HFR1, a phytochrome A-signalling component,acts in a separate pathway from HY5, downstream of COP1 in Arabidopsis thaliana

HFR1, a basic helix-loop-helix protein, is known to be required for a subset of phytochrome A (phyA)-dependent photoresponses. To investigate the role of HFR1 in light signalling, we have examined the genetic interaction between HFR1 and HY5, a positive regulator of light signalling, and COP1, a repressor of photomorphogenesis. Double mutant analysis suggests that HFR1 mediates phyA-dependent inhibition of hypocotyl elongation independently of HY5. HFR1 was shown to be necessary for a subset of cop1-triggered photomorphogenic phenotypes in the dark, including inhibition of hypocotyl elongation, gravitropic hypocotyl growth, and expression of the light-inducible genes CAB and RBCS. Phenotypic analysis of the triple mutant cop1hy5hfr1 indicated that both HFR1 and HY5 are required for cop1-mediated photomorphogenic seedling development in darkness, consistent with their additive roles in phyA-dependent signalling. Taken together, these results suggest that HFR1 might act downstream of COP1, in a separate pathway from HY5, to mediate photomorphogenesis in Arabidopsis.     

UV-B-induced photomorphogenesis in Arabidopsis thaliana

Relatively little is known about the types of photomorphogenic responses and signal transduction pathways that plants employ in response to ultraviolet-B (UV-B, 290–320 nm) radiation. In wild-type Arabidopsis seedlings, hypocotyl growth inhibition and cotyledon expansion were both reproducibly promoted by continuous UV-B. The fluence rate response of hypocotyl elongation was examined and showed a biphasic response. Whereas photomorphogenic responses were observed at low doses, higher fluences resulted in damage symptoms. In support of our theory that photomorphogenesis, but not damage, occurs at low doses of UV-B, photomorphogenic responses of UV-B sensitive mutants were indistinguishable from wild-type plants at the low dose. This allowed us to examine UV-B-induced photomorphogenesis in photoreceptor deficient plants and constitutive photomorphogenic mutants. The cry1 cryptochrome structural gene mutant, and phytochrome deficient hy1, phyA and phyB mutant seedlings resembled wild-type seedlings, while phyA/phyB double mutants were less sensitive to the photomorphogenic effects of UV-B. These results suggest that either phyA or phyB is required for UV-B-induced photomorphogenesis. The constitutive photomorphogenic mutants cop1 and det1 did not show significant inhibition of hypocotyl growth in response to UV-B, while det2 was strongly affected by UV-B irradiation. This suggests that COP1 and DET1 work downstream of the UV-B signaling pathway.     

Molecular and phenotypic specificity of an antisense PHYB gene in Arabidopsis

The family of phytochrome photoreceptors plays an essential role in regulating plant growth and development in response to the light environment. An antisense PHYB transgene has been introduced into wild-type Arabidopsis and shown to inhibit expression of the PHYB sense mRNA and the phyB phytochrome protein 4- to 5-fold. This inhibition is specific to phyB in that the levels of the four other phytochromes, notably the closely related phyD and phyE phytochromes, are unaffected in the antisense lines. Antisense-induced reduction in phyB causes alterations of red light effects on seedling hypocotyl elongation, rosette leaf morphology, and chlorophyll content, similar to the phenotypic changes caused by phyB null mutations. However, unlike the phyB mutants, the antisense lines do not flower early compared to the wild type. Furthermore, unlike the phyB mutants, the antisense lines do not show a reduction in phyC level compared to the wild type, making it possible to unequivocally associate several of the photomorphogenic effects seen in phyB mutants with phytochrome B alone. These results indicate that an antisense transgene approach can be used to specifically inhibit the expression and activity of a single member of the phytochrome family and to alter aspects of shade avoidance responses in a targeted manner.     

The regulatory role of c‐MYC on HDAC2 and PcG expression in human multipotent stem cells

Myelocytomatosis oncogene (c‐MYC) is a well‐known nuclear oncoprotein having multiple functions in cell proliferation, apoptosis and cellular transformation. Chromosomal modification is also important to the differentiation and growth of stem cells. Histone deacethylase (HDAC) and polycomb group (PcG) family genes are well‐known chromosomal modification genes. The aim of this study was to elucidate the role of c‐MYC in the expression of chromosomal modification via the HDAC family genes in human mesenchymal stem cells (hMSCs). To achieve this goal, c‐MYC expression was modified by gene knockdown and overexpression via lentivirus vector. Using the modified c‐MYC expression, our study was focused on cell proliferation, differentiation and cell cycle. Furthermore, the relationship of c‐MYC with HDAC2 and PcG genes was also examined. The cell proliferation and differentiation were checked and shown to be dramatically decreased in c‐MYC knocked‐down human umbilical cord blood‐derived MSCs, whereas they were increased in c‐MYC overexpressing cells. Similarly, RT‐PCR and Western blotting results revealed that HDAC2 expression was decreased in c‐MYC knocked‐down and increased in c‐MYC overexpressing hMSCs. Database indicates presence of c‐MYC binding motif in HDAC2 promoter region, which was confirmed by chromatin immunoprecipitation assay. The influence of c‐MYC and HDAC2 on PcG expression was confirmed. This might indicate the regulatory role of c‐MYC over HDAC2 and PcG genes. c‐MYCs’ regulatory role over HDAC2 was also confirmed in human adipose tissue‐derived MSCs and bone‐marrow derived MSCs. From this finding, it can be concluded that c‐MYC plays a vital role in cell proliferation and differentiation via chromosomal modification.     

