Plant 115-kDa actin-filament bundling protein, P-115-ABP, is a homologue of plant villin and is widely distributed in cells

In many cases, actin filaments are arranged into bundles and serve as tracks for cytoplasmic streaming in plant cells. We have isolated an actin-filament bundling protein, which is composed of 115-kDa polypeptide (P-115-ABP), from the germinating pollen of lily, Lilium longiflorum [Nakayasu et al. (1998) BIOCHEM: Biophys. Res. Commun. 249: 61]. P-115-ABP shared similar antigenicity with a plant 135-kDa actin-filament bundling protein (P-135-ABP), a plant homologue of villin. A full-length cDNA clone (ABP115; accession no. AB097407) was isolated from an expression cDNA library of lily pollen by immuno-screening using antisera against P-115-ABP and P-135-ABP. The amino acid sequence of P-115-ABP deduced from this clone showed high homology with those of P-135-ABP and four villin isoforms of Arabidopsis thaliana (AtVLN1, AtVLN2, AtVLN3 and AtVLN4), especially AtVLN4, indicating that P-115-ABP can also be classified as a plant villin. The P-115-ABP isolated biochemically from the germinating lily pollen was able to arrange F-actin filaments with uniform polarity into bundles and this bundling activity was suppressed by Ca2+-calmodulin (CaM), similar to the actin-filament bundling properties of P-135-ABP. The P-115-ABP type of plant villin was widely distributed in plant cells, from algae to land plants. In root hair cells of Hydrocharis dubia, this type of plant villin was co-localized with actin-filament bundles in the transvacuolar strands and the sub-cortical regions. Microinjection of the antiserum against P-115-ABP into living root hair cells caused the disappearance of transvaculor strands and alteration of the route of cytoplasmic streaming. In internodal cells of Chara corallina in which the P-135-ABP type of plant villin is lacking, the P-115-ABP type showed co-localization with actin-filament cables anchored on the intracellular surface of chloroplasts. These results indicated that plant villins are widely distributed and involved in the organization of actin filaments into bundles throughout the plant kingdom.     

Arabidopsis VILLIN4 is involved in root hair growth through regulating actin organization in a Ca2+-dependent manner

? Villin is one of the major actin filament bundling proteins in plants. The function of Arabidopsis VILLINs (AtVLNs) is still poorly understood in living cells. In this report, the biochemical activity and cellular function of AtVLN4 were examined. ? The biochemical property of AtVLN4 was characterized by co-sedimentation assays, fluorescence microscopy and spectroscopy of pyrene fluorescence. The in vivo function of AtVLN4 was analysed by ectopically expressing it in tobacco pollen and examining the phenotypes of its T-DNA insertional plants. ? Recombinant AtVLN4 protein exhibited multiple activities on actin, including actin filament bundling, calcium (Ca(2+))-dependent filament severing and barbed end capping. Expression of AtVLN4 in tobacco pollen induced the formation of supernumerary actin cables and reduced pollen tube growth. Loss of function of AtVLN4 resulted in slowing of root hair growth, alteration in cytoplasmic streaming routes and rate, and reduction of both axial and apical actin bundles. ? Our results demonstrated that AtVLN4 is involved in root hair growth through regulating actin organization in a Ca(2+)-dependent manner.     

ACTIN BINDING PROTEIN 29 from Lilium pollen plays an important role in dynamic actin remodeling

Villin/gelsolin/fragmin superfamily proteins have been shown to function in tip-growing plant cells. However, genes encoding gelsolin/fragmin do not exist in the Arabidopsis thaliana and rice (Oryza sativa) databases, and it is possible that these proteins are encoded by villin mRNA splicing variants. We cloned a 1006-bp full-length cDNA from Lilium longiflorum that encodes a 263-amino acid predicted protein sharing 100% identity with the N terminus of 135-ABP (Lilium villin) except for six C-terminal amino acids. The deduced 29-kD protein, Lilium ACTIN BINDING PROTEIN29 (ABP29), contains only the G1 and G2 domains and is the smallest identified member of the villin/gelsolin/fragmin superfamily. The purified recombinant ABP29 accelerates actin nucleation, blocks barbed ends, and severs actin filaments in a Ca(2+)- and/or phosphatidylinositol 4,5-bisphosphate-regulated manner in vitro. Microinjection of the protein into stamen hair cells disrupted transvacuolar strands whose backbone is mainly actin filament bundles. Transient expression of ABP29 by microprojectile bombardment of lily pollen resulted in actin filament fragmentation and inhibited pollen germination and tube growth. Our results suggest that ABP29 is a splicing variant of Lilium villin and a member of the villin/gelsolin/fragmin superfamily, which plays important roles in rearrangement of the actin cytoskeleton during pollen germination and tube growth.     

