Screening of secondary metabolites and antioxidant potential of endophytic fungus Penicillium citrinum and host Digitaria bicornis by spectrophotometric and electrochemical methods.

Perennial grasses are hosts to an extremely diverse assemblage of endophytic fungi, but their significance is still underexplored. In the present study, an endophytic fungus was isolated from the aerial regions of Digitaria bicornis (Lam.) Roem. & Schult. and was characterized by morphological and molecular methods (ITS rDNA region), as Penicillium citrinum Thom. The crude extracts of endophytic fungus and host were recovered and evaluated for their antioxidant potential by spectrophotometric and electrochemical methods. The present study was also an attempt to compare the anti-radical power of extracts by spectrophotometric (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), hydrogen peroxide and nitric oxide radical scavenging assay) and electrochemical (cyclic voltammetry) methods and suggested that cyclic voltammetry could be used routinely instead of assaying by more number of spectrophotometric methods. The results indicated that the ethyl acetate extract of P. citrinum and methanolic extract of D. bicornis has potential compounds with antioxidant and other pharmaceutical activities. Nine and 17 antioxidant biomolecules, respectively, in P. citrinum and D. bicornis extracts were detected by OHR-LC-MS and the presence of function group of the bioactive compounds was confirmed by FTIR spectroscopy. Finally, the study also reported that the extracts of P. citrinum and D. bicornis have several bioactive compounds with application in commercial pharmaceutical industries.

Polymeric Nanoparticles as a Metolachlor Carrier: Water-Based Formulation for Hydrophobic Pesticides and Absorption by Plants.

Pesticide formulation is highly desirable for effective utilization of pesticide and environmental pollution reduction. Studies of pesticide delivery system such as microcapsules are developing prosperously. In this work, we chose polymeric nanoparticles as a pesticide delivery system and metolachlor was used as a hydrophobic pesticide model to study water-based mPEG-PLGA nanoparticle formulation. Preparation, characterization results showed that the resulting nanoparticles enhanced "water solubility" of hydrophobic metolachlor and contained no organic solvent or surfactant, which represent one of the most important sources of pesticide pollution. After the release study, absorption of Cy5-labeled nanoparticles into rice roots suggested a possible transmitting pathway of this metolachlor formulation and increased utilization of metolachlor. Furthermore, the bioassay test demonstrated that this nanoparticle showed higher effect than non-nano forms under relatively low concentrations on Oryza sativa, Digitaria sanguinalis. In addition, a simple cytotoxicity test involving metolachlor and metolachlor-loaded nanoparticles was performed, indicating toxicity reduction of the latter to the preosteoblast cell line. All of these results showed that those polymeric nanoparticles could serve as a pesticide carrier with lower environmental impact, comparable effect, and effective delivery.

Acetyl-CoA carboxylase overexpression in herbicide-resistant large crabgrass (Digitaria sanguinalis).

BACKGROUND: The occurrence of herbicide-resistant weed biotypes is increasing and this report of an acetyl-CoA carboxylase (ACCase) inhibitor-resistant Digitaria sanguinalis L. Scop. from southwestern Ontario is another example. The identified weed escaped control in an onion and carrot rotation in which graminicides were used for several consecutive years. Our goal was to characterize the level and mechanism of resistance of the biotype. RESULTS: The biotype was resistant to all five ACCase inhibitor herbicides tested. Gene-expression profiling was performed because none of the mutations known to confer resistance in the ACCase gene were detected. RNASeq and quantitative reverse-transcriptase PCR (qRT-PCR) results indicated that transcription of ACCase was 3.4-9.3 times higher in the resistant biotype than the susceptible biotype. ACCase gene copy number was determined by qPCR to be five to seven times higher in the resistant compared with the susceptible biotype. ACCase gene overexpression was directly related to the increase of the ACCase gene copy number. CONCLUSION: Our results are consistent with the hypothesis that overexpression of the herbicide target gene ACCase confers resistance to the herbicide. This is the first reported case of target gene duplication conferring resistance to a herbicide other than glyphosate. © 2017 Society of Chemical Industry See related Article.

