At the molecular scale, adaptive advantages during plant growth and development rely on modulation of gene expression, primarily provided by epigenetic machinery. One crucial part of this machinery is histone posttranslational modifications, which form a flexible system, driving transient changes in chromatin, and defining particular epigenetic states. Posttranslational modifications work in concert with replication-independent histone variants further adapted for transcriptional regulation and chromatin repair. However, little is known about how such complex regulatory pathways are orchestrated and interconnected in cells. In this work, we demonstrate the utility of mass spectrometry-based approaches to explore how different epigenetic layers interact in Arabidopsis mutants lacking certain histone chaperones. We show that defects in histone chaperone function (e.g., chromatin assembly factor-1 or nucleosome assembly protein 1 mutations) translate into an altered epigenetic landscape, which aids the plant in mitigating internal instability. We observe changes in both the levels and distribution of H2A.W.7, altogether with partial repurposing of H3.3 and changes in the key repressive (H3K27me1/2) or euchromatic marks (H3K36me1/2). These shifts in the epigenetic profile serve as a compensatory mechanism in response to impaired integration of the H3.1 histone in the fas1 mutants. Altogether, our findings suggest that maintaining genome stability involves a two-tiered approach. The first relies on flexible adjustments in histone marks, while the second level requires the assistance of chaperones for histone variant replacement.

• Klíčová slova
• Arabidopsis, chromatin remodeling, histone chaperone complex, histone variants, immunochemistry, mass spectrometry, post-translational modifications,
• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• epigeneze genetická * MeSH
• faktor 1 pro uspořádání chromatinu metabolismus   genetika   MeSH
• histonové chaperony * metabolismus   genetika   MeSH
• histony * metabolismus   MeSH
• mutace MeSH
• posttranslační úpravy proteinů MeSH
• proteiny huseníčku * metabolismus   genetika   MeSH
• regulace genové exprese u rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• faktor 1 pro uspořádání chromatinu MeSH
• histonové chaperony * MeSH
• histony * MeSH
• proteiny huseníčku * MeSH

Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.

• Klíčová slova
• A. thaliana, cell biology, centremeres, centromeric histone, chromosomes, gene expression, meiosis, micronuclei, spindle assembly checkpoint,
• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• centromera metabolismus   MeSH
• histony metabolismus   MeSH
• kinetochory metabolismus   MeSH
• meióza MeSH
• reakce na tepelný šok MeSH
• rostliny genetika   MeSH
• segregace chromozomů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• histony MeSH

Double haploid production is the most effective way to create true-breeding lines in a single generation. In Arabidopsis, haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when the mutant is outcrossed to the wild-type, and the wild-type genome remains in the haploid progeny. However, factors that affect haploid induction are still poorly understood. Here, we report that a mutant of the cenH3 assembly factor Kinetochore Null2 (KNL2) can be used as a haploid inducer when pollinated by the wild-type. We discovered that short-term temperature stress of the knl2 mutant increased the efficiency of haploid induction 10-fold. We also demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid-inducing lines, suggesting that haploid-inducing lines in crops can be identified in a naturally occurring or chemically induced mutant population, avoiding the generic modification (GM) approach at any stage. Furthermore, a cenh3-4 mutant functioned as a haploid inducer in response to short-term heat stress, even though it did not induce haploids under standard conditions. Thus, we identified KNL2 as a new target gene for the generation of haploid-inducer lines and showed that exposure of centromeric protein mutants to high temperature strongly increases their haploid induction efficiency.

• Klíčová slova
• CENPC-k, cenh3-4, centromere, haploid induction, kinetochore null 2, temperature stress,
• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• centromera genetika   MeSH
• haploidie MeSH
• kinetochory MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Meiosis in angiosperm plants is followed by mitotic divisions to form multicellular haploid gametophytes. Termination of meiosis and transition to gametophytic development is, in Arabidopsis, governed by a dedicated mechanism that involves SMG7 and TDM1 proteins. Mutants carrying the smg7-6 allele are semi-fertile due to reduced pollen production. We found that instead of forming tetrads, smg7-6 pollen mother cells undergo multiple rounds of chromosome condensation and spindle assembly at the end of meiosis, resembling aberrant attempts to undergo additional meiotic divisions. A suppressor screen uncovered a mutation in centromeric histone H3 (CENH3) that increased fertility and promoted meiotic exit in smg7-6 plants. The mutation led to inefficient splicing of the CENH3 mRNA and a substantial decrease of CENH3, resulting in smaller centromeres. The reduced level of CENH3 delayed formation of the mitotic spindle but did not have an apparent effect on plant growth and development. We suggest that impaired spindle re-assembly at the end of meiosis limits aberrant divisions in smg7-6 plants and promotes formation of tetrads and viable pollen. Furthermore, the mutant with reduced level of CENH3 was very inefficient haploid inducer indicating that differences in centromere size is not the key determinant of centromere-mediated genome elimination.