Identification and functional characterization of the BAG protein family in Arabidopsis thaliana

The genes that control mammalian programmed cell death are conserved across wide evolutionary distances. Although plant cells can undergo apoptosis-like cell death, plant homologs of mammalian regulators of apoptosis have, in general, not been found. This is in part due to the lack of primary sequence conservation between animal and putative plant regulators of apoptosis. Thus, alternative approaches beyond sequence similarities are required to find functional plant homologs of apoptosis regulators. Here, we present the results of using advanced bioinformatic tools to uncover the Arabidopsis family of BAG proteins. The mammalian BAG (Bcl-2-associated athanogene) proteins are a family of chaperone regulators that modulate a number of diverse processes ranging from proliferation to growth arrest and cell death. Such proteins are distinguished by a conserved BAG domain that directly interacts with Hsp70 and Hsc70 proteins to regulate their activity. Our searches of the Arabidopsis thaliana genome sequence revealed seven homologs of the BAG protein family. We further show that plant BAG family members are also multifunctional and remarkably similar to their animal counterparts, as they regulate apoptosis-like processes ranging from pathogen attack to abiotic stress and development.     

Plant defensins

Plant defensins are small, basic peptides that have a characteristic three-dimensional folding pattern that is stabilized by eight disulfide-linked cysteines. They are termed plant defensins because they are structurally related to defensins found in other types of organism, including humans. To date, sequences of more than 80 different plant defensin genes from different plant species are available. In Arabidopsis thaliana, at least 13 putative plant defensin genes (PDF) are present, encoding 11 different plant defensins. Two additional genes appear to encode plant defensin fusions. Plant defensins inhibit the growth of a broad range of fungi but seem nontoxic to either mammalian or plant cells. Antifungal activity of defensins appears to require specific binding to membrane targets. This review focuses on the classification of plant defensins in general and in Arabidopsis specifically, and on the mode of action of plant defensins against fungal pathogens.     

A tomato metacaspase gene is upregulated during programmed cell death in<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>-infected leaves

Programmed cell death (PCD) in plant cells is often accompanied by biochemical and morphological hallmarks similar to those of animal apoptosis. However, orthologs of animal caspases, cysteinyl aspartate-specific proteases that constitute the core component of animal apoptosis, have not yet been identified in plants. Recent studies have revealed the presence of a family of genes encoding proteins with distant homology to mammalian caspases, designated metacaspases, in the Arabidopsis thaliana genome. Here, we describe the isolation of LeMCA1, a type-II metacaspase cDNA clone from tomato (Lycopersicon esculentum Mill.). BLAST analysis demonstrated that the LeMCA1 gene is located in close vicinity of several genes that have been linked with PCD. Southern analysis indicated the existence of at least one more metacaspase in the tomato genome. LeMCA1 mRNA levels rapidly increased upon infection of tomato leaves with Botrytis cinerea, a fungal pathogen that induces cell death in several plant species. LeMCA1 was not upregulated during chemical-induced PCD in suspension-cultured tomato cells.     

Selective autophagy in the aid of plant germination and response to nutrient starvation

Selective autophagy, mediated by Atg8 binding proteins, has not been extensively studied in plants. Plants possess a large gene family encoding multiple isoforms of the Atg8 protein. We have recently reported the identification of two new, closely homologous Arabidopsis thaliana plant proteins that bind the Arabidopsis Atg8f protein isoform. These two proteins are specific to plants and have no homologs in nonplant organisms. The expression levels of the genes encoding these proteins are elevated during carbon starvation and also during late stages of seed development. Exposure of young seedlings to carbon starvation induces the production of a newly identified compartment decorated by these Atg8-binding proteins. This compartment dynamically moves along the endoplasmic reticulum membrane and is also finally transported into the vacuole. Enhanced or suppressed expression of these Atg8-binding proteins respectively enhances or suppresses seed germination under suboptimal germination conditions, indicating that they contribute to seed germination vigor.     

