Interactions of phytochromes A,B1 and B2 in light-induced competence for adventitious shoot formation in hypocotyl of tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.)

The interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phytochrome B2 (phyB2) in light-dependent shoot regeneration from the hypocotyl of tomato was analysed using all eight possible homozygous allelic combinations of the null mutants. The donor plants were pre-grown either in the dark or under red or far-red light for 8 days after sowing; thereafter hypocotyl segments (apical, middle and basal portions) were transferred onto hormone-free medium for culture under different light qualities. Etiolated apical segments cultured in vitro under white light showed a very high frequency of regeneration for all of the genotypes tested besides phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants. Evidence is provided of a specific interference of phyB2 with phyA-mediated HIR to far-red and blue light in etiolated explants. Pre-treatment of donor plants by growth under red light enhanced the competence of phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants for shoot regeneration, whereas pre-irradiation with far-red light enhanced the frequency of regeneration only in the phyAphyB1 mutant. Multiple phytochromes are involved in red light- and far-red light-dependent acquisition of competence for shoot regeneration. The position of the segments along the hypocotyl influenced the role of the various phytochromes and the interactions between them. The culture of competent hypocotyl segments under red, far-red or blue light reduced the frequency of explants forming shoots compared to those cultured under white light, with different genotypes having different response patterns.Abbreviations HIR: High irradiance response - LFR: Low fluence response - Pfr: Far-red absorbing form of phytochrome - phyA: Phytochrome A - phyB1: Phytochrome B1 - phyB2: Phytochrome B2 - phyA(B1, B2): Phytochrome mutant deficient in phyA (B1, B2) - phyAphyB1(B1B2,AB2): Double phytochrome mutant deficient in phyA and phyB1(B1, B2) - phyAphyB1phyB2: Triple mutant deficient in phyA, phyB1 and phyB2 - VLFR: Very low fluence response - WT: Wild-type tomato Communicated by R. Reski     

A role for ABCB19‐mediated polar auxin transport in seedling photomorphogenesis mediated by cryptochrome 1 and phytochrome B

During seedling establishment, blue and red light suppress hypocotyl growth through the cryptochrome 1 (cry1) and phytochrome B (phyB) photosensory pathways, respectively. How these photosensory pathways integrate with growth control mechanisms to achieve the appropriate degree of stem elongation was investigated by combining cry1 and phyB photoreceptor mutations with genetic manipulations of a multidrug resistance‐like membrane protein known as ABCB19 that influenced auxin distribution within the plant, as evidenced by a combination of reporter gene assays and direct auxin measurements. Auxin signaling and ABCB19 protein levels, hypocotyl growth rates, and apical hook opening were measured in mutant and wild‐type seedlings exposed to a range of red and blue light conditions. Ectopic/overexpression of ABCB19 (B19OE) greatly increased auxin in the hypocotyl, which reduced the sensitivity of hypocotyl growth specifically to blue light in long‐term assays and red light in high‐resolution, short‐term assays. Loss of ABCB19 partially suppressed the cry1 hypocotyl growth phenotype in blue light. Hypocotyl growth of B19OE seedlings in red light was very similar to phyB mutants. Altered auxin distribution in B19OE seedlings also affected the opening of the apical hook. The cry1 and phyB photoreceptor mutations both increased ABCB19 protein levels at the plasma membrane, as measured by confocal microscopy. The B19OE plant proved to be a useful tool for determining aspects of the mechanism by which light, acting through cry1 or phyB, influences the auxin transport process to control hypocotyl growth during de‐etiolation.     

