Biochemical properties and chloroplast fine-structure of a viridis mutant of Gateway barley are described. The mutant was deficient in chlorophyll and carotenoids when young but developed nearly normal levels of pigment with age (a virescens type). Etiolated mutant seedlings were low in protochlorophyll. In the young mutant, chloroplasts were small and irregular in shape and they contained large vesicles but no normal lamellae or grana. The level of free amino acids, particularly of serine, was extremely high. These disturbances were overcome with age. It is suggested that the mutation alters the lipoprotein of lamellar structures in the plastid, and that other effects are consequential.

The polyamines putrescine, spermidine, and spermine prevent the loss of chlorophyl normaDly associated with senescence of excised leaf tissue maintained in darkness on water (control). Retention of chlorophyl in barley leaf discs was in the range of 90% 4 days after excision and placement on effective polyamine solutions. In contrast, the loss of soluble protein was hastened with 0.5 miLimolar spermidine and spermine treatments but it was retarded by 0.5 millimolar putrescine.Photosystem I and II activities of chloroplasts from polyamine-treated leaf discs declined more rapidly as compared to the control. Chloroplast ultrastructural changes resulting from the polyamine treatments included the apparent destruction of the envelope, preservation of thylakoid membrane structure, and reduced accumulation of osmiophilic bodies. The influence of polyamines on senescence-related processes may be due to their cationic nature.Natural and artificially induced senescence of leaf tissue have been used to study the effects of a number of metabolites and growth regulators on protein metabolism and on the structure and photochemical properties of chloroplasts (9,16,18,23,26,32,36). In this respect it has been found that kinetin can retard the characteristic loss of protein and pigments during leaf senescence (7,15,22) as well as preserve chloroplast structure (7) and the integrity of the electron transport chain (15). Poovaiah and Leopold (27) have reported that inorganic cations, particularly Ca, can also defer the symptoms of leaf senescence, an effect they attributed to the maintenance of membrane integrity.The stabilization of membrane integrity by Ca and the naturally occurring organic cations, the polyamines, has been shown with osmotically sensitive forms of different bacteria (19,30). More recently, polyamines have also been found to be capable of retarding the progressive senescence of protoplasts isolated from oat leaves (1, 17). There are many examples from both prokaryotes and eukaryotes of polyamine involvement in the regulation of macromolecular synthesis (6, 10, 13, 31), and it has been shown that polyamines can replace the Mg2" requirement for protein synthesis in a barley in vitro system (12). The present study was undertaken to determine the effects of polyamines on Chl and protein retention, photochemical activity, and chloroplast ultrastructure of barley leaf discs during senescence.

The effect of spermine on photochemical activity and polypeptide conposition of chloroplasts from barley leaf discs during senescence in the dark was studied. Chloroplast membranes did not show pbotosystem II activity after spermine treatment when water was the electron donor, but in the presence of diphenylcarbazide, this activity was observed. The diphenylcarbazide-stimulated photoreduction of dichloroindophenol was 3-fold greater in leaf discs incubated for 72 hours in spermine than in water.Photosystem I activity was reduced by about 90% within the first 24 hours in the spermine-treated samples. This reduction, however, was not due to a decrease in the photosynthetic unit size. A preferential lss of polypeptides other than those associated with photosystem II was observed during senescence of the leaf discs in water, but this loss was reduced by spermine. Spermine treatment also prevented the appearance of several additional chlorophyll proteins found in the controls during senescence. The results have been interpreted on the basis of the interaction of spermine with thylakoid membranes resulting in stabilization of membrane function during senescence.The role of polyamines in plant cell metabolism is not fully understood, although their significance in biochemical processes such as protein synthesis (6,11,13) and RNA degradation (19) has been recognized. Polyamines have been shown to inhibit senescence in oat leaf protoplasts (1) and in whole leaves of a number ofplant species (18), and to stabilize bacterial spheroplasts against lysis (29). In the preceding study (12) we found that of the polyamines tested spermine was the most effective in reducing Chl destruction and maintaining the thylakoid system within the chloroplasts of barley leaf discs during stress-induced senescence. It was suggested that the cationic polyamines exert their influence through interaction with the negatively charged loci on the membranes. We extended the investigation to examine the photoreductive activities and thylakoid membrane polypeptide composition of barley leaf discs incubated in spermine. The results, in general, provide further evidence that polyamines stabilize the chloroplast membranes, thereby preventing the loss of Chl during senescence.

The development of photochemical activity in relation to pigment and membrane protein accumulation in chloroplasts of greening wild-type barley (Hordeum vulgare L. cv. Gateway) and its virescens mutant were studied. The rate of chlorophyll accumulation per plastid was faster in the wild-type than in the mutant seedlings upon illumination after 6 days of etiolation, but was not different after 8 days. Although the protein content per plastid did not vary during greening, there was a change in the sodium dodecyl sulfate-polyacrylamide gel polypeptide profiles. High molecular weight proteins of %,000 and 66,000 decreased whereas those at 34,000, 27,000 and 22,000 increased in relative quantity as a function of greening. The fully greened mutant seedlings were not deficient in the light-harvesting chlorophyll protein complex (LHC) or the reaction centers of photosystem I and photosystem 11. Photosystem I-associated photochemical activities appeared within the first hour of plastid development and photosystem 11 associated activities and 02 evolution within the next 6 hours. In all cases, the developmental rates per unit protein were slower in the mutant following 6 days of etiolation, but no differences between the two genotypes could be seen after 8 days due to a decrease in the developmental rate of the wild-type chloroplasts. An increase in photosynthetic unit size associated with plastid morphogenesis was faster in the wild-type seedlings after 6 days, but again the difference was negligible after 8 days. It was concluded that no single measured photochemical parameter is affected by this mutation, but rather, all aspects of chloroplast development are affected similarly by an overall reduction in the rate of chloroplast morphogenesis. This mutant, therefore, undergoes the normal pattern of proplastid to chloroplast development, but at a markedly reduced rate.

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