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Summary: The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We previously developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a double‐strand break and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell‐specific expression of the potent but less broadly applicable SaCas9 nuclease. In this study, we now tested whether we could improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20–80‐fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for gene targeting (GT) and chromosomal repeat‐induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower non‐homologous end‐joining (NHEJ) induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT‐rich PAM broadens the range of ipGT drastically, particularly when targeting in CG‐deserts like promoters and introns. Significance Statement: The introduction of pre‐designed changes in the plant genome by homologous recombination (HR) ('gene targeting' GT) still has room for improvement. Although we were recently able to improve the technology by egg cell‐specific expression of SaCas9, the number of addressable genomic sites was limited. Here we demonstrate that LbCas12a, which is able to target more and different sites than SaCas9, can be used efficiently for in planta GT, making the technology more efficient and widely applicable. [ABSTRACT FROM AUTHOR]

SUMMARY: The RTR (RecQ/Top3/Rmi1) complex has been elucidated as essential for ensuring genome stability in eukaryotes. Fundamental for the dissolution of Holliday junction (HJ)‐like recombination intermediates, the factors have been shown to play further, partly distinct roles in DNA repair and homologous recombination. Across all kingdoms, disruption of this complex results in characteristic phenotypes including hyper‐recombination and sensitivity to genotoxins. The type IA topoisomerase TOP3α has been shown as essential for viability in various animals. In contrast, in the model plant species Arabidopsis, the top3α mutant is viable. rmi1 mutants are deficient in the repair of DNA damage. Moreover, as opposed to other eukaryotes, TOP3α and RMI1 were found to be indispensable for proper meiotic progression, with mutants showing severe meiotic defects and sterility. We now established mutants of both TOP3α and RMI1 in tomato using CRISPR/Cas technology. Surprisingly, we found phenotypes that differed dramatically from those of Arabidopsis: the top3α mutants proved to be embryo‐lethal, implying an essential role of the topoisomerase in tomato. In contrast, no defect in somatic DNA repair or meiosis was detectable for rmi1 mutants in tomato. This points to a differentiation of function of RTR complex partners between plant species. Our results indicate that there are relevant differences in the roles of basic factors involved in DNA repair and meiosis within dicotyledons, and thus should be taken as a note of caution when generalizing knowledge regarding basic biological processes obtained in the model plant Arabidopsis for the entire plant kingdom. Significance Statement: We were analysing the biological function of members of the RTR (RecQ/Top3/Rmi1) complex, which is required for safeguarding genome stability in all eukaryotes. Surprisingly, phenotypes differ drastically between tomato and Arabidopsis mutants of the same genes. Our results should be taken as a note of caution when generalizing knowledge regarding basic biological processes obtained in the model plant Arabidopsis for the entire plant kingdom. [ABSTRACT FROM AUTHOR]

SUMMARY: Non‐homologous end joining (NHEJ) plays a major role in repairing DNA double‐strand breaks and is key to genome stability and editing. The minimal core NHEJ proteins, namely Ku70, Ku80, DNA ligase IV and XRCC4, are conserved, but other factors vary in different eukaryote groups. In plants, the only known NHEJ proteins are the core factors, while the molecular mechanism of plant NHEJ remains unclear. Here, we report a previously unidentified plant ortholog of PAXX, the crystal structure of which showed a similar fold to human 'PAXX'. However, plant PAXX has similar molecular functions to human XLF, by directly interacting with Ku70/80 and XRCC4. This suggests that plant PAXX combines the roles of mammalian PAXX and XLF and that these functions merged into a single protein during evolution. This is consistent with a redundant function of PAXX and XLF in mammals. Significance Statement: Non‐homologous end joining (NHEJ) is a major pathway to repair DNA double‐strand breaks and therefore is important for genome maintenance and editing. Using an evolutional analysis, we identified a plant ortholog of an NHEJ protein PAXX and contributed to understanding the exact molecular mechanism of NHEJ in plants. [ABSTRACT FROM AUTHOR]

SUMMARY: Chromosomal rearrangements (CRs) may occur in newly formed polyploids due to compromised meiotic fidelity. Moreover, CRs can be more readily tolerated in polyploids allowing their longer‐term retention and hence potential spreading/fixation within a lineage. The direct functional consequences of CRs in plant polyploids remain unexplored. Here, we identified a heterozygous individual from a synthetic allohexaploid wheat in which the terminal parts of the long‐arms of chromosomes 2D (approximately 193 Mb) and 4A (approximately 167 Mb) were reciprocally translocated. Five homogeneous translocation lines including both unbalanced and balanced types were developed by selfing fertilization of the founder mutant (RT [2DL; 4AL]‐ter/1, reciprocal translocation). We investigated impacts of these translocations on phenotype, genome‐wide gene expression and metabolome. We find that, compared with sibling wild‐type, CRs in the form of both unbalanced and balanced translocations induced substantial changes of gene expression primarily via trans‐regulation in the nascent allopolyploid wheat. The CRs also manifested clear phenotypic and metabolic consequences. In particular, the genetically balanced, stable reciprocal translocations lines showed immediate enhanced reproductive fitness relative to wild type. Our results underscore the profound impact of CRs on gene expression in nascent allopolyploids with wide‐ranging phenotypic and metabolic consequences, suggesting CRs are an important source of genetic variation that can be exploited for crop breeding. Significance Statement: Our results have revealed the profound impact of chromosomal rearrangements on gene expression in nascent allopolyploids with phenotypic and metabolic consequences likely relevant to polyploid crop breeding. [ABSTRACT FROM AUTHOR]

Summary: Gene targeting (GT), the programmed change of genomic sequences by homologous recombination (HR), is still a major challenge in plants. We previously developed an in planta GT strategy by simultaneously releasing from the genome a dsDNA donor molecule and creating a double‐stranded break (DSB) at a specific site within the targeted gene. Using Cas9 form Streptococcus pyogenes (SpCas9) under the control of a ubiquitin gene promoter, we obtained seeds harbouring GT events, although at a low frequency. In the present research we tested different developmentally controlled promotors and different kinds of DNA lesions for their ability to enhance GT of the acetolactate synthase (ALS) gene of Arabidopsis. For this purpose, we used Staphylococcus aureus Cas9 (SaCas9) nuclease and the SpCas9 nickase in various combinations. Thus, we analysed the effect of single‐stranded break (SSB) activation of a targeted gene and/or the HR donor region. Moreover, we tested whether DSBs with 5′ or 3′ overhangs can improve in planta GT. Interestingly, the use of the SaCas9 nuclease controlled by an egg cell‐specific promoter was the most efficient: depending on the line, in the very best case 6% of all seeds carried GT events. In a third of all lines, the targeting occurred around the 1% range of the tested seeds. Molecular analysis revealed that in about half of the cases perfect HR of both DSB ends occurred. Thus, using the improved technology, it should now be feasible to introduce any directed change into the Arabidopsis genome at will. [ABSTRACT FROM AUTHOR]

SUMMARY: Structural maintenance of chromosomes (SMC) complexes are molecular machines ensuring chromatin organization at higher levels. They play direct roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of long‐armed SMC, kleisin, and kleisin‐associated subunits. Additional factors, like NSE6 within SMC5/6, bind to SMC core complexes and regulate their activities. In the human HsNSE6/SLF2, we recently identified a new CANIN domain. Here we tracked down its sequence homology to lower plants, selected the bryophyte Physcomitrium patens, and analyzed PpNSE6 protein–protein interactions to explore its conservation in detail. We identified a previously unrecognized core sequence motif conserved from yeasts to humans within the NSE6 CANIN domain. This motif mediates the interaction between NSE6 and its NSE5 partner in yeasts and plants. In addition, the CANIN domain and its preceding PpNSE6 sequences bind both PpSMC5 and PpSMC6 arms. Interestingly, we mapped the PpNSE6‐binding site at the PpSMC5 arm right next to the PpNSE2‐binding surface. The position of NSE6 at SMC arms suggests its role in the regulation of SMC5/6 dynamics. Consistent with the regulatory role of NSE6 subunits, Ppnse6 mutant lines were viable and sensitive to the DNA‐damaging drug bleomycin and lost a large portion of rDNA copies. These moss mutants also exhibited reduced growth and developmental aberrations. Altogether, our data showed the conserved function of the NSE6 subunit and architecture of the SMC5/6 complex across species. [ABSTRACT FROM AUTHOR]

