Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.

A significant number of proteins possess sizable intrinsically disordered regions (IDRs). Due to the dynamic nature of IDRs, NMR spectroscopy is often the tool of choice for characterizing these segments. However, the application of NMR to IDRs is often hindered by their instability, spectral overlap and resonance assignment difficulties. Notably, these challenges increase considerably with the size of the IDR. In response to these issues, here we report the use of sortase-mediated ligation (SML) for segmental isotopic labeling of IDR-containing samples. Specifically, we have developed a ligation strategy involving a key segment of the large IDR and adjacent folded headpiece domain comprising the C-terminus of A. thaliana villin 4 (AtVLN4). This procedure significantly reduces the complexity of NMR spectra and enables group identification of signals arising from the labeled IDR fragment, a process we refer to as segmental assignment. The validity of our segmental assignment approach is corroborated by backbone residue-specific assignment of the IDR using a minimal set of standard heteronuclear NMR methods. Using segmental assignment, we further demonstrate that the IDR region adjacent to the headpiece exhibits nonuniform spectral alterations in response to temperature. Subsequent residue-specific characterization revealed two segments within the IDR that responded to temperature in markedly different ways. Overall, this study represents an important step toward the selective labeling and probing of target segments within much larger IDR contexts. Additionally, the approach described offers significant savings in NMR recording time, a valuable advantage for the study of unstable IDRs, their binding interfaces, and functional mechanisms.

Synthesis and activity of the archazolid western hemisphere.

A convergent and scalable synthesis of the archazolid western hemisphere has been completed. The V-ATPase inhibitory activity of this compound along with a previously prepared eastern domain was then tested using a convenient Arabidopsis-based V-ATPase assay.

Molecular characterization of mutant Arabidopsis plants with reduced plasma membrane proton pump activity.

Arabidopsis mutants containing gene disruptions in AHA1 and AHA2, the two most highly expressed isoforms of the Arabidopsis plasma membrane H(+)-ATPase family, have been isolated and characterized. Plants containing homozygous loss-of-function mutations in either gene grew normally under laboratory conditions. Transcriptome and mass spectrometric measurements demonstrate that lack of lethality in the single gene mutations is not associated with compensation by increases in RNA or protein levels. Selected reaction monitoring using synthetic heavy isotope-labeled C-terminal tryptic peptides as spiked standards with a triple quadrupole mass spectrometer revealed increased levels of phosphorylation of a regulatory threonine residue in both isoforms in the mutants. Using an extracellular pH assay as a measure of in vivo ATPase activity in roots, less proton secreting activity was found in the aha2 mutant. Among 100 different growth conditions, those that decrease the membrane potential (high external potassium) or pH gradient (high external pH) caused a reduction in growth of the aha2 mutant compared with wild type. Despite the normal appearance of single mutants under ideal laboratory growth conditions, embryos containing homozygous double mutations are lethal, demonstrating that, as expected, this protein is absolutely essential for plant cell function. In conclusion, our results demonstrate that the two genes together perform an essential function and that the effects of their single mutations are mostly masked by overlapping patterns of expression and redundant function as well as by compensation at the post-translational level.

AUXIN RESPONSE FACTOR1 and AUXIN RESPONSE FACTOR2 regulate senescence and floral organ abscission in Arabidopsis thaliana.

In plants, both endogenous mechanisms and environmental signals regulate developmental transitions such as seed germination, induction of flowering, leaf senescence and shedding of senescent organs. Auxin response factors (ARFs) are transcription factors that mediate responses to the plant hormone auxin. We have examined Arabidopsis lines carrying T-DNA insertions in AUXIN RESPONSE FACTOR1 (ARF1) and ARF2 genes. We found that ARF2 promotes transitions between multiple stages of Arabidopsis development. arf2 mutant plants exhibited delays in several processes related to plant aging, including initiation of flowering, rosette leaf senescence, floral organ abscission and silique ripening. ARF2 expression was induced in senescing leaves. ARF2 regulated leaf senescence and floral organ abscission independently of the ethylene and cytokinin response pathways. arf1 mutations enhanced many arf2 phenotypes, indicating that ARF1 acts in a partially redundant manner with ARF2. However, unlike arf2 mutations, an arf1 mutation increased transcription of Aux/IAA genes in Arabidopsis flowers, supporting previous biochemical studies that indicated that ARF1 is a transcriptional repressor. Two other ARF genes, NPH4/ARF7 and ARF19, were also induced by senescence, and mutations in these genes enhanced arf2 phenotypes. NPH4/ARF7 and ARF19 function as transcriptional activators, suggesting that auxin may control senescence in part by activating gene expression.

