Biology subjects

Ruwe, H.

Publications and source records attributed to Ruwe, H..

5 recordsLinked to original sources

bioRxiv2026

Virus-induced genome editing in the parasitic plant Phtheirospermum japonicum

Phtheirospermum japonicum is a genetic model for parasitic Orobanchaceae, a plant family that includes noxious parasitic weeds from the genera Striga, Orobanche, and Phelipanche (Ishida et al., 2011). Striga species alone cause billions of dollars in annual losses by reducing yields of major crops (Pennisi, 2010). The lack of stable transgenesis protocols often hinders heritable CRISPR/Cas9 genome editing for gene function analysis in crops and species beyond standard model plants, including parasitic Orobanchaceae (Steinberger and Voytas, 2025). Here, we adapted a virus-mediated delivery system for ultracompact TnpB nucleases, enabling genome editing independently of tissue regeneration or floral dip transformation in the parasitic plant P. japonicum (Nagalakshmi et al., 2025; Weiss et al., 2025).

plant biology↗

bioRxiv2023

A role for chloroplast RNA binding protein CP29A in rbcL expression during cold acclimation

The chloroplast genome encodes key components of the photosynthetic light reaction machinery as well as the large subunit of the enzyme central for carbon fixation, RuBisCo. Its expression is predominantly regulated post-transcriptionally, with nuclear-encoded RNA binding proteins (RBPs) playing a key role. Mutants of chloroplast gene expression factors often exhibit impaired chloroplast biogenesis, especially in cold conditions. Low temperatures pose a challenge for plants as this leads to electron imbalances and oxidative damage. A well-known response of plants to this problem is to increase the production of RuBisCo and other Calvin Cycle enzymes in the cold, but how this is achieved is unclear. The chloroplast RBP CP29A has been shown to be essential for cold resistance in growing leaf tissue of Arabidopsis thaliana. Here, we examined CP29A-RNA interaction sites at nucleotide resolution. We discovered that CP29A preferentially binds to the 5-UTR of rbcL, downstream of the binding site of the pentatricopeptide repeat (PPR) protein MRL1. MRL1 is an RBP known to be necessary for the accumulation of rbcL. In Arabidopsis mutants lacking CP29A, we were unable to observe significant effects on rbcL, possibly due to CP29As restricted role in a limited number of cells at the base of leaves. In contrast, CRISPR/Cas9-induced mutants of tobacco NtCP29A exhibit cold-dependent photosynthetic deficiencies throughout the entire leaf blade. This is associated with a parallel reduction in rbcL mRNA and RbcL protein accumulation. Our work unravels the molecular player behind cold acclimation of the photosynthetic dark reaction. Significance StatementThis study unveils the critical role of CP29A, a chloroplast-localized RNA binding protein, in facilitating plants acclimation to cold environments. Through advanced molecular techniques, we discovered that CP29A specifically targets the rbcL mRNA, vital for the production of RuBisCo--a key enzyme in photosynthesis and the most abundant protein on Earth. Our findings elucidate a previously unknown mechanism of how plants adjust to cold stress by regulating RuBisCo levels, highlighting the intricate interplay between nuclear and chloroplast genomes. This research not only advances our understanding of plant cold acclimation but also provides insights that could help enhance plant resilience and productivity when facing temperature challenges.

plant biology↗

bioRxiv2023

MatK impacts Differential Chloroplast Translation of Ribosomal and Photosynthetic genes by limiting spliced tRNA-K(UUU) abundance

The protein levels of chloroplast photosynthetic genes and genes related to the chloroplast genetic apparatus vary to adapt to different conditions. However, the underlying mechanisms governing these variations remain unclear. The chloroplast intron Maturase K is encoded within the trnK intron and has been suggested to be required for splicing several group IIA introns, including the trnK intron. In this study, we employed RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq) to identify MatKs preference for binding to group IIA intron domains I and VI within target transcripts. Importantly, these domains are crucial for branch point selection, and we discovered alternative branch points in three MatK target introns, the first observed instances of alternative splicing in chloroplasts. The alternative trnK lariat structure showed increased accumulation during heat acclimation. The cognate codon of tRNA-K(UUU) is highly enriched in mRNAs encoding ribosomal proteins and ribosome profiling in a trnK-matK over-expressor exhibited elevated levels of the spliced tRNA-K(UUU). Our analysis revealed a significant up-shift in the translation of ribosomal proteins compared to photosynthetic genes. Our findings suggest the existence of a novel regulatory mechanism linked to the abundance of tRNA-K(UUU), enabling the differential expression of functional chloroplast gene groups.

plant biology↗

bioRxiv2021

The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs involved in Chloroplast Development

The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7 or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL and their barley homologs and to the function of the putative cTPs of CIA2 and CIL. HIGHLIGHTThe nucleus-localized CCT domain proteins CIA2 and CIL in Arabidopsis and the homologous chloroplast-localized HvCMF3 and HvCMF7 in barley retained partially overlapping functions in chloroplast development.

plant biology↗

bioRxiv2020

The chloroplast ribonucleoprotein CP33B quantitatively binds the psbA mRNA

Chloroplast RNAs are stabilized and processed by a multitude of nuclear-encoded RNA binding proteins, often in response to external stimuli like light and temperature. A particularly interesting RNA based regulation occurs with the psbA mRNA, which shows light-dependent translation. Recently, the chloroplast ribonucleoprotein CP33B was identified as a ligand of the psbA mRNA. We here characterized the interaction of CP33B with chloroplast RNAs in greater detail using a combination of RIP-chip, quantitative dot-blot, and RNA-Bind-n-Seq experiments. We demonstrate that CP33B prefers psbA over all other chloroplast RNAs and associates with vast majority of the psbA transcript pool. The RNA sequence target motif determined in vitro does not fully explain CP33Bs preference for psbA, suggesting that there are other determinants of specificity in vivo.

molecular biology↗