Expression of heterologous phytochromes A, B or C in transgenic tobacco plants alters vegetative development and flowering time

In this study, oat phytochrome A (phyA), Arabidopsis phytochrome B (phyB) or Arabidopsis phytochrome C (phyC) were expressed in both day-neutral and photo-period-sensitive (short-day) tobacco (Nicotiana tabacum cv. Hicks). Introgression of the Maryland Mammoth (MM) gene into cv Hicks was used to confer short-day photo-periodic sensitivity. Expression of oat phyA led to characteristic hypersensitivity of hypocotyls to red light (R) and far-red light (FR) and an overall dwarfing of the mature plant. Expression of Arabidopsis phyB enhanced the sensitivity of hypocotyls to R and caused even more marked dwarfing of the mature plant. In contrast, the expression of Arabidopsis phyC had no detectable consequences for the photocontrol of hypocotyl elongation. However, phyC expression did lead to a R-dependent increase in cotyledon expansion in de-etiolating seedlings and to a significant increase in leaf area in mature plants. This provides the first experimental evidence that phyC is biologically active. The flowering time of cv Hicks plants grown under 8 h photoperiods was virtually unaffected by a 30 min white light (W) night break given 8 h into the dark period. In contrast, cv Hicks MM plants responded to a night break with a delay in flowering. Expression of phyA or phyB led to a night break-dependent delay in flowering in cv Hicks plants. For cv Hicks MM plants, the expression of any of phyA, phyB or phyC caused a marked enhancement of the flower-delaying effect of a night break. These observations indicate that transgenic phyA, phyB or phyC can interact with the endogenous mechanisms controlling flowering time in tobacco.     

Absence of phyB inhibits hypocotyl elongation in dark-grown Ih cucumber seedlings: an active role for PrB

Cucumber (Cucumis sativus L.) seedlings carrying the long hypocotyl (Ih) mutation, which confers a lack of B-type phytochrome (phyB), were significantly shorter than their near-isogenic wild-type counterparts when grown in complete darkness. Relative growth rates determined for 5 mm hypocotyl regions were lower in Ih seedlings in all growing regions, and the zone of elongation was less extensive in Ih hypocotyls. Digital imaging microscopy revealed that the pattern of epidermal cell lengths along the stem axis differed between the Ih mutant and the iso-genic wild-type. These findings (and the fact that experiments were conducted under conditions where phytochrome photoconversion to the far-red-absorbing form does not occur) suggest that the red-absorbing form of phyB (PrB) is an active positive regulator of development in etiolated plants.     

Temperature-dependent internode elongation in vegetative plants of Arabidopsis thaliana lacking phytochrome B and cryptochrome 1

 Vegetative plants of Arabidopsis thaliana (L.) Heynh. form a compact rosette of leaves in which internode growth is virtually arrested. Rapid extension of the internodes occurs after flower buds are present in the reproductive apex. Under natural radiation, continuous light from fluorescent lamps, or short photoperiods of light from fluorescent lamps, plants of the phyB cry1 double mutant (lacking both phytochrome B and cryptochrome 1) did not form normal rosettes because all the internodes showed some degree of elongation. Internode elongation was weak in the phyB single mutant and absent in the cry1 mutant, indicating redundancy between phytochrome B and cryptochrome 1. The absence of phytochrome A caused no effects. The failure to form normal rosettes was conditional because internode elongation was arrested at low temperatures in all the mutant combinations. In contrast, the temperature dependence of phytochrome B and cryptochrome 1 effects on hypocotyl growth was weak. The elongation of the internodes in phyB cry1 was not accompanied by early flowering as showed by the lack of effects on the final number of leaves. Apex dissection indicated that in phyB cry1 double mutants internode elongation anticipated the transition from the vegetative to the reproductive stage. Thus, stem growth in Arabidopsis thaliana is not fully dependent on the program of reproductive development. Received: 2 June 1999 / Accepted: 13 August 1999     

Cathemerality in wild ring-tailed lemurs (Lemur catta) in the spiny forest of Tsimanampetsotsa National Park: camera trap data and preliminary behavioral observations

