Assaying proteasomal degradation in a cell-free system in plants.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions. The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Disassembly of synthetic Agrobacterium T-DNA-protein complexes via the host SCF(VBF) ubiquitin-ligase complex pathway.

One the most intriguing, yet least studied, aspects of the bacterium-host plant interaction is the role of the host ubiquitin/proteasome system (UPS) in the infection process. Increasing evidence indicates that pathogenic bacteria subvert the host UPS to facilitate infection. Although both mammalian and plant bacterial pathogens are known to use the host UPS, the first prokaryotic F-box protein, an essential component of UPS, was identified in Agrobacterium. During its infection, which culminates in genetic modification of the host cell, Agrobacterium transfers its T-DNA--as a complex (T-complex) with the bacterial VirE2 and host VIP1 proteins--into the host cell nucleus. There the T-DNA is uncoated from its protein components before undergoing integration into the host genome. It has been suggested that the host UPS mediates this uncoating process, but there is no evidence indicating that this activity can unmask the T-DNA molecule. Here we provide support for the idea that the plant UPS uncoats synthetic T-complexes via the Skp1/Cullin/F-box protein VBF pathway and exposes the T-DNA molecule to external enzymatic activity.

Expression of complete metabolic pathways in transgenic plants.

Plant genetic engineering emerged as a methodology to introduce only few transgenes into the plant genome. Following fast-paced developments of the past few decades, engineering of much larger numbers of transgenes became a reality, allowing to introduce full metabolic pathways from other organisms into plants and generate transgenics with startling new traits. From the advent of the classical plant genetic engineering, the transgenes were introduced into the nuclear genome of the plant cell, and this strategy still is quite successful when applied to few transgenes. However, for introducing large number of transgenes, we advocate that the chloroplast genome is a superior choice, especially for engineering of new complete metabolic pathways into plants. The ability to genetically engineer plants with complex and fully functional metabolic pathways from other organisms bears a substantial promise in generation of pharmaceuticals, i.e., biopharming, and new agricultural crops with that traits never existed before, leading to enhancement in quality of human life.

Involvement of KDM1C histone demethylase-OTLD1 otubain-like histone deubiquitinase complexes in plant gene repression.

Covalent modifications of histones, such as acetylation, methylation and ubiquitination, are central for regulation of gene expression. Heterochromatic gene silencing, for example, is associated with hypoacetylation, methylation and demethylation, and deubiquitination of specific amino acid residues in histone molecules. Many of these changes can be effected by histone-modifying repressor complexes that include histone lysine demethylases, such as KDM1 in animals and KDM1C in plants. However, whereas KDM1-containing repressor complexes have been implicated in histone demethylation, methylation and deacetylation, whether or not they can also mediate histone deubiquitination remains unknown. We identify an Arabidopsis otubain-like deubiquitinase OTLD1 which directly interacts with the Arabidopsis KDM1C in planta, and use one target gene to exemplify that both OTLD1 and KDM1C are involved in transcriptional gene repression via histone deubiquitination and demethylation. We also show that OTLD1 binds plant chromatin and has enzymatic histone deubiquitinase activity, specific for the H2B histone. Thus, we suggest that, during gene repression, lysine demethylases can directly interact and function in a protein complex with histone deubiquitinases.

Plant defense pathways subverted by Agrobacterium for genetic transformation.

The soil phytopathogen Agrobacterium has the unique ability to introduce single-stranded transferred DNA (T-DNA) from its tumor-inducing (Ti) plasmid into the host cell in a process known as horizontal gene transfer. Following its entry into the host cell cytoplasm, the T-DNA associates with the bacterial virulence (Vir) E2 protein, also exported from Agrobacterium, creating the T-DNA nucleoprotein complex (T-complex), which is then translocated into the nucleus where the DNA is integrated into the host chromatin. VirE2 protects the T-DNA from the host DNase activities, packages it into a helical filament, and interacts with the host proteins, one of which, VIP1, facilitates nuclear import of the T-complex and its subsequent targeting to the host chromatin. Although the VirE2 and VIP1 protein components of the T-complex are vital for its intracellular transport, they must be removed to expose the T-DNA for integration. Our recent work demonstrated that this task is aided by an host defense-related F-box protein VBF that is induced by Agrobacterium infection and that recognizes and binds VIP1. VBF destabilizes VirE2 and VIP1 in yeast and plant cells, presumably via SCF-mediated proteasomal degradation. VBF expression in and export from the Agrobacterium cell lead to increased tumorigenesis. Here, we discuss these findings in the context of the "arms race" between Agrobacterium infectivity and plant defense.