The possibility to predict the outcome of targeted DNA double-stranded break ( DSB) repair would be desirable for genome editing. Furthermore the consequences of mis-repair of potentially cell-lethal DSBs and the underlying pathways are not yet fully understood. Here we study the clustered regularly interspaced short palindromic repeats ( CRISPR)/Cas9-induced mutation spectra at three selected endogenous loci in Arabidopsis thaliana by deep sequencing of long amplicon libraries. Notably, we found sequence-dependent genomic features that affected the DNA repair outcome. Deletions of 1-bp to <1000-bp size and/or very short insertions, deletions >1 kbp (all due to NHEJ) and deletions combined with insertions between 5-bp to >100 bp [caused by a synthesis-dependent strand annealing ( SDSA)-like mechanism] occurred most frequently at all three loci. The appearance of single-stranded annealing events depends on the presence and distance between repeats flanking the DSB. The frequency and size of insertions is increased if a sequence with high similarity to the target site was available in cis. Most deletions were linked to pre-existing microhomology. Deletion and/or insertion mutations were blunt-end ligated or via de novo generated microhomology. While most mutation types and, to some degree, their predictability are comparable with animal systems, the broad range of deletion mutations seems to be a peculiar feature of the plant A. thaliana. [ABSTRACT FROM AUTHOR]

Production of mutants of crop plants by the use of chemical or physical genotoxins has a long tradition. These factors induce the natural DNA repair machinery to repair damage in an error-prone way. In the case of radiation, multiple double-strand breaks ( DSBs) are induced randomly in the genome, leading in very rare cases to a desirable phenotype. In recent years the use of synthetic, site-directed nucleases ( SDNs) - also referred to as sequence-specific nucleases - like the CRISPR/Cas system has enabled scientists to use exactly the same naturally occurring DNA repair mechanisms for the controlled induction of genomic changes at pre-defined sites in plant genomes. As these changes are not necessarily associated with the permanent integration of foreign DNA, the obtained organisms per se cannot be regarded as genetically modified as there is no way to distinguish them from natural variants. This applies to changes induced by DSBs as well as single-strand breaks, and involves repair by non-homologous end-joining and homologous recombination. The recent development of SDN-based ' DNA-free' approaches makes mutagenesis strategies in classical breeding indistinguishable from SDN-derived targeted genome modifications, even in regard to current regulatory rules. With the advent of new SDN technologies, much faster and more precise genome editing becomes available at reasonable cost, and potentially without requiring time-consuming deregulation of newly created phenotypes. This review will focus on classical mutagenesis breeding and the application of newly developed SDNs in order to emphasize similarities in the context of the regulatory situation for genetically modified crop plants. [ABSTRACT FROM AUTHOR]

SUMMARY: In eukaryotes, meiotic recombination maintains genome stability and creates genetic diversity. The conserved Ataxia‐Telangiectasia Mutated (ATM) kinase regulates multiple processes in meiotic homologous recombination, including DNA double‐strand break (DSB) formation and repair, synaptonemal complex organization, and crossover formation and distribution. However, its function in plant meiotic recombination under stressful environmental conditions remains poorly understood. In this study, we demonstrate that ATM is required for the maintenance of meiotic genome stability under heat stress in Arabidopsis thaliana. Using cytogenetic approaches we determined that ATM does not mediate reduced DSB formation but does ensure successful DSB repair, and thus meiotic chromosome integrity, under heat stress. Further genetic analysis suggested that ATM mediates DSB repair at high temperature by acting downstream of the MRE11–RAD50–NBS1 (MRN) complex, and acts in a RAD51‐independent but chromosome axis‐dependent manner. This study extends our understanding on the role of ATM in DSB repair and the protection of genome stability in plants under high temperature stress. Significance Statement: Heat stress affects meiotic recombination and thus influences genome diversity and stability in plants. In eukaryotes, the conserved kinase ATM regulates multiple meiotic recombination processes; however, its role in meiotic recombination in plants under high‐temperature conditions remains unknown. In this study, we report that ATM, acting downstream of the MRN complex, mediates a RAD51‐independent but chromosome axis‐dependent DSB repair mechanism to protect meiotic genome integrity in Arabidopsis under heat stress. [ABSTRACT FROM AUTHOR]

It is possible to target individual sequence motives within genomes by using synthetic DNA-binding domains. This one-dimensional approach has been used successfully in plants to induce mutations or for the transcriptional regulation of single genes. When the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system was discovered, a tool became available allowing the extension of this approach from one to three dimensions and to construct at least partly synthetic entities on the genome, epigenome and transcriptome levels. The second dimension can be obtained by targeting the Cas9 protein to multiple unique genomic sites by applying multiple different single guiding (sg) RNAs, each defining a different DNA-binding site. Finally, the simultaneous use of phylogenetically different Cas9 proteins or sg RNAs that harbour different types of protein binding motives, allows for a third dimension of control. Thus, different types of enzyme activities - fused either to one type of Cas9 orthologue or to one type of RNA-binding domain specific to one type of sg RNA - can be targeted to multiple different genomic sites simultaneously. Thus, it should be possible to induce quantitatively different levels of expression of certain sets of genes and at the same time to repress other genes, redefining the nuclear transcriptome. Likewise, by the use of different types of histone-modifying and/or DNA (de)methylating activities, the epigenome of plants should be reprogrammable. On our way to synthetic plant genomes, the next steps will be to use complex genome engineering approaches within or between species borders to restructure and recombine natural or artificial chromosomes. [ABSTRACT FROM AUTHOR]

SUMMARY: Somatic mutations may alter important traits in tree fruits, such as fruit color, size and maturation date. Autumn Gala (AGala), a somatic mutation from apple cultivar Gala, matures 4 weeks later than Gala. To understand the mechanisms underlying the delayed maturation, RNA‐seq analyses were conducted with fruit sampled at 13 (Gala) and 16 (AGala) time‐points during their growth and development. Weighted gene co‐expression network analysis (WGCNA) of 23 372 differentially expressed genes resulted in 25 WGCNA modules. Of these, modules 1 (r = −0.98, P = 2E‐21) and 2 (r = −0.52, P = 0.004), which were suppressed in AGala, were correlated with fruit maturation date. Surprisingly, 77 of the 152 member genes in module 1 were harbored in a 2.8‐Mb genomic region on chromosome 6 that was deleted and replaced by a 10.7‐kb gypsy‐like retrotransposon (Gy‐36) from chromosome 7 in AGala. Among the 77 member genes, MdACT7 was the most suppressed (by 10.5‐fold) in AGala due to a disruptive 2.5‐kb insertion in coding sequence. Moreover, MdACT7 is the exclusive apple counterpart of ArabidopsisACT7 known of essential roles in plant development, and the functional allele MdACT7, which was lost to the deletion in AGala, was associated with early fruit maturation in 268 apple accessions. Overexpressing alleles MdACT7 and Mdact7 in an Arabidopsis act7 line showed that MdACT7 largely rescued its stunted growth and delayed initial flowering while Mdact7 did not. Therefore, the 2.8‐Mb hemizygous deletion is largely genetically causal for fruit maturation delay in AGala, and the total loss of MdACT7 might have contributed to the phenotype. Significance Statement: Fruit maturation date is a developmental trait of great horticultural importance. However, the trait remains poorly understood in apple. By sequencing the fruit mRNA of Gala and its somatic mutant, Autumn Gala (AGala), we revealed that a large deletion that occurred concurrently with a retrotransposon insertion is the primary genetic cause for fruit maturation delay in AGala. We also provide evidence that MdACT7, an actin encoding gene, is likely an important factor. [ABSTRACT FROM AUTHOR]

The CRISPR/ Cas nuclease is becoming a major tool for targeted mutagenesis in eukaryotes by inducing double-strand breaks ( DSBs) at pre-selected genomic sites that are repaired by non-homologous end joining ( NHEJ) in an error-prone way. In plants, it could be demonstrated that the Cas9 nuclease is able to induce heritable mutations in Arabidopsis thaliana and rice. Gene targeting ( GT) by homologous recombination ( HR) can also be induced by DSBs. Using a natural nuclease and marker genes, we previously developed an in planta GT strategy in which both a targeting vector and targeting locus are activated simultaneously via DSB induction during plant development. Here, we demonstrate that this strategy can be used for natural genes by CRISPR/ Cas-mediated DSB induction. We were able to integrate a resistance cassette into the ADH1 locus of A. thaliana via HR. Heritable events were identified using a PCR-based genotyping approach, characterised by Southern blotting and confirmed on the sequence level. A major concern is the specificity of the CRISPR/ Cas nucleases. Off-target effects might be avoided using two adjacent sg RNA target sequences to guide the Cas9 nickase to each of the two DNA strands, resulting in the formation of a DSB. By amplicon deep sequencing, we demonstrate that this Cas9 paired nickase strategy has a mutagenic potential comparable with that of the nuclease, while the resulting mutations are mostly deletions. We also demonstrate the stable inheritance of such mutations in A. thaliana. [ABSTRACT FROM AUTHOR]

