Elongated mesocotyl1, a phytochrome-deficient mutant of maize

To begin the functional dissection of light signal transduction pathways of maize (Zea mays), we have identified and characterized the light-sensing mutant elm1 (elongated mesocotyl1). Seedlings homozygous for elm1 are pale green, show pronounced elongation of the mesocotyl, and fail to de-etiolate under red or far-red light. Etiolated elm1 mutants contain no spectrally active phytochrome and do not deplete levels of phytochrome A after red-light treatment. High-performance liquid chromatography analyses show that elm1 mutants are unable to convert biliverdin IX alpha to 3Z-phytochromobilin, preventing synthesis of the phytochrome chromophore. Despite the impairment of the phytochrome photoreceptors, elm1 mutants can be grown to maturity in the field. Mature plants retain aspects of the seedling phenotype and flower earlier than wild-type plants under long days. Thus, the elm1 mutant of maize provides the first direct evidence for phytochrome-mediated modulation of flowering time in this agronomically important species.     

Chlorophyll Deficiency in the Maize elongated mesocotyl2 Mutant Is Caused by a Defective Heme Oxygenase and Delaying Grana Stacking

Background

Etiolated seedlings initiate grana stacking and chlorophyll biosynthesis in parallel with the first exposure to light, during which phytochromes play an important role. Functional phytochromes are biosynthesized separately for two components. One phytochrome is biosynthesized for apoprotein and the other is biosynthesized for the chromophore that includes heme oxygenase (HO).

Methodology/Principal Finding

We isolated a ho1 homolog by map-based cloning of a maize elongated mesocotyl2 (elm2) mutant. cDNA sequencing of the ho1 homolog in elm2 revealed a 31 bp deletion. De-etiolation responses to red and far-red light were disrupted in elm2 seedlings, with a pronounced elongation of the mesocotyl. The endogenous HO activity in the elm2 mutant decreased remarkably. Transgenic complementation further confirmed the dysfunction in the maize ho1 gene. Moreover, non-appressed thylakoids were specifically stacked at the seedling stage in the elm2 mutant.

Conclusion

The 31 bp deletion in the ho1 gene resulted in a decrease in endogenous HO activity and disrupted the de-etiolation responses to red and far-red light. The specific stacking of non-appressed thylakoids suggested that the chlorophyll biosynthesis regulated by HO1 is achieved by coordinating the heme level with the regulation of grana stacking.     

Succinate dehydrogenase in Arabidopsis thaliana is regulated by light via phytochrome A

The effect of light on succinate dehydrogenase (SDH) activity and mRNA content was studied in Arabidopsis thaliana plants. The transition from darkness to light caused a short transient increase in the SDH activity followed by a decrease to a half of the original activity. The white or red light were found to be down-regulating factors for the mRNA content of the sdh1-2 and sdh2-3 genes and SDH catalytic activity both in A. thaliana wild-type plants and in the mutant deficient in the phytochrome B gene, but not in the mutant deficient in the phytochrome A gene, while the far-red light of 730 nm reversed the red light effect. It is concluded that phytochrome A participates in the regulation of mitochondrial respiration through effect on SDH expression.     

The Arabidopsis ELF3 gene regulates vegetative photomorphogenesis and the photoperiodic induction of flowering

