The endogenous endoprotease inhibitors of barley and malt and their roles in malting and brewing

Endoproteases play an important role in barley germination by controlling the hydrolysis of the grain's storage proteins into peptides and amino acids that are needed by the young plant. During malting, the commercial version of this process, many high M_r barley biopolymers are converted into malt nutrients that can be utilized by yeasts during brewing. However, barley and malt both contain endogenous proteins that inhibit the enzymatic activities of these proteases. High levels of these inhibitors can cause brewing problems by preventing the proteases from producing optimal levels of soluble proteins and amino acids. Both high and low M_r inhibitors of cysteine proteases occur in barley and malt. Two of the high M_r inhibitors, lipid transfer protein 1 (LTP1) and LTP2, have been purified and studied. Recently, members of the trypsin/alpha-amylase inhibitor protein family (CM proteins) have been shown to inhibit the activity of SEP-1, a purified serine class barley protease. No inhibitors of aspartic proteases or metalloproteases have yet been purified, but it has been reported that endogenous metalloprotease inhibitors do exist. The inhibitors of the cysteine proteases and metalloproteases are probably the ones most important for brewing, because members of these two protease classes apparently catalyse most of the protein hydrolysis that occurs during malt mashing and, presumably, also during malting. More biochemical studies are needed to clarify how these proteins interact with the proteases to control protein hydrolysis during germination.     

The endogenous endoprotease inhibitors of barley and malt and their roles in malting and brewing

Endoproteases play an important role in barley germination by controlling the hydrolysis of the grain's storage proteins into peptides and amino acids that are needed by the young plant. During malting, the commercial version of this process, many high M_r barley biopolymers are converted into malt nutrients that can be utilized by yeasts during brewing. However, barley and malt both contain endogenous proteins that inhibit the enzymatic activities of these proteases. High levels of these inhibitors can cause brewing problems by preventing the proteases from producing optimal levels of soluble proteins and amino acids. Both high and low M_r inhibitors of cysteine proteases occur in barley and malt. Two of the high M_r inhibitors, lipid transfer protein 1 (LTP1) and LTP2, have been purified and studied. Recently, members of the trypsin/alpha-amylase inhibitor protein family (CM proteins) have been shown to inhibit the activity of SEP-1, a purified serine class barley protease. No inhibitors of aspartic proteases or metalloproteases have yet been purified, but it has been reported that endogenous metalloprotease inhibitors do exist. The inhibitors of the cysteine proteases and metalloproteases are probably the ones most important for brewing, because members of these two protease classes apparently catalyse most of the protein hydrolysis that occurs during malt mashing and, presumably, also during malting. More biochemical studies are needed to clarify how these proteins interact with the proteases to control protein hydrolysis during germination.     

Effect of hydrogen peroxide and ozone treatment on improving the malting quality

The effect of hydrogen peroxide (HP) and ozone (O₃) treatment during barley steeping on the quality of malt produced from two barley varieties (GrangeR and AC Metcalfe) by micro-malting was investigated. The two steeping oxidation treatments that was observed to promote barley acrospire growth. Ozone treatment improved the malt enzyme activity of endo-protease, α-amylase, free beta-amylase and total limit dextrinase to differing extents, with GrangeR improving to a greater degree. HP treatment contributed to the increase of α-amylase, β-glucanase and endo-protease. Surprisingly, HP or ozone oxidation during malting resulted in different and novel outcomes for total beta-amylase in GrangeR and AC Metcalfe. In GrangeR, total beta-amylase activity reduced with respect to the control in both treatments. In comparison with AC Metcalfe there was a substantial increase of 78% with HP and 90% O₃ in total beta-amylase activity. Malt quality including wort free amino nitrogen, β-glucan, turbidity and diastatic power was differentially increased by the oxidation induction treatment during steeping in malting. Gene expression analysis indicated that the effects of the steep oxidation treatments on enzyme and malt quality were putatively linked with the up-regulation of certain genes involved in GA synthesis (GA20ox1) and ABA catabolism (ABA8′OH). Barley grain germination assay results also showed that moderate HP induction could improve barley germination tolerance to the ABA effect. Malting including steep oxidation induction was shown to be beneficial to malt quality by improving the resultant wort quality and the efficiency of the beer brewing process. These observations point the way towards improving malt quality and the efficiency of the malting process.     

