Crystal structures of two homologous pathogenesis-related proteins from yellow lupine

Pathogenesis-related class 10 (PR10) proteins are restricted to the plant kingdom where they are coded by multigene families and occur at high levels. In spite of their abundance, their physiological role is obscure although members of a distantly related subclass (cytokinin-specific binding proteins) are known to bind plant hormones. PR10 proteins are of special significance in legume plants where their expression patterns are related to infection by the symbiotic, nitrogen-fixing bacteria. Here we present the first crystal structures of classic PR10 proteins representing two homologues from one subclass in yellow lupine. The general fold is similar and, as in a birch pollen allergen, consists of a seven-stranded beta-sheet wrapped around a long C-terminal helix. The mouth of a large pocket formed between the beta-sheet and the helix seems a likely site for ligand binding. The shape of the pocket varies because, in variance with the rigid beta-sheet, the helix shows unusual conformational variability consisting in bending, disorder, and axial shifting. A surface loop, proximal to the entrance to the internal cavity, shows an unusual structural conservation and rigidity in contrast to the high glycine content in its sequence. The loop is different from the so-called glycine-rich P-loops that bind phosphate groups of nucleotides, but it is very likely that it does play a role in ligand binding in PR10 proteins.     

NMCP/LINC proteins: Putative lamin analogs in plants?

Lamins are the main components of the metazoan lamina, and while the organization of the nuclear lamina of metazoans and plants is similar, there are apparently no genes encoding lamins or most lamin-binding proteins in plants. Thus, the plant lamina is not lamin-based and the proteins that form this structure are still to be characterized. Members of the plant NMCP/LINC/CRWN protein family share the typical tripartite structure of lamins, although the 2 exhibit no sequence similarity. However, given the many similarities between NMCP/LINC/CRWN proteins and lamins (structural organization, position of conserved regions, sub-nuclear distribution, solubility, and pattern of expression), these proteins are good candidates to carry out the functions of lamins in plants. Moreover, functional analysis of NMCP/LINC mutants has revealed their involvement in maintaining nuclear size and shape, another activity fulfilled by lamins. This review summarizes the current understanding of NMCP/LINC proteins and discusses future studies that will be required to demonstrate definitively that these proteins are plant analogs of lamins.     

Higher plant microtubule-associated proteins (MAPs): A survey

Summary— Microtubule-associated proteins (MAPs) are one of the factors which regulate the different properties of microtubules during cell cycle and differentiation. They have been characterized as proteins which promote tubulin assembly in a concentration-dependent manner and bind to the outer surface of the polymers in vitro. Most of our knowledge comes from studies of neural microtubule-associated proteins and recent results highlight their implication in neuronal morphogenesis. In contrast, until recently, few data are available about the proteins that associate with plant tubulins. This is due principally to the fact that plant microtubule-associated proteins cannot be purified by the standard procedures used for neural microtubule-associated proteins. First, we will describe methods which have been used to isolate these proteins in plant cells. We will then discuss the biochemical and immunological properties of the plant microtubule-associated proteins which have been isolated. From these results, putative functions can be proposed for these proteins n the particular plant cytoskeleton activities.     

The Role of Thionins in Plant Protection

Thionins are a group of small (5000 Da), sulfur-rich plant proteins found mainly in cereals and mistletoes. Their three-dimensional structures are very compact and amphipathic, stabilized by three or four disulfide bridges. Thionins are usually basic and exert toxicity in various biological systems by destroying membranes. Thionins are synthesized as preproproteins and secreted into vacuoles, protein bodies, and the cell wall. Their antibacterial and antifungal activities point to a role as plant defense proteins. Support for this possible function comes particularly from work on the leaf thionins of barley, showing that these proteins can be induced by several stress factors. Infection of barley with mildew, one of its most devastating pathogens, leads to an incorporation of leaf thionins into papillae in incompatible interactions. The possible role of thionins to enhance the resistance of crop plants by genetic engineering is discussed.     

