ABA-responsive RNA-binding proteins are involved in chloroplast and stromule function in Arabidopsis seedlings

The phytohormone abscisic acid (ABA) regulates essential growth and developmental processes in plants. Recently, RNA-binding proteins have been described as components of ABA signaling during germination. We have identified ten ABA-regulated RNA-binding proteins in Arabidopsis seedlings. Among those genes, AtCSP41B and cpRNP29 are highly expressed in seedlings. Using promoter:reporter gene analyses, we showed that both AtCSP41B and cpRNP29 were in particular expressed in photosynthetically active organs like green cotyledons, leaves, and petioles. The analysis of CFP-fusion proteins demonstrates that cpRNP29 localized to chloroplasts and AtCSP41B to chloroplasts and stromules. Whereas RNA-binding of cpRNP29 has previously been shown, we demonstrated through in vitro RNA-binding assays that recombinant AtCSP41B binds to RNA, and that chloroplast petD RNA can serve as a target of AtCSP41B. Developmental or environmental stimuli affected the expression of AtCSP41B and cpRNP29 in seedlings. Both genes were repressed during senescence, but only AtCSP41B was significantly repressed upon water stress. In addition, AtCSP41B and cpRNP29 exhibited low expression in etiolated seedlings compared to green seedlings, and cpRNP29 was regulated during the day photoperiod. Homozygous T-DNA insertion lines were isolated, characterized on the molecular level, and monitored for phenotypic changes. Taken together, the data show that both proteins are regulated during processes that are known to involve ABA signaling. Their localization in chloroplasts and RNA-binding activity suggest a role in chloroplast RNA metabolism in Arabidopsis seedlings.     

Multiple plant RNA binding proteins identified by PCR: expression of cDNAs encoding RNA binding proteins targeted to chloroplasts in Nicotiana plumbaginifolia

Summary Pre-mRNA processing in eukaryotic cells requires the participation of multiple protein factors and ribonucleoprotein particles. One class of proteins involved in this process are RNA-binding proteins, which contain a domain of ca. 90 amino acids with a characteristic ribonucleoprotein consensus sequence (RNP-CS). A PCR approach that is suitable for the characterization of RNP-CS-type proteins is described. Fifteen different RNA-binding domains were amplified from Nicotiana tabacum (tobacco) using oligonucleotide primers specific for the sequences (K/R)G(F/Y)(G/A)FVX(F/Y) and (L/I/V)(F/Y)(V/I)(G/K)(N/G)L, which are conserved in known RNP-CS proteins. Using the tobacco domains as probes, cDNAs encoding two RNA-binding proteins, each containing two RNP-CS-type domains, were characterized in N. plumbaginifolia. The proteins, designated CP-RBP30 and CP-RBP31, are targeted to chloroplasts as demonstrated by expression of epitope-tagged cDNAs in transfected protoplasts, followed by indirect immunofluorescence. High levels of mRNA for each protein were found in leaves but not in roots, and expression of the CP-RBP31 mRNA was strongly regulated by light. The N. plumbaginifolia proteins described in this work are distinct from chloroplast RNA-binding proteins characterized recently in tobacco and spinach.     

The cp genome characterization of Adenium obesum: Gene content,repeat organization and phylogeny

Adenium obesum (Forssk.) Roem. & Schult. belonging to the family Apocynaceae, is remarkable for its horticultural and ornamental values, poisonous nature, and medicinal uses. In order to have understanding of cp genome characterization of highly valued medicinal plant, and the evolutionary and systematic relationships, the complete plastome / chloroplast (cp) genome of A. obesum was sequenced. The assembled cp genome of A. obesum was found to be 154,437 bp, with an overall GC content of 38.1%. A total of 127 unique coding genes were annotated including 96 protein-coding genes, 28 tRNA genes, and 3 rRNA genes. The repeat structures were found to comprise of only mononucleotide repeats. The SSR loci are compososed of only A/T bases. The phylogenetic analysis of cp genomes revealed its proximity with Nerium oleander.     

Characterization of the chloroplast genome sequence of oil palm (Elaeis guineensis Jacq.)

Oil palm (Elaeis guineensis Jacq.) is an economically important crop, which is grown for oil production. To better understand the molecular basis of oil palm chloroplasts, we characterized the complete chloroplast (cp) genome sequence obtained from 454 pyrosequencing. The oil palm cp genome is 156,973 bp in length consisting of a large single-copy region of?85,192 bp flanked on each side by inverted repeats of 27,071 bp with a small single-copy region of 17,639 bp joining the?repeats. The genome contains 112 unique genes: 79 protein-coding genes, 4 ribosomal RNA genes and 29 tRNA genes. By aligning the cp?genome sequence with oil palm cDNA sequences, we observed 18 non-silent and 10 silent RNA editing events among 19 cp protein-coding genes. Creation of an initiation codon by RNA editing in rpl2 has been reported in several monocots and was also found in the oil palm cp genome. Fifty common chloroplast protein-coding genes from 33 plant taxa were used to construct ML and MP?phylogenetic trees. Their topologies are similar and strongly support for the position of E. guineensis as the sister of closely related species Phoenix dactylifera in Arecaceae (palm families) of monocot subtrees.     

