Cytonuclear integration and co-evolution.

The partitioning of genetic material between the nucleus and cytoplasmic (mitochondrial and plastid) genomes within eukaryotic cells necessitates coordinated integration between these genomic compartments, with important evolutionary and biomedical implications. Classic questions persist about the pervasive reduction of cytoplasmic genomes via a combination of gene loss, transfer and functional replacement - and yet why they are almost always retained in some minimal form. One striking consequence of cytonuclear integration is the existence of 'chimeric' enzyme complexes composed of subunits encoded in two different genomes. Advances in structural biology and comparative genomics are yielding important insights into the evolution of such complexes, including correlated sequence changes and recruitment of novel subunits. Thus, chimeric cytonuclear complexes provide a powerful window into the mechanisms of molecular co-evolution.

Rapid sequence evolution is associated with genetic incompatibilities in the plastid Clp complex.

KEY MESSAGE: Replacing the native clpP1 gene in the Nicotiana plastid genome with homologs from different donor species showed that the extent of genetic incompatibilities depended on the rate of sequence evolution. The plastid caseinolytic protease (Clp) complex plays essential roles in maintaining protein homeostasis and comprises both plastid-encoded and nuclear-encoded subunits. Despite the Clp complex being retained across green plants with highly conserved protein sequences in most species, examples of extremely accelerated amino acid substitution rates have been identified in numerous angiosperms. The causes of these accelerations have been the subject of extensive speculation but still remain unclear. To distinguish among prevailing hypotheses and begin to understand the functional consequences of rapid sequence divergence in Clp subunits, we used plastome transformation to replace the native clpP1 gene in tobacco (Nicotiana tabacum) with counterparts from another angiosperm genus (Silene) that exhibits a wide range in rates of Clp protein sequence evolution. We found that antibiotic-mediated selection could drive a transgenic clpP1 replacement from a slowly evolving donor species (S. latifolia) to homoplasmy but that clpP1 copies from Silene species with accelerated evolutionary rates remained heteroplasmic, meaning that they could not functionally replace the essential tobacco clpP1 gene. These results suggest that observed cases of rapid Clp sequence evolution are a source of epistatic incompatibilities that must be ameliorated by coevolutionary responses between plastid and nuclear subunits.

Wide-ranging transcriptome remodelling mediated by alternative polyadenylation in response to abiotic stresses in Sorghum.

Alternative polyadenylation (APA) regulates diverse developmental and physiological processes through its effects on gene expression, mRNA stability, translatability, and transport. Sorghum is a major cereal crop in the world and, despite its importance, not much is known about the role of post-transcriptional regulation in mediating responses to abiotic stresses in Sorghum. A genome-wide APA analysis unveiled widespread occurrence of APA in Sorghum in response to drought, heat, and salt stress. Abiotic stress treatments incited changes in poly(A) site choice in a large number of genes. Interestingly, abiotic stresses led to the re-directing of transcriptional output into non-productive pathways defined by the class of poly(A) site utilized. This result revealed APA to be part of a larger global response of Sorghum to abiotic stresses that involves the re-direction of transcriptional output into non-productive transcriptional and translational pathways. Large numbers of stress-inducible poly(A) sites could not be linked with known, annotated genes, suggestive of the existence of numerous unidentified genes whose expression is strongly regulated by abiotic stresses. Furthermore, we uncovered a novel stress-specific cis-element in intronic poly(A) sites used in drought- and heat-stressed plants that might play an important role in non-canonical poly(A) site choice in response to abiotic stresses.

Transcriptome Analysis of Drought-Resistant and Drought-Sensitive Sorghum (Sorghum bicolor) Genotypes in Response to PEG-Induced Drought Stress.

Drought is a major limiting factor of crop yields. In response to drought, plants reprogram their gene expression, which ultimately regulates a multitude of biochemical and physiological processes. The timing of this reprogramming and the nature of the drought-regulated genes in different genotypes are thought to confer differential tolerance to drought stress. Sorghum is a highly drought-tolerant crop and has been increasingly used as a model cereal to identify genes that confer tolerance. Also, there is considerable natural variation in resistance to drought in different sorghum genotypes. Here, we evaluated drought resistance in four genotypes to polyethylene glycol (PEG)-induced drought stress at the seedling stage and performed transcriptome analysis in seedlings of sorghum genotypes that are either drought-resistant or drought-sensitive to identify drought-regulated changes in gene expression that are unique to drought-resistant genotypes of sorghum. Our analysis revealed that about 180 genes are differentially regulated in response to drought stress only in drought-resistant genotypes and most of these (over 70%) are up-regulated in response to drought. Among these, about 70 genes are novel with no known function and the remaining are transcription factors, signaling and stress-related proteins implicated in drought tolerance in other crops. This study revealed a set of drought-regulated genes, including many genes encoding uncharacterized proteins that are associated with drought tolerance at the seedling stage.

