David C. Higgs

Transcription and translation in chloroplasts

Chloroplast gene expression is primarily regulated at the post-transcriptional level, where a variety of complex mechanism have evolved to govern the interaction between the chloroplast and nuclear genomes. These rely on different nuclear-encoded proteins that act on chloroplast mRNAs, including RNA polymerase subunits, RNA stability factors and translational activators. Such control mechanisms must be viewed in the light of the evolutionary heritage of the chloroplast, as an independent unicellular organism. Although some features of prokaryotic gene expression still occur in chloroplasts, their multiple genetic mechanisms for synthesizing RNA and protein are new, and appear to have arisen to provide flexibility of responding to the developmental or environmental state of the plant.

The Chloroplast Gene ycf9 Encodes a Photosystem II (PSII) Core Subunit, PsbZ, That Participates in PSII Supramolecular Architecture

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.

Small cis-Acting Sequences That Specify Secondary Structures in a Chloroplast mRNA Are Essential for RNA Stability and Translation

ABSTRACT Nucleus-encoded proteins interact with cis-acting elements in chloroplast transcripts to promote RNA stability and translation. We have analyzed the structure and function of three such elements within the Chlamydomonas petD 5′ untranslated region; petD encodes subunit IV of the cytochromeb 6/f complex. These elements were delineated by linker-scanning mutagenesis, and RNA secondary structures were investigated by mapping nuclease-sensitive sites in vitro and by in vivo dimethyl sulfate RNA modification. Element I spans a maximum of 8 nucleotides (nt) at the 5′ end of the mRNA; it is essential for RNA stability and plays a role in translation. This element appears to form a small stem-loop that may interact with a previously described nucleus-encoded factor to block 5′→3′ exoribonucleolytic degradation. Elements II and III, located in the center and near the 3′ end of the 5′ untranslated region, respectively, are essential for translation, but mutations in these elements do not affect mRNA stability. Element II is a maximum of 16 nt in length, does not form an obvious secondary structure, and appears to bind proteins that protect it from dimethyl sulfate modification. Element III spans a maximum of 14 nt and appears to form a stem-loop in vivo, based on dimethyl sulfate modification and the sequences of intragenic suppressors of element III mutations. Furthermore, mutations in element II result in changes in the RNA structure near element III, consistent with a long-range interaction that may promote translation.

Microarray Analysis Confirms the Specificity of a Chlamydomonas reinhardtii Chloroplast RNA Stability Mutant

The expression of chloroplast and mitochondrial genes depends on nucleus-encoded proteins, some of which control processing, stability, and/or translation of organellar RNAs. To test the specificity of one such RNA stability factor, we used two known Chlamydomonas reinhardtii nonphotosynthetic mutants carrying mutations in the Mcd1 nuclear gene (mcd1-1 and mcd1-2). We previously reported that these mutants fail to accumulate the chloroplast petD mRNA and its product, subunit IV of the cytochrome b6/f complex, which is essential for photosynthesis. Such mutants are generally presumed to be gene specific but are not tested rigorously. Here, we have used microarray analysis to assess changes in chloroplast, mitochondrial, and nuclear RNAs, and since few other RNAs were significantly altered in these mutants, conclude that Mcd1 is indeed specifically required for petD mRNA accumulation. In addition, a new unlinked nuclear mutation was discovered in mcd1-2, which greatly reduced chloroplast atpA mRNA accumulation. Genetic analyses showed failure to complement mda1-ncc1, where atpA-containing transcripts are similarly affected (D. Drapier, J. Girard-Bascou, D.B. Stern, F.-A. Wollman [2002] Plant J 31: 687–697), and we have named this putative new allele mda1-2. We conclude that DNA microarrays are efficient and useful for characterizing the specificity of organellar RNA accumulation mutants.

A nucleus-encoded suppressor defines a new factor which can promote petD mRNA stability in the chloroplast of Chlamydomonas reinhardtii

Abstract Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chloroplast petD mRNA, which encodes subunit IV of the cytochrome b6/f complex. The MCD1 gene product is thought to interact with the mRNA 5′ end to protect it from degradation by a 5′ → 3′ exoribonuclease and may also have a role in translation initiation. Here we report the isolation and characterization of a semidominant, allele-specific, nucleus-encoded suppressor of the mcd1-2 mutation. The suppressor mutation, which defines a new locus MCD2, allows accumulation of 10% of the wild-type level of petD mRNA and as much as 50% of the wild-type subunit IV level. Taken together, these results suggest the suppressor mutation restores photosynthetic growth by stabilizing petD mRNA. In addition, it may promote increased translational efficiency, an inference supported by direct measurements of the subunit IV synthesis rate. Thus, both MCD1 and MCD2 may participate in both chloroplast RNA stability and translation initiation.

Regulatory sequences of orthologous petD chloroplast mRNAs are highly specific among Chlamydomonas species.