Flowering in Arabidopsis thaliana is promoted by long-day (LD) photoperiods such that plants grown in LD flower earlier, and after the production of fewer leaves, than plants grown in short-day (SD) photoperiods. The early-flowering 3 ( elf 3) mutant of Arabidopsis , which is insensitive to photoperiod with regard to floral initiation has been characterized. elf 3 mutants are also altered in several aspects of vegetative photomorphogenesis, including hypocotyl elongation. When inhibition of hypocotyl elongation was measured, elf 3 mutant seedlings were less responsive than wild-type to all wavelengths of light, and most notably defective in blue and green light-mediated inhibition. When analyzed for the flowering-time phenotype, elf 3 was epistatic to mutant alleles of the blue-light receptor encoding gene, HY 4. However, when elf 3 mutants were made deficient for functional phytochrome by the introduction of hy 2 mutant alleles, the elf 3 hy 2 double mutants displayed the novel phenotype of flowering earlier than either single mutant while still exhibiting photoperiod insensitivity, indicating that a phytochrome-mediated pathway regulating floral initiation remains functional in elf 3 single mutants. In addition, the inflorescences of one allelic combination of elf 3 hy 2 double mutants form a terminal flower similar to the structure produced by tfl 1 single mutants. These results suggest that one of the signal transduction pathways controlling photoperiodism in Arabidopsis is regulated, at least in part, by photoreceptors other than phytochrome, and that the activity of the Arabidopsis inflorescence and floral meristem identity genes may be regulated by this same pathway.     

The control of morphogenesis in Saccharomyces cerevisiae by Elm1 kinase is responsive to RAS/cAMP pathway activity and tryptophan availability

Many fungi undergo a morphological transition to filamentous growth in response to limiting nutrient conditions. Constitutively elongated Saccharomyces cerevisiae mutants ( elm ) have been isolated; the ELM1 gene encodes a putative serine/threonine protein kinase. A novel allele, elm1-15 , has been isolated in an S288C-derived strain, which causes a pleiotropic phenotype, including media-specific growth effects, abnormal morphology and altered stress response, in cells that are auxotrophic for tryptophan. elm1-15 trp1 cells cannot use many nitrogen sources, are sensitive to amino acid analogues, have very low general amino acid permease activity and do not accumulate trehalose. In contrast, haploid elm1-15 TRP1 cells grow well in budding form on all media, are stress resistant and overaccumulate trehalose. Several lines of evidence suggest that Elm1 acts on functions related to the RAS /cAMP pathway. Overexpression of Elm1 partially rescues the ts phenotype of cdc25 and cyr1 mutants. Deletion of ELM1 in low PKA activity mutants increased the severity of their phenotypes, and activation of Ras2 decreases the cell elongation phenotype of elm1 mutants. A 'signal integration' model for the complex relationship of Elm1 and the RAS/ cAMP pathway in controlling morphogenesis in response to nutrients is proposed.     

Making light of it: the role of plant haem oxygenases in phytochrome chromophore synthesis

The haem oxygenase (HO) enzyme catalyses the oxidation of haem to biliverdin IX alpha, CO and Fe(2+), and performs a wide variety of roles in Nature, including degradation of haem from haemoglobin, iron acquisition and phycobilin biosynthesis. In plants, HOs are required for the synthesis of the chromophore of the phytochrome family of photoreceptors. There are four HO genes in the Arabidopsis genome. Analysis of a mutant deficient in HO1 (the hy1 mutant) has demonstrated that this plastid-localized protein is the major HO in the phytochrome chromophore synthesis pathway. HO2 may also have a minor role in this pathway, but our understanding of the divergent roles of this small gene family is still far from complete.     

Photomorphogenic responses in maize seedling development

As an emerging maize (Zea mays) seedling senses light, there is a decrease in the rate of mesocotyl elongation, an induction of root growth, and an expansion of leaves. In leaf tissues, mesophyll and bundle sheath cell fate is determined, and the proplastids of each differentiate into the dimorphic chloroplasts typical of each cell type. Although it has been inferred from recent studies in several model plant species that multiple photoreceptor systems mediate this process, surprisingly little is known of light signal transduction in maize. Here, we examine two photomorphogenic responses in maize: inhibition of mesocotyl elongation and C4 photosynthetic differentiation. Through an extensive survey of white, red, far-red, and blue light responses among a diverse collection of germplasm, including a phytochrome-deficient mutant elm1, we show that light response is a highly variable trait in maize. Although all inbreds examined appear to have a functional phytochrome signal transduction pathway, several lines showed reduced sensitivity to blue light. A significant correlation was observed between light response and subpopulation, suggesting that light responsiveness may be a target of artificial selection. An examination of C4 gene expression patterns under various light regimes in the standard W22 inbred and elm1 indicate that cell-specific patterns of C4 gene expression are maintained in fully differentiated tissues independent of light quality. To our knowledge, these findings represent the first comprehensive survey of light response in maize and are discussed in relation to maize breeding strategies.     

