Changes in Endopeptidase Activity during Photosynthetic Declination in Rice Leaf

Two japonica rice varieties, Wuyujing 3 and 97-7, were used to study the changes in contents of soluble protein, free amino acids and endopeptidase activity during photosynthetic declination. The content of soluble protein in flag leaf of cv.Wuyujing 3 was higher than that of cv. 97-7, but decreased rapidly in Wuyujing 3. Free amino acids in flag leaf and the thirteenth leaf of Wuyujing 3 started to increase 10 days before the turning point of photosynthetic declination (TPPD), while it occurred just 1-2 days before TPPD in the flag leaf and the thirteenth leaf of 97-7. During reversible phase of photosynthetic declination,endopeptidase activity remained at a low level and increased slightly only in the later part of this phase. Then it rose up rapidly at irreversible decline phase and reached a very high level. For Wuyujing 3, the change in endopeptidase activity in the thirteenth leaf was parallel to that in flag leaf. However, for 97-7, the rapid increase of endopeptidase activity in the thirteenth leaf started later than that of flag leaf. The results implied that the rate of protein breakdown and conversion to transportable nitrogen in leaves of 97-7 was slower than that in leaves of Wuyujing 3 during photosynthetic declination and it led to relatively lower seed setting rate and fully filling grains rate in 97-7. This may be one of the important reasons why 97-7 could not bring the high yield potentiality into play and the findings may be taken into consideration while breeding for high potential varieties in future.     

用凝胶电泳法研究小麦叶片衰老期间的内肽酶同工酶

采用SDS－聚丙烯酰胺凝胶电泳（SDS－PAGE）和梯度凝胶电泳方法，研究了小麦旗叶暗诱导衰老过程中内肽酶同工酶的变化。发现用SDS－PAGE法几科检测不到新的内肽酶同工酶；用梯度凝胶电泳法则能检测到6种新的同工酶，而且在第一叶自然衰老过程中也能检测到。表明在这两种衰老过程中内肽酶酶谱变化基本相似。     

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。     

Early Morphological and Physiological Events Occurring During Germination of Maize Seeds

The early morphological and physiological events occurring during maize （Zea mays cv. Nongda 108） seed imbibition and germination were studied. Water uptake of seeds exhibited a triphasic pattern with a marked increase during the initial phase of imbibition, and then a slow increase, followed by a second substantial increase. Imbibition time for 10 and 50% of seed germination was about 26 and 46 h at 30℃, respectively. The relative conductivity of maize seeds dramatically decreased during the initial phase of imbibition, followed by a substantial increase. Respiratory rate of seeds gradually increased with imbibition. Length of root cap cells decreased during the initial phase and then increased; those of meristematic zone cells increased during the initial phase and then decreased; and those of elongation zone cells and of the whole elongation zone of the radicle gradually increased during germination. The contents of soluble sugars and starch in embryos gradually decreased as the activities of α- and β-amylase strikingly increased with imbibition. In the meantime, protein contents of embryos gradually decreased and free amino acid content increased. The activities of aminopeptidase and endopeptidase increased until 12 h of imbibition and then decreased. It is concluded that germination of maize seeds is mainly completed by extension of cells in the elongation zone of the radicle, and that mobilization of stored reserves in the embryo during the initial phase of imbibition is also an early event during seed germination.     

小麦叶片衰老期间内肽酶同工酶的变化

以扬麦158为试验材料,采用改进的以血红蛋白为底物的梯度凝胶电泳结合内肽酶同工酶染色方法,探讨在自然衰老和暗诱导衰老条件下,叶片内肽酶(EP)活性和同工酶酶谱的变化模式。结果显示小麦叶片暗诱导衰老和自然衰老过程中,内肽酶活力均先上升后下降;在叶片暗诱导衰老过程中先后出现4种新的内肽酶同工酶,而在自然衰老过程中先后出现5种新的内肽酶同工酶,其中EP3只在自然衰老末期才能检测到。表明自然衰老与暗诱导衰老过程中的内肽酶同工酶谱变化基本相似,因此暗诱导衰老可以作为研究叶片自然衰老期间内肽酶的一种模式。     

苜蓿中金属肽酶对青贮过程中蛋白降解的作用

为探讨金属肽酶在苜蓿青贮过程中对蛋白的降解作用,以苜蓿（Medicago stativa L.）绿汁发酵液模拟青贮发酵过程,添加金属肽酶抑制剂后,测定发酵后第0.5、1、1.5、2、2.5、3、4、5、7和14天时绿汁发酵液的蛋白组分。结果表明,在苜蓿发酵液发酵过程中添加金属肽酶抑制剂能够降低pH值、非蛋白氮、游离氨基酸和氨态氮含量,增加乳酸含量;发酵第14天时,抑制剂处理组非蛋白氮、游离氨基酸和氨态氮质量分数为483.98 g/kg TN,256.98 g/kg TN和26.11 g/kg TN,与对照组相比分别降低28.92%,38.59%和67.08%。金属肽酶对苜蓿发酵液蛋白降解起着一定的作用;添加金属肽酶抑制剂能够提高发酵液发酵品质。     

