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Cordycepin (3′dAdo), an inhibitor of RNA synthesis and poly(A) addition to pre-mRNA, inhibits protein synthesis and the appearance of enzyme activity thought to come from stored mRNA in germinating cotton cotyledons. 3′dCyd and Actinomycin D, other RNA synthesis inhibitors, have no effect on early germination. The period of sensitivity to 3′dAdo is short, comprising the 6th–30th hour of germination. This is consistent with the idea that the stored mRNA of cotton cotyledons is not processed until germination.

Any study of ribosomes and polyribosomes from the point of view of tissue development demands a quantitative extraction of all classes of these particles from the tissue. The use of bentonite, a commonly used inhibitor of RNase, has been found to influence considerably the total yield of the ribosomal complement of germinating oat tissues, and to influence the yield of monomeric ribosomes relative to polyribosomes. To prepare ribosomes and polyribosomes from these tissues, oat seeds were germinated in darkness for 72 hours after which time the coleoptile sheath and its enclosed primary leaf were excised from the plantlet at the coleoptilar node. This material, hereafter referred to as sections, was immediately homogenized with a loose fitting, motor driven Dual1 homogenizer at l-2°C. Generally 100 to ZOO sections were utilized for each preparation and homogenized in 15 to 30 ml of medium respectively. The homogenizing medium was composed of 0.1 M trissuccinate buffer, pH, 7.6 at 5"C, 0.005 M MgC12, 0.001 PI spermidine. 0.25 M sucrose and other additives as mentioned in the text. This homogenate was centrifuged for 20 minutr-, at 10,000 gravities and sodium deoxycholate (Na Dot) added to the supernatant to give a concentration of 0.5%. The supematant was next layered over a concentrated sucrose solution (5 ml over 7 ml) in Spinco #40 rotor centrifuge tubes. If the entire ribosome population Igas to be sedimented in the subsequent centrifugation, the sucrose concentration was 1 molar; if polyribosomes, predominately free of monomeric ribosomes, were to be collected, the sucrose con

Two isoaccepting chloroplastic and one cytoplasmic tRNA Met species have been separated from germinating cotton cotyledons. The methionylated form of one of the chloroplastic species (but none of the other or of the cytoplasmic tRNA Met ) can be formylated either by an endogenous transformylase or by Escherichia coli transformylase.

Cotton cotyledons have been found to synthesize de novo during germination two enzymes, a carboxypeptidase and isocitratase. The mRNA for this synthesis is transcribed in embryogenesis at a point when the embryos have reached two-thirds of their final size. The translation of the mRNA is prevented during the completion of embryogenesis by the plant hormone abscisic acid, which is produced by the tissues surrounding the embryo and absorbed by the embryo. The inhibition of the translation of the mRNA appears to require the concomitant synthesis of nonribosomal RNA. The mRNA for the two enzymes can be prematurely induced in younger cotyledons by excision of the embryo from the mother plant. This also stops DNA synthesis and cell division in this tissue. The synthesis of the mRNA and the cessation of DNA synthesis and cell division normally occur at the time in embryogenesis when the vascular connection between the mother plant and the incipient seed degenerates. It is postulated that these events are the result of a loss of vascular factors supplied by the mother plant. The synthesis of the two enzymes appears to be identically regulated, and they may represent a body of enzymes that are required for germination and whose synthesis is under the same coordinated regulation.

We have measured the amount of tRNA in cotton cotyledons that can be charged with each of the 20 amino acids at four developmental stages in cotyledon embryogenesis and germination. We have examined the number and relative levels of isoaccepting tRNA species for 15 amino acids at the same developmental stages by RPC-2 (Freon) chromatography and by the chromatography of RNAse T1 digests of aminoacyl-tRNA. We have made the same measurements with tRNA from cotton root tissue and from partially purified cotton chloroplasts. The results suggest that there are no differences in the levels of cytoplasmic tRNA species between cotyledons of various developmental stages nor between cotyledons and roots. There is, however, a large increase in the amount of chloroplastic tRNA per cell that occurs during early germination, and this increase is not induced by a light stimulus. Chloroplast tRNA exists in young embryogenic cotyledons and roots, and the amounts of the individual chloroplastic isoaccepting tRNA species do not change relative to each other as they increase in the cotyledon cells during germination. It is concluded from these data that developmental changes in cotton seedlings are not accompanied by changes in the capacity for translating individual code words.

The control of the translation inhibition of germination mRNAs during cotton embryogenesis has been studied by determining the events necessary to remove the translation inhibition during precocious germination. The results indicate that the plant hormone abscisic acid may control translation inhibition by a mechanism involving RNA synthesis.

Cotton embryos after 72 hours of germination synthesize ribosomal RNA that does not become incorporated into ribosomes and that is very stable. This RNA has many characteristics similar to those of newly synthesized ribosomal RNA of bacteria.

This communication describes the isolation, purification and the amino acid, iron and sulfur composition of a type I ferredoxin from cotton.

In germinating cotton embryos the partial inhibition of RNA synthesis by actinomycin D does not inhibit the incorporation of leucine-(14)C into soluble protein nor cause a loss of polyribosomes during the first 16 hours of germination. This suggests that the protein synthesis observed during this period is directed by messenger RNA which exists in the mature seed and which is not rapidly degraded and resynthesized.

The synthesis of RNA has been mapped during the initial (dark) phase of germination in cotton seed. During the first 16 hours whole embryos synthesize new ribosomes and protein. Treatment of whole embryos with cycloheximide during this period inhibits protein synthesis and stops further development. Actinomycin D inhibits the synthesis of new ribosomes. This inhibition does not decrease the total ribosomal population, inhibit the synthesis of protein, or retard normal development during this time. These observations suggest that the protein synthesis observed during this period is directed by stable messenger RNA and ribosomes which exist preformed in the mature seed. The synthesis of ribosomes ceases after about 40 hours in the cotyledons and 48 hours in the roots and stems. After this time the synthesis of ribosomal RNA continues but it does not become incorporated into ribosomes. Rather, this RNA is incorporated into a particle, detectable only by radioactivity, which sediments more slowly than monomeric ribosomes on a sucrose density-gradient. On prolonged incubation, the radioactivity associated with the particle moves into mature ribosomes. The particle appears to be an intermediate in the synthesis of ribosomes and has properties similar to those reported for ribosomal precursor particles in bacteria. Its synthesis is completely suppressed by light. During the period of precursor particle synthesis in the roots and stems, protein and RNA synthesis is necessary for further development as determined by inhibition studies with cycloheximide and actinomycin D. The required RNA synthesis is possibly messenger RNA. In cotyledons, during this period, protein synthesis appears to be required for normal development. Whether this protein synthesis is dependent upon concurrent RNA synthesis cannot be evaluated by the use of actinomycin D, probably due to an inability to get the antibiotic into the cells during this period.