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Pre-mRNA splicing is a fundamental process in eukaryotic gene expression, and the mechanism of intron definition, involving the recognition of the canonical GU (5'-splice site) and AG (3'-splice site) dinucleotides by splicing factors, has been postulated for most cases of splicing initiation in plants. Splice site mutations have played crucial roles in unraveling the mechanism of pre-mRNA splicing in planta. Typically, splice site mutations abolish splicing events or activate one or more cryptic splice sites surrounding the mutated region. In this report, we investigated the splicing pattern of the EGY1 gene in an Ar-ion-induced egy1-4 allele of Arabidopsis thaliana. egy1-4 has an AG-to-AC mutation in the 3'-end of intron 3, along with 4-bp substitutions and a 5-bp deletion in adjacent exon 4. RT-PCR, cDNA cloning, and amplicon sequencing analyses of EGY1 revealed that while most wild-type EGY1 mRNAs had a single splicing pattern, egy1-4 mRNAs had multiple splicing defects. Almost half of EGY1 transcripts showed 'intron retention' at intron 3, while the other half exhibited activation of 3' cryptic splice sites either upstream or downstream of the original 3'-splice site. Unexpectedly, around 8% of EGY1 transcripts in egy1-4 exhibited activation of cryptic 5'-splice sites positioned upstream of the authentic 5'-splice site of intron 3. Whole genome resequencing of egy1-4 indicated that it has no other known impactful mutations. These results may provide a rare, but real case of activation of cryptic 5'-splice sites by downstream 3'-splice site/exon mutations in planta. [ABSTRACT FROM AUTHOR]

Plastids in vascular plants have various differentiated forms, among which amyloplasts are crucial for starch storage and plant productivity. Despite the vast knowledge of the binary-fission mode of chloroplast division, our understanding of the replication of non-photosynthetic plastids, including amyloplasts, remains limited. Recent studies have suggested the involvement of stromules (stroma-filled tubules) in plastid replication when the division apparatus is faulty. However, details of the underlying mechanism(s) and their relevance to normal processes have yet to be elucidated. Here, we developed a live analysis system for studying amyloplast replication using Arabidopsis (Arabidopsis thaliana) ovule integuments. We showed the full sequence of amyloplast development and demonstrated that wild-type amyloplasts adopt three modes of replication, binary fission, multiple fission, and stromule-mediated fission, via multi-way placement of the FtsZ ring. The minE mutant, with severely inhibited chloroplast division, showed marked heterogeneity in amyloplast size, caused by size-dependent but wild-type modes of plastid fission. The dynamic properties of stromules distinguish the wild-type and minE phenotypes. In minE cells, extended stromules from giant amyloplasts acquired stability, allowing FtsZ ring assembly and constriction, as well as the growth of starch grains therein. Despite hyper-stromule formation, amyloplasts did not proliferate in the ftsZ null mutant. These data clarify the differences between amyloplast and chloroplast replication and demonstrate that the structural plasticity of amyloplasts underlies the multiplicity of their replication processes. Furthermore, this study shows that stromules can generate daughter plastids via the assembly of the FtsZ ring. Live analysis of the Arabidopsis outer ovule integument reveals the regulatory basis of amyloplast replication is complex and differs from that of chloroplast replication. [ABSTRACT FROM AUTHOR]

Foeniculoxin is a major phytotoxin produced by Italian strains of Phomopsis foeniculi. The first total synthesis is described utilizing the ene reaction and Sonogashira cross‐coupling reaction as key steps. The absolute configuration of the C6' was determined using chiral separation and an advanced Mosher's method. The phytotoxicity of the synthesized compound was demonstrated via syringe‐based infiltration into Chenopodium album and Arabidopsis thaliana leaves. Synthetic foeniculoxin induced various defects in A. thaliana leaf cells before lesion formation, including protein leakage into the cytoplasm from both chloroplasts and mitochondria and mitochondrial rounding and swelling. Furthermore, foeniculoxin and the antibiotic hygromycin B caused similar agglomeration of mitochondria around chloroplasts, highlighting this event as a common component in the early stages of plant cell death. [ABSTRACT FROM AUTHOR]

