B-box containing proteins play an important role in light signaling in plants. Here, we identify LIGHT-REGULATED ZINC FINGER1/SALT TOLERANCE HOMOLOG3 (STH3), a B-box encoding gene that genetically interacts with two key regulators of light signaling, ELONGATED HYPOCOTYL5 (HY5) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). STH3 physically interacts with HY5 in vivo and shows a COP1-dependent localization to nuclear speckles when coexpressed with COP1 in plant cells. A T-DNA insertion mutant, sth3, is hyposensitive to high fluence blue, red, and far-red light and has elongated hypocotyls under short days. Analyses of double mutants between sth3, sth2, and hy5 suggest that they have partially overlapping functions. Interestingly, functional assays in protoplasts suggest that STH3 can activate transcription both independently and together with STH2 through the G-box promoter element. Furthermore, sth3 suppresses the cop1 hypocotyl phenotype in the dark as well as the anthocyanin accumulation in the light. Finally, COP1 ubiquitinates STH3 in vitro, suggesting that STH3 is regulated by COP1. In conclusion, we have identified STH3 as a positive regulator of photomorphogenesis acting in concert with STH2 and HY5, while also being a target of COP1-mediated ubiquitination.

SUMMARYInositol pyrophosphates are unique cellular signaling molecules with recently discovered roles in energy sensing and metabolism. Studies in eukaryotes have revealed that these compounds have a rapid turnover, and thus only small amounts accumulate. Inositol pyrophosphates have not been the subject of investigation in plants even though seeds produce large amounts of their precursor, myo-inositol hexakisphosphate (InsP 6 ). Here, we report that Arabidopsis and maize InsP 6 transporter mutants have elevated levels of inositol pyrophosphates in their seed, providing unequivocal identification of their presence in plant tissues. We also show that plant seeds store a little over 1% of their inositol phosphate pool as InsP 7 and InsP 8 . Many tissues, including, seed, seedlings, roots and leaves accumulate InsP 7 and InsP 8 , thus synthesis is not confined to tissues with high InsP 6 . We have identified two highly similar Arabidopsis genes, AtVip1 and AtVip2, which are orthologous to the yeast and mammalian VIP kinases. Both AtVip1 and AtVip2 encode proteins capable of restoring InsP 7 synthesis in yeast mutants, thus AtVip1 and AtVip2 can function as bonafide InsP 6 kinases. AtVip1 and AtVip2 are differentially expressed in plant tissues, suggesting nonredundant or non-overlapping functions in plants. These results contribute to our knowledge of inositol phosphate metabolism and will lay a foundation for understanding the role of InsP 7 and InsP 8 in plants.

Peroxisomes are single membrane-delimited subcellular organelles that carry out numerous vital metabolic reactions in nearly all eukaryotes. Peroxisomes alter their morphology, abundance, and enzymatic constituents in response to environmental cues, yet little is known about the underlying mechanisms. In this work, we investigated the regulatory role of light in peroxisome proliferation in Arabidopsis (Arabidopsis thaliana). We provide evidence that light induces proliferation of peroxisomes in Arabidopsis seedlings and that the peroxisomal protein PEX11b plays an important role in mediating this process. The far-red light receptor phytochrome A (phyA) and the bZIP transcription factor HY5 HOMOLOG (HYH) are both required for the up-regulation of PEX11b in the light. We further demonstrate that the phyA and hyh mutants exhibit reduced peroxisome abundance, a phenotype that can be rescued by overexpressing PEX11b in these plants. The HYH protein is able to bind to the promoter of PEX11b, suggesting that the PEX11b gene is a direct target of HYH. We conclude that HYH and PEX11b constitute a novel branch of the phyA-mediated light signaling cascade, which promotes peroxisome proliferation during seedling photomorphogenesis.

Quantitative reverse transcriptase PCR (RT-PCR) is a sensitive method for the measurement of mRNA copy number. However, the methodology has gained a reputation for poor reproducibility, leading to concern over the validity of much of the data generated using this technique. We have developed two variants of quantitative competitive RT-PCR using a synthesized RNA as an internal standard to measure precisely the relative levels of alpha-, beta- and gamma-fibrinogen mRNAs in the four lobes of the rat liver. In the first of these variants we altered only the amount of total RNA in the RT-PCR reaction, keeping the amount of internal standard RNA and the number of PCR cycles constant. In the second variant only the number of PCR cycles was altered, and the amounts of total RNA and standard RNA were kept constant. Both variants of RT-PCR allowed calculation of the number of mRNA copies, which did not differ significantly between the two techniques. Of the two variants, the second gave better reproducibility, and the intra-assay coefficient of variation for this technique was 14% (n = 20). Using these two variants we have shown that there are different numbers of fibrinogen mRNAs in the four liver lobes for each of the three genes (alpha-fibrinogen F = 14.64, P = 0.0003; beta-fibrinogen F = 3.74, P = 0.04; gamma-fibrinogen F = 3.75, P = 0.04). In conclusion, by using two variants of quantitative competitive RT-PCR we have shown that this technique can be used to give reproducible results, and the low intra-assay coefficient of variation suggests that quantitative RT-PCR should be the technique of choice for accurate measurement of mRNA copy number.

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