Nuclear and plastid genetic engineering of plants: comparison of opportunities and challenges.

Plant genetic engineering is one of the key technologies for crop improvement as well as an emerging approach for producing recombinant proteins in plants. Both plant nuclear and plastid genomes can be genetically modified, yet fundamental functional differences between the eukaryotic genome of the plant cell nucleus and the prokaryotic-like genome of the plastid will have an impact on key characteristics of the resulting transgenic organism. So, which genome, nuclear or plastid, to transform for the desired transgenic phenotype? In this review we compare the advantages and drawbacks of engineering plant nuclear and plastid genomes to generate transgenic plants with the traits of interest, and evaluate the pros and cons of their use for different biotechnology and basic research applications, ranging from generation of commercial crops with valuable new phenotypes to 'bioreactor' plants for large-scale production of recombinant proteins to research model plants expressing various reporter proteins.

Agrobacterium induces expression of a host F-box protein required for tumorigenicity.

Agrobacterium exports DNA into plant cells, eliciting neoplastic growths on many plant species. During this process, a Skp1-Cdc53-cullin-F-box (SCF) complex that contains the bacterial virulence F-box protein VirF facilitates genetic transformation by targeting for proteolysis proteins, the Agrobacterium protein VirE2 and the host protein VIP1, that coat the transferred DNA. However, some plant species do not require VirF for transformation. Here, we show that Agrobacterium induces expression of a plant F-box protein, which we designated VBF for VIP1-binding F-box protein, that can functionally replace VirF, regulating levels of the VirE2 and VIP1 proteins via a VBF-containing SCF complex. When expressed in Agrobacterium and exported into the plant cell, VBF functionally complements tumor formation by a strain lacking VirF. VBF expression is known to be induced by diverse pathogens, suggesting that Agrobacterium has co-opted a plant defense response and that bacterial VirF and plant VBF both contribute to targeted proteolysis that promotes plant genetic transformation.

Proteasomal degradation in plant-pathogen interactions.

The ubiquitin/26S proteasome pathway is a basic biological mechanism involved in the regulation of a multitude of cellular processes. Increasing evidence indicates that plants utilize the ubiquitin/26S proteasome pathway in their immune response to pathogen invasion, emphasizing the role of this pathway during plant-pathogen interactions. The specific functions of proteasomal degradation in plant-pathogen interactions are diverse, and do not always benefit the host plant. Although in some cases, proteasomal degradation serves as an effective barrier to help plants ward off pathogens, in others, it is used by the pathogen to enhance the infection process. This review discusses the different roles of the ubiquitin/26S proteasome pathway during interactions of plants with pathogenic viruses, bacteria, and fungi.

Regulation of root elongation by histone acetylation in Arabidopsis.

Transcriptional repression by histone modification represents a universal mechanism that underlies critical biological processes, such as neurogenesis and hematopoietic differentiation, in animals. In plants, however, the extent to which these regulatory pathways are involved in development and morphogenesis is not well defined. SWP1/LDL1 is a component of a plant corepressor complex that is involved in regulation of flower timing. Here, we report that SWP1 also plays a role in the regulation of root elongation by repressing a root-specific gene lateral root primordium 1 (LRP1) via histone deacetylation. A null mutation in SWP1 results in hyperacetylation of histones H3 and H4 in LRP1 chromatin, elevation of LRP1 expression, and increased root elongation. This effect of SWP1 knockout on the root phenotype is mimicked by transgenic expression of LRP1, which bypasses the SWP1-mediated suppression of the native gene. Thus, SWP1 likely functions as a regulator of developmental events both in the shoot and in the root meristem.

Yeast-plant coupled vector system for identification of nuclear proteins.

Nuclear proteins are involved in many critical biological processes within plant cells and, therefore, are in the focus of studies that usually begin with demonstrating that the protein of interest indeed exhibits nuclear localization. Thus, studies of plant nuclear proteins would be facilitated by a convenient experimental system for identification of proteins that are actively imported into the cell nucleus and visualization of their nuclear accumulation in vivo. To this end, we developed a system of vectors that allows screening for cDNAs coding for nuclear proteins in a simple genetic assay in yeast cells, and verification of nuclear accumulation in planta following one-step transfer and autofluorescent tagging of the identified clones into a multiple cloning site-compatible and reading frame-compatible plant expression vector. In a recommended third experimental step, the plant expression cassette containing the identified clone can be transferred, also by a one-step cloning, into a binary multigene expression vector for transient or stable coexpression with any other proteins.