TIR-X and TIR-NBS proteins: two new families related to disease resistance TIR-NBS-LRR proteins encoded in Arabidopsis and other plant genomes

The Toll/interleukin-1 receptor (TIR) domain is found in one of the two large families of homologues of plant disease resistance proteins (R proteins) in Arabidopsis and other dicotyledonous plants. In addition to these TIR-NBS-LRR (TNL) R proteins, we identified two families of TIR-containing proteins encoded in the Arabidopsis Col-0 genome. The TIR-X (TX) family of proteins lacks both the nucleotide-binding site (NBS) and the leucine rich repeats (LRRs) that are characteristic of the R proteins, while the TIR-NBS (TN) proteins contain much of the NBS, but lack the LRR. In Col-0, the TX family is encoded by 27 genes and three pseudogenes; the TN family is encoded by 20 genes and one pseudogene. Using massively parallel signature sequencing (MPSS), expression was detected at low levels for approximately 85% of the TN-encoding genes. Expression was detected for only approximately 40% of the TX-encoding genes, again at low levels. Physical map data and phylogenetic analysis indicated that multiple genomic duplication events have increased the numbers of TX and TN genes in Arabidopsis. Genes encoding TX, TN and TNL proteins were demonstrated in conifers; TX and TN genes are present in very low numbers in grass genomes. The expression, prevalence, and diversity of TX and TN genes suggests that these genes encode functional proteins rather than resulting from degradation or deletions of TNL genes. These TX and TN proteins could be plant analogues of small TIR-adapter proteins that function in mammalian innate immune responses such as MyD88 and Mal.     

Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.     

A villin-like protein in the intestinal brush border of Calliphora vicina R.D. larvae (Diptera : Calliphoridae)

The brush border of the larval midgut and hindgut in Calliphora vicina (Diptera : Calliphoridae) shows a positive staining with a rabbit antiserum directed against pig villin. This protein has been extensively studied in chicken and in several mammals; it is known to bind together the actin filaments of the microvillar cytoskeleton. The presence of a villin-like protein in insect microvilli has never been reported. The immunodetection of villin in both hind- and midgut points out that villin exists in ectodermal tissues as well as in endodermal or mesodermal ones. Moreover, the “insect villin” seems to present the polypeptidic sequence of 13 amino acids, which has been found in both mammalian and avian villin. That may indicate that villin is a conserved molecule in several taxa.     

丝瓜的三聚体G蛋白的初步研究

根据拟南芥（ａｒａｂｉｄｏｐｓｉｓｔｈａｈｌｉａｎａ）ＧＰＡ１的保守区段Ａ设计一对特异引物（５′ｃｔｇｇｇｇａａｔｃｔｇｇａａａａｔｃ３′,５′ｃａｃａｇｃｔｇｔａｃａｃｃｔｃａａａｃ３′）通过ＰＣＲ从丝瓜核基因组中扩增植物的三聚体Ｇ蛋白α亚基编码基因,获得了２个片段（ＬＦＧ１,ＬＦＧ２）,并已克隆和测序（已在ＥＭＢＬ数据库中登记,登记号为：ｙ１５２７０,ｙ１５２７１）．序列分析表明ＬＦＧ１和ＬＦＧ２分别由１５１５ｂｐ和７３２ｂｐ构成,都含有三聚体Ｇ蛋白α亚基编码基因的保守区段Ａ,但也都含有内含子．根据片段的大小及ＰＣＲ的特性,ＬＦＧ１可能是丝瓜三聚体Ｇ蛋白α亚基编码基因上的片段．     

Selective autophagy in the aid of plant germination and response to nutrient starvation

Selective autophagy, mediated by Atg8 binding proteins, has not been extensively studied in plants. Plants possess a large gene family encoding multiple isoforms of the Atg8 protein. We have recently reported the identification of two new, closely homologous Arabidopsis thaliana plant proteins that bind the Arabidopsis Atg8f protein isoform. These two proteins are specific to plants and have no homologs in nonplant organisms. The expression levels of the genes encoding these proteins are elevated during carbon starvation and also during late stages of seed development. Exposure of young seedlings to carbon starvation induces the production of a newly identified compartment decorated by these Atg8-binding proteins. This compartment dynamically moves along the endoplasmic reticulum membrane and is also finally transported into the vacuole. Enhanced or suppressed expression of these Atg8-binding proteins respectively enhances or suppresses seed germination under suboptimal germination conditions, indicating that they contribute to seed germination vigor.     