Differential gene expression for Curvularia eragrostidis pathogenic incidence in crabgrass (Digitaria sanguinalis) revealed by cDNA-AFLP analysis.

Gene expression profiles of Digitaria sanguinalis infected by Curvularia eragrostidis strain QZ-2000 at two concentrations of conidia and two dew durations were analyzed by cDNA amplified fragment length polymorphisms (cDNA-AFLP). Inoculum strength was more determinant of gene expression than dew duration. A total of 256 primer combinations were used for selective amplification and 1214 transcript-derived fragments (TDFs) were selected for their differential expression. Of these, 518 up-regulated differentially expressed TDFs were identified. Forty-six differential cDNA fragments were chosen to be cloned and 35 of them were successfully cloned and sequenced, of which 25 were homologous to genes of known function according to the GenBank database. Only 6 genes were up-regulated in Curvularia eragrostidis-inoculated D. sanguinalis, with functions involved in signal transduction, energy metabolism, cell growth and development, stress responses, abscisic acid biosynthesis and response. It appears that a few pathways may be important parts of the pathogenic strategy of C. eragrostidis strain QZ-2000 on D. sanguinalis. Our study provides the fundamentals to further study the pathogenic mechanism, screen for optimal C. eragrostidis strains as potential mycoherbicide and apply this product to control D. sanguinalis.

Protein enrichment and digestion improvement of napiergrass and pangolagrass with solid-state fermentation.

BACKGROUND AND PURPOSE: Napiergrass (Pennisetum purpureum Schumacher) and pangolagrass (Digitaria decumbens Stent) are two major forage grasses for cow feeding. They possess high yields and high regeneration properties. Inoculation of cellulolytic microbes on herbage could enhance the protein content of herbage and promote digestibility in chickens. METHODS: Cellulolytic microbes were isolated from various sources and cultivated on napiergrass and pangolagrass with solid-state fermentation for protein enrichment and in vitro digestion improvement.The fermented napiergrass and pangolagrass were used as the main protein source in chicken diets to assess the feasibility for non-ruminants feed. RESULTS: After a 42-day fermentation period, napiergrass showed higher protein contents (13.4-13.9%) than those of pangolagrass(11.1-11.7%). The in vitro digestibility of pangolagrass increased from 5.29% to 20.4%, whereas that of napiergrass increased from 5.29% to 19.0%. The average feed conversion efficiencies of chickens were close to the traditional fodder using corn as the main ingredient. CONCLUSION: Inoculation of appropriate cellulolytic microbes to enrich protein content and improve in vitro digestibility of herbage with solid-state fermentation for chicken feed is the prospective technique for agriculture, animal husbandry, and substantial management.

Protein enrichment, cellulase production and in vitro digestion improvement of pangolagrass with solid state fermentation.

BACKGROUND AND PURPOSE: Pangolagrass, Digitaria decumbens Stent, is a major grass for cow feeding, and may be a good substrate for protein enrichment. To improve the quality of pangolagrass for animal feeding, cellulolytic microbes were isolated from various sources and cultivated with solid state fermentation to enhance the protein content, cellulase production and in vitro digestion. The microbes, culture conditions and culture media were studied. METHODS: Cellulolytic microbes were isolated from pangolagrass and its extracts, and composts. Pangolagrass supplemented with nitrogen and minerals was used to cultivate the cellulolytic microbes with solid state fermentation. The optimal conditions for protein enrichment and cellulase activity were pangolagrass substrate at initial moisture 65-70%, initial pH 6.0-8.0, supplementation with 2.5% (NH(4))(2)SO(4), 2.5% KH(2)PO(4) and K(2)HPO(4) mixture (2:1, w/w) and 0.3% MgSO(4).7H(2)O and cultivated at 30(o)C for 6 days. RESULTS: The protein content of fermented pangolagrass increased from 5.97-6.28% to 7.09-16.96% and the in vitro digestion improved from 4.11-4.38% to 6.08-19.89% with the inoculation of cellulolytic microbes by solid state fermentation. Each 1 g of dried substrate yielded Avicelase 0.93-3.76 U, carboxymethylcellulase 1.39-4.98 U and ß-glucosidase 1.20-6.01 U. The isolate Myceliophthora lutea CL3 was the strain found to be the best at improving the quality of pangolagrass for animal feeding with solid state fermentation. CONCLUSION: Solid state fermentation of pangolagrass inoculated with appropriate microbes is a feasible process to enrich protein content, increase in vitro digestibility and improve the quality for animal feeding.

