The underlying research themes in the Stern laboratory are chloroplast biology, bioenergy and nuclear-cytoplasmic interactions. Within this framework, we study how chloroplast genes and metabolic activities are regulated by the products of nuclear genes, usually acting at the transcriptional or post-transcriptional level. Areas of emphasis include the roles of ribonucleases and RNA-binding proteins and assembly of the carbon-fixing enzyme Rubisco. We are also using molecular and genetic techniques to adjust chloroplast metabolism for the production of useful hydrocarbons.
Germain, A., Kim, S.H., Gutierrez, R. and Stern, D.B. 2012. Ribonuclease II preserves chloroplast RNA homeostasis by increasing mRNA decay rates, and cooperates with polynucleotide phosphorylase in 3' end maturation.. The Plant journal : for cell and molecular biology 0: 960–971
Hotto , A.M., Germain, A. and Stern, D.B. 2012. Plastid non-coding RNAs: emerging candidates for gene regulation. Trends Plant Sci 17: 737-744
Wostrikoff, K., Clark, A., Sato, S., Clemente, T. and Stern, D. 2012. Ectopic expression of Rubisco subunits in maize mesophyll cells does not overcome barriers to cell type-specific accumulation. Plant Physiol 160: 419-432
Sharwood, R.E., Hotto, A.M., Bollenbach, T.J. and Stern, D.B. 2011. Overaccumulation of the chloroplast antisense RNA AS5 is correlated with decreased abundance of 5S rRNA in vivo and inefficient 5S rRNA maturation in vitro. RNA 17: 230-243
Sharwood, R.E., Halpert, M., Luro, S., Schuster , G. and Stern, D.B. 2011. Chloroplast RNase J compensates for inefficient transcription termination by removal of antisense RNA. RNA 17: 2165-2176
Hotto, A.M., Schmitz, R.J., Fei, Z., Ecker, J.E. and Stern, D.B. 2011. Unexpected diversity of chloroplast noncoding RNAs as revealed by deep sequencing of the Arabidopsis transcriptome G3. 1: 559-570
Germain, A., Herlich, S., Larom, S., Kim, S.H., Schuster, G. and Stern, D.B. 2011. Mutational analysis of Arabidopsis chloroplast polynucleotide phosphorylase reveals roles for both RNase PH core domains in polyadenylation, RNA 3'-end maturation and intron degradation. Plant J 67: 381-394
Nishimura, Y. and Stern, D.B. 2010. Differential replication of two chloroplast genome forms in heteroplasmic Chlamydomonas reinhardtii gametes contributes to alternative inheritance patterns. Genetics 185: 1167-1181
Johnson, X., Wostrikoff, K., Finazzi, G., Kuras, R., Schwarz, C., Bujaldon, S., Nickelsen, J., Stern, D.B., Wollman, F.A. and Vallon, O. 2010. MRL1, a Conserved Pentatricopeptide Repeat Protein, Is Required for Stabilization of rbcL mRNA in Chlamydomonas and Arabidopsis. Plant Cell 22: 234-248
Hotto, A.M., Huston, Z.E. and Stern, D.B. 2010. Overexpression of a natural chloroplast-encoded antisense RNA in tobacco destabilizes 5S rRNA and retards plant growth. BMC Plant Biol 10: 213
Alverson, A.J., Wei, X.X., Rice, D.W., Stern, D.B., Barry, K. and Palmer, J.D. 2010. Insights into the evolution of mitochondrial genome size from complete sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae). Molecular Biology and Evolution 27: 1436-1448
Sattarzadeh, A., Fuller, J., Moguel, S., Wostrikoff, K., Sato, S., Covshoff, S., Clemente, T., Hanson, M. and Stern, D.B. 2010. Transgenic maize lines with cell-type specific expression of fluorescent proteins in plastids. Plant Biotechnology Journal 8: 112-125
Sharpe, R.M., Mahajan, A., Takacs, E.M., Stern, D.B. and Cahoon, A.B. 2010. Developmental and cell type characterization of bundle sheath and mesophyll chloroplast transcript abundance in maize. Curr Genet 0:
Stern, D.B., Goldschmidt-Clermont, M. and Hanson, M.R. 2010. Chloroplast RNA metabolism. Annu Rev Plant Biol 61: 125-155
Williams-Carrier, R., Stiffler, N., Belcher, S., Kroeger, T., Stern, D.B., Monde, R.A., Coalter, R. and Barkan, A. 2010. Use of Illumina sequencing to identify transposon insertions underlying mutant phenotypes in high-copy Mutator lines of maize. Plant Journal 63: 167-177