Cathemerality consists of discrete periods of activity during both the day and night. Though uncommon within Primates, cathemerality is prevalent in some lemur genera, such as Eulemur, Hapalemur, and Prolemur. Several researchers have also reported nighttime activity in Lemur catta, yet these lemurs are generally considered “strictly diurnal”. We used behavioral observations and camera traps to examine cathemerality of L. catta at the Tsimanampetsotsa National Park, Madagascar. Nighttime activity occurred throughout the study period (September 2010–April 2011), and correlated with warm overnight temperatures but not daytime temperatures. Animals spent 25 % of their daytime active behaviors on the ground, but appeared to avoid the ground at night, with only 5 % of their time on the ground. Furthermore, at night, animals spent the majority of their active time feeding (53 % nighttime, 43 % daytime). These findings imply that both thermoregulation and diet play a role in the adaptive significance of cathemerality. Additionally, predator avoidance may have influenced cathemerality here, in that L. catta may limit nighttime activity as a result of predation threat by forest cats (Felis sp.) or fossa (Cryptoprocta ferox). Further data are needed on cathemeral lemurs generally, but particularly in L. catta if we are to fully understand the evolutionary mechanisms of cathemerality in the Lemuridae.     

Roles for the N- and C-terminal domains of phytochrome B in interactions between phytochrome B and cryptochrome signaling cascades

Plants fine-tune light responses through interactions betweenphotoreceptors. We have previously reported that the greeningof Arabidopsis thaliana roots is regulated synergistically byphytochromes and cryptochromes. In the present study, we investigatedthe functions of the N- and C-terminal domains of phytochromeB (phyB) in the interactions between phyB and cryptochrome signalingcascades. Transgenic Arabidopsis expressing the phyB N-terminaldomain fused to green fluorescent protein (GFP), ß-glucuronidase(GUS) and the nuclear localization signal (NLS) showed intenseroot greening under blue light, indicating that the C-terminaldomain was dispensable for the synergistic interaction in theinduction of root greening. However, root greening under redlight was substantially reduced in the absence of the C-terminaldomain. This effect was opposite to the previous observationthat removal of the C-terminal domain enhanced the signalingactivity of phyB in the inhibition of hypocotyl elongation.In addition, we found that overexpression of the isolated C-terminaldomain of phyB enhanced the blue light response not only forroot greening but also for the inhibition of hypocotyl elongation.Analysis of this activity on various photoreceptor mutant backgroundsdemonstrated that the isolated C-terminal domain enhanced cryptochromesignaling. In summary, these results demonstrate that differentdomains of phyB can play various roles which are dependent onlight conditions as well as on the specific physiological response.     

Overexpressed phytochrome C has similar photosensory specificity to phytochrome B but a distinctive capacity to enhance primary leaf expansion

Phytochrome C (phyC) is a low-abundance member of the five-membered phytochrome family of photoreceptors in Arabidopsis. Towards developing an understanding of the photosensory and physiological functions of phyC, transgenic Arabidopsis plants were generated that overexpress cDNA-encoded phyC and seedling responses to continuous white, red, or far-red light (Wc, Rc or FRc, respectively) were examined. Transgenic seedlings overexpressing phyC displayed enhanced inhibition of hypocotyl elongation in Rc, but were unchanged in responsiveness to FRc relative to wild-type. These data indicate that phyC has photosensory specificity that is similar to that of phyB and thus distinct from that of phyA. phyC overexpressors with levels only 3 to 4 times the level of endogenous phyC exhibited enhanced primary leaf expansion in Wc. This is in contrast to phyA or phyB overexpressors which respectively have levels that are 500-and 100-fold that of overexpressed phyC but showed no enhancement of primary leaf expansion. Therefore, phyC may have some physiological roles that are different to those of phyA and phyB in the control of seedling responses to light signals.     

