Immunological relatedness of cytoplasmic and membrane-bound acid phosphatases from Dioscorea cayenensis rotundata

Antibodies were raised against one cytoplasmic and two membrane-bound acid phosphatases purified from yam tubers (Dioscorea cayenensis rotundata). Experiments of immunoinactivation and immunoelectrophoresis revealed cross-immunological reactions between the cytoplasmic enzyme (acid phosphatase A) and one of two membrane-bound counterparts (acid phosphatase B) suggesting that these molecules share common antigenic determinants. The antibodies raised against the other membrane-bound enzyme (acid phosphatase C) only inhibited and precipitated this enzyme.     

Purification and characterization of a lectin from wild sunflower (Helianthus tuberosus L.) tubers

A lectin (HTTL) was isolated from Helianthus tuberosus L. (wild sunflower) tubers using ion-exchange chromatography, gel filtration, and affinity chromatography. The lectin agglutinated both untreated and trypsin-treated rabbit erythrocytes and did not agglutinate human blood cells of groups A, B, and O. The gel filtration showed the native molecular mass of 72 kDa and subunit molecular masses of 17 and 18.5 kDa on 12% SDS-PAGE. The lectin activity was inhibited by D-mannose. The tetrameric protein revealed a unique characteristic by forming a broad zone of protein in native PAGE at pH 8.3, which dissociated into seven subunits of varying e/m ratios on acid gel at pH 4.3. These seven bands revealed two polypeptide species of molecular masses 17 and 18.5 kDa on 12% SDS-PAGE, as in the case of the native protein. The result indicated that of the seven subunits, three were homotetramers of 17 kDa, one was a homotetramer of 18.5 kDa, and three were heterotetramers of 17 and 18.5 kDa. The lectin was thermostable with broad pH optima (pH 4-8) and had no requirement for divalent metal cations for its activity. The amino acid composition showed that the lectin contained higher amounts of glycine, alanine, and lysine, but no methionine. The sugar content was estimated to be 5.3% mannose equivalent. The HTTL was mitogenic to mouse spleen (total) cells at 25 microg/ml concentration. The lectin showed characteristics different from those of the earlier reported H. tuberosus tuber lectins and hence opens up a new avenue to investigate the structure-function relationship of lectin in Helianthus species.     

热碱预处理对菊芋茎秆组成和酶水解影响

为了深入了解菊芋茎秆用于生物能源转化的潜力,在对菊芋茎秆的全秆、韧皮以及髓芯的组成分析基础上,采用不同浓度的Na OH在121℃对菊芋茎秆进行预处理,并对预处理后的茎秆进行酶水解。结果表明:菊芋茎秆具有较高木质素含量(32.0%),且韧皮中木质素含量最高;茎秆中碳水化合物总含量与传统农作物秸秆相当,但纤维素含量相对较高(40.5%),半纤维含量相对较低(19.6%)。经不同浓度Na OH预处理后,相对于未处理茎秆,全秆、韧皮以及髓芯中木质素含量分别降低13.1%–13.4%、8.3%–13.5%和19.9%–27.2%,半纤维素含量分别降低了87.8%–96.9%、87.6%–95.0%和74.0%–90.2%。纤维素含量在全秆、韧皮和髓芯中相应增加了56.5%–60.2%、52.2%–55.4%和62.7%–73.2%。酶水解的结果显示,增加预处理过程中Na OH的浓度,全秆和韧皮的水解率可被提高2.3–2.6倍和10.3–18.5倍。虽然热Na OH预处理可以有效地改善髓芯水解性能,但经过高浓度的Na OH(2.0 mol/L)预处理,髓芯的水解性能下降明显。由此可见,菊芋用于生物能源转化技术中,热碱法可较好地适用于菊芋秸秆预处理。提高碱浓度,有利于半纤维素和木质素的去除,并实现酶水解糖化产率的提高。但鉴于碱浓度过高会造成髓芯糖产率降低,热碱预处理菊芋秸秆工艺条件需进一步优化。     

