A. Bruce Cahoon

yellow-in-the-dark Mutants of Chlamydomonas Lack the CHLL Subunit of Light-Independent Protochlorophyllide Reductase

Light-independent protochlorophyllide reduction leading to chlorophyll formation in the dark requires both chloroplast and nuclear gene expression in Chlamydomonas reinhardtii. Mutations in any one of the plastid (chlL, chlN, and chlB) or nuclear (y-1 to y-10) genes required for this process result in the phenotype of the yellow-in-the-dark or y mutants. Analysis of the chlL, chlN, and chlB transcript levels in both light- and dark-grown wild-type and y mutant cells showed that the y mutations have no effect on the transcription of these plastid genes. Protein gel blot analysis showed that the CHLN and CHLB proteins are present in similar amounts in light- and dark-grown wild-type cells, whereas CHLL is present only in wild-type cells grown in the dark or at light intensities ≤15 μmol m–2 sec–1. Analysis of chlL transcript distribution on polysome profiles and rates of protein turnover in chloramphenicol-treated cells suggested that CHLL formation is most probably blocked at translation initiation or elongation. Furthermore, treatment of cells with metabolic inhibitors and uncouplers of photosynthetic electron transport showed that regulation of CHLL formation is linked to the physiologic status of the chloroplast. Similar to wild-type cells, y mutants contain nearly identical amounts of CHLN and CHLB when grown in either light or darkness. However, no CHLL is present in any of the y mutants except y-7, which contains an immunoreactive CHLL smaller than the expected size. Our findings indicate that CHLL translation is negatively photoregulated by the energy state or redox potential within the chloroplast in wild-type cells and that nuclear y genes are required for synthesis or accumulation of the CHLL protein.

Analysis of developing maize plastids reveals two mRNA stability classes correlating with RNA polymerase type.

The plastid genome is transcribed by two distinct RNA polymerases, the PEP encoded by the plastid genome and the NEP encoded in the nucleus. Initial models of plastid transcription held that the NEP is responsible for the transcription of housekeeping genes needed early in development, and that the PEP transcribes genes required for photosynthesis. Recently, this model was challenged by the discovery that all plastid genes are transcribed by NEP in PEP‐deficient tobacco plastids, suggesting that mRNA turnover may have a strong role in previously observed transcription patterns. In this study, we provide evidence that the NEP enzyme level decreases as plastids mature. In contrast, production of mRNAs by NEP increases as plastids mature, yet their accumulations remain constant. These results suggest that as plastids mature NEP may become more active, and that mRNA turnover varies between transcripts synthesized by NEP and PEP.

Plastid transcription: a menage à trois?

Abstract Two extracellular transcriptional compartments, mitochondria and plastids, are active in plants, depending mainly on nucleus-encoded proteins for transcription. Several nuclear genes encoding RNA polymerases have been cloned from a variety of organisms and their subcellular localization studied in detail. Surprisingly, the transit peptide of a recently published phage-type RNA polymerase from Arabidopsis (RpoT2) possesses dual targeting properties to both mitochondria and chloroplasts, inferring involvement in the transcription of two different genomes. Possible implications of such a complex system are discussed in this article.

Nuclear, chloroplast, and mitochondrial transcript abundance along a maize leaf developmental gradient

In maize, the chloroplast chromosome encodes 104 genes whose roles are primarily in photosynthesis and gene expression. The 2,000–3,000 nuclear gene products that localize to plastids are required both to encode and regulate plastid gene expression as well as to underpin each aspect of plastid physiology and development. We used a new “three-genome” maize biogenesis cDNA microarray to track abundance changes in nuclear, chloroplast and mitochondrial transcripts in stage 2 semi-emerged leaf blades of one month-old maize plants. We report the detection and quantification of 433 nuclear, 62 chloroplast, and 27 mitochondrial transcripts, with the majority of the nuclear transcripts predicted or known to encode plastid proteins. The data were analyzed as ratios of expression of individual transcripts in the green tip (mature chloroplasts) versus the yellow base of the leaf (etioplasts). According to the microarray data at least 51 plastid genes and 121 nuclear genes are expressed at least two-fold higher in the tip of the leaf. Almost all (25) mitochondrial and 177 nuclear transcripts were expressed at least 2–fold higher in the leaf base. Independent quantification of a subset of each transcript population by RNA gel blot analysis and/or quantitative real time RT-PCR concurred with the transcript ratios determined by the array. Ontological distribution of the transcripts suggests that photosynthesis-related RNAs were most highly abundant in the leaf tip and that energy use genes were most highly expressed in the base. Transcripts whose products are used in plastid translation constituted the largest single ontological group with relatively equal numbers of genes in the three expression categories, defined as higher in tip, higher in base, or equally expressed in tip and base.