Genome editing approaches: manipulating of lovastatin and taxol synthesis of filamentous fungi by CRISPR/Cas9 system.

Filamentous fungi are prolific repertoire of structurally diverse secondary metabolites of remarkable biological activities such as lovastatin and paclitaxel that have been approved by FDA as drugs for hypercholesterolemia and cancer treatment. The clusters of genes encoding lovastatin and paclitaxel are cryptic at standard laboratory cultural conditions (Kennedy et al. Science 284:1368-1372, 1999; Bergmann et al. Nature Chem Biol 3:213-217, 2007). The expression of these genes might be triggered in response to nutritional and physical conditions; nevertheless, the overall yield of these metabolites does not match the global need. Consequently, overexpression of the downstream limiting enzymes and/or blocking the competing metabolic pathways of these metabolites could be the most successful technologies to enhance their yield. This is the first review summarizing the different strategies implemented for fungal genome editing, molecular regulatory mechanisms, and prospective of clustered regulatory interspaced short palindromic repeat/Cas9 system in metabolic engineering of fungi to improve their yield of lovastatin and taxol to industrial scale. Thus, elucidating the putative metabolic pathways in fungi for overproduction of lovastatin and taxol was the ultimate objective of this review.

Identification of an intronic splicing regulatory element involved in auto-regulation of alternative splicing of SCL33 pre-mRNA.

In Arabidopsis, pre-mRNAs of serine/arginine-rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis-elements and trans-acting proteins involved in regulating AS. Using a splicing reporter (GFP-intron-GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis-elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis-elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP-intron-GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto-regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis-element involved in AS of a plant SR gene, and elucidated a mechanism for auto-regulation of AS of this intron.

Metabolic engineering of Arabidopsis for butanetriol production using bacterial genes.

1,2,4-butanetriol (butanetriol) is a useful precursor for the synthesis of the energetic material butanetriol trinitrate and several pharmaceutical compounds. Bacterial synthesis of butanetriol from xylose or arabinose takes place in a pathway that requires four enzymes. To produce butanetriol in plants by expressing bacterial enzymes, we cloned native bacterial or codon optimized synthetic genes under different promoters into a binary vector and stably transformed Arabidopsis plants. Transgenic lines expressing introduced genes were analyzed for the production of butanetriol using gas chromatography coupled to mass spectrometry (GC-MS). Soil-grown transgenic plants expressing these genes produced up to 20 µg/g of butanetriol. To test if an exogenous supply of pentose sugar precursors would enhance the butanetriol level, transgenic plants were grown in a medium supplemented with either xylose or arabinose and the amount of butanetriol was quantified. Plants expressing synthetic genes in the arabinose pathway showed up to a forty-fold increase in butanetriol levels after arabinose was added to the medium. Transgenic plants expressing either bacterial or synthetic xylose pathways, or the arabinose pathway showed toxicity symptoms when xylose or arabinose was added to the medium, suggesting that a by-product in the pathway or butanetriol affected plant growth. Furthermore, the metabolite profile of plants expressing arabinose and xylose pathways was altered. Our results demonstrate that bacterial pathways that produce butanetriol can be engineered into plants to produce this chemical. This proof-of-concept study for phytoproduction of butanetriol paves the way to further manipulate metabolic pathways in plants to enhance the level of butanetriol production.

Copper homeostasis.

Copper (Cu) is a cofactor in proteins that are involved in electron transfer reactions and is an essential micronutrient for plants. Copper delivery is accomplished by the concerted action of a set of evolutionarily conserved transporters and metallochaperones. As a result of regulation of transporters in the root and the rarity of natural soils with high Cu levels, very few plants in nature will experience Cu in toxic excess in their tissues. However, low Cu bioavailability can limit plant productivity and plants have an interesting response to impending Cu deficiency, which is regulated by an evolutionarily conserved master switch. When Cu supply is insufficient, systems to increase uptake are activated and the available Cu is utilized with economy. A number of Cu-regulated small RNA molecules, the Cu-microRNAs, are used to downregulate Cu proteins that are seemingly not essential. On low Cu, the Cu-microRNAs are upregulated by the master Cu-responsive transcription factor SPL7, which also activates expression of genes involved in Cu assimilation. This regulation allows the most important proteins, which are required for photo-autotrophic growth, to remain active over a wide range of Cu concentrations and this should broaden the range where plants can thrive.

