A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests

Cystatin CsC, a cysteine proteinase inhibitor from chestnut (Castanea sativa) seeds, has been purified and characterized. Its full-length cDNA clone was isolated from an immature chestnut cotyledon library. The inhibitor was expressed in Escherichia coli and purified from bacterial extracts. Identity of both seed and recombinant cystatin was confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis, two-dimensional electrophoresis and N-terminal sequencing. CsC has a molecular mass of 11275 Da and pI of 6.9. Its amino acid sequence includes all three motifs that are thought to be essential for inhibitory activity, and shows significant identity to other phytocystatins, especially that of cowpea (70%). Recombinant CsC inhibited papain (Ki 29 nM), ficin (Ki 65 nM), chymopapain (Ki 366 nM), and cathepsin B (Ki 473 nM). By contrast with most cystatins, it was also effective towards trypsin (Ki 3489 nM). CsC is active against digestive proteinases from the insect Tribolium castaneum and the mite Dermatophagoides farinae, two important agricultural pests. Its effects on the cysteine proteinase activity of two closely related mite species revealed the high specificity of the chestnut cystatin.     

Occurrence of Free Ceramide in Acetobacter xylinum

Oryzacystatin, a proteinaceous cysteine proteinase inhibitor (cystatin) from rice seeds and probably the first well-defined cystatin superfamily member of plant origin, was immunologically investigated for its occurrence in rice seeds during maturation and germination. The enzyme-linked immunosorbent assay (ELISA) using anti-oryzacystatin Immunoglobulin G showed that all the investigated 23 cultivars of rice, Oryza sativa L. japónica, contained oryzacystatin at 1 ~ 4 mg % in their seeds. Particularly, oryzacystatin levels were high in precocious cultivars and low in sticky rice cultivars. The use of the ELISA method for the representative rice cultivar, Nipponbare, gave the result that in the seed maturation process, oryzacystatin was synthesized in precedence to total seed protein. In the germination process, oryzacystatin tended to decrease in accordance with degradation of total seed protein.     

Hg2+ 与POD 复合处理对小麦萌发及幼苗生长的影响

通过水培实验研究了0、10、20、50、70和100 mg.L-1 Hg2+和65 U.mL-1的过氧化物酶(POD)混合浸种对小麦(Triticum aestivum L.)萌发及幼苗生长过程中的10个形态和生理生化指标的影响。结果表明：施加外源POD可明显提高种子发芽率、植株日均增重和幼苗叶片的可溶性蛋白含量,增加幼苗叶片内源超氧物歧化酶(SOD)和POD的活性,拮抗Hg2+胁迫对种子发芽率、苗高、日均增重及叶片可溶性蛋白含量的不利影响,Hg2+浓度较高时(≥50 mg.L-1),对种子发芽率和日均增重的拮抗作用更明显,并对较低浓度Hg2+(≤20 mg.L-1)胁迫引起的叶片SOD活性的上升和低于100 mg.L-1的Hg2+胁迫引起的叶片 POD活性的上升有进一步的促进作用;然而对幼苗平均最长根长度、侧根数和幼苗叶片叶绿素含量则无明显影响。     

Gene expression programs during Brassica oleracea seed maturation, osmopriming, and germination are indicators of progression of the germination process and the stress tolerance level

During seed maturation and germination, major changes in physiological status, gene expression, and metabolic events take place. Using chlorophyll sorting, osmopriming, and different drying regimes, Brassica oleracea seed lots of different maturity, stress tolerance, and germination behavior were created. Through careful physiological analysis of these seed lots combined with gene expression analysis using a dedicated cDNA microarray, gene expression could be correlated to physiological processes that occurred within the seeds. In addition, gene expression was studied during early stages of seed germination, prior to radicle emergence, since very little detailed information of gene expression during this process is available. During seed maturation expression of many known seed maturation genes, such as late-embryogenesis abundant or storage-compound genes, was high. Notably, a small but distinct subgroup of the maturation genes was found to correlate to seed stress tolerance in osmoprimed and dried seeds. Expression of these genes rapidly declined during priming and/or germination in water. The majority of the genes on the microarray were up-regulated during osmopriming and during germination on water, confirming the hypothesis that during osmopriming, germination-related processes are initiated. Finally, a large group of genes was up-regulated during germination on water, but not during osmopriming. These represent genes that are specific to germination in water. Germination-related gene expression was found to be partially reversible by physiological treatments such as slow drying of osmoprimed seeds. This correlated to the ability of seeds to withstand stress.     

Spatial and temporal expression of a maize lipid transfer protein gene.