How pollen tubes grow.

Sexual reproduction of flowering plants depends on delivery of the sperm to the egg, which occurs through a long, polarized projection of a pollen cell, called the pollen tube. The pollen tube grows exclusively at its tip, and this growth is distinguished by very fast rates and reaches extended lengths. Thus, one of the most fascinating aspects of pollen biology is the question of how enough cell wall material is produced to accommodate such rapid extension of pollen tube, and how the cell wall deposition and structure are regulated to allow for rapid changes in the direction of growth. This review discusses recent advances in our understanding of the mechanism of pollen tube growth, focusing on such basic cellular processes as control of cell shape and growth by a network of cell wall-modifying enzymes, molecular motor-mediated vesicular transport, and intracellular signaling by localized gradients of second messengers.

Biological systems of the host cell involved in Agrobacterium infection.

Genetic transformation of plants by Agrobacterium, which in nature causes neoplastic growths, represents the only known case of trans-kingdom DNA transfer. Furthermore, under laboratory conditions, Agrobacterium can also transform a wide range of other eukaryotic species, from fungi to sea urchins to human cells. How can the Agrobacterium virulence machinery function in such a variety of evolutionarily distant and diverse species? The answer to this question lies in the ability of Agrobacterium to hijack fundamental cellular processes which are shared by most eukaryotic organisms. Our knowledge of these host cellular functions is critical for understanding the molecular mechanisms that underlie genetic transformation of eukaryotic cells. This review outlines the bacterial virulence machinery and provides a detailed discussion of seven major biological systems of the host cell-cell surface receptor arrays, cellular motors, nuclear import, chromatin targeting, targeted proteolysis, DNA repair, and plant immunity--thought to participate in the Agrobacterium-mediated genetic transformation.

Pollen-specific pectin methylesterase involved in pollen tube growth.

Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation.

Two types of FtsH protease subunits are required for chloroplast biogenesis and Photosystem II repair in Arabidopsis.

FtsH protease is important in chloroplast biogenesis and thylakoid maintenance. Although bacteria contain only one essential FTSH gene, multiple genes exist in cyanobacteria and higher plants. However, the functional significance of FTSH multiplication in plants is unclear. We hypothesized that some FTSH genes may be redundant. To test this hypothesis, we generated double mutant combinations among the different FTSH genes in Arabidopsis thaliana. A double mutant of ftsh1 and ftsh8 showed no obvious phenotypic alterations, and disruption of either FTSH1 or FTSH5 enhanced the phenotype of the ftsh2 mutant. Unexpectedly, new phenotypes were recovered from crosses between ftsh2 and ftsh8 and between ftsh5 and ftsh1, including albinism, heterotrophy, disruption of flowering, and severely reduced male fertility. These results suggest that the duplicated genes, FTSH1 and FTSH5 (subunit type A) and FTSH2 and FTSH8 (subunit type B), are redundant. Furthermore, they reveal that the presence of two types of subunits is essential for complex formation, photosystem II repair, and chloroplast biogenesis.

Developmental and light effects on the accumulation of FtsH protease in Arabidopsis chloroplasts--implications for thylakoid formation and photosystem II maintenance.