盐胁迫下不结球白菜幼苗叶片内肽酶同工酶变化及生化特性研究

研究了不同盐浓度胁迫下不结球白菜暑绿幼苗叶片中可溶性蛋白质含量、内肽酶同工酶及其活性的变化和内肽酶同工酶生化特性.结果表明,暑绿叶片中存在5种内肽酶同工酶(EP1～EP5),其中,EP3和EP5为半胱氨酸型内肽酶,EP1和EP2为丝氨酸型内肽酶,而EP4几乎不被所试的各种内肽酶抑制剂所抑制;内肽酶的最适pH为6.0,最适温度为50 ℃;EP3、EP4和EP5的热稳定性较好,在60 ℃下保温1 h后还保留较高活性.100 mmol·L-1 NaCl处理暑绿4 d后,EP4活性增强,其他4种同工酶活性变化不明显;200 mmol·L-1 NaCl处理相同时间,叶片1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco)等蛋白发生明显降解,EP2、EP4、EP5活性随着时间推进不断升高,而EP1、EP3活性明显降低,显示EP2、EP4、EP5可能在盐胁迫诱导的暑绿叶片衰老中起主要作用.     

Molecular cloning and expression analysis of the main gliadin-degrading cysteine endopeptidase EP8 from triticale

During the development and maturation of cereal grains, storage proteins are accumulated in the starchy endosperm. The enzymes responsible for the mobilisation of stored proteins during seed germination are cysteine endopeptidases and serine carboxypeptidases. In our previous study, we purified the major gliadin-degrading cysteine, endopeptidase EP8, from germinating triticale kernels. We confirmed in an experiment with exogenous gibberellic acid (GA₃) that this enzyme is synthesised in aleurone during germination. In this study, the nucleotide sequence of EP8, a barley EP-A (HvEPA) homologue, and the expression pattern during grain development and germination have been analysed. Additionally, by comparing the activity of purified EP8 with the activity pattern of crude enzymatic extracts, the lack of enzyme activity in some tissues and during certain developmental stages has been demonstrated. The correlation of the results of the EP8 expression analysis with the activity pattern of this endopeptidase allowed us to formulate a hypothesis regarding the mechanism of EP8 activity regulation. We believe that the absence of active EP8 until the third day of germination may be associated with high levels of endogenous cystatin TrcC-4, which has the ability to inhibit EP8 in vitro.     

Gene Localization of Aspartate Aminotransferase and Endopeptidase Isozymes in Wheat and Rye Using Developmental and Organ-Specific Patterns

Wheat, rye and wheat-rye addition lines have been investigated regarding their developmental and organ-specific isozyme patterns of aspartate amino-transferase (AAT) and endopeptidase (EP). Evidence is given, that development-specific isozymes of AAT are encoded by chromosomes 3R and 4R of ‘Imperial’ rye which can be used as biochemical markers up to leaf age of 14 days. Organ-specific increase of intensity of bands for AAT in stems could be assigned to genes or alleles of chromosome 3A of ‘Chinese Spring’ wheat. For EP new markers were localized on chromosomes 4R and 6R of ‘Imperial’ rye showing variability. Utilization of these markers is possible at all developmental stages of the leaves. Mechanisms of gene regulation are discussed.     

Malt hydrolysates for gluten-free applications: Autolytic and proline endopeptidase assisted removal of prolamins from wheat,barley and rye

Cereal based products intended for gluten sensitive individuals, particularly to celiac disease patients, tend to have poor organoleptic qualities and they contain low levels of healthy whole grain compounds. Adding whole grain ingredients, such as malt hydrolysates, could compensate these defects provided that the ingredients are adequately free from toxic prolamin epitopes. Here we demonstrate that the level of toxic prolamin epitopes in the malt autolysates (wheat, barley, rye) were substantially lower than in the native malts but too high to allow “very low in gluten” labelling. To further eliminate the residual levels of toxic prolamin epitopes, a proline-specific endoprotease from Aspergillus niger was added to the malt autolysates. In the resulting malt hydrolysates (of wheat and rye but not barley), the prolamins were indeed greatly reduced and were below the very low gluten limit of 100 mg/kg. Malt hydrolysates with adequately low gluten levels may potentially be used as novel ingredients within gluten-free foods.