Research overview

Plastids are semi-autonomous organelles with a relatively small (120-180 kb), highly polyploid genome present in 1,000 to 10,000 copies per cell. The best-known plastids, chloroplasts, convert sunlight into chemical energy. Plastid engineering, in contrast to nuclear engineering, offers higher protein yields, the opportunity to express several genes controlling complex traits, and natural containment that prevents transgene flow via pollen. During the past twenty years we developed protocols for transformation of the tobacco (Nicotiana tabacum) plastid genome, efficient post-transformation excision of the marker genes, and high-level expression of recombinant proteins. Patents based on research from our laboratory cover all aspects of the genetic manipulation of plastid genomes.

Currently, we pursue research in the following areas:

PPR proteins to regulate plastid transgene expression

Most nuclear-encoded pentatricopeptide repeat (PPR) proteins are targeted to plastids and mitochondria, and bind specific RNA sequences via a modular recognition mechanism. Study of plant mutants suggests that the P-type PPR proteins are involved in regulating mRNA translation and stability. Our objective is to explore the feasibility of regulating plastid transgene expression by incorporating PPR binding sites in plastid transgenes and regulating the expression of cognate PPR proteins from nuclear transgenes.

Plastid engineering in Arabidopsis

Arabidopsis thaliana, an important model plant species, is recalcitrant to plastid transformation. To enable early identification of transplastomic events, we developed a novel marker system that is selectively expressed in chloroplasts. Plastid transformation in Arabidopsis in the appropriate genetic background is now as efficient as in tobacco. We now conduct systematic experimentation to tie shoot regeneration into a streamlined seed production pipeline as part of the refinement of the Arabidopsis plastid transformation protocol. Plastid genome engineering will open up the unique genomic resources of Arabidopsis for studies of plastid-nucleus interactions and for improving crop productivity by engineering the photosynthetic machinery.

Inter-cellular movements of DNA-containing organelles in plants

To detect organelle movement between cells, we graft two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. We initiate tissue culture from sliced graft junctions and select for clonal lines in which the nuclear gentamycin resistance marker of one line is combined with the plastid-encoded spectinomycin resistance marker of the second. We obtained evidence for cell-to-cell movement of the entire 161-kb plastid genome in the absence of the movement of chromosomes or mitochondrial DNA. In some of the clones, mitochondrial DNA movement was also detected by restoration of pollen fertility in the cytoplasmic male sterile (CMS) graft partner. Homologous recombination yielded fertile and sterile mitochondrial genomes due to recombination at alternative sites, linking CMS to a unique open reading frame in CMS mitochondria. We are now interested in stripping plastid genomes from species-specific features so that the same engineered plastid genome can be utilized in multiple hosts.