Exploitation of Acha (Digitaria exiliis) and Iburu (Digitaria iburua) flours: chemical characterization and their use for sourdough fermentation.

This study aimed at characterizing the chemical and microbiological composition of Acha (Digitaria exiliis) and Iburu (Digitaria iburua) flours and at exploiting their potential for sourdough fermentation. Both the flours had a gross composition similar to other cereals. As shown by two-dimensional electrophoresis analysis, Acha flour had a higher number of prolamins with respect to Iburu flour. The opposite was found for the number of glutelin spots. The concentration of total free amino acids of Iburu flour was higher than that of Acha flour (1348±34 vs. 100±20 mg/kg). Pediococcus pentosaceus was the dominant species in both the flours. Several isolates were used to ferment Acha or Iburu flours. After 8h fermentation at 30°C, pH ranged from 3.41 to 3.83 and from 4.20 to 4.66 for Acha and Iburu sourdoughs, respectively. The highest values of TTA and concentration of lactic acid were found in almost all Iburu sourdoughs. The synthesis of acetoin and γ-amino butyric acid (GABA) was only found in Iburu sourdoughs. Data from the kinetics of acidification, synthesis of lactic and acetic acids, acetoin, and liberation of total free amino acids were elaborated by Principal Component Analysis. Sourdoughs from Acha and Iburu flour were clearly differentiated.

Accelerated metolachlor degradation in soil by zerovalent iron and compost amendments.

Soil incubation and germination tests were conducted to assess zerovalent iron (ZVI), organic compost, moisture and their combinations on metolachlor degradation in soil. The ZVI alone degraded 91% of metolachlor in soil within 40 days following bi-phasic kinetics. Organic amendment alone facilitated metolachlor degradation in soil up to 60% after 40 days depending on the amendment rate. However, the combination of ZVI with compost amendment at 30 ton ha(-1) and 30% moisture content accelerated metolachlor degradation to 90% after 3 days and 98% after 40 days. The half life (t (1/2)) of metolachlor degradation with ZVI, compost at 30 ton ha(-1), and 30% moisture was about 1 day, which was faster than ZVI treatment alone and 98% faster than controls. Germination and growth of lettuce (Lactuca sativa) and crabgrass (Digitaria sanguinalis L. Scop.) were severely inhibited in unamended metolachlor-contaminated soils but when these soils were amended with ZVI, germination and growth was comparable to controls (metolachlor free soil). Metolachlor degradation was greatest when ZVI, compost and moisture were used together, suggesting that these treatments will maximize in situ remediation of metolachlor-contaminated soils in the field.

Mesotrione control and pigment concentration of large crabgrass (Digitaria sanguinalis) under varying environmental conditions.