Stromules, or stroma‐filled tubules, are thin extensions of the plastid envelope membrane that are most frequently observed in undifferentiated or non‐mesophyll cells. The formation of stromules is developmentally regulated and responsive to biotic and abiotic stress; however, the physiological roles and molecular mechanisms of the stromule formation remain enigmatic. Accordingly, we attempted to obtain Arabidopsis thaliana mutants with aberrant stromule biogenesis in the leaf epidermis. Here, we characterize one of the obtained mutants. Plastids in the leaf epidermis of this mutant were giant and pleomorphic, typically having one or more constrictions that indicated arrested plastid division, and usually possessed one or more extremely long stromules, which indicated the deregulation of stromule formation. Genetic mapping, whole‐genome resequencing‐aided exome analysis, and gene complementation identified PARC6/CDP1/ARC6H, which encodes a vascular plant‐specific, chloroplast division site‐positioning factor, as the causal gene for the stromule phenotype. Yeast two‐hybrid assay and double mutant analysis also identified a possible interaction between PARC6 and MinD1, another known chloroplast division site‐positioning factor, during the morphogenesis of leaf epidermal plastids. To the best of our knowledge, PARC6 is the only known A. thaliana chloroplast division factor whose mutations more extensively affect the morphology of plastids in non‐mesophyll tissue than in mesophyll tissue. Therefore, the present study demonstrates that PARC6 plays a pivotal role in the morphology maintenance and stromule regulation of non‐mesophyll plastids. [ABSTRACT FROM AUTHOR]

The article presents a study that investigates the participation of AtMinE1 in the plastid morphogenesis of several tissues of Arabidopsis thaliana. It states that plastids of cotyledons, roots, and floral organs were examined in the Arabidopsis thaliana, and were compared with those in the wild type. The study showed that the plastid size in the atmine1 mutant is smaller than those in the wild type and the plastid number per cell in the atmine1 is comparably smaller than those in the wild type.

The article presents a study that examines the localization and functionality of Arabidopsis thaliana MinD (AtMinD1) fused with a dual hemagglutinin epitope (dHA) or a yellow fluorescent protein (YFP). It used the wildtype Arabidopsis thaliana to provide a basis of MinD function and localization in chloroplast division. Results say that AtMinD1-dHA successfully complements the arc11/atminD1 mutant phenotype and is located at the envelope membrane and the mid-chloroplast constriction site.

Chloroplast division is mediated by the coordinated action of a prokaryote-derived division system(s) and a host eukaryote-derived membrane fission system(s). The evolutionary conserved prokaryote-derived system comprises several nucleus-encoded proteins, two of which are thought to control division site placement at the midpoint of the organelle: a stromal ATPase MinD and a topological specificity factor MinE. Here, we show that arc11, one of 12 recessive accumulation and replication of chloroplasts (arc) mutants in Arabidopsis, contains highly elongated and multiple-arrayed chloroplasts in developing green tissues. Genomic sequence analysis revealed that arc11 contains a missense mutation in α-helix 11 of the chloroplast-targeted AtMinD1 changing an Ala at position 296 to Gly (A296G). Introduction of wild-type AtMinD1 restores the chloroplast division defects of arc11 and quantitative RT-PCR analysis showed that the degree of complementation was highly dependent on transgene expression levels. Overexpression of the mutant ARC11/AtMinD1 in transgenic plants results in the inhibition of chloroplast division, showing that the mutant protein has retained its division inhibition activity. However, in contrast to the defined and punctate intraplastidic localization patterns of an AtMinD1-YFP fusion protein, the single A296G point mutation in ARC11/AtMinD1 results in aberrant localization patterns inside chloroplasts. We further show that AtMinD1 is capable of forming homodimers and that this dimerization capacity is abolished by the A296G mutation in ARC11/AtMinD1. Our data show that arc11 is a loss-of-function mutant of AtMinD1 and suggest that the formation of functional AtMinD1 homodimers is paramount for appropriate AtMinD1 localization, ultimately ensuring correct division machinery placement and chloroplast division in plants. [ABSTRACT FROM AUTHOR]

The dioecious plant Silene latifolia has heteromorphic sex chromosomes, and comparison of the positions of sex-linked genes indicates that at least three large inversions have occurred during the evolution of the Y chromosome. In this article, we describe the isolation of a new sex-linked gene from S. latifolia, which provides new information on the evolution of this plant's young sex chromosomes. By using reverse-transcription polymerase chain reaction degenerate primers based on the Arabidopsis thaliana sequence of WUSCHEL, a flower-development gene, we found two copies in S. latifolia, which we named SlWUS1 and SlWUS2. Southern blot and genetic segregation analysis showed that SlWUS1 is located on the X chromosome and SlWUS2 is autosomal. No Y-linked copy of SlWUS1 was found by either Southern blot analysis under low-stringency conditions or polymerase chain reaction with degenerate primers, so we conclude that SlWUS1 probably has no Y-linked homolog. It is unknown whether the Y chromosome lost the SlWUS1 copy by degeneration of this individual gene or whether deletion of a larger genome region was involved. Several tests lead us to conclude that dosage compensation has not evolved for this sex-linked gene. We mapped the ortholog in the nondioecious relative S. vulgaris (SvWUS1), to compare the location in a species that has no history of having sex chromosomes. SvWUS1 maps to the same linkage group as other fully X-linked genes, indicating that it was not added to the X, but was lost from the Y. Its location differs in the maps from the two species, raising the possibility that the X chromosome, as well as the Y, may have been rearranged.