Bollenbach, T.J., Sharwood, R.E., Gutierrez, R., Lerbs-Mache, S. and Stern, D.B. 2009. The RNA-binding proteins CSP41a and CSP41b may regulate transcription and translation of chloroplast-encoded RNAs in Arabidopsis. Plant Molecular Biology 69: 541-552
Jiang, X. and Stern, D. 2009. Mating and tetrad separation of Chlamydomonas reinhardtii for genetic analysis. J Vis Exp 0:
Newton, K.J., Stern, D.B. and Gabay-Laughnan, S. 2009. Mitochondria and Chloroplasts. In Handbook of Maize (Bennetzen, J.L. and Hake, S.C. eds). 0: Springer
Marchive, C., Yehudai-Resheff, S., Germain, A., Fei, Z.J., Jiang, X.S., Judkins, J., Wu, H., Fernie, A.R., Fait, A. and Stern, D.B. 2009. Abnormal physiological and molecular mutant phenotypes link chloroplast polynucleotide phosphorylase to the phosphorus deprivation response in Arabidopsis. Plant Physiology 151: 905-924
Schuster, G. and Stern, D. 2009. RNA polyadenylation and decay in mitochondria and chloroplasts. Prog Mol Biol Transl 85: 393-422
Bozkurt, A., Gilmour, R.F., Sinha, A., Stern, D. and Lal, A. 2009. Insect-machine interface based neurocybernetics. Ieee T Bio-Med Eng 56: 1727-1733
Zimmer, S.L., Schein, A., Zipor, G., Stern, D.B. and Schuster, G. 2009. Polyadenylation in Arabidopsis and Chlamydomonas organelles: the input of nucleotidyltransferases, poly(A) polymerases and polynucleotide phosphorylase. Plant Journal 59: 88-99
Stern, D.B. 2008. Organellar and metabolic processes. In The Chlamydomonas Sourcebook (Stern, D.B. ed. Oxford 0: Elsevier
Cahoon, A.B., Takacs, E.M., Sharpe, R.M. and Stern, D.B. 2008. Nuclear, chloroplast, and mitochondrial transcript abundance along a maize leaf developmental gradient. Plant Molecular Biology 66: 33-46
Zimmer, S.L., Fei, Z.J. and Stern, D.B. 2008. Genome-based analysis of Chlamydomonas reinhardtii exoribonucleases and poly(A) polymerases predicts unexpected organellar and exosomal features. Genetics 179: 125-136
Yehudai-Resheff S, Zimmer SL, Komine Y, Stern DB. 2007. Integration of chloroplast nucleic acid metabolism into the phosphate deprivation response in Chlamydomonas reinhardtii. Plant Cell 19: 1023-1038
Wostrikoff K and, Stern DB. 2007. Rubisco large subunit translation is autoregulated in response to its assembly state in tobacco chloroplasts. Proc. Natl. Acad. Sci. USA 104: 6466-6471
Merchant SS, Prochnik SE et al. 2007. The evolution of key animal and plant functions is revealed by analysis of the Chlamydomonas genome. Science 318: 245-250
Cahoon AB, Takacs EM, Sharpe RM, Stern DB. 2007. Nuclear, chloroplast, and mitochondrial transcript abundance along a maize leaf developmental gradient. Plant Mol. Biol 66: 33-46
Rymarquis, L. A., D. C. Higgs, D. B. Stern. 2006. Nuclear Suppressors Define Three Factors that Participate in Both 5’ and 3’ End Processing of mRNAs in Chlamydomonas chloroplasts. The Plant Journal 46: 448-461
Cui, L., J. Leebens-Mack, L. S. Wang, J. Tang, L. Rymarquis, D. B. Stern, C. W. dePamphilis. 2006. Adaptive Evolution of Chloroplast Genome Structure Inferred Using a Parametric Bootstrap Approach. BMC Evolutionary Biology 6: 13
Bohne, A. V., V. Ihrimovitch, A. Weihe, D. B. Stern. 2006. Chlamydomonas reinhardtii Encodes a Single sigma70-like Factor Which Likely Functions in Chloroplast Transcription. Current Genetics 49: 333-340
Irihimovitch, V., D. B. Stern. 2006. The sulfur acclimation SAC3 kinase is required for chloroplast transcriptional repression under sulfur limitation in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 103: 7911-7916
Rymarquis, L. A., J. M. Handley, M. Thomas, D. B. Stern. 2005. Beyond Complementation: Map-Based Cloning in Chlamydomonas reinhardtii. Plant Physiology 137: 557-566
Murakami, S., K. Kuehnle, D. B. Stern. 2005. A Spontaneous tRNA Suppressor of a Mutation in the Chlamydomonas reinhardtii Nuclear MCD1 Gene Required for Stability of the Chloroplast petD mRNA. Nucleic Acids Research 33: 3372-3380
Erickson, B., D. B. Stern, D. C. Higgs. 2005. Microarray Analysis Confirms the Specificity of a Chlamydomonas reinhardtii Chloroplast RNA Stability Mutant. Plant Physiology 137: 534-544