SUMMARY: High efficiency site‐directed chromosomal integration of exogenous DNA in plants remains a challenge despite recent advances in genome editing technologies. One approach to mitigate this problem is to increase the effective concentration of the donor DNA at the target site of interest. HUH endonucleases (ENs) coordinate rolling circle replication. In vitro, they can form stable covalent bonds with DNA that carries their recognition motifs. When fused to a CRISPR‐associated endonuclease, HUH ENs may improve integration rates by increasing the local donor concentration through tethering of the donor to the CRISPR nuclease. We tested this hypothesis by using chimeric proteins between LbCas12a as a CRISPR‐associated endonuclease and the HUH EN from Faba Bean Necrotic Yellow Virus in soybean (Glycine max). Two fusion protein configurations were tested to integrate a 70‐nt oligonucleotide donor into a commercially important target site using protoplasts and in planta transformation. Site‐directed integration rates of the donor DNA, when tethered to the fusion protein, reached about 26% in plants and were up to four‐fold higher than in untethered controls. Integrations via canonical homology‐directed repair or non‐homologous end joining were promoted by tethering in a similar fashion. This study is the first demonstration of HUH EN‐associated tethering to improve site‐directed DNA integration in plants. Significance Statement: An exceptionally high rate of site‐directed integration of exogenous DNA into the soybean (Glycine max) genome was achieved using a novel method to increase the local donor DNA concentration around the CRISPR target site of interest. A donor DNA was covalently tethered to a fusion protein between LbCas12a and the HUH endonuclease of Faba Bean Necrotic Yellow Virus, which was delivered into soybean protoplasts and plants as part of a ribonucleoprotein complex. [ABSTRACT FROM AUTHOR]

SUMMARY: Through the inactivation of genes that act during meiosis it is possible to direct the genetic make‐up of plants in subsequent generations and optimize breeding schemes. Offspring may show higher recombination of parental alleles resulting from elevated crossover (CO) incidence, or by omission of meiotic divisions, offspring may become polyploid. However, stable mutations in genes essential for recombination, or for either one of the two meiotic divisions, can have pleiotropic effects on plant morphology and line stability, for instance by causing lower fertility. Therefore, it is often favorable to temporarily change gene expression during meiosis rather than relying on stable null mutants. It was previously shown that virus‐induced gene silencing (VIGS) can be used to transiently reduce CO frequencies. We asked if VIGS could also be used to modify other processes throughout meiosis and during pollen formation in Arabidopsis thaliana. Here, we show that VIGS‐mediated knock‐down of FIGL1, RECQ4A/B, OSD1 and QRT2 can induce (i) an increase in chiasma numbers, (ii) unreduced gametes and (iii) pollen tetrads. We further show that VIGS can target both sexes and different genetic backgrounds and can simultaneously silence different gene copies. The successful knock‐down of these genes in A. thaliana suggests that VIGS can be exploited to manipulate any process during or shortly after meiosis. Hence, the transient induction of changes in inheritance patterns can be used as a powerful tool for applied research and biotechnological applications. Significance Statement: The present work demonstrates that VIGS is an efficient tool to quickly accomplish breeding aims such as an increase in crossover recombination, the generation of polyploid offspring and modification of pollen‐specific processes. [ABSTRACT FROM AUTHOR]

By inducing double-strand breaks ( DSB), it is possible to initiate DNA recombination. For a long time, it was not possible to use DSB induction for efficient genome engineering due to the lack of a means to target DSBs to specific sites. This limitation was overcome by development of modified meganucleases and synthetic DNA-binding domains. Domains derived from zinc-finger transcription factors or transcription activator-like effectors may be designed to recognize almost any DNA sequence. By fusing these domains to the endonuclease domains of a class II restriction enzyme, an active endonuclease dimer may be formed that introduces a site-specific DSB. Recent studies demonstrate that gene knockouts via non-homologous end joining or gene modification via homologous recombination are becoming routine in many plant species. By creating a single genomic DSB, complete knockout of a gene, sequence-specific integration of foreign DNA or subtle modification of individual amino acids in a specific protein domain may be achieved. The induction of two or more DSBs allows complex genomic rearrangements such as deletions, inversions or the exchange of chromosome arms. The potential for controlled genome engineering in plants is tremendous. The recently discovered RNA-based CRISPR/Cas system, a new tool to induce multiple DSBs, and sophisticated technical applications, such as the in planta gene targeting system, are further steps in this development. At present, the focus remains on engineering of single genes; in the future, engineering of whole genomes will become an option. [ABSTRACT FROM AUTHOR]

SUMMARY: Mediator 17 (MED17) is a subunit of the Mediator complex that regulates transcription initiation in eukaryotic organisms. In yeast and humans, MED17 also participates in DNA repair, physically interacting with proteins of the nucleotide excision DNA repair system, but this function in plants has not been investigated. We studied the role of MED17 in Arabidopsis plants exposed to UV‐B radiation. Our results demonstrate that med17 and OE MED17 plants have altered responses to UV‐B, and that MED17 participates in various aspects of the DNA damage response (DDR). Comparison of the med17 transcriptome with that of wild‐type (WT) plants showed that almost one‐third of transcripts with altered expression in med17 plants were also changed by UV‐B exposure in WT plants. Increased sensitivity to DNA damage after UV‐B in med17 plants could result from the altered regulation of UV‐B responsive transcripts but MED17 also physically interacts with DNA repair proteins, suggesting a direct role of this Mediator subunit during repair. Finally, we show that MED17 is necessary to regulate the DDR activated by ataxia telangiectasia and Rad3 related (ATR), and that programmed cell death 5 (PDCD5) overexpression reverts the deficiencies in DDR shown in med17 mutants. Our data demonstrate that MED17 is an important regulator of DDR after UV‐B irradiation in Arabidopsis. Significance Statement: In Arabidopsis, MED17 regulates the DNA damage response after UV‐B exposure transcriptionally modulating the expression of genes and possibly also physically interacting with DNA repair proteins. [ABSTRACT FROM AUTHOR]

SUMMARY: Plant cysteine oxidases (PCOs) are plant O2‐sensing enzymes. They catalyse the O2‐dependent step which initiates the proteasomal degradation of Group VII ethylene response transcription factors (ERF‐VIIs) via the N‐degron pathway. When submerged, plants experience a reduction in O2 availability; PCO activity therefore decreases and the consequent ERF‐VII stabilisation leads to upregulation of hypoxia‐responsive genes which enable adaptation to low O2 conditions. Resulting adaptations include entering an anaerobic quiescent state to maintain energy reserves and rapid growth to escape floodwater and allow O2 transport to submerged tissues. Stabilisation of ERF‐VIIs has been linked to improved survival post‐submergence in Arabidopsis, rice (Oryza sativa) and barley (Hordeum vulgare). Due to climate change and increasing flooding events, there is an interest in manipulating the PCO/ERF‐VII interaction as a method of improving yields in flood‐intolerant crops. An effective way of achieving this may be through PCO inhibition; however, complete ablation of PCO activity is detrimental to growth and phenotype, likely due to other PCO‐mediated roles. Targeting PCOs will therefore require either temporary chemical inhibition or careful engineering of the enzyme structure to manipulate their O2 sensitivity and/or substrate specificity. Sufficient PCO structural and functional information should make this possible, given the potential to engineer site‐directed mutagenesis in vivo using CRISPR‐mediated base editing. Here, we discuss the knowledge still required for rational manipulation of PCOs to achieve ERF‐VII stabilisation without a yield penalty. We also take inspiration from the biocatalysis field to consider how enzyme engineering could be accelerated as a wider strategy to improve plant stress tolerance and productivity. Significance Statement: Plant cysteine oxidase enzymes sense oxygen availability such that when oxygen levels drop, as occurs when plants are flooded, they initiate an adaptive response to help the plant survive until oxygen becomes available again. Manipulating these enzymes to alter their oxygen‐sensing and/or substrate‐binding characteristics has the potential to prolong flood tolerance in plants, illustrating that enzyme engineering could be adopted more widely as a strategy to improve plant stress resistance and productivity. [ABSTRACT FROM AUTHOR]