BACKGROUND: Mesotrione is a carotenoid biosynthesis-inhibiting herbicide currently labeled for crabgrass (Digitaria spp.) control. Mesotrione control of large crabgrass has been reported to vary with temperature and relative humidity; however, the effect of irradiance on mesotrione efficacy has not previously been reported. Likewise, little is known about pigment concentrations of Digitaria spp. The present research investigated the effects of mesotrione on large crabgrass, Digitaria sanguinalis (L.) Scop., control and pigment concentrations under varying irradiance at three temperatures. RESULTS: Mesotrione (0.28 kg ha(-1)) control of large crabgrass did not differ between temperature levels (18, 26 and 32 degrees C). Control was similar at tested irradiance levels (600, 1100 and 1600 micromol m(-2) s(-1)). Mesotrione reduced large crabgrass chlorophyll a, chlorophyll b and total carotenoid concentrations, as well as chlorophyll a to b ratios. Treated plant bleaching was highest 7 days after treatment (DAT) but decreased by 21 DAT. Treated plants were less than 10% necrotic 3 and 7 DAT but nearly 35% necrotic 21 DAT. Treated large crabgrass bleaching was highest and photochemical efficiency was lowest 7 DAT. These results indicate that some plant recovery occurs prior to 21 DAT. CONCLUSION: Although mesotrione efficacy has previously been reported to vary according to environmental factors, mesotrione control of large crabgrass did not vary with measured temperature and irradiance levels in this study. On account of crabgrass convalescence, secondary applications of mesotrione may control large crabgrass more effectively when applied prior to 21 DAT.

Novel cysteine-rich peptides from Digitaria ciliaris and Oryza sativa enhance tolerance to cadmium by limiting its cellular accumulation.

By means of functional screening using the cadmium (Cd)-sensitive ycf1 yeast mutant, we have isolated a novel cDNA clone, DcCDT1, from Digitaria ciliaris growing in a former mining area in northern Japan, and have shown that it confers Cd tolerance to the yeast cells, which accumulated almost 2-fold lower Cd levels than control cells. The 521 bp DcCDT1 cDNA contains an open reading frame of 168 bp and encodes a deduced peptide, DcCDT1, that is 55 amino acid residues in length, of which 15 (27.3%) are cysteine residues. Five DcCDT1 homologs (here termed OsCDT1-OsCDT5) have been identified in rice, and all of them were up-regulated to varying degrees in the above-ground tissues by CdCl(2) treatment. Localization of green fluorescent protein fusions suggests that DcCDT1 and OsCDT1 are targeted to both cytoplasmic membranes and cell walls of plant cells. Transgenic Arabidopsis thaliana plants overexpressing DcCDT1 or OsCDT1 displayed a Cd-tolerant phenotype and, consistent with our yeast data, accumulated lower amounts of Cd when grown on CdCl(2). Collectively, our data suggest that DcCDT1 and OsCDT1 function to prevent entry of Cd into yeast and plant cells and thereby enhance their Cd tolerance.

Effect of crabgrass (Digitaria ciliaris) hay harvest interval on forage quality and performance of growing calves fed mixed diets.

Twelve 0.81-ha crabgrass (Digitaria ciliaris [Retz.] Koel.) hay fields were harvested at 21, 35, and 49 d of regrowth (average phonological growth stage of 30, 51, and 56, respectively). Increased harvest interval exhibited a linear decrease (P < 0.01) in CP (14.1, 13.7, and 10.6% of DM, respectively) and increase (P < 0.01) in NDF (65.3, 70.6, and 70.2% of DM, respectively) and ADF (35.7, 38.9, and 42.7% of DM, respectively). Hays were incorporated into 3 diets that contained 20% (DM basis) crabgrass hay, ground corn (33%), and soybean hulls (32%). Diets contained 14.4, 14.4, and 13.6% CP; 1.83, 1.72, and 1.81 Mcal of NE(m)/kg; and 1.21, 1.10, and 1.17 Mcal of NE(g)/kg; respectively. Diets were fed to beef calves in 12 pens at a rate of 2.3% (DM basis) of BW in 1 experiment (n = 120, initial BW 210 +/- 4.4 kg) and ad libitum in another experiment (n = 60, initial BW 207 +/- 4.4 kg). To measure passage rate of the hay and concentrate portions of the diets, 12 heifer calves (BW = 145 +/- 4.5 kg) were individually fed at 2.3% of BW for 14 d and dosed with Dy-labeled soybean hulls and Yb-labeled hay. In situ DM digestibility of the hays and diets were determined using 3 ruminally cannulated steers (BW = 584 +/- 10.4 kg). Harvest interval did not affect (P > or = 0.11) ADG of limit-fed calves during the diet acclimation or growing phases (average 0.32 and 0.80 kg, respectively) or ADG of calves fed ad libitum (average 1.21 kg). Dry matter intake of calves fed ad libitum averaged 7.9 kg/d (3.28% of BW) and was not affected (P > or = 0.22) by harvest interval. Gain:feed was not affected (P > or = 0.20) by harvest interval (0.13 and 0.15 for limit-fed and ad libitum-fed calves, respectively). Increased harvest interval linearly increased (P < 0.01) ruminal retention time of the hay and tended (P = 0.06) to linearly increase ruminal retention time of the concentrate portions of the diet. Harvest interval linearly decreased (P < or = 0.05) the extent of degradability and effective degradability of DM and NDF of hays, but DM disappearance of the total diet did not differ (P > or = 0.35). In the conditions of this study, increasing harvest interval of crabgrass hay from 21 to 49 d had no deleterious impact on animal performance or efficiency of gain when fed to growing calves in a high-concentrate mixture.