Copper cofactor delivery in plant cells.

Copper (Cu) is a micronutrient that has roles in photosynthesis, respiration, antioxidant activity, cell wall metabolism and hormone perception. Excess Cu is toxic and therefore its delivery has to be tightly regulated. Recent progress in the study of Cu homeostasis has revealed not only components of the Cu delivery machinery but also regulatory systems that control Cu-protein expression and coordinate the activity of Cu-delivery systems. The response of photosynthetic organisms to Cu deficiency indicates the existence of cross-talk between metal cofactor delivery pathways. Next to its well-established roles in plant metabolism, a novel function for Cu, first discovered in plants, is in the biogenesis of molybdenum cofactor. Defects in Cu delivery factors also suggest important roles for Cu in cell expansion.

Expression of the Intracellular COPT3-Mediated Cu Transport Is Temporally Regulated by the TCP16 Transcription Factor.

Copper is an essential element in plants. When scarce, copper is acquired from extracellular environment or remobilized from intracellular sites, through members of the high affinity copper transporters family COPT located at the plasma membrane and internal membrane, respectively. Here, we show that COPT3 is an intracellular copper transporter, located at a compartment of the secretory pathway, that is mainly expressed in pollen grains and vascular bundles. Contrary to the COPT1 plasma membrane member, the expression of the internal COPT3 membrane transporter was higher at 12 h than at 0 h of a neutral photoperiod day under copper deficiency. The screening of a library of conditionally overexpressed transcription factors implicated members of the TCP family in the COPT3 differential temporal expression pattern. Particularly, in vitro, TCP16 was found to bind to the COPT3 promoter and down-regulated its expression. Accordingly, TCP16 was mainly expressed at 0 h under copper deficiency and induced at 12 h by copper excess. Moreover, TCP16 overexpression resulted in increased sensitivity to copper deficiency, whereas the tcp16 mutant was sensitive to copper excess. Both copper content and the expression of particular copper status markers were altered in plants with modified levels of TCP16. Consistent with TCP16 affecting pollen development, the lack of COPT3 function led to altered pollen morphology. Furthermore, analysis of copt3 and COPT3 overexpressing plants revealed that COPT3 function exerted a negative effect on TCP16 expression. Taken together, these results suggest a differential daily regulation of copper uptake depending on the external and internal copper pools, in which TCP16 inhibits copper remobilization at dawn through repression of intracellular transporters.

Biogenesis of iron-sulfur cluster proteins in plastids.

Iron-sulfur (Fe-S) clusters are co-factors of proteins that perform a number of biological roles, including electron transfer, redox and non-redox catalysis, regulation of gene expression, and as sensors within all living organisms, prokaryotes and eukaryotes. These clusters are thought to be among the oldest structures found in biological cells. In chloroplasts, Fe-S clusters play a key role in photosynthetic electron transport as well as nitrogen and sulfur assimilation. The capacity of the Fe atom in Fe-S clusters to take up an electron reversibly provides the required electron carrier capacity in these pathways. Iron and sulfur limitation both affect plant primary production and growth. It has long been known that iron deficiency leads to defects in photosynthesis and bleaching in young leaves, phenomena that are closely linked to a defect in chloroplastic photosystem-I (PSI) accumulation, a major Fe-S containing protein complex in plants. Although the functional importance of Fe-S cluster proteins is evident and isolated chloroplasts have been shown to be able to synthesize their own Fe-S clusters, much is yet to be learned about the biosynthesis of Fe-S proteins in plastids. The recent discovery of a NifS-like protein in plastids has hinted to the existence of an assembly machinery related to bacterial Fe-S assembly systems. This chapter aims to summarize what we presently know about the assembly of Fe-S clusters in plants with an emphasis on green plastids.

Production of Phloroglucinol, a Platform Chemical, in Arabidopsis using a Bacterial Gene.

Phloroglucinol (1,3,5-trihydroxybenzene; PG) and its derivatives are phenolic compounds that are used for various industrial applications. Current methods to synthesize PG are not sustainable due to the requirement for carbon-based precursors and co-production of toxic byproducts. Here, we describe a more sustainable production of PG using plants expressing a native bacterial or a codon-optimized synthetic PhlD targeted to either the cytosol or chloroplasts. Transgenic lines were analyzed for the production of PG using gas and liquid chromatography coupled to mass spectroscopy. Phloroglucinol was produced in all transgenic lines and the line with the highest PhlD transcript level showed the most accumulation of PG. Over 80% of the produced PG was glycosylated to phlorin. Arabidopsis leaves have the machinery to glycosylate PG to form phlorin, which can be hydrolyzed enzymatically to produce PG. Furthermore, the metabolic profile of plants with PhlD in either the cytosol or chloroplasts was altered. Our results provide evidence that plants can be engineered to produce PG using a bacterial gene. Phytoproduction of PG using a bacterial gene paves the way for further genetic manipulations to enhance the level of PG with implications for the commercial production of this important platform chemical in plants.

