, phyD, and phyE phytochrome apoproteins show higher amino acid sequence similarity to each other than to phyA or phyC, they are the most recently evolved members of this photoreceptor family, and they may interact in regulating photomorphogenesis. The expression patterns of translational fusions of the 5[prime] upstream regions of the PHYB, PHYD, and PHYE genes to the [beta]-glucuronidase (GUS) coding sequence were compared. PD-GUS and PE-GUS fusions were 5- to 10-fold less active than a PB-GUS fusion, but all three promoter regions drove expression of the reporter gene in all stages of the plants life cycle. Over the first 10 d of seedling growth, the PHYB and PHYD promoters were more active in the dark than in the light, whereas the opposite was true of the PHYE promoter. Unlike the PB-GUS construct, which was expressed in most parts of seedlings and mature plants, the PD-GUS and PE-GUS transgenes showed differential expression, notably in leaves, flower organs, and root tips. Tissue sections showed that the three promoters are coexpressed in at least some leaf cells. Hence, the PHYB, PHYD, and PHYE genes differ in expression pattern but these patterns overlap and interaction of these receptor forms within individual cells is possible.

The out of phase 1 Mutant Defines a Role for PHYB in Circadian Phase Control in Arabidopsis

Arabidopsis displays circadian rhythms in stomatal aperture, stomatal conductance, and CO2 assimilation, each of which peaks around the middle of the day. The rhythmic opening and closing of stomata confers a rhythm in sensitivity and resistance, respectively, to the toxic gas sulfur dioxide. Using this physiological assay as a basis for a mutant screen, we isolated mutants with defects in circadian timing. Here, we characterize one mutant, out of phase 1 (oop1), with the circadian phenotype of altered phase. That is, the timing of the peak (acrophase) of multiple circadian rhythms (leaf movement, CO2 assimilation, andLIGHT-HARVESTING CHLOROPHYLL a/b-BINDING PROTEINtranscription) is early with respect to wild type, although all circadian rhythms retain normal period length. This is the first such mutant to be characterized in Arabidopsis. oop1 also displays a strong photoperception defect in red light characteristic ofphytochrome B (phyB) mutants. Theoop1 mutation is a nonsense mutation ofPHYB that results in a truncated protein of 904 amino acids. The defect in circadian phasing is seen in seedlings entrained by a light-dark cycle but not in seedlings entrained by a temperature cycle. Thus, PHYB contributes light information critical for proper determination of circadian phase.

Obligate Heterodimerization of Arabidopsis Phytochromes C and E and Interaction with the PIF3 Basic Helix-Loop-Helix Transcription Factor

Phytochromes are dimeric chromoproteins that regulate plant responses to red (R) and far-red (FR) light. The Arabidopsis thaliana genome encodes five phytochrome apoproteins: type I phyA mediates responses to FR, and type II phyB–phyE mediate shade avoidance and classical R/FR-reversible responses. In this study, we describe the complete in vivo complement of homodimeric and heterodimeric type II phytochromes. Unexpectedly, phyC and phyE do not homodimerize and are present in seedlings only as heterodimers with phyB and phyD. Roles in light regulation of hypocotyl length, leaf area, and flowering time are demonstrated for heterodimeric phytochromes containing phyC or phyE. Heterodimers of phyC and chromophoreless phyB are inactive, indicating that phyC subunits require spectrally intact dimer partners to be active themselves. Consistent with the obligate heterodimerization of phyC and phyE, phyC is made unstable by removal of its phyB binding partner, and overexpression of phyE results in accumulation of phyE monomers. Following a pulse of red light, phyA, phyB, phyC, and phyD interact in vivo with the PHYTOCHROME INTERACTING FACTOR3 basic helix-loop-helix transcription factor, and this interaction is FR reversible. Therefore, most or all of the type I and type II phytochromes, including heterodimeric forms, appear to function through PIF-mediated pathways. These findings link an unanticipated diversity of plant R/FR photoreceptor structures to established phytochrome signaling mechanisms.

Spotlight on phytochrome nomenclature.

