Arabidopsis thaliana mutants dysfunctional in the evolutionarily conserved protein complex chromatin assembly factor-1 (CAF-1), which deposits the canonical histone H3 variant H3.1 during DNA synthesis-dependent chromatin assembly, display complex phenotypic changes including meristem and growth alterations, sensitivity to DNA-damaging agents, and reduced fertility. We reported previously that mutants in the FAS1 subunit of CAF-1 progressively lose telomere and 45S rDNA repeats. Here we show that multiple aspects of the fas phenotype are recovered immediately on expression of a reintroduced FAS1 allele, and are clearly independent of the recovery of rDNA copy-numbers and telomeres. In reverted lines, 45S rDNA genes are recovered to diverse levels with a strikingly different representation of their variants, and the typical association of nucleolar organizing region 4 with the nucleolus is perturbed. One of 45S rDNA variants (VAR1), which is silenced in wild-type (WT) plants without mutation history (Col-0 WT), dominates the expression pattern, whereas VAR2 is dominant in Col-0 WT plants. We propose an explanation for the variability of telomere and 45S rDNA repeats associated with CAF-1 function, suggesting that the differences in nuclear partitioning and expression of the rDNA variants in fas mutants and their revertants provide a useful experimental system to study genetic and epigenetic factors in gene dosage compensation.

• Klíčová slova
• rDNA, CAF-1, chromatin assembly, genome instability, rRNA gene regulation, telomeres,
• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• chromatin genetika   metabolismus   MeSH
• genom rostlinný genetika   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• ribozomální DNA genetika   metabolismus   MeSH
• telomery genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• proteiny huseníčku MeSH
• ribozomální DNA MeSH

Fas (CD95/APO-1) belongs to the TNF receptor (TNFR) family. Fas ligand binding followed by Fas-receptor oligomerisation leads to formation of a death-inducing signal complex starting with recruitment of the Fas-adapter protein (FADD). Components of this initiation complex (Fas, Fas-L, FADD) were correlated with apoptotic cells, detected by specific DNA fragmentation and morphological criteria. Apoptotic cells can be detected throughout the embryonic development of molar teeth. Restricted temporospatial distribution suggests several important roles for apoptosis in tooth morphogenesis. However, the mechanisms employed in dental apoptosis remain unclear. Frontal sections of the field vole at stage 13.5-15.5 of embryonic development were exploited to investigate and correlate location of Fas, Fas-ligand, FADD molecules and apoptosis in developing first molars by immunohistochemistry. During these stages the primary enamel knot appears and is gradually terminated by apoptosis. Initially, apoptotic cells were demonstrated in the most superficial layer of the dental lamina. The number of TUNEL-positive cells expanded from late bud to cap stages. Restricted areas of apoptotic cells were found in the stalk and primary enamel knot. Fas, Fas-L and FADD were co-localised, particularly in the primary enamel knot, and the stalk, correlating with the occurrence of apoptosis in these areas. Fas-L, however, was also found in proliferating parts of the developing tooth germ, such as in the cervical loops. Interestingly, FADD molecules were also observed in areas, where Fas protein was not detected. According to the immunohistochemical data, Fas-mediated signalling may have a triggering or enhancing role in dental apoptosis. This remains to be functionally confirmed.

• MeSH
• antigeny CD95 analýza   metabolismus   MeSH
• apoptóza fyziologie   MeSH
• Arvicolinae embryologie   metabolismus   MeSH
• desaturasy mastných kyselin analýza   metabolismus   MeSH
• imunohistochemie MeSH
• koncové značení zlomů DNA in situ MeSH
• moláry MeSH
• odontogeneze fyziologie   MeSH
• orgán skloviny metabolismus   MeSH
• proteiny huseníčku analýza   metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD95 MeSH
• desaturasy mastných kyselin MeSH
• Fad7 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH

