Biology subjects

Kleine, T.

Publications and source records attributed to Kleine, T..

8 recordsLinked to original sources

bioRxiv2024

Arabidopsis GENOMES UNCOUPLED PROTEIN1 binds to plastid RNAs and promotes their maturation

Plastid biogenesis and the coordination of plastid and nuclear genome expression through anterograde and retrograde signaling are essential for plant development. GENOMES UNCOUPLED1 (GUN1) plays a central role in retrograde signaling during early plant development. The putative function of GUN1 has been extensively studied, but its molecular function remains controversial. Here, we evaluate published transcriptome data and generate our own data from gun1 mutants grown under signaling relevant conditions to show that editing and splicing are not relevant for GUN1-dependent retrograde signaling. Our study of the plastid (post)-transcriptome of gun1 seedlings with white and pale cotyledons demonstrates that GUN1 deficiency significantly alters the entire plastid transcriptome. By combining this result with a PPR code-based prediction and experimental validation by RNA immunoprecipitation experiments, several targets of GUN1 were identified, including 23S rRNA, tRNAs and RNAs derived from ycf1.2 and the ndhH-ndhA-ndhI-ndhG-ndhE-psaC-ndhD gene cluster. The absence of plastid rRNAs and the significant reduction of almost all plastid transcripts in white gun1 mutants account for the cotyledon phenotype. Our study identifies RNA binding and maturation as the long-sought molecular function of GUN1 and resolves long-standing controversies. We anticipate that our findings will serve as a basis for subsequent studies investigating the mechanism of plastid gene expression and will facilitate the elucidation of GUN1s function in retrograde signaling.

plant biology↗

bioRxiv2023

A pgr5 suppressor screen uncovers a distinct mechanism safeguarding the cytochrome b6f complex from damage through PGR5

PROTON GRADIENT REGULATION5 (PGR5) is thought to promote cyclic electron flow (CEF) and its deficiency causes increased photosensitivity of photosystem I (PSI), leading to lethality under fluctuating light (FL). By screening for suppressor mutations that rescue FL lethality of pgr5 plants, we identified a portfolio of mutations affecting 12 photosynthesis-related proteins. Six are required for proper PSII function, one (CcdA) promotes cytochrome (cyt) b6f assembly, and another (PAA1) provides plastocyanin with its copper cofactor. Two other mutations are associated with the chloroplast FBPase cFBP1. This, together with targeted knockout of other genes in the pgr5 background, suggests three pathways to restore FL viability: (i) reduced electron flow to PSI due to defects in PSII, cyt b6f or plastocyanin but not PSI, (ii) increased electron flow from PSI due to inactivation of ACHT2, a regulator of cFBP1 activity, and (iii) hyperactivity of the NDH-dependent CEF due to inactivation of cFBP1. The remaining two suppressor mutations affected the cyt b6f complex. PFSC1 controls cyt b6f accumulation at early developmental stages. DEIP1/NTA1, previously suggested to be essential for cyt b6f assembly, appears to protect cyt b6f from deleterious effects of PGR5, since plants lacking both DEIP1/NTA1 and PGR5 are viable and accumulate cyt b6f.

plant biology↗

bioRxiv2023

Identification of a highly drought-resistant pp7l hda6 mutant

Plants have evolved efficient strategies to cope with drought stress, including stomata closure, significant changes in nuclear gene expression, and epigenetic mechanisms. Previously, we identified Arabidopsis thaliana PROTEIN PHOSPHATASE7-LIKE (PP7L) as an extrachloroplastic protein that promotes chloroplast development and high light, and salt tolerance. Here, we demonstrate that the pp7l mutant can withstand prolonged periods of drought stress. Interestingly, chloroplast development in pp7l recovers under drought conditions, despite growth of the mutant is impaired under normal growth conditions. To assess the (post)transcriptional changes occurring in the pp7l mutant under different durations of drought exposure, we used long non-coding RNA-sequencing. Compared to the previously reported drought-responsive changes in the wild type, the drought-responsive changes detected in the pp7l mutant were negligible. Our analysis of data generated in this study and previously motivated us to create a pp7l hda6 mutant, which exhibits remarkable drought resistance. Notably, the growth penalty associated with pp7l was alleviated in the double mutant, ruling out a dwarf effect on the drought-tolerant trait of this genotype.

plant biology↗

bioRxiv2023

Natural genetic variation in GLK1-mediated photosynthetic acclimation in response to light

GOLDEN-like (GLK) transcription factors are central regulators of chloroplast biogenesis in Arabidopsis and other species. Findings from Arabidopsis show that these factors also contribute to photosynthetic acclimation, e.g. to variation in light intensity, and are controlled by retrograde signals emanating from the chloroplast. However, the natural variation of GLK1-centered gene-regulatory networks is largely unexplored. By evaluating the activities of GLK1 target genes and GLK1 itself in vegetative leaves of natural Arabidopsis accessions grown under standard conditions, we uncovered a remarkable variation in the activity of GLK1 centered regulatory networks. This is linked with ecogeographic origin of the accessions, and can be associated with a complex genetic variation across loci acting in different functional pathways, including photosynthesis, ROS and brassinosteroid pathways. Our results identify candidate upstream regulators that contribute to GLK1 activity in rosette leaves. Indeed, accessions with higher GLK1 activity, arising from habitats with a high monthly variation in solar radiation levels, may show lower levels of photoinhibition at higher light intensities. Our results provide evidence for natural variation in GLK1 regulatory activities in vegetative leaves. This variation is associated with ecogeographic origin and can contribute to acclimation to high light conditions.

plant biology↗

bioRxiv2023

Spliceosomal complex components are critical for adjusting the C:N balance during high-light acclimation