Biochemical and Biological Evaluation of an L-Asparaginase from Isolated Escherichia coli MF-107 as an Anti-Tumor Enzyme on MCF7 Cell Line

Background: One of the most widely used anticancer agents is microbial L-ASNase. Herein, we assessed the biochemical and biological properties of an isolated L-ASNase from a Gram-negative bacteria strain, Escherichia coli MF-107. Methods:Using garden asparagus, we obtained several bacterial isolates. These strains were further screened for L-ASNase activity. A promising bacterial isolate was selected for L-ASNase production and subsequent purification. The molecular weight of purified L-ASNase was determined. The MTT assay was applied to assess the cytotoxic effect of the purified enzyme. Also, for caspase activity determination and the apoptotic effect of purified enzyme on in cells, we conducted a real-time PCR method. Results:The molecular weight of the enzyme was approximately 37 kDa. In the pH range of 7.5 to 8, the enzyme had considerable stability. At 35 °C, the purified L-ASNase optimum activity was recorded. The cytotoxic effect of the enzyme on treated cells was dose-dependent with an IC₅₀ value of 5.7 IU/ml. The Bax gene expression considerably raised by 5.75-fold (p < 0.001) upon L-ASNase treatment. On the other hand, the anti-apoptotic Bcl-2 gene expression showed a 2.63-fold increase compared to the control (p < 0.05). It was detected that the mRNA levels of caspase-3 and p53 were considerably upregulated (5.93 and 1.85-fold, respectively). We did not find any alternation in the caspase-8 activity of the treated cells compared to untreated cells. Conclusion:In this research, the proliferation of the breast cancer cells remarkably inhibited via the cytotoxic effect of isolated L-ASNase from microbial sources.Key Words: Apoptosis, Breast cancer, Escherichia coli, MCF7 cell line     

Development of proteolytic activities during barley malting and their localization in the green malt kernel

The water-insoluble storage proteins of barley seeds reside in the starchy endosperm tissue. This tissue, when expressed from germinating barley, has a pH of 4·8. The hydrolysis of storage proteins during germination (malting) occurs mainly in the endosperm, so proteinases that are located in endosperm and are active at pH 4·8 are probably important to the storage protein hydrolytic process. This study reports our continued investigations of the endoproteinases of germinating barley (Hordeum vulgare L., cv. Morex) with a two-dimensional gel separation method that uses isoelectric focusing (IEF) and non-denaturing polyacrylamide gel electrophoresis (PAGE) in gels containing incorporated substrate protein. We identified the endoproteinases that were active at pH 4·8 and determined when they appeared during germination and where they were located in 4-day germinated barley (green malt). A total of nine cysteine, four aspartic, and two serine class proteolytic activities that were active at pH 4·8 were extracted from the endosperm tissue of green malt. It seems probable that some or all of these endosperm endoproteinases, especially proteinases C7, C8, C11, D3, E3 and E4, are the ones most intimately involved in hydrolyzing the storage proteins during malting.     

NADPH Oxidases Act as Key Enzyme on Germination and Seedling Growth in Barley (Hordeum vulgare L.)

