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<jats:title>SUMMARY</jats:title><jats:p>Pungent capsaicinoid is synthesized only in chili pepper (<jats:italic>Capsicum</jats:italic> spp.). The production of vanillylamine from vanillin is a unique reaction in the capsaicinoid biosynthesis pathway. Although <jats:italic>putative aminotransferase</jats:italic> (<jats:italic>pAMT</jats:italic>) has been isolated as the vanillylamine synthase gene, it is unclear how <jats:italic>Capsicum</jats:italic> acquired <jats:italic>pAMT</jats:italic>. Here, we present a phylogenetic overview of <jats:italic>pAMT</jats:italic> and its homologs. The <jats:italic>Capsicum</jats:italic> genome contained 5 homologs, including <jats:italic>pAMT</jats:italic>, <jats:italic>CaGABA‐T1</jats:italic>, <jats:italic>CaGABA‐T3</jats:italic>, and two pseudogenes. Phylogenetic analysis indicated that <jats:italic>pAMT</jats:italic> is a member of the Solanaceae cytoplasmic <jats:italic>GABA‐Ts</jats:italic>. Comparative genome analysis found that multiple copies of <jats:italic>GABA‐T</jats:italic> exist in a specific Solanaceae genomic region, and the cytoplasmic <jats:italic>GABA‐Ts</jats:italic> other than <jats:italic>pAMT</jats:italic> are located in the region. The cytoplasmic <jats:italic>GABA‐T</jats:italic> was phylogenetically close to pseudo‐<jats:italic>GABA‐T</jats:italic> harboring a plastid transit peptide (<jats:italic>pseudo‐GABA‐T3</jats:italic>). This suggested that Solanaceae cytoplasmic <jats:italic>GABA‐T</jats:italic>s occurred via duplication of a chloroplastic <jats:italic>GABA‐T</jats:italic> ancestor and subsequent loss of the plastid transit signal. The cytoplasmic <jats:italic>GABA‐T</jats:italic> may have been translocated from the specific Solanaceae genomic region during <jats:italic>Capsicum</jats:italic> divergence, resulting in the current <jats:italic>pAMT</jats:italic> locus. A recombinant protein assay demonstrated that pAMT had higher vanillylamine synthase activity than those of other plant GABA‐Ts. <jats:italic>pAMT</jats:italic> was expressed exclusively in the placental septum of mature green fruit, whereas tomato orthologs SlGABA‐T2/4 exhibit a ubiquitous expression pattern in plants. These findings suggested that both the increased catalytic efficiency and transcriptional changes in <jats:italic>pAMT</jats:italic> may have contributed to establish vanillylamine synthesis in the capsaicinoid biosynthesis pathway. This study provides insights into the establishment of pungency in the evolution of chili peppers.</jats:p>

<jats:title>SUMMARY</jats:title><jats:p>The importance of RNA‐binding proteins (RBPs) for plant responses to environmental stimuli and development is well documented. Insights into the portfolio of RNAs they recognize, however, clearly lack behind the understanding gathered in non‐plant model organisms. Here, we characterize binding of the circadian clock‐regulated <jats:italic>Arabidopsis thaliana</jats:italic> GLYCINE‐RICH RNA‐BINDING PROTEIN 7 (<jats:italic>At</jats:italic>GRP7) to its target transcripts. We identified novel RNA targets from individual‐nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) data using an improved bioinformatics pipeline that will be broadly applicable to plant RBP iCLIP data. 2705 transcripts with binding sites were identified in plants expressing <jats:italic>At</jats:italic>GRP7‐GFP that were not recovered in plants expressing an RNA‐binding dead variant or GFP alone. A conserved RNA motif enriched in uridine residues was identified at the <jats:italic>At</jats:italic>GRP7 binding sites. NMR titrations confirmed the preference of <jats:italic>At</jats:italic>GRP7 for RNAs with a central U‐rich motif. Among the bound RNAs, circadian clock‐regulated transcripts were overrepresented. Peak abundance of the <jats:italic>LHCB1.1</jats:italic> transcript encoding a chlorophyll‐binding protein was reduced in plants overexpressing <jats:italic>At</jats:italic>GRP7 whereas it was elevated in <jats:italic>atgrp7</jats:italic> mutants, indicating that <jats:italic>LHCB1.1</jats:italic> was regulated by <jats:italic>At</jats:italic>GRP7 in a dose‐dependent manner. In plants overexpressing <jats:italic>At</jats:italic>GRP7, the <jats:italic>LHCB1.1</jats:italic> half‐life was shorter compared to wild‐type plants whereas in <jats:italic>atgrp7</jats:italic> mutant plants, the half‐life was significantly longer. Thus, <jats:italic>At</jats:italic>GRP7 modulates circadian oscillations of its in vivo binding target <jats:italic>LHCB1.1</jats:italic> by affecting RNA stability.</jats:p>