A plasma membrane H+-ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana.

The plasma membrane in plant cells is energized with an electrical potential and proton gradient generated through the action of H+ pumps belonging to the P-type ATPase superfamily. The Arabidopsis genome encodes 11 plasma membrane H+ pumps. Auto-inhibited H+-ATPase isoform 10 (AHA10) is expressed primarily in developing seeds. Here we show that four independent gene disruptions of AHA10 result in seed coats with a transparent testa (tt) phenotype (light-colored seeds). A quantitative analysis of extractable flavonoids in aha10 seeds revealed an approximately 100-fold reduction of proanthocyanidin (PA), one of the two major end-product pigments in the flavonoid biosynthetic pathway. In wild-type seed coat endothelial cells, PA accumulates in a large central vacuole. In aha10 mutants, the formation of this vacuole is impaired, as indicated by the predominance of multiple small vacuoles observed by fluorescence microscopy using a vacuole-specific dye, 5-(and -6)-carboxy 2',7'-dichlorofluorescein diacetate. A similar vacuolar defect was also observed for another tt mutant, tt12, a proton-coupled multidrug and toxic compound extrusion transporter potentially involved in loading provacuoles with a flavonoid intermediate required for PA production. The endothelial cells in aha10 mutants are otherwise healthy, as indicated by the lack of a significant decrease in (i) the accumulation of other flavonoid pathway end products, such as anthocyanins, and (ii) mRNA levels for two endothelium-specific transcripts (TT12 and BAN). Thus, the specific effect of aha10 on vacuolar and PA biogenesis provides genetic evidence to support an unexpected endomembrane function for a member of the plasma membrane H+-ATPase family.

An Arabidopsis thaliana plasma membrane proton pump is essential for pollen development.

The plasma membrane proton pump (H(+)-ATPase) found in plants and fungi is a P-type ATPase with a polypeptide sequence, structure, and in vivo function similar to the mammalian sodium pump (Na(+), K(+)-ATPase). Despite its hypothetical importance for generating and maintaining the proton motive force that energizes the carriers and channels that underlie plant nutrition, genetic evidence for such a central function has not yet been reported. Using a reverse genetic approach for investigating each of the 11 isoforms in the Arabidopsis H(+)-ATPase (AHA) gene family, we found that one member, AHA3, is essential for pollen formation. A causative role for AHA3 in male gametogenesis was proven by complementation with a normal transgenic gene and rescue of the mutant phenotype back to wild type. We also investigated the requirement for phosphorylation of the penultimate threonine, which is found in most members of the AHA family and is thought to be involved in regulating catalytic activity. We demonstrated that a T948D mutant form of the AHA3 gene rescues the mutant phenotype in knockout AHA3 plants, but T948A does not, providing the first in planta evidence in support of the model in which phosphorylation of this amino acid is essential.

Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis.

A key component of a sound functional genomics infrastructure is the availability of a knockout mutant for every gene in the genome. A fruitful approach to systematically knockingout genes in the plant Arabidopsis thaliana has been the use of transferred-DNA (T-DNA) from Agrobacterium tumefaciens as an insertional mutagen. One of the assumptions underlying the use of T-DNA as a mutagen is that the insertion of these DNA elements into the Arabidopsis genome occurs at randomly selected locations. We have directly investigated the distribution of T-DNA insertions sites in populations of transformed Arabidopsis using two different approaches. To begin with, we utilized a polymerase chain reaction (PCR) procedure to systematically catalog the precise locations of all the T-DNA elements inserted within a 65 kb segment of chromosome IV. Of the 47 T-DNA insertions identified, 30% were found within the coding regions of genes. We also documented the insertion of T-DNA elements within the centromeric region of chromosome IV. In addition to these targeted T-DNA screens, we also mapped the genomic locations of 583 randomly chosen T-DNA elements by sequencing the genomic DNA flanking the insertion sites from individual T-DNA-transformed lines. 35% of these randomly chosen T-DNA insertions were located within the coding regions of genes. For comparison, coding sequences account for 44% of the Arabidopsis genome. Our results demonstrate that there is a small bias towards recovering T-DNA insertions within intergenic regions. However, this bias does not limit the utility of T-DNA as an effective insertional mutagen for use in reverse-genetic strategies.