Reactive oxygen species (ROS) play an important role in seed germination. Although hydrogen peroxide (H₂O₂), a type of ROS, enhances the germination rate of various plant seeds, little is known about the mechanism. NADPH oxidases catalyze the production of superoxide anion (O₂^-) that is one of the ROS and the enzymes regulate plant development. We, therefore, investigated the role of NADPH oxidases in seed germination and seedling growth in barley (Hordeum vulgare L.). The production of O₂^- was observed both in embryo and aleurone layers in barley seeds treated with distilled water (DW). However, it was suppressed in seeds treated with diphenylene iodonium (DPI) chloride, NADPH oxidase inhibitor. Moreover, DPI markedly delayed germination and remarkably suppressed α-amylase activity in barley seeds, indicating the importance of NADPH oxidases in germination of barley seeds. The gene expression and the enzyme activity of NADPH oxidases gradually increased after imbibition, and the enzyme activities were closely correlated with seedling growth after imbibition. Besides, DPI markedly suppressed the seedling growth. These results indicated that NADPH oxidases perform a crucial function in germination and seedling growth in barley. These facts clearly reveal that O₂^- produced by NADPH oxidases after imbibition regulates seed germination and seedling growth in barley.     

Kunitz Soybean Trypsin Inhibitor is Modified at its C-terminus by Novel Soybean Thiol Protease (Protease T1)

Abstract

Kunitz soybean trypsin inhibitor (KSTI) is hydrolyzed during seed germination to yield amino acids needed to support initial seedling growth. The type of KSTI from Glycine max (L.) Merrill cv. Toyokomachi is KSTI-Ti ^b. The KSTI-Ti ^b from 4-day-old post-germination cotyledons (KSTI-Ti ^b’) has 3 or 4 amino acid residues cleaved off at the C-terminus. This KSTI modification is important to understand the mechanism of degradation in seed reserve proteins by proteases. Protease K1 also cleaves amino acid residues at the C-terminus of KSTI but it removes 5 amino acid residues. Therefore, we presumed the KSTI-Ti ^b’ was produced by a protease other than protease K1. In this study, the protease T1 responsible for cleavage of KSTI-Ti ^b at the C-terminus was purified. The enzyme was estimated to have a molecular mass of 33 kDa from its mobility on SDS-PAGE gels. The N-terminal amino acid sequence of the purified protease T1 corresponded to amino acids Phe-73 to Phe-92 of both thiol protease isoforms A and B from the soybean leaf, and shared 83% identity with the partial amino acid sequence of the membrane-associated cysteine protease from mung bean seedlings, a protease known to perform post-translational cleavage of C-terminal peptides of target proteins. Finally, this enzyme was shown to convert KSTI-Ti ^b to KSTI-Ti ^b’.     

Purification and Characterization of a Glutenin Hydrolysing Proteinase from Wheat Damaged by the New Zealand Wheat Bug, Nysius huttoni

A glutenin hydrolysing enzyme (bug proteinase), present in New Zealand wheat damaged by Nysius huttoni, was purified 50000-fold by anion exchange, hydrophobic interaction, immobilized metal ion affinity and gel filtration chromatography. The enzyme had an apparent M_r of 14·1k as determined by gel filtration chromatography. SDS-PAGE showed a major protein band of M_r 30k and six minor bands of M_r 13·2-28·5k, none of which was a glycoprotein. Isoelectric focusing revealed two major enzyme active bands (pI 9·6 and 9·2) and three minor activity bands (pI 9·9, 8·8 and 8·2). IEF showed no protein contaminants in the most purified sample. The enzymes had optimum activity at pH 8·9 and 45°C. The activity was stable in the pH range 4·5-11 and at 50°C for 20 min at pH 8·9. The bug proteinase was shown to be a serine proteinase by inhibition with phenylmethylsulphonyl fluoride and potato proteinase inhibitors (POT-IC and POT-ID). Thirty other proteinaceous serine proteinase inhibitors did not inhibit the enzyme. Bread baking with partially purified enzyme produced loaves with the poor quality characteristics of loaves made with bug-damage wheat.     