Recent Publications

Gurdon, C, Svab Z, Feng Y, Kumar D, Maliga P.  2016.  Cell-to-cell movement of mitochondria in plants. Proc Natl Acad Sci U S A. 113:3395-400. AbstractWebsite
We report cell-to-cell movement of mitochondria through a graft junction. Mitochondrial movement was discovered in an experiment designed to select for chloroplast transfer fromNicotiana sylvestrisintoNicotiana tabacumcells. The alloplasmicN. tabacumline we used carriesNicotiana undulatacytoplasmic genomes, and its flowers are male sterile due to the foreign mitochondrial genome. Thus, rare mitochondrial DNA transfer fromN. sylvestristoN. tabacumcould be recognized by restoration of fertile flower anatomy. Analyses of the mitochondrial genomes revealed extensive recombination, tentatively linking male sterility toorf293, a mitochondrial gene causing homeotic conversion of anthers into petals. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of the mitochondrial genome by DNA transmitted from a sexually incompatible species. Conversion of anthers into petals is a visual marker that can be useful for mitochondrial transformation.
Wang, W, Zhang W, Wu Y, Maliga P, Messing J.  2015.  RNA Editing in Chloroplasts of Spirodela polyrhiza, an Aquatic Monocotelydonous Species. PLoS One. 10:e0140285. AbstractWebsite
RNA editing is the post-transcriptional conversion from C to U before translation, providing a unique feature in the regulation of gene expression. Here, we used a robust and efficient method based on RNA-seq from non-ribosomal total RNA to simultaneously measure chloroplast-gene expression and RNA editing efficiency in the Greater Duckweed, Spirodela polyrhiza, a species that provides a new reference for the phylogenetic studies of monocotyledonous plants. We identified 66 editing sites at the genome-wide level, with an average editing efficiency of 76%. We found that the expression levels of chloroplast genes were relatively constant, but 11 RNA editing sites show significant changes in editing efficiency, when fronds turn into turions. Thus, RNA editing efficiency contributes more to the yield of translatable transcripts than steady state mRNA levels. Comparison of RNA editing sites in coconut, Spirodela, maize, and rice suggests that RNA editing originated from a common ancestor.
Lutz, KA, Martin C, Khairzada S, Maliga P.  2015.  Steroid-inducible BABY BOOM system for development of fertile Arabidopsis thaliana plants after prolonged tissue culture. Plant Cell Rep. 34:1849-56. AbstractWebsite
KEY MESSAGE: We describe a steroid-inducible BABY BOOM system that improves plant regeneration in Arabidopsis leaf cultures and yields fertile plants. Regeneration of Arabidopsis thaliana plants for extended periods of time in tissue culture may result in sterile plants. We report here a novel approach for A. thaliana regeneration using a regulated system to induce embryogenic cultures from leaf tissue. The system is based on BABY BOOM (BBM), a transcription factor that turns on genes involved in embryogenesis. We transformed the nucleus of A. thaliana plants with BBM:GR, a gene in which the BBM coding region is fused with the glucocorticoid receptor (GR) steroid-binding domain. In the absence of the synthetic steroid dexamethasone (DEX), the BBM:GR fusion protein is localized in the cytoplasm. Only when DEX is included in the culture medium does the BBM transcription factor enter the nucleus and turn on genes involved in embryogenesis. BBM:GR plant lines show prolific shoot regeneration from leaf pieces on media containing DEX. Removal of DEX from the culture media allowed for flowering and seed formation. Therefore, use of BBM:GR leaf tissue for regeneration of plants for extended periods of time in tissue culture will facilitate the recovery of fertile plants.
Bosacchi, M, Gurdon C, Maliga P.  2015.  Plastid Genotyping Reveals Uniformity of cms-T Maize Cytoplasms.. Plant Physiology. Abstract
Cytoplasmic male sterile (CMS) lines in maize have been classified by their response to specific restorer genes into three categories: cms-C, cms-S, and cms-T. A mitochondrial genome representing each of the CMS cytotypes has been sequenced and male sterility in the cms-S and cms-T cytotypes is linked to chimeric mitochondrial genes. To identify markers for plastid genotyping, we sequenced the plastid genomes (ptDNA) of three fertile maize lines (B37, B73, A188) and the B37 cms-C, cms-S, and cms-T cytoplasmic substitution lines. We found that the plastid genomes of B37 and B73 lines are identical. Furthermore, the fertile and CMS plastid genomes are conserved, differing only by 0-3 single nucleotide polymorphisms (SNPs) in coding regions and 8-22 SNPs and 10-21 short insertions/deletions in noncoding regions. To gain insight into the origin and transmission of the cms-T trait, we identified three SNPs unique to the cms-T plastids, and tested the three diagnostic SNPs in 27 cms-T lines, representing the HA, I, Q, RS and T male sterile cytoplasms. We report that each of the tested 27 cms-T group accessions have the same three diagnostic plastid SNPs indicating a single origin and maternal co-transmission of the cms-T mitochondria and plastids to the seed progeny. Our data exclude exceptional pollen transmission of organelles or multiple horizontal gene transfer events as the source of the urf13-T gene in the cms-T cytoplasms. Plastid genotyping enables a reassessment of evolutionary relationships of cytoplasms in cultivated maize.