The microtubule plus-end tracking proteins SPR1 and EB1b interact to maintain polar cell elongation and directional organ growth in Arabidopsis.

The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1 proteins, setting SPR1 apart from most studied +TIPs in animals and fungi. Moreover, we found that the severity of defects in microtubule dynamics in spr1 eb1b mutant hypocotyl cells correlated well with the severity of growth defects. These data indicate that SPR1 and EB1b have complex interactions as they load onto microtubule plus ends and direct polar cell expansion and organ growth in response to directional cues.

Identification and suppression of the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L.

Grasses, such as Zea mays L. (maize), contain relatively high levels of p-coumarates (pCA) within their cell walls. Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols. To understand the role of pCA in maize development, the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase (pCAT) was isolated and purified from maize stems. Purified pCAT was subjected to partial trypsin digestion, and peptides were sequenced by tandem mass spectrometry. TBLASTN analysis of the acquired peptide sequences identified a single full-length maize cDNA clone encoding all the peptide sequences obtained from the purified enzyme. The cDNA clone was obtained and used to generate an RNAi construct for suppressing pCAT expression in maize. Here we describe the effects of suppression of pCAT in maize. Primary screening of transgenic maize seedling leaves using a new rapid analytical platform was used to identify plants with decreased amounts of pCA. Using this screening method, mature leaves from fully developed plants were analyzed, confirming reduced pCA levels throughout plant development. Complete analysis of isolated cell walls from mature transgenic stems and leaves revealed that lignin levels did not change, but pCA levels decreased and the lignin composition was altered. Transgenic plants with the lowest levels of pCA had decreased levels of syringyl units in the lignin. Thus, altering the levels of pCAT expression in maize leads to altered lignin composition, but does not appear to alter the total amount of lignin present in the cell walls.

Progressive multifocal leukoencephalopathy genetic risk variants for pharmacovigilance of immunosuppressant therapies.

Background: Progressive multifocal leukoencephalopathy (PML) is a rare and often lethal brain disorder caused by the common, typically benign polyomavirus 2, also known as JC virus (JCV). In a small percentage of immunosuppressed individuals, JCV is reactivated and infects the brain, causing devastating neurological defects. A wide range of immunosuppressed groups can develop PML, such as patients with: HIV/AIDS, hematological malignancies (e.g., leukemias, lymphomas, and multiple myeloma), autoimmune disorders (e.g., psoriasis, rheumatoid arthritis, and systemic lupus erythematosus), and organ transplants. In some patients, iatrogenic (i.e., drug-induced) PML occurs as a serious adverse event from exposure to immunosuppressant therapies used to treat their disease (e.g., hematological malignancies and multiple sclerosis). While JCV infection and immunosuppression are necessary, they are not sufficient to cause PML. Methods: We hypothesized that patients may also have a genetic susceptibility from the presence of rare deleterious genetic variants in immune-relevant genes (e.g., those that cause inborn errors of immunity). In our prior genetic study of 184 PML cases, we discovered 19 candidate PML risk variants. In the current study of another 152 cases, we validated 4 of 19 variants in both population controls (gnomAD 3.1) and matched controls (JCV+ multiple sclerosis patients on a PML-linked drug ≥ 2 years). Results: The four variants, found in immune system genes with strong biological links, are: C8B, 1-57409459-C-A, rs139498867; LY9 (alias SLAMF3), 1-160769595-AG-A, rs763811636; FCN2, 9-137779251-G-A, rs76267164; STXBP2, 19-7712287-G-C, rs35490401. Carriers of any one of these variants are shown to be at high risk of PML when drug-exposed PML cases are compared to drug-exposed matched controls: P value = 3.50E-06, OR = 8.7 [3.7-20.6]. Measures of clinical validity and utility compare favorably to other genetic risk tests, such as BRCA1 and BRCA2 screening for breast cancer risk and HLA-B*15:02 pharmacogenetic screening for pharmacovigilance of carbamazepine to prevent Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis. Conclusion: For the first time, a PML genetic risk test can be implemented for screening patients taking or considering treatment with a PML-linked drug in order to decrease the incidence of PML and enable safer use of highly effective therapies used to treat their underlying disease.