Plant acclimation to an ever-changing environment is decisive for growth, reproduction and survival. Light availability limits biomass production on both ends of the intensity spectrum. Therefore, the adjustment of plant metabolism is central to high-light (HL) acclimation, and accumulation of photoprotective anthocyanins is commonly observed. However, mechanisms and factors regulating the HL acclimation response are less clear. Two Arabidopsis mutants of spliceosome components exhibiting a pronounced anthocyanin overaccumulation in HL were isolated from a forward genetic screen for new factors crucial for plant acclimation. Time-resolved physiological, transcriptome and metabolome analysis revealed a vital function of the spliceosome components for rapidly adjusting gene expression and metabolism. Deficiency of INCREASED LEVEL OF POLYPLOIDY1 (ILP1), NTC-RELATED PROTEIN1 (NTR1), and PLEIOTROPIC REGULATORY LOCUS1 (PRL1) resulted in a marked overaccumulation of carbohydrates and strongly diminished amino acid biosynthesis in HL. While not generally limited in N-assimilation, ilp1, ntr1, and prl1 mutants showed higher glutamate levels and reduced amino acid biosynthesis in HL. The comprehensive analysis reveals a function of the spliceosome in the conditional regulation of the carbon:nitrogen-balance and the accumulation of anthocyanins during HL acclimation. The importance of gene expression, metabolic regulation, and re-direction of carbon towards anthocyanin biosynthesis for HL acclimation are discussed.

molecular biology↗

bioRxiv2023

Arabidopsis thaliana BBX14 is a target of GLK1 and involved in high-light acclimation, photomorphogenesis and GUN-type retrograde signaling

Development of photosynthetically competent seedlings requires both light and retrograde biogenic signaling pathways. The transcription factor GLK1 functions at the interface between these pathways, and receives input from the biogenic-signaling integrator GUN1. BBX14 was previously identified, together with GLK1, in a core module that mediates the response to high light levels and biogenic signaling. To gain insight into the function of BBX14, we generated BBX14 overexpressors and CRISPR/Cas-mediated bbx14 mutant plants, conducted high-light, RT-qPCR and ChIP-Seq experiments, measured photosynthetic parameters, chlorophyll contents and growth rates, and analyzed alterations in transcriptomics. We found that, although overexpression of BBX14 is deleterious under normal growth conditions, BBX14 is needed to acclimate plants to high light stress. BBX14 is a direct target of GLK1, and RNA-Seq analysis suggests that BBX14 is involved in the circadian clock. Knockout of BBX14 results in a long-hypocotyl phenotype that depends on a retrograde signal, and BBX14 expression during biogenic signaling requires GUN1. Finally, we clarify the role of BBX14 in GUN-type biogenic signaling. We conclude that BBX14 is an integrator of photomorphogenetic and biogenic signals, and suggest that BBX14 is a nuclear target of retrograde signals downstream of the GUN1/GLK1 module.

plant biology↗

bioRxiv2022

Response of the organellar and nuclear (post)transcriptomes of Arabidopsis to drought stress

Plants have evolved sophisticated mechanisms to cope with drought, which involve massive changes in nuclear gene expression. However, little is known about the roles of post-transcriptional processing of nuclear or organellar transcripts and how meaningful these changes are. To address these issues, we used long non-coding RNA-sequencing to monitor (post)transcriptional changes during different times of drought exposure in Arabidopsis Col-0 and a mutant (protein phosphatase 7-like, pp7l), from which we demonstrated that it can survive long periods of drought stress. The changes detected in the pp7l mutant were marginal, while in the wild type chloroplast transcript levels were globally reduced, editing efficiency dropped, but splicing was not affected. Mitochondrial transcripts were slightly elevated, while editing and splicing were unchanged. Also, transcriptional activation of transposable elements played only a minor role. Conversely, alternative splicing (AS) affected nearly 2,000 genes (11% of expressed nuclear genes). Of these, 25% underwent isoform switching, and 15% were regulated solely at the level of AS, representing transcripts that would have gone unnoticed in a microarray-based approach. Our data show that AS enhances proteome diversity to counteract drought stress and represent a valuable resource that will facilitate the development of new strategies to improve plant performance under drought. Moreover, altering the relative contributions of spliced isoforms might enhance drought resistance. For instance, our data imply that accumulation of a non-functional FLM (FLOWERING LOCUS M) isoform - and not the ratio of functional isoforms as suggested for temperature responses - accounts for the early-flowering phenotype under drought conditions.

plant biology↗

bioRxiv2021

The RNA-binding protein RBP45D of Arabidopis plays a role in epigenetic control of flowering time and DCL3-independent RNA-directed DNA methylation

RNA-directed DNA methylation (RdDM) helps to defend plants against invasive nucleic acids. In the canonical form of RdDM, 24-nt small interfering RNAs (siRNAs) are produced by DICER-LIKE 3 (DCL3). Here, we describe the Arabidopsis thaliana prors1 (LUC) transgenic system, in which transcriptional gene silencing (TGS) is independent of DLC3. A forward genetics screen performed with this system identified both known components of RdDM, and the RNA-binding protein RBP45D. RBP45D promotes DNA methylation, and its loss delays flowering, especially at high temperature, presumably mediated by elevated FLC levels. RBP45D is localized to the nucleus, where it is associated with snRNAs and snoRNAs. RBP45D maintains siRNA production originating from the LUC transgene, but does not alter mRNA levels or affect processing of transcripts of known RdDM genes. We suggest that RBPD45 facilitates DCL3-independent siRNA production by stabilising either the precursor RNA or the - as yet unidentified - slicer protein.

plant biology↗