<jats:title>SUMMARY</jats:title><jats:p>Sugar beet and its wild relatives share a base chromosome number of nine and similar chromosome morphologies. Yet, interspecific breeding is impeded by chromosome and sequence divergence that is still not fully understood. Since repetitive DNAs are among the fastest evolving parts of the genome, we investigated, if repeatome innovations and losses are linked to chromosomal differentiation and speciation. We traced genome and chromosome‐wide evolution across 13 beet species comprising all sections of the genera <jats:italic>Beta</jats:italic> and <jats:italic>Patellifolia</jats:italic>. For this, we combined short and long read sequencing, flow cytometry, and cytogenetics to build a comprehensive framework that spans the complete scale from DNA to chromosome to genome. Genome sizes and repeat profiles reflect the separation into three gene pools with contrasting evolutionary patterns. Among all repeats, satellite DNAs harbor most genomic variability, leading to fundamentally different centromere architectures, ranging from chromosomal uniformity in <jats:italic>Beta</jats:italic> and <jats:italic>Patellifolia</jats:italic> to the formation of patchwork chromosomes in <jats:italic>Corollinae</jats:italic>/<jats:italic>Nanae</jats:italic>. We show that repetitive DNAs are causal for the genome expansions and contractions across the beet genera, providing insights into the genomic underpinnings of beet speciation. Satellite DNAs in particular vary considerably between beet genomes, leading to the evolution of distinct chromosomal setups in the three gene pools, likely contributing to the barriers in beet breeding. Thus, with their isokaryotypic chromosome sets, beet genomes present an ideal system for studying the link between repeats, genomic variability, and chromosomal differentiation and provide a theoretical fundament for understanding barriers in any crop breeding effort.</jats:p>

<jats:title>SUMMARY</jats:title><jats:p>The 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 signal integrator GUN1. BBX14 was previously identified, together with GLK1, in a core module that mediates the response to high light (HL) levels and biogenic signals, which was studied by using inhibitors of chloroplast development. Our chromatin immunoprecipitation‐Seq experiments revealed that <jats:italic>BBX14</jats:italic> is a direct target of GLK1, and RNA‐Seq analysis suggests that BBX14 may function as a regulator of the circadian clock. In addition, BBX14 plays a role in chlorophyll biosynthesis during early onset of light. Knockout of <jats:italic>BBX14</jats:italic> results in a long hypocotyl phenotype dependent on a retrograde signal. Furthermore, the expression of <jats:italic>BBX14</jats:italic> and <jats:italic>BBX15</jats:italic> during biogenic signaling requires GUN1. Investigation of the role of BBX14 and BBX15 in GUN‐type biogenic (<jats:italic>gun</jats:italic>) signaling showed that the overexpression of BBX14 or BBX15 caused de‐repression of <jats:italic>CA1</jats:italic> mRNA levels, when seedlings were grown on norflurazon. Notably, transcripts of the <jats:italic>LHCB1.2</jats:italic> marker are not de‐repressed. Furthermore, BBX14 is required to acclimate plants to HL stress. We propose that BBX14 is an integrator of biogenic signals and that BBX14 is a nuclear target of retrograde signals downstream of the GUN1/GLK1 module. However, we do not classify BBX14 or BBX15 overexpressors as <jats:italic>gun</jats:italic> mutants based on a critical evaluation of our results and those reported in the literature. Finally, we discuss a classification system necessary for the declaration of new <jats:italic>gun</jats:italic> mutants.</jats:p>