Ruminal in situ disappearance kinetics of nitrogen and neutral detergent insoluble nitrogen from common crabgrass forages sampled on seven dates in northern Arkansas.

Southern crabgrass (Digitaria ciliaris [Retz.] Koel.) is often an undesirable species in field and forage crops, but visual observations suggest that livestock prefer it to many other summer forages. The objectives of this study were to assess the nutritive value of crabgrass sampled weekly between July 11 and August 22, 2001 and then to determine ruminal in situ disappearance kinetics of N and neutral detergent insoluble N (NDIN) for these forages. A secondary objective was to compare these kinetic estimates for crabgrass with those of alfalfa (Medicago sativa L.), bermudagrass (Cynodon dactylon [L.] Pers.), and or-chardgrass (Dactylis glomerata L.) as control hays. All kinetic evaluations were conducted with 5 ruminally cannulated Gelbvieh x Angus x Brangus steers (383 +/- 22.7 kg). Concentrations of N for crabgrass decreased linearly (P < or = 0.002) across sampling dates for leaf, stem, and whole-plant tissues. Conversely, percentages of the total N pool within NDIN and ADIN fractions generally increased over sampling dates in mostly linear patterns. For crabgrass, the immediately soluble portion of the total N pool (fraction A; overall mean = 54.6% of N) was greater (P < 0.001) than for all control hays. Crabgrass exhibited a more rapid N disappearance rate (overall mean = 0.093/h; expressed as a proportion disappearing/h) than that of bermudagrass (0.046/h; P < 0.001), but the disappearance rate for alfalfa N (0.223/h) was considerably faster (P < 0.001) than for crabgrass. The effective ruminal disappearance of N was greater (P < 0.001) for crabgrass (overall mean = 85.4%) than for the alfalfa (83.3%), bermudagrass (72.3%), or orchardgrass (76.0%) control hays. For alfalfa, the ruminal disappearance rate of NDIN (0.150/h) was more rapid (P < 0.001) than for crabgrass (overall mean = 0.110/h); however, the disappearance rate for crabgrass was faster than that for bermudagrass (0.072/h; P < 0.001) or for orchardgrass (0.098/h; P = 0.010). Effective ruminal disappearance of NDIN was greater (P < 0.001) for crabgrass (overall mean = 72.0%) than for the bermudagrass (69.0%) or alfalfa hays (50.5%), but there was no difference (P = 0.865) between crabgrass and orchardgrass (72.1%). Although crabgrass forages exhibited concentrations of total N that were comparable with those of alfalfa and rates of ruminal N disappearance that were < 50% of those for the alfalfa hay control, improvements in N use efficiency relative to alfalfa are questionable because of the excessively large Fraction A for crabgrass.

Photochemical and antioxidant responses in the leaves of Xerophyta viscosa Baker and Digitaria sanguinalis L. under water deficit.