The 5′ untranslated regions (UTR) of chloroplast mRNAs often contain regulatory sequences that control RNA stability and/or translation. The petD chloroplast mRNA in Chlamydomonas reinhardtii has three such essential regulatory elements in its 362-nt long 5′ UTR. To further analyze these elements, we compared 5′ UTR sequences from four Chlamydomonas species (C. reinhardtii, C. incerta, C. moewusii and C. eugametos) and five independent strains of C. reinhardtii. Overall, these petD 5′ UTRs have relatively low sequence conservation across these species. In contrast, sequences of the three regulatory elements and their relative positions appear partially conserved. Functionality of the 5′ UTRs was tested in C. reinhardtii chloroplasts using β-glucuronidase reporter genes, and the nearly identical C. incerta petD functioned for mRNA stability and translation in C. reinhardtii chloroplasts while the more divergent C. eugametos petD did not. This identified what may be key features in these elements. We conclude that these petD regulatory elements, and possibly the corresponding trans-acting factors, function via mechanisms highly specific and surprisingly sensitive to minor sequence changes. This provides a new and broader perspective of these important regulatory sequences that affect photosynthesis in these algae.

β-glucuronidase gene expression and mRNA stability in oat protoplasts.

SummaryProtoplasts derived from oat (Avena sativa L.) suspension culture cells (7 days after subculturing) were electroporated with plasmid DNA containing the Escherichia coli uidA gene encoding the ß-glucuronidase reporter enzyme. Consistently high enzyme activity was observed with electroporation conditions of 500 μF and 1125 volts/cm. Enzyme activity and mRNA accumulation time courses were determined. The maximum enzyme activity was detected at 24 hours after electroporation, while the maximum mRNA level was detected at 12 hours after electroporation. ß-glucuronidase mRNA was in vitro synthesized with and without a 5′ methylated cap and then electroporated into protoplasts. Only capped mRNA produced significant enzyme activity. By electroporating radiolabeled, in vitro synthesized mRNA, the ß-glucuronidase mRNA half-life was estimated to be ∼35 minutes in oat protoplasts.

The Chloroplast Genome

Publisher Summary The chloroplast genomes from Chlamydomonas species have provided important insights into the function and evolution of this essential organelle. This chapter focuses on Chlamydomonas cpDNA structure, sequence, gene content, replication, and evolution, and it is not intended to be comprehensive for all Chlamydomonas species. The chloroplast genomes from Chlamydomonas species have provided important insights into the function and evolution of this essential organelle. It shows the pronounced number of predicted rearrangements for C. reinhardtii and Chlorella vulgaris. The complete Chlamydomonas chloroplast genome sequence is a resource that complements the many available genetic techniques. It discusses the weakness of using only complete genome sequence from a few taxa versus the more traditional method of using more taxa but with fewer select gene/protein sequences. In this chapter, the complete sequence of C. reinhardtii cpDNA is the reference genome. The cumulative data have and will continue to contribute to our understanding of chloroplast evolution and gene function through comparative genomics and gene manipulation. Examples of genes where analysis is incomplete are the unusual split genes rpoB, rpoC1, rpoC2 , rps2, and clpP, whose structures in some cases are apparently unique to Chlamydomonas. Other areas ripe for investigation are the chloroplast transcriptional, translational, and DNA replication machinery.

Implementation and Assessment of a Molecular Biology and Bioinformatics Undergraduate Degree Program

The Department of Biological Sciences at the University of Wisconsin‐Parkside has developed and implemented an innovative, multidisciplinary undergraduate curriculum in Molecular Biology and Bioinformatics (MBB). The objective of the MBB program is to give students a hands‐on facility with molecular biology theories and laboratory techniques, an understanding of mathematical and physical concepts, an ability to apply these concepts to MBB, and a proficiency with the computational tools and skills related to bioinformatics. We hypothesized that a greater exposure to bioinformatics methods, more rigorous requirements in math and computer science, and a constant demand for integrating information in hands‐on laboratory courses would help students develop better analytical skills. Indeed, the assessment data support these predictions. Interestingly, 80% of MBB majors apply and are accepted into graduate schools.

ABUNDANCE AND HALF-LIFE OF THE DISTINCT OAT PHYTOCHROME A3 AND A4 MRNAS

Gene-preferential oligonucleotide probes were used to determined the relative abundance and half-lives of distinct oat phytochrome A (PHYA) mRNAs. Oat PHYA mRNAs are highly conserved in the 5′-untranslated region and the coding region, but the 3′-untranslated region has an overall lower sequence conservation and was the source of gene-preferential probes. PHYA3 mRNA was estimated to be ca. 61% of the oat PHYA mRNA pool present in poly(A)+ RNA from dark-grown seedlings. The half-lives for PHYA3 and PHYA4 mRNAs were both estimated to be ca. 30 min, and a similar short half-life was estimated for the average PHYA mRNA. Sequence comparisons of PHYA mRNAs from four grass species identified conserved sequences within the 5′- and 3′-untranslated regions that might be important for PHYA mRNA degradation.