菊芋粕菊粉的酶法提取、组成及抗氧化活性研究

本研究拟应用酶法提取技术解决菊芋菊粉工业化生产中的废弃物——菊芋粕再利用程度低的问题,并评价菊芋粕菊粉的抗氧化活性功效。对菊芋粕菊粉的果胶酶酶法提取的最佳条件通过响应面法进行了优化,并对菊芋初次水提菊粉(primary water-extracted inulin,PWI)和二次酶提菊粉(secondary enzymatic-extracted inulin,SEI)的组成成分和抗氧化活性进行了比较分析。响应面法优化确定的菊芋粕菊粉最佳酶法提取条件为:pH4.5、提取温度50 ℃、酶底比7.5 U/g、提取时间2 h,该提取方法所得菊芋粕菊粉的得率为35.30%±0.85%,与传统热水浸提法相比菊粉得率提高38.16%。组成分析结果显示,SEI的总糖和菊粉含量均显著高于PWI( P <0.05)。在菊粉聚合度方面,PWI中蔗果三糖和蔗果四糖含量较高,而SEI中蔗果五糖、蔗果六糖及其以上聚合度菊粉的含量较高。此外,SEI的抗氧化活性优于PWI。因此,果胶酶辅助提取方法有望为菊芋粕菊粉的再利用难题提供新的解决思路。     

Phosphoenolpyruvate metabolism in Jerusalem artichoke mitochondria

We report here initial studies on phosphoenolpyruvate metabolism in coupled mitochondria isolated from Jerusalem artichoke tubers. It was found that:

(1)

phosphoenolpyruvate can be metabolized by Jerusalem artichoke mitochondria by virtue of the presence of the mitochondrial pyruvate kinase, shown both immunologically and functionally, located in the inner mitochondrial compartments and distinct from the cytosolic pyruvate kinase as shown by the different pH and inhibition profiles.

(2)

Jerusalem artichoke mitochondria can take up externally added phosphoenolpyruvate in a proton compensated manner, in a carrier-mediated process which was investigated by measuring fluorimetrically the oxidation of intramitochondrial pyridine nucleotide which occurs as a result of phosphoenolpyruvate uptake and alternative oxidase activation.

(3)

The addition of phosphoenolpyruvate causes pyruvate and ATP production, as monitored via HPLC, with their efflux into the extramitochondrial phase investigated fluorimetrically. Such an efflux occurs via the putative phosphoenolpyruvate/pyruvate and phosphoenolpyruvate/ATP antiporters, which differ from each other and from the pyruvate and the adenine nucleotide carriers, in the light of the different sensitivity to non-penetrant compounds. These carriers were shown to regulate the rate of efflux of both pyruvate and ATP. The appearance of citrate and oxaloacetate outside mitochondria was also found as a result of phosphoenolpyruvate addition.

     

Optimization of simultaneously enzymatic fructo- and inulo-oligosaccharide production using co-substrates of sucrose and inulin from Jerusalem artichoke

Prebiotic substances are extracted from various plant materials or enzymatic hydrolysis of different substrates. The production of fructo-oligosaccharide (FOS) and inulo-oligosaccharide (IOS) was performed by applying two substrates, sucrose and inulin; oligosaccharide yields were maximized using central composite design to evaluate the parameters influencing oligosaccharide production. Inulin from Jerusalem artichoke (5–15% w/v), sucrose (50–70% w/v), and inulinase from Aspergillus niger (2–7 U/g) were used as variable parameters for optimization. Based on our results, the application of sucrose and inulin as co-substrates for oligosaccharide production through inulinase hydrolysis and synthesis is viable in comparative to a method using a single substrate. Maximum yields (674.82?mg/g substrate) were obtained with 5.95% of inulin, 59.87% of sucrose, and 5.68 U/g of inulinase, with an incubation period of 9?hr. The use of sucrose and inulin as co-substrates in the reaction simultaneously produced FOS and IOS from sucrose and inulin. Total conversion yield was approximately 67%. Our results support the high value-added production of oligosaccharides using Jerusalem artichoke, which is generally used as a substrate in prebiotics and/or bioethanol production.     