Identification of two loci involved in phytochrome expression in Nicotiana plumbaginifolia and lethality of the corresponding double mutant

Four Nicotiana plumbaginifolia mutants exhibiting long hypocotyls and chlorotic cotyledons under white light, have been isolated from M2 seeds following mutagenesis with ethyl methane sulphonate. In each of these mutants, this partly etiolated in white light (pew) phenotype is due to a recessive nuclear mutation at a single locus. Complementation analysis indicates that three mutants, dap5, ems28 and ems3-6-34, belong to a single complementation group called pew1, while dap1 defines the pew2 locus. The mutants at pew1 contain normal levels of immunochemically detectable apoprotein of the phytochrome that is relatively abundant in etiolated seedlings, but are deficient in spectrophotometrically detectable phytochrome, whether seedlings are grown in darkness or light. Moreover, biliverdin, a precursor of the phytochrome chromophore, restores light-regulated responses in pew1 mutants and increases their level of photoreversible phytochrome when grown in darkness. These results indicate that the pew1 locus may be involved in chromophore biosynthesis. The mutant at the pew2 locus displays no photoreversible phytochrome in etiolated seedlings, but does contain normal levels of photoreversible phytochrome when grown in the light. Biliverdin had little effect on light-regulated responses in this mutant. In addition, biliverdin did not alter the level of phytochrome in etiolated seedlings. These observations lead us to propose that this mutant could be affected in the phyA gene itself. We have also obtained the homozygous double mutant at the pew1 and pew2 loci. This double mutant is lethal at an early stage of development, consistent with a critical role for phytochrome in early development of higher plants.     

High pigment1 mutation negatively regulates phototropic signal transduction in tomato seedlings

Phototropins and phytochromes are the major photosensory receptors in plants and they regulate distinct photomorphogenic responses. The molecular mechanisms underlying functional interactions of phototropins and phytochromes remain largely unclear. We show that the tomato (Lycopersicon esculentum) phytochrome A deficient mutant fri lacks phototropic curvature to low fluence blue light, indicating requirement for phytochrome A for expression of phototropic response. The hp1 mutant that exhibits hypersensitive responses to blue light and red light reverses the impairment of second-positive phototropic response in tomato in phytochrome A-deficient background. Physiological analyses indicate that HP1 functions as a negative regulator of phototropic signal transduction pathway, which is removed via action of phytochrome A. The loss of HP1 gene product in frihp1 double mutant allows the unhindered operation of phototropic signal transduction chain, obviating the need for the phytochrome action. Our results also indicate that the role of phytochrome in regulating phototropism is restricted to low fluence blue light only, and at high fluence blue light, the phytochrome A-deficient fri mutant shows the normal phototropic response.     