抗冻蛋白及其在植物抗冻基因工程的应用

从应用的角度系统综述了抗冻蛋白（ＡＦＰs)的特性、活性、用途、生化特征、在细菌中的表达,在植物抗冻生理中的作用及其基因工程,简洁地讨论了抗冻蛋白的研究现状和最新进展。     

119种植物种子蛋氨酸含量分析

豆科植物蛋白中含硫氨基酸尤其是蛋氨酸含量低,影响其蛋白质的营养价值。为开发出更高甲硫氨酸含量蛋白质基因资源,采用酸水解法,对我国亚热带常见的119种森林植物种子的蛋氨酸含量进行了测定。结果表明:93%以上植物种子的蛋氨酸含量较低,只有八仙花、榕叶冬青、山苍子、锌树、龙葵、栾树、商陆和盐肤木的蛋氨酸含量在10.00mg/g以上,其中盐肤木的蛋氨酸含量为119种植物中最高,达36.89mg/g,可以作为进一步开发的新型高蛋氨酸蛋白基因资源。     

植物生物反应器表达药用蛋白研究新进展

植物生物反应器被称为"分子农田",它具有无限生产重组蛋白的巨大潜力。利用转基因植物表达的重组蛋白具备原有的理化性质和生物活性,从而为人类提供了一种大量生产药用蛋白的安全可靠、经济、方便的新生产体系。目前已广泛运用于工业、农业尤其是生命科学以及医学制造领域。用植物生物反应器产重组疫苗、重组抗体和其他药用蛋白已成为国内外基因工程研究热点之一。然而,转基因植物产物的表达量、下游加工等问题却也成为利用植物生物反应器应用的限制因素。本文就其优势、近三年内国内外转基因植物生产药用蛋白的研究进展、存在问题及对策作一综述。     

植物类整合素蛋白研究进展

整合素是动物细胞膜上普遍存在的一类胞外基质受体,它所介导的粘附作用参与调节多种细胞功能.近年来的研究发现在植物细胞中可能也存在类整合素.综述了在植物类整合素检测、定位、组成、结构、基因以及生理功能研究方面所取得的初步进展与存在的问题,并与动物整合素的研究作了比较.     

植物过敏性蛋白质及其生物学功能

在引起I型超敏反应的变应原中 ,植物的花粉、果实和汁液可以分别作为吸入性变应原 (inhalentallergen)、食入性变应原 (ingestentallergen)、接触性变应原 (contactentallergen)使过敏者患上鼻炎、哮喘、枯草热等疾病。而其中引起这些超敏反应的植物类蛋白质本身在植物体内亦行使着特定的生物学功能。对这些植物类过敏性蛋白质的研究不仅在植物学本身研究中具有一定意义 ,同时在变态反应性疾病的免疫治疗中亦具有重要的应用价值。目前 ,这类涉及植物学、免疫学和变态反应学的研究逐渐形成了一个新的交叉研究领域。     

Vacuolar sorting determinants within a plant storage protein trimer act cumulatively

The mechanism for vacuolar sorting of seed storage proteins is as yet poorly understood and no receptor has been identified to date. The homotrimeric glycoprotein phaseolin, which is the major storage protein of the common bean, requires a transient tetrapeptide at the C-terminus for its vacuolar sorting. A mutated construct without the tetrapeptide is secreted. We show here that coexpression of wild-type phaseolin and the mutated, secreted form in transgenic tobacco results in the formation of mixed trimers and partial vacuolar delivery of the mutated polypeptides and partial secretion of wild-type polypeptides. This indicates that the sorting signal has a cumulative effect within a phaseolin trimer. The result is discussed in the light of the hypothesized mechanisms for vacuolar sorting of seed storage proteins.     

Cloning of Rac and Rho-GDI from tobacco using an heterologous two-hybrid screen

To examine whether molecular similarities exist between the animal and plant Rho GTPase signaling pathways, we have developed a heterologous two-hybrid screening method. By this technique, we have cloned a cDNA encoding a tobacco Rac-like protein able to interact with a mammalian Rho-GDI. In a second screen this tobacco Rac was used as a bait and a tobacco homologue of Rho-GDI was identified. These results show that some components of the animal and plant Rac signaling pathways are similar enough to allow their interaction in an heterologous approach. Moreover these data suggest a similar regulation of Rho GTPases in animals and plants.     