Dysfunction of chromatin assembly factor 1 in FASCIATA mutants (fas) of Arabidopsis thaliana results in progressive loss of telomeric DNA. Although replicative telomere shortening is typically associated with incomplete resynthesis of their ends by telomerase, no change in telomerase activity could be detected in vitro in extracts from fas mutants. Besides a possible telomerase malfunction, the telomere shortening in fas mutants could presumably be due to problems with conventional replication of telomeres. To distinguish between the possible contribution of suboptimal function of telomerase in fas mutants under in vivo conditions and problems in conventional telomere replication, we crossed fas and tert (telomerase reverse transcriptase) knockout mutants and analyzed telomere shortening in segregated fas mutants, tert mutants, and double fas tert mutants in parallel. We demonstrate that fas tert knockouts show greater replicative telomere shortening than that observed even in the complete absence of telomerase (tert mutants). While the effect of tert and fas mutations on telomere lengths in double mutants is additive, manifestations of telomere dysfunction in double fas tert mutants (frequency of anaphase bridges, onset of chromosome end fusions, and common involvement of 45S rDNA in chromosome fusion sites) are similar to those in tert mutants. We conclude that in addition to possible impairment of telomerase action, a further mechanism contributes to telomere shortening in fas mutants.

• MeSH
• Arabidopsis enzymologie   genetika   metabolismus   MeSH
• chromozomy rostlin genetika   metabolismus   MeSH
• faktor 1 pro uspořádání chromatinu genetika   metabolismus   MeSH
• mutace * MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• sestřihové faktory MeSH
• telomerasa genetika   metabolismus   MeSH
• telomery genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• At2g20020 protein, Arabidopsis MeSH Prohlížeč
• faktor 1 pro uspořádání chromatinu MeSH
• FAS protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• sestřihové faktory MeSH
• telomerasa MeSH
• TERT protein, Arabidopsis MeSH Prohlížeč

Deficiency in chromatin assembly factor-1 (CAF-1) in plants through dysfunction of its components, FASCIATA1 and 2 (FAS1, FAS2), leads to the specific and progressive loss of rDNA and telomere repeats in plants. This loss is attributed to defective repair mechanisms for the increased DNA breaks encountered during replication, a consequence of impaired replication-dependent chromatin assembly. In this study, we explore the role of KU70 in these processes. Our findings reveal that, although the rDNA copy number is reduced in ku70 mutants when compared with wild-type plants, it is not markedly affected by diverse KU70 status in fas1 mutants. This is consistent with our previous characterisation of rDNA loss in fas mutants as a consequence part of the single-strand annealing pathway of homology-dependent repair. In stark contrast to rDNA, KU70 dysfunction fully suppresses the loss of telomeres in fas1 plants and converts telomeres to their elongated and heterogeneous state typical for ku70 plants. We conclude that the alternative telomere lengthening pathway, known to be activated in the absence of KU70, overrides progressive telomere loss due to CAF-1 dysfunction.

• Klíčová slova
• 45S rDNA, ALT, Arabidopsis thaliana, CAF‐1, FAS1, KU70, genome instability, telomeres,
• MeSH
• Arabidopsis * genetika   metabolismus   fyziologie   MeSH
• chromozomy rostlin metabolismus   MeSH
• DNA vazebné proteiny * MeSH
• faktor 1 pro uspořádání chromatinu * metabolismus   genetika   MeSH
• homeostáza telomer * MeSH
• mutace MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• ribozomální DNA genetika   metabolismus   MeSH
• telomery metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• At2g20020 protein, Arabidopsis MeSH Prohlížeč
• DNA vazebné proteiny * MeSH
• faktor 1 pro uspořádání chromatinu * MeSH
• FAS protein, Arabidopsis MeSH Prohlížeč
• KU70 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku * MeSH
• ribozomální DNA MeSH

Histone chaperones mediate the assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-function of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1 (CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. CAF-1 dysfunction can lead to altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage, clearly demonstrating its severity. How chromatin composition is maintained without functional CAF-1 remains elusive. Here we show that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) suppresses the FAS1 loss-of-function phenotype. The quadruple mutant fas1 nap1;1 nap1;2 nap1;3 shows wild-type growth, decreased sensitivity to genotoxic stress and suppression of telomere and 45S rDNA loss. Chromatin of fas1 nap1;1 nap1;2 nap1;3 plants is less accessible to micrococcal nuclease and the nuclear H3.1 and H3.3 histone pools change compared to fas1. Consistently, association between NAP1 and H3 occurs in the cytoplasm and nucleus in vivo in protoplasts. Altogether we show that NAP1 proteins play an essential role in DNA repair in fas1, which is coupled to nucleosome assembly through modulation of H3 levels in the nucleus.