We studied the temporal and spatial pattern of lipid transfer protein (LTP) gene expression, as well as the localization of this protein, in maize. Using an LTP gene, we observed an accumulation of LTP mRNA in embryos and endosperms during seed maturation. LTP gene expression was also investigated in young seedlings. After germination, the level of LTP mRNA in the coleoptile increased, with a maximum at 7 days, whereas LTP mRNA levels were low in the scutellum and negligible in roots. The high levels of LTP mRNA found in coleoptiles and embryos were confirmed by in situ hybridization. Moreover, LTP gene expression appeared to be localized in the external cellular layers and around the leaf veins. Using immunogold methods, we also observed that LTP was distributed heterogeneously in the different cells of coleoptiles and leaves. The highest concentrations of LTP were found in the outer epidermis of the coleoptiles as well as the leaf veins. Together, our observations indicate that LTP gene expression is not only organ specific and time specific but also cell specific.     

Molecular and biochemical characterisation of two aspartic proteinases TcAP1 and TcAP2 from Theobroma cacao seeds

Aspartic proteinase (EC 3.4.23) activity plays a pivotal role in the degradation of Theobroma cacao L. seed proteins during the fermentation step of cacao bean processing. Therefore, this enzyme is believed to be critical for the formation of the peptide and amino acid cocoa flavor precursors that occurs during fermentation. Using cDNA cloning and northern blot analysis, we show here that there are at least two distinct aspartic proteinase genes ( TcAP1 and TcAP2) expressed during cacao seed development. Both genes are expressed early during seed development and their mRNA levels decrease towards the end of seed maturation. TcAP2 is expressed at a much higher level than TcAP1, although the expression of TcAP1 increases slightly during germination. The proteins encoded by TcAP1 and TcAP2 are relatively different from each other (73% identity). This, and the fact that the two corresponding genes have different expression patterns, suggests that the TcAP1 and TcAP2 proteins may have different functions in the maturing seeds and during germination. Because the TcAP2 gene is expressed at a much higher level during seed development than TcAP1, it is likely that the TcAP2 protein is primarily responsible for the majority of the industrially important protein hydrolysis that occurs during cacao bean fermentation. Finally, TcAP2 has been functionally expressed in the yeast Yarrowia lipolytica. The secreted recombinant protein is able to hydrolyse bovine haemoglobin at acidic pH and is sensitive to pepstatin A, confirming that TcAP2 encodes an aspartic proteinase, and strongly suggests that this gene encodes the well-characterized aspartic proteinase of mature cacao seeds.     

板栗“燕山早丰”幼苗光合与碳氮代谢对干旱胁迫的响应

干旱是影响燕山地区板栗树生长和产量的主要因子。为了在整株水平上研究板栗幼苗对干旱胁迫的响应,本试验以盆栽“燕山早丰”板栗幼苗为研究对象,通过模拟自然干旱处理22 d,测定叶片光合特性,根、茎、叶生物量、脯氨酸、丙二醛、碳、氮等生理指标变化。结果表明: 与正常浇水相比,干旱胁迫下幼苗根、茎、叶含水量分别显著下降18.3%、29.0%和62.8%,脯氨酸(355.0%～1586.7%)和丙二醛(41.1%～81.3%)含量显著上升(茎中丙二醛除外),但叶部非光化学淬灭系数和净光合速率分别显著下降49.4%和77.4%;同时,茎和叶中非结构碳水化合物含量分别显著增加21.4%和69.5%,根中增加幅度未达显著差异水平;根和叶中硝态氮含量分别显著增加28.9%和26.8%,茎中增加幅度未达显著差异水平;根、茎、叶中铵态氮含量分别增加了16.2%、12.9%、217.6%,但仅在叶部差异显著。综上,干旱胁迫对燕山早丰板栗幼苗产生了较严重的伤害,显著抑制了其光合性能,但能够通过增强体内碳氮代谢来提高其适应干旱环境的能力。本研究结果可为当地板栗抗旱性资源选育和栽培提供参考。     

Amino Acids Content in Germinating Seeds and Seedlings from Castanea sativa L

During germination the chestnut (Castanea sativa L.) var ecotype 33 accumulates a large amount of asparagine in the cotyledons. This compound also accumulates in the growing axis:shoots and roots. In the cotyledons, γ-aminobutyrate (GABA) represents a major amino compound during germination and early seedling growth. In young seedlings, 35 days old, arginine predominates over the other soluble amino acids, particularly in roots. Five enzymic activities involved in arginine and GABA have been measured in the storage organ of the seed: arginase and ornithine carbamyltransferase decrease during germination indicating the slowing down of the urea cycle. In contrast, ornithine aminotransferase increases. Glutamate decarboxylase is particularly active about 21 days after imbibition and GABA aminotransferase activity decreases during germination. These two activities are in good agreement with the likely transport of GABA from cotyledons to growing axis. Asparagine, arginine, and GABA are the three amino compounds obviously involved in the mobilization of nitrogen reserves in the germinating chestnut seeds Castanea sativa.     