For many years after its discovery over four decades ago, the regulatory photoreceptor phytochrome was widely considered, implicitly or otherwise, to be a single molecular species (Sage, 1992). However, steadily accumulating evidence from physiological, spectrophotometric, biochemical, and immunochemical studies made it increasingly difficult to reconcile the diversity of phytochrome-mediated responses with the action of asingle photoreceptor species (Smith and Whitelam, 1990; Quail, 1991). There is now direct molecular evidence that the phytochrome polypeptide is encoded by multiple, divergent genes, at least in higher plants (Quail, 1994). Moreover, studies with photomorphogenic mutants and transgenic plants overexpressing different phytochromes have established that individual family members perform discrete photosensory functions in interpreting and processing information from the light environment (Somers et al., 1991; McCormac et al., 1992, 1993; Smith, 1992; Dehesh et al., 1993; Nagatani et al., 1993; Parks and Quail, 1993; Reed et al., 1993, 1994; Whitelam et al., 1993). In Arabidopsis, the phytochrome polypeptide is encoded by a family of five genes (Sharrock and Quail, 1989; Clack et al., 1994). These genes were initially designated phyA, phyB, phyC, phyD, and phyf (note lower case), based on the bacteria1 system of gene nomenclature (Sharrock and Quail, 1989; Quail, 1991, 1994). However, as increasing numbers of publications on the different phytochrome family members have appeared from multiple laboratories, a variety of nomenclature systems and symbols have been used to describe not only the genes themselves, but also the gene products and photochemical forms of the individual family members. We believe that it is timely, therefore, to implement a more standardized terminology to describe the phytochrome system. In addition, the recent demonstration that a series of photomorphogenic mutants of Arabidopsis, isolated independently in

Arabidopsis phytochromes C and E have different spectral characteristics from those of phytochromes A and B.

The red/far‐red light absorbing phytochromes play a major role as sensor proteins in photomorphogenesis of plants. In Arabidopsis the phytochromes belong to a small gene family of five members, phytochrome A (phyA) to E (phyE). Knowledge of the dynamic properties of the phytochrome molecules is the basis of phytochrome signal transduction research. Beside photoconversion and destruction, dark reversion is a molecular property of some phytochromes. A possible role of dark reversion is the termination of signal transduction. Since Arabidopsis is a model plant for biological and genetic research, we focussed on spectroscopic characterization of Arabidopsis phytochromes, expressed in yeast. For the first time, we were able to determine the relative absorption maxima and minima for a phytochrome C (phyC) as 661/725 nm and for a phyE as 670/724 nm. The spectral characteristics of phyC and E are strictly different from those of phyA and B. Furthermore, we show that both phyC and phyE apoprotein chromophore adducts undergo a strong dark reversion. Difference spectra, monitored with phycocyanobilin and phytochromobilin as the apoproteins chromophore, and in vivo dark reversion of the Arabidopsis phytochrome apoprotein phycocyanobilin adducts are discussed with respect to their physiological function.

The light-induced reduction of the gravitropic growth-orientation of seedlings of Arabidopsis thaliana (L.) Heynh. is a photomorphogenic response mediated synergistically by the far-red-absorbing forms of phytochromes A and B

Hypocotyls of dark-grown seedlings of Ara bidosis thaliana exhibit a strong negative gravitropism, which is reduced by red and also by long-wavelength, far-red light treatments. Light treatments using phytochrome A (phyA)- and phytochrome B (phyB)-deficient mutants showed that this response is controlled by phyB in a red/far-red reversible way, and by phyA in a non-reversible, very-low-fluence response. Crosses of the previously analyzed phyB-1 allele (in the ecotype Landsberg erecta background) to the ecotype Nossen wild-type (WT) background resulted in a WT-like negative gravitropism in darkness, indicating that the previously described gravitropic randomization observed with phyB-1 in the dark is likely due to a second mutation independent of that in the PHYB gene.

Distinct Light and Clock Modulation of Cytosolic Free Ca2+ Oscillations and Rhythmic CHLOROPHYLL A/B BINDING PROTEIN2 Promoter Activity in Arabidopsis

Plants have circadian oscillations in the concentration of cytosolic free calcium ([Ca2+]cyt). To dissect the circadian Ca2+-signaling network, we monitored circadian [Ca2+]cyt oscillations under various light/dark conditions (including different spectra) in Arabidopsis thaliana wild type and photoreceptor and circadian clock mutants. Both red and blue light regulate circadian oscillations of [Ca2+]cyt. Red light signaling is mediated by PHYTOCHROME B (PHYB). Blue light signaling occurs through the redundant action of CRYPTOCHROME1 (CRY1) and CRY2. Blue light also increases the basal level of [Ca2+]cyt, and this response requires PHYB, CRY1, and CRY2. Light input into the oscillator controlling [Ca2+]cyt rhythms is gated by EARLY FLOWERING3. Signals generated in the dark also regulate the circadian behavior of [Ca2+]cyt. Oscillations of [Ca2+]cyt and CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are dependent on the rhythmic expression of LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK-ASSOCIATED1, but [Ca2+]cyt and CAB2 promoter activity are uncoupled in the timing of cab1 (toc1-1) mutant but not in toc1-2. We suggest that the circadian oscillations of [Ca2+]cyt and CAB2 promoter activity are regulated by distinct oscillators with similar components that are used in a different manner and that these oscillators may be located in different cell types in Arabidopsis.