• MeSH
• aparát dělícího vřeténka MeSH
• Arabidopsis genetika   fyziologie   MeSH
• fertilita genetika   MeSH
• meióza genetika   MeSH
• messenger RNA genetika   MeSH
• mutace * MeSH
• proteiny huseníčku genetika   MeSH
• rostlinné geny * MeSH
• transportní proteiny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• proteiny huseníčku MeSH
• SMG7 protein, Arabidopsis MeSH Prohlížeč
• transportní proteiny MeSH

Determining the function of proteins remains a key task of modern biology. Classical genetic approaches to knocking out protein function in plants still face limitations, such as the time-consuming nature of generating homozygous transgenic lines or the risk of non-viable loss-of-function phenotypes. We aimed to overcome these limitations by acting downstream of the protein level. Chimeric E3 ligases degrade proteins of interest in mammalian cell lines, Drosophila melanogaster embryos, and transgenic tobacco. We successfully recruited the 26S proteasome pathway to directly degrade a protein of interest located in plant nuclei. This success was achieved via replacement of the interaction domain of the E3 ligase adaptor protein SPOP (Speckle-type POZ adapter protein) with a specific anti-GFP nanobody (VHHGFP4). For proof of concept, the target protein CENH3 of A. thaliana fused to EYFP was subjected to nanobody-guided proteasomal degradation in planta. Our results show the potential of the modified E3-ligase adapter protein VHHGFP4-SPOP in this respect. We were able to point out its capability for nucleus-specific protein degradation in plants.

• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• buněčné linie MeSH
• geneticky modifikované rostliny genetika   metabolismus   MeSH
• histony genetika   metabolismus   MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• proteinové inženýrství * metody   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteolýza * MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• tabák genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ATP dependent 26S protease MeSH Prohlížeč
• histony MeSH
• proteasomový endopeptidasový komplex MeSH
• proteiny huseníčku MeSH
• rekombinantní fúzní proteiny MeSH

Several histone variants are posttranslationally phosphorylated. Little is known about phosphorylation of the centromere-specific histone 3 (CENH3) variant in plants. We show that CENH3 of Arabidopsis thaliana is phosphorylated in vitro by Aurora3, predominantly at serine 65. Interaction of Aurora3 and CENH3 was found by immunoprecipitation (IP) in A. thaliana and by bimolecular fluorescence complementation. Western blotting with an anti-CENH3 pS65 antibody showed that CENH3 pS65 is more abundant in flower buds than elsewhere in the plant. Substitution of serine 65 by either alanine or aspartic acid resulted in a range of phenotypic abnormalities, especially in reproductive tissues. We conclude that Aurora3 phosphorylates CENH3 at S65 and that this post-translational modification is required for the proper development of the floral meristem.

• Klíčová slova
• Arabidopsis, Aurora kinase, CENH3, floral meristem, phosphorylation,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Sugar beet (Beta vulgaris) is an important crop of temperate climate zones, which provides nearly 30 % of the world's annual sugar needs. From the total genome size of 758 Mb, only 567 Mb were incorporated in the recently published genome sequence, due to the fact that regions with high repetitive DNA contents (e.g. satellite DNAs) are only partially included. Therefore, to fill these gaps and to gain information about the repeat composition of centromeres and heterochromatic regions, we performed chromatin immunoprecipitation followed by sequencing (ChIP-Seq) using antibodies against the centromere-specific histone H3 variant of sugar beet (CenH3) and the heterochromatic mark of dimethylated lysine 9 of histone H3 (H3K9me2). RESULTS: ChIP-Seq analysis revealed that active centromeres containing CenH3 consist of the satellite pBV and the Ty3-gypsy retrotransposon Beetle7, while heterochromatin marked by H3K9me2 exhibits heterogeneity in repeat composition. H3K9me2 was mainly associated with the satellite family pEV, the Ty1-copia retrotransposon family Cotzilla and the DNA transposon superfamily of the En/Spm type. In members of the section Beta within the genus Beta, immunostaining using the CenH3 antibody was successful, indicating that orthologous CenH3 proteins are present in closely related species within this section. CONCLUSIONS: The identification of repetitive genome portions by ChIP-Seq experiments complemented the sugar beet reference sequence by providing insights into the repeat composition of poorly characterized CenH3-chromatin and H3K9me2-heterochromatin. Therefore, our work provides the basis for future research and application concerning the sugar beet centromere and repeat-rich heterochromatic regions characterized by the presence of H3K9me2.

• Klíčová slova
• Beta vulgaris, CenH3, Centromere, ChIP-Seq, H3K9me2, Heterochromatin, Repeats,
• MeSH
• Beta vulgaris genetika   metabolismus   MeSH
• centromera metabolismus   MeSH
• chromatin genetika   metabolismus   MeSH
• chromatinová imunoprecipitace MeSH
• heterochromatin genetika   metabolismus   MeSH
• histony metabolismus   MeSH
• lysin metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sekvenční analýza DNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• heterochromatin MeSH
• histony MeSH
• lysin MeSH
• rostlinné proteiny MeSH