转精氨酸酶基因ZmARG玉米后代株系的遗传稳定性分析和功能验证

以T4、T5代转ZmARG基因玉米株系AKF65和受体自交系KF513为试材,对目标基因的整合表达、酶活性及产量相关农艺性状进行检测。结果表明,PCR、Southern和RT-PCR检测证明,ZmARG基因以单拷贝的形式插入基因组,在转录水平上能够稳定表达,且两个世代中稳定遗传。转基因株系灌浆期叶片中的精氨酸酶活力显著高于对照,萌发种子和苗期根中极显著高于对照,种子萌发时酶活力最高。转基因株系百粒重、穗重和小区产量显著高于对照,穗长极显著高于对照,其他各产量相关农艺性状差异不显著。     

Sulfated Polysaccharides from Seaweed Strandings as Renewable Source for Potential Antivirals against Herpes simplex Virus 1

Herpes simplex virus 1 (HSV-1) remains a prominent health concern widespread all over the world. The increasing genital infections by HSV-1 that might facilitate acquisition and transmission of HIV-1, the cumulative evidence that HSV-1 promotes neurodegenerative disorders, and the emergence of drug resistance signify the need for new antiviral agents. In this study, the in vitro anti-herpetic activity of sulfated polysaccharides (SPs) extracted by enzyme or hot water from seaweeds collected in France and Mexico from stranding events, were evaluated. The anti-herpetic activity evaluation of the semi-refined-polysaccharides (sr-SPs) and different ion exchange purified fractions showed a wide range of antiviral activity. Among them, the sr-SPs from the Rhodophyta Halymenia floresii showed stronger activity EC₅₀ 0.68 μg/mL with SI 1470, without cytotoxicity. Further, the antiviral activity of the sr-SPs evaluated at different treatment schemes showed a high EC₅₀ of 0.38 μg/mL during the viral adsorption assays when the polysaccharide and the virus were added simultaneously, whilst the protection on Vero cell during the post-infection assay was effective up to 1 h. The chemical composition, FTIR and ¹H NMR spectroscopic, and molecular weights of the sr-SPs from H. floresii were determined and discussed based on the anti-herpetic activity. The potential utilization of seaweed stranding as a source of antiviral compounds is addressed.     

Thermostability variation in alleles of barley beta-amylase

Thermostability assays in conjunction with IEF and molecular mapping were used to identify three beta-amylase alleles (Bmyl-Sd1, -Sd2L, -Sd2H) in cultivated barley and an additional allele (Bmy1-Sd3) in an accession of wild barley Hordeum vulgare ssp. spontaneum. The four forms of beta-amylase exhibit different rates of thermal inactivation in barley extracts. This variation was shown to persist after the proteolytic processing of the enzyme that occurs during germination. Three forms of beta-amylase representing the range of thermostabilities were purified and shown to have T₅₀ temperatures of 56·8°C for the Sd2L enzyme, 58·5°C for the Sd1 enzyme, and 60·8°C for the Sd3 beta-amylase from wild barley. Analysis of the relationship between beta-amylase thermostability and fermentability, i.e. the yield of fermentable sugars obtained from starch hydrolysis during brewing in 42 commercial malt samples suggests that increased thermostability results in more efficient starch degradation. Screening for specific beta-amylase alleles is proposed as a method for increasing fermentability in malting barley.     

Identification of a GDSL-motif carboxylester hydrolase from rice bran (Oryza sativa L.)

