Institutional Publications

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PHYTOCHROME INTERACTING FACTOR1 interactions leading to the completion or prolongation of seed germination
(Taylor & Francis Group, 2018) Dirk, Lynnette M. A.; Kumar, Santosh; Majee, Manoj; Downie, A. Bruce
In Arabidopsis thaliana, the basic Helix Loop Helix transcription factor, PHYTOCHROME INTERACTING FACTOR1 (PIF1) is known to orchestrate the seed transcriptome such that, ultimately, proteins repressing the completion of germination are produced in darkness. While PIF1-mediated control of abscisic acid (ABA) and gibberellic acid (GA) anabolism/catabolism is indirect, PIF1 action favors ABA while discriminating against GA, firmly establishing ABA’s repressive influence on the completion of germination. The result is tissue that is more sensitive to and producing more ABA; and is less responsive to and deficient in GA. Illumination of the appropriate wavelength activates phytochrome which enters the nucleus, and binds to PIF1, initiating PIF1’s phosphorylation by diverse kinases, subsequent polyubiquitination, and hydrolysis. One mechanism by which phosphorylated PIF1 is eliminated from the cells of the seed upon illumination involves an F-BOX protein, COLD TEMPERATURE GERMINATING10 (CTG10). Discovered in an unbiased screen of activation tagged lines hastening the completion of seed germination at 10°C, one indirect consequence of CTG10 action in reducing PIF1 titer, should be to enhance the transcription of genes whose products work to increase bioactive GA titer, shifting the intracellular milieu from one that is repressive to, toward one conducive to, the completion of seed germination. We have tested this hypothesis using a variety of Arabidopsis lines altered in CTG10 amounts. Here we demonstrate using bimolecular fluorescence complementation that PIF1 interacts with CTG10 and show that, in light exposed seeds, PIF1 is more persistent in ctg10 relative to WT seeds while it is less stable in seeds over-expressing CTG10. These results are congruent with the relative transcript abundance from three genes whose products are involved in bioactive GA accumulation. We put forth a model of how PIF1 interactions in imbibed seeds change during germination and how a permissive light signal influences these changes, leading to the completion of germination of these positively photoblastic propagules.
Kelch f-box protein positively influences Arabidopsis seed germination by targeting phytochrome-interacting factor1
(PNAS, USA, 2018) Majee, Manoj; Kumar, Santosh; Kathare, Praveen Kumar; Wu, Shuiqin; Gingerich, Derek; Nayak, Nihar R.; Salaita, Louai; Dinkins, Randy; Martin, Kathleen; Goodin, Michael; Dirk, Lynnette M. A.; Lloyd, Taylor D.; Zhu, Ling; Chappell, Joseph; Hunt, Arthur G.; Vierstra, Richard; Huq, Enamul; Downie, A. Bruce
Seeds employ sensory systems that assess various environmental cues over time to maximize the successful transition from embryo to seedling. Here we show that the Arabidopsis F-BOX protein COLD TEMPERATURE-GERMINATING (CTG)-10, identified by activation tagging, is a positive regulator of this process. When overexpressed (OE), CTG10 hastens aspects of seed germination. CTG10 is expressed predominantly in the hypocotyl, and the protein is localized to the nucleus. CTG10 interacts with PHYTOCHROME-INTERACTING FACTOR 1 (PIF1) and helps regulate its abundance in planta. CTG10-OE accelerates the loss of PIF1 in light, increasing germination efficiency, while PIF1-OE lines fail to complete germination in darkness, which is reversed by concurrent CTG10-OE. Double-mutant (pif1 ctg10) lines demonstrated that PIF1 is epistatic to CTG10. Both CTG10 and PIF1 amounts decline during seed germination in the light but reaccumulate in the dark. PIF1 in turn down-regulates CTG10 transcription, suggesting a feedback loop of CTG10/PIF1 control. The genetic, physiological, and biochemical evidence, when taken together, leads us to propose that PIF1 and CTG10 coexist, and even accumulate, in the nucleus in darkness, but that, following illumination, CTG10 assists in reducing PIF1 amounts, thus promoting the completion of seed germination and subsequent seedling development.
A misannotated locus positively influencing Arabidopsis seed germination is deconvoluted using multiple methods, including surrogate splicing
(Elsevier B.V., 2017) Majee, Manoj; Wu, Shuiqin; Salaita, Louai; Gingerich, Derek; Dirk, Lynnette M.A.; Chappell, Joseph; Hunt, Art G.; Vierstra, Richard; Downie, A. Bruce
A screen of activation tagged lines of Arabidopsis thaliana retrieved COLD TEMPERATURE GERMINATING10-D(tag) (CTG10-D(tag)) seeds, capable of radicle protrusion in advance of wild type (WT) at suboptimal- and optimal-temperatures. Genomic walking revealed T-DNA in the intragenic region that upregulates expression of the At4g19330 locus previously predicted to encode an F-BOX protein. A combination of surrogate splicing, primer scanning, RACE, and Illumina PolyAdenylation Tag (PAT) sequencing in petunia (Petunia X hybrida) and Arabidopsis were required to demonstrate that the region around At4g19330 was misannotated and is actually compromised of two separate genes. Even though homologous regions nearby and elsewhere on chromosome 4 are confounding elements in the molecular characterization of the locus, we could determine that the 5′ entity encodes a ribonucleoprotein of unknown function whereas the 3′ gene includes the promoter and full coding region of an F-BOX protein. Although both genes were upregulated, only independently-transformed lines over-expressing the F-Box exhibited enhanced completion of seed germination. We named it CTG10, and a single, poorly penetrant, mutant line of ctg10 manifested the expected reduced completion of seed germination. Whereas CTG10-OE lines are hyposensitive to the gibberellin biosynthetic inhibitor paclobutrazol, the ctg10 mutant line is hypersensitive; a phenotype which could be alleviated when transgenically rescued with CTG10. The F-Box moiety promoted association of CTG10 with ASK proteins in yeast two hybrid assays, indicating that it likely assembles into an SCF-type ubiquitin ligase to promote the ubiquitination of one or more substrates. In this capacity CTG10 might target a protein repressing seed germination for polyubiquitination and subsequent proteasomal degradation.

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