The role of Rab GTPases in cell wall metabolism

The synthesis and modification of the cell wall must involve the production of new cell wall polymers and enzymes. Their targeted secretion to the apoplast is one of many potential control points. Since Rab GTPases have been strongly implicated in the regulation of vesicle trafficking, a review of their involvement in cell wall metabolism should throw light on this possibility. Cell wall polymer biosynthesis occurs mainly in the Golgi apparatus, except for cellulose and callose, which are made at the plasma membrane by an enzyme complex that cycles through the endomembrane system and which may be regulated by this cycling. Several systems, including the growth of root hairs and pollen tubes, cell wall softening in fruit, and the development of root nodules, are now being dissected. In these systems, secretion of wall polymers and modifying enzymes has been documented, and Rab GTPases are highly expressed. Reverse genetic experiments have been used to interfere with these GTPases and this is revealing their importance in regulation of trafficking to the wall. The role of the RabA (or Rab11) GTPases is particularly exciting in this respect.     

果实多聚半乳糖醛酸酶分子生物学研究进展

多聚半乳糖醛酸酶（PG酶）是一种在植物细胞壁降解中起重要的作用的酶。作者介绍了PG酶在果实成熟软化中的作用。概述了PG酶基因及其表达调控,评述了乙烯对PG酶合成的影响。     

间歇低温胁迫对桃果实细胞壁代谢的影响

桃果实在成熟过程中细胞壁干物质不断减少,随着共价结合果胶质和离子结合果胶质减少,水溶性果胶质明显增加,纤维素也逐渐减少,但半纤维素含量变化较小.低温胁迫造成果胶质和纤维素的降解过程受阻,从而造成较高分子量果胶质的积累,果汁粘度升高.中途加温则能促进果胶质和纤维素的增溶和解聚,引导细胞进行与果实成熟有关的细胞壁代谢.14C-蔗糖标记试验表明,在细胞壁不断降解的同时,也进行着合成.在果实成熟的启动阶段,细胞壁的合成能力加强.果实衰老过程与细胞壁合成减少有着直接的联系.受到低温伤害的果实细胞壁物质含量高于正常果实的原因,并不是其合成水平的升高,而是其降解的减慢.     

PG在番茄果实成熟中的作用及二价金属离子与乙烯对PG活性的影响

对采后番茄果实的电镜观察表明:当果实成熟衰老时,叶绿体数量减少,多数基粒结构丧失;成熟果实胞壁中胶层水解成中空的电子透明区,初生壁的纤丝也发生一定程度的水解,相邻细胞分离;外源 PG(多聚半乳糖醛酸酶)提取物处理绿熟期果实组织,也可引起胞壁结构和叶绿体发生与正常衰老相同的变化。Ca~(2+)、Mg~(2+)、Co~(2+)二价金属离子处理果实,可明显降低番茄红素含量和 PG 活性,延缓果实软化。外源乙烯处理果实,可促进番茄红素的形成,提高 PG活性,并能解除钙对 PG 活性的抑制。本文也对 PG 在乙烯和 Ca~(2+)调节果实成熟中的作用进行了讨论。     

The use of transgenic and naturally occurring mutants to understand and manipulate tomato fruit ripening

In the years since we last reviewed the use of mutants to study tomato fruit ripening ( Grierson et al. 1987 ), considerable information has been gained by the cloning, sequencing and identification of many mRNAs implicated in this developmental process. Genes involved in cell wall degradation, colour change and ethylene synthesis have been cloned, and antisense techniques have been developed and used to produce genetically engineered mutant fruit deficient in these aspects of ripening (see Gray et al. 1992 ). Recently, a previously cloned ripening gene has been used to complement a naturally occurring fruit colour mutant, yellow flesh ( Fray & Grierson 1993a ), and a ripening impaired mutant, ripening inhibitor, has been used to identify several new ripening-related mRNAs ( Picton et al. 1993b ). The chromosomal region bearing the ripening inhibitor mutation has been subjected to high-resolution mapping ( Churchill, Giovannoni & Tanksley 1993 ) and chromosome walking experiments are in progress to identify this gene.     