SUMMARY: Being sessile organisms, plants are ubiquitously exposed to stresses that can affect the DNA replication process or cause DNA damage. To cope with these problems, plants utilize DNA damage response (DDR) pathways, consisting of both highly conserved and plant‐specific elements. As a part of this DDR, cell cycle checkpoint control mechanisms either pause the cell cycle, to allow DNA repair, or lead cells into differentiation or programmed cell death, to prevent the transmission of DNA errors in the organism through mitosis or to its offspring via meiosis. The two major DDR cell cycle checkpoints control either the replication process or the G2/M transition. The latter is largely overseen by the plant‐specific SOG1 transcription factor, which drives the activity of cyclin‐dependent kinase inhibitors and MYB3R proteins, which are rate limiting for the G2/M transition. By contrast, the replication checkpoint is controlled by different players, including the conserved kinase WEE1 and likely the transcriptional repressor RBR1. These checkpoint mechanisms are called upon during developmental processes, in retrograde signaling pathways, and in response to biotic and abiotic stresses, including metal toxicity, cold, salinity, and phosphate deficiency. Additionally, the recent expansion of research from Arabidopsis to other model plants has revealed species‐specific aspects of the DDR. Overall, it is becoming evidently clear that the DNA damage checkpoint mechanisms represent an important aspect of the adaptation of plants to a changing environment, hence gaining more knowledge about this topic might be helpful to increase the resilience of plants to climate change. Significance Statement: To cope with stresses that might impede the replication process or cause DNA breaks, plants utilize DNA damage‐responsive cell cycle checkpoint mechanisms that help them adapt to the stress while maintaining genome integrity. Understanding these mechanisms might be pivotal to increase the resilience of plants to climate change. [ABSTRACT FROM AUTHOR]

SUMMARY: Global agriculture is dominated by a handful of species that currently supply a huge proportion of our food and feed. It additionally faces the massive challenge of providing food for 10 billion people by 2050, despite increasing environmental deterioration. One way to better plan production in the face of current and continuing climate change is to better understand how our domestication of these crops included their adaptation to environments that were highly distinct from those of their centre of origin. There are many prominent examples of this, including the development of temperate Zea mays (maize) and the alteration of day‐length requirements in Solanum tuberosum (potato). Despite the pre‐eminence of some 15 crops, more than 50 000 species are edible, with 7000 of these considered semi‐cultivated. Opportunities afforded by next‐generation sequencing technologies alongside other methods, including metabolomics and high‐throughput phenotyping, are starting to contribute to a better characterization of a handful of these species. Moreover, the first examples of de novo domestication have appeared, whereby key target genes are modified in a wild species in order to confer predictable traits of agronomic value. Here, we review the scale of the challenge, drawing extensively on the characterization of past agriculture to suggest informed strategies upon which the breeding of future climate‐resilient crops can be based. Significance Statement: In this article we summarize recent advances in the understanding of the genetic basis of crop domestication and how this knowledge, combined with state‐of‐the‐art and future tools for gene editing or classical breeding, could open new avenues for establishing crops better suited to future climate conditions. [ABSTRACT FROM AUTHOR]

SUMMARY: MicroRNAs negatively regulate gene expression by promoting target mRNA cleavage and/or impairing its translation, thereby playing a crucial role in plant development and environmental stress responses. In Arabidopsis, the MIR840 gene is located within the overlapping 3′UTR of the PPR and WHIRLY3 (WHY3) genes, both being predicted targets of miR840* and miR840, the short maturation products of MIR840. Gain‐ and loss‐of‐function of MIR840 in Arabidopsis resulted in opposite senescence phenotypes. The highest expression levels of the MIR840 precursor transcript pre‐miR840 were observed at senescence initiation, and pre‐miR840 expression is significantly correlated with a reduction in PPR, but not WHY3, transcript levels. Although a reduction of transcript level of PPR, but not WHY3 transcript levels were not significantly affected by MIR840 overexpression, its protein levels were strongly reduced. Mutating the cleavage sites or replacing the target sequences abolishes the miR840*/miR840‐mediated degradation of PPR transcripts and accumulation of WHY3 protein. In support for this, concurrent knockdown of both PPR and WHY3 in wild‐type plants resulted in a senescence phenotype resembling that of the MIR840‐overexpressing plant. This indicates that both PRR and WHY3 are targets in the MIR840‐mediated senescence pathway. Moreover, single knockout mutants of PPR and WHY3 show a convergent upregulated subset of senescence‐associated genes, which are also found among those induced by MIR840 overexpression. Our data provide evidence for a regulatory role of MIR840 in plant senescence. [ABSTRACT FROM AUTHOR]

SUMMARY: Agrobacterium tumefaciens‐mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T‐DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)‐mediated end joining (TMEJ). Targeting of the T‐DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T‐DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Polθ. Whereas in TMEJ‐proficient plants we predominantly found GT events accompanied by random T‐DNA integrations, GT events obtained in the teb mutant background lacked additional T‐DNA copies, corroborating the essential role of Polθ in T‐DNA integration. Polθ deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high‐quality GT events in crop plants. Significance Statement: We find that homologous recombination‐mediated gene targeting (GT) in polymerase theta (Polθ)‐deficient Arabidopsis exclusively produces plants that contain true GT events and are devoid of random T‐DNA integration. These observations corroborate the crucial role for Polθ in T‐DNA integration, and reveal that Polθ‐mediated end joining is essential for ectopic GT, which is a prominent outcome in Polθ‐proficient plants. [ABSTRACT FROM AUTHOR]

Heavy metal-rich toxic soils and ordinary soils are both natural habitats of Arabidopsis halleri, a diploid perennial and obligate outcrosser in the sister clade of the genetic model plant Arabidopsis thaliana. The molecular divergence underlying survival in sharply contrasting environments is unknown. Here we comparatively address metal physiology and transcriptomes of A. halleri originating from the most highly heavy metal-contaminated soil in Europe, Ponte Nossa, Italy (Noss), and from non-metalliferous (NM) soils. Plants from Noss exhibit enhanced hypertolerance and attenuated accumulation of cadmium (Cd), and their transcriptomic Cd responsiveness is decreased, compared to plants of NM soil origin. Among the conditionindependent transcriptome characteristics of Noss, the most highly overrepresented functional class of 'meiotic cell cycle' comprises 21 transcripts with elevated abundance in vegetative tissues, in particular Argonaute 9 (AGO9) and the synaptonemal complex transverse filament protein-encoding ZYP1a/b. Increased AGO9 transcript levels in Noss are accompanied by decreased long terminal repeat retrotransposon expression. Similar to Noss, plants from other highly metalliferous sites in Poland and Germany share elevated somatic AGO9 transcript levels in comparison to plants originating from NM soils in their respective geographic regions. Transcript levels of Iron-Regulated Transporter 1 (IRT1) are very low and transcript levels of Heavy Metal ATPase 2 (HMA2) are strongly elevated in Noss, which can account for its altered Cd handling. We conclude that in plants adapted to the most extreme abiotic stress, broadly enhanced functions comprise genes with likely roles in somatic genome integrity maintenance, accompanied by few alterations in stress-specific functional networks. [ABSTRACT FROM AUTHOR]

SUMMARY: Cell walls, especially secondary cell walls (SCWs), maintain cell shape and reinforce wood, but their structure and shape can be altered in response to gravity. In hardwood trees, tension wood is formed along the upper side of a bending stem and contains wood fiber cells that have a gelatinous layer (G‐layer) inside the SCW. In a previous study, we generated nst/snd quadruple‐knockout aspens (Populus tremula × Populus tremuloides), in which SCW formation was impaired in 99% of the wood fiber cells. In the present study, we produced nst/snd triple‐knockout aspens, in which a large number of wood fibers had thinner SCWs than the wild type (WT) and some had no SCW. Because SCW layers are always formed prior to G‐layer deposition, the nst/snd mutants raise interesting questions of whether the mutants can form G‐layers without SCW and whether they can control their postures in response to changes in gravitational direction. The nst/snd mutants and the WT plants showed growth eccentricity and vessel frequency reduction when grown on an incline, but the triple mutants recovered their upright growth only slightly, and the quadruple mutants were unable to maintain their postures. The mutants clearly showed that the G‐layers were formed in SCW‐containing wood fibers but not in those lacking the SCW. Our results indicate that SCWs are essential for G‐layer formation and posture control. Furthermore, each wood fiber cell may be able to recognize its cell wall developmental stage to initiate the formation of the G‐layer as a response to gravistimulation. [ABSTRACT FROM AUTHOR]

