EARLY STARVATION 1 Is a Functionally Conserved Protein Promoting Gravitropic Responses in Plants by Forming Starch Granules.

Starch granules in the endodermis of plant hypocotyls act as statoliths that promote hypocotyl negative gravitropism-the directional growth of hypocotyls against gravity-in the dark. To identify the molecular components that regulate hypocotyl negative gravitropism, we performed a mutagenesis screen and isolated reduced gravitropic 1 (rgv1) mutants that lack starch granules in their hypocotyl endodermis and show reduced hypocotyl negative gravitropism in the dark. Using whole genome sequencing, we identified three different rgv1 mutants that are allelic to the previously reported early starvation 1 mutant, which is rapidly depleted of starch just before the dawn. ESV1 orthologs are present in starch-producing green organisms, suggesting ESV1 is a functionally conserved protein necessary for the formation of starch granules. Consistent with this, we found that liverwort and rice ESV1 can complement the Arabidopsis ESV1 mutant phenotype for both starch granules and hypocotyl negative gravitropism. To further investigate the function of ESV1 in other plants, we isolated rice ESV1 mutants and found that they show reduced levels of starch in their leaves and loosely packed starch granules in their grains. Both Arabidopsis and rice ESV1 mutants also lack starch granules in root columella and show reduced root gravitropism. Together, these results indicate ESV1 is a functionally conserved protein that promotes gravitropic responses in plants via its role in starch granule formation.

PHYTOCHROME INTERACTING FACTOR8 Inhibits Phytochrome A-Mediated Far-Red Light Responses in Arabidopsis.

PHYTOCHROME INTERACTING FACTORs (PIFs) are a group of basic helix-loop-helix (bHLH) transcription factors that repress plant light responses. PIF8 is one of the less-characterized Arabidopsis (Arabidopsis thaliana) PIFs, whose putative orthologs are conserved in other plant species. PIF8 possesses a bHLH motif and an active phytochrome B motif but not an active phytochrome A motif. Consistent with this motif composition, PIF8 binds to G-box elements and interacts with the Pfr form of phyB but only very weakly, if at all, with that of phyA. PIF8 differs, however, from other PIFs in its protein accumulation pattern and functional roles in different light conditions. First, PIF8 inhibits phyA-induced seed germination, suppression of hypocotyl elongation, and randomization of hypocotyl growth orientation in far-red light, but it does not inhibit phyB-induced red light responses. Second, PIF8 protein accumulates more in far-red light than in darkness or red light. This is distinct from the pattern observed with PIF3, which accumulates more in darkness. This PIF8 accumulation pattern requires degradation of PIF8 by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in darkness, inhibition of COP1 by phyA in far-red light, and promotion of PIF8 degradation by phyB in red light. Together, our results indicate that PIF8 is a genuine PIF that represses phyA-mediated light responses.

Phytochrome Regulation of Seed Germination.

Seed germination assays consist of counting the number of germinated seeds, defined as seeds in which the radicle has ruptured the endosperm and emerged from the seed coat. In Arabidopsis seed germination assays, Arabidopsis seeds are surface-sterilized, plated on agar plates containing test compounds, and incubated at specific temperatures under specific light conditions, after which the germinated seeds are counted, either with the naked eye or under a microscope. This chapter describes step-by-step protocols for Arabidopsis seed germination assays under phytochrome-dependent conditions.

Epidermal Phytochrome B Inhibits Hypocotyl Negative Gravitropism Non-Cell-Autonomously.

Seedling hypocotyls display negative gravitropism in the dark but agravitropism in the light. The Arabidopsis thaliana pif quadruple mutant (pifQ), which lacks four PHYTOCHROME-INTERACTING FACTORS (PIFs), is agravitropic in the dark. Endodermis-specific expression of PIF1 rescues gravitropism in pifQ mutant seedlings. Since phytochromes induce light responses by inhibiting PIFs and the COP1-SPA ubiquitin E3 ligase complex in the nucleus, we asked whether phyB can cell autonomously inhibit hypocotyl negative gravitropism in the endodermis. We found that while epidermis-specific expression of PHYB rescues hypocotyl negative gravitropism and all other phyB mutant phenotypes, endodermis-specific expression of PHYB does not. Epidermal phyB induces the phosphorylation and degradation of endodermal PIFs in response to red light. This induces a global gene expression pattern similar to that induced by red light treatment of seedlings expressing PHYB under the control of its own endogenous promoter. Our results imply that epidermal phyB generates an unidentified mobile signal that travels to the endodermis where it promotes PIF degradation and inhibits hypocotyl negative gravitropism.

Reaction and stabilizing mechanism of the cross-type macromonomers in the dispersion polymerization of styrene.

The cross-type (C-VUM) and linear-type (L-VUM) bifunctional vinyl urethane macromonomers and polystyrene (PS) using these macromonomers were synthesized in the dispersion polymerization in ethanol, and the reaction and stabilizing mechanism of the macromonomers was proposed. The structural verification of the macromonomers and PS was studied using (1)H NMR. The weight-average particle size of C-PS (PS prepared with C-VUM) and L-PS (PS prepared with L-VUM) decreased from 4.41 to 1.36 microm and from 3.56 to 1.52 microm, whereas the average-molecular weight of those increased from 34,100 to 100,500 g/mol and from 32,200 to 71,800 g/mol, respectively. The XPS result showed that the C-PS was anchored with a larger amount of PEG than that of the L-PS on the particle surface. Thus, the reaction and stabilizing mechanism of the macromonomers for the formation of PS particles is proposed as the following. The particle surface of the C-PS is surrounded by a large amount of tail shaped macromonomers leading to higher molecular weights and smaller particle sizes. On the other hand, the particle surface of the L-PS is comprised of relatively small amounts of loop shaped macromonomers inducing lower molecular weights and larger particles of L-PS than C-PS.