Molecular characterization of CER2, an Arabidopsis gene involved in cuticular wax accumulation

Cuticular waxes are complex mixtures of very long chain fatty acids (VLCFAs) and their derivatives. The CER2 locus of Arabidopsis is involved in cuticular wax accumulation on stems, siliques, and hypocotyls. The CER2 gene was cloned via chromosome walking. This cloned sequence is able to complement the cer2 mutant phenotype. It is a single-copy sequence in the Arabidopsis genome and encodes a novel protein with a predicted mass of 47 kD. Anti-CER2 polyclonal antibodies detected a 47 kD polypeptide. Cell fractionation and immunoblot analyses demonstrated that the CER2 protein is not membrane-bound and localized in nuclei. These results suggest that CER2 might have a regulatory role in cuticular wax accumulation. The expression patterns of the CER2 gene were studied by in situ RNA hybridization and the analysis of Arabidopsis transgenic plants harboring an in-frame fusion of -1009/+234 of the CER2 gene to [beta]-glucuronidase (CER2-GUS). These analysis demonstrate that CER2 gene expression is developmentally regulated and organ- and tissue-specific. Consistence with the visible phenotype associated with cer2 mutants, the CER2 gene is highly expressed only on the epidermal cells of stems, siliques, and hypocotyls. In addition, CER2 expression was observed in guard cells, trichomes, petioles, sepals, petals, ovaries, pedicels, the tapetum layer of anthers, and pollen grains. CER2-GUS expression was not detected in roots or in the pavement cells of leaves. The observation of CER2 expression in anthers and pollen grains is in agreement with the fact that the CER2 gene is involved in pollen fertility. Light, drought, high osmotic pressure, heat or cold shock, and wounding were not be found to cause visible changes in CER2-GUS expression patterns. However, exogenous application of the cytokinin, BAP, induced ectopic expression of CER2-GUS in leaves, suggesting that CER2 gene expression might be mediated by endogenous cytokinins

NAD tagSeq reveals that NAD+-capped RNAs are mostly produced from a large number of protein-coding genes in Arabidopsis.

The 5' end of a eukaryotic mRNA transcript generally has a 7-methylguanosine (m7G) cap that protects mRNA from degradation and mediates almost all other aspects of gene expression. Some RNAs in Escherichia coli, yeast, and mammals were recently found to contain an NAD+ cap. Here, we report the development of the method NAD tagSeq for transcriptome-wide identification and quantification of NAD+-capped RNAs (NAD-RNAs). The method uses an enzymatic reaction and then a click chemistry reaction to label NAD-RNAs with a synthetic RNA tag. The tagged RNA molecules can be enriched and directly sequenced using the Oxford Nanopore sequencing technology. NAD tagSeq can allow more accurate identification and quantification of NAD-RNAs, as well as reveal the sequences of whole NAD-RNA transcripts using single-molecule RNA sequencing. Using NAD tagSeq, we found that NAD-RNAs in Arabidopsis were produced by at least several thousand genes, most of which are protein-coding genes, with the majority of these transcripts coming from <200 genes. For some Arabidopsis genes, over 5% of their transcripts were NAD capped. Gene ontology terms overrepresented in the 2,000 genes that produced the highest numbers of NAD-RNAs are related to photosynthesis, protein synthesis, and responses to cytokinin and stresses. The NAD-RNAs in Arabidopsis generally have the same overall sequence structures as the canonical m7G-capped mRNAs, although most of them appear to have a shorter 5' untranslated region (5' UTR). The identification and quantification of NAD-RNAs and revelation of their sequence features can provide essential steps toward understanding the functions of NAD-RNAs

Molecular characterization of CER2, an Arabidopsis gene involved in cuticular wax accumulation

Cuticular waxes are complex mixtures of very long chain fatty acids (VLCFAs) and their derivatives. The CER2 locus of Arabidopsis is involved in cuticular wax accumulation on stems, siliques, and hypocotyls. The CER2 gene was cloned via chromosome walking. This cloned sequence is able to complement the cer2 mutant phenotype. It is a single-copy sequence in the Arabidopsis genome and encodes a novel protein with a predicted mass of 47 kD. Anti-CER2 polyclonal antibodies detected a 47 kD polypeptide. Cell fractionation and immunoblot analyses demonstrated that the CER2 protein is not membrane-bound and localized in nuclei. These results suggest that CER2 might have a regulatory role in cuticular wax accumulation. The expression patterns of the CER2 gene were studied by in situ RNA hybridization and the analysis of Arabidopsis transgenic plants harboring an in-frame fusion of -1009/+234 of the CER2 gene to [beta]-glucuronidase (CER2-GUS). These analysis demonstrate that CER2 gene expression is developmentally regulated and organ- and tissue-specific. Consistence with the visible phenotype associated with cer2 mutants, the CER2 gene is highly expressed only on the epidermal cells of stems, siliques, and hypocotyls. In addition, CER2 expression was observed in guard cells, trichomes, petioles, sepals, petals, ovaries, pedicels, the tapetum layer of anthers, and pollen grains. CER2-GUS expression was not detected in roots or in the pavement cells of leaves. The observation of CER2 expression in anthers and pollen grains is in agreement with the fact that the CER2 gene is involved in pollen fertility. Light, drought, high osmotic pressure, heat or cold shock, and wounding were not be found to cause visible changes in CER2-GUS expression patterns. However, exogenous application of the cytokinin, BAP, induced ectopic expression of CER2-GUS in leaves, suggesting that CER2 gene expression might be mediated by endogenous cytokinins.</p

Ssk1p-Independent Activation of Ssk2p Plays an Important Role in the Osmotic Stress Response in Saccharomyces cerevisiae: Alternative Activation of Ssk2p in Osmotic Stress

In Saccharomyces cerevisiae, external high osmolarity activates the HOG MAPK pathway, which controls various aspects of osmoregulation. MAPKKK Ssk2 is activated by Ssk1 in the SLN1 branch of the osmoregulatory HOG MAPK pathway under hyperosmotic stress. We observed that Ssk2 can be activated independent of Ssk1 upon osmotic shock by an unidentified mechanism. The domain for the Ssk1p-independent activation was identified to be located between the amino acids 177,240. This region might be involved in the binding of an unknown regulator to Ssk2 which in turn activates Ssk2p without Ssk1p under hyperosmotic stress. The osmotic stress response through the Ssk1p-independent Ssk2p activation is strong, although its duration is short compared with the Ssk1p-dependent activation. The alternative Ssk2p activation is also important for the salt resistance

Ssk2p can be activated independent of Ssk1p under severe osmotic stress.

<p>A. Hog1p was phosphorylated in the <i>ste11Δssk1Δssk22Δ</i> mutant under severe osmotic stress (higher than 0.5 M sorbitol). B. Hog1p could not be phosphorylated in the <i>ste11Δssk1Δssk2Δ</i> mutant under 0.4 M or 1.0 M sorbitol. C. Actin disassembly did not activate the HOG pathway through Ssk2p. Within Lat B treatment, wild type strain and <i>ste11Δssk1Δ</i> mutant did not display activation of Hog1p. D.The effect of Lat B on actin structures in yeast cells. Rd-phalloidin was used to observe the effects of Lat B addition to yeast cells. Both the wild type cells and <i>ste11Δssk1Δ</i> mutant cells were incubated in the absence of Lat B and for 20 min in the presence of 200 mM Lat B. E. The osmosensitivity phenotype of budding yeast HOG pathway mutants. Serial dilutions (from left to right in each panel) of indicated strains were spotted onto YPD and salt plates and growth was scored after 3 days.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p