The chloroplast ATP-dependent metalloprotease FtsH is involved in the degradation of unassembled proteins, the repair of photosystem II (PSII) from photoinhibition, and, apparently, the formation of thylakoids. In Arabidopsis, it is encoded by a family of 12 genes. However, the products of only four of them, FtsH1, 2, 5 and 8, have been found in chloroplasts to date. Mutations in two of these, FtsH2 and 5, demonstrate a visible phenotype of variegated leaves, with the phenotype of the FtsH2 mutant being more pronounced. Moreover, the degree of variegation appears to be dependent on developmental stage and environmental factors, suggesting an intricate relationship between the different gene products. To explore this, developmental and light effects on the accumulation of FtsH protease were studied in wild-type (WT) and FtsH2-mutant plants. Whereas cotyledons of the mutant were indistinguishable from those of the WT, the first true leaves were almost completely white. Subsequent leaves contained increasing proportions of green sectors. Analysis of the mRNA of the four FtsH genes, in cotyledons, first and second leaves of WT and mutant plants, revealed that: (i) transcript level increases during development, and (ii) transcript level in the mutant is higher than in the WT. FtsH protein level in the mutant was ca. 50% of that found in the WT, whereas the levels of other thylakoid proteins were the same. In individual leaves, the level of FtsH protein increased during development as well. Exposure of seedlings to different light intensities did not affect the degree of variegation, suggesting that it is due to a defect in chloroplast development rather than photobleaching. Examination of FtsH protein during exposure to high light revealed a decrease in its level, concomitant with a decrease in PSII potential, suggesting that the kinetics of photoinhibition reflects not only photodamage to PSII and induction of protective mechanisms, but also a decrease in repair capacity due to a reduction in the level of FtsH protease.

Expression in multigene families. Analysis of chloroplast and mitochondrial proteases.

The proteolytic machinery of chloroplasts and mitochondria in Arabidopsis consists primarily of three families of ATP-dependent proteases, Clp, Lon, and FtsH, and one family of ATP-independent proteases, DegP. However, the functional significance of the multiplicity of their genes is not clear. To test whether expression of specific isomers could be differently affected by growth conditions, we analyzed transcript abundance following short-term exposure to different environmental stimuli, using 70-mer oligonucleotide arrays. This analysis revealed variability in the response to high light and different temperatures within members of each family. Thirty out of the 41 tested genes were up-regulated in response to high light, including both chloroplast and mitochondrial isozymes, whereas only six and five genes responded to either high or low temperature, respectively. The extent of response was variable, ranging from 2- to 20-fold increase in the steady-state levels. Absolute transcript levels of the tested genes, compiled from one-channel arrays, were also variable. In general, transcripts encoding mitochondrial isozymes were accumulated to a lower level than chloroplastic ones. Within the FtsH family, transcript abundance of most genes correlated with the severity of mutant phenotypes in the relevant genes. This correlation was also evident at the protein level. Analysis of FtsH isozymes revealed that FtsH2 was the most abundant species, followed by FtsH5 and 8, with FtsH1 being accumulated to only 10% of FtsH2 level. These results suggest that, unlike previous expectations, the relative importance of different chloroplast protease isozymes, evidenced by mutant phenotypes at least in the FtsH family, is determined by their abundance, and not necessarily by different specific functions or specialized expression under certain conditions.

Coordinated regulation and complex formation of yellow variegated1 and yellow variegated2, chloroplastic FtsH metalloproteases involved in the repair cycle of photosystem II in Arabidopsis thylakoid membranes.

Arabidopsis yellow variegated1 (VAR1) and VAR2 are separate loci that encode similar chloroplast FtsH proteases. To date, FtsH is the best-characterized protease in thylakoid membranes involved in the turnover of photosynthetic protein complexes. It comprises a protein family that is encoded by 12 different nuclear genes in Arabidopsis. We show here that nine FtsH proteins are located in the chloroplasts. Mutations in either VAR1 or VAR2 cause typical leaf variegation and sensitivity to photoinhibition. By contrast, none of these phenotypes was observed in T-DNA insertion mutants in other ftsH genes (ftsh1, ftsh6, and ftsh8) closely related to VAR1 and VAR2. This finding suggests that VAR1 and VAR2 play a predominant role in the photosystem II repair cycle in thylakoid membranes. By generating VAR1- and VAR2-specific antibodies, we found that loss of either VAR1 or VAR2 results in the decreased accumulation of the other. Thus, the genetic nonredundancy between VAR1 and VAR2 could be attributed to their coordinated regulation at the protein level. These observations led us to examine whether VAR1 and VAR2 form a complex. Sucrose density gradient and gel filtration analyses revealed a complex of approximately 400 to 450 kD, probably representing a hexamer. Furthermore, VAR1 and VAR2 were shown to coprecipitate by immunoprecipitation using VAR1- and VAR2-specific antibodies. The majority of VAR1 appears to exist as heterocomplexes with VAR2, whereas VAR2 may be present as homocomplexes as well. Based on these results, we conclude that VAR1 and VAR2 are the major components of an FtsH complex involved in the repair of photodamaged proteins in thylakoid membranes.