The CBM48 domain-containing protein FLO6 regulates starch synthesis by interacting with SSIVb and GBSS in rice.

KEY MESSAGE: FLO6 is involved in starch synthesis by interacting with SSIVb and GBSS in rice. Starch synthesized and stored in plastids including chloroplasts and amyloplasts plays a vital role in plant growth and provides the major energy for human diet. However, the molecular mechanisms by which regulate starch synthesis remain largely unknown. In this study, we identified and characterized a rice floury endosperm mutant M39, which exhibited defective starch granule formation in pericarp and endosperm, accompanied by the decreased starch content and amylose content. The abnormal starch accumulation in M39 pollen grains caused a significant decrease in plant fertility. Chloroplasts in M39 leaves contained no or only one large starch granule. Positional cloning combined with complementary experiment demonstrated that the mutant phenotypes were restored by the FLOURY ENDOSPERM6 (FLO6). FLO6 was generally expressed in various tissues, including leaf, anther and developing endosperm. FLO6 is a chloroplast and amyloplast-localized protein that is able to bind to starch by its carbohydrate-binding module 48 (CBM48) domain. Interestingly, we found that FLO6 interacted with starch synthase IVb (SSIVb) and granule-bound starch synthase (GBSSI and GBSSII). Together, our results suggested that FLO6 plays a critical role in starch synthesis through cooperating with several starch synthesis enzymes throughout plant growth and development.

Comparative genome analysis revealed gene inversions, boundary expansions and contractions, and gene loss in the Stemona sessilifolia (Miq.) Miq. chloroplast genome.

Stemona sessilifolia (Miq.) Miq., commonly known as Baibu, is one of the most popular herbal medicines in Asia. In the Chinese Pharmacopoeia, Baibu has multiple authentic sources and there are many similar herbs sold as Baibu in herbal medicine markets. The existence of counterfeits of Baibu brings challenges to its identification. To assist in its accurate identification, we sequenced and analyzed the complete chloroplast genome of S. sessilifolia using next-generation sequencing technology. The genome was found to be 154,037 bp in length, possessing a typical quadripartite structure consisting of a pair of inverted repeats (IRs: 27,090 bp) separated by a large single copy (LSC: 81,949 bp) and a small single copy (SSC: 17,908 bp). A total of 112 unique genes were identified, including 80 protein-coding, 28 transfer RNA and four ribosomal RNA genes. In addition, 45 tandem, 27 forward, 23 palindromic and 104 simple sequence repeats were detected in the genome by repeated analysis. Compared with its counterfeits (Asparagus officinalis and Carludovica palmata) we found that IR expansion and SSC contraction events of S. sessilifolia resulted in two copies of the rpl22 gene in the IR regions and a partial duplication of the ndhF gene in the SSC region. An approximately 3-kb-long inversion was also identified in the LSC region, leading to the petA and cemA genes being presented in the complementary strand of the chloroplast DNA molecule. Comparative analysis revealed some highly variable regions, including trnF-GAA_ndhJ, atpB_rbcL, rps15_ycf1, trnG-UCC_trnR-UCU, ndhF_rpl32, accD_psaI, rps2_rpoC2, trnS-GCU_trnG-UCC, trnT-UGU_trnL-UAA and rps16_trnQ-UUG. Finally, gene loss events were investigated in the context of phylogenetic relationships. In summary, the complete plastome of S. sessilifolia will provide valuable information for the distinction between Baibu and its counterfeits and assist in elucidating the evolution of S. sessilifolia.

Complete chloroplast genome features and phylogenetic analysis of Eruca sativa (Brassicaceae).