A major esterase (designated OsEST1) showing high activity using 1-naphthyl acetate as a substrate was identified from rice bran and purified approximately 239-fold to near-homogeniety. The purified enzyme migrated as a single polypeptide band on native and SDS-polyacrylamide gels and had a molecular mass of 25 kDa under denaturing conditions. Analysis of its tryptic peptides by MALDI-TOF-MS and subsequent data mining identified a corresponding cDNA OsEST1 consisting of 714 nucleotides and encoding a 238 amino acid protein. Analysis of its primary sequence indicated that OsEST1 is a GDSL-motif carboxylester hydrolase belonging to the SGNH protein subfamily in containing the putative catalytic triad of Ser¹¹, Asp¹⁸⁷, and His¹⁹⁰. OsEST1 showed the highest catalytic activity at approximately pH 8.0–8.5 and at 45 °C with K_m and V_max values for 1-naphthyl acetate of 172 μM and 63.7 μmol/min/mg protein, respectively. However, OsEST1 showed no activity with triacylglycerol. Alignment of the primary sequence of OsEST1 and other rice GDSL-motif esterases/lipases showed that OsEST1 aligns with a specific family of plant SGNH esterases involved in response to dehydration and cuticle formation. These results suggest that OsEST1 is not a lipase but an esterase activity which has some other function in rice, especially during seed development.     

Differential expression of defence-related proteins in Vigna unguiculata (L. Walp.) seedlings after infection with Fusarium oxysporum

This work aimed to study the induction of defence proteins in cowpea seedlings during the first days after infection with the fungi Fusarium oxysporum f. sp. cubenses and F. oxysporum f. sp. phaseoli. Cowpea seeds, after disinfection, were transferred to Petri dishes containing 0.5% agar and, after germination, were infected with a drop of a suspension containing 0.5 × 10⁴ spores ml⁻¹. Seedlings were collected at 24, 48, 72 and 96 h after infection and were dissected into leaves, hypocotyls, roots, cotyledons and teguments, which were measured and weighed for morphometric analysis. The agar medium was also analyzed. Enzymatic assays of proteic extracts yielded antimicrobial peptides detected by Western blotting. The cowpea seedlings showed a complex pattern of induction and repression of defence proteins in response to infection by both pathogens. Furthermore, morphometric analysis showed differences between infected and control seedlings. Infected samples did not at any time exhibit chitinase activity, but did exhibit different β-1,3-glucanase and peroxidase activities. Western blotting for lipid transfer protein (LTP) demonstrated its presence in all parts of the infected seedlings. Exuded proteins, also obtained from cowpea seeds in the germination medium, were separated by SDS-PAGE and tricine gel electrophoresis. The analysis showed that some proteins were exuded from moistened cowpea seeds, particularly after F. oxysporum infection.     

Protein composition of grains of theleguminosae

The main seed proteins of Pisum and faba beans are the storage proteins synthesised in the seed and subsequently utilised to supply nitrogen and carbon for biosynthesis during germination and seedling development. There are two major families of storage proteins, legumin and vicilin. Legumins are hexameric proteins comprising of 6M _r 60 000 subunits which, as isolated from mature seeds, show considerable charge and size heterogeneity. Vicilins are a less well-defined group of proteins consisting largely ofM _r 50 000 subunits and non-equimolar amounts of lower molecular weight subunits. A third storage protein, convicilin, is also present in peas and beans. It is a vicilin-type protein with vicilin immunological determinants. Proteins homologous to legumin have been found in Arachis, Glycine,Vigna unguiculata, Lupinus, Cicer, Lens and Lathyrus. Equivalent vicilin-type proteins occur inPhaseolus vulgaris, Glycine, Arachis andVigna unguiculata. The storage proteins have an unusual amino acid composition related to their biological role. They are multimeric and readily undergo association/dissociation reactions. They show a high degree of charge and size heterogeneity, partly caused by co and post-translational modifications and partly due to gene divergence.     

Wheat Superoxide Dismutase Isoenzymes

Evidence was obtained indicating that the superoxide dismutase (SOD) A and B isoenzymes of the wheat cultivar Tonic are of the Cu/Zn type, whereas the SOD–C group are similar to manganese containing dismutases. Heat inactivation plots showed that the enzymic activity in crude extracts was relatively stable up to 50°C. The thermodynamic parameters enthalpy, ΔH#, free energy, ΔG#, and entropy, ΔS#, were estimated for the thermal inactivation of extracted SOD activity and a purified SOD–C isoenzyme. The SOD-C isoenzyme was purified extensively and shown to contain two closely related tetrametic isozymes (pI6·0 and 6·1) ofM_r80 000. AM_rof 20 000 has been calculated for the subunits. TheN-terminal amino acid sequence of the purified SOD–C could be aligned with that of the Mn–SOD enzyme of maize and showed 57% homology.     