Deep sequencing of the tobacco mitochondrial transcriptome reveals expressed ORFs and numerous editing sites outside coding regions

BackgroundThe purpose of this study was to sequence and assemble the tobacco mitochondrial transcriptome and obtain a genomic-level view of steady-state RNA abundance. Plant mitochondrial genomes have a small number of protein coding genes with large and variably sized intergenic spaces. In the tobacco mitogenome these intergenic spaces contain numerous open reading frames (ORFs) with no clear function.ResultsThe assembled transcriptome revealed distinct monocistronic and polycistronic transcripts along with large intergenic spaces with little to no detectable RNA. Eighteen of the 117 ORFs were found to have steady-state RNA amounts above background in both deep-sequencing and qRT-PCR experiments and ten of those were found to be polysome associated. In addition, the assembled transcriptome enabled a full mitogenome screen of RNA C→U editing sites. Six hundred and thirty five potential edits were found with 557 occurring within protein-coding genes, five in tRNA genes, and 73 in non-coding regions. These sites were found in every protein-coding transcript in the tobacco mitogenome.ConclusionThese results suggest that a small number of the ORFs within the tobacco mitogenome may produce functional proteins and that RNA editing occurs in coding and non-coding regions of mitochondrial transcripts.

The complete chloroplast genome of tall fescue (Lolium arundinaceum; Poaceae) and comparison of whole plastomes from the family Poaceae.

In this paper, we describe the complete chloroplast genome of Lolium arundinaceum. This sequence is the culmination of a long-term project completed by >400 undergraduates who took general genetics at Middle Tennessee State University from 2004-2007. It was undertaken in an attempt to introduce these students to an open-ended experiential/exploratory lesson to produce and analyze novel data. The data they produced should provide the necessary information for both phylogenetic comparisons and plastome engineering of tall fescue. The fescue plastome (GenBank FJ466687) is 136048 bp with a typical quadripartite structure and a gene order similar to other grasses; 56% of the plastome is coding region comprised of 75 protein-coding genes, 29 tRNAs, four rRNAs, and one hypothetical coding region (ycf). Comparisons of Poaceae plastomes reveal size differences between the PACC (subfamilies Panicoideae, Arundinoideae, Centothecoideae, and Chloridoideae) and BOP (subfamilies Bambusoideae, Oryzoideae, and Pooideae) clades. Alignment analysis suggests that several potentially conserved large deletions in previously identified intergenic length polymorphic regions are responsible for the majority of the size discrepancy. Phylogenetic analysis using whole plastome data suggests that fescue closely aligns with Lolium perenne. Some unique features as well as phylogenetic branch length calculations, however, suggest that a number of changes have occurred since these species diverged.

Developmental and cell type characterization of bundle sheath and mesophyll chloroplast transcript abundance in maize

The C4 grass Zea mays separates light and light-independent photosynthetic processes into two leaf cell types: bundle sheath (BS) and mesophyll (M). When mature, BS and M cells have anatomically and biochemically distinct chloroplasts that must cooperate to complete the process of photosynthesis. This report compares changes in transcript abundance between young and mature maize BS and M chloroplasts from specific segments of the leaf developmental gradient. Representative transcripts encoding components of Photosystem I, Photosystem II, Cytochrome b6f, thylakoidal NADH dehydrogenase; and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase as well as nine nuclear-coded transcripts encoding chloroplast proteins were measured using quantitative RT-PCR. In addition, 887 nuclear genes encoding plastid-localized proteins, as well as 64 chloroplast and 34 mitochondrial genes were assayed utilizing a cDNA microarray. In 9 out of the 18 chloroplast-encoded genes and 84 genes from the 985 element microarray revealed greater than twofold transcript abundance differences between developmental stages and/or cell types. Patterns for transcripts associated with operons and gene clusters suggest differing regulatory mechanisms for particular polycistronic stretches. In summary, this report provides evidence that cell type-specific transcript abundance varies more in the young developing chloroplast, and differences plateau or subside as chloroplasts mature.