Involvement of a site-specific trans-acting factor and a common RNA-binding protein in the editing of chloroplast mRNAs: development of a chloroplast in vitro RNA editing system

RNA editing in higher plant chloroplasts involves C-->U conversion at approximately 30 specific sites. An in vitro system supporting accurate editing has been developed from tobacco chloroplasts. Mutational analysis of substrate mRNAs derived from tobacco chloroplast psbL and ndhB mRNAs confirmed the participation of cis-acting elements that had previously been identified in vivo. Competition analysis revealed the existence of site-specific trans-acting factors interacting with the corresponding upstream cis-elements. A chloroplast protein of 25 kDa was found to be specifically associated with the cis-element involved in psbL mRNA editing. Immunological analyses revealed that an additional factor, the chloroplast RNA-binding protein cp31, is also required for RNA editing at multiple sites. This combination of site-specific and common RNA-binding proteins recognizes editing sites in chloroplasts.     

Complete chloroplast genome sequences of Dipterygium glaucum and Cleome chrysantha and other Cleomaceae Species,comparative analysis and phylogenetic relationships

This current study presents, for the first time, the complete chloroplast genome of two Cleomaceae species: Dipterygium glaucum and Cleome chrysantha in order to evaluate the evolutionary relationship. The cp genome is 158,576 bp in length with 35.74% GC content in D. glaucum and 158,111 bp with 35.96% GC in C. chrysantha. Inverted repeats IR 26,209 bp, 26,251 bp each, LSC of 87,738 bp, 87,184 bp and SSC of 18,420 bp, 18,425 bp respectively. There are 136 genes in the genome, which includes 80 protein coding genes, 31 tRNA genes and four rRNA genes were observed in both chloroplast genomes. 117 genes are unique while the remaining 19 genes are duplicated in IR regions. The analysis of repeats shows that the cp genome includes all types of repeats with more frequent occurrences of palindromic; Also, this analysis indicates that the total number of simple sequence repeats (SSR) were 323 in D. glaucum, and 313 in C. chrysantha, of which the majority of the SSRs in these plastid genomes were mononucleotide repeats A/T which are located in the intergenic spacer. Moreover, the comparative analysis of the four cp sequences revealed four hotspot genes (atpF, rpoC2, rps19, and ycf1), these variable regions could be used as molecular makers for the species authentication as well as resources for inferring phylogenetic relationships of the species. All the relationships in the phylogenetic tree are with high support, this indicate that the complete chloroplast genome is a useful data for inferring phylogenetic relationship within the Cleomaceae and other families. The simple sequence repeats identified will be useful for identification, genetic diversity, and other evolutionary studies of the species. This study reported the first cp genome of the genus Dipterygium and Cleome. The finding of this study will be beneficial for biological disciplines such as evolutionary and genetic diversity studies of the species within the core Cleomaceae.     

Constancy of organellar genome copy numbers during leaf development and senescence in higher plants

In higher plants, plastid and mitochondrial genomes occur at high copy numbers per cell. Several recent publications have suggested that, in higher plants like Arabidopsis and maize, chloroplast DNA is virtually absent in mature and old leaves. This conclusion was mainly based on DAPI staining of isolated chloroplasts. If correct, the finding that chloroplasts in mature leaves lack DNA would change dramatically our understanding of gene expression, mRNA stability and protein stability in chloroplasts. In view of the wide implications that the disposal of chloroplast DNA during leaf development would have, we have reinvestigated the age dependency of genome copy numbers in chloroplasts and, in addition, tested for possible changes in mitochondrial genome copy number during plant development. Analyzing chloroplast and mitochondrial DNA amounts in Arabidopsis and tobacco plants, we find that organellar genome copy numbers remain remarkably constant during leaf development and are present in essentially unchanged numbers even in the senescing leaves. We conclude that, during leaf development, organellar gene expression in higher plants is not significantly regulated at the level of genome copy number and we discuss possible explanations for the failure to detect DNA in isolated chloroplasts stained with DAPI.     

Effect of salt stress on genes encoding translation-associated proteins in Arabidopsis thaliana

Salinity negatively affects plant growth and disturbs chloroplast integrity. Here, we aimed at identifying salt-responsive translation-related genes in Arabidopsis thaliana with an emphasis on those encoding plastid-located proteins. We used quantitative real-time PCR to test the expression of 170 genes after short-term salt stress (up to 24 h) and identified several genes affected by the stress including: PRPL11, encoding plastid ribosomal protein L11, ATAB2, encoding a chloroplast-located RNA-binding protein presumably functioning as an activator of translation, and PDF1B, encoding a peptide deformylase involved in N-formyl group removal from nascent proteins synthesized in chloroplasts. These genes were previously shown to have important functions in chloroplast biology and may therefore represent new targets for biotechnological optimization of salinity tolerance.     

