Modulators of transit peptide activity in targeting and translocation of precursors into plastides : the mature domain, lipids and Toc-Tic components

The import of nuclear-encoded precursor proteins into chloroplasts occurs via a cleavable, N-terminal targeting sequence known as the transit peptide. To test the influence of the mature domain of the small subunit of Rubisco during import in vitro, the precursor (prSSU), the mature domain (mSSU), the transit peptide (SStp) and three Cterminal deletion mutants (Δ52, Δ67, and Δ74) of prSSU were expressed and purified from Escherichia coli. Activity was then evaluated by inhibition of import of 35S-prSSU to show that removal of C-terminal prSSU sequences inhibits its interaction with the translocation apparatus. Import studies demonstrated that prSSU and Δ52 were processed and accumulated within the chloroplast, whereas Δ67 and Δ74 were rapidly degraded. Import-competent proteins were also able to induce anion channel closure for PIRAC (Protein Import Related Iconic C,/u\u3ehannel). Although the C-terminal deletion mutants were less effective at inducing channel closure upon import, they did not affect the mean duration of channel closure. In addition the same proteins, as well as the precursors to the 33 and 23 kD subunits of the oxygen evolving complex of photosystem II, prOE33 and prOE23, respectively, were used in liposome dye release assays to investigate the interaction between precursors and chloroplast outer membrane lipids. Chloroplast precursor proteins do interact with liposomes mimicking the chloroplast outer envelope lipids through a process mediated through the transit peptide and requiring the presence of non-bilayer forming lipids and anionic lipids. The interaction of the transit peptide with liposomes involves electrostatic interactions between the peptide and the anionic lipids in the liposome. From this study, two additional precursors were shown to be membrane active, prOE33 and prOE23 Finally to investigate the in vivo activity of prSSU transit peptide, we designed green fluorescent protein (GPP) chimeras with the precursor to the small subunit of Rubisco (prSSU-GFP). GFP alone or prSSU-GFP could be expressed in a transient assay in onion epidermal cells. These experiments lay the groundwork for expression of mutant precursors fused to GFP in either onion epidermal to explore the effect mutations in the transit peptide have on targeting and import relative to the wildtype precursor

Root Hair Single Cell Type Specific Profiles of Gene Expression and Alternative Polyadenylation Under Cadmium Stress

Transcriptional networks are tightly controlled in plant development and stress responses. Alternative polyadenylation (APA) has been found to regulate gene expression under abiotic stress by increasing the heterogeneity at mRNA 3′-ends. Heavy metals like cadmium pollute water and soil due to mining and industry applications. Understanding how plants cope with heavy metal stress remains an interesting question. The Arabidopsis root hair was chosen as a single cell model to investigate the functional role of APA in cadmium stress response. Primary root growth inhibition and defective root hair morphotypes were observed. Poly(A) tag (PAT) libraries from single cell types, i.e., root hair cells, non-hair epidermal cells, and whole root tip under cadmium stress were prepared and sequenced. Interestingly, a root hair cell type-specific gene expression under short term cadmium exposure, but not related to the prolonged treatment, was detected. Differentially expressed poly(A) sites were identified, which largely contributed to altered gene expression, and enriched in pentose and glucuronate interconversion pathways as well as phenylpropanoid biosynthesis pathways. Numerous genes with poly(A) site switching were found, particularly for functions in cell wall modification, root epidermal differentiation, and root hair tip growth. Our findings suggest that APA plays a functional role as a potential stress modulator in root hair cells under cadmium treatment

The Role of Native Cysteine Residues in the Oligomerization of KCNQ1 Channels

KCNQ1, the major component of the slow-delayed rectifier potassium channel, is responsible for repolarization of cardiac action potential. Mutations in this channel can lead to a variety of diseases, most notably long QT syndrome. It is currently unknown how many of these mutations change channel function and structure on a molecular level. Since tetramerization is key to proper function and structure of the channel, it is likely that mutations modify the stability of KCNQ1 oligomers. Presently, the C-terminal domain of KCNQ1 has been noted as the driving force for oligomer formation. However, truncated versions of this protein lacking the C-terminal domain still tetramerize. Therefore, we explored the role of native cysteine residues in a truncated construct of human KCNQ1, amino acids 100-370, by blocking potential interactions of cysteines with a nitroxide based spin label. Mobility of the spin labels was investigated with continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. The oligomerization state was examined by gel electrophoresis. The data provide information on tetramerization of human KCNQ1 without the C-terminal domain. Specifically, how blocking the side chains of native cysteines residues reduces oligomerization. A better understanding of tetramer formation could provide improved understanding of the molecular etiology of long QT syndrome and other diseases related to KCNQ1

Root Hair Single Cell Type Specific Profiles of Gene Expression and Alternative Polyadenylation Under Cadmium Stress

Transcriptional networks are tightly controlled in plant development and stress responses. Alternative polyadenylation (APA) has been found to regulate gene expression under abiotic stress by increasing the heterogeneity at mRNA 3′-ends. Heavy metals like cadmium pollute water and soil due to mining and industry applications. Understanding how plants cope with heavy metal stress remains an interesting question. The Arabidopsis root hair was chosen as a single cell model to investigate the functional role of APA in cadmium stress response. Primary root growth inhibition and defective root hair morphotypes were observed. Poly(A) tag (PAT) libraries from single cell types, i.e., root hair cells, non-hair epidermal cells, and whole root tip under cadmium stress were prepared and sequenced. Interestingly, a root hair cell type-specific gene expression under short term cadmium exposure, but not related to the prolonged treatment, was detected. Differentially expressed poly(A) sites were identified, which largely contributed to altered gene expression, and enriched in pentose and glucuronate interconversion pathways as well as phenylpropanoid biosynthesis pathways. Numerous genes with poly(A) site switching were found, particularly for functions in cell wall modification, root epidermal differentiation, and root hair tip growth. Our findings suggest that APA plays a functional role as a potential stress modulator in root hair cells under cadmium treatment

Characterization of three members of the Arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family

Arabidopsis thaliana has nine genes that constitute a family of putative carotenoid cleavage dioxygenases (CCDs). While five members of the family are believed to be involved in synthesis of the phytohormone abscisic acid, the functions of the other four enzymes are less clear. Recently two of the enzymes, CCD7/MAX3 and CCD8/MAX4, have been implicated in synthesis of a novel apocarotenoid hormone that controls lateral shoot growth. Here, we report on the molecular and genetic interactions between CCD1, CCD7/MAX3 and CCD8/ MAX4. CCD1 distinguishes itself from other reported CCDs as being the only member not targeted to the plastid. Unlike ccd7/max3 and ccd8/max4, both characterized as having highly branched phenotypes, ccd1 loss-of-function mutants are indistinguishable from wild-type plants. Thus, even though CCD1 has similar enzymatic activity to CCD7/MAX3, it does not have a role in synthesis of the lateral shoot growth inhibitor. Rather, it may have a role in synthesis of apocarotenoid flavor and aroma volatiles, especially in maturing seeds where loss of function leads to significantly higher carotenoid levels