SUMMARY: An analysis of over 10 000 plant genome sizes (GSs) indicates that most species have smaller genomes than expected given the incidence of polyploidy in their ancestries, suggesting selection for genome downsizing. However, comparing ancestral GS with the incidence of ancestral polyploidy suggests that the rate of DNA loss following polyploidy is likely to have been very low (4–70 Mb/million years, 4–482 bp/generation). This poses a problem. How might such small DNA losses be visible to selection, overcome the power of genetic drift and drive genome downsizing? Here we explore that problem, focussing on the role that double‐strand break (DSB) repair pathways (non‐homologous end joining and homologous recombination) may have played. We also explore two hypotheses that could explain how selection might favour genome downsizing following polyploidy: to reduce (i) nitrogen (N) and phosphate (P) costs associated with nucleic acid synthesis in the nucleus and the transcriptome and (ii) the impact of scaling effects of GS on cell size, which influences CO2 uptake and water loss. We explore the hypothesis that losses of DNA must be fastest in early polyploid generations. Alternatively, if DNA loss is a more continuous process over evolutionary time, then we propose it is a byproduct of selection elsewhere, such as limiting the damaging activity of repetitive DNA. If so, then the impact of GS on photosynthesis, water use efficiency and/or nutrient costs at the nucleus level may be emergent properties, which have advantages, but not ones that could have been selected for over generational timescales. Significance Statement: Despite an astonishing 2400‐fold range in angiosperm genome sizes, a key unanswered question is why most plant genomes are small, in spite of the abundance of repeats and the prevalence of polyploidy. This review outlines our current understanding of the mechanisms and processes responsible for genome size diversity, predicts typical rates of DNA loss following polyploidy and explores how selection might act to drive genome downsizing. [ABSTRACT FROM AUTHOR]

SUMMARY: Genome‐editing technologies consisting of targeted mutagenesis and gene targeting enable us to modify genes of interest rapidly and precisely. The discovery in 2012 of CRISPR/Cas9 systems and their development as sequence‐specific nucleases has brought about a paradigm shift in biology. Initially, CRISPR/Cas9 was applied in targeted mutagenesis to knock out a target gene. Thereafter, advances in genome‐editing technologies using CRISPR/Cas9 developed rapidly, with base editing systems for transition substitution using a combination of Cas9 nickase and either cytidine or adenosine deaminase being reported in 2016 and 2017, respectively, and later in 2021 bringing reports of transversion substitution using Cas9 nickase, cytidine deaminase and uracil DNA glycosylase. Moreover, technologies for gene targeting and prime editing systems using DNA or RNA as donors have also been developed in recent years. Besides these precise genome‐editing strategies, reports of successful chromosome engineering using CRISPR/Cas9 have been published recently. The application of genome editing to crop breeding has advanced in parallel with the development of these technologies. Genome‐editing enzymes can be introduced into plant cells, and there are now many examples of crop breeding using genome‐editing technologies. At present, it is no exaggeration to say that we are now in a position to be able to modify a gene precisely and rearrange genomes and chromosomes in a predicted way. In this review, we introduce and discuss recent highlights in the field of precise gene editing, chromosome engineering and genome engineering technology in plants. Significance Statement: New developments in genome‐editing technologies allow not only gene knockout but also precise editing and chromosome rearrangements. This review summarizes current trends in genome‐editing technology and its application in plants. [ABSTRACT FROM AUTHOR]

SUMMARY: Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR‐associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR‐Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target‐specific and a screening process is usually required to identify those most effective. Plant virus‐derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus‐induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX‐based vector, Nicotianabenthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus‐free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR‐Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops. [ABSTRACT FROM AUTHOR]

SUMMARY: Genome editing by RNA‐guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron‐optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene‐free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2, respectively). We developed novel counter‐selection markers for N. benthamiana, most importantly Sl‐FAST2, comparable to the well‐established Arabidopsis seed fluorescence marker, and FCY‐UPP, based on the production of toxic 5‐fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY‐UPP for selection of non‐transgenic T1, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher‐order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. [ABSTRACT FROM AUTHOR]

SUMMARY: Indole‐3‐carbinol (I3C), a hydrolysis product of indole‐3‐methylglucosinolate, is toxic to herbivorous insects and pathogens. In mammals, I3C is extensively studied for its properties in cancer prevention and treatment. Produced in Brassicaceae, I3C reversibly inhibits root elongation in a concentration‐dependent manner. This inhibition is partially explained by the antagonistic action of I3C on auxin signaling through TIR1. To further elucidate the mode of action of I3C in plants, we have identified and characterized a novel Arabidopsis mutant tolerant to I3C, ICT1. This mutant was identified following screening of the Full‐length cDNA Over‐eXpression library (FOX) seed collection for root growth in the presence of exogenous I3C. ICT1 carries the AT2G19750 gene, which encodes an S30 ribosomal protein. Overexpression, but not knockout, of the S30 gene causes tolerance to I3C. The tolerance is specific to I3C, since ICT1 did not exhibit pronounced tolerance to other indole or benzoxazinoid molecules tested. ICT1 maintains I3C‐induced antagonism of auxin signaling, indicating that the tolerance is due to an auxin‐independent mechanism. Transcript profiling experiments revealed that ICT1 is transcriptionally primed to respond to I3C treatment. Significance Statement: Overexpression of the ribosomal S30 gene in Arabidopsis causes tolerance to the glucosinolate breakdown product indole‐3‐carbinol by an extra‐ribosomal, auxin‐independent mechanism. Transcript profiling experiments revealed that ICT1 is transcriptionally primed to respond to I3C treatment, as well as to oxidative and biotic challenges. [ABSTRACT FROM AUTHOR]

The first line of plant defense occurs when a plant pattern recognition receptor (PRR) recognizes microbe‐associated molecular patterns. Plant PRRs are either receptor‐like kinases (RLKs), which have an extracellular domain for ligand binding, a single‐pass transmembrane domain, and an intracellular kinase domain for activating downstream signaling, or receptor‐like proteins (RLPs), which share the same overall structure but lack an intracellular kinase domain. The tomato (Solanum lycopersicum) LeEIX2 is an RLP that binds ethylene‐inducing xylanase (EIX), a fungal elicitor. To identify LeEIX2 receptor interactors, we conducted a yeast two‐hybrid screen and found a tomato protein that we termed SlRLK‐like. The interaction of LeEIX2 with SlRLK‐like was verified using co‐immunoprecipitation and bimolecular fluorescence complementation assays. The defense responses induced by EIX were markedly reduced when SlRLK‐like was overexpressed in Nicotiana benthamiana or Nicotiana tabacum, and knockout of SlRLK‐like using the CRISPR/Cas9 system increased EIX‐induced ethylene production and 1‐aminocyclopropane‐1‐carboxylate synthase (SlACS2) gene expression in tomato. Co‐expression of SlRLK‐like with LeEIX2 led to a reduction in its abundance, apparently through an endoplasmic reticulum‐associated degradation process. Notably, truncation of SlRLK‐like protein revealed that the malectin‐like domain is sufficient and essential for its function. Moreover, SlRLK‐like associated with the RLK FLS2, resulting in its degradation and concomitantly a reduction of the flagellin 22 (flg22)‐induced burst of reactive oxygen species. In addition, SlRLK‐like co‐expression with other RLPs, Ve1 and AtRLP23, also led to a reduction in their abundance. Our findings suggest that SlRLK‐like leads to a decreased stability of various PRRs, leading to a reduction in their abundance and resulting in attenuation of defense responses. [ABSTRACT FROM AUTHOR]

