Phytochrome in Pharbitis nil during and after de-etiolation

Phytochrome (P) was measured by in vivo spectrophotometry in the cotyledons of Pharbitis nil Choisy cv. Violet. In etiolated plants exposure to red light (R) results in a rapid fall in P content by destruction of the far-red absorbing form of phytochrome (Pfr). In continuous R or white light the P content falls as a result of destruction to a stable 3% of that originally present. In Norflurazon-treated white light de-etiolated plants, Pfr undergoes reversion in darkness to the red absorbing form of phytochrome (Pr) with a half life of 4 h at 19°C, while total P remains constant. Synthesis of Pr after de-etiolation occurs at a slow rate and is enhanced by terminating the de-etiolation light treatment with far-red light. The results are discussed in relation to the P control of flowering.     

Phytochrome A, phytochrome B and HY4 are involved in hypocotyl growth responses to natural radiation in Arabidopsis: weak de-etiolation of the phyA mutant under dense canopies

The roles of phytochrome A (phyA), phytochrome B (phyB) and a putative blue-light (BL) photoreceptor (HY4) in the control of hypocotyl growth by natural radiation were investigated using phyA, phyB and hy4 mutants of Arabidopsis thaliana. Full sunlight inhibited hypocotyl growth to a larger extent in wild-type (WT) than in phyA, phyB and, particularly, hy4 seedlings. In WT seedlings, hypocotyl growth was promoted by selectively lowering BL irradiance, lowering red-light (R) plus far-red-light (FR) irradiance or lowering the R/FR ratio (which was achieved either by increasing FR or by reducing R). The effects of lowering BL were reduced in hy4 and exaggerated in phyA seedlings. The effects of lowering R+FR were reduced in phyA and exaggerated in hy4 seedlings. Neither phyB nor hy4 mutants responded to low R/FR ratios. Neighbouring plants reflecting FR without shading caused subtle reductions of the R/FR ratio. This signal promoted hypocotyl growth in WT but not in phyA, phyB or hy4 seedlings. Intermediate canopy shade produced similar effects in all genotypes. Under deep shade, de-etiolation was severely impaired in phyA seedlings, which died prematurely. Thus, the FR ‘high-irradiance reaction’ mediated by phyA could be important for seedling survival under dense canopies.     

The SPA1-like proteins SPA3 and SPA4 repress photomorphogenesis in the light

Suppressor of phyA-105 (SPA1) is a phytochrome A-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. SPA1 is part of a small gene family comprising three genes: SPA1-related 2 (SPA2), SPA1-related 3 (SPA3), and SPA1-related 4 (SPA4). Here, we investigate the functions of SPA3 and SPA4, two very closely related genes coding for proteins with 74% identical amino acids. Seedlings with mutations in SPA3 or SPA4 exhibit enhanced photomorphogenesis in the light, but show no phenotype in darkness. While there are small differences between the effects of spa3 and spa4 mutations, it is apparent that SPA3 and SPA4 function to inhibit light responses in continuous far-red, red, and blue light. Phytochrome A is necessary for all aspects of the spa4 mutant phenotype, suggesting that SPA4, like SPA1, acts specifically in phytochrome A signaling. Enhanced photoresponsiveness of spa3 mutants is also fully dependent on phytochrome A in far-red and blue light, but not in red light. Hence, SPA3 function in red light may be dependent on other phytochromes in addition to phytochrome A. Using yeast two-hybrid and in vitro interaction assays, we further show that SPA3 as well as SPA4 can physically interact with the constitutive repressor of light signaling COP1. Deletion analyses suggest that SPA3 and SPA4, like SPA1, bind to the coiled-coil domain of COP1. Taken together, our results have identified two new loci coding for negative regulators that may be involved in fine tuning of light responses by interacting with COP1.     

Interactive signalling by phytochromes and cryptochromes generates de-etiolation homeostasis in Arabidopsis thaliana

Single, double, triple and quadruple mutants of phyA, phyB, cry1 and cry2 were exposed to different sunlight irradiances and photoperiods to investigate the roll played by phytochrome A, phytochrome B, cryptochrome 1 and cryptochrome 2 during de-etiolation of Arabidopsis thaliana seedlings under natural radiation. Even the quadruple mutant retained some hypocotyl-growth inhibition by sunlight. Hypocotyl length was strongly affected by interactions among photoreceptors. Double phyA phyB, phyA cry1, and cry1 cry2 mutants were taller than expected from the additive action of single mutations. Some of these redundant interactions required the presence of phytochromes A and/or B. Interactions among photoreceptors resulted in a 44% reduction of the response to irradiance and a 70% reduction of the response to photoperiod. The complex network of interactions among photoreceptors is proposed to buffer de-etiolation against changes in irradiance and photoperiod, i.e light fluctuations not related to the positions of the shoot above or below soil level     

The effect of red and far-red light in the high irradiance reaction of phytochrome (hypocotyl growth in dark-grown Sinapis alba L.)