Overexpression of a Sugarcane BAHD Acyltransferase Alters Hydroxycinnamate Content in Maize Cell Wall.

The purification of hydroxycinnamic acids [p-coumaric acid (pCA) and ferulic acid (FA)] from grass cell walls requires high-cost processes. Feedstocks with increased levels of one hydroxycinnamate in preference to the other are therefore highly desirable. We identified and conducted expression analysis for nine BAHD acyltransferase ScAts genes from sugarcane. The high conservation of AT10 proteins, together with their similar gene expression patterns, supported a similar role in distinct grasses. Overexpression of ScAT10 in maize resulted in up to 75% increase in total pCA content. Mild hydrolysis and derivatization followed by reductive cleavage (DFRC) analysis showed that pCA increase was restricted to the hemicellulosic portion of the cell wall. Furthermore, total FA content was reduced up to 88%, resulting in a 10-fold increase in the pCA/FA ratio. Thus, we functionally characterized a sugarcane gene involved in pCA content on hemicelluloses and generated a C4 plant that is promising for valorizing pCA production in biorefineries.

Grass Cell Walls: A Story of Cross-Linking.

Cell wall matrices are complex composites mainly of polysaccharides, phenolics (monomers and polymers), and protein. We are beginning to understand the synthesis of these major wall components individually, but still have a poor understanding of how cell walls are assembled into complex matrices. Valuable insight has been gained by examining intact components to understand the individual elements that make up plant cell walls. Grasses are a prominent group within the plant kingdom, not only for their important roles in global agriculture, but also for the complexity of their cell walls. Ferulate incorporation into grass cell wall matrices (C3 and C4 types) leads to a cross-linked matrix that plays a prominent role in the structure and utilization of grass biomass compared to dicot species. Incorporation of p-coumarates as part of the lignin structure also adds to the complexity of grass cell walls. Feruoylation results in a wall with individual hemicellulosic polysaccharides (arabinoxylans) covalently linked to each other and to lignin. Evidence strongly suggests that ferulates not only cross-link arabinoxylans, but may be important factors in lignification of the cell wall. Therefore, the distribution of ferulates on arabinoxylans could provide a means of structuring regions of the matrix with the incorporation of lignin and have a significant impact upon localized cell wall organization. The role of other phenolics in cell wall formation such as p-coumarates (which can have concentrations higher than ferulates) remains unknown. It is possible that p-coumarates assist in the formation of lignin, especially syringyl rich lignin. The uniqueness of the grass cell wall compared to dicot sepcies may not be so much in the gross composition of the wall, but how the distinctive individual components are organized into a functional wall matrix. These features are discussed and working models are provided to illustrate how changing the organization of feruoylation and p-coumaroylation could lead to differing cell wall properties.

Cell wall composition and digestibility alterations in Brachypodium distachyon achieved through reduced expression of the UDP-arabinopyranose mutase.

Nucleotide-activated sugars are essential substrates for plant cell-wall carbohydrate-polymer biosynthesis. The most prevalent grass cell wall (CW) sugars are glucose (Glc), xylose (Xyl), and arabinose (Ara). These sugars are biosynthetically related via the UDP-sugar interconversion pathway. We sought to target and generate UDP-sugar interconversion pathway transgenic Brachypodium distachyon lines resulting in CW carbohydrate composition changes with improved digestibility and normal plant stature. Both RNAi-mediated gene-suppression and constitutive gene-expression approaches were performed. CWs from 336 T0 transgenic plants with normal appearance were screened for complete carbohydrate composition. RNAi mutants of BdRGP1, a UDP-arabinopyranose mutase, resulted in large alterations in CW carbohydrate composition with significant decreases in CW Ara content but with minimal change in plant stature. Five independent RNAi-RGP1 T1 plant lines were used for in-depth analysis of plant CWs. Real-time PCR analysis indicated that gene expression levels for BdRGP1, BdRGP2, and BdRGP3 were reduced in RNAi-RGP1 plants to 15-20% of controls. CW Ara content was reduced by 23-51% of control levels. No alterations in CW Xyl and Glc content were observed. Corresponding decreases in CW ferulic acid (FA) and ferulic acid-dimers (FA-dimers) were observed. Additionally, CW p-coumarates (pCA) were decreased. We demonstrate the CW pCA decrease corresponds to Ara-coupled pCA. Xylanase-mediated digestibility of RNAi-RGP1 Brachypodium CWs resulted in a near twofold increase of released total carbohydrate. However, cellulolytic hydrolysis of CW material was inhibited in leaves of RNAi-RGP1 mutants. Our results indicate that targeted manipulation of UDP-sugar biosynthesis can result in biomass with substantially altered compositions and highlights the complex effect CW composition has on digestibility.