A survey of the sorghum transcriptome using single-molecule long reads.

Alternative splicing and alternative polyadenylation (APA) of pre-mRNAs greatly contribute to transcriptome diversity, coding capacity of a genome and gene regulatory mechanisms in eukaryotes. Second-generation sequencing technologies have been extensively used to analyse transcriptomes. However, a major limitation of short-read data is that it is difficult to accurately predict full-length splice isoforms. Here we sequenced the sorghum transcriptome using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and APA sites. Our analysis reveals transcriptome-wide full-length isoforms at an unprecedented scale with over 11,000 novel splice isoforms. Additionally, we uncover APA of ∼11,000 expressed genes and more than 2,100 novel genes. These results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop. The TAPIS pipeline will serve as a useful tool to analyse Iso-Seq data from any organism.

AtCCS is a functional homolog of the yeast copper chaperone Ccs1/Lys7.

In plant chloroplasts two superoxide dismutase (SOD) activities occur, FeSOD and Cu/ZnSOD, with reciprocal regulation in response to copper availability. This system presents a unique model to study the regulation of metal-cofactor delivery to an organelle. The Arabidopsis thaliana gene AtCCS encodes a functional homolog to yeast Ccs1p/Lys7p, a copper chaperone for SOD. The AtCCS protein was localized to chloroplasts where it may supply copper to the stromal Cu/ZnSOD. AtCCS mRNA expression levels are upregulated in response to Cu-feeding and senescence. We propose that AtCCS expression is regulated to allow the most optimal use of Cu for photosynthesis.

Biochemical stability and molecular dynamic characterization of Aspergillus fumigatus cystathionine γ-lyase in response to various reaction effectors.

Cystathionine γ-lyase (CGL) is a key enzyme in the methionine-cysteine cycle in all living organisms forming cysteine, α-ketobutyrate and ammonia via homocysteine and cystathionine intermediates. Although, human and plant CGLs have been extensively studied at the molecular and mechanistic levels, there has been little work on the molecular and catalytic properties of fungal CGL. Herein, we studied in detail for the first time the molecular and catalytic stability of Aspergillus fumigatus CGL, since conformational instability, inactivation and structural antigenicity are the main limitations of the PLP-dependent enzymes on various therapeutic uses. We examined these properties in response to buffer compositions, stabilizing and destabilizing agents using Differential Scanning Fluorometery (DSF), steady state and gel-based fluorescence of the intrinsic hydrophobic core, stability of internal aldimine linkage and catalytic properties. The activity of the recombinant A. fumigatus CGL was 13.8U/mg. The melting temperature (Tm) of CGL in potassium phosphate buffer (pH 7.0-8.0) was 73.3°C, with ∼3°C upshifting in MES and sodium phosphate buffers (pH 7.0). The conformational thermal stability was increased in potassium phosphate, sodium phosphate and MES buffers, in contrast to Tris-HCl, HEPES (pH 7.0) and CAPS (pH 9.0-10.0). The thermal stability and activity of CGL was slightly increased in the presence of trehalose and glycerol that might be due to hydration of the enzyme backbone, unlike the denaturing effect of GdmCl and urea. Modification of surface CGL glutamic and aspartic acids had no significant effect on the enzyme conformational and catalytic stability. Molecular modeling and dynamics simulations unveil the high conformational stability of the overall scaffold of CGL with high flexibility at the non-structural regions. CGL structure has eight buried Trp residues, which are reoriented to the enzyme surface and get exposed to the solvent under perturbation of destabilizers. Furthermore, electrostatic calculations of selected snapshots of CGL 3D structure under different experimental conditions showed a remarkable differences on the polarity of the enzyme surface.

CpKLP1: A CALMODULIN-BINDING KINESIN-LIKE PROTEIN FROM CYANOPHORA PARADOXA (GLAUCOPHYTA).

