Active DNA Demethylation during Gametogenesis Regulates Gene Imprinting and Transposon Silencing in Arabidopsis

Arabidopsis thaliana carboxyl-terminal domain (CTD) phosphatase-like 1 (CPL1) is a protein that regulates plant transcriptional responses to diverse stress signals. Unlike prototypical CTD phosphatases, which contain a phospho-peptide-binding Breast Cancer 1 C-terminal (BRCT) domain, CPL1 contains 2 double-stranded RNA binding motifs (dsRBM) at its C-terminus. While some dsRBMs can bind to dsRNA and/or other protein partners, the function of CPL1 dsRBMs has been obscure. A biochemical approach using tandem-affinity purification followed by mass spectrometry analyses of CPL1 complex identified CPL1-Associaiting Protein 1 (CAKH1), which contained multiple K homology domains important for binding to single-stranded DNA/RNA. Yeast two-hybrid and luciferase complementation imaging analyses has established that CPL1 and CAKH1 strongly associate in vivo, and dsRBM1 of CPL1 and KH3/KH4 domains of CAKH1 mediate their interaction. Mapping of functional regions in CPL1 indicated importance of dsRBM1, as well as catalytic activity and nuclear targeting of CPL1 for its proper in vivo function. These results suggest that tethering CPL1 to CAKH1 via dsRBM1 is required for CPL1 to regulate stress-responsive transcription, and nuclear targeting of CPL1 is likely prerequisite for the interaction. Gene expression profiles of rcf3 alleles of cakh1 mutants overlapped with, but were distinct from that of cpl1. This suggests that CAKH1 regulats only a subset of CPL1-regulated transcripts, which is likely involved in the negative feedback of osmotic stress signaling. A Novel Role for Extracellular Nucleotides in Early Growth and Development of Ceratopteris Spores Ashley E. Cannon, Thomas J. Bushart, Kathy Mostajeran, Greg Clark, and Stanley J. Roux Molecular, Cell, and Developmental Biology, The University of Texas at Austin, Austin, TX 78712 Abstract: Calcium channels play a critical role in the polarized development of Ceratopteris spores, just as they Calcium channels play a critical role in the polarized development of Ceratopteris spores, just as they do in the polarized growth of pollen tubes and root hairs. Recent data indicate that calcium channels in plants can be regulated by extracellular nucleotides and that the concentration of these nucleotides is regulated by ectoapyrases. Since apyrase activity is needed for the germination and polarized growth of pollen tubes and for the emergence and polarized growth of root hairs, the observed expression of a gene encoding an apyrase-like enzyme in Ceratopteris spores suggested the hypothesis that apyrases and extracellular nucleotides could play a regulatory role in the germination and early polarized development of spores. Our documentation that applied nucleotides can alter polar axis alignment and rhizoid growth in Ceratopteris spores, and that a purinoceptor antagonist can alter the gravity response of rhizoids, is consistent with this hypothesis. Furthermore, our observation that Ceratopteris spores release ATP as they germinate and grow is consistent with the postulate that extracellular ATP could influence calcium transport in the spores. A hypothetical model that could explain how ATP and PPADS could alter the gravity response would include gravity causing stretch-activated channels to open preferentially along the bottom of the spore. In animal and Arabidopsis cells, these channels release ATP. If ATP release was mainly along the bottom of the spore, this ATP could induce the opening of calcium channels preferentially at the pole. The added ATP disrupts this gradient and potentially disrupts the asymmetry of calcium entry. Collectively, these results and hypothetical model describe a novel role for extracellular nucleotides in early growth and development of Ceratopteris spores. Differential temperature operation of plant immune responses Cheng Cheng 1 , Xiquan Gao 2 , Baomin Feng 1 , Jen Sheen 3 , Libo Shan 2 and Ping He 1 1 Department of Biochemistry and Biophysics, and Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX 77843 3 Department of Genetics, Harvard Medical School, and Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, MA 02114, USA Abstract: Microbe and host have co-evolved dynamically in their arms race for fitness and survival. Environmental factors often influence the physiological responses in both sides and have profound impacts on microbial invasion and host evasion. Temperature fluctuates both daily and seasonally, and has long been considered as one of