Phytochrome A and phytochrome B1 control the acquisition of competence for shoot regeneration in tomato hypocotyl

 We analysed the light-dependent acquisition of competence for adventitious shoot formation in hypocotyls of phytochrome A (fri) and phytochrome B1 (tri) mutants of tomato and their wild type by pre-growing the seedlings under different light quality. The regenerative response in vitro of explants from etiolated seedlings was reduced in comparison to that displayed by light-grown ones. Our results indicate that the light-dependent acquisition of competence for shoot regeneration in the tomato hypocotyl is regulated by phytochrome and antagonistically by a blue-light receptor. By using phytochrome mutants and narrow wave band light we showed that it is mediated at least by two distinct phytochrome species: phytochrome B1 and phytochrome A. The action of phytochrome B1 during seedling growth was sufficient to induce the full capacity of the subsequent regenerative response in vitro in explants from all positions along the hypocotyls. In contrast far-red light acting through phytochrome A did not induce the full capability of shoot regeneration from middle and basal segments of the hypocotyl when phytochrome B1 was absent (tri mutant). A few middle and basal hypocotyl explants pre-grown in blue light regenerated shoots. Received: 12 April 1999 / Revision received: 5 July 1999 · Accepted: 6 August 1999     

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.     

Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl

A majority of the cells in the Arabidopsis hypocotyl undergo endoreduplication. The number of endocycles in this organ is partially controlled by light. Up to two cycles occur in light-grown hypocotyls, whereas in the dark about 30% of the cells go through a third cycle. Is the inhibition of the third endocycle in the light an indirect result of the reduced cell size in the light-grown hypocotyl, or is it under independent light control? To address this question, the authors examined the temporal and spacial patterns of endoreduplication in light- or dark-grown plants and report here on the following observations: (i) during germination two endocycles take place prior to any significant cell expansion; (ii) in the dark the third cycle is completed very early during cell growth; and (iii) a mutation that dramatically reduces cell size does not interfere with the third endocycle. The authors then used mutants to study the way light controls the third endocycle and found that the third endocycle is completely suppressed in far red light through the action of phytochrome A and, to a lesser extent, in red light by phytochrome B. Furthermore, no 16C nuclei were observed in dark-grown constitutive photomorphogenic 1 seedlings. And, finally the hypocotyl of the cryptochrome mutant, hy4, grown in blue light was about three times longer than that of the wild-type without a significant difference in ploidy levels. Together, the results support the view that the inhibition of the third endocycle in light-grown hypocotyls is not the consequence of a simple feed-back mechanism coupling the number of cycles to the cell volume, but an integral part of the phytochrome-controlled photomorphogenic program.     

Photomorphogenic development of the Arabidopsisshy2–1D mutation and its interaction with phytochromes in darkness

We previously reported a photomorphogenic mutation of Arabidopsis thaliana, shy2–1D, as a dominant suppressor of a hy2 mutation. Here, we report that shy2–1D confers various photo-responsive phenotypes in darkness and the dark phenotypes of the mutant are affected by phytochrome deficiency. Dark-grown seedlings of the mutant developed several photomorphogenic characteristics such as short hypocotyls, cotyledon expansion and opening, and partial differentiation of plastids. When grown further in darkness, the mutant plant underwent most of the developmental stages of a light-grown wild-type plant, including development of foliar leaves, an inflorescence stem with cauline leaves, and floral organs. In addition, two light-inducible genes, the nuclear-encoded CAB and the plastid-encoded PSBA genes, were highly expressed in the dark-grown mutant seedlings. Furthermore, reduced gravitropism, a phytochrome-modulated response, was observed in the mutant hypocotyl in darkness. Thus, shy2–1D is one of the most pleiotropic photomorphogenic mutations identified so far. The results indicate that SHY2 may be a key component regulating photomorphogenesis in Arabidopsis. Surprisingly, double mutants of the shy2–1D mutant with the phytochrome-deficient mutants hy2, hy3 (phyB-1) and fre1–1 (phyA-201) showed reduced photomorphogenic response in darkness with a longer hypocotyl, a longer inflorescence stem, and a lower level expression of the CAB gene than the shy2–1D single mutant. These results showed that phytochromes function in darkness in the shy2–1D mutant background. The implications of these results are discussed.