Cohen, I., J. A. Knopf, V. Irihimovitch, M. Shapira. 2005. A Proposed Mechanism for the Inhibitory Effects of Oxidative Stress on Rubisco Assembly and Its Subunit Expression. Plant Physiology 137: 738-746
Bollenbach, T. J., H. Lange, R. Gutierrez, D. B. Stern, D. Gagliardi. 2005. RNR1, a 3’-5’ Exoribonuclease Belonging to the RNR Superfamily, Catalyzes 3’ Maturation of Chloroplast Ribosomal RNAs in Arabidopsis thaliana. Nucleic Acids Research 33: 2751-2763
Stern, D. B., M. R. Hanson, A. Barkan. 2004. Genetics and Genomics of Chloroplast Biogenesis: Maize as a Model System. Trends in Plant Science 9: 293-301
Nishimura, Y., E. A. Kikis, S. L. Zimmer, Y. Komine, D. B. Stern. 2004. Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas chloroplasts. Plant Cell 16: 2849-2869
feature released -2011
Gene expression is a very complicated process, especially in the chloroplast. For chloroplast genes to be expressed, the DNA sequence is transcribed (“copied”) into an RNA sequence that is either functional as an RNA molecule (for example, tRNA and rRNA), or is further translated to a protein counterpart. In addition, many chloroplast RNAs require modification(s) from a precursor form to a functional, mature RNA, such as trimming off extra sequence at the ends and cutting long stretches of RNA into their individual components. The regulation of these steps (DNA > RNA > protein) is known to be orchestrated by numerous proteins originating from nuclear genes, and imported into the chloroplast, such as ribonucleases and polymerases. Recently, the concept of RNA molecules (â€śnoncodingâ€ť RNAs) having a regulatory role in both transcription and translation has been proposed for bacteria, plants and humans. David Sternâ€™s laboratory is uncovering the function of these regulatory RNAs in the chloroplast. One such RNA, named AS5 for antisense to the 5S rRNA, was found to have a functional role in the maturation and stabilization of the 5S rRNA. This was determined using two different experimental approaches. First, postdoctoral associate Amber Hotto overexpressed AS5 in tobacco chloroplasts. This resulted in plants that grew slower than the wild-type tobacco, and had reduced accumulation of 5S rRNA. Second, postdoctoral associate Robert Sharwood synthesized numerous variations of AS5 and 5S rRNA precursor transcripts in the test tube, and incubated them together with an extract from spinach chloroplasts that contains the proteins necessary for RNA processing and degradation. Robert observed that adding increased amounts of AS5 to the 5S rRNA precursor with the spinach extract resulted in a decreased accumulation of the mature 5S rRNA. These results are a first step in deciphering the role of chloroplast noncoding RNAs. The lab continues to investigate the function of other noncoding RNAs that have recently been identified.
How do plants regulate the enzyme that fixes CO2?
feature released -2008
Through photosynthesis, plants use sunlight to convert carbon dioxide and water into sugar and then release oxygen into the air. This process is critical to the plantâ€™s production of energy, and to the balance of CO2 and oxygen in the earthâ€™s atmosphere. If the ability of plants to absorb and sequester CO2 could be improved beyond their natural capacity, plants could play an even more important role in helping to mitigate global warming.
In one research project, David Sternâ€™s laboratory has concentrated on understanding the molecular regulation of CO2 fixation, which occurs in the chloroplasts of plant cells. It has long been known that plants rely on an enzyme called ribulose bisphosphate carboxylase/oxygenase (Rubisco) to sequester CO2, and that a tremendous amount of the plantâ€™s energy is required to produce the enzyme. It was also known that to conserve energy, plants carefully calibrate how much Rubisco they need at any point in time. What was not known â€“ and recently investigated by the Stern laboratory â€“ is how Rubisco production is regulated in the chloroplast.