Arabidopsis PHYTOCHROME INTERACTING FACTOR Proteins Promote Phytochrome B Polyubiquitination by COP1 E3 Ligase in the Nucleus

Many plant photoresponses from germination to shade avoidance are mediated by phytochrome B (phyB). In darkness, phyB exists as the inactive Pr in the cytosol but upon red (R) light treatment, the active Pfr translocates into nuclei to initiate signaling. Degradation of phyB Pfr likely regulates signal termination, but the mechanism is not understood. Here, we show that phyB is stable in darkness, but in R, a fraction of phyB translocates into nuclei and becomes degraded by 26S proteasomes. Nuclear phyB degradation is mediated by COP1 E3 ligase, which preferentially interacts with the PhyB N-terminal region (PhyB-N). PhyB-N polyubiquitination by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in vitro can be enhanced by different PHYTOCHROME INTERACTING FACTOR (PIF) proteins that promote COP1/PhyB interaction. Consistent with these results, nuclear phyB accumulates to higher levels in pif single and double mutants and in cop1-4. Our results identify COP1 as an E3 ligase for phyB and other stable phytochromes and uncover the mechanism by which PIFs negatively regulate phyB levels.     

The role of phytochrome B,D and E in thermoperiodic responses of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The objective of this work was to study the role of the phytochromes (phy) B, D and E in the thermoperiodic control of elongation and flowering time in Arabidopsis thaliana. WT, and phyB, phyD and phyE single mutants, and phyB phyD and phyB phyE double mutants, were grown under day/night temperatures (DT/NT) of 12/22°C, 17/17°C or 22/12°C (negative, zero and positive DIF, respectively) for inflorescence stem length measurements, and under DT/NT 17/25°C or 25/17°C (negative and positive DIF, respectively) for leaf morphology and flowering time measurements. In WT final length of the stem, petiole and leaf blade were longer under positive DIF compared to negative DIF. The temperature effect was stronger in the leaf petiole than the stem, whereas only a slight change was seen in the leaf blade length direction and none in the width direction. The temperature effect on stem and petiole elongation was reduced or nearly eliminated in the genotypes lacking phyB, while a phyD or a phyE mutation had no influence or a slightly positive influence on the temperature effect, respectively. These results suggest that phyB, and not phyD or phyE, is needed for a complete thermoperiodic control of elongation growth in A. thaliana. For all genotypes tested, plants flowered earlier at negative DIF than positive DIF, suggesting that none of the three phytochromes B, D, or E is needed for a thermoperiodic control of flowering time in A. thaliana.     

Altered dark- and photoconversion of phytochrome B mediate extreme light sensitivity and loss of photoreversibility of the phyB-401 mutant

The phyB-401 mutant is 10(3) fold more sensitive to red light than its wild-type analogue and shows loss of photoreversibility of hypocotyl growth inhibition. The phyB-401 photoreceptor displays normal spectral properties and shows almost no dark reversion when expressed in yeast cells. To gain insight into the molecular mechanism underlying this complex phenotype, we generated transgenic lines expressing the mutant and wild-type phyB in phyB-9 background. Analysis of these transgenic lines demonstrated that the mutant photoreceptor displays a reduced rate of dark-reversion but normal P(fr) to P(r) photoconversion in vivo and shows an altered pattern of association/dissociation with nuclear bodies compared to wild-type phyB. In addition we show (i) an enhanced responsiveness to far-red light for hypocotyl growth inhibition and CAB2 expression and (ii) that far-red light mediated photoreversibility of red light induced responses, including inhibition of hypocotyl growth, formation of nuclear bodies and induction of CAB2 expression is reduced in these transgenic lines. We hypothesize that the incomplete photoreversibility of signalling is due to the fact that far-red light induced photoconversion of the chromophore is at least partially uncoupled from the P(fr) to P(r) conformation change of the protein. It follows that the phyB-401 photoreceptor retains a P(fr)-like structure (P(r) (*)) for a few hours after the far-red light treatment. The greatly reduced rate of dark reversion and the formation of a biologically active P(r) (*) conformer satisfactorily explain the complex phenotype of the phyB-401 mutant and suggest that amino acid residues surrounding the position 564 G play an important role in fine-tuning phyB signalling.     