Eruca sativa Mill. (Brassicaceae) is an important edible vegetable and a potential medicinal plant due to the antibacterial activity of its seed oil. Here, the complete chloroplast (cp) genome of E. sativa was de novo assembled with a combination of long PacBio reads and short Illumina reads. The E. sativa cp genome had a quadripartite structure that was 153,522 bp in size, consisting of one large single-copy region of 83,320 bp and one small single-copy region of 17,786 bp which were separated by two inverted repeat (IRa and IRb) regions of 26,208 bp. This complete cp genome harbored 113 unique genes: 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Forty-nine long repetitive sequences and 69 simple sequence repeats were identified in the E. sativa cp genome. A codon usage analysis of the E. sativa cp genome showed a bias toward codons ending in A/T. The E. sativa cp genome was similar in size, gene composition, and linearity of the structural region when compared with other Brassicaceae cp genomes. Moreover, the analysis of the synonymous (Ks) and non-synonymous (Ka) substitution rates demonstrated that protein-coding genes generally underwent purifying selection pressure, expect ycf1, ycf2, and rps12. A phylogenetic analysis determined that E. sativa is evolutionarily close to important Brassica species, indicating that it may be possible to transfer favorable E. sativa alleles into other Brassica species. Our results will be helpful to advance genetic improvement and breeding of E. sativa, and will provide valuable information for utilizing E. sativa as an important resource to improve other Brassica species.

Characterization and Comparative Analysis of Two Rheum Complete Chloroplast Genomes.

Rheum species present a significant economic value. Traditional Chinese medicine rhubarb is an important medicinal material in China. It has a long history of use, with a record of use as early as two thousand years ago. Here, we determined the complete chloroplast genome sequences of Rheum nobile and Rheum acuminatum and comprehensively compared them to two other available Rheum cp genomes at the genome scale. The results revealed cp genomes ranging in size from 159,051 to 161,707 bp with a similar typical quadripartite and circular structure. The genome organization, gene numbers, gene order, and GC contents of these four Rheum cp genomes were similar to those of many angiosperm cp genomes. Repeats and microsatellites were detected in the R. nobile and R. acuminatum cp genomes. The Mauve alignment revealed that there were no rearrangements in the cp genomes of the four Rheum species. Thirteen mutational hotspots for genome divergence were identified, which could be utilized as potential markers for phylogenetic studies and the identification of Rheum species. The phylogenetic relationships of the four species showed that the members of Rheum cluster into a single clade, indicating their close relationships. Our study provides valuable information for the taxonomic, phylogenetic, and evolutionary analysis of Rheum.

Chloroplast genome sequences of Artemisia maritima and Artemisia absinthium: Comparative analyses, mutational hotspots in genus Artemisia and phylogeny in family Asteraceae.

Artemisia L. is a complex genus of medicinal importance. Publicly available chloroplast genomes of few Artemisia species are insufficient to resolve taxonomic discrepancies at species level. We report chloroplast genome sequences of two further Artemisia species: A. maritima (151,061 bp) and A. absinthium (151,193 bp). Both genomes possess typical quadripartite structure comprising of a large single copy, a small single copy and a pair of long inverted repeats. The two genomes exhibited high similarities in genome sizes, gene synteny, GC content, synonymous and non-synonymous substitutions, codon usage, amino acids frequencies, RNA editing sites, microsatellites, and oligonucleotide repeats. Transition to transversion ratio was <1. Maximum likelihood tree showed Artemisia a monophyletic genus, sister to genus Chrysanthemum. We also identified 20 highly polymorphic regions including rpoC2-rps2, trnR-UCU-trnG-UCC, rps18-rpl20, and trnL-UAG-rpl32 that could be used to develop authentic and cost-effective markers to resolve taxonomic discrepancies and infer phylogenetic relationships among Artemisia species.

Calmodulin is involved in the dual subcellular location of two chloroplast proteins.

Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids.

Full-Length Multi-Barcoding: DNA Barcoding from Single Ingredient to Complex Mixtures.

DNA barcoding has been used for decades, although it has mostly been applied to somesingle-species. Traditional Chinese medicine (TCM), which is mainly used in the form ofcombination-one type of the multi-species, identification is crucial for clinical usage.Next-generation Sequencing (NGS) has been used to address this authentication issue for the pastfew years, but conventional NGS technology is hampered in application due to its short sequencingreads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) vialong-read sequencing, is employed for the identification of biological compositions in herbalcompound formulas in adequate and well controlled studies. By directly sequencing the full-lengthamplicons of ITS2 and psbA-trnH through single-molecule real-time (SMRT) technology, thebiological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the sametime, clone-dependent Sanger sequencing was carried out as a parallel control. Further, anotherformula-Sanwei-Jili-San (SJS)-was analyzed with genes of ITS2 and CO1. All the ingredients inthe samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenousspecies were also checked, some of which were considered as common contaminations in theseproducts. Methodology analysis demonstrated that this method was sensitive, accurate andreliable. FLMB, a superior but feasible approach for the identification of biological complexmixture, was established and elucidated, which shows perfect interpretation for DNA barcodingthat could lead its application in multi-species mixtures.