key determinants for disease epidemics. In contrast to animals that maintain a fairly constant body temperature, plant body temperature oscillates on a daily basis modulated by the environment. It remains largely elusive how plants operate inducible defense programs in response to the ambient temperature changes. We report Microbe and host have co-evolved dynamically in their arms race for fitness and survival. Environmental factors often influence the physiological responses in both sides and have profound impacts on microbial invasion and host evasion. Temperature fluctuates both daily and seasonally, and has long been considered as one of key determinants for disease epidemics. In contrast to animals that maintain a fairly constant body temperature, plant body temperature oscillates on a daily basis modulated by the environment. It remains largely elusive how plants operate inducible defense programs in response to the ambient temperature changes. We report here that ambient temperature changes lead to pronounced shifts of two distinct plant immune responses: pathogen/microbe-associated molecular pattern (P/MAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). Plants preferentially activate ETI signaling via intracellular nucleotide-binding domain leucine-rich repeat (NLR) immune receptors at relatively lower temperatures (10~23 ◦ C). However, the plant defense strategy is switched to PTI signaling mediated by cell surface receptor-like kinase (RLK) receptors at moderately elevated temperatures (23~32 ◦ C). Furthermore, the high temperature transcriptional mimicing and phenocopying Arabidopsis mutants, arp6 and hta9hta11 deficient in incorporating histone H2A.Z into nucleosomes exhibit enhanced PTI responses and yet reduced ETI responses compared with wild-type plants. Our findings suggest that plants co-evolved distinct immune strategies under ambient temperature oscillation to counteract the physiological changes of pathogens: multiply vigorously at elevated temperatures (25~30 ◦ C), accompanied with increased MAMP production; whereas the secretion of bacterial pathogenic effectors favors low temperatures (16~23 ◦ C). Pullulanase Activity is Associated with Formation of Vitreous Endosperm in Quality Protein Maize Wu, Hao; Clay, Kasi; Thompson, Stephanie S.; Love, Sterling; Gibbon, Bryan C. Baylor University Abstract: The opaque2 (o2) mutation of maize increases lysine and tryptophan content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called “Quality Protein Maize” (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. Four starch biosynthesis genes, SSIIa, SSIIb, SSIII and Zpu1, have been discovered to have unique alleles in mo2 lines; therefore these genes may play a role in formation of vitreous endosperm. qPCR analysis of recombinant inbred lines (RILs) derived from a cross of QPM and soft o2 lines showed a significant increase in expression of the QPM-derived ZpuI allele. Quantitative enzyme activity assays showed that QPM lines had higher pullulanase activity than o2 and wild type. Furthermore, pullulanase activity was positively correlated with kernel vitreousness in the RILs. Differential scanning calorimetry showed that the thermal properties of starch from the RILs correlated well with the presence of the QPM-derived allele of Zpu1, which had decreased onset and peak endotherm temperatures while total enthalpy of gelatinization was unchanged. Pullulanase activity was negatively The opaque2 (o2) mutation of maize increases lysine and tryptophan content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called “Quality Protein Maize” (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. Four starch biosynthesis genes, SSIIa, SSIIb, SSIII and Zpu1, have been discovered to have unique alleles in mo2 lines; therefore these genes may play a role in formation of vitreous endosperm. qPCR analysis of recombinant inbred lines (RILs) derived from a cross of QPM and soft o2 lines showed a significant increase in expression of the QPM-derived ZpuI allele. Quantitative enzyme activity assays showed that QPM lines had higher pullulanase activity than o2 and wild type. Furthermore, pullulanase activity was positively correlated with kernel vitreousness in the RILs. Differential scanning calorimetry showed that the thermal properties of starch from the RILs correlated well with the presence of the QPM-derived allele of Zpu1, which had decreased onset and peak endotherm temperatures while total enthalpy of gelatinization was unchanged. Pullulanase activity was negatively correlated with the onset and peak endotherm