Photocontrol of anthocyanin biosynthesis in tomato

Juvenile anthocyanin biosynthesis has been studied in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. During a subsequent 24-hr period of monochromatic irradiation at different fluence rates of red light (R) the fluence-rate response relationships for induction of anthocyanin in all the WTs are similar, yet complex, showing a response at low fluence rates (LFRR) followed by a fluence rate-dependent high irradiance response (HIR). In the hypocotyl this response is restricted to the sub-epidermal layer of cells. The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both response components. Theatroviolacea (atv) mutant shows strongest amplification of the HIR component. In contrast, a transgenic line overexpressing an oat phytochrome A gene (PHYA3 ⁺) shows a most dramatic amplification of the LFRR component. The far-red light (FR)-insensitive (fri) mutant, deficient in phytochrome A (phyA), lacks the LFRR component whilst retaining a normal HIR. The temporarily R-insensitive (tri) mutant, deficient in phytochrome B1 (phyB1) retains the LFRR, but lacks the HIR. Thehp-1,fri andhp-1,tri double mutant, exhibit amplified, yet qualitatively similar responses to the monogenicfri andtri mutants. Thefri,tri double mutant lacks both response components in R, but a residual response to blue light (B) remains. Similarly, theaurea (au) mutant deficient in phytochrome chromophore biosynthesis and presumably all phytochromes, lacks both response components in the R and FR regions of the spectrum. Experiments at other wavelengths demonstrate that while there is only a small response in the FR spectral region (729 nm) in tomato, there is an appreciable HIR response in the near FR at 704 nm, which is retained in thetri mutant. This suggests that the labile phyA pool participates in the HIR at this wavelength. The intense pigmentation (Ip) mutant appears to be specifically deficient in the B1 induced anthocyanin biosynthesis. Adult plants, grown under fluorescent light/dark cycles, show a reduction of anthocyanin content of young developing leaves upon application of supplemtary or end-of-day FR. The involvement of different phytochrome species in anthocyanin biosynthesis based on micro-injection studies into theau mutant and studies using type specific phytochrome mutants is discussed.     

New lv Mutants of Pea Are Deficient in Phytochrome B

The lv-1 mutant of pea (Pisum sativum L.) is deficient in responses regulated by phytochrome B (phyB) in other species but has normal levels of spectrally active phyB. We have characterized three further lv mutants (lv-2, lv-3, and lv-4), which are all elongated under red (R) and white light but are indistinguishable from wild type under far-red light. The phyB apoprotein present in the lv-1 mutant was undetectable in all three new lv mutants. The identification of allelic mutants with and without phyB apoprotein suggests that Lv may be a structural gene for a B-type phytochrome. Furthermore, it indicates that the lv-1 mutation results specifically in the loss of normal biological activity of this phytochrome. Red-light-pulse and fluence-rate-response experiments suggest that lv plants are deficient in the low-fluence response (LFR) but retain a normal very-low-fluence-rate-dependent response for leaflet expansion and inhibition of stem elongation. Comparison of lv alleles of differing severity indicates that the LFR for stem elongation can be mediated by a lower level of phyB than the LFR for leaflet expansion. The retention of a strong response to continuous low-fluence-rate R in all four lv mutants suggests that there may be an additional phytochrome controlling responses to R in pea. The kinetics of phytochrome destruction and reaccumulation in the lv mutant indicate that phyB may be involved in the light regulation of phyA levels.     

White mutants of Chlamydomonas reinhardtii are defective in phytoene synthase

Carotenoids play an integral and essential role in photosynthesis and photoprotection in plants and algae. A collection of Chlamydomonas reinhardtii mutants lacking carotenoids was characterized for pigment and tocopherol (vitamin E) composition, growth phenotypes under different light conditions, and the molecular basis of their mutant phenotype. The carotenoid-less mutants, or "white" mutants, were also deficient in chlorophylls but had approximately twice the tocopherol content of the wild type. White mutants grew in the dark but were unable to survive in the light, even under very low light conditions on acetate-containing medium. Genetic crosses and recombination tests revealed that all individual white mutants in the collection are alleles of a single gene, lts1, and the white phenotype was closely linked to a marker located in the phytoene synthase gene. DNA sequencing of the phytoene synthase gene from each of the mutants revealed nonsense, missense, frameshift, and splice site mutations. Transformation with a wild-type copy of the phytoene synthase gene was able to complement the lts1-210 mutation. Together, these results show that all the white mutants examined in this work are affected in the phytoene synthase gene.     