In this study, photochemical and antioxidant responses of the monocotyledonous resurrection plant Xerophyta viscosa Baker and the crab grass Digitaria sanguinalis L. under water deficit were investigated as a function of time. Water deficit was imposed by withholding irrigation for 21 d. Gas exchange and chlorophyll a fluorescence analyses indicated that the dehydration treatment caused photoinhibition in both species. The reduction in the photosynthesis rate in both species during water deficit probably contributed to the decline in the photochemical efficiency of PSII and electron transport rate. However, the stomatal conductance of both species did not change during treatment whereas the intercellular CO2 pressure increased after 10 d of water deficit treatment. These observations could be related to nonstomatal limitations. The increasing net transpiration rate of both species may have contributed to leaf cooling because of water limitations. Prolonged water deficit resulted in photosynthetic pigment chlorophyll (a + b) and carotenoids content loss in only D. sanguinalis. Both species especially D. sanguinalis had increased the level of anthocyanin after 15 d of treatment, possibly to prevent the damaging effect of photooxidation. The total SOD activity of D. sanguinalis was significantly different from X. viscosa during the treatment. The total peroxidase activity in D. sanguinalis was significantly higher than in X. viscosa. X. viscosa acclimated to water deficit with no ultimate apparent oxidative damage due to endogenous protective mechanisms of resurrection. In case of D. sanguinalis, water deficit induced considerable stress and possibly caused some oxidative damage, despite the upregulation of protection mechanisms.

Ruminal in situ disappearance kinetics of dry matter and fiber in growing steers for common crabgrass forages sampled on seven dates in northern Arkansas.

Southern crabgrass (Digitaria ciliaris [Retz.] Koel.) is often viewed as an undesirable weed, largely because it encroaches upon field and forage crops, gardens, and lawns. However, visual observations of livestock grazing mixed-species pastures suggest that cattle seem to prefer crabgrass to many other summer forages. The objectives of this study were to assess the nutritive value of crabgrass sampled weekly between July 11, and August 22, 2001, and then to determine ruminal in situ disappearance kinetics of DM and NDF for these crabgrass forages. A secondary objective was to compare these kinetic estimates with those of alfalfa (Medicago sativa L.), bermudagrass (Cynodon dactylon [L.] Pers.), and orchardgrass (Dactylis glomerata L.) control hays. All forages were evaluated in situ using five (383 +/- 22.7 kg) ruminally cannulated crossbred (Gelbvieh x Angus x Brangus) steers. Whole-plant crabgrass exhibited more rapid (P < or = 0.002) ruminal disappearance rates of DM (overall range = 0.069 to 0.084 h(-1)) than did bermudagrass (0.054 h(-1)) and orchardgrass (0.060 h(-1)) hays, but disappearance rates were slower (P < 0.001) for crabgrass than for alfalfa hay (0.143 h(-1)). Effective ruminal disappearance of DM was greater (P < 0.001) for crabgrass (overall range = 69.3 to 75.4%) than for all the control hays. Similarly, disappearance rates of NDF for crabgrass (overall range = 0.069 to 0.086 h(-1)) were more rapid (P < 0.001) than observed for bermudagrass and orchardgrass hays; however, NDF in alfalfa disappeared at a faster (P < 0.001) rate (0.107 h(-1)) than crabgrass. These results indicate that crabgrass offers greater effective ruminal degradability of DM and NDF than orchardgrass or alfalfa of moderate quality. More importantly, it potentially offers faster and more extensive ruminal disappearance than perennial warm-season grasses typically found throughout the southeastern United States, and it should likely support improved performance by ruminant livestock in this region.

The effect of a condensed tannin-containing forage on methane emission by goats.