<jats:title>SUMMARY</jats:title><jats:p>The allopolyploid okra (<jats:italic>Abelmoschus esculentus</jats:italic>) unveiled telomeric repeats flanking distal gene‐rich regions and short interstitial TTTAGGG telomeric repeats, possibly representing hallmarks of chromosomal speciation. Ribosomal RNA (rRNA) genes organize into 5S clusters, distinct from the 18S–5.8S–28S units, indicating an S‐type rRNA gene arrangement. The assembly, in line with cytogenetic and cytometry observations, identifies 65 chromosomes and a 1.45 Gb genome size estimate in a haploid sibling. The lack of aberrant meiotic configurations implies limited to no recombination among sub‐genomes. k‐mer distribution analysis reveals 75% has a diploid nature and 15% heterozygosity. The configurations of Benchmarking Universal Single‐Copy Ortholog (BUSCO), k‐mer, and repeat clustering point to the presence of at least two sub‐genomes one with 30 and the other with 35 chromosomes, indicating the allopolyploid nature of the okra genome. Over 130 000 putative genes, derived from mapped IsoSeq data and transcriptome data from public okra accessions, exhibit a low genetic diversity of one single nucleotide polymorphisms per 2.1 kbp. The genes are predominantly located at the distal chromosome ends, declining toward central scaffold domains. Long terminal repeat retrotransposons prevail in central domains, consistent with the observed pericentromeric heterochromatin and distal euchromatin. Disparities in paralogous gene counts suggest potential sub‐genome differentiation implying possible sub‐genome dominance. Amino acid query sequences of putative genes facilitated phenol biosynthesis pathway annotation. Comparison with manually curated reference KEGG pathways from related Malvaceae species reveals the genetic basis for putative enzyme coding genes that likely enable metabolic reactions involved in the biosynthesis of dietary and therapeutic compounds in okra.</jats:p>

<jats:title>SUMMARY</jats:title><jats:p>Phenotyping of model organisms grown on Petri plates is often carried out manually, despite the procedures being time‐consuming and laborious. The main reason for this is the limited availability of automated phenotyping facilities, whereas constructing a custom automated solution can be a daunting task for biologists. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time‐lapse photographs of up to four vertically oriented Petri plates in a single experiment, corresponding to 192 seedlings for a typical root growth assay and up to 2500 seeds for a germination assay. SPIRO is catered specifically to biologists' needs, requiring no engineering or programming expertise for assembly and operation. Its small footprint is optimized for standard incubators, the inbuilt green LED enables imaging under dark conditions, and remote control provides access to the data without interfering with sample growth. SPIRO's excellent image quality is suitable for automated image processing, which we demonstrate on the example of seed germination and root growth assays. Furthermore, the robot can be easily customized for specific uses, as all information about SPIRO is released under open‐source licenses. Importantly, uninterrupted imaging allows considerably more precise assessment of seed germination parameters and root growth rates compared with manual assays. Moreover, SPIRO enables previously technically challenging assays such as phenotyping in the dark. We illustrate the benefits of SPIRO in proof‐of‐concept experiments which yielded a novel insight on the interplay between autophagy, nitrogen sensing, and photoblastic response.</jats:p>

<jats:title>SUMMARY</jats:title><jats:p>RNA editing is a crucial post‐transcriptional modification process in plant organellar RNA metabolism. rRNA removal‐based total RNA‐seq is one of the most common methods to study this event. However, the lack of commercial kits to remove rRNAs limits the usage of this method, especially for non‐model plant species. DSN‐seq is a transcriptome sequencing method utilizing duplex‐specific nuclease (DSN) to degrade highly abundant cDNA species especially those from rRNAs while keeping the robustness of transcript levels of the majority of other mRNAs, and has not been applied to study RNA editing in plants before. In this study, we evaluated the capability of DSN‐seq to reduce rRNA content and profile organellar RNA editing events in plants, as well we used commercial Ribo‐off‐seq and standard mRNA‐seq as comparisons. Our results demonstrated that DSN‐seq efficiently reduced rRNA content and enriched organellar transcriptomes in rice. With high sensitivity to RNA editing events, DSN‐seq and Ribo‐off‐seq provided a more complete and accurate RNA editing profile of rice, which was further validated by Sanger sequencing. Furthermore, DSN‐seq also demonstrated efficient organellar transcriptome enrichment and high sensitivity for profiling RNA editing events in <jats:italic>Arabidopsis thaliana</jats:italic>. Our study highlights the capability of rRNA removal‐based total RNA‐seq for profiling RNA editing events in plant organellar transcriptomes and also suggests DSN‐seq as a widely accessible RNA editing profiling method for various plant species.</jats:p>