SUMMARY: Meiotic recombination is initiated by formation of DNA double‐strand breaks (DSBs). This involves a protein complex that includes in plants the two similar proteins, SPO11‐1 and SPO11‐2. We analysed the sequences of SPO11‐2 in hexaploid bread wheat (Triticum aestivum), as well as in its diploid and tetraploid progenitors. We investigated its role during meiosis using single, double and triple mutants. The three homoeologous SPO11‐2 copies of hexaploid wheat exhibit high nucleotide and amino acid similarities with those of the diploids, tetraploids and Arabidopsis. Interestingly, however, two nucleotides deleted in exon‐2 of the A copy lead to a premature stop codon and suggest that it encodes a non‐functional protein. Remarkably, the mutation was absent from the diploid A‐relative Triticum urartu, but present in the tetraploid Triticum dicoccoides and in different wheat cultivars indicating that the mutation occurred after the first polyploidy event and has since been conserved. We further show that triple mutants with all three copies (A, B, D) inactivated are sterile. Cytological analyses of these mutants show synapsis defects, accompanied by severe reductions in bivalent formation and numbers of DMC1 foci, thus confirming the essential role of TaSPO11‐2 in meiotic recombination in wheat. In accordance with its 2‐nucleotide deletion in exon‐2, double mutants for which only the A copy remained are also sterile. Notwithstanding, some DMC1 foci remain visible in this mutant, suggesting a residual activity of the A copy, albeit not sufficient to restore fertility. Significance Statement: Analysis of the three homoeologous copies of TaSPO11‐2 of bread wheat and of meiosis in the corresponding mutants reveals that TaSPO11‐2‐7A is non‐functional due to a two‐nucleotide deletion in exon‐2 that occurred after the first polyploidization event. Consequently, numbers of DMC1‐foci (and presumably of double‐strand breaks) are strongly reduced in triple and double Taspo11‐2 mutants, accompanied by strong meiotic defects and confirming the essential role of TaSPO11‐2 in meiosis. [ABSTRACT FROM AUTHOR]

SUMMARY: We previously developed a large‐scale genome restructuring technology called the TAQing system. It can induce genomic rearrangements by introducing transient and conditional formation of DNA double‐strand breaks (DSBs) via heat activation of a restriction enzyme TaqI, which can cleave DNA at 5′‐TCGA‐3′ sequences in the genome at higher temperatures (37–42°C). Such heat treatment sometimes confers lethal damage in certain plant species and TaqI cannot induce rearrangements in AT‐rich regions. To overcome such problems we developed an extended TAQing (Ex‐TAQing) system, which enables the use of a wider range of restriction enzymes active at standard plant‐growing temperatures. We established the Ex‐TAQing system using MseI that can efficiently cleave DNA at room temperature (at temperatures ranging from 22 to 25°C) and the 5′‐TTAA‐3′ sequence which is highly abundant in the Arabidopsis genome. A synthetic intron‐spanning MseI gene, which was placed downstream of a heat‐shock‐inducible promoter, was conditionally expressed upon milder heat treatment (33°C) to generate DSBs in Arabidopsis chromosomes. Genome resequencing revealed various types of genomic rearrangements, including copy number variations, translocation and direct end‐joining at MseI cleavage sites. The Ex‐TAQing system could induce large‐scale rearrangements in diploids more frequently (17.4%, n = 23) than the standard TAQing system. The application of this system to tetraploids generated several strains with chromosomal rearrangements associated with beneficial phenotypes, such as high salinity stress tolerance and hypersensitivity to abscisic acid. We have developed the Ex‐TAQing system, allowing more diverse patterns of genomic rearrangements, by employing various types of endonucleases and have opened a way to expand the capacity for artificial genome reorganization. Significance Statement: We have developed an extended TAQing (Ex‐TAQing) system to overcome weaknesses concerning plant fertility and the efficiency of genomic rearrangement in the TAQing system. The Ex‐TAQing system can induce massive genomic rearrangements at normal growth temperatures and extend the variety of plant genome beyond genome ploidy. Ex‐TAQing is able to contribute to screening for useful traits in polyploid plants, providing insight into the relationship between the rearranged genome and phenotypic change. [ABSTRACT FROM AUTHOR]

SUMMARY: The manipulation of meiotic recombination in crops is essential to develop new plant varieties rapidly, helping to produce more cultivars in a sustainable manner. One option is to control the formation and repair of the meiosis‐specific DNA double‐strand breaks (DSBs) that initiate recombination between the homologous chromosomes and ultimately lead to crossovers. These DSBs are introduced by the evolutionarily conserved topoisomerase‐like protein SPO11 and associated proteins. Here, we characterized the homoeologous copies of the SPO11‐1 protein in hexaploid bread wheat (Triticum aestivum). The genome contains three SPO11‐1 gene copies that exhibit 93–95% identity at the nucleotide level, and clearly the A and D copies originated from the diploid ancestors Triticum urartu and Aegilops tauschii, respectively. Furthermore, phylogenetic analysis of 105 plant genomes revealed a clear partitioning between monocots and dicots, with the seven main motifs being almost fully conserved, even between clades. The functional similarity of the proteins among monocots was confirmed through complementation analysis of the Oryza sativa (rice) spo11‐1 mutant by the wheat TaSPO11‐1‐5D coding sequence. Also, remarkably, although the wheat and Arabidopsis SPO11‐1 proteins share only 55% identity and the partner proteins also differ, the TaSPO11‐1‐5D cDNA significantly restored the fertility of the Arabidopsis spo11‐1 mutant, indicating a robust functional conservation of the SPO11‐1 protein activity across distant plants. These successful heterologous complementation assays, using both Arabidopsis and rice hosts, are good surrogates to validate the functionality of candidate genes and cDNA, as well as variant constructs, when the transformation and mutant production in wheat is much longer and more tedious. Significance Statement: We analyzed the three homoeologous copies of the SPO11‐1 gene found in the bread wheat genome. They are very similar but not identical, revealing their parental origin from diploid wheats. These SPO11‐1 proteins contain seven essential and evolutionarily conserved motifs, now found in 105 plant species. The coding sequence of the wheat D copy significantly complemented the rice and Arabidopsis spo11‐1 mutants, indicating strong functional conservation between highly divergent species. [ABSTRACT FROM AUTHOR]

SUMMARY: The role of rice (Oryza sativa) COM1 in meiotic homologous recombination (HR) is well understood, but its part in somatic double‐stranded break (DSB) repair remains unclear. Here, we show that for rice plants COM1 conferred tolerance against DNA damage caused by the chemicals bleomycin and mitomycin C, while the COM1 mutation did not compromise HR efficiencies and HR factor (RAD51 and RAD51 paralogues) localization to irradiation‐induced DSBs. Similar retarded growth at the post‐germination stage was observed in the com1‐2 mre11 double mutant and the mre11 single mutant, while combined mutations in COM1 with the HR pathway gene (RAD51C) or classic non‐homologous end joining (NHEJ) pathway genes (KU70, KU80, and LIG4) caused more phenotypic defects. In response to γ‐irradiation, COM1 was loaded normally onto DSBs in the ku70 mutant, but could not be properly loaded in the MRE11RNAi plant and in the wortmannin‐treated wild‐type plant. Under non‐irradiated conditions, more DSB sites were occupied by factors (MRE11, COM1, and LIG4) than RAD51 paralogues (RAD51B, RAD51C, and XRCC3) in the nucleus of wild‐type; protein loading of COM1 and XRCC3 was increased in the ku70 mutant. Therefore, quite differently to its role for HR in meiocytes, rice COM1 specifically acts in an alternative NHEJ pathway in somatic cells, based on the Mre11–Rad50–Nbs1 (MRN) complex and facilitated by PI3K‐like kinases. NHEJ factors, not HR factors, preferentially load onto endogenous DSBs, with KU70 restricting DSB localization of COM1 and XRCC3 in plant somatic cells. Significance Statement: Double‐stranded breaks (DSB) in genomic DNA are potentially lethal lesions that separate parts of chromosome arms from their centromeres. Quite differently to its role in homologous recombination (HR) for meiocytes, here we show that rice COM1 specifically acts in an alternative non‐homologous end joining (alt‐NHEJ) pathway in somatic cells, based on the Mre11–Rad50–Nbs1 (MRN) complex and facilitated by PI3K‐like kinases, and that the NHEJ factors (MRE11, COM1, and LIG4), not the HR factors (RAD51B, RAD51C, and XRCC3), preferentially load onto endogenous DSBs, with the KU70 restricting DSB localization of COM1 and XRCC3 in plant somatic cells. [ABSTRACT FROM AUTHOR]

