Quantitative Changes in In Vitro and In Vivo Protein Synthesis in Aging and Rejuvenated Soybean

Cotyledons of Light-grown soybean (GbvJne mar L. var Wayne) seedlings were used as a model system to study the possibility that aging requires qualitative changes in protein synthesis. Cotyledons reached a final stage of senescence and then abscised about 22 days after imbibition. Cotyledon senescence was reversed at 20 days after germination by epicotyl removal. Thereafter, the cotyledons regained much of the chlorophyll, RNA, protein, and polyribosomes lost during aging. Total poly(A)mRNA was extracted from 4-, 12., 20-day-old, and rejuvenated cotyledons and translated in a wheat germ system. Comparison of translation products on two-dimensional O'Farrell gels showed that many translation products increased in quantity during aging, while roughly half as many decreased. Rejuvenation returned the translation products to approximately 4-day-old levels in roughly halfof those products which were diminished with age. Conversely, almost one-third of the products which had increased with age decreased with rejuvenation. None ofthe translation products were totally lost nor were newly synthesized products detected during aging. Therefore, aging in this system probably does not involve complete gene repression or depression. The observation that epicotyl removal causes a reversal in the levels of various proteins synthesized in vitro was corroborated by similar observations following in vivo labeling of cotyledon sections and analysis by SDS-polyacrylamide gel electrophoresis and fluorography. Densitometric scans of fluorograms revealed a gradual shift in profiles of both in vitro and in vivo translation products during aging. Rejuvenated cotyledon proteins had a proffle resembling that of 4day-old cotyledons. The oVerall level of 135Simethionine incorporation into protein in vivo declined gradually during aging but was restored to 4-dayold levels within 2 days after epicotyl removal. Many theories have been advanced to explain aging in plants and animals. A major concern is whether aging is controlled by genetically programmed mechanisms. Indirect evidence supports the concept that aging in plants is programmed (15, 24) while direct evidence is available for human cells (9, 16). Changes in the ability to transcribe, process or protect RNA may result in declining protein synthesis, leaving cells unable to perform vital functions. Selective alterations in various transcription factors, tRNA turn-over rates, or aminoacyl tRNA synthetase activities may lead to qualitative changes in protein synthesis 1This work represents a portion of research submitted by Ronald W. Skadsen to Purdue University in partial fulfillment of the requirements for the Ph.D. degree. This paper is Journal Paper No. 8904 of the Purdue Agricultural Experiment Station. 2Present address: Department of Genetics, P. 0. Box 5487, North Carolina State University, Raleigh, NC 27650. 3To whom reprint requests should be addressed. during aging (3, 22). Changes in translation at these levels, as well as in rates of mRNA transcription, could reduce the synthesis of vital proteins or even favor the production of destructive proteins. The initial agent which triggers the senescence process is unknown. Cytokinins appear to regulate the decline in various macromolecules (protein, nucleic acid, etc.) associated with aging in plants (6, 10, 13, 18), and the loss of membrane permeability (20, 23, 25). We have examined changes in the spectrum of proteins synthesized during the aging and rejuvenation of soybean cotyledons. The aging of soybean cotyledons can be reversed simply by epicotyl removal, even after roughly 90%o of the nucleic acids and proteins have been lost (11). Following epicotyl removal, Chl, RNA, protein, and polyribosome levels are increased (Skadsden and Cherry, unpublished results). If aging results from changes in genetic programs, a difference in detectable protein synthesis should be observed by two-dimensional polyacrylamide gels. If, for example, aging is regulated by specific proteins which increase with age, they should decline upon rejuvenation. Alternatively, if aging results from the loss of critical proteins, they should be replenished upon rejuvenation. In the following study, the in vitro translation products from total poly(A)mRNA from aging and rejuvenated cotyledons were compared. Proteins were also labeled with [35S]methionine in vivo via cotyledon sections to assess the validity of the in vitro experiments. MATERIALS AND METHODS Plant Material. Soybeans (Glycine max L. var. Wayne) were imbibed for 6 h in tap water and sown in 53x 26x 6-cm trays in a mixture of Vermiculite:Perlite (2:1). Each tray contained about 400 seedlings. Plants were grown in a greenhouse with day temperatures of26 to 37°C and nights of 16 to 23°C. Supplemental overhead lighting was provided for 16 h during the day. Light intensities ranged from 200 to 2,000 AE/m2.s. Rejuvenation was accomplished by excising the epicotyls of 20-d-old plants (20 d after imbibition) and leaving the plants an additional 20 d before harvesting the cotyledons. Axillary shoots were continuously pruned to prevent resumption of cotyledon aging. Cotyledons were excised, rinsed with deionized H20, and stored at -55°C. mRNA Isolation. Total RNA was isolated by the method of Hall et aL (8), with the following changes. Cotyledons were ground in liquid N2 to a fine powder. Proteinase K (EM Laboratories, Germany) was added to 0.5 mg/ml for 4-d-old cotyledons and 0.2 mg/ml for 12-, 20-d-old, and rejuvenated cotyledons. The suspension were incubated at 37 to 40°C with shaking for 1 h. A 10-ml solution of 0.2 M KCI was added, and the suspension was allowed to cool on ice prior to centrifugation at 12,000g for 10 min. The extracted RNA was redissolved in 10 mm Tris acetate buffer (pH 7.5), and the A2w/AlA was determined. Poly(A)mRNA was prepared according to Aviv and Leder (1) with several modifications. Oligo(dT) cellulose type III (0.5 g, from Collaborative Research, Inc.) was equilibrated with binding