• Klíčová slova
• DNA repair, chromatin, histone chaperones, histone variants,
• MeSH
• adenosintrifosfatasy genetika   metabolismus   MeSH
• Arabidopsis genetika   metabolismus   MeSH
• chromatin genetika   metabolismus   MeSH
• histonové chaperony genetika   metabolismus   MeSH
• mutace genetika   MeSH
• nestabilita genomu genetika   fyziologie   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• chromatin MeSH
• FAS protein, Arabidopsis MeSH Prohlížeč
• histonové chaperony MeSH
• NAP1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH

Chromatin Assembly Factor 1 (CAF1) is a three-subunit H3/H4 histone chaperone responsible for replication-dependent nucleosome assembly. It is composed of CAC 1-3 in yeast; p155, p60, and p48 in humans; and FASCIATA1 (FAS1), FAS2, and MULTICOPY SUPPRESSOR OF IRA1 in Arabidopsis thaliana. We report that disruption of CAF1 function by fas mutations in Arabidopsis results in telomere shortening and loss of 45S rDNA, while other repetitive sequences (5S rDNA, centromeric 180-bp repeat, CACTA, and Athila) are unaffected. Substantial telomere shortening occurs immediately after the loss of functional CAF1 and slows down at telomeres shortened to median lengths around 1 to 1.5 kb. The 45S rDNA loss is progressive, leaving 10 to 15% of the original number of repeats in the 5th generation of mutants affecting CAF1, but the level of the 45S rRNA transcripts is not altered in these mutants. Increasing severity of the fas phenotype is accompanied by accumulation of anaphase bridges, reduced viability, and plant sterility. Our results show that appropriate replication-dependent chromatin assembly is specifically required for stable maintenance of telomeres and 45S rDNA.

• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• DNA rostlinná genetika   metabolismus   MeSH
• faktor 1 pro uspořádání chromatinu genetika   metabolismus   MeSH
• inzerční mutageneze MeSH
• mutace MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• restrukturace chromatinu MeSH
• ribozomální DNA genetika   metabolismus   MeSH
• RNA ribozomální genetika   metabolismus   MeSH
• telomery metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• faktor 1 pro uspořádání chromatinu MeSH
• FAS protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• ribozomální DNA MeSH
• RNA ribozomální MeSH
• RNA, ribosomal, 45S MeSH Prohlížeč

Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability.

• Klíčová slova
• 45S rDNA, Arabidopsis thaliana, DNA repair, FAS1, FAS2, RAD51B, chromatin assembly factor 1, genome instability,
• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• faktor 1 pro uspořádání chromatinu genetika   metabolismus   MeSH
• nestabilita genomu genetika   fyziologie   MeSH
• oprava DNA genetika   fyziologie   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• ribozomální DNA genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktor 1 pro uspořádání chromatinu MeSH
• FAS protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• RAD51B protein, Arabidopsis MeSH Prohlížeč
• ribozomální DNA MeSH

The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1KO and ATRKO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of-function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that are described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.

• MeSH
• Arabidopsis genetika   fyziologie   MeSH
• ATM protein genetika   metabolismus   MeSH
• fyziologický stres MeSH
• genom rostlinný MeSH
• nestabilita genomu MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• protoonkogenní proteiny c-myb genetika   metabolismus   MeSH
• replikace DNA * MeSH
• signální transdukce * MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ATM protein, Arabidopsis MeSH Prohlížeč
• ATM protein MeSH
• ATR1 protein, Arabidopsis MeSH Prohlížeč
• FAS protein, Arabidopsis MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• proteiny huseníčku MeSH
• protoonkogenní proteiny c-myb MeSH
• SOG1 protein, Arabidopsis MeSH Prohlížeč
• transkripční faktory MeSH
• WEE1 protein, Arabidopsis MeSH Prohlížeč