Maize BMS cultured cell lines survive with massive plastid gene loss

Abstract As part of developing an ex planta model system for the study of maize plastid and mitochondrial gene expression, a series of established Black Mexican Sweet (BMS) suspension cell lines was characterized. Although the initial assumption was that their organelle biochemistry would be similar enough to normal in planta cells to facilitate future work, each of the three lines was found to have plastid DNA (ptDNA) differing from control maize plants, in one case lacking as much as 70% of the genome. The other two BMS lines possessed either near-wild-type ptDNA or displayed an intermediate state of gene loss, suggesting that these clonal lines are rapidly evolving. Gene expression profiles of BMS cells varied dramatically from those in maize leaf chloroplasts, but resembled those of albino plants lacking plastid ribosomes. In spite of lacking most plastid gene expression and apparently mature rRNAs, BMS cells appear to import proteins from the cytoplasm in a normal manner. The regions retained in BMS ptDNAs point to a set of tRNA genes universally preserved among even highly reduced plastid genomes, whereas the other preserved regions may illuminate which plastid genes are truly indispensable for plant cell survival.

Plasmolysis facilitates the accumulation of protein and DNA into extra-plasmalemma spaces of intact plant cells

Abstract Epidermal cells of Allium cepa hort. yellow and suspension culture cells of Petunia hybrida and Oryza sativa were plasmolysed by various concentrations of NaCl, CaCl2, sucrose, and manitol. The relative void areas (defined as the area between the plasmalemma and cell wall) in plasmolysed cells were measured and the numbers of living cells remaining after plasmolysis were counted. The loss of viability after deplasmolysis was related closely to the types of solute used, and was associated only slightly with the sizes of relative void area. Fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA), autofluorescence c-phycocyanin, and ethidium bromide-stained calf thymus DNA (ct-DNA) were used as fluorescence markers to monitor the movements of macromolecules into and through the cell walls during the plasmolysis. A significant larger number of cells showed the fluorescence in their void areas when the cells were plasmolysed in the presence of these DNA and protein markers. When the cells were plasmolysed before the addition of these macromolecules, no fluorescence was observed in the void area. We conclude that plasmolysis facilitated the passage of macromolecules through the cell wall and accumulation in the void areas. It is suggested that plasmolysis of intact plant cells can be used as a tool to create ‘temporary protoplast’ within the cell wall to allow macromolecular uptake or gene transfer.

A plastome primer set for comprehensive quantitative real time RT-PCR analysis of Zea mays: a starter primer set for other Poaceae species.

BackgroundQuantitative Real Time RT-PCR (q2(RT)PCR) is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids. Primer design and optimization is an essential yet time consuming aspect of using q2(RT)PCR. In this paper we describe the design and empirical optimization of primers to amplify and quantify plastid RNAs from Zea mays that are robust enough to use with other closely related species.ResultsPrimers were designed and successfully optimized for 57 of the 104 reported genes in the maize plastome plus two nuclear genes. All 59 primer pairs produced single amplicons after end-point reverse transcriptase polymerase chain reactions (RT-PCR) as visualized on agarose gels and subsequently verified by q2(RT)PCR. Primer pairs were divided into several categories based on the optimization requirements or the uniqueness of the target gene. An in silico test suggested the majority of the primer sets should work with other members of the Poaceae family. An in vitro test of the primer set on two unsequenced species (Panicum virgatum and Miscanthus sinensis) supported this assumption by successfully producing single amplicons for each primer pair.ConclusionDue to the highly conserved chloroplast genome in plant families it is possible to utilize primer pairs designed against one genomic sequence to detect the presence and abundance of plastid genes or transcripts from genomes that have yet to be sequenced. Analysis of steady state transcription of vital system genes is a necessary requirement to comprehensively elucidate gene expression in any organism. The primer pairs reported in this paper were designed for q2(RT)PCR of maize chloroplast genes but should be useful for other members of the Poaceae family. Both in silico and in vitro data are presented to support this assumption.