UBIQUITIN-PHYTOCHROME CONJUGATES - POOL DYNAMICS DURING INVIVO PHYTOCHROME DEGRADATION

  • 25 March 1989
    • journal article
    • research article
    • Vol. 264  (9) , 4998-5005
Abstract
The plant photoreceptor chromoprotein, phytochrome, is rapidly degraded in vivo after photoconversion from a stable red light-absorbing form (Pr) to a far-red light-absorbing form (Pfr). Previously, we demonstrated that during Pfr degradation in etiolated oat seedlings, ubiquitin-phytochrome conjugates, (Ub-P), appear and disappear suggesting that phytochrome is degraded via a ubiquitin-dependent proteolytic pathway (Shanklin, J., Jabben, M., and Vierstra, R. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 359-363). Here, we provide additional kinetic and localization data consistent with this hypothesis by exploiting the unique ability to photoregulate phytochrome degradation in vivo. An assay for the quantitation of Ub-P was developed involving immunoprecipitation of total conjugates with anti-ubiquitin antibodies, followed by the detection of Ub-P with anti-phytochrome antibodies. Using this immunoassay, we found that Ub-P will accumulate to approximately 5% of initial phytochrome during Pfr degradation induced by a saturating red light pulse. Reducing the amount of Pfr produced initially by attenuating the red light pulse, lowered the amount of phytochrome degraded in the following dark period and concomitantly reduced the maximal accumulation of Ub-P. Continuous far-red irradiations that maintained only 4% of phytochrome as Pfr induced rapid phytochrome degradation similar to that induced by a red light pulse converting 86% of Pr to Pfr. The appearance and disappearance of Ub-P were similar for each irradiation indicating that Ub-P accumulation is independent of the level of Pfr provided rapid phytochrome degradation is maintaned. Pulse-chase studies employing continuous far-red light followed by darkness showed that Ub-P are continuously synthesized during phytochrome degradation and rapidly disappear once degradation ceases. Ub-P also accumulated during "cycled Pr" degradation induced by the transformation of Pr to Pfr and back to Pr. The commitment to degrade cycled Pr and form Ub-P occurred within seconds after Pfr formation making the cause(s) underlying this phenomenon one of the fastest phytochrome reactions known. Within seconds after Pfr formation, a majority of phytochrome is also known to aggregate in vivo (previously defined as sequestered or pelletable), with aggregated phytochrome preferentially lost during phytochrome degradation. In vitro analysis of aggregated phytochrome indicated that they contain most of the Ub-P. Moreover, the appearance of Ub-P in the aggregated and soluble fractions correlated with the time that phytochrome disappeared from that fraction. Taken together, these observations suggest that light-induced phytochrome degradation involves a step after Pfr formation that can rapidly commit phytochrome to degradation regardless of the final spectral form. This event is followed by conjugation of ubiquitin to the chromoprotein and finally catabolism of the conjugated protein. Both kinetic and localization data suggest that light-induced phytochrome aggregation represents an early step in degrading this chromoprotein.