<jats:title>SUMMARY</jats:title><jats:p>The intercellular space or apoplast constitutes the main interface in plant–pathogen interactions. Apoplastic subtilisin‐like proteases—subtilases—may play an important role in defence and they have been identified as targets of pathogen‐secreted effector proteins. Here, we characterise the role of the Solanaceae‐specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen <jats:italic>Ralstonia solanacearum</jats:italic>. <jats:italic>R. solanacearum</jats:italic> infection post‐translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to <jats:italic>R. solanacearum</jats:italic>. <jats:italic>In vitro</jats:italic> experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D‐deficient tomato plants were more susceptible to bacterial wilt and transient expression of <jats:italic>P69B</jats:italic>, <jats:italic>D</jats:italic> and <jats:italic>G</jats:italic> in <jats:italic>Nicotiana benthamiana</jats:italic> limited proliferation of <jats:italic>R. solanacearum</jats:italic>. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to <jats:italic>R. solanacearum</jats:italic> but not to other vascular pathogens like <jats:italic>Fusarium oxysporum</jats:italic>.</jats:p>

<jats:title>SUMMARY</jats:title><jats:p><jats:italic>Sclerotinia sclerotiorum</jats:italic> causes white mold or stem rot in a wide range of economically important plants, bringing significant yield losses worldwide. Control of this pathogen is difficult as its resting structure sclerotia can survive in soil for years, and no <jats:italic>Resistance</jats:italic> genes have been identified in <jats:italic>S. sclerotiorum</jats:italic> hosts. Host‐induced gene silencing (HIGS) has shown promising effects in controlling many fungal pathogens, including <jats:italic>S. sclerotiorum</jats:italic>. However, better molecular genetic understanding of signaling pathways involved in its development and pathogenicity is needed to provide effective HIGS gene targets. Here, by employing a forward genetic screen, we characterized an evolutionarily conserved mitogen‐activated protein kinase (MAPK) cascade in <jats:italic>S. sclerotiorum</jats:italic>, consisting of SsSte50‐SsSte11‐SsSte7‐Smk1, which controls mycelial growth, sclerotia development, compound appressoria formation, virulence, and hyphal fusion. Moreover, disruption of the putative downstream transcription factor SsSte12 led to normal sclerotia but deformed appressoria and attenuated host penetration, as well as impaired apothecia formation, suggestive of diverged regulation downstream of the MAPK cascade. Most importantly, targeting <jats:italic>SsSte50</jats:italic> using host‐expressed double‐stranded RNA resulted in largely reduced virulence of <jats:italic>S. sclerotiorum</jats:italic> on both <jats:italic>Nicotiana benthamiana</jats:italic> leaves and transgenic <jats:italic>Arabidopsis thaliana</jats:italic> plants. Therefore, this MAPK signaling cascade is generally needed for its growth, development, and pathogenesis and can serve as ideal HIGS targets for mitigating economic damages caused by <jats:italic>S. sclerotiorum</jats:italic> infection.</jats:p>

<jats:title>SUMMARY</jats:title><jats:p>Petals in rapeseed (<jats:italic>Brassica napus</jats:italic>) serve multiple functions, including protection of reproductive organs, nutrient acquisition, and attraction of pollinators. However, they also cluster densely at the top, forming a thick layer that absorbs and reflects a considerable amount of photosynthetically active radiation. Breeding genotypes with large, small, or even petal‐less varieties, requires knowledge of primary genes for allelic selection and manipulation. However, our current understanding of petal‐size regulation is limited, and the lack of markers and pre‐breeding materials hinders targeted petal‐size breeding. Here, we conducted a genome‐wide association study on petal size using 295 diverse accessions. We identified 20 significant single nucleotide polymorphisms and 236 genes associated with petal‐size variation. Through a cross‐analysis of genomic and transcriptomic data, we focused on 14 specific genes, from which molecular markers for diverging petal‐size features can be developed. Leveraging CRISPR‐Cas9 technology, we successfully generated a quadruple mutant of <jats:italic>Far‐Red Elongated Hypocotyl 3</jats:italic> (<jats:italic>q‐bnfhy3</jats:italic>), which exhibited smaller petals compared to the wild type. Our study provides insights into the genetic basis of petal‐size regulation in rapeseed and offers abundant potential molecular markers for breeding. The <jats:italic>q‐bnfhy3</jats:italic> mutant unveiled a novel role of <jats:italic>FHY3</jats:italic> orthologues in regulating petal size in addition to previously reported functions.</jats:p>