The Role of Polygalacturonase (PG) in Tomato Fruit Ripening and Effects of Divalent Metal Ions and Ethylene on PG Activity

It has been reported that PG is a key enzyme related to the tomato fruit ripening. In this study tomato fruits were harvested at the mature-green stage and stored at room temperature. The cell ultrastructure of pericarp tissue was observed at different ripening stages, and the effects of treatments with ethylene and calcium on PG activity and fruit ripening were examined. The object of this study is to elucidate the role of PG in regulation of tomato fruit ripening by ethylene and calcium. PG activity, was undetectable at mature-green stage, but it rose rapidly as fruif ripening. The rise in PG activity was coincided with the dechnmg of fruit firmness during ripening of tomato fruits. The observation of cell ultrastructure showed that the most of grana in chloroplast were lost and the mitochondrial cristae decreased as fruit ripening. Striking changes of cell wall structure was most noted, beginning with dissolution of the middle lamella and eventual disruption of primary cell wall. A similar pattern of changes of cell wall and chloroplast have been observed in pericarp tissue treated with PG extract. In fruits treated with calcium and other divalent metal ions atmature-green stage, the lycopene content and PG activity decreased dramatically. Ethylene application enhanced the formation of lycopene and PG activity. The inhibition of Ca2+ on PG ac ivity was removed by ethylene. Based on the above results, it was demonstrated that PG played a major role in ripening of tomato fruits, and suggested that the regulation of fruit ripening by ethylene and Ca2+ was all mediated by PG. PG induced the hydrolysis of cell wall and released the other hydrolytic enzymes, then effected the ripening processes follow up.     

Rab GTPase Mediated Procollagen Trafficking in Ascorbic Acid Stimulated Osteoblasts

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly up-regulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and diseases involving defects in bone protein secretion and deposition. Protein trafficking along the exocytic and endocytic pathways is aided by many molecules, with Rab GTPases being master regulators of vesicle targeting. In this study, we used microarray analysis to identify the Rab GTPases that are up-regulated during a 5-day AA differentiation of OBs, namely Rab1, Rab3d, and Rab27b. Further, we investigated the role of identified Rabs in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane utilizing Rab dominant negative (DN) expression. We also observed that experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs partially alleviated this negative feedback mechanism and resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels was also observed implicating the functional importance of Rab1, Rab3d and Rab27b in these major collagen-producing cells.     

Plant Rabs: Characterization,Functional Diversity,and Role in Stress Tolerance

Rab proteins form the largest family of small guanosine triphosphate (GTP)-binding proteins. The Rab family in plants is divided into eight subfamilies, Rab1, Rab2, Rab5, Rab6, Rab7, Rab8, Rab11, and Rab18. Phylogenetic analyses of amino acid sequence of Rab GTPases suggest their segregation into subfamilies on the basis of their localization and/or function in membrane trafficking. The Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane-trafficking pathways. The Rab proteins show highly conserved structural features with a great functional versatility. They play an important role in regulating hormone signaling during fruit ripening and apical dominance, brassinosteroid biosynthesis, pollen and nodule development, and in response to both abiotic and biotic stresses.     

An expansin gene expressed in ripening strawberry fruit

Tissue softening accompanies the ripening of many fruit and initiates the processes of irreversible deterioration. Expansins are plant cell wall proteins proposed to disrupt hydrogen bonds within the cell wall polymer matrix. Expression of specific expansin genes has been observed in tomato (Lycopersicon esculentum) meristems, expanding tissues, and ripening fruit. It has been proposed that a tomato ripening-regulated expansin might contribute to cell wall polymer disassembly and fruit softening by increasing the accessibility of specific cell wall polymers to hydrolase action. To assess whether ripening-regulated expansins are present in all ripening fruit, we examined expansin gene expression in strawberry (Fragaria x ananassa Duch.). Strawberry differs significantly from tomato in that the fruit is derived from receptacle rather than ovary tissue and strawberry is non-climacteric. A full-length cDNA encoding a ripening-regulated expansin, FaExp2, was isolated from strawberry fruit. The deduced amino acid sequence of FaExp2 is most closely related to an expansin expressed in early tomato development and to expansins expressed in apricot fruit rather than the previously identified tomato ripening-regulated expansin, LeExp1. Nearly all previously identified ripening-regulated genes in strawberry are negatively regulated by auxin. Surprisingly, FaExp2 expression was largely unaffected by auxin. Overall, our results suggest that expansins are a common component of ripening and that non-climacteric signals other than auxin may coordinate the onset of ripening in strawberry.     

Developmental abnormalities and reduced fruit softening in tomato plants expressing an antisense Rab11 GTPase gene

A cDNA clone from tomato fruit encodes a protein with strong homology with the rab11/YPT3 class of small GTPases that is thought to be involved in the control of protein trafficking within cells. The gene, LeRab11a, showed a pattern consistent with a single copy in DNA gel blots. The corresponding mRNA was developmentally regulated during fruit ripening, and its expression was inhibited in several ripening mutants. Its reduced expression in the Never-ripe mutant indicates that it may be induced by ethylene in fruit. The ripening-induced expression in tissues that are undergoing cell wall loosening immediately suggests a possible role in trafficking of cell wall-modifying enzymes. The message also was produced in leaves and flowers but not in roots. Antisense transformation was used to generate a "mutant phenotype." Antisense fruit changed color as expected but failed to soften normally. This was accompanied by reduced levels of two cell wall hydrolases, pectinesterase and polygalacturonase. There were other phenotypic effects in the plants, including determinate growth, reduced apical dominance, branched inflorescences, abnormal floral structure, and ectopic shoots on the leaves. In some plants, ethylene production was reduced. These data suggest an alternative or additional role in exocytosis or endocytosis of homeotic proteins, hormone carriers, or receptors.     