Cell wall composition throughout development for the model grass Brachypodium distachyon.

Temperate perennial grasses are important worldwide as a livestock nutritive energy source and a potential feedstock for lignocellulosic biofuel production. The annual temperate grass Brachypodium distachyon has been championed as a useful model system to facilitate biological research in agriculturally important temperate forage grasses based on phylogenetic relationships. To physically corroborate genetic predictions, we determined the chemical composition profiles of organ-specific cell walls throughout the development of two common diploid accessions of Brachypodium distachyon, Bd21-3 and Bd21. Chemical analysis was performed on cell walls isolated from distinct organs (i.e., leaves, sheaths, stems, and roots) at three developmental stages of (1) 12-day seedling, (2) vegetative-to-reproductive transition, and (3) mature seed fill. In addition, we have included cell wall analysis of embryonic callus used for genetic transformations. Composition of cell walls based on components lignin, hydroxycinnamates, uronosyls, neutral sugars, and protein suggests that Brachypodium distachyon is similar chemically to agriculturally important forage grasses. There were modest compositional differences in hydroxycinnamate profiles between accessions Bd21-3 and Bd21. In addition, when compared to agronomical important C3 grasses, more mature Brachypodium stem cell walls have a relative increase in glucose of 48% and a decrease in lignin of 36%. Though differences exist between Brachypodium and agronomical important C3 grasses, Brachypodium distachyon should be still a useful model system for genetic manipulation of cell wall composition to determine the impact upon functional characteristics such as rumen digestibility or energy conversion efficiency for bioenergy production.

Protein antigen expression in Escherichia coli for antibody production.

Escherichia coli is a frequently used expression system for the generation of protein encoded by genes from diverse kingdoms and, thus, it is well suited for the production of protein antigens for antibody generation. It is a system of choice for many due to factors such as (1) the commercial availability of a vast array of reagents and materials needed for cloning, expression, and purification and (2) the potential high protein yields that can be acquired in a timely and cost-effective manner. This chapter will focus on (1) the general principles to keep in mind when choosing an antigen to express and (2) the use of a modified pGEX vector system (Rancour et al., J. Biol. Chem. 279:54264-54274, 2004) to use in its expression. Simplified protocols are provided for (1) assessing the expression of your protein, (2) testing whether your protein is or is not expressed as a soluble product, (3) performing bulk purifications of soluble or insoluble E. coli-expressed protein to acquire enough to be used for a complete immunization protocol, and (4) an optional procedure for epitope tag removal from your expressed protein of interest in order to avoid the unnecessary and sometimes unwanted production of antibodies against the fusion protein affinity chromatography tag. These four procedures have been used extensively and successfully in our lab as a basis for the production of recombinant protein and subsequent antibody production.

Protein domain-domain interactions and requirements for the negative regulation of Arabidopsis CDC48/p97 by the plant ubiquitin regulatory X (UBX) domain-containing protein, PUX1.