Investigation of the response to salinity of transgenic potato plants overexpressing the transcription factor StERF94.

Salinity is one of the most important constraints threatening the cultivation of potato plants (Solanum tuberosum L.). It affects plant growth and leads to significant yield loss. Consequently, it is important to improve the tolerance of potato plants to salinity. In this context, we investigated the involvement of a potato ethylene responsive factor (StERF94) in plant response to salinity, since our previous genome-wide analysis showed that it may be related to biotic and abiotic stress response. ERF proteins belong to a large family of transcription factors that participate in plant response to abiotic stresses. We have previously identified the StERF94 gene which shows increased expression in potato plants submitted to salt treatment. In this study, transgenic potato plants overexpressing StERF94 were produced and submitted to salt treatment (100 mM NaCl) in vitro and under greenhouse culture conditions. StERF94 transgenic lines showed lower decrease of stem elongation under salt treatment in comparison to non-transgenic wild-type plants. Moreover, these plants showed a low level of H2O2 and Malondialdehyde content, and an increase in catalase and GPX (Gluthation peroxidase) activities compared to non-transgenic plants. In a second step, enhanced expression of some target genes for example CuZn-SOD, DHN25 (Dehydrin) and ERD (Early Responsive to Dehydration) was noted in the StERF94 transgenic plants, submitted to salt treatment. The StERF94 factor was also involved in the activation of osmoprotectant synthesis. Taken together, all these data suggest that overexpression of the StERF94 transcription factor increases the tolerance of potato plants to salinity by improving plant growth, osmoprotectant synthesis and antioxidant activityleading to low oxidative stress damage.

Nucleus- and plastid-targeted annexin 5 promotes reproductive development in Arabidopsis and is essential for pollen and embryo formation.

BACKGROUND: Pollen development is a strictly controlled post-meiotic process during which microspores differentiate into microgametophytes and profound structural and functional changes occur in organelles. Annexin 5 is a calcium- and lipid-binding protein that is highly expressed in pollen grains and regulates pollen development and physiology. To gain further insights into the role of ANN5 in Arabidopsis development, we performed detailed phenotypic characterization of Arabidopsis plants with modified ANN5 levels. In addition, interaction partners and subcellular localization of ANN5 were analyzed to investigate potential functions of ANN5 at cellular level. RESULTS: Here, we report that RNAi-mediated suppression of ANN5 results in formation of smaller pollen grains, enhanced pollen lethality, and delayed pollen tube growth. ANN5 RNAi knockdown plants also displayed aberrant development during the transition from the vegetative to generative phase and during embryogenesis, reflected by delayed bolting time and reduced embryo size, respectively. At the subcellular level, ANN5 was delivered to the nucleus, nucleolus, and cytoplasm, and was frequently localized in plastid nucleoids, suggesting a likely role in interorganellar communication. Furthermore, ANN5-YFP co-immunoprecipitated with RABE1b, a putative GTPase, and interaction in planta was confirmed in plastidial nucleoids using FLIM-FRET analysis. CONCLUSIONS: Our findings let us to propose that ANN5 influences basal cell homeostasis via modulation of plastid activity during pollen maturation. We hypothesize that the role of ANN5 is to orchestrate the plastidial and nuclear genome activities via protein-protein interactions however not only in maturing pollen but also during the transition from the vegetative to the generative growth and seed development.

PAP genes are tissue- and cell-specific markers of chloroplast development.

