Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase Activity of Nuclei and Plastids from Etiolated Peas and their Response to Red and Far Red Light in Vivo

Abstract

Common wealth Scien tific a)1d Inidlustrial ResearchABSTRlACT DNA-depenidenit RINA polymiierase activity has lbeell found in both the iucleci anid etioplasts of (lark-grownvi pea seedIiiigs (Pisurn satirurn). Althliotugh thiese en-izymiies lhad simiiilar over-all clharacteristics witlh respect to silIstrate, pll, anid iinhiibitor responises, tlhey could be distiniguiished by tlhei differenit sensitivities to sonicationi.Brief irradiation of the seedlings witlh red liglht resulted in ani increase in the activity of botli the iutnclear anid plastid enizymiies, btut the timiie couirse of this response shiowedi it to be too slow to be considered aii early-resuilt of phyitocliromiie control of imietabolismii. Far red lighit follow-ving red treatient largely prevenited the inierease in thie activity of tile inuiclear eizym-ltie. The plastid enzymie responded to far red light in a imianniier simiiilar to its responise to red liglht so that no reversal was observ-ed wlheni far redl treatmiienit followed re(l. The emission intensities of the two sources are shown in Figure 1.Isolation of Nuclei and Plastids. During isolation and assay, all solutions contained 10 mm mercaptoethanol and 25 g,g ml chloramphenicol. The chloramphenicol had no detectable effect on the enzyme activity. During isolation all solutions were kept at 0 to 4 C. Approximately 10 g of terminal buds of the etiolated peas were harvested, cooled in ice, and chopped with a razor blade in 20 ml of 0.05 M tris (pH 8.3 at 4 C) containing 10 mm MgCI2 and 0.4 mI sucrose, by the method of Spencer and Wildman (20). After filtration through two layers of Miracloth (Chicopee Mills, Inc.), the sLIspension was centrifuged at 700 g for 10 min. The pellet was resuspended in 5 ml of the 0.4 M sucrose-tris-MgCl. chopping buffer and layered on a discontinuous sucrose gradient consisting of 5 ml each of 1.4 M, 1.1 M, 1.0 M, 0.8 M, and 0.6 M sucrose in 0.05 M tris (pH 8.3 at 4 C) containing 10 mM MgC12. The gradient was centrifuged at 10OOg for 15 min, after which time the intact etioplasts were recovered from the 1.0 M sucrose layer and the nuclei from the pellet at the bottom of the tube. Carotenoid Estimation. A 0.2-ml aliquot of the plastid suspension was diluted with 0.8 ml of acetone, and the solution was shaken, warmed, and centrifuged to clarify it. The absorbance of the supernatant at 473 nm was determined, and the carotenoid content was calculated assuming Ell,c to be 2500 (6).

Parental Age Affects Somatic Mutation Rates in the Progeny of Flowering Plants

“…One possible explanation is that certain unknown bioactive compounds or epigenetic changes that accumulate with age are inherited to the progeny and affect somatic mutation rates. Previous work has revealed that DNA polymerase activity decreases in older plants (Bottomley, 1970;Golubov et al, 2010), and if this age-related down-regulation is epigenetically transmitted to the progeny, it may affect somatic mutation rates. Such a down-regulation of gene expression may result from the inheritance of DNA methylation patterns, histone modifications, or small RNAs that mediate gene silencing (Brennecke et al, 2008;Boyko and Kovalchuk, 2010).…”

Some Effects of Triton X-100 on Pea Etioplasts

“…No increase in the activity of the enzyme in the supernatant fractions was observed on addition of DNA. Like the etioplast polymerase, it was rapidly destroyed by sonication (Table I), thus distinguishing it from nuclear RNA polymerase which is stable to sonication in the presence or absence of Triton (1). Treatment of the nuclei with Triton X-100 caused no loss of RNA polymerase activity.…”

“…No direct evidence for the origin of the DNA, RNA, and protein recovered after Triton treatment was obtained. However, the small nuclear contamination in the preparations (1) indicates that at least the latter two are largely of plastid origin.…”

“…Most of the methods used have been described previously (1). These include the isolation of the etioplasts and nuclei from buds of Pisum sativum cv.…”

“…DNA-dependent RNA Polymerase Acitivity. This activity was determined by measuring the incorporation of 3H-ATP into the acid-insoluble fraction as previously described (1). Each Triton X-100 Treatment.…”