Density labeling of proteins with 13C-labeled amino acids: no accumulation of an inactive alpha-amylase precursor in barley aleurone cells.

Gibberellic acid enhances α-amylase (EC 3.2.1.1) production in isolated barley aleurone layers after a lag period of 4 to 8 h, and most of the enzyme is produced after 12 h of hormone treatment. Amino acids necessary for protein synthesis in barley aleurone layers are derived from the degradation of storage proteins in this tissue. Since bromate is an inhibitor of barley protease, in the presence of bromate the production of α-amylase in aleurone layers becomes dependent on exogenous amino acids. We have incubated aleurone layers with bromate plus ¹³C-labeled amino acids and [³H]leucine from 0 to 24, 0 to 12, and 12 to 24 h after the application of gibberellic acid. The chemical quantity of [³H]leucine was negligible in comparison to that of ¹³C-labeled amino acids. Therefore, any density shift of proteins observed must be due to the incorporation of ¹³C-labeled amino acids. The density shift of α-amylase and that of newly synthesized proteins (radioactivity profile) were determined by isopycnic centrifugation in CsCl density gradients. The density shift of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 12 to 24 h after the addition of hormone was much larger than that of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 0 to 12 h of hormone treatment. By comparing the density shift of α-amylase with that of newly synthesized proteins, it is apparent that essentially all the amylase molecules are de novo synthesized. We can conclude that there is little or no accumulation of an inactive α-amylase precursor in barley aleurone cells between the time of the application of gibberellic acid and the time of the rapid increase in α-amylase activity.     

Free proline content and sensitivity to desiccation and heat during yeast sporulation and spore germination.

Ascospores of a strain of Saccharomyces cerevisiae Hansen were less sensitive to desiccation and heat than vegetative cells. Desiccation resistance was acquired earlier during sporulation and lost later during spore germination than heat resistance. As spores matured, resistance to both stresses increased. With the exception of the first few hours in sporulation medium, when proline appeared to be utilized, the intracellular free proline content increased during sporulation and decreased during spore germination. Not all the proline lost could be detected in the germination medium, indicating that some was metabolically utilized by the germinating spores. Since exogenous proline supplied to vegetative or sporulating cells before desiccation increased their survival, it is suggested that the high level of free proline in mature spores may protect against desiccation stress.     

Nitrite-reducing activity of modified cytochrome c-553 from the red alga Porphyra yezoensis Ueda

Crystalline cytochrome c-553 was obtained from Porphyra yezoensisUeda. The cytochrome in areduced form was modified to show anitrite-reducing activity after appropriate treatment with heat,hydrogen peroxide, or photooxidation using methylene blue asthe electron acceptor, but the reducing activity was far lowerthan that of the nitrite reductase isolated from this alga.The modified cytochrome c-553 was autooxidizable and showedan absorption spectrum resembling that of cytochrome c-553 inthe oxidized form except for slight shifts of the absorptionmaximumin the -band region toward shorter wavelengths. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

AvrRps4 effector family processing and recognition in lettuce

During pathogenesis, effector proteins are secreted from the pathogen to the host plant to provide virulence activity for invasion of the host. However, once the host plant recognizes one of the delivered effectors, effector‐triggered immunity activates a robust immune and hypersensitive response (HR). In planta, the effector AvrRps4 is processed into the N‐terminus (AvrRps4^N) and the C‐terminus (AvrRps4^C). AvrRps4^C is sufficient to trigger HR in turnip and activate AtRRS1/AtRPS4‐mediated immunity in Arabidopsis; on the other hand, AvrRps4^N induces HR in lettuce. Furthermore, AvrRps4^N‐mediated HR requires a conserved arginine at position 112 (R112), which is also important for full‐length AvrRps4 (AvrRps4^F) processing. Here, we show that effector processing and effector recognition in lettuce are uncoupled for the AvrRps4 family. In addition, we compared effector recognition by lettuce of AvrRps4 and its homologues, HopK1 and XopO. Interestingly, unlike for AvrRps4 and HopK1, mutation of the conserved R111 in XopO by itself was insufficient to abolish recognition. The combination of amino acid substitutions arginine 111 to leucine with glutamate 114 to lysine abolished the XopO‐mediated HR, suggesting that AvrRps4 family members have distinct structural requirements for perception by lettuce. Together, our results provide an insight into the processing and recognition of AvrRps4 and its homologues.     

TEB/POLQ plays dual roles in protecting Arabidopsis from NO-induced DNA damage

Nitric oxide (NO) is a key player in numerous physiological processes. Excessive NO induces DNA damage, but how plants respond to this damage remains unclear. We screened and identified an Arabidopsis NO hypersensitive mutant and found it to be allelic to TEBICHI/POLQ, encoding DNA polymerase θ. The teb mutant plants were preferentially sensitive to NO- and its derivative peroxynitrite-induced DNA damage and subsequent double-strand breaks (DSBs). Inactivation of TEB caused the accumulation of spontaneous DSBs largely attributed to endogenous NO and was synergistic to DSB repair pathway mutations with respect to growth. These effects were manifested in the presence of NO-inducing agents and relieved by NO scavengers. NO induced G2/M cell cycle arrest in the teb mutant, indicative of stalled replication forks. Genetic analyses indicate that Polθ is required for translesion DNA synthesis across NO-induced lesions, but not oxidation-induced lesions. Whole-genome sequencing revealed that Polθ bypasses NO-induced base adducts in an error-free manner and generates mutations characteristic of Polθ-mediated end joining. Our experimental data collectively suggests that Polθ plays dual roles in protecting plants from NO-induced DNA damage. Since Polθ is conserved in higher eukaryotes, mammalian Polθ may also be required for balancing NO physiological signaling and genotoxicity.