PHOTOSYNTHETIC SENSITIVITY TO TEMPERATURE IN POPULATIONS OF TWO C4 BOUTELOUA (POACEAE) SPECIES NATIVE TO DIFFERENT ALTITUDES

The relationships between photosynthesis, flowering, and growth temperatures were examined experimentally in four populations of the C₄ grass genus Bouteloua. Field-collected plants were grown under two temperature regimes, cool (20 C day/6 C night) and warm (30/16), representative of the extreme populations. Populations collected from the warm climates had significantly lower photosynthetic capacity when grown in the cool chamber relative to the warm chamber, while photosynthetic capacity in the cool climate populations did not differ between the growth conditions. Additionally, exposure to a 2-day cold temperature treatment (10/-2), representative of late-season frosts in high altitude sites, resulted in further reductions in photosynthesis in the warm climate plants, but not in the cool climate plants. This effect was greater for plants grown in the cool growth chamber. Flowering was reduced by 70% in the warm climate plants grown in the cool chamber, and was correlated with photosynthetic inhibition following the short-term cold temperature treatment. These results indicate that genetic differentiation for photosynthetic temperature sensitivity has occurred in the cool climate populations, and that long-term exposure to cool temperatures coupled with short-term relatively extreme low temperatures results in greater photosynthetic inhibition in nontolerant populations.     

The pineal complex and melatonin affect the expression of the daily rhythm of behavioral thermoregulation in the green iguana

Daily variation in the body temperature of the green iguana (Iguana iguana) was studied by telemetry in laboratory photo-thermal enclosures under a 12Light12Dark (LD) photoperiod. The lizards showed robust daily rhythms of thermoregulation maintaining their body temperatures (T_b) at higher levels during the day than during the night. Some animals maintained rhythmicity when kept in constant darkness. On lightdark cycles parietalectomy produced only a transient increase of median T_b in the first or second night following the operation. Pinealectomized lizards on the other hand maintained their body temperatures at significantly lower levels during the day and at significantly higher levels during the night than did sham-operated or intact lizards. This effect was apparently permanent, since one month after pinealectomy lizards still displayed the altered pattern. Plasma melatonin levels in intact animals were high during the night and low during the day and were unaffected by parietalectomy. Pinealectomized lizards showed low levels of plasma melatonin during both the day and the night. A daily intraperitoneal injection of melatonin in pinealectomized animals given a few minutes after the light to dark transition decreased the body temperatures selected by the lizards during the night and increased the body temperatures selected during the following day. Control injections of saline solution had no effect. The significance of these results is discussed in relation to the role of the pineal complex and melatonin in the mediation of thermoregulatory behavior.Abbreviations LD LightDark - T _b body temperature - PAR-X parietalectomy - PIN-X pinealectomy     

Control of hypocotyl elongation in Arabidopsis thaliana by photoreceptor interaction

In order to test the interaction of different phytochromes and blue-light receptors, etiolated seedlings of wild-type Arabidopsis thaliana (L.) Heynh., a phytochrome (phy) B-overexpressor line (ABO), and the photoreceptor mutants phyA-201, phyB-5, hy4-2.23n, fha-1, phyA-201/phyB-5, and phyA-201/hy4-2.23n were exposed to red and far-red light pulses after various preirradiations. The responsiveness to the inductive red pulses is primarily mediated by phyB which is rather stable in its far-red-absorbing form as demonstrated by a very slow loss of reversibility. Without preirradiation the red pulses had an impact on hypocotyl elongation only in PHYA mutants but not in the wild type. This indicates a suppression of phyB function by the presence of phyA. Preirradiation with either far-red or blue light resulted in an inhibition of hypocotyl elongation by red pulses in the wild type. Responsiveness amplification by far-red light is mediated by phyA and disappears slowly in the dark. The extent of responsiveness amplification by blue light was identical in the wild type and in the absence of phyA, or the cryptochromes cryl (hy4-2.23n) or cry2 (fha-1). Therefore, we conclude that stimulation of phyB by blue light preirradiation is either mediated by an additional still-unidentified blue-light-absorbing pigment or that phyA, cry1 and cry2 substitute for each other completely. Both blue and red preirradiation established responsiveness to red pulses in phyA-201/phyB-5 double mutants. These results demonstrate that inhibition of hypocotyl elongation by red pulses is not only mediated by phyB but also by a phytochrome(s) other than phyA and phyB. Received: 21 July 1998 / Accepted: 7 December 1998     