Genetic analysis of the roles of phytochromes A and B1 in the reversed gravitropic response of the lz-2 tomato mutant

The lz-2 mutation in tomato ( Lycopersicon esculentum ) causes conditional reversal of shoot gravitropism by light. This response is mediated by phytochrome. To further elicit the mechanism by which phytochrome regulates the lz-2 phenotype, phytochrome-deficient lz-2 plants were generated. Introduction of au alleles, which severely block chromophore biosynthesis, eliminated the reversal of hypocotyl gravitropism in continuous red and far-red light. The fri ¹ and tri ¹ alleles were introduced to specifically deplete phytochromes A and B1, respectively. In dark-grown seedlings, phytochrome A was necessary for response to high-irradiance far-red light, a complete response to low fluence red light, and also mediated the effects of blue light in a far-red reversible manner. Loss of phytochrome B1 alone did not significantly affect the behaviour of lz-2 plants under any light treatment tested. However, dark-grown lz-2 plants lacking both phytochrome A and B1 exhibited reduced responses to continuous red and were less responsive to low fluence red light and high fluence blue light than plants that were deficient for phytochrome A alone. In high light, full spectrum greenhouse conditions, lz-2 plants grew downward regardless of the phytochrome deficiency. These results indicate that phytochromes A and B1 play significant roles in mediating the lz-2 phenotype and that at least one additional phytochrome is involved in reversing shoot gravitropism in this mutant.     

Functional analysis of ZmCOP1 and ZmHY5 reveals conserved light signaling mechanism in maize and Arabidopsis

Plants have evolved light signaling mechanisms to optimally adapt developmental patterns to the ambient light environments. CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and LONG HYPOCOTYL5 (HY5) are two critical components in the light signaling pathway in Arabidopsis thaliana. COP1 acts as an E3 ubiquitin ligase that targets positive regulators, such as HY5, leading to their degradation in darkness. However, functional analysis of the COP1-HY5 module in maize (Zea mays) has not been reported. Here, we investigated the expression patterns and roles of the COP1 and HY5 orthologs, ZmCOP1 and ZmHY5, in regulating photomorphogenesis. These two genes have high amino acid identities with their Arabidopsis homolog and were both regulated by light. Subcellular localization assay showed that ZmCOP1 was distributed in the cytosol and ZmHY5 localized in the nucleus. Exogenous expression of ZmCOP1 rescued the physiological defects of the cop1-4 mutant, and expression of ZmHY5 complemented the long hypocotyl phenotype of the hy5-215 mutant in Arabidopsis. Yeast two-hybrid and fluorescence resonance energy transfer assays showed that ZmCOP1 interacted with ZmHY5. Our study gains insight into the conserved function and regulatory mechanism of the COP1-HY5 signaling pathway in maize and Arabidopsis.     

Mechanistic studies of the phytochromobilin synthase HY2 from Arabidopsis

Phytochromobilin (PPhiB) is an open chain tetrapyrrole molecule that functions as the chromophore of light-sensing phytochromes in plants. Derived from heme, PPhiB is synthesized through an open chain tetrapyrrole intermediate, biliverdin IXalpha (BV), in the biosynthesis pathway. BV is subsequently reduced by the PPhiB synthase HY2 in plants. HY2 is a ferredoxin-dependent bilin reductase that catalyzes the reduction of the A-ring 2,3,3(1),3(2)-diene system to produce an ethylidene group for assembly with apophytochromes. In this study, we sought to determine the catalytic mechanism of HY2. Data from UV-visible and EPR spectroscopy showed that the HY2-catalyzed BV reaction proceeds via a transient radical intermediate. Site-directed mutagenesis showed several ionizable residues that are involved in the catalytic steps. Detailed analysis of these site-directed mutants highlighted a pair of aspartate residues central to proton donation and substrate positioning. A mechanistic prediction for the HY2 reaction is proposed. These results support the hypothesis that ferredoxin-dependent bilin reductases reduce BV through a radical mechanism, but their double bond specificity is decided by strategic placement of different proton-donating residues surrounding the bilin substrate in the active sites.     

