Reprogramming of Protein Synthesis from a Developmental to a Germinative Mode Induced by Desiccation of the Axes of Phaseolus vulgaris

Abstract

Immature seeds of Phaseolus vulgaris cv Taylor's Horticultural removed from the pod at 32 days of development do not germinate unless first subjected to desiccation. Our results show that premature drying not only redirects metabolism from a developmental to a germination program but it does so permanently, thus effecting an irreversible switch. This is shown by in vitro protein synthesis, and analysis of poly(A)' mRNA with a cDNA probe specific for phaseolin message. For example, the pattern of proteins synthesized in vitro by the mRNA fraction from fresh and prematurely dried axes show strong similarities; on the other hand, the mRNA population from rehydrated axes code for a different set of proteins. Also, the message for phaseolin is preserved following the normal maturation process and premature desiccation of seeds. Following rehydration of immature seeds at the desiccation-tolerant stage, this message is no longer detectable in the axes.For most seeds, maturation drying is the terminal event in development, leading to a state of metabolic quiescence. Prior to this event, the metabolism of the seed is geared towards the completion of developmental events, in particular the biosynthesis and deposition of stored reserves. Following drying, on the other hand, the seed's metabolism is exclusively related to germination and growth; reserve deposition ceases and hydrolytic processes resulting in the degradation of the storage reserves are instituted (2,14,21,25,28 with a cloned cDNA probe for phaseolin, the major storage protein, we have shown that the changes occur in the protein profiles in response to premature desiccation of Phaseolus vulgaris. The changes are due to differences in the translatable message population between developing and germinating axes, and mimic those changes which occur during the normal course of maturation drying. MATERIALS AND METHODSPhaseolus vulgaris seeds (cv Taylor's Horticultural) were purchased from Asgrow Canada Ltd., Brantford, Ontario, and stored at 4°C. Developing seeds were harvested from plants grown under natural and artificial daylight conditions in a greenhouse. Seeds were pooled according to their developmental stages, as described by Walbot et al. (35), and used either in the fresh state or after drying in screw-cap jars over activated silica gel at room temperature for 48 h. Where required, seeds were imbibed in sterile distilled H20 in darkness at 22C. For RNA extraction, axes of imbibed mature seeds and fresh immature seeds were frozen in liquid N2 and stored at -70°C.Extraction of RNA. Bulk RNA from embryo axes was prepared by phenol:chloroform extraction (7). The plant tissue was ground to a fine powder with liquid N2 in a mortar and pestle. Two g of powder were suspended in an emulsion that was prepared by mixing 10 ml of phenol with 10 ml of 0.1 M Tris-HCl buffer (pH 9.0) containing 100 mM NaCl, 2 mm EDTA, and 1% SDS [w/v]. The suspension was blended at room temperature by five 8-s bursts ofa Teckman Tissuemizer (John's Scientific, Toronto) and the r...

Seed development and maturation inPhaseolus vulgarisI. Ability to germinate and to tolerate desiccation

“…In discussing desiccation tolerance, it is necessary to be clear as to the moisture contents to which seeds are dried before they are rehydrated. Much of the work on desiccation tolerance in many species, including bean (Dasgupta et al, 1982;Misra and Bewley, 1985;Kermode et al, 1986), is unclear as to the precise moisture contents after drying. Recent work on Norway maple (Acer platanoides L.) (Hong and Ellis, 1992) and rice (Oryza sativa L.) (Ellis et al, 1993;Ellis and Hong, 1994) has emphasized that the development of desiccation tolerance to low moisture contents (5%) can lag considerably behind that for tolerance of desiccation to higher moisture contents.…”

Vicilin and Napin Storage-Protein Gene Promoters Are Responsive to Abscisic Acid in Developing Transgenic Tobacco Seed but Lose Sensitivity following Premature Desiccation