The first characterised wheat (Triticum aestivum L.) member of the nudix hydrolase family shows specificity for NAD(P)(H) and FAD

We cloned the first nudix hydrolase of wheat (Triticum aestivum L.) (TaNUDT1) and expressed it using Escherichia coli as expression host. Properties of the purified His₆-tagged protein were studied. The sequence codes for a nudix hydrolase with a molecular mass of around 43x10³ and a predicted pI of 5.68. The characteristic residues of the nudix box were highly conserved in the wheat enzyme sequence, and TaNUDT1 is most probably targeted to the peroxisomes. In the presence of dithiothreitol and MnCl₂, the enzyme hydrolyses the nicotinamide coenzymes NAD(P)(H) as could be predicted by the presence of a conserved amino acid array C-terminal to the nudix box, and the enzyme has higher affinity towards the reduced forms of the coenzymes. In light of previous reports of nudix enzymes and the physiological concentration of their substrates, we found its K_m values towards some of the coenzymes to be relatively high. As NAD(P)(H) and FAD play crucial roles in cell metabolism, extensive hydrolysis could be detrimental to cell functioning. We speculate that a decreased enzyme affinity may point to a built-in restriction on coenzyme hydrolysis in vivo. Furthermore, by hydrolysing the cofactors of several redox enzymes, this enzyme may well affect several wheat based processes.     

Avenanthramide content and related enzyme activities in oats as affected by steeping and germination

Samples from three different oat (Avena sativa L.) cultivars, Vista, Gem and Dane were steeped and germinated in a pilot plant malting system. The content of avenanthramides, antioxidant compounds unique to oats, and some unknown compounds as well as the activities of the avenanthramide-synthesising enzyme hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) and the avenanthramide-oxidizing enzyme phenoloxidase (PO) were measured. An increase in avenanthramide content of germinated seeds, as compared to raw grains, was observed for Dane (125%, p < 0.001) and for Vista (29%, p = 0.007). The HHT activity increased 62% (p = 0.014) in Dane, whereas no change was detected in Vista and Gem. The PO activity decreased slightly during the germination process for Gem (p < 0.001) and Vista (p = 0.005). Many of the unknown compounds increased significantly during germination in all three cultivars, and one of them was identified to be the avenanthramide N-(4′-hydroxy-3′-methoxy-(E)-cinnamoyl)-5-hydroxy-4-methoxyanthranilic acid. This study indicates that a highly controlled steeping and germination process can be a valuable method to increase the content of endogenous avenanthramides in oats.     

Some Characteristics of β-D-Xylopyranosidases, α-L-Arabinofuranosidases and an Arabinoxylan α-L-arabinofuranohydrolase from Wheat Bran and Germinated Wheat

Enzymes from wheat bran and germinated wheat involved in the degradation of arabinoxylan and arabinoxylooligosaccharides were investigated. Fourp-nitrophenyl-α-l-arabinofuranoside hydrolysing activities (ArafI-IV) and threep-nitrophenyl-β-d-xylopyranoside hydrolysing activities (XylpI-III) were identified in wheat kernels and germinating wheat. Two of these activities, ArafI and XylpII, were purified about 10 000-fold from wheat bran. Both enzymes were inactive towards polymeric arabinoxylan. ArafI produced no arabinose but some xylose from arabinoxylooligosaccharides, while XylpII gave only xylose upon incubation with this substrate. An arabinoxylan arabinofuranohydrolase (AXH) was found in wheat bran and germinated wheat. This enzyme was active towards the polymeric substrate, but was unable to hydrolysep-nitrophenyl-α-l-arabinofuranoside. TheM_rs of these enzymes were determined by size exclusion chromatography and were in the range of 40–50 000, except for ArafIII, for which aM_rof 104 000 was determined. During germination, the levels of these enzymes increased markedly between the third and fifth day, after which some of them decreased again by the seventh day. ArafI-IV were inhibited strongly by arabinonic acid-γ-lactone, while xylonic acid-γ-lactone was a good inhibitor of XylpI-III. The latter lactone also inhibited ArafI. Neither of these lactones inhibited AXH. Endoxylanase activity was demonstrated but not quantified.     