MAIN CONCLUSION: Expression of PAP genes is strongly coordinated and represents a highly selective cell-specific marker associated with the development of chloroplasts in photosynthetically active organs of Arabidopsis seedlings and adult plants. Transcription in plastids of plants depends on the activity of phage-type single-subunit nuclear-encoded RNA polymerases (NEP) and a prokaryotic multi-subunit plastid-encoded RNA polymerase (PEP). PEP is comprised of the core subunits α, ß, ß' and ß″ encoded by rpoA, rpoB/C1/C2 genes located on the plastome. This core enzyme needs to interact with nuclear-encoded sigma factors for proper promoter recognition. In chloroplasts, the core enzyme is surrounded by additional 12 nuclear-encoded subunits, all of eukaryotic origin. These PEP-associated proteins (PAPs) were found to be essential for chloroplast biogenesis as Arabidopsis inactivation mutants for each of them revealed albino or pale-green phenotypes. In silico analysis of transcriptomic data suggests that PAP genes represent a tightly controlled regulon, whereas wetlab data are sparse and correspond to the expression of individual genes mostly studied at the seedling stage. Using RT-PCR, transient, and stable expression assays of PAP promoter-GUS-constructs, we do provide, in this study, a comprehensive expression catalogue for PAP genes throughout the life cycle of Arabidopsis. We demonstrate a selective impact of light on PAP gene expression and uncover a high tissue specificity that is coupled to developmental progression especially during the transition from skotomorphogenesis to photomorphogenesis. Our data imply that PAP gene expression precedes the formation of chloroplasts rendering PAP genes a tissue- and cell-specific marker of chloroplast biogenesis.

Chloroplast genome structures in Gentiana (Gentianaceae), based on three medicinal alpine plants used in Tibetan herbal medicine.

The genus Gentiana is the largest in the Gentianaceae family with ca. 400 species. However, with most species growing on the Qinghai-Tibet plateau, the processes of adaptive evolution and speciation within the genus is not clear. Also, the genomic analyses could provide important information. So far, the complete chloroplast (cp) genome data of the genus are still deficient. As the second and third sequenced members within Gentianaceae, we report the construction of complete cp sequences of Gentiana robusta King ex Hook. f. and Gentiana crassicaulis Duthie ex Burk., and describe a comparative study of three Gentiana cp genomes, including the cp genome of Gentiana straminea Maxim. published previously. These cp genomes are highly conserved in gene size, gene content, and gene order and the rps16 pseudogene with exon2 missing was found common. Three repeat types and five SSR types were investigated, and the number and distribution are similar among the three genomes. Sixteen genome divergent hotspot regions were identified across these cp genomes that could provide potential molecular markers for further phylogenetic studies in Gentiana. The IR/SC boundary organizations in Gentianales cp genomes were compared and three different types of boundaries were observed. Six data partitions of cp genomes in Gentianales were used for phylogenetic analyses and different data partitions were largely congruent with each other. The ML phylogenetic tree was constructed based on the fragments in cp genomes commonly available in 33 species from Lamiids, including 12 species in Gentianales, 1 in Boraginaceae, 10 in Solanales, and 10 in Lamiales. The result strongly supports the position of Boraginaceae (Ehretia acuminata) as the sister of Solanales, with the bootstrap values of 97 %. This study provides a platform for further research into the molecular phylogenetics of species in the order Gentianales (family Gentianaceae) notably in respect of speciation and species identification.

The Complete Chloroplast Genome Sequence of the Medicinal Plant Swertia mussotii Using the PacBio RS II Platform.

Swertia mussotii is an important medicinal plant that has great economic and medicinal value and is found on the Qinghai Tibetan Plateau. The complete chloroplast (cp) genome of S. mussotii is 153,431 bp in size, with a pair of inverted repeat (IR) regions of 25,761 bp each that separate an large single-copy (LSC) region of 83,567 bp and an a small single-copy (SSC) region of 18,342 bp. The S. mussotii cp genome encodes 84 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. The identity, number, and GC content of S. mussotii cp genes were similar to those in the genomes of other Gentianales species. Via analysis of the repeat structure, 11 forward repeats, eight palindromic repeats, and one reverse repeat were detected in the S. mussotii cp genome. There are 45 SSRs in the S. mussotii cp genome, the majority of which are mononucleotides found in all other Gentianales species. An entire cp genome comparison study of S. mussotii and two other species in Gentianaceae was conducted. The complete cp genome sequence provides intragenic information for the cp genetic engineering of this medicinal plant.

The complete chloroplast genome sequence of the medicinal plant Glehnia littoralis F.Schmidt ex Miq. (Apiaceae).

