The identification and characterization of seedlings hyper-responsive to light 2 (SHL2), a gene implicated in developmental responses to light

Mutants showing developmental hyper-responsiveness to limited light were screened and designated as seedlings hyper-responsive to light (shl). These mutants showed an etiolated phenotype similar to wild type in the dark, yet had shorter hypocotyls, larger cotyledons, and more advanced development of true leaves than wild type in low light. The SHL genes act (genetically) as light-dependent negative regulators of photomorphogenesis, possibly in a downstream signaling or developmental pathway that is shared by the major photoreceptor genes (CRY1, PHYA, and PHYB) and other photoreceptors (CRY2, PHYC, PHYD, and PHYE). shl1 and shl2 were shown to be partially dependent on HY5 activity for their light-hyperresponsive phenotypes. shl1-1 showed a defect in responding to auxin in its root development in both white and yellow light conditions, and showed a defect in responding to auxin in hypocotyl elongation in yellow light. Compared to wild type, both shl1-1 and shl2-2 showed increased hypocotyl length in response to cytokinin in white light. Gibberellin (GA) partially recovered shl1-1 mutant phenotype in yellow light, whereas showed no effect on hypocotyl elongation of shl2-2 in this light condition. These altered responses of shl1-1 and shl2-2 to multiple phytohormones in different light regimes suggests that cross-talks among light and hormones regulate SHL1 and SHL2. One of the SHL genes, SHL2 was cloned by map-based positional cloning and shown to be allelic to the previously identified locus designated murus3(mur3) and katamari1(kam1). MUR3/KAM1 encodes a XyG galactosyltransferase. Sequence analysis demonstrated that our original EMS generated reference allele shl2-2 is probably not a null mutant, therefore the phenotypes of T-DNA insertion null mutant in SHL2, SALK_074435 were studied in different light conditions. Unlike shl2-2, SALK_074435 had a slightly short hypocotyl phenotype in the dark (though not to the extent of the det/cop/fus mutants). A consideration of the phenotypes and molecular lesions of shl2-2 and mur3 alleles, along with the phenotypes of null alleles kam1 and SALK_74435, suggests that SHL2/MUR3/KAM1 may be involved in hypocotyl elongation in low light through the modification of xyloglucan in the plant cell wall, and may play a role in hypocotyl elongation in the dark through proper organization of the endomembrane

DFL2, a New Member of the Arabidopsis GH3 Gene Family, is Involved in Red Light-Specific Hypocotyl Elongation

A new GH3-related gene, designated DFL2, causes a short hypocotyl phenotype when overexpressed under red and blue light and a long hypocotyl when antisensed under red light conditions. Higher expression of this gene was observed in continuous white, blue and far-red light but the expression level was low in red light and darkness. DFL2 gene expression was induced transiently with red light pulse treatment. DFL2 transgenic plants exhibited a normal root phenotype including primary root elongation and lateral root formation, although primary root elongation was inhibited in antisense transgenic plants only under red light. The adult phenotypes of sense and antisense transgenic plants were not different from that of wild type. DFL2 promoter activity was observed in the hypocotyl. Our results suggest that DFL2 is located downstream of red light signal transduction and determines the degree of hypocotyl elongation.publishersversionPeer reviewe

slim shady is a novel allele of PHYTOCHROME B present in the T-DNA line SALK_015201