Summary: Gene‐editing techniques are currently revolutionizing biology, allowing far greater precision than previous mutagenic and transgenic approaches. They are becoming applicable to a wide range of plant species and biological processes. Gene editing can rapidly improve a range of crop traits, including disease resistance, abiotic stress tolerance, yield, nutritional quality and additional consumer traits. Unlike transgenic approaches, however, it is not facile to forensically detect gene‐editing events at the molecular level, as no foreign DNA exists in the elite line. These limitations in molecular detection approaches are likely to focus more attention on the products generated from the technology than on the process in itself. Rapid advances in sequencing and genome assembly increasingly facilitate genome sequencing as a means of characterizing new varieties generated by gene‐editing techniques. Nevertheless, subtle edits such as single base changes or small deletions may be difficult to distinguish from normal variation within a genotype. Given these emerging scenarios, downstream 'omics' technologies reflective of edited affects, such as metabolomics, need to be used in a more prominent manner to fully assess compositional changes in novel foodstuffs. To achieve this goal, metabolomics or 'non‐targeted metabolite analysis' needs to make significant advances to deliver greater representation across the metabolome. With the emergence of new edited crop varieties, we advocate: (i) concerted efforts in the advancement of 'omics' technologies, such as metabolomics, and (ii) an effort to redress the use of the technology in the regulatory assessment for metabolically engineered biotech crops. Significance Statement: Unlike with transgenic approaches, it is not facile to detect gene‐editing events forensically at the molecular level, as no foreign DNA exists in gene‐edited lines. The limitation in molecular detection approaches are likely to focus more attention on the products generated from the technology than on the process itself. Here, we highlight the use of metabolomics as a means to characterize (and in some cases identify) gene‐edited material and champion its use for regulatory purposes. [ABSTRACT FROM AUTHOR]

Summary: Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are crucial for the maintenance of genome integrity. In most plants, telomeres consist of conserved tandem repeat units comprising the TTTAGGG motif. Recently, non‐canonical telomeres were described in several plants and plant taxons, including the carnivorous plant Genlisea hispidula (TTCAGG/TTTCAGG), the genus Cestrum (Solanaceae; TTTTTTAGGG), and plants from the Asparagales order with either a vertebrate‐type telomere repeat TTAGGG or Allium genus‐specific CTCGGTTATGGG repeat. We analyzed epigenetic modifications of telomeric histones in plants with canonical and non‐canonical telomeres, and further in telomeric chromatin captured from leaves of Nicotiana benthamiana transiently transformed by telomere CRISPR‐dCas9‐eGFP, and of Arabidopsis thaliana stably transformed with TALE_telo C‐3×GFP. Two combinatorial patterns of telomeric histone modifications were identified: (i) an Arabidopsis‐like pattern (A. thaliana, G. hispidula, Genlisea nigrocaulis, Allium cepa, Narcissus pseudonarcissus, Petunia hybrida, Solanum tuberosum, Solanum lycopersicum) with telomeric histones decorated predominantly by H3K9me2; (ii) a tobacco‐like pattern (Nicotiana tabacum, N. benthamiana, C. elegans) with a strong H3K27me3 signal. Our data suggest that epigenetic modifications of plant telomere‐associated histones are related neither to the sequence of the telomere motif nor to the lengths of the telomeres. Nor the phylogenetic position of the species plays the role; representatives of the Solanaceae family are included in both groups. As both patterns of histone marks are compatible with fully functional telomeres in respective plants, we conclude that the described specific differences in histone marks are not critical for telomere functions. Significance Statement: In plants, besides the canonical telomeric repeat (TTTAGGG)n, other sequences have been described in several taxa. We identified two patterns of telomeric histone modifications in plants with canonical and non‐canonical telomeres that were related neither to the respective telomere sequences or lengths, nor to the phylogenetic relationship of plants. Thus, the proper function of plant telomeres does not correlate with the exclusive pattern of histone epigenetic marks. [ABSTRACT FROM AUTHOR]

Summary: Many plant genomes display high levels of repetitive sequences. The assembly of these complex genomes using short high‐throughput sequence reads is still a challenging task. Underestimation or disregard of repeat complexity in these datasets can easily misguide downstream analysis. Detection of repetitive regions by k‐mer counting methods has proved to be reliable. Easy‐to‐use applications utilizing k‐mer counting are in high demand, especially in the domain of plants. We present Kmasker plants, a tool that uses k‐mer count information as an assistant throughout the analytical workflow of genome data that is provided as a command‐line and web‐based solution. Beside its core competence to screen and mask repetitive sequences, we have integrated features that enable comparative studies between different cultivars or closely related species and methods that estimate target specificity of guide RNAs for application of site‐directed mutagenesis using Cas9 endonuclease. In addition, we have set up a web service for Kmasker plants that maintains pre‐computed indices for 10 of the economically most important cultivated plants. Source code for Kmasker plants has been made publically available at https://github.com/tschmutzer/kmasker. The web service is accessible at https://kmasker.ipk-gatersleben.de. Significance Statement: Whole‐genome shotgun sequencing is producing billions of sequencing reads which can be processed as short sequence words, called k‐mers, that have the power to reveal differences between species or detect sequence regions with striking patterns. Kmasker plants is a tool that utilizes k‐mer count information to perform various applications, allowing sequence data scientists as well as non‐bioinformatics experts to perform k‐mer analysis on whole‐genome shotgun sequencing data for their species of interest. [ABSTRACT FROM AUTHOR]

Summary: Genome wide screening of pooled pollen samples from a single interspecific F1 hybrid obtained from a cross between tomato, Solanum lycopersicum and its wild relative, Solanum pimpinellifolium using linked read sequencing of the haploid nuclei, allowed profiling of the crossover (CO) and gene conversion (GC) landscape. We observed a striking overlap between cold regions of CO in the male gametes and our previously established F6 recombinant inbred lines (RILs) population. COs were overrepresented in non‐coding regions in the gene promoter and 5′UTR regions of genes. Poly‐A/T and AT rich motifs were found enriched in 1 kb promoter regions flanking the CO sites. Non‐crossover associated allelic and ectopic GCs were detected in most chromosomes, confirming that besides CO, GC represents also a source for genetic diversity and genome plasticity in tomato. Furthermore, we identified processed break junctions pointing at the involvement of both homology directed and non‐homology directed repair pathways, suggesting a recombination machinery in tomato that is more complex than currently anticipated. Significance Statement: This paper reports a technical advance of using linked‐read sequencing to identify meiotic recombination events, both crossovers and non‐crossovers, from the pollen of an F1 tomato hybrid. Meiotic recombination is of basic importance, and also has strategic relevance to crop improvement, particularly in crop species such as tomato where large regions of the chromosomes are non‐recombining. [ABSTRACT FROM AUTHOR]

Summary: Repetitive DNA sequences and some genes are epigenetically repressed by transcriptional gene silencing (TGS). When genetic mutants are not available or problematic to use, TGS can be suppressed by chemical inhibitors. However, informed use of epigenetic inhibitors is partially hampered by the absence of any systematic comparison. In addition, there is emerging evidence that epigenetic inhibitors cause genomic instability, but the nature of this damage and its repair remain unclear. To bridge these gaps, we compared the effects of 5‐azacytidine (AC), 2′‐deoxy‐5‐azacytidine (DAC), zebularine and 3‐deazaneplanocin A (DZNep) on TGS and DNA damage repair. The most effective inhibitor of TGS was DAC, followed by DZNep, zebularine and AC. We confirmed that all inhibitors induce DNA damage and suggest that this damage is repaired by multiple pathways with a critical role of homologous recombination and of the SMC5/6 complex. A strong positive link between the degree of cytidine analog‐induced DNA demethylation and the amount of DNA damage suggests that DNA damage is an integral part of cytidine analog‐induced DNA demethylation. This helps us to understand the function of DNA methylation in plants and opens the possibility of using epigenetic inhibitors in biotechnology. Significance Statement: We show that 2′‐deoxy‐5‐azacytidine is the most potent chemical inhibitor of DNA methylation in plants, and demonstrate that azacytidine‐like drugs cause DNA damage, which is signaled by ATM and ATR kinases and requires repair by homologous recombination. [ABSTRACT FROM AUTHOR]

Summary: Correct chloroplast development and function require co‐ordinated expression of chloroplast and nuclear genes. This is achieved through chloroplast signals that modulate nuclear gene expression in accordance with the chloroplast's needs. Genetic evidence indicates that GUN1, a chloroplast‐localized pentatricopeptide repeat (PPR) protein with a C‐terminal Small MutS‐Related (SMR) domain, is involved in integrating multiple developmental and stress‐related signals in both young seedlings and adult leaves. Recently, GUN1 was found to interact physically with factors involved in chloroplast protein homeostasis, and with enzymes of tetrapyrrole biosynthesis in adult leaves that function in various retrograde signalling pathways. Here we show that following perturbation of chloroplast protein homeostasis: (i) by growth in lincomycin‐containing medium; or (ii) in mutants defective in either the FtsH protease complex (ftsh), plastid ribosome activity (prps21‐1 and prpl11‐1) or plastid protein import and folding (cphsc70‐1), GUN1 influences NEP‐dependent transcript accumulation during cotyledon greening and also intervenes in chloroplast protein import. Significance Statement: Correct chloroplast development and function require co‐ordinated regulation of chloroplast and nuclear gene expression. Here, we propose that GUN1, a main player of chloroplast‐to‐nucleus retrograde communication, influences NEP‐dependent transcript accumulation during cotyledon greening and also intervenes in chloroplast protein import. [ABSTRACT FROM AUTHOR]

