Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells.

Cas12a is a promising addition to the CRISPR toolbox, offering versatility due to its TTTV-protospacer adjacent motif (PAM) and the fact that it induces double-stranded breaks (DSBs) with single-stranded overhangs. We characterized Cas12a-mediated genome editing in tomato using high-throughput amplicon sequencing on protoplasts. Of the three tested variants, Lachnospiraceae (Lb) Cas12a was the most efficient. Additionally, we developed an easy and effective Golden-Gate-based system for crRNA cloning. We compared LbCas12a to SpCas9 by investigating on-target efficacy and specificity at 35 overlapping target sites and 57 (LbCas12a) or 100 (SpCas9) predicted off-target sites. We found LbCas12a an efficient, robust addition to SpCas9, with similar overall though target-dependent efficiencies. LbCas12a induced more and larger deletions than SpCas9, which can be advantageous for specific genome editing applications. Off-target activity for LbCas12a was found at 10 out of 57 investigated sites. One or two mismatches were present distal from the PAM in all cases. We conclude that Cas12a-mediated genome editing is generally precise as long as such off-target sites can be avoided. In conclusion, we have determined the mutation pattern and efficacy of Cas12a-mediated CRISPR mutagenesis in tomato and developed a cloning system for the routine application of Cas12a for tomato genome editing.

A gene regulatory network critical for axillary bud dormancy directly controlled by Arabidopsis BRANCHED1.

The Arabidopsis thaliana transcription factor BRANCHED1 (BRC1) plays a pivotal role in the control of shoot branching as it integrates environmental and endogenous signals that influence axillary bud growth. Despite its remarkable activity as a growth inhibitor, the mechanisms by which BRC1 promotes bud dormancy are largely unknown. We determined the genome-wide BRC1 binding sites in vivo and combined these with transcriptomic data and gene co-expression analyses to identify bona fide BRC1 direct targets. Next, we integrated multi-omics data to infer the BRC1 gene regulatory network (GRN) and used graph theory techniques to find network motifs that control the GRN dynamics. We generated an open online tool to interrogate this network. A group of BRC1 target genes encoding transcription factors (BTFs) orchestrate this intricate transcriptional network enriched in abscisic acid-related components. Promoter::ß-GLUCURONIDASE transgenic lines confirmed that BTFs are expressed in axillary buds. Transient co-expression assays and studies in planta using mutant lines validated the role of BTFs in modulating the GRN and promoting bud dormancy. This knowledge provides access to the developmental mechanisms that regulate shoot branching and helps identify candidate genes to use as tools to adapt plant architecture and crop production to ever-changing environmental conditions.

TM3 and STM3 Promote Flowering Together with FUL2 and MBP20, but Act Antagonistically in Inflorescence Branching in Tomato.

The moment at which a plant transitions to reproductive development is paramount to its life cycle and is strictly controlled by many genes. The transcription factor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) plays a central role in this process in Arabidopsis. However, the role of SOC1 in tomato (Solanum lycopersicum) has been sparsely studied. Here, we investigated the function of four tomato SOC1 homologs in the floral transition and inflorescence development. We thoroughly characterized the SOC1-like clade throughout the Solanaceae and selected four tomato homologs that are dynamically expressed upon the floral transition. We show that of these homologs, TOMATO MADS 3 (TM3) and SISTER OF TM3 (STM3) promote the primary and sympodial transition to flowering, while MADS-BOX PROTEIN 23 (MBP23) and MBP18 hardly contribute to flowering initiation in the indeterminate cultivar Moneyberg. Protein-protein interaction assays and whole-transcriptome analysis during reproductive meristem development revealed that TM3 and STM3 interact and share many targets with FRUITFULL (FUL) homologs, including cytokinin regulators. Furthermore, we observed that mutating TM3/STM3 affects inflorescence development, but counteracts the inflorescence-branching phenotype of ful2 mbp20. Collectively, this indicates that TM3/STM3 promote the floral transition together with FUL2/MBP20, while these transcription factors have opposite functions in inflorescence development.

High-throughput sgRNA testing reveals rules for Cas9 specificity and DNA repair in tomato cells.