N-Glycoprotein biosynthesis in plants: recent developments and future trends

N-glycosylation is a major modification of proteins in plant cells. This process starts in the endoplasmic reticulum by the co-translational transfer of a precursor oligosaccharide to specific asparagine residues of the nascent polypeptide chain. Processing of this oligosaccharide into high-mannose-type, paucimannosidic-type, hybrid-type or complex-type N-glycans occurs in the secretory pathway as the glycoprotein moves from the endoplasmic reticulum to its final destination. At the end of their maturation, some plant N-glycans have typical structures that differ from those found in their mammalian counterpart by the absence of sialic acid and the presence of (1,2)-xylose and (1,3)-fucose residues. Glycosidases and glycosyltransferases that respectively catalyse the stepwise trimming and addition of sugar residues are generally considered as working in a co-ordinated and highly ordered fashion to form mature N-glycans. On the basis of this assembly line concept, fast progress is currently made by using N-linked glycan structures as milestones of the intracellular transport of proteins along the plant secretory pathway. Further developments of this approach will need to more precisely define the topological distribution of glycosyltransferases within a plant Golgi stack. In contrast with their acknowledged role in the targeting of lysosomal hydrolases in mammalian cells, N-glycans have no specific function in the transport of glycoproteins into the plant vacuole. However, the presence of N-glycans, regardless of their structures, is necessary for an efficient secretion of plant glycoproteins. In the biotechnology field, transgenic plants are rapidly emerging as an important system for the production of recombinant glycoproteins intended for therapeutic purposes, which is a strong motivation to speed up research in plant glycobiology. In this regard, the potential and limits of plant cells as a factory for the production of mammalian glycoproteins will be illustrated.     

Arabinogalactan proteins: actors or spectators during abiotic and biotic stress in plants?

Arabinogalactan proteins (AGPs) are a family of hydroxyproline-rich glycoproteins (HRGP) ubiquitous in the plant kingdom. They are probably one of the most heterogeneous and complex families of macromolecules, making them able to perform different and multiple functions. Located at the plasma membrane–cell wall interface, AGPs are involved in several processes, from plant growth and development to reproduction. An additional function of AGPs in response to biotic and abiotic stress has been suggested by several studies. The purpose of this review is to summarize critically and analytically the available knowledge on the effects of abiotic stress (low and high temperatures, drought, flooding, anoxia and metal deficiency/toxicity) and biotic stress (bacteria, fungi, nematodes and viruses) on AGPs. A deeper understanding of the role of AGPs during these conditions can be an important tool for understanding AGP biology and for the possible development of efficient breeding strategies.     

The targeting of the oxysterol-binding protein ORP3a to the endoplasmic reticulum relies on the plant VAP33 homolog PVA12

In plants, sterols play fundamental roles as membrane constituents in the biosynthesis of steroid hormones, and act as precursors for cell wall deposition. Sterols are synthesized in the endoplasmic reticulum (ER), but mainly accumulate in the plasma membrane. How sterols are trafficked in plant cells is largely unknown. In non-plant systems, oxysterol-binding proteins have been involved in sterol trafficking and homeostasis. There are at least twelve homologs of oxysterol-binding proteins in the Arabidopsis genome, but the biology of these proteins remains for the most part obscure. Here, we report our analysis of the targeting requirements and the sterol-binding properties of a small Arabidopsis oxysterol-binding protein, ORP3a. We have determined that ORP3a is a bona fide sterol-binding protein with sitosterol-binding properties. Live-cell imaging analyses revealed that ORP3a is localized at the ER, and that binding to this organelle depends on a direct interaction with PVA12, a member of the largely uncharacterized VAP33 family of plant proteins. Molecular modeling analyses and site-directed mutagenesis led to the identification of a novel protein domain that is responsible for the PVA12–ORP3a interaction. Disruption of the integrity of this domain caused redistribution of ORP3a to the Golgi apparatus, suggesting that ORP3a may cycle between the ER and the Golgi. These results represent new insights into the biology of sterol-binding proteins in plant cells, and elucidate a hitherto unknown relationship between members of oxysterol-binding protein and VAP33 families of plant proteins in the early plant secretory pathway.     