Abstract. Fluence-rate response curves were determined for the inhibition of hypocotyl growth in 54-h-old dark-grown Sinapis alba L. seedlings by continuous or hourly 5 min far-red light irradiation (24 h). Just as in red light ( Heim & Schäfer, 1982 ), a fluence-rate dependence was observed for both kinds of irradiations, even if only 35% of the continuous light effect could be substituted for by hourly far-red pulses. The same total fluence was used for the two different light regimes. Measurements of Pfr and Pfr/Ptot showed a strong fluence-rate dependence under continuous light which only partially paralleled the fluence-rate response curves for the inhibition of the hypocotyl growth. It was concluded that neither spectrophotometrically determined levels of Pfr nor Pfr/Ptot can be the only light-dependent factor controlling hypocotyl lengthening under continuous irradiation.     

Phytochrome A, phytochrome B and other phytochrome(s) regulate ATHB-2 gene expression in etiolated and green Arabidopsis plants

The expression of the Arabidopsis ATHB-2 gene is light-regulated both in seedlings and in adult plants. The gene is expressed at high levels in rapidly elongating etiolated seedlings and is down-regulated by a pulse of red light (R) through the action of a phytochrome other than phytochrome A or B, or by a pulse of far-red light (FR) through the action of phytochrome A. In green plants, the expression of the ATHB-2 gene is rapidly and strongly enhanced by lowering the R:FR ratio perceived by a phytochrome other than A or B. Returning the plant to a high R:FR ratio results in an equally rapid decrease of the ATHB-2 mRNA. Consistently, plants overproducing ATHB-2 show developmental phenotypes characteristic of plants grown in low R:FR: elongated petioles, reduced leaf area, early flowering, and reduced number of rosette leaves. Taken together, the data strongly suggest a direct involvement of ATHB-2 in light-regulated growth phenomena throughout Arabidopsis development.     

Branching of the PIF3 regulatory network in Arabidopsis: Roles of PIF3-regulated MIDAs in seedling development in the dark and in response to light

Plants need to accurately adjust their development after germination in the underground darkness to ensure survival of the seedling, both in the dark and in the light upon reaching the soil surface. Recent studies have established that the photoreceptors phytochromes and the bHLH phytochrome interacting factors PIFs regulate seedling development to adjust it to the prevailing light environment during post-germinative growth. However, complete understanding of the downstream regulatory network implementing these developmental responses is still lacking. In a recent work, published in The Plant Cell, we report a subset of PIF3-regulated genes in dark-grown seedlings that we have named MIDAs (MISREGULATED IN DARK). Analysis of their functional relevance using mutants showed that four of them present phenotypic alterations in the dark, and that each affected a particular facet of seedling development, suggesting organ-specific branching in the signal that PIF3 relays downstream. Furthermore, our results also showed an altered response to light in seedlings with an impaired PIF3/MIDA regulatory network, indicating that these factors might also be essential to initiate and optimize the developmental adjustment of the seedling to the light environment.     

Stem extension-growth responses to blue light require Pfr in tomato seedlings but are not reduced by the low phytochrome levels of the aurea mutant

The effects of blue light (B) on stem extension growth were investigated in wild-type (WT) and aurea (au ) mutant seedlings of tomato. The au mutant has reduced phytochrome levels. Etiolated seedlings were grown under background red light (R) or far-red light (FR) with or without B. Hypocotyl growth was inhibited by B added to R but not by B added to FR, both in WT and au seedlings. The levels of B and/or R reaching the stem of fully de-etiolated seedlings grown in a glasshouse were reduced by means of collars around it. Both in WT and au -mutant seedlings the responses to B were larger at high than at low R/FR quantum ratios. In etiolated and light-grown au seedlings, changing the levels of phytochrome-absorbable radiation did not cause the same effect as changing B levels, indicating the action of specific BL/UV-A photoreceptor(s) (BAP). The responses to B are reduced by the low calculated levels of Pfr established by light treatments but not by the low levels of phytochrome present in the au mutant. The au mutant appears to be deficient in a phytochrome pool that is not essential for the interdependent co-action observed between phytochrome and BAP in the control of stem extension growth in tomato.     