The blue-light receptor cryptochrome 1 shows functional dependence on phytochrome A or phytochrome B in Arabidopsis thaliana

Blue-light responses in higher plants are mediated by specific photoreceptors, which are thought to be flavoproteins; one such flavin-type blue-light receptor, CRY1 (for cryptochrome), which mediates inhibition of hypocotyl elongation and anthocyanin biosynthesis, has recently been characterized. Prompted by classical photobiological studies suggesting possible co-action of the red/far-red absorbing photoreceptor phytochrome with blue-light photoreceptors in certain plant species, the role of phytochrome in CRY1 action in Arabidopsis was investigated. The activity of the CRY1 photoreceptor can be substantially altered by manipulating the levels of active phytochrome (Pfr) with red or far-red light pulses subsequent to blue-light treatments. Furthermore, analysis of severely phytochrome-deficient mutants showed that CRY1-mediated blue-light responses were considerably reduced, even though Western blots confirmed that levels of CRY1 photoreceptor are unaffected in these phytochrome-deficient mutant backgrounds. It was concluded that CRY1-mediated inhibition of hypocotyl elongation and anthocyanin production requires active phytochrome for full expression, and that this requirement can be supplied by low levels of either phyA or phyB.     

Nocturnal gibberellin biosynthesis is carbon dependent and adjusts leaf expansion rates to variable conditions

Optimal plant growth performance requires that the presence and action of growth signals, such as gibberellins (GAs), are coordinated with the availability of photo-assimilates. Here, we studied the links between GA biosynthesis and carbon availability, and the subsequent effects on growth. We established that carbon availability, light and dark cues, and the circadian clock ensure the timing and magnitude of GA biosynthesis and that disruption of these factors results in reduced GA levels and expression of downstream genes. Carbon-dependent nighttime induction of gibberellin 3-beta-dioxygenase 1 (GA3ox1) was severely hampered when preceded by reduced daytime light availability, leading specifically to reduced bioactive GA₄ levels, and coinciding with a decline in leaf expansion rate during the night. We attributed this decline in leaf expansion mostly to reduced photo-assimilates. However, plants in which GA limitation was alleviated had significantly improved leaf expansion, demonstrating the relevance of GAs in growth control under varying carbon availability. Carbon-dependent expression of upstream GA biosynthesis genes (Kaurene synthase and gibberellin 20 oxidase 1, GA20ox1) was not translated into metabolite changes within this short timeframe. We propose a model in which the extent of nighttime biosynthesis of bioactive GA₄ by GA3ox1 is determined by nighttime consumption of starch reserves, thus providing day-to-day adjustments of GA responses.

GA-sugar matching occurs specifically at night and determines day to day adjustment of GA levels and subsequent growth.     

Cold-acclimation of Hibiscus rosa-sinensis L. and Hibiscus syriacus L. in natural and controlled environments

Abstract Seasonal cold-acclimation patterns and the effects of photoperiod and temperature on cold-hardiness of Hibiscus rosa-sinensis L. and Hibiscus syriacus L. were determined. Field-grown H. rosasinensis consistently failed to survive freezing at - 2°C. Two genotypes of field- and container-grown H. syriacus initiated cold-acclimation in mid September, in response to decreasing daylength, and continued to an ultimate midwinter hardiness level of - 27°C in early February. Controlled environment experiments using combinations of short days (SD) and cool day/night temperatures were unable to induce even minimal cold acclimation of H. rosasinensis. In controlled environments, H. syriacus attained a moderate amount of cold tolerance at warm temperatures and long days (LD). Low night temperature combined with LD, warm day produced the same degree of cold-acclimation as the SD treatments. While not essential, SD enhanced H. syriacus cold-acclimation in controlled environments. A - 5°C frost treatment of intact plants did not enhance cold-hardiness of H. syriacus.