KCBP (kinesin-like calmodulin [CaM]-binding proteins), a member of the carboxy-terminal kinesin-like proteins (KLPs), is unique among KLPs in having a CaM-binding domain (CBD). CaM-binding KLPs have been identified from flowering plants and the sea urchin. To determine if CaM-binding KLP is present in phylogenetically divergent protists, we probed Cyanophora paradoxa protein extract with affinity-purified KCBP antibody. The KCBP antibody detected a polypeptide with a molecular mass of about 133 kDa in the crude extract. In a CaM-Sepharose column-purified fraction, the same band was detected with both KCBP antibody and biotinylated CaM. In a PCR reaction using degenerate primers corresponding to two conserved regions in the motor domain of kinesin, a 500-bp fragment (CpKLP1) was amplified from a cDNA library. The predicted amino acid sequence of CpKLP1 showed significant sequence similarity with KCBPs. In phylogenetic analysis, CpKLP1 fell into the KCBP group within the carboxy-terminal subfamily. These biochemical data, sequence, and phylogenetic analysis strongly suggest the presence of a calmodulin-binding KLP in C. paradoxa and that it is related to Ca2 +/calmodulin regulated KLPs from plants. This is the first report on identification of any motor protein in C. paradoxa. Furthermore, our data suggest that CaM-binding KLPs may have evolved long before the divergence of plants and animals.

Arabidopsis thaliana PGR7 encodes a conserved chloroplast protein that is necessary for efficient photosynthetic electron transport.

A significant fraction of a plant's nuclear genome encodes chloroplast-targeted proteins, many of which are devoted to the assembly and function of the photosynthetic apparatus. Using digital video imaging of chlorophyll fluorescence, we isolated proton gradient regulation 7 (pgr7) as an Arabidopsis thaliana mutant with low nonphotochemical quenching of chlorophyll fluorescence (NPQ). In pgr7, the xanthophyll cycle and the PSBS gene product, previously identified NPQ factors, were still functional, but the efficiency of photosynthetic electron transport was lower than in the wild type. The pgr7 mutant was also smaller in size and had lower chlorophyll content than the wild type in optimal growth conditions. Positional cloning located the pgr7 mutation in the At3g21200 (PGR7) gene, which was predicted to encode a chloroplast protein of unknown function. Chloroplast targeting of PGR7 was confirmed by transient expression of a GFP fusion protein and by stable expression and subcellular localization of an epitope-tagged version of PGR7. Bioinformatic analyses revealed that the PGR7 protein has two domains that are conserved in plants, algae, and bacteria, and the N-terminal domain is predicted to bind a cofactor such as FMN. Thus, we identified PGR7 as a novel, conserved nuclear gene that is necessary for efficient photosynthetic electron transport in chloroplasts of Arabidopsis.

Essential transition metal homeostasis in plants.

The homeostasis of the essential transition metals copper, iron, manganese and zinc requires balanced activities of transporters that mediate import into the cell, distribution to organelles and export from the cell. Transcriptional control is important for the regulation of cellular homeostasis. In the case of Fe and Cu much progress has been made in uncovering the regulatory networks that mediate homeostasis, and key transcription factors have now been described. A master regulator of Cu homeostasis in Arabidopsis thaliana, AtSPL7, is related to the Chlamydomonas master regulator CCR1, suggesting that the key switch is conserved between the two systems even though different sets of targets are regulated in the two systems.

Copper delivery by the copper chaperone for chloroplast and cytosolic copper/zinc-superoxide dismutases: regulation and unexpected phenotypes in an Arabidopsis mutant.

Copper (Cu) is an important mineral nutrient found in chloroplasts as a cofactor associated with plastocyanin and Cu/Zn superoxide dismutase (Cu/ZnSOD). Superoxide dismutases are metallo-enzymes found in most oxygenic organisms with proposed roles in reducing oxidative stress. Several recent studies in Arabidopsis have shown that microRNAs and a SQUAMOSA promoter binding protein-like7 (SPL7) transcription factor function to down-regulate the expression of many Cu-proteins, including Cu/ZnSOD in both plastids and the cytosol, during growth on low Cu. Plants contain the Cu Chaperone for SOD (CCS) that delivers Cu to Cu/ZnSODs, and, in Arabidopsis, both cytosolic and plastidic CCS versions are encoded by one gene. In this study, we demonstrate that Arabidopsis CCS transcript levels are regulated by Cu, mediated by microRNA 398 that was not previously predicted to target CCS. The microRNA target site is conserved in CCS of Oryza sativa. The data suggest that Cu-regulated microRNAs may have more mRNA targets than was previously predicted. A CCS null mutant has no measurable SOD activity in the chloroplast and cytosol, indicating an absolute requirement for CCS. When the CCS null mutant was grown on high Cu media, it lacked both Fe superoxide dismutase (FeSOD) and Cu/ZnSOD activity. However, this did not lead to a visual phenotype and no photosynthetic deficiencies were detected, even after high light stress. These results indicate that Cu/ZnSOD is not a pivotal component of the photosynthetic anti-oxidant system during growth in laboratory conditions.