The Rubisco molecule is composed of eight large subunits (LS) produced in the chloroplast and eight small subunits (SS) produced in the cytoplasm of the cell. The SS molecules migrate into the chloroplast from the cytoplasm where they join with the LS molecules to form Rubisco. But how does the plant regulate Rubisco production when LS and SS are produced by two different genes in two structurally separate parts of the cell?
In research with transgenic tobacco plants, Stern and his colleagues showed that when an LS molecule cannot find an SS molecule to combine with, the LS molecule binds to its own messenger RNA,which in turn blocks, or prevents, the RNA from translating into more LS. In effect, the LS molecule shuts down its own production to save energy needed for other processes. This is called auto-regulation, and it controls the amount of Rubisco a plant produces.
Other scientists had encountered problems when they attempted to over-produce Rubisco, because their efforts resulted in a Rubisco-production shutdown that was not fully understood. With the labâ€™s discovery of the regulatory process, scientists may one day be able to modify plants to produce extra Rubisco,which would enable them to sequester more CO2. The result should be increased yields and, perhaps, reduced CO2 in the atmosphere.
How to Make a Ribosome.
feature released -2007
The ribosome is a strange beast. Often likened to a factory, the ribosome can also be seen as a translator, using the four-letter code of RNA to build proteins with 20 kinds of amino acids. To accomplish this, the ribosome needs its many components-that is, dozens of proteins and a few specialized RNA molecules-to work together like a well-oiled machine.
The situation is even more complicated for mitochondria and chloroplasts, cellular compartments with their own genes and ribosomes. Of the 54 proteins in a chloroplast’s ribosome, 33 come from genes in the nucleus of the cell, while the chloroplast’s own genes code for the rest. David Stern’s lab studies how the chloroplasts and nuclei of plant cells accomplish ribosome construction and other feats of cooperation.
In one experiment, research associate Tom Bollenbach investigated the origin of the four RNA molecules incorporated into chloroplast ribosomes. He knew that all four came from genes adjacent to each other on the chloroplast chromosome, and were copied into RNA in one long stretch. Bollenbach wanted to learn how that stretch of RNA gets cut up into useful form.
He and other lab members started by identifying three nuclear plant genes that looked similar to a bacterial gene known to be involved in RNA processing. Using microscopy, they found that only one of the three acted in the chloroplast. The lab next knocked out this gene, RNR1, and got white plants that couldn’t photosynthesize: their chloroplasts weren’t working. Uncut ribosomal RNAs were building up in these mutant chloroplasts, Bollenbach found, and their ribosomes weren’t coming together. So the RNR1 gene must code for an enzyme that processes the ribosomal RNA.
Intriguingly, cells sense the buildup of unprocessed RNAs in the absence of RNR1, and scale back production of the chloroplast proteins needed to make the ribosome. The lab is working to determine how plants manage this regulation.
The Meaning of Teamwork.
feature released -2006
A long time ago, in a land of primordial goo, two bacteria lent new meaning to the word “teamwork.” It didnâ€™t start out that way, most scientists think: One bacterium probably ate the other. But it turned out that the two survived better together than they had independently, and their progeny evolved into a single, more sophisticated organism. This process happened at least twice, yielding the specialized cellular compartments chloroplasts and mitochondria.
David Stern’s lab studies chloroplasts, light-harvesting factories unique to plants and algae. While structures called nuclei contain most genes in plant cells, chloroplasts hold a few of their own. Most of these genes have to do with harnessing energy from sunlight to make sugars, a process agricultural scientists hope can be made more efficient through genetic engineering. But since the nucleus controls how the chloroplast’s genes operate, understanding their interactionâ€”the theme of Sternâ€™s researchâ€”is essential.
In one project, the Stern lab used a mutant single-celled algae to find how chloroplast gene activity decreases when cells are starved of sulfur. The mutation occurred in a gene in the nucleus that scaled down copying of DNA to RNA under low-sulfur conditions. By comparing the mutant with normal algae, lab members found that the protein affected gene copying not only in the nucleus (as previously thought), but also in the chloroplast. Since plants need sulfur-containing proteins to process light energy and avoid its potentially harmful effects, this system may protect a sulfur-starved cell by cutting how much light it absorbs.
This mechanism is just one step in the delicate choreography between nuclei, chloroplasts, and the environment. Future experiments on their relationship will add a novel chapter to our understanding of evolutionâ€”one that explains how three organisms became one.
Senior Research Specialist