The Phytochrome-Deficient pcd1 Mutant of Pea Is Unable to Convert Heme to Biliverdin IX[alpha

We isolated a new pea mutant that was selected on the basis of pale color and elongated internodes in a screen under white light. The mutant was designated pcd1 for phytochrome chromophore deficient. Light-grown pcd1 plants have yellow-green foliage with a reduced chlorophyll (Chl) content and an abnormally high Chl a/Chl b ratio. Etiolated pcd1 seedlings are developmentally insensitive to far-red light, show a reduced response to red light, and have no spectrophotometrically detectable phytochrome. The phytochrome A apoprotein is present at the wild-type level in etiolated pcd1 seedlings but is not depleted by red light treatment. Crude phytochrome preparations from etiolated pcd1 tissue also lack spectral activity but can be assembled with phycocyanobilin, an analog of the endogenous phytochrome chromophore phytochromobilin, to yield a difference spectrum characteristic of an apophytochrome-phycocyanobilin adduct. These results indicate that the pcd1-conferred phenotype results from a deficiency in phytochrome chromophore synthesis. Furthermore, etioplast preparations from pcd1 seedlings can metabolize biliverdin (BV) IX[alpha] but not heme to phytochromobilin, indicating that pcd1 plants are severely impaired in their ability to convert heme to BV IX[alpha]. This provides clear evidence that the conversion of heme to BV IX[alpha] is an enzymatic process in higher plants and that it is required for synthesis of the phytochrome chromophore and hence for normal photomorphogenesis.     

The Expression of Light-Regulated Genes in the High-Pigment-1 Mutant of Tomato

Three light-regulated genes, chlorophyll a/b-binding protein (CAB), ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, and chalcone synthase (CHS), are demonstrated to be up-regulated in the high-pigment-1 (hp-1) mutant of tomato (Lycopersicon esculentum Mill.) compared with wild type (WT). However, the pattern of up-regulation of the three genes depends on the light conditions, stage of development, and tissue studied. Compared with WT, the hp-1 mutant showed higher CAB gene expression in the dark after a single red-light pulse and in the pericarp of immature fruits. However, in vegetative tissues of light-grown seedlings and adult plants, CAB mRNA accumulation did not differ between WT and the hp-1 mutant. The ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit mRNA accumulated to a higher level in the hp-1 mutant than WT under all light conditions and tissues studied, whereas CHS gene expression was up-regulated in de-etiolated vegetative hp-1-mutant tissues only. The CAB and CHS genes were shown to be phytochrome regulated and both phytochrome A and B1 play a role in CAB gene expression. These observations support the hypothesis that the HP-1 protein plays a general repressive role in phytochrome signal transduction.     

Growth analysis of wild-type and photomorphogenic-mutant tomato plants

A custom designed growth-measuring apparatus, controlled by a microcomputer has been used to study extension growth kinetics of wild-type and photomorphogenic-mutant tomato ( Lycopersicon esculentum Mill.) plants with and without end-of-day farred light (EODFR). The following photomorphogenic mutants were used. Far-red insensitive ( fri ^.1): deficient in phytochrome A (phyA); temporarily red light-insensitive ( tri ^.3): deficient in phytochrome Bl (phyB1), and their isogenic wild type (WT) cv. MoneyMaker. aurea (au) : deficient in phytochrome chromophore biosynthesis; high-pigment-1 ( hp-1 ): exhibiting exaggerated phytochrome responses, and their isogenic WT cv. Ailsa Craig. The stem elongation rate (SER) during a 24-h period of all the genotypes studied shows a similar pattern, having two dramatic transients, one shortly after the onset of the light period (a sharp decline in SER) and another shortly after the start of the dark period (a sharp increase in SER). These transients are probably associated with water relations as a consequence of opening and closure of the stomata. The fastest SER occurs during the dramatic oscillations early in the dark period. Between the genotypes there are large quantitative differences in SER. All the genotypes tested exhibited a strong EODFR response, resulting in a relative promotion of SER during the first period after the start of EODFR and in the subsequent light and dark periods. These results indicate that phyA, absent in the fri ^.1 mutant, does not play a major role in SER of light-grown tomato plants, whereas phyB 1, absent in the tri ^.3 mutant, is partly responsible for the compact stature of WT plants. An additional phytochrome other than phy A and phy B1 must therefore be capable of eliciting the EODFR response.