Rab’ing tumor cell migration and invasion: Focal adhesion disassembly driven by Rab5

The small GTPase Rab5 has been extensively studied in the context of endocytic trafficking because it is critical in the regulation of early endosome dynamics. In addition to this canonical role, evidence obtained in recent years implicates Rab5 in the regulation of cell migration. This novel role of Rab5 is based not only on an indirect relationship between cell migration and endosomal trafficking as separate processes, but also on the direct regulation of signaling proteins implicated in cell migration. However, the precise mechanisms underlying this connection have remained elusive. Recent studies have shown that the activation of Rab5 is a critical event for maintaining the dynamics of focal adhesions, which is fundamental in regulating not only cell migration but also tumor cell invasion.     

Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy

Background

One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening.

Scope

In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected.     

Loss of tomato cell wall galactan may involve reduced rate of synthesis

Changes in the galactose content of the noncellulosic polysaccharides of tomato (Mill) fruit cell walls were analyzed under various conditions. On the plant, galactan decreased gradually during fruit growth. As normal fruits ripened, the loss of galactan increased sharply; this was not observed in attached rin fruits beyond the fully mature stage. The ability to produce new wall galactan in vitro was retained in mature fruit tissue but declined with ripening. Normal tomatoes ripening on the plant showed a transient increase in galactan content at the climacteric. It is suggested that the decline in wall galactan is partly due to reduced synthesis in senescing, normal fruits and in detached rin tomatoes.     

In vivo and in vitro swelling of cell walls during fruit ripening

Swelling properties of the cell walls of nine temperate fruit species, selected for their different ripening and textural characteristics, were studied during ripening. Cell wall swelling was examined in intact fruit using microscopy techniques and in vitro, using cell wall material isolated from fruit tissue. In fruit which ripened to a soft melting texture (persimmon, avocado, blackberry, strawberry, plum), wall swelling was pronounced, particularly in vitro. In-vivo swelling was marked only in avocado and blackberry. Fruit which ripened to a crisp, fracturable texture [apple (two cultivars), nashi pear, watermelon] did not show either in-vivo or in-vitro swelling of the cell wall. There was a correlation between swelling and the degree of pectin solubilisation, suggesting that wall swelling occurred as a result of changes to the viscoelastic properties of the cell wall during pectin solubilisation. Chemical and enzymatic removal of pectin from kiwifruit cell wall material supported the idea that swelling is associated with movement of water into voids left in the cellulose-hemicellulose network by the solubilised pectin. However, the results also suggested that swelling in vivo was more complex than this, and that the physicochemical changes which led to swelling included other elements of cell wall modification involving the site and mechanism of pectin solubilisation and-or the cellulose-xyloglucan complex. Received: 28 January 1997 / accepted: 11 March 1997     

Compositional changes in cell wall polymers during mango fruit ripening

Softening of mango fruit has been investigated by analysis of ripening related changes in the composition of the fruit cell walls. There is an apparent overall loss of galactosyl and deoxyhexosyl residues during ripening, the latter indicating degradation of the pectin component of the wall. The loss of galactose appears to be restricted to the chelator soluble fraction of the wall pectin, whilst loss of deoxyhexose seems to be more evenly distributed amongst the pectin. The chelator soluble pectin fraction is progressively depolymerised and becomes more polydisperse during ripening. These changes are similar to those occurring in other fruit and are related to the action of wall hydrolases during ripening.     

Xyloglucan endotransglucosylase/hydrolases (XTHs) during tomato fruit growth and ripening