Summary: DNA damage responses are crucial for plant growth under genotoxic stress. Accumulating evidence indicates that DNA damage responses differ between plant cell types. Here, quantitative shotgun phosphoproteomics provided high‐throughput analysis of the DNA damage response network in callus cells. MS analysis revealed a wide network of highly dynamic changes in the phosphoprotein profile of genotoxin‐treated cells, largely mediated by the ATAXIA TELANGIECTASIA MUTATED (ATM) protein kinase, representing candidate factors that modulate plant growth, development and DNA repair. A C‐terminal dual serine target motif unique to H2AX in the plant lineage showed 171‐fold phosphorylation that was absent in atm mutant lines. The physiological significance of post‐translational DNA damage signalling to plant growth and survival was demonstrated using reverse genetics and complementation studies of h2ax mutants, establishing the functional role of ATM‐mediated histone modification in plant growth under genotoxic stress. Our findings demonstrate the complexity and functional significance of post‐translational DNA damage signalling responses in plants and establish the requirement of H2AX phosphorylation for plant survival under genotoxic stress. Significance Statement: This work demonstrates the central role for ATM in DNA damage signalling and establishes the functional requirement for H2AX phosphorylation in plant resistance to genotoxic stress. These results revealed the physiological significance of ATM‐dependent post‐translational protein modification in the plant DNA damage response in mitigating the deleterious cellular consequences of genome damage. [ABSTRACT FROM AUTHOR]

Summary: Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non‐vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co‐delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss‐of‐function mutants of multiple genes from different gene families. Significance Statement: In Physcomitrella patens, functional analysis, especially the analysis of genes from different gene families or genes with redundant functions in the same gene family, is hampered to some extent by the complexity and redundancy of these genes. The multiplex gene editing method reported here facilitates the creation of loss‐of‐function mutants of multiple genes from different gene families, without the need for a laborious hybridization step. [ABSTRACT FROM AUTHOR]

Summary: Meiosis is critical for sexual reproduction and the generation of new allelic variations in most eukaryotes. In this study, we report the isolation of a meiotic gene, DLC1, using a map‐based cloning strategy. The dlc1 mutant is sterile in both male and female gametophytes due to an earlier defect in the leptotene chromosome and subsequent abnormalities at later stages. DLC1 is strongly expressed in the pollen mother cells (PMCs) and tapetum and encodes a nucleus‐located rice type‐B response regulator (RR) with transcriptional activity. Further investigations showed that DLC1 interacts with all five putative rice histidine phosphotransfer proteins (HPs) in yeast and planta cells, suggesting a possible participation of the two‐component signalling systems (TCS) in rice meiosis. Our results demonstrated that DLC1 is required for rice meiosis and fertility, providing useful information for the role of TCS in rice meiosis. Significance Statement: As a member of the two‐component signalling system (TCS), DLC1 participates in rice meiosis and is required for leptotene chromosome organization and fertility. [ABSTRACT FROM AUTHOR]

Summary: Telomeres and ribosomal RNA genes (rDNA) are essential for cell survival and particularly sensitive to factors affecting genome stability. Here, we examine the role of RAD51 and its antagonist, RTEL1, in the moss Physcomitrella patens. In corresponding mutants, we analyse their sensitivity to DNA damage, the maintenance of telomeres and rDNA, and repair of double‐stranded breaks (DSBs) induced by genotoxins with various modes of action. While the loss of RTEL1 results in rapid telomere shortening, concurrent loss of both RAD51 genes has no effect on telomere lengths. We further demonstrate here the linked arrangement of 5S and 45S rRNA genes in P. patens. The spacer between 5S and 18S rRNA genes, especially the region downstream from the transcription start site, shows conspicuous clustering of sites with a high propensity to form quadruplex (G4) structures. Copy numbers of 5S and 18S rDNA are reduced moderately in the pprtel1 mutant, and significantly in the double pprad51‐1‐2 mutant, with no progression during subsequent cultivation. While reductions in 45S rDNA copy numbers observed in pprtel1 and pprad51‐1‐2 plants apply also to 5S rDNA, changes in transcript levels are different for 45S and 5S rRNA, indicating their independent transcription by RNA polymerase I and III, respectively. The loss of SOL (Sog One‐Like), a transcription factor regulating numerous genes involved in DSB repair, increases the rate of DSB repair in dividing as well as differentiated tissue, and through deactivation of G2/M cell‐cycle checkpoint allows the cell‐cycle progression manifested as a phenotype resistant to bleomycin. Significance Statement: To explore roles of RAD51, RTEL1 and SOL in the maintenance of the Physcomitrella patens genome stability, we characterize sensitivity of the respective mutants to genotoxic treatments and compare the rates of repair of double strand DNA breaks to the wild type moss. We demonstrate a linked arrangement of 5S‐45S rDNA loci, whose copies are reduced in RTEL1 and RAD51 mutants, and observe a rapid telomere shortening in RTEL1 mutants. [ABSTRACT FROM AUTHOR]

Summary: During the evolution of plant genomes, sequence inversions occurred repeatedly making the respective regions inaccessible for meiotic recombination and thus for breeding. Therefore, it is important to develop technologies that allow the induction of inversions within chromosomes in a directed and efficient manner. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), we were able to obtain scarless heritable inversions with high efficiency in the model plant Arabidopsis thaliana. Via deep sequencing, we defined the patterns of junction formation in wild‐type and in the non‐homologous end‐joining (NHEJ) mutant ku70‐1. Surprisingly, in plants deficient of KU70, inversion induction is enhanced, indicating that KU70 is required for tethering the local broken ends together during repair. However, in contrast to wild‐type, most junctions are formed by microhomology‐mediated NHEJ and thus are imperfect with mainly deletions, making this approach unsuitable for practical applications. Using egg‐cell‐specific expression of Cas9, we were able to induce heritable inversions at different genomic loci and at intervals between 3 and 18 kb, in the percentage range, in the T1 generation. By screening individual lines, inversion frequencies of up to the 10% range were found in T2. Most of these inversions had scarless junctions and were without any sequence change within the inverted region, making the technology attractive for use in crop plants. Applying our approach, it should be possible to reverse natural inversions and induce artificial ones to break or fix linkages between traits at will. Significance Statement: During the evolution of plant genomes, sequence inversions occurred repeatedly making the regions inaccessible for breeders to transfer certain traits to cultivated relatives. Using the CRISPR/Cas nuclease of S. aureus, we developed a methodology for not only inducing such intrachromosomal rearrangements in a precise way, but also for their transfer to the next generation. Applying the technology, it should now be possible to fix or break genetic linkages between traits for breeding. [ABSTRACT FROM AUTHOR]

Summary: Aluminium (Al) ions are one of the primary growth‐limiting factors for plants on acid soils, globally restricting agriculture. Despite its impact, little is known about Al action in planta. Earlier work has indicated that, among other effects, Al induces DNA damage. However, the loss of major DNA damage response regulators, such SOG1, partially suppressed the growth reduction in plants seen on Al‐containing media. This raised the question whether Al actually causes DNA damage and, if so, how. Here, we provide cytological and genetic data corroborating that exposure to Al leads to DNA double‐strand breaks. We find that the Al‐induced damage specifically involves homology‐dependent (HR) recombination repair. Using an Al toxicity assay that delivers higher Al concentrations than used in previous tests, we find that sog1 mutants become highly sensitive to Al. This indicates a multi‐level response to Al‐induced DNA damage in plants. Significance Statement: Aluminium (Al) is considered to be one of the primary growth‐limiting factors for agriculture especially in developing countries. Here, we explore a recently discovered aspect of Al, i.e. that it causes DNA damage, and show that this damage requires homology‐dependent DNA repair to sustain plant growth on Al‐containing media. [ABSTRACT FROM AUTHOR]