Robert A. Kennedy

Constitutive and inducible aerobic and anaerobic stress proteins in the Echinochloa complex and rice

Anaerobic stress resulted in a change in the protein accumulation patterns in shoots of several Echinochloa (barnyard grass) species and Oryza sativa (L.) (rice) as resolved by two-dimensional gel electrophoresis. Of the six Echinochloa species investigated, E. phyllopogon (Stev.) Koss, E. muricata (Beauv.) Fern, E. oryzoides (Ard.) Fritsch Clayton, and E. crus-galli (L.) Beauv. are tolerant of anaerobiosis and germinate in the absence of oxygen, as does rice. In contrast, E. crus-pavonis (H.B.K.) Schult and E. colonum (L.) Link are intolerant and do not germinate without oxygen. Computer analysis of the protein patterns from the four tolerant species and rice indicated that the anaerobic response is of five classes: class 1 proteins, enhanced under anaerobiosis (9 to 13 polypeptides ranging from 16–68 kD); class 2 proteins, unique to anaerobiosis (1 to 5 polypeptides ranging from 17–69 kD); class 3 proteins, remained constant under aerobiosis and anaerobiosis; class 4 proteins, prominent only in air and repressed under anoxia (3 to 7 polypeptides ranging from 19–45 kD); and class 5 proteins, unique to aerobiosis (1 to 4 polypeptides ranging from 18–63 kD). In the intolerant species, E. colonum and E. crus-pavonis, no polypeptides were enhanced or repressed under anoxia (class 1 and class 4, respectively), whereas in the tolerant Echinochloa species and rice, a total of at least 9 to 13 anaerobic stress proteins and 4 to 7 “aerobic” proteins were noted. Immunoblotting identified two of the major anaerobic stress proteins as fructose-1,6-bisphosphate aldolase and pyruvate decarboxylase. Based on the differential response of the intolerant species to anaerobiosis, we suggest that another set of genes, whose products may not necessarily be among the major anaerobic stress polypeptides, might confer tolerance in Echinochloa under prolonged anaerobic stress.

Ecotypic differences in the C3 and C4 photosynthetic activity in Mollugo verticillata, a C3−C4 intermediate

Four populations of Mollugo verticillata L. were compared on the basis of their photosynthetic products, photosynthetic rates, enhancement under low oxygen concentration, and CO2 compensation points. In addition, pulse-chase labeling experiments were conducted using one of the four populations. Depending on the plant population, C4 acids ranged from 40% to 11% of the primary products under short-term exposure to 14CO2. These compounds were also metabolized during pulse-chase experiments. All four populations had significantly different photosynthetic rates and those rates were correlated with the amounts of labelled C4 acids produced and C4-acid turnover. Three populations of M. verticillata had similar compensation points (40 μl/l) and degrees of photosynthetic enhancement under low [O2] (20%), the fourth population was much lower in both characteristics (CO2 compensation, 25 μl/l; low-O2 enhancement, 12%). The results verify the intermediate nature of photosynthesis in this species, and illustrate populational differences in its photosynthetic and photorespiratory carbon metabolism.

Relationship between leaf development, carboxylase enzyme activities and photorespiration in the C4-plant Portulaca oleracea L.

SummaryRibulose diphosphate (RuDP) and (PEP) phosphoenolpyruvate carboxylase enzyme activities were studied in young, mature, and senescent Portulaca oleracea leaves. While the absolute amount of both the C3 (RuDP) and C4 (PEP) carboxylase is less in senescent leaves than in mature leaves, RuDP carboxylase activity is reduced to a lesser degree. In senescent leaves, PEP carboxylase activity equals 10% of that in mature tissue, but RuDP carboxylase is 27% of that in mature leaves. The same ontogenetic series was also used to determine photorespiration rates and responses to several gas treatments. Young and mature leaves were unaffected by changes in the light regime or oxygen concentrations, and exhibited typical C4-plant light/dark 14CO2 evolution ratios. Senescent leaves, on the other hand, have photorespiration ratios similar to C3-plants. In addition, senescent leaves were affected by minus CO2, 100% O2 and N2 in a manner expected of C3-plants, but not C4-plants. These results are discussed in terms of a relative increase in activity of the C3 cycle in later developmental stages in this plant.

Photosynthetic carbon metabolism during leaf ontogeny in Zea mays L.: Enzyme studies.

The activities of several enzymes, including ribulose-1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured as a function of leaf age in Z. mays. Mature leaf tissue had a RuDP-carboxylase activity of 296.7 μmol CO2 g-1 fresh weight h-1 and a PEP-carboxylase activity of 660.6 μmol CO2 g-1 fresh weight h-1. In young corn leaves the activity of the two enzymes was 11 and 29%, respectively, of the mature leaves. In senescent leaf tissue, RuDP carboxylase activity declined more rapidly than that of any of the other enzymes assayed. On a relative basis the activities of NADP malic enzyme (EC 1.1.1.40), aspartate (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2), and NAD malate dehydrogenase (EC 1.1.1.37) exceeded those of both PEP and RuDP carboxylase in young and senescent leaf tissue. Pulse-chase labeling experiments with mature and senescent leaf tissue show that the predominant C4 acid differs between the two leaf ages. Labeling of alanine in senescent tissue never exceeded 4% of the total 14C remaining during the chase period, while in mature leaf tissue alanine accounted for 20% of the total after 60 s in 12CO2. The activity of RuDP carboxylase during leaf ontogeny in Z. mays parallels the development of the activity of this enzyme in C3 plants.