temperatures but was not correlated with enthalpy. Additionally, pullulanase activity was negatively correlated with the sensitivity of starch Metabolic engineering of a green microalgae for autotrophic production of isoprenoid-type advanced hydrocarbon fuels. Ryan Syrenne, Shangxian Xie, Ugur Uzuner, Susie Y. Dai, and Joshua S. Yuan 1, Department of Plant Pathology and Microbiology, Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX 77843 Abstract: The monoterpene limonene could potentially replace jet fuel (JP-8), gasoline and diesel as an alternative ‘drop-in’ liquid hydrocarbon fuel source. Synthetic biology-guided metabolic engineering of microalgae for the direct production of terpenoid-type hydrocarbon could provide the ultimate enablement of third-generation biofuels. Traditional microbial platforms used to produce hydrocarbon molecules similar to petroleum derived fuels rely on tractable heterotrophic hosts, such as E. coli or yeast, and essentially trade sustainability for high product yields. This study demonstrates the heterologous expression of a rice monoterpene synthase gene (OsTPS26) in the eukaryotic green algae Chlamydomonas reinhardtii (cc503) to autotrophically produce the high-volumetric energy dense hydrocarbon limonene. Using a modified nuclear transformation strategy, we engineered nuclear transformants expressing a functional rice limonene synthase localized to the plastid. Although microalgae are not known to poses monoterpene synthases, the precursor substrate geranyl diphsophate (GPP) pool is available as it’s required for downstream sesquiterpenes and other larger terpenoid molecules. Additionally, we show that limonene is emitted from the algal biomass into the headspace region of enclosed photobioreactors – eliminating the requirement of cell harvesting, dewatering, and biomass processing. Chlamydomonas mutant lines were cultured in enclosed photobioreactors enabling a purge-and-trap method of trapped volatile limonene in the culture head-space. Limonene analyte adsorbed on to volatile collection traps were subjected to gas-chromatography/mass-spectrometry (GC/MS) for product identification and quantification analyses. We show limonene production from a single transformant line at ~1 μg/g dry biomass in 72 hrs. In total, ten transformant lines were evaluated for limonene production; mRNA abundance was quantified by qPCR and limonene analyte in the head-space was quantified by The monoterpene limonene could potentially replace jet fuel (JP-8), gasoline and diesel as an alternative ‘drop-in’ liquid hydrocarbon fuel source. Synthetic biology-guided metabolic engineering of microalgae for the direct production of terpenoid-type hydrocarbon could provide the ultimate enablement of third-generation biofuels. Traditional microbial platforms used to produce hydrocarbon molecules similar to petroleum derived fuels rely on tractable heterotrophic hosts, such as E. coli or yeast, and essentially trade sustainability for high product yields. This study demonstrates the heterologous expression of a rice monoterpene synthase gene (OsTPS26) in the eukaryotic green algae Chlamydomonas reinhardtii (cc503) to autotrophically produce the high-volumetric energy dense hydrocarbon limonene. Using a modified nuclear transformation strategy, we engineered nuclear transformants expressing a functional rice limonene synthase localized to the plastid. Although microalgae are not known to poses monoterpene synthases, the precursor substrate geranyl diphsophate (GPP) pool is available as it’s required for downstream sesquiterpenes and other larger terpenoid molecules. Additionally, we show that limonene is emitted from the algal biomass into the headspace region of enclosed photobioreactors – eliminating the requirement of cell harvesting, dewatering, and biomass processing. Chlamydomonas mutant lines were cultured in enclosed photobioreactors enabling a purge-and-trap method of trapped volatile limonene in the culture head-space. Limonene analyte adsorbed on to volatile collection traps were subjected to gas-chromatography/mass-spectrometry (GC/MS) for product identification and quantification analyses. We show limonene production from a single transformant line at ~1 μg/g dry biomass in 72 hrs. In total, ten transformant lines were evaluated for limonene production; mRNA abundance was quantified by qPCR and limonene analyte in the head-space was quantified by GC/MS analysis. Conceptually, determining baseline capability of monoterpene production in algae will pave the way for advanced autotrophic hydrocarbon production.