GA3 and Proline Promote Germination of Wheat Seeds by Stimulating α-Amylase at Unfavorable Temperatures

abstract

The effects of various constant temperatures (4, 9, 14, 19, 24, 29, 34, and 38°C) on the germination of winter wheat seed ( Triticum aestivum L. cv. ‘Koyuki’) in a dark condition were studied. The maximum germination percentage was 98% at 24°C. The speed of germination was fastest at 29°G. These results indicate that the most suitable temperature for germination was in the range of 24 to 29°G. α-Amylase expression during germination was also high at higher temperature, and maximum expression occurred at 29°C, although a high temperature of 38°C prevented the synthesis of α-amylase. The close correlation between germination and α-amylase activity at various temperatures indicates that α-amylase is an essential factor for the temperature-dependent germination of wheat seed. In contrast, accumulation of proline increased at a lower temperature, and was the highest at 4°G. We also studied the effects of gibberellin (GA₃) and proline, a compatible osmotica in alleviating the effect of low and high temperature stresses. Pre-soaking treatment with GA3 and proline was effective in promoting germination and increasing α-amylase expression at a low (4°C) and high (38°C) temperature. These results suggest that GA₃ and proline exhibit positive effects on stress alleviation through the stimulation of α-amylase expression.     

开放式空气CO2浓度增高和施氮量对水稻结实期叶片内肽酶活力的影响

 利用农田开放式空气CO2浓度增高 (free air CO2 enrichment,FACE) 系统,CO2浓度设正常CO2 (ambient, AMB) 和高CO2 (FACE,AMB + 200 μmol/mol) 2个水平,施N量设低氮 (LN,15 g/m2,以纯氮计)、中氮 (NN,25 g/m2)和高氮 (HN,35 g / m2) 3个水平,对水稻品种武香粳14结实期剑叶和倒2叶的内肽酶活力变化情况进行了研究。结果表明: 1) 同AMB相比,FACE处理使剑叶抽穗后10 d、20 d以及倒2叶抽穗到抽穗后20 d内肽酶活力明显提高,但使剑叶成熟期内肽酶活力明显下降;使剑叶抽穗后10 d、20 d和成熟期以及倒2叶抽穗到成熟期内肽酶比活力明显提高;FACE对抽穗后10 d、20 d叶片内肽酶活力和比活力影响较大,而对抽穗期和成熟期的影响较小。同LN相比,HN使灌浆前期叶片内肽酶活力明显降低,灌浆中后期则呈增加趋势;HN降低了叶片各期内肽酶比活力,灌浆前期降幅大于后期。 2) 同AMB相比,FACE使叶片抽穗后10 d至成熟期可溶性蛋白含量明显降低;同LN相比,HN明显减缓FACE处理对抽穗后10 d至成熟期功能叶片可溶性蛋白含量的影响。 3）除成熟期叶片可溶性蛋白含量与内肽酶活力呈正相关外,结实期剑叶及倒2叶可溶性蛋白含量与对应时期内肽酶活力呈显著负相关。上述结果说明,FACE处理下水稻剑叶及倒2叶结实中后期可溶性蛋白含量明显下降与结实期叶片内肽酶活力的变化关系密切。