CDC48/p97 is an essential AAA-ATPase chaperone that functions in numerous diverse cellular activities through its interaction with specific adapter proteins. The ubiquitin regulatory X (UBX)-containing protein, PUX1, functions to regulate the hexameric structure and ATPase activity of AtCDC48. To characterize the biochemical mechanism of PUX1 action on AtCDC48, we have defined domains of both PUX1 and AtCDC48 that are critical for interaction and oligomer disassembly. Binding of PUX1 to AtCDC48 was mediated through a region containing both the UBX domain and the immediate C-terminal flanking amino acids (UBX-C). Like other UBX domains, the primary binding site for the UBX-C of PUX1 is the N(a) domain of AtCDC48. Alternative plant PUX protein UBX domains also bind AtCDC48 through the N terminus but were found not to be able to substitute for the action imparted by the UBX-C of PUX1 in hexamer disassembly, suggesting unique features for the UBX-C of PUX1. We propose that the PUX1 UBX-C domain modulates a second binding site on AtCDC48 required for the N-terminal domain of PUX1 to interact with and promote dissociation of the AtCDC48 hexamer. Utilizing Atcdc48 ATP hydrolysis and binding mutants, we demonstrate that PUX1 binding was not affected but that hexamer disassembly was significantly influenced by the ATP status of AtCDC48. ATPase activity in both the D1 and the D2 domains was critical for PUX1-mediated AtCDC48 hexamer disassembly. Together these results provide new mechanistic insight into how the hexameric status and ATPase activity of AtCDC48 are modulated.

The dynamin-like protein ADL1C is essential for plasma membrane maintenance during pollen maturation.

Dynamin-related GTPases regulate a wide variety of dynamic membrane processes in eukaryotes. Here, we investigated the function of ADL1C, a member of the Arabidopsis 68 kDa dynamin-like protein family. Analysis of heterozygous adl1C-1 indicates that the mutation specifically affects post-meiotic male gametogenesis. Fifty percent of the mature pollen from heterozygous adl1C-1 androecia are shriveled and fail to germinate in vitro. During microspore maturation, adl1C-1 pollen grains display defects in the plasma membrane and intine morphology, suggesting that ADL1C is essential for the formation and maintenance of the pollen cell surface and viability during desiccation. Consistent with a role in cell-surface dynamics, immunofluorescence microscopy indicates that ADL1C is localized to the cell plate of dividing somatic cells and to the tip of expanding root hairs. We propose that ADL1C functions in plasma membrane dynamics, and we discuss the role of the ADL1 family in plant growth and development.

Plant UBX domain-containing protein 1, PUX1, regulates the oligomeric structure and activity of arabidopsis CDC48.

p97/CDC48 is a highly abundant hexameric AAA-ATPase that functions as a molecular chaperone in numerous diverse cellular activities. We have identified an Arabidopsis UBX domain-containing protein, PUX1, which functions to regulate the oligomeric structure of the Arabidopsis homolog of p97/CDC48, AtCDC48, as well as mammalian p97. PUX1 is a soluble protein that co-fractionates with non-hexameric AtCDC48 and physically interacts with AtCDC48 in vivo. Binding of PUX1 to AtCDC48 is mediated through the UBX-containing C-terminal domain. However, disassembly of the chaperone is dependent upon the N-terminal domain of PUX1. These findings provide evidence that the assembly and disassembly of the hexameric p97/CDC48 complex is a dynamic process. This new unexpected level of regulation for p97/CDC48 was demonstrated to be critical in vivo as pux1 loss-of-function mutants display accelerated growth relative to wild-type plants. These results suggest a role for AtCDC48 and PUX1 in regulating plant growth.

Characterization of AtCDC48. Evidence for multiple membrane fusion mechanisms at the plane of cell division in plants.

The components of the cellular machinery that accomplish the various complex and dynamic membrane fusion events that occur at the division plane during plant cytokinesis, including assembly of the cell plate, are not fully understood. The most well-characterized component, KNOLLE, a cell plate-specific soluble N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein receptor (SNARE), is a membrane fusion machine component required for plant cytokinesis. Here, we show the plant ortholog of Cdc48p/p97, AtCDC48, colocalizes at the division plane in dividing Arabidopsis cells with KNOLLE and another SNARE, the plant ortholog of syntaxin 5, SYP31. In contrast to KNOLLE, SYP31 resides in defined punctate membrane structures during interphase and is targeted during cytokinesis to the division plane. In vitro-binding studies demonstrate that AtCDC48 specifically interacts in an ATP-dependent manner with SYP31 but not with KNOLLE. In contrast, we show that KNOLLE assembles in vitro into a large approximately 20S complex in an Sec18p/NSF-dependent manner. These results suggest that there are at least two distinct membrane fusion pathways involving Cdc48p/p97 and Sec18p/NSF that operate at the division plane to mediate plant cytokinesis. Models for the role of AtCDC48 and SYP31 at the division plane will be discussed.