Water channels in the plant plasma membrane cloned by immunoselection from a mammalian expression system

Expression in mammalian COS cells and an efficient microtiter-based strategy for immunoselection was used in a novel approach to identify genes encoding plant membrane proteins. COS cells were transfected with an Arabidopsis thaliana root cDNA library constructed in a bacterial mammalian shuttle vector and screened with an antiserum raised against purified deglycosylated integral plasma membrane proteins from A. thaliana roots. Antibodies directed against a prominent 27 kDa antigen led to the identification of five different genes. They comprised two subfamilies related to the major intrinsic protein (MIP) superfamily and were named plasma membrane intrinsic proteins, PIP1 and PIP2, since the cellular localization of PIP1 and most probably PIP2 proteins in the plasma membrane was independently confirmed by their co-segregation with marker enzymes during aequeous two-phase partitioning. Surprisingly, expression in Xenopus laevis oocytes revealed that all five PIP mRNAs coded for Hg²⁺-sensitive water transport facilitating activities. There had been no previous evidence of the existence of water channels in the plasma membrane of plant cells and the high diffusional water permeability of the lipid bilayer was considered to be sufficient for water exchange. Nevertheless, Northern and Western analyses showed that the PIP genes are constitutively and possibly even redundantly expressed from the small A. thaliana genome.     

Intracellular sorting of the tail-anchored protein cytochrome b5 in plants: a comparative study using different isoforms from rabbit and Arabidopsis

Tail-anchored (TA) proteins are bound to membranes by a hydrophobic sequence located very close to the C-terminus, followed by a short luminal polar region. Their active domains are exposed to the cytosol. TA proteins are synthesized on free cytosolic ribosomes and are found on the surface of every subcellular compartment, where they play various roles. The basic mechanisms of sorting and targeting of TA proteins to the correct membrane are poorly characterized. In mammalian cells, the net charge of the luminal region determines the sorting to the correct target membrane, a positive charge leading to mitochondria and negative or null charge to the endoplasmic reticulum (ER). Here sorting signals of TA proteins were studied in plant cells and compared with those of mammalian proteins, using in vitro translation-translocation and in vivo expression in tobacco protoplasts or leaves. It is shown that rabbit cytochrome b5 (cyt b5) with a negative charge is faithfully sorted to the plant ER, whereas a change to a positive charge leads to chloroplast targeting (instead of to mitochondria as observed in mammalian cells). The subcellular location of two cyt b5 isoforms from Arabidopsis thaliana (At1g26340 and At5g48810, both with positive net charge) was then determined. At5g48810 is targeted to the ER, and At1g26340 to the chloroplast envelope. The results show that the plant ER, unlike the mammalian ER, can accommodate cytochromes with opposite C-terminal net charge, and plant cells have a specific and as yet uncharacterized mechanism to sort TA proteins with the same positive C-terminal charge to different membranes.     

Evolutionary implications of the family of 14-3-3 brain protein homologs in Arabidopsis thaliana

The GF14 family of proteins in Arabidopsis thaliana consists of a homologous group of polypeptides ranging in size from 27 kDa to 32 kDa. As a group, GF14 proteins are also homologous to a family of mammalian proteins most commonly referred to as 14-3-3 proteins. Several distinct and different biochemical activities have been historically attributed to the various isoforms of the mammalian 14-3-3 proteins. These data present the possibility that the various activities are performed by functionally distinct lineages of the gene family. Here we present phylogenetic analyses based on the derived amino acid sequences of five GF14 isoforms expressed in Arabidopsis suspension-cultured cells. A high degree of sequence integrity is apparent in the various Arabidopsis isoforms, and the overall structures of the plant forms are quite conserved with regard to the structures of the known mammalian forms. These gene phylogenies indicate no evolutionary conservation of specific isoform lineages within both plants and animals. Rather, the evolutionary history of this protein appears to be characterized by a separate radiation of plant and animal forms from a common ancestral sequence. Even though the plant and animal forms have evolved independently since that ancestral split, large domains are conserved in both major lineages.