The objective of this study was to compare methane emission by goats consuming the condensed tannin-containing forage sericea lespedeza (Les-pedeza cuneata) or a mixture of crabgrass (Digitaria ischaemum) and Kentucky 31 tall fescue (Festuca arundinacea). Two groups of 12 Angora does (initial average BW = 41.5 +/- 2.7 kg) that previously grazed a pasture of sericea lespedeza or crabgrass/tall fescue for approximately 4 mo were used. After 1 wk of adaptation to metabolism cages, gas exchange was measured for 24 h in an open-circuit respiration calorimetry system with four head boxes. Forage harvested daily from the previously grazed pastures was consumed ad libitum. Crude protein concentration was 10.3 and 13.0%, IVDMD was 64.5 and 75.3%, and the level of condensed tannins was 17.7 and 0.5% for sericea lespedeza and crabgrass/tall fescue, respectively. Dry matter intake (1.11 vs. 0.67 kg/d) and digestible DMI (estimated from IVDMD; 0.71 vs. 0.51 kg/d) were greater (P < 0.01) for sericea lespedeza than for crabgrass/tall fescue. Ruminal ammonia N (3.7 and 9.9 mg/dL; P < 0.001) and plasma urea-N concentrations (16.7 and 20.9 mg/dL; P = 0.07) were lower for sericea lespedeza than for crabgrass/tall fescue. Concentrations of individual and total VFA and the acetate-to-propionate ratio in ruminal fluid did not differ between treatments (P > 0.19). Despite higher DMI by goats fed sericea lespedeza, daily energy expenditure (409 vs. 431 kJ/kg BW(0.75)), heart rate (70 vs. 73 beats/min), and the ratio of energy expenditure to heart rate (5.82 vs. 5.94) did not differ between sericea lespedeza and crabgrass/tall fescue, respectively (P > 0.13). Methane emission expressed as both quantity per day or relative to DMI was lower (P <0.001) for sericea lespedeza than for crabgrass/tall fescue (7.4 vs. 10.6 g/d and 6.9 vs. 16.2 g/kg DMI). Substantial differences between the forages in condensed tannins concentration and methane emission by Angora goats suggest that condensed tannins decreased methane emission.

A Ca(2+)-dependent protein kinase with characteristics of protein kinase C in leaves and mesophyll cell protoplasts from Digitaria sanguinalis: possible involvement in the C(4)-phosphoenolpyruvate carboxylase phosphorylation cascade.

In mesophyll cells (MC) of Digitaria sanguinalis, the C(4)-phosphoenolpyruvate carboxylase (C(4)-PEPC) initiating the photosynthetic pathway is controlled by a complex light-dependent phosphorylation process. We showed previously that the transduction cascade involves the phosphoinositide pathway and a Ca(2+)-dependent step, which precedes the upregulation of the PEPC kinase (PEPCk). We have now further characterized the cascade component requiring Ca(2+). A Ca(2+)-dependent protein kinase that shows several characteristics of the conventional type of mammalian protein kinase C (PKC) was detected in protein extracts from mesophyll cell protoplasts (MCPs). It catalyzed the in vitro phosphorylation of the C1-peptide PKC substrate and was markedly inhibited by a PKC-specific pseudosubstrate domain. However, it was only modestly activated by the phospholipids phosphatidylserine and lysophosphatidylcholine, while choline, oleyl acetylglycerol, phosphatidylinositol, and the phorbol ester phorbol 12-myristate 13-acetate did not show any effect. Nevertheless, its activity was found to be associated with a polypeptide of 75kDa that was recognized by a PKC antibody raised against the C-terminus of rabbit PKCbeta II. In addition, this protein kinase was also inhibited by the Ca(2+)-dependent protein kinase (CDPK)/PKC inhibitors W7, H7, and staurosporine. Surprisingly, it was found to be phosphorylated in dark-adapted MCPs, albeit to a low extent, and this did not change during protoplast induction by light. W7, H7, and staurosporine were shown to markedly inhibit C(4)-PEPC phosphorylation in light-treated MCPs. These results support the view that this protein kinase is a good candidate to represent the Ca(2+)-activated component of the C(4)-PEPC phosphorylation cascade.