José Javier Pueyo

Photoinhibition of Photosystem II from Higher Plants EFFECT OF COPPER INHIBITION

Strong illumination of Cu(II)-inhibited photosystem II membranes resulted in a faster loss of oxygen evolution activity compared with that of the intact samples. The phenomenon was oxygen- and temperature-dependent. However, D1 protein degradation rate was similar in both preparations and slower than that found in non-oxygen evolving PSII particles (i.e. Mn-depleted photosystem II). These results seem to indicate that during illumination Cu(II)-inhibited samples do not behave as a typical non-oxygen evolving photosystem II. Cytochrome b559 was functional in the presence of Cu(II). The effect of Cu(II) inhibition decreased the amount of photoreduced cytochrome b559 and slowed down the rate of its photoreduction. The presence of Cu(II) during illumination seems to protect P680 against photodamage as occurs in photosystem II reaction centers when the acceptor side is protected. The data were consistent with the finding that production of singlet oxygen was highly reduced in the preparations treated with Cu(II). EPR spin trapping experiments showed that inactivation of Cu(II)-treated samples was dominated by hydroxyl radical, and the loss of oxygen evolution activity was diminished by the presence of superoxide dismutase and catalase. These results indicate that the rapid loss of oxygen evolution activity in the presence of Cu(II) is mainly due to the formation of ˙OH radicals from superoxide ion via a Cu(II)-catalyzed Haber-Weiss mechanism. Considering that this inactivation process was oxygen-dependent, we propose that the formation of superoxide occurs in the acceptor side of photosystem II by interaction of molecular oxygen with reduced electron acceptor species, and thus, the primarily Cu(II)-inhibitory site in photosystem II is on the acceptor side.

Activation of Bean (Phaseolus vulgaris) [alpha]-Amylase Inhibitor Requires Proteolytic Processing of the Proprotein

Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the [alpha]-amylases of mammals and insects. This [alpha]-amylase inhibitor ([alpha]AI) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (Mr) 15,000 to 18,000. We report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, we found that antibodies to [alpha]AI recognize large (Mr 30,000–35,000) polypeptides as well as typical [alpha]AI processing products (Mr 15,000–18,000). [alpha]AI activity was found in all extracts that had the typical [alpha]AI processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, we made a mutant [alpha]AI in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-[alpha]AI when the gene is expressed in tobacco. When pro-[alpha]AI was separated from mature [alpha]AI by gel filtration, pro-[alpha]AI was found not to have [alpha]-amylase inhibitory activity. We interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. We suggest that the polypeptide cleavage removes a conformational constraint on the precursor to produce the biochemically active molecule.

Degradation of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in the vacuole

To understand how plant cells exert quality control over the proteins that pass through the secretory system we examined the transport and accumulation of the bean (Phaseolus vulgaris L.) vacuolar storage protein phaseolin, structurally modified to contain a helix-breaking epitope and carboxyterminal HDEL, an endoplasmic reticulum (ER)-retention signal. The constructs were expressed in tobacco (Nicotiana tabacum L.) with a seedspecific promoter. The results show that phaseolin-HDEL accumulates in the protein-storage vacuoles, indicating that HEDL does not contain sufficient information for retention in the ER. However, the ER of seeds expressing the phaseolin-HDEL construct contain relatively more phaseolin-HDEL compared to phaseolin in the ER of seeds expressing the phaseolin construct. This result indicates that the flow out of the ER is retarded but not arrested by the presence of HDEL. Introduction into phaseolin of the epitope “himet” (Hoffman et al., 1988, Plant Mol. Biol. 11, 717–729) greatly reduces the accumulation of HiMet phaseolin compared to normal phaseolin. However, the increased abundance within the ER is similar for both phaseolin-HDEL and HiMet phaseolin-HDEL. Using immunocytochemistry with specific antibodies, HiMet phaseolin was found in the ER, the Golgi stack, and in transport vesicles indicating that it was transport competent. It was also present at an early stage of seed development in the protein-storage vacuoles, but was not found there at later stages of seed development. Together these results support the conclusion that the HiMet epitope did not alter the structure of the protein sufficiently to make it transport incompetent. However, the protein was sufficiently destabilized to be degraded by vacuolar proteases.