Differential localization of two acid proteinases in germinating barley (Hordeum vulgare) seed

A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in ungerminated barley ( Hordeum vulgare ) seed while a 30 kDa cysteine endoproteinase (EC 3.4.22) is one of the proteinases synthesized de novo in the germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by immunomicroscopy. The results confirm that they have completely different functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggestions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32 + 16 kDa) was found in all the living tissues, whereas the low molecular mass form (29 + 11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, and later in the vacuoles which are formed by fusion of the protein bodies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or roots. The 30 kDa cysteine proteinase was detected in the Golgi apparatus and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine proteinase which is apparently one of the proteases initiating the hydrolysis of storage proteins in the starchy endosperm.     

Ultra-low gossypol cottonseed: generational stability of the seed-specific, RNAi-mediated phenotype and resumption of terpenoid profile following seed germination

Cottonseed, containing 22.5% protein, remains an under-utilized and under-valued resource because of the presence of toxic gossypol. RNAi-knockdown of δ-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseed (ULGCS). In the original study, we observed that RNAi plants, a month or older, maintain normal complement of gossypol and related terpenoids in the roots, foliage, floral organs, and young bolls. However, the terpenoid levels and profile of the RNAi lines during the early stages of germination, under normal conditions and in response to pathogen exposure, had not been examined. Results obtained in this study show that during the early stages of seed germination/seedling growth, in both non-transgenic and RNAi lines, the tissues derived directly from bulk of the seed kernel (cotyledon and hypocotyl) synthesize little, if any new terpenoids. However, the growing root tissue and the emerging true leaves of RNAi seedlings showed normal, wild-type terpenoid levels. Biochemical and molecular analyses showed that pathogen-challenged parts of RNAi seedlings are capable of launching a terpenoid-based defence response. Nine different RNAi lines were monitored for five generations. The results show that, unlike the unstable nature of antisense-mediated low seed-gossypol phenotype, the RNAi-mediated ULGCS trait exhibited multi-generational stability.     

Accumulation and degradation of thiamin-binding protein and level of thiamin in wheat seeds during seed maturation and germination

Changes in the levels of thiamin-binding globulin and thiamin in wheat seeds during maturation and germination were studied. The thiamin-binding activity of the seed proteins increased with seed development after flowering. The thiamin content of the seeds also increased with development. Thiamin-binding activity decreased during seed germination. On the other hand, immunological analysis using an antibody directed against the thiamin-binding protein isolated from wheat seeds showed that the thiamin-binding globulin accumulated in the aleurone layer of the seeds during maturation, and then the protein was degraded and disappeared during seed germination. These results suggested that the thiamin-binding globulin of wheat seeds was synthesized and accumulated in the aleurone layer of the seeds with seed development, similar to the thiamin-binding albumin in sesame seeds, and that thiamin bound to the thiamin-binding globulin in the dormant wheat seeds for germ growth during germination.     

紫茎泽兰对三种岩溶地区木本植物种子萌发的化感作用

采用生物测定法研究了紫茎泽兰叶和根水浸提液对任豆、香椿和龙须藤等三种岩溶木本植物种子萌发的化感作用。结果表明,除最低浓度(0.0125 g/mL)外,其它浓度的叶浸提液均能显著抑制香椿种子的萌发及幼苗生长,浓度越高抑制作用越大;对任豆和龙须藤种子萌发也有一定的化感作用,但抑制作用相对较弱,有时为促进作用。紫茎泽兰叶浸提液对受试植物的化感作用强于根水浸提液。     

栽培大豆和野生大豆耐盐性及离子效应的比较

以国际上常用的耐盐大豆（Glycine max L.)品种Lee68为对照,在发芽期和苗期两个阶段,利用发芽指数、指害指数和耐盐系数等指标对一年生具盐腺野生大豆（Glycine soja L.)和部分栽培大豆（Glycine max L.)及某些野生大豆品系或品种的耐盐性进行了比较,讨论了耐盐指标的可行性。从离子效应方面比较了Na^ 和Cl^-对大豆发芽率的影响,并对具盐腺野生大豆的耐盐机理进行了初步分析。结果表明,大豆品种的耐盐性在发芽期和苗期无一致相关性。轻度等渗胁迫下,Na^ 对种子发芽率的抑制作用大于Cl^-,而重度等渗胁迫下则相反。通过减少由根系吸收的Na^ 、Cl^-向叶片的运输,维持叶片中较高含量的K^ ,减轻盐离子毒害,可能是具盐腺野生大事耐盐的主要生理机制之一。