Glehnia littoralis F. Schmidt ex Miq is an oriental medicinal herb belonging to Apiaceae family, and its dried roots and rhizomes are known to show various pharmacological effects. The complete chlorplast genome of G. littoralis was generated by de novo assembly using whole genome sequencing data. The chloroplast genome of G. littoralis was 147 467 bp in length and divided into four distinct regions: large single copy region (93 493 bp), small single copy region (17 546 bp) and a pair of inverted repeat regions (18 214 bp). A total of 114 genes including 80 protein-coding genes, 30 tRNA genes and 4 rRNA genes were predicted and accounted for 57.1% of the chloroplast genome. Phylogenetic analysis with the reported chloroplast genomes revealed that G. littoralis is an herbal species closely related to Ledebouriella seseloides, an herbal medicinal plant.

Complete chloroplast genome sequence of Fritillaria unibracteata var. wabuensis based on SMRT Sequencing Technology.

Fritillaria unibracteata var. wabuensis is an important medicinal plant used for the treatment of cough symptoms related to the respiratory system. The chloroplast genome of F. unibracteata var. wabuensis (GenBank accession no. KF769142) was assembled using the PacBio RS platform (Pacific Biosciences, Beverly, MA) as a circle sequence with 151 009 bp. The assembled genome contains 133 genes, including 88 protein-coding, 37 tRNA, and eight rRNA genes. This genome sequence will provide important resource for further studies on the evolution of Fritillaria genus and molecular identification of Fritillaria herbs and their adulterants. This work suggests that PacBio RS is a powerful tool to sequence and assemble chloroplast genomes.

Inhibitory mechanisms of Acacia mearnsii extracts on the growth of Microcystis aeruginosa.

Our previous work revealed that Acacia mearnsii extract can inhibit the growth of Microcystis aeruginosa, the common species forming toxic cyanobacterial blooms in eutrophic freshwater. In the present study, we demonstrated that this plant extract can significantly increase cell membrane permeability and Ca²âº/Mg²âº-ATPase activity on the membrane. Long-term exposure to concentrations of 20 ppm A. mearnsii extract led to algal cell membrane leakage or even lysis. Comparison of expression of three photosynthesis-related genes (rbcL, psaB and psbD) in M. aeruginosa with and without plant extract treatment revealed that their expression was remarkably reduced in the presence of the extract. Down-regulation of photosynthesis-related genes could indicate the inhibition of the photosynthetic process. Thus, our results suggested that both photosynthetic systems and membranes of M. aeruginosa are potentially damaged by A. mearnsii extract.

Flower development, pollen fertility and sex expression analyses of three sexual phenotypes of Coccinia grandis.

BACKGROUND: Coccinia grandis is a dioecious species of Cucurbitaceae having heteromorphic sex chromosomes. The chromosome constitution of male and female plants is 22 + XY and 22 + XX respectively. Y chromosome of male sex is conspicuously large and plays a decisive role in determining maleness. Sex modification has been studied in hypogynous Silene latifolia (Caryophyllaceae) but there is no such report in epigynous Coccinia grandis. Moreover, the role of organ identity genes during sex expression in Coccinia has not been evaluated earlier. Investigations on sexual phenotypes of C. grandis including a rare gynomonoecious (GyM) form and AgNO3 mediated sex modification have added a new dimension to the understanding of sex expression in dioecious flowering plants. RESULTS: Morphometric analysis showed the presence of staminodes in pistillate flowers and histological study revealed the absence of carpel initials in male flowers. Though GyM plant had XX sex chromosomes, the development of stamens occurred in hermaphrodite flowers but the pollens were not fertile. Silver nitrate (AgNO3) application enhanced stamen growth in wild type female flowers like that of GyM plant but here also the pollens were sterile. Differential expression of CgPI could be involved in the development of different floral phenotypes. CONCLUSIONS: The three principle factors, Gynoecium Suppression (SuF), Stamen Promoting Factor (SPF) and Male Fertility (mF) that control sex expression in dioecious C. grandis assumed to be located on Y chromosome, play a decisive role in determining maleness. However, the characteristic development of stamens in hermaphrodite flowers of GyM plant having XX sex chromosomes indicates that Y-linked SPF regulatory pathway is somehow bypassed. Our experimental findings together with all other previous chromosomal and molecular cytogenetical data strongly support the view that C. grandis could be used as a potential model system to study sex expression in dioecious flowering plant.

Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed.

The plastidic DEAD-box RNA helicase 22, HS3, is essential for plastid functions both in seed development and in seedling growth.