Chloroplast ribonucleoprotein-like proteins of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis> are not involved in RNA stability and RNA editing

Many RNA recognition motif (RRM)-containing proteins are known to exist in chloroplasts. Major members of the RRM protein family, which are chloroplast ribonucleoproteins (cpRNPs), have been investigated in seed plants, including tobacco and Arabidopsis thaliana, but never in early land plants, such as bryophytes. In this study, we surveyed RRM proteins encoded in the moss Physcomitrella patens genome and predicted 25 putative chloroplast RRM proteins. Among them, two RRM-containing proteins, PpRBP2a and PpRBP2b, resembled cpRNPs and were thus referred to as cpRNP-like proteins. However, knockout mutants of either one or two PpRBP2 genes exhibited a wild type-like phenotype. Unlike Arabidopsis cpRNPs, the levels of mRNA accumulation in chloroplasts were not affected in the PpRBP2 knockout mutants. In addition, the efficiency of RNA editing was also not altered in the mutants. This suggests that PpRBP2a and 2b may be functionally distinct from Arabidopsis cpRNPs.     

药用植物华重楼(黑药花科)叶绿体全基因组研究

为探究华重楼(Paris polyphylla var. chinensis)的叶绿体基因组特征,利用叶绿体系统发育基因组学方法,对华重楼与其它百合目植物的叶绿体全基因组进行了比较。结果表明,华重楼的叶绿体全基因组长158307 bp,由4个区组成,包括2个反向重复区(IRA和IRB,27473 bp)、1个小单拷贝区(SSC,18175 bp)和1个大单拷贝区(LSC,85187 bp)。其叶绿体基因组有115个基因,包括81个编码蛋白质基因、30个转运RNA基因和4 个核糖体RNA基因。11种百合目植物的叶绿体全基因组的基因组成和基因顺序相似。华重楼的cemA基因是假基因,其起始密码子后有多聚核苷酸poly(A)及CA双核苷酸重复序列,编码序列中出现多个终止密码子, 且与北重楼(Paris verticillata)的cemA编码序列中的终止密码子位置不同。因此,华重楼叶绿体基因组比较保守;cemA结构及假基因化现象可能具有重要的进化与系统发育信息,其编码序列中的终止密码子可以区分华重楼和北重楼。     

The Complete Chloroplast Genome Sequence of the Medicinal Plant Salvia miltiorrhiza

Salvia miltiorrhiza is an important medicinal plant with great economic and medicinal value. The complete chloroplast (cp) genome sequence of Salvia miltiorrhiza, the first sequenced member of the Lamiaceae family, is reported here. The genome is 151,328 bp in length and exhibits a typical quadripartite structure of the large (LSC, 82,695 bp) and small (SSC, 17,555 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,539 bp). It contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs and four rRNAs. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Four forward, three inverted and seven tandem repeats were detected in the Salvia miltiorrhiza cp genome. Simple sequence repeat (SSR) analysis among the 30 asterid cp genomes revealed that most SSRs are AT-rich, which contribute to the overall AT richness of these cp genomes. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the non-coding regions, indicating an uneven distribution of SSRs within the cp genomes. Entire cp genome comparison of Salvia miltiorrhiza and three other Lamiales cp genomes showed a high degree of sequence similarity and a relatively high divergence of intergenic spacers. Sequence divergence analysis discovered the ten most divergent and ten most conserved genes as well as their length variation, which will be helpful for phylogenetic studies in asterids. Our analysis also supports that both regional and functional constraints affect gene sequence evolution. Further, phylogenetic analysis demonstrated a sister relationship between Salvia miltiorrhiza and Sesamum indicum. The complete cp genome sequence of Salvia miltiorrhiza reported in this paper will facilitate population, phylogenetic and cp genetic engineering studies of this medicinal plant.     

De novo assembly and characterization of the complete chloroplast genome of radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) is an edible root vegetable crop that is cultivated worldwide and whose genome has been sequenced. Here we report the complete nucleotide sequence of the radish cultivar WK10039 chloroplast (cp) genome, along with a de novo assembly strategy using whole genome shotgun sequence reads obtained by next generation sequencing. The radish cp genome is 153,368 bp in length and has a typical quadripartite structure, composed of a pair of inverted repeat regions (26,217 bp each), a large single copy region (83,170 bp), and a small single copy region (17,764 bp). The radish cp genome contains 87 predicted protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Sequence analysis revealed the presence of 91 simple sequence repeats (SSRs) in the radish cp genome.