Ubiquitin conjugation to endogenous proteins in the dormant tuber of Helianthus tuberosus and during the first cell cycle

Ubiquitin is a small protein involved in an ATP-dependent proteolytic pathway in all eukaryotes. This pathway has been demonstrated to be required for both the bulk degradation of cellular proteins and the targeted proteolysis of specific regulatory proteins. We have investigated the presence of ubiquitin (Ub) and the ubiquitin-conjugating system in dormant and activated tubers of Helianthus tuberosus L. cv. OB 1 that represent a widely used model system for studies on the cell cycle in plants. Immunoblot experiments revealed the presence of free ubiquitin and ubiquitin conjugates. Furthermore, the presence of an active ubiquitin-conjugating system, both time- and ATP-dependent, was demonstrated by incubation with ¹²⁵I-labeled ubiquitin. A few proteins able to form thiol esters with ¹²⁵I-Ub and probably corresponding to ubiquitin-conjugating enzymes, E1 and E2s, were also found. During the first cell cycle, several proteins become ubiquitinated. In particular a large amount of protein conjugates was present at 6 h when the lowest content of free ubiquitin was found. Subsequently, a dramatic decrease in ubiquitin conjugates occurred. It is well known that cell cycle progression in eukaryotes depends on cyclin levels and cyclin B degradation is ubiquitin- and ATP-dependent. By immunoblot experiments we showed that cyclin B in H. tuberosus is present as at least two protein bands of 50 and 54 kDa and that their amounts undergo profound changes during the cell cycle. The 54-kDa band was also recognized by an anti-ubiquitin antibody. These data seem to indicate that in H. tuberosus activated tuber slices, the ATP-dependent ubiquitin proteolytic pathway is involved in the dedifferentiation process occurring after the artificial break of dormancy when the cells acquire the characteristics linked to the meristematic state.     

Tissue Differentiation of the Regeneration Rind in Jerusalem artichoke Stem

In trees, after removal of the bark, the vascular tissues of the newly-formed bark usually developed as a continuous layer. However, the stem of the herbaceous Jerusalem artichoke, after girdled, gives rise to regeneration of many irregularly arranged vascular bundles. Early July is the best time for girdling as the vascular bundles are well-developed, One week after girdling, some small groups of vascular tissues appeared in callus. Later on the vascular bundles eventually grew close together sooner or later, yet there were some wide pith rays which separated the various sized vascular bundles and exhibited irregularly contours. From these experiments, it is further evidenced that tile stem of herbaceous plants can also be girdled and regenerates a new rind. Furthermore, the girdled portion of this plant regenerates the vascular tissues which in a rather different way from all the plants that previously studied.     

Improved production of potato microtubers using a temporary immersion system

A temporary immersion system for potato microtuber production was designed using 4-l vessels. This culture technique showed several advantages compared to solid cultures: i.e., three fold increase in shoot length, more internodes per plant and improved vigor. In the tuber induction stage, microtubers can be induced at all plant nodes, indicating that the tuberization is not restricted to specific regions. For both cultivars tested, Desiree and Atlantic, an average of 3.1 and 2.8 tubers per single node cutting was achieved after 9 weeks in culture. The size and weight of the tubers were higher than on solid media. Scale up was performed with cv. Atlantic in 10-l polycarbonate flasks and 12 units were mounted containing 150 single nodal cuttings each. An average of 2.6 tubers per inoculated cutting was obtained, with 1.3 g fresh weight per microtuber. Temporary immersion is a valuable option for potato microtuber production, as well as for shoot production during the planting season. This revised version was published online in June 2006 with corrections to the Cover Date.