Depolymerization of cell wall xyloglucan has been proposed to be involved in tomato fruit softening, along with the xyloglucan modifying enzymes. Xyloglucan endotransglucosylase/hydrolases (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151) have been proposed to have a dual role integrating newly secreted xyloglucan chains into an existing wall-bound xyloglucan, or restructuring the existing cell wall material by catalyzing transglucosylation between previously wall-bound xyloglucan molecules. Here, 10 tomato (Solanum lycopersicum) SlXTHs were studied and grouped into three phylogenetic groups to determine which members of each family were expressed during fruit growth and fruit ripening, and the ways in which the expression of different SlXTHs contributed to the total XET and XEH activities. Our results showed that all of the SlXTHs studied were expressed during fruit growth and ripening, and that the expression of all the SlXTHs in Group 1 was clearly related to fruit growth, as were SlXTH12 in Group 2 and SlXTH6 in Group 3-B. Only the expression of SlXTH5 and SlXTH8 from Group 3-A was clearly associated with fruit ripening, although all 10 of the different SlXTHs were expressed at the red ripe stage. Both total XET and XEH activities were higher during fruit growth, and decreased during fruit ripening. Ethylene production during tomato fruit growth was low and experienced a significant increase during fruit ripening, which was not correlated either with SlXTH expression or with XET and XEH activities. We suggest that the role of XTH during fruit development could be related to the maintenance of the structural integrity of the cell wall, and the decrease in XTHs expression, and the subsequent decrease in activity during ripening may contribute to fruit softening, with this process being regulated through different XTH genes.     

Temporal Sequence of Cell Wall Disassembly in Rapidly Ripening Melon Fruit

The Charentais variety of melon (Cucumis melo cv Reticulatus F1 Alpha) was observed to undergo very rapid ripening, with the transition from the preripe to overripe stage occurring within 24 to 48 h. During this time, the flesh first softened and then exhibited substantial disintegration, suggesting that Charentais may represent a useful model system to examine the temporal sequence of changes in cell wall composition that typically take place in softening fruit. The total amount of pectin in the cell wall showed little reduction during ripening but its solubility changed substantially. Initial changes in pectin solubility coincided with a loss of galactose from tightly bound pectins, but preceded the expression of polygalacturonase (PG) mRNAs, suggesting early, PG-independent modification of pectin structure. Depolymerization of polyuronides occurred predominantly in the later ripening stages, and after the appearance of PG mRNAs, suggesting the existence of PG-dependent pectin degradation in later stages. Depolymerization of hemicelluloses was observed throughout ripening, and degradation of a tightly bound xyloglucan fraction was detected at the early onset of softening. Thus, metabolism of xyloglucan that may be closely associated with cellulose microfibrils may contribute to the initial stages of fruit softening. A model is presented of the temporal sequence of cell wall changes during cell wall disassembly in ripening Charentais melon.     

Apoplastic pH and inorganic ion levels in tomato fruit: A potential means for regulation of cell wall metabolism during ripening

Apoplastic pH and ionic conditions exert strong influence on cell wall metabolism of many plant tissues; however, the nature of the apoplastic environment of ripening fruit has been the subject of relatively few studies. In this report, a pressure-bomb technique was used to extract apoplastic fluid from tomato fruit ( Lycopersicon esculentum Mill.) pericarp at several developmental stages. pH and the levels of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻ and P were determined and compared with the values for the bulk pericarp and locule tissues. The pH of the apoplastic fluid from pericarp tissue decreased from 6.7 in immature and mature-green fruits to 4.4 in fully-ripe fruit. During the same period, the K⁺ concentration increased from 13 to 37 m M . The levels of Na⁺ and divalent cations did not change, whereas the anions P and Cl⁻ increased in ripe fruit. Ca²⁺ levels remained relatively constant during ripening at 4–5 m M , concentrations that effectively limit pectin solubilization. The electrical conductivity of the apoplastic liquid increased 3-fold during ripening, whereas osmotically active solutes increased 2-fold. Pressure-treated fruit retained the capacity to ripen. The decline in apoplastic pH and increase in ionic strength during tomato fruit ripening may regulate the activity of cell wall hydrolases. The potential role of apoplastic changes in fruit ripening and softening is discussed.     

Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking

The closely related Rab6 isoforms, Rab6A and Rab6A', have been shown to regulate vesicular trafficking within the Golgi and post-Golgi compartments, but studies using dominant active or negative mutant suggested conflicting models. Here, we report that reduction in the expression of Rab6 isoform using specific small interfering RNA reveals noticeable differences in the Rab6A and Rab6A' biological functions. Surprisingly, Rab6A seems to be largely dispensable in membrane trafficking events, whereas knocking down the expression of Rab6A' hampers the intracellular transport of the retrograde cargo marker, the Shiga Toxin B-subunit along the endocytic pathway, and causes defects in Golgi- associated protein recycling through the endoplasmic reticulum. We also showed that Rab6A' is required for cell cycle progression through mitosis and identify Ile(62) as a key residue for uncoupling Rab6A' functions in mitosis and retrograde trafficking. Thus, our work shows that Rab6A and Rab6A' perform different functions within the cell and suggests a novel role for Rab6A' as the major Rab6 isoform regulating previously described Rab6-dependent transport pathways.