植物营养贮存蛋白及其生物学功能研究进展

植物营养贮存蛋白（vegetative storage proteins）是广泛存在于植物营养组织且含量丰富的蛋白,最初是作为植物氮源的临时贮存形式而被人们认识。然而,不同植物中的营养贮存蛋白的生化来源和生物学特性并不相同,并且除了营养贮存功能外,更重要的是这类蛋白在植物防御中也承担着多种多样的重要角色,或具有抗虫活性,或能够抑制病原细菌和病原真菌的生长,或参与植物防御过程中的信号转导等。对植物营养贮存蛋白在植物防御中作用机制的深入研究将使这类蛋白在新型生物农药的开发和植物抗病基因工程中具有广阔的应用前景。     

Actin polymerization processes in plant cells

Growing evidence shows that the actin cytoskeleton is a key effector of signal transduction, which controls and maintains the shape of plant cells, as well as playing roles in plant morphogenesis. Recently, several signaling pathways, including those triggered by hormones, Ca(2+), and cAMP, have been reported to be connected to the reorganization of the actin cytoskeleton. The molecular mechanisms involved in such signaling cascades are, however, largely unknown. The Arabidopsis genome sequence is a valuable tool for identifying some of the highly conserved molecules that are involved in such signaling cascades. Recent work has begun to unravel these complex pathways using a panoply of techniques, including genetic analysis, live-cell imaging of intracellular actin dynamics, in vivo localization of factors that are involved in the control of actin dynamics, and the biochemical characterization of how these factors function.     

用美洲拟鲽抗冻蛋白基因转化植物的问题

评估了我们用美洲拟鲽抗冻蛋白基因转化烟草和国内有人用同一基因转化蕃茄的研究 ,提出植物耐寒性状的提高与转基因植物中抗冻蛋白含量呈正相关是转基因植物成功的关键性指标。同时 ,美洲拟鲽抗冻蛋白的空间结构与冷诱导蛋白相似而且都保护细胞膜免受冻害 ,因而提出了用冷诱导蛋白调节元的顺式作用元件和反式作用因子的调控序列来控制抗冻蛋白基因在植物中冷诱导表达的思路。     

Tomato cystatin SlCYS8 as a stabilizing fusion partner for human serpin expression in plants

Studies have reported the usefulness of fusion proteins to bolster recombinant protein yields in plants. Here, we assess the potential of tomato SlCYS8, a Cys protease inhibitor of the cystatin protein superfamily, as a stabilizing fusion partner for human alpha‐1‐antichymotrypsin (α1ACT) targeted to the plant cell secretory pathway. Using the model expression platform Nicotiana benthamiana, we show that the cystatin imparts a strong stabilizing effect when expressed as a translational fusion with α1ACT, allowing impressive accumulation yields of over 2 mg/g of fresh weight tissue for the human serpin, a 25‐fold improvement on the yield of α1ACT expressed alone. Natural and synthetic peptide linkers inserted between SlCYS8 and α1ACT have differential effects on protease inhibitory potency of the two protein partners in vitro. They also have a differential impact on the yield of α1ACT, dependent on the extent to which the hybrid protein may remain intact in the plant cell environment. The stabilizing effect of SlCYS8 does not involve Cys protease inhibition and can be partly reproduced in the cytosol, where peptide linkers are less susceptible to degradation. The effect of SlCYS8 on α1ACT yields could be explained by: (i) an improved translation of the human protein coding sequence; and/or (ii) an overall stabilization of its tertiary structure preventing proteolytic degradation and/or polymerization. These findings suggest the potential of plant cystatins as stabilizing fusion partners for recombinant proteins in plant systems. They also underline the need for an empirical assessment of peptide linker functions in plant cell environments.