Overexpression of Flavodoxin in Bacteroids Induces Changes in Antioxidant Metabolism Leading to Delayed Senescence and Starch Accumulation in Alfalfa Root Nodules

Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis flavodoxin, a protein that is involved in the response to oxidative stress. Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control bacteria. The decline of nitrogenase activity and the nodule structural and ultrastructural alterations that are associated with nodule senescence were significantly delayed in flavodoxin-expressing nodules. Substantial changes in nodule antioxidant metabolism, involving antioxidant enzymes and ascorbate-glutathione cycle enzymes and metabolites, were detected in flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in flavodoxin-expressing nodules than in control nodules. The observed amelioration of the oxidative balance suggests that the delay in nodule senescence was most likely due to a role of the protein in reactive oxygen species detoxification. Flavodoxin overexpression also led to high starch accumulation in nodules, without reduction of the nitrogen-fixing activity.

Induced New Mutation of D1 Serine-268 in Soybean Photosynthetic Cell Cultures Produced Atrazine Resistance, Increased Stability of S2QB - and S3QB - States, and Increased Sensitivity to Light Stress

We have isolated several herbicide-resistant cell lines from photosynthetic cell suspensions of soybean (Glycine max) that possessed different levels of herbicide resistance, photosystem II activity, and chlorophyll a/b ratio. We have further studied the STR7 mutant, which showed the highest level of resistance to atrazine as well as a cross-resistance to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (50- and 3-fold, respectively, compared with the wild type). Sequencing of the psbA gene (coding for the D1 polypeptide of photosystem II) from this mutant revealed a single change, serine-268 to proline, in the D1 protein. To our knowledge, this substitution has not previously been described in any photosynthetic organism. In addition to affecting atrazine resistance, this single amino acid change caused a decrease in the electron transfer rate between the secondary acceptors QA and QB and a stabilization of the S2QB- and S3QB- states. The mutant also showed a larger antenna size, an increase in non-QB-reducing centers, and a higher sensitivity to light stress. The unusual stability of the S2QB- and S3QB- states indicates that STR7 belongs to a new class of QB-site mutants.

Effects of bean and wheat α‐amylase inhibitors on α‐amylase activity and growth of stored product insect pests*

Insect α‐amylase inhibiting and/or growth inhibiting activities of proteinaceous inhibitors from red kidney bean (Phaseolus vulgaris) and hard red winter wheat (Triticum aestivum) were examined. The bean inhibitor was most effective in vitro against α‐amylases from the red flour beetle (Tribolium castaneum) and the confused flour beetle (T. confusum), followed by those from the rice weevil (Sitophilus oryzae) and yellow mealworm (Tenebrio molitor). The insect enzymes were from two‐ to 50‐fold more susceptible than human salivary α‐amylase. When the inhibitors were added at a 1% level to a wheat flour plus germ diet, the growth of red flour beetle larvae was slowed relative to that of the control group of larvae, with the bean inhibitor being more effective than the wheat inhibitor. Development of both the red flour beetle and flat grain beetle (Cryptolestes pusillus) was delayed by 1% bean inhibitor, but development of the sawtoothed grain beetle (Oryzaephilus surinamensis) and lesser grain borer (Rhyzopertha dominica) was not affected by either the bean or wheat inhibitor at the 1% level. Rice weevil adults fed a diet containing 1% bean or wheat inhibitor exhibited more mortality than weevils fed the control diet. When the wheat amylase inhibitor was combined with a cysteine protease inhibitor, E‐64, and fed to red flour beetle larvae, a reduction in the growth rate and an increase in the time required for adult eclosion occurred relative to larvae fed either of the inhibitors separately. The bean inhibitor was just as effective alone as when it was combined with the protease inhibitor. These results demonstrate that plant inhibitors of insect digestive enzymes act as growth inhibitors of insects and possibly as plant defense proteins, and open the way to the use of the genes of these inhibitors for genetically improving the resistance of cereals to storage pests.