CRISPR/Cas9 technology has the potential to significantly enhance plant breeding. To determine the specificity and the mutagenic spectrum of SpCas9 in tomato, we designed 89 g(uide) RNAs targeting genes of the tomato MYB transcription factor family with varying predicted specificities. Plasmids encoding sgRNAs and Cas9 were introduced into tomato protoplasts, and target sites as well as 224 predicted off-target sites were screened for the occurrence of mutations using amplicon sequencing. Algorithms for the prediction of efficacy of the sgRNAs had little predictive power in this system. The analysis of mutations suggested predictable identity of single base insertions. Off-target mutations were found for 13 out of 89 sgRNAs and only occurred at positions with one or two mismatches (at 14 and 3 sites, respectively). We found that PAM-proximal mismatches do not preclude low frequency off-target mutations. Off-target mutations were not found at all 138 positions that had three or four mismatches. We compared off-target mutation frequencies obtained with plasmid encoding sgRNAs and Cas9 with those induced by ribonucleoprotein (RNP) transfections. The use of RNPs led to a significant decrease in relative off-target frequencies at 6 out of 17, no significant difference at 9, and an increase at 2 sites. Additionally, we show that off-target sequences with insertions or deletions relative to the sgRNA may be mutated, and should be considered during sgRNA design. Altogether, our data help sgRNA design by providing insight into the Cas9-induced double-strand break repair outcomes and the occurrence of off-target mutations.

HD-ZIP Transcription Factors and Brassinosteroid Signaling Play a Role in Capitulum Patterning in Chrysanthemum.

Chrysanthemum is a genus in the Asteraceae family containing numerous cut flower varieties with high ornamental value. It owes its beauty to the composite flower head, which resembles a compact inflorescence. This structure is also known as a capitulum, in which many ray and disc florets are densely packed. The ray florets are localized at the rim, are male sterile, and have large colorful petals. The centrally localized disc florets develop only a small petal tube but produce fertile stamens and a functional pistil. Nowadays, varieties with more ray florets are bred because of their high ornamental value, but, unfortunately, this is at the expense of their seed setting. In this study, we confirmed that the disc:ray floret ratio is highly correlated to seed set efficiency, and therefore, we further investigated the mechanisms that underlie the regulation of the disc:ray floret ratio. To this end, a comprehensive transcriptomics analysis was performed in two acquired mutants with a higher disc:ray floret ratio. Among the differentially regulated genes, various potential brassinosteroid (BR) signaling genes and HD-ZIP class IV homeodomain transcription factors stood out. Detailed follow-up functional studies confirmed that reduced BR levels and downregulation of HD-ZIP IV gene Chrysanthemum morifolium PROTODERMAL FACTOR 2 (CmPDF2) result in an increased disc:ray floret ratio, thereby providing ways to improve seed set in decorative chrysanthemum varieties in the future.

Identification of the rice Rc gene as a main regulator of seed survival under dry storage conditions.

Seed deterioration during storage results in poor germination, reduced vigour, and non-uniform seedling emergence. The aging rate depends on storage conditions and genetic factors. This study aims to identify these genetic factors determining the longevity of rice (Oryza sativa L.) seeds stored under experimental aging conditions mimicking long-term dry storage. Genetic variation for tolerance to aging was studied in 300 Indica rice accessions by storing dry seeds under an elevated partial pressure of oxygen (EPPO) condition. A genome-wide association analysis identified 11 unique genomic regions for all measured germination parameters after aging, differing from those previously identified in rice under humid experimental aging conditions. The significant single nucleotide polymorphism in the most prominent region was located within the Rc gene, encoding a basic helix-loop-helix transcription factor. Storage experiments using near-isogenic rice lines (SD7-1D (Rc) and SD7-1d (rc) with the same allelic variation confirmed the role of the wildtype Rc gene, providing stronger tolerance to dry EPPO aging. In the seed pericarp, a functional Rc gene results in accumulation of proanthocyanidins, an important sub-class of flavonoids having strong antioxidant activity, which may explain the variation in tolerance to dry EPPO aging.

Dual specificity and target gene selection by the MADS-domain protein FRUITFULL.

How transcription factors attain their target gene specificity and how this specificity may be modulated, acquiring different regulatory functions through the development of plant tissues, is an open question. Here we characterized different