Arabidopsis (Arabidopsis thaliana) plants can produce photosynthetic tissue with active chloroplasts at temperatures as low as 4°C, and this process depends on the presence of the nuclear-encoded, chloroplast-localized RNA-binding protein CP29A. In this study, we demonstrate that CP29A undergoes phase separation in vitro and in vivo in a temperature-dependent manner, which is mediated by a prion-like domain (PLD) located between the two RNA recognition motif (RRM) domains of CP29A. The resulting droplets display liquid-like properties and are found near chloroplast nucleoids. The PLD is required to support chloroplast RNA splicing and translation in cold-treated tissue. Together, our findings suggest that plant chloroplast gene expression is compartmentalized by inducible condensation of CP29A at low temperatures, a mechanism that could play a crucial role in plant cold resistance.

Chloroplast RNA processing requires a large number of nuclear-encoded RNA binding proteins (RBPs) that are imported post-translationally into the organelle. Most of these RBPs are highly specific for one or few target RNAs. By contrast, members of the chloroplast ribonucleoprotein family (cpRNPs) have a wider RNA target range. We here present a quantitative analysis of RNA targets of the cpRNP CP31A using digestion-optimized RNA co-immunoprecipitation with deep sequencing (DO-RIP-seq). This identifies the mRNAs coding for subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex as main targets for CP31A. We demonstrate using whole-genome gene expression analysis and targeted RNA gel blot hybridization that the ndh mRNAs are all down-regulated in cp31a mutants. This diminishes the activity of the NDH complex. Our findings demonstrate how a chloroplast RNA binding protein can combine functionally related RNAs into one post-transcriptional operon.

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, Ribulose-1,5-bisphosphat-carboxylase/-oxygenase (RuBisCo). Its expression is predominantly regulated posttranscriptionally, 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 found that CP29A preferentially binds to the 5′-untranslated region of rbcL , downstream of the binding site of the pentatricopeptide repeat protein MATURATION OF RBCL 1 (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 CP29A’s 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 indicates that a chloroplast RNA–binding protein contributes to cold acclimation of RbcL production.

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 the vast majority of the psbA transcript pool. The RNA sequence target motif, determined in vitro, does not fully explain CP33B’s preference for psbA, suggesting that there are other determinants of specificity in vivo.

Arabidopsis thaliana is capable of producing photosynthetic tissue with active chloroplasts at temperatures as low as 4°C, and this process depends on the presence of the nuclear-encoded, chloroplast-localized RNA-binding protein CP29A. In this study, we demonstrate that CP29A undergoes phase separation in vitro and in vivo in a temperature-dependent manner, which is mediated by a prion-like domain (PLD) located between the two RNA recognition motif (RRM) domains of CP29A. The resulting droplets display liquid-like properties and are found in close proximity to chloroplast nucleoids. The PLD is required to support chloroplast RNA splicing and translation in cold-treated tissue. Together, our findings suggest that plant chloroplast gene expression is compartmentalized by inducible condensation of CP29A at low temperatures, a mechanism that could play a crucial role for plant cold resistance.

Flowering that requires a period of cold (vernalization) is a key trait in many crops, but the underlying regulatory pathways are often poorly understood. Taraxacum koksaghyz is a rubber-producing dandelion of the family Asteraceae, which also includes other economically important crops such as chicory and lettuce. Most T. koksaghyz plants require cold exposure to induce flowering, but plants that have lost the dependence on vernalization are more suitable for domestication and breeding. To provide insight into the molecular basis of mandatory vernalization in T. koskaghyz , we identified three FLOWERING LOCUS T ( FT ) homologs ( TkFT1–3 ) that are differentially expressed under varying environmental conditions. TkFT1 and TkFT2 are expressed under long-day conditions at ambient temperatures whereas only TkFT1 is weakly expressed under short-day conditions. Exposure to cold was shown to repress TkFT1 but induce TkFT3 . Overexpression experiments revealed that TkFT1–3 bypass the vernalization requirement in T. koksaghyz and its close relative T. officinale , and promote early flowering in vernalization-independent T. brevicorniculatum . We also identified two FRUITFULL homologs ( TkFUL1 and TkFUL2 ) as downstream targets, which were upregulated in TkFT overexpression lines. Our findings suggest that TkFT1 promotes vernalization-independent flowering, whereas TkFT3 expression during the cold period is needed to promote vernalization-dependent flowering. This study explicates the regulatory network controlling flowering time in T. koksaghyz , contributing to a broader understanding of flowering in the family Asteraceae and providing knowledge that can be used in the future to facilitate domestication and breeding. Key message: In Taraxacum koksaghyz , FT homologs function as floral inducers that upregulate FRUITFULL homologs, with TkFT1 linked to vernalization-independent flowering and TkFT3 associated with the acquisition of flowering competence during vernalization.