The plastid-localized phosphoglucose isomerase isoform PGI1 is an important determinant of growth in Arabidopsis thaliana, likely due to its involvement in the biosynthesis of plastidial isoprenoid-derived hormones. Here, we investigated whether PGI1 also influences seed yields. PGI1 is strongly expressed in maturing seed embryos and vascular tissues. PGI1-null pgi1-2 plants had ∼60% lower seed yields than wild-type plants, with reduced numbers of inflorescences and thus fewer siliques and seeds per plant. These traits were associated with low bioactive gibberellin (GA) contents. Accordingly, wild-type phenotypes were restored by exogenous GA application. pgi1-2 seeds were lighter and accumulated ∼50% less fatty acids (FAs) and ∼35% less protein than wild-type seeds. Seeds of cytokinin-deficient plants overexpressing CYTOKININ OXIDASE/DEHYDROGENASE1 (35S:AtCKX1) and GA-deficient ga20ox1 ga20ox2 mutants did not accumulate low levels of FAs, and exogenous application of the cytokinin 6-benzylaminopurine and GAs did not rescue the reduced weight and FA content of pgi1-2 seeds. Seeds from reciprocal crosses between pgi1-2 and wild-type plants accumulated wild-type levels of FAs and proteins. Therefore, PGI1 is an important determinant of Arabidopsis seed yield due to its involvement in two processes: GA-mediated reproductive development and the metabolic conversion of plastidial glucose-6-phosphate to storage reserves in the embryo.

• MeSH
• Arabidopsis enzymologie   metabolismus   MeSH
• gibereliny metabolismus   MeSH
• glukosa-6-fosfát metabolismus   MeSH
• glukosa-6-fosfátisomerasa genetika   metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• oxidoreduktasy působící na CH-NH vazby genetika   metabolismus   MeSH
• plastidy metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• semena rostlinná enzymologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CKX1 protein, Arabidopsis MeSH Prohlížeč
• gibereliny MeSH
• glukosa-6-fosfát MeSH
• glukosa-6-fosfátisomerasa MeSH
• membránové proteiny MeSH
• oxidoreduktasy působící na CH-NH vazby MeSH
• proteiny huseníčku MeSH

Chromatin is assembled by histone chaperones such as chromatin assembly factor CAF-1. We had noticed that vigor of Arabidopsis thaliana CAF-1 mutants decreased over several generations. Because changes in mutant phenotype severity over generations are unusual, we asked how repeated selfing of Arabidopsis CAF-1 mutants affects phenotype severity. CAF-1 mutant plants of various generations were grown, and developmental phenotypes, transcriptomes and DNA cytosine-methylation profiles were compared quantitatively. Shoot- and root-related growth phenotypes were progressively more affected in successive generations of CAF-1 mutants. Early and late generations of the fasciata (fas)2-4 CAF-1 mutant displayed only limited changes in gene expression, of which increasing upregulation of plant defense-related genes reflects the transgenerational phenotype aggravation. Likewise, global DNA methylation in the sequence context CHG but not CG or CHH (where H = A, T or C) changed over generations in fas2-4. Crossing early and late generation fas2-4 plants established that the maternal contribution to the phenotype severity exceeds the paternal contribution. Together, epigenetic rather than genetic mechanisms underlie the progressive developmental phenotype aggravation in the Arabidopsis CAF-1 mutants and preferred maternal transmission reveals a more efficient reprogramming of epigenetic information in the male than the female germline.

• Klíčová slova
• Arabidopsis thaliana, CAF-1, Chromatin, DNA methylation, Development, epigenetics, histone,
• MeSH
• alely MeSH
• Arabidopsis genetika   MeSH
• epigeneze genetická * MeSH
• fenotyp MeSH
• fyziologický stres genetika   MeSH
• genová ontologie MeSH
• metylace DNA genetika   MeSH
• mutace genetika   MeSH
• neplodnost rostlin MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• sekvence nukleotidů MeSH
• semena rostlinná embryologie   MeSH
• sestřihové faktory genetika   metabolismus   MeSH
• transkriptom genetika   MeSH
• typy dědičnosti genetika   MeSH
• vajíčko rostlin embryologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• At2g20020 protein, Arabidopsis MeSH Prohlížeč
• FAS protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• sestřihové faktory MeSH