“…Some evidence lends support to this contention, and at the transcriptional level, some of the cis-regulatory sequences responsive to ABA within developmental genes (e.g. the sunflower helianthinin storage-protein gene) have been identified (reviewed by Thomas et al,199 1; Thomas, 1993;Nunberg et al, 1994). Our previous studies show that the 5' upstream regions of napin and vicilin storage-protein genes are responsive to desiccation in transgenic tobacco seed (Jiang and Kermode, 1994;Jiang et al, 1995a).…”

“…Similarly, in wheat, alfalfa, rape, and soybean embryos, the decline in endogenous ABA during late development is accompanied by a corresponding decrease in tissue sensitivity to exogenous ABA, as measured by its capacity to inhibit germination and to maintain or enhance expression of developmental storageprotein genes (Eisenberg and Mascarenhas, 1985;Finkelstein et al, 1985;Williamson et al, 1985;Quatrano, 1986;Xu and Bewley, 1991). When developing transgenic tobacco seeds (expressing a helianthinin 5' GUS chimeric gene) are incubated on a basal medium containing 1OP6 M ABA, they exhibit enhanced GUS activities, as compared to control seeds placed on a basal medium minus ABA (Thomas et al, 1991;Nunberg et al, 1994). However, the responsiveness of GUS expression to ABA declines as seed development progresses.…”

“…When desiccation is imposed prematurely at certain stages prior to the completion of development, a switch in synthetic events is elicited; events unique to development, such as synthesis of storage protein, are terminated, and syntheses associated with germination and growth are initiated (Evans et al, 1975;Dasgupta and Bewley, 1982;Adams et al, 1983;Kermode and Bewley, 1985a, 1985b, 1986bKermode et al, 1985;Misra and Bewley, 1985;Cornford et al, 1986;Rosenberg and Rinne, 1987;Oishi and Bewley, 1992). In effecting this switch in gene expression, desiccation appears to act primarily at the transcriptional and posttranscriptional levels (Misra and Bewley, 1985;Cornford et al, 1986;Kermode et al, 1989c;Jiang and Kermode, 1994;Jiang et al, 1995aJiang et al, , 1995b. We are interested in examining events at the molecular leve1 caused by drying, which ultimately result in the off-or down-regulation of development-related genes and the onregulation of germinative and postgerminative genes.…”

Role of desiccation in the termination of expression of genes for storage proteins

“…Although there are important exceptions, changes in the extent of synthesis of storage protein during seed development appear to be related to the amount of appropriate mRNAs available (Goldberg et al, 1981a, b), which is characteristic of regulatory processes effected primarily at the level of transcription (mRNA synthesis) and mRNA stability (reviewed in Higgins, 1984, Muntz, 1987, Goldberg et al, 1989, Bewley and Marcus, 1990 The changes in protein synthesis from a developmental to a germinative/ growth mode are indicative of a switch in genome activity This results in permanent suppression of developmental protein synthesis and induction of germination-and growth-related proteins The molecular level of control at which desiccation effects this switch in metabolic activity has been investigated (Misra and Bewley, 1985, Cornford et al, 1986, Bewley et al, 1989, Kermode et al, 1989c by examining the effects of premature desiccation and rehydration upon the fate and stability of the mRNA populations in the seed A switch in message population is induced by premature desiccation and rehydration of P vulgans axes (at a tolerant stage of development) For example, the mRNAs for several major developmental proteins (including the storage protein phaseohn) decline during early rehydration after premature drying New mRNAs, coding for germination proteins, are available for utilization in these rehydrated axes, messages which were not present either before drying or in the dry state (Misra and Bewley, 1985) Desiccation of P vulgans axes at 22 DAP (the intolerant stage) does not lead to any significant change in their complement of translatable messages The mRNAs present upon rehydration code largely for developmental proteins, germination messages are not produced in detectable amounts Thus, when the seed is still desiccation intolerant, drying neither elicits 'germination-gene' activity nor leads to a decline in developmental messages after rehydration…”