Léon Hirth

Enzymes of the phenylpropanoid pathway and the necrotic reaction of hypersensitive tobacco to tobacco mosaic virus

Abstract Pronounced increases in activity of phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (CAH) and caffeic acid- O -methyltransferase (OMT)

Biosynthesis of the coumarins: Scopoletin formation in tobacco tissue cultures

Abstract Feeding experiments of tobacco tissue cultures with (U)- 14 C-phenylalanine, 2- 14 C-cinnamic acid, 2- 14 C-glucosidoferulic acid and methyl- 14 C-methionine were carried out over periods from 3 min to 10 hr. The use of short feedings enabled us to study the kinetic aspect of the biosynthesis and to demonstrate the following main pathway: phenylalanine → free cinnamic acid → free p -coumaric acid → free caffeic acid → free ferulic acid → scopoletin → scopolin. Turn-over rates of free forms were shown to be much higher than those of the corresponding bound forms.

Changes in Protein Synthesis during the Initial Stage of Life of Tobacco Protoplasts

The biosynthesis of Fraction I protein in isolated protoplasts is compared with that in the plant. Radioactive precursors were incorporated into isolated protoplasts (“in vitro” labeling) and into leaves, from which the protoplasts were isolated later (“in situ” labeling). The biosynthesis of Fraction I protein stopped almost completely as soon as the protoplasts were incubated in the culture medium.

Partial reconstitution of tobacco mosaic virus

Abstract TMV reconstitutes rapidly in vitro at 24°; a yield of 30% was obtained after 30 min of reconstitution. The maximum yield was around 60%, reached after 7 hours. Electron micrographs of reconstituted TMV suspensions show particles of predominant lengths 700 A and 3000 A. The two populations were separated by rate zonal centrifugation on a linear sucrose gradient: the bottom zone contained infective 3000 A particles; the top zone contained whole infective RNA molecules partially coated to form a nucleoprotein of mean length 700 A. These incompletely coated particles were able to produce infective 3000 A particles when they were mixed with native viral protein. These results support a polar reconstitution.

A virus specified protein produced upon infection by cauliflower mosaic virus (CaMV)

A virus specific protein produced during infection by the cauliflower mosaic virus (CaMV) is characterized: it is a 62 Kd protein. It is constitutive of inclusion bodies or ⪡ viroplasms ⪢. This protein is coded by the viral genome: its polyadenylated mRNA hybridizes with the viral DNA. Its gene is located between the EcoRI-B and EcoRI-E sites on the restriction map.

Changes in phenylalanine ammonia-lyase during the hypersensitive reaction of tobacco to TMV.

Abstract Changes in phenylalanine ammonia-lyase (PAL) levels in TMV-infected Samsun NN leaves have been investigated in relation to two basic characteristics of hypersensitivity: necrosis and acquired systemic resistance. Higher levels of enzymatic activity are detectable before the symptoms are visible. A sharp increase occurs at the time of appearance and during subsequent development of the local necrotic lesions. The stimulated PAL activity is not related to virus multiplication but to necrosis since it depends markedly on the number of local lesions and since it is localized in narrow rings around the local lesions. In uninoculated leaves PAL activity remains at the preinoculation level during induction and development of systemic resistance. Resistant and nonresistant TMV challenge-inoculated leaves show PAL levels that, are of the same order of magnitude. The relative differences are independent of the degree of resistance and are probably caused by differences in the number of local lesions induced by challenge inoculation. Thus, phenylalanine ammonia-lyase does not seem to be involved in acquired systemic resistance.

Effect of elevated temperatures on the development of two strains of tobacco mosaic virus

Abstract The optimal temperature for multiplication of a common strain TMV(C) in submerged leaf disks was close to 24 °C. At 28 ° virus concentration decreases around the ninth day after inoculation. At 35 ° multiplication of the common strain is very low and capsids accumulate, but there is no parallel accumulation of free viral RNA. This suggests that at 35 ° and to a lesser degree at other temperatures that are not optimal, replication of viral RNA is inhibited. A “thermophilic” strain, TMV(LB), which develops actively at 36 ° was isolated. At 24 ° the multiplication of this strain was accompanied by a significant accumulation of capsids. At this same temperature the plants ( Nicotiana tabacum var. Samsun) inoculated with the thermophilic strain did not develop symptoms.

O-diphenol O-methyltransferases of healthy and tobacco-mosaic-virus-infected hypersensitive tobacco

Three distinct o-diphenol O-methyltransferases (OMTs) were found in leaves of Nicotiana tabacum, variety Samsun NN. They could be clearly distinguished by differences in elution pattern upon chromatography on DEAE-cellulose and in specificity towards 16 diphenolic substrates. The phenylpropanoids caffeic acid and 5-hydroxyferulic acid, whose importance as lignin precursors is well known, were the best substrates of OMT I, but they were also efficiently methylated by the two other OMTs that showed a broader substrate specificity. The highest rates of methylation were observed by assaying these latter enzymes with catechol, homocatechol and protocatechuic aldehyde. The flavonoid quercetin, the major o-diphenol of tobacco leaves, was a good substrate for OMTs II and III, but was also methylated significantly by OMT I. The tobacco OMTs showed both para-and meta-directing activities with protocatechuic acid, protocatechuic aldehyde and esculetin as substrates. Para-O-methylation of the former substrate arose almost exclusively from OMT I whereas that of the two latter substrates from all three enzymes. In healthy leaves the total O-methylating activity varied very much with the batch of plants whereas the relative contributions of the three enzymes were rather constant. On an average, OMTs I, II and III acounted towards caffeic acid, respectively. In tobacco mosaic virus-infected leaves carrying local necrotic lesions we found the same three OMTs with the same substrate specificities, but with increased activities. The degree of stimulation of both OMTs II and III was 2–3 times greater than that of OMT I when the leaves had a moderate number of lesions, and 3–5 times greater with large number of lesions. It is very likely that the changes in both the pattern of the O-methylating enzymes and the concentrations of the naturally occuring o-diphenolic substrates are related to an increased biosynthesis of lignins and of lignin-like compounds. These aromatic polymers could be involved in the cell wall thickening associated with the hypersensitive reaction and with the resistance to virus spread that occur in the cells surrounding the local lesions.

Translation of alfalfa mosaic virus RNA's in mammalian cell-free systems

Abstract Four alfalfa mosaic virus (AMV) RNAs have been isolated by electrophoresis on polyacrylamide gels and used as messengers in the in vitro protein-synthesizing systems prepared from rabbit reticulocyte lysates and Krebs-II ascites cells. These two systems have been shown to be capable of translating all of the RNAs. The translation product of the 12 S RNA has been found to be identical to the AMV coat protein by molecular weight estimation, serology and fingerprint analysis. The major product of 17 S RNA consists of a polypeptide of 35,000 daltons, which only represents a partial translation of this RNA. For 20 and 24 S RNAs, several specific polypeptides can be found in both cell-free systems. In contrast to the Krebs-II ascites cell system, the reticulocyte system, which is devoid of RNase activity, seems capable of producing polypeptides of high molecular weight. Moreover, in both systems, with the exception of 17 S RNA, a polypeptide corresponding to the translation of the whole length of the RNA can be found, thus suggesting a monocistronic messenger behavior for each of the three other RNAs.

Comparison of proteins synthesized in vivo and in vitro by mRNA from isolated protoplasts.

Studies of proteins synthesized in vitro by messenger RNA (mRNA) extracted from tobacco protoplasts showed that the changes in protein synthesis and especially the lack of certain proteins observed previously in isolated protoplasts did not result from a failure of translation.