Comprehensive Analysis of Plastid Gene Expression During Fruit Development and Ripening of Kiwifruit

A serious limitation in the application of plastid biotechnology is the low-level expression of transgene in non-green plastids like chromoplasts compared with photosynthetically active chloroplasts. Unlike other fruits, not all chloroplasts are transformed into chromoplast during ripening of red-fleshed kiwifruit ( Actinidia chinensis vs Hongyang) fruits, which may make kiwifruit as an ideal horticultural plant for oral vaccine production by plastid engineering. To identify cis -elements potentially triggering high-level transgene expression in edible tissues of the ‘Hongyang’ kiwifruit, here we report a comprehensive analysis of kiwifruit plastid gene transcription in the green leaves and fruits at three different developing stages. While transcripts of a few photosynthesis-related genes and most genetic system genes were substantially upregulated in green fruits compared with leaves, nearly all plastid genes were significantly downregulated at the RNA level during fruit development. Expression of a few genes remained unchanged, including psbA , the gene encoding the D1 polypeptide of photosystem II. However, PsbA protein accumulation decreased continuously during chloroplast-to-chromoplast differentiation. Analysis of post-transcriptional steps in mRNA maturation, including intron splicing and RNA editing, revealed that splicing and editing may contribute to regulating plastid gene expression. Altogether, 40 RNA editing sites were verified, and five of them were newly discovered. Taken together, this study has generated a valuable resource for the analysis of plastid gene expression, and provides cis -elements for future efforts to engineer the plastid genome of kiwifruit.

New molecular and phenotypic data on Mayamaea Arida (Bock) Lange-Bertalot (Bacillariophyta)

Mayamaea arida (Bock) Lange-Bertalot is a rare species, previously recorded from four locations in Europe only. The aim of this study is to report additional molecular, phenotypic and geographic data on M. arida. The strain of this species was isolated from the soil sample collected on 04.08.2018 in Iturup Island (Sakhalin Region, Russia) in Haplic Cambisol. The strain was identified using light and scanning electron microscopy. Phylogenetic analysis revealed that the M. arida was a member of the strongly supported Mayamaea generic clade and resolved as a basal lineage in the subclade comprised by M. atomus and M. terrestris. New data on partial rbcL plastid gene, chloroplasts morphology and nucleus, lower limit of the cell length, higher number of striae in 10 urn, shape index (length/width), teratological forms and distribution of species are presented.

Full Length Transcriptome Highlights the Coordination of Plastid Transcript Processing

Plastid gene expression involves many post-transcriptional maturation steps resulting in a complex transcriptome composed of multiple isoforms. Although short-read RNA-Seq has considerably improved our understanding of the molecular mechanisms controlling these processes, it is unable to sequence full-length transcripts. This information is crucial, however, when it comes to understanding the interplay between the various steps of plastid gene expression. Here, we describe a protocol to study the plastid transcriptome using nanopore sequencing. In the leaf of Arabidopsis thaliana, with about 1.5 million strand-specific reads mapped to the chloroplast genome, we could recapitulate most of the complexity of the plastid transcriptome (polygenic transcripts, multiple isoforms associated with post-transcriptional processing) using virtual Northern blots. Even if the transcripts longer than about 2,500 nucleotides were missing, the study of the co-occurrence of editing and splicing events identified 42 pairs of events that were not occurring independently. This study also highlighted a preferential chronology of maturation events with splicing happening after most sites were edited.

Phosphatidylglycerol synthesis facilitates plastid gene expression and light induction of nuclear photosynthetic genes

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development with decreased chlorophyll accumulation, impaired thylakoid formation, and downregulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects the gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated the growth and transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species-responsive genes were upregulated in pgp1-2. Decreased growth light did not alleviated the impaired leaf development and the downregulation of photosynthesis-associated genes in pgp1-2, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered downregulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor regulating chloroplast development, in pgp1-2 upregulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in the plastid gene expression and plant development.

A hybrid capture RNA bait set for resolving genetic and evolutionary relationships in angiosperms from deep phylogeny to intraspecific lineage hybridization

Novel multi-gene targeted capture probes have been developed with the objective of obtaining multi-locus high quality sequence reads across any angiosperm lineage. Using existing genomic and transcriptomic data, two independent single assay probe/bait sets have been developed, the first targeting conserved exons from 20 low copy nuclear genes (OzBaits_NR V1.0) and the second, 19 plastid gene regions (OZBaits_CP V1.0). These universal bait sets can efficiently generate DNA sequence data that are suitable for systematics and evolutionary studies of flowering plants. The bait sets can be ordered as Daicel-Arbor Sciences custom myBaits. We demonstrate the utility of the bait set in consistently recovering the targeted genomic regions across an evolutionarily broad range of angiosperm taxa.

Full Length Transcriptome Highlights the Coordination of Plastid Transcript Processing

Plastid gene expression involves many post-transcriptional maturation steps resulting in a complex transcriptome composed of multiple isoforms. Although short read RNA-seq has considerably improved our understanding of the molecular mechanisms controlling these processes, it is unable to sequence full-length transcripts. This information is however crucial when it comes to understand the interplay between the various steps of plastid gene expression. Here, the study of the Arabidopsis leaf plastid transcriptome using Nanopore sequencing showed that many splicing and editing events were not independent but co-occurring. For a given transcript, maturation events also appeared to be chronologically ordered with splicing happening after most sites are edited.

Guanosine tetraphosphate (ppGpp) accumulation inhibits chloroplast gene expression and promotes super grana formation in the moss Physcomitrium (Physcomitrella) patens

The nucleotides guanosine tetraphosphate and pentaphosphate (or ppGpp) are implicated in the regulation of chloroplast function in plants. ppGpp signalling is best understood in the model vascular plant Arabidopsis thaliana where it acts to regulate plastid gene expression to influence photosynthesis, plant development and immunity. However, little is known about the conservation or diversity of ppGpp signaling in other land plants. Here, we studied the function of ppGpp in the moss Physcomitrium (previously Physcomitrella) patens using an inducible system for triggering ppGpp accumulation. We used this approach to investigate the effects of ppGpp on chloroplast function, photosynthesis and growth. We demonstrate that ppGpp accumulation causes a dramatic drop in photosynthetic capacity by inhibiting chloroplast gene expression. This was accompanied by the unexpected reorganisation of the thylakoid system into super grana. Surprisingly, these changes did not affect gametophore growth, suggesting that bryophytes and vascular plants may have different tolerances to defects in photosynthesis. Our findings point to the existence of both highly conserved and more specific targets of ppGpp signalling in the land plants that may reflect different growth strategies.