Amino-acid biosynthesis in the cotyledons of Sinapis alba L. in darkness and far-red light studied by deuterium labelling and mass spectrometry

The incorporation of deuterium from deuterium oxide into the free amino acids of the cotyledons of Sinapis alba L. was studied by gas chromatography-mass spectrometry and was similar, both qualitatively and quantitatively, after incubation of the seedlings in darkness or far-red light. The results support studies which show that phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is synthesised de novo, rather than activated, in response to far-red light.Abbreviations GC-MS Gas chromatography-mass spectrometry - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - HFB n-propyl heptafluorobutyryl n-propyl     

Avena phytochrome a overexpressed in transgenic tobacco seedlings differentially affects red/far-red reversible and very-low-fluence responses (cotyledon unfolding) during de-etiolation

Etiolated seedlings of tobacco (Nicotiana tabacum L.) were exposed to single light pulses predicted to establish different proportions of phytochrome in its far-red absorbing form (Pfr/P). The angle between the cotyledons was compared in wild-type and transgenic seedling overexpressing Avena phytochrome A over the range of both very low-fluence responses (VLFR) and low-fluence responses (LFR). The unfolding of the cotyledons increased linearly for 24 h after the light pulse. At this time the Pfr/P-response curve showed two linear segments. The segment below a calculated Pfr/P = 3% (i.e. VLFR) was steeper than the segment above 3% (i.e. LFR). In the VLFR range the slope was almost threefold higher in transgenic than wild-type seedlings. However, in the LFR range the difference was less than 50%. From these data we propose that Avena phytochrome A makes a higher contribution to VLFR than LFR in etiolated tobacco seedlings.Abbreviations FR far-red light - LFR low-fluence response - Pfr/P proportion of phytochrome (P) in its FR-absorbing form (Pfr) - R red light - VLFR very low-fluence response Financial support was provided by the University of Buenos Aires and Fundación Antorchas (Argentina) to J.J.C., CONICET (Argentina) to R.A.S. and the U.S. Department of Energy (DE-FG02-88ER13968) to R.D.V.     

Temporal and light regulation of the expression of three phytochromes in germinating seeds and young seedlings of Avena sativa L.

An oat (Avena sativa L.) plant contains at least three phytochromes, which have monomeric masses of 125, 124, and 123 kilodaltons (kDa) (Wang et al., 1991, Planta 184, 96–104). The 124-kDa phytochrome is most abundant in dark-grown seedlings, while the other two phytochromes predominate in light-grown seedlings. Using three monoclonal antibodies, each specific to one of the three phytochromes, we have monitored by immunoblot assay the expression of these three phytochromes in the 5 d following onset of imbibition of seeds. On a per-organism basis, each of these three phytochromes increased in abundance for the first 3 d in the light, or for the first 4 d in darkness, after which they each began to decrease in quantity. When 3-d-old dark-grown seedlings were transferred to the light, the abundance of each of these three phytochromes decreased both in absolute amount and relative to the phytochrome levels in control seedlings kept in darkness. In contrast, when 3-d-old light-grown seedlings were transferred to darkness, the abundance of the 124-kDa and 125-kDa phytochromes increased while that of 123-kDa phytochrome remained unchanged. In each case, the level of phytochrome was greater than that of control seedlings maintained in the light. Thus, in addition to temporal regulation, all three phytochromes exhibit photoregulated expression at the protein level, although the magnitude of this photoregulation varies substantially. We thank Drs. Elizabeth Williams and Tammy Sage (Botany Department, University of Georgia, USA) for generously permitting us to use their image-analysis system. This research was supported by USDA NRICGP grant 91-37100-6490.     

Differences and similarities in the photoregulation of gibberellin metabolism between rice and dicots

In rice seedlings, elongation of leaf sheaths is suppressed by light stimuli. The response is mediated by two classes of photoreceptors, phytochromes and cryptochromes. However, it remains unclear how these photoreceptors interact in the process. Our recent study using phytochrome mutants and novel cryptochrome RNAi lines revealed that cryptochromes and phytochromes function cooperatively, but independently to reduce active GA contents in seedlings in visible light. Blue light captured by cryptochrome 1 (cry1a and cry1b) induces robust expression of GA 2-oxidase genes (OsGA2ox4-7). In parallel, phytochrome B with auxiliary action of phytochrome A mediates repression of GA 20-oxidase genes (OsGA20ox2 and OsGA20ox4). The independent effects cumulatively reduce active GA contents, leading to a suppression of leaf sheath elongation. These regulatory mechanisms are distinct from phytochrome B function in dicots. We discuss reasons why the distinct system appeared in rice, and advantages of the rice system in early photomorphogenesis.     

A unique system for regulating mitochondrial mRNA poly(A) status and stability in plants

Poly(A) status is the major determinant of mRNA stability, even in endosymbiotic organelles. Poly(A) specific ribonuclease (PARN) is distributed widely among eukaryotes and has been shown to regulate the poly(A) status of cytoplasmic mRNA in various organisms. Surprisingly, our recent study revealed that PARN also directly regulates poly(A) status of mitochondrial mRNA in Arabidopsis. In this addendum, we discuss whether this mitochondrial function of PARN is common in plants and why PARN has been assigned such a unique function.