Plants accumulate large amounts of storage products in seeds to provide an energy reserve and to supply nutrients for germination and post-germinative growth. Arabidopsis thaliana belongs to the Brassica family, and oil is the main storage product in Arabidopsis seeds. To elucidate the regulatory mechanisms of oil biosynthesis in seeds, we screened for high density seeds (heavy seed) that have a low oil content. HS3 (heavy seed 3) encodes the DEAD-box RNA helicase 22 that is localized to plastids. The triacylglycerol (TAG) content of hs3-1 seeds was 10% lower than that of wild-type (WT) seeds, while the protein content was unchanged. The hs3-1 plants displayed a pale-green phenotype in developing seeds and seedlings, but not in adult leaves. The HS3 expression level was high in developing seeds and seedlings, but was low in stems, rosette leaves and flowers. The plastid gene expression profile of WT developing seeds and seedlings differed from that of hs3-1 developing seeds and seedlings. The expression of several genes was reduced in developing hs3-1 seeds, including accD, a gene that encodes the ß subunit of carboxyltransferase, which is one component of acetyl-CoA carboxylase in plastids. In contrast, no differences were observed between the expression profiles of WT and hs3-1 rosette leaves. These results show that HS3 is essential for proper mRNA accumulation of plastid genes during seed development and seedling growth, and suggest that HS3 ensures seed oil biosynthesis by maintaining plastid mRNA levels.

Motif analysis unveils the possible co-regulation of chloroplast genes and nuclear genes encoding chloroplast proteins.

Chloroplasts play critical roles in land plant cells. Despite their importance and the availability of at least 200 sequenced chloroplast genomes, the number of known DNA regulatory sequences in chloroplast genomes are limited. In this paper, we designed computational methods to systematically study putative DNA regulatory sequences in intergenic regions near chloroplast genes in seven plant species and in promoter sequences of nuclear genes in Arabidopsis and rice. We found that -35/-10 elements alone cannot explain the transcriptional regulation of chloroplast genes. We also concluded that there are unlikely motifs shared by intergenic sequences of most of chloroplast genes, indicating that these genes are regulated differently. Finally and surprisingly, we found five conserved motifs, each of which occurs in no more than six chloroplast intergenic sequences, are significantly shared by promoters of nuclear-genes encoding chloroplast proteins. By integrating information from gene function annotation, protein subcellular localization analyses, protein-protein interaction data, and gene expression data, we further showed support of the functionality of these conserved motifs. Our study implies the existence of unknown nuclear-encoded transcription factors that regulate both chloroplast genes and nuclear genes encoding chloroplast protein, which sheds light on the understanding of the transcriptional regulation of chloroplast genes.

Purification of an Arabidopsis chloroplast extract with in vitro RNA processing activity on psbA and petD 3'-untranslated regions.

The mature 3'-end of many chloroplast mRNAs is generated by the processing of the 3'-untranslated region (3'-UTR), which is a mechanism that involves the removal of a segment located downstream an inverted repeat sequence that forms a stem-loop structure. Nuclear-encoded chloroplast RNA binding proteins associate with the stem-loop to process the 3'-UTR or to influence mRNA stability. A spinach chloroplast processing extract (CPE) has been previously generated and used to in vitro dissect the biochemical mechanism underlying 3'-UTR processing. Being Arabidopsis thaliana an important genetic model, the development of a CPE allowing to correlate 3'-UTR processing activity with genes encoding proteins involved in this process, would be of great relevance. Here, we developed a purification protocol that generated an Arabidopsis CPE able to correctly process a psbA 3'-UTR precursor. By UV crosslinking, we characterized the protein patterns generated by the interaction of RNA binding proteins with Arabidopsis psbA and petD 3'-UTRs, finding that each 3'-UTR bound specific proteins. By testing whether Arabidopsis CPE proteins were able to bind spinach ortholog 3'-UTRs, we also found they were bound by specific proteins. When Arabidopsis CPE 3'-UTR processing activity on ortholog spinach 3'-UTRs was assessed, stable products appeared: for psbA, a smaller size product than the expected mature 3'-end, and for petD, low amounts of the expected product plus several others of smaller sizes. These results suggest that the 3'-UTR processing mechanism of these chloroplast mRNAs might be partially conserved in Arabidopsis and spinach.