Relationship between early photosynthetic products, photorespiration, and stage of leaf development in Zea mays

Summary Early photosynthetic products were determined for three stages of leaf development in Zea mays. After a 10 second exposure to 14CO2, the major percentage of label resides in the four-carbon acids, malate and aspartate, in each leaf age examined. There is a decrease in the amount of C4 acids labeled during leaf ontogeny but this decrease does not lead to an increase in C3 cycle intermediates. Several minor photosynthetic products increase slightly in labeling. Photorespiratory activities for the ontogenetic series were also determined using a 14CO2 assay. Young and mature tissue had light to dark 14CO2 evolution ratios of less that 1.0, as expected of C4 plants. Senescent leaf tissue had a light to dark ratio of 3.3, a value typical for C3 plants, and the amount of 14CO2 evolved in the light was directly influenced by oxygen concentrations. The data indicates that there are physiological changes which occur during development of Zea mays leaves, particularly with respect to photorespiration.

The effects of NaCl-, polyethyleneglycol-, and naturally-induced water stress on photosynthetic products, photosynthetic rates, and CO2compensation points in C4 plants

Summary This study investigated the effects of water stress on the photosynthetic products, photosynthetic rates and CO2 compensation points in two C4 plants, Zea mays and Portulaca oleracea. Three methods of experimentally inducing water stress were also compared to determine their relative effect on the photosynthetic characteristics listed. Thus, low leaf water potentials were obtained by growing the plants in solutions of NaCl or polyethyleneglycol, or by withholding water. In most instances, four carbon acid percentages were the lowest in both plants with increased levels of water deficit. Radioactivity located in C3 cycle products, phosphoglyceric acid, sugar phosphates, and several miscellaneous compounds, increased under these conditions. The only exception to this was when Portulaca was grown in the presence of NaCl. Sodium chloride also resulted in increased labeling of amino acids in the dark in Portulaca and in the light in both Portulaca and Zea mays. Photosynthetic rates were adversely affected by water stress, the amount of the reduction being dependent on the water potential of the leaves, the osmoticum used, and the basis of calculation. Low leaf water potentials also resulted in increased CO2 compensation points in both C4 plants, and possible explanations for these increases — photorespiration, mitochondrial respiration, or other factors — are discussed.

Effect of Aerobic Priming on the Response of Echinochloa crus-pavonis to Anaerobic Stress (Protein Synthesis and Phosphorylation)

Echinochloa species differ in their ability to germinate and grow in the absence of oxygen. Seeds of Echinochloa crus-pavonis (H.B.K.) Schult do not germinate under anoxia but remain viable for extended periods (at least 30 d) when incubated in an anaerobic environment. E. crus-pavonis can be induced to germinate and grow in an anaerobic environment if the seeds are first subjected to a short (1–18 h) exposure to aerobic conditions (aerobic priming). Changes in polypeptide patterns (constitutive and de novo synthesized) and protein phosphorylation induced by aerobic priming were investigated. In the absence of aerobic priming protein degradation was not evident under anaerobic conditions, although synthesis of a 20-kD polypeptide was induced. During aerobic priming, however, synthesis of 37- and 55-kD polypeptides was induced and persisted upon return of the seeds to anoxia. Furthermore, phosphorylation of two 18-kD polypeptides was observed only in those seeds that were labeled with 32PO4 during the aerobic priming period. Subsequent chasing in an anaerobic environment resulted in a decrease in phosphorylation of these polypeptides. Likewise, phosphorylation of the 18-kD polypeptides was not observed if the seeds were labeled in an anaerobic atmosphere. These results suggest that the regulated induction of the 20-, 37-, and 55-kD polypeptides may be important for anaerobic germination and growth of E. crus-pavonis and that the specific phosphorylation of the 18-kD polypeptides may be a factor in regulating this induction.

Biochemical Adaptations to Anoxia in Barnyard Grass

Although higher plants require oxygen for growth, they frequently experience low oxygen environments which occur in natural wetlands and during flooding or irrigation. Plants tolerate these conditions for only a short period of time before irreversible damage occurs. We have been studying a group of rice weeds which, like cultivated rice (Oryza sativa), can germinate and grow without oxygen. Echinochloa crus-galli var. oryzicola (hereafter oryzicola) metabolizes its seed reserves under N2 and produces a larger seedling from a smaller seed than rice (Kennedy, Rumpho and VanderZee, 1983b). In response to temperature, oryzicola and rice exhibit similar germination characteristics under aerobic conditions. Under anaerobic conditions, however, oryzicola germinates better and tolerates colder temperatures than rice. Thus, both Echinochloa and rice provide excellent opportunities to study metabolic adaptations to low oxygen stress. In addition, the Echinochloa ‘complex’ is composed of several species that differ in their ability to germinate under anoxia (Kennedy et al., 1983b), each exhibiting a full range of habitat preference and weediness in rice cultivation — an ideal natural system for comparative studies on the biochemistry of these important weed species.