Auxin is a hormone that is required for hypocotyl elongation during seedling development. In response to auxin rapid changes in transcript and protein abundance occur in hypocotyls and some auxin responsive gene expression is linked to hypocotyl growth. To functionally validate proteomic studies, a reverse genetics screen was performed on mutants in auxin-regulated proteins to identify novel regulators of plant growth. This uncovered a long hypocotyl mutant, which we called slim shady, in an annotated insertion line in IMMUNOREGULATORY RNA-BINDING PROTEIN (IRR). Overexpression of the IRR gene failed to rescue the slim shady phenotype and characterization of a second T-DNA allele of IRR found that it had a wild-type hypocotyl length. The slim shady mutant has an elevated expression of numerous genes associated with the brassinosteroid-auxin-phytochrome (BAP) regulatory module compared to wild-type, including transcription factors that regulate brassinosteroid, auxin and phytochrome pathways. Additionally, slim shady seedlings fail to exhibit a strong transcriptional response to auxin. Using whole genome sequence and transcriptomics data for SALK_015201C we determined that a novel single nucleotide polymorphism in PHYTOCHROME B was responsible for the slim shady phenotype. This is predicted to convert induce a frameshift and premature stop codon at leucine 1125, within the histidine kinase-related domain of the carboxy terminus of PHYB, which is required for phytochrome signaling and function. Genetic complementation analyses with phyb-9 confirmed that slim shady is a mutant allele of PHYB. This study advances our understanding of the molecular mechanisms in seedling development, by furthering our understanding of how light signaling is linked to auxin dependent cell elongation. Furthermore, this study highlights the importance of confirming the genetic identity of research material before attributing phenotypes to known mutations sourced from T-DNA stocks

The identification and characterization of seedlings hyper-responsive to light 2 (SHL2), a gene implicated in developmental responses to light

Mutants showing developmental hyper-responsiveness to limited light were screened and designated as seedlings hyper-responsive to light (shl). These mutants showed an etiolated phenotype similar to wild type in the dark, yet had shorter hypocotyls, larger cotyledons, and more advanced development of true leaves than wild type in low light. The SHL genes act (genetically) as light-dependent negative regulators of photomorphogenesis, possibly in a downstream signaling or developmental pathway that is shared by the major photoreceptor genes (CRY1, PHYA, and PHYB) and other photoreceptors (CRY2, PHYC, PHYD, and PHYE). shl1 and shl2 were shown to be partially dependent on HY5 activity for their light-hyperresponsive phenotypes. shl1-1 showed a defect in responding to auxin in its root development in both white and yellow light conditions, and showed a defect in responding to auxin in hypocotyl elongation in yellow light. Compared to wild type, both shl1-1 and shl2-2 showed increased hypocotyl length in response to cytokinin in white light. Gibberellin (GA) partially recovered shl1-1 mutant phenotype in yellow light, whereas showed no effect on hypocotyl elongation of shl2-2 in this light condition. These altered responses of shl1-1 and shl2-2 to multiple phytohormones in different light regimes suggests that cross-talks among light and hormones regulate SHL1 and SHL2. One of the SHL genes, SHL2 was cloned by map-based positional cloning and shown to be allelic to the previously identified locus designated murus3(mur3) and katamari1(kam1). MUR3/KAM1 encodes a XyG galactosyltransferase. Sequence analysis demonstrated that our original EMS generated reference allele shl2-2 is probably not a null mutant, therefore the phenotypes of T-DNA insertion null mutant in SHL2, SALK_074435 were studied in different light conditions. Unlike shl2-2, SALK_074435 had a slightly short hypocotyl phenotype in the dark (though not to the extent of the det/cop/fus mutants). A consideration of the phenotypes and molecular lesions of shl2-2 and mur3 alleles, along with the phenotypes of null alleles kam1 and SALK_74435, suggests that SHL2/MUR3/KAM1 may be involved in hypocotyl elongation in low light through the modification of xyloglucan in the plant cell wall, and may play a role in hypocotyl elongation in the dark through proper organization of the endomembrane

The role of COP1/SPA in light signaling: Growth control, cell-cell communication and functional conservation in plants

Light is one of the most important environmental factors affecting almost all stages of plant growth and development. Arabidopsis SPA and COP1 proteins act as repressors of light signaling in darkness. Members of the SPA protein family (SPA1-SPA4) can physically interact with COP1 and, together, they constitute a functional E3-ubiquitin ligase complex. The COP1/SPA complex regulates seedling development, stomata differentiation, leaf size and photoperiodic flowering in Arabidopsis by targeting transcription factors such as HY5, HFR1, CO etc. for degradation. In the present study, I investigated in which tissues SPA1 needs to be expressed to regulate different plant developmental processes. To this end, I expressed a GUS-SPA1 fusion protein under the control of various tissue-specific promoters (phloem, leaf-mesophyll, epidermis, meristem and root) in a spa mutant background and analyzed the transgenic plants for complementation of the spa mutant phenotype. The results show that SPA1 functions exclusively in the phloem to regulate photoperiodic flowering suggesting that SPA1 acts cell-autonomously in the phloem to target its substrate CO for degradation. To regulate the leaf size, SPA1 acts in both the phloem and the leaf mesophyll, but not in the epidermis indicating non-cell autonomous effects in SPA1-dependent leaf size regulation. Moreover, phloem-specific expression of SPA1 has major effects on seedling development in both darkness and light. Eventually, stomata differentiation and epidermal pavement cell shape are also regulated by phloem-specific functions of SPA1. These results indicate that cell-cell communication plays a very important role in SPA1-regulated plant developmental processes. SPA proteins and, therefore, the COP1/SPA complexes are plant specific. However, the function of COP1 and SPA proteins are not known in plant species other than the dicot Arabidopsis. In a second project, I examined the functionality of the COP1 and SPA proteins from the moss Physcomitrella and the monocot rice in Arabidopsis. To this end, I expressed the open reading frames of rice and Physcomitrella COP1 and SPA homologs in Arabidopsis cop1 and spa mutant plants, respectively, and then analyzed the transgenic plants for complementation of the respective mutant phenotypes. Rice and Physcomitrella COP1 homologs were functional in Arabidopsis, whereas SPA homologs from these species were not functional, suggesting a conserved basic mechanism of action of COP1, but functional divergence of SPA proteins during plant evolution. Interestingly, Physcomitrella COP1 and SPA proteins interact in vitro suggesting the possibility of formation of a COP1/SPA complex early in evolution

Ultraviolet – B -mediated control of PHYTOCHROME INTERACTING FACTOR (PIF) transcription in Arabidopsis thaliana

Plants, as sessile autotrophic organisms, rely on light cues not only as a source of energy, but also to regulate developmental responses to cope with their everchanging environment. Physiological changes triggered by light vary according to the light quality that is perceived by specific specialized photoreceptors, including phytochromes, cryptochromes and UV RESISTANCE LOCUS 8 (UVR8). These photoreceptors transduce the light cues to regulate PHYTOCHROME-INTERACTING FACTORS (PIFs). PIFs are a small subset of transcription factors of the basic helix-loop-helix (bHLH) subfamily, which act as a cellular signalling hub that integrates multiple signals, including light and temperature, to regulate plant morphogenesis. The mechanisms underlying transcriptional regulation of PIFs are poorly understood in comparison to their posttranscriptional regulation. This thesis examines the transcriptional regulation of PIFs in response to low dose ULTRAVIOLET-B (UV-B) light. UV-B is shown to suppress the transcript abundance of PIF3, PIF4 and PIF5 by inhibition of promoter activity, in a UVR8- dependent manner. Evidence supporting a role for COP1 in the suppression of PIF4 and PIF5transcript abundance in UV-B is also presented. Three different mechanisms controlling UV-B - mediated suppression of PIF transcript abundance are investigated. The first involves the plant hormones, brassinosteroids (BR). This thesis shows that BR signals are not involved in the UV-B - mediated suppression of PIF4 transcript at high temperatures, but support a role for BR signalling in the UV-B-mediated suppression of thermomorphogenesis. The second involves a potential autoregulatory loop involving UV-B-mediated degradation of PIF protein. Data suggest that UV-B - mediated PIF4 degradation may occur via an alternative pathway to PIF5. The third investigates the role of MYB30 in regulating PIF transcript abundance. Data show that MYB30 is suppressed by UV-B in a UVR8-dependent manner and promotes PIF7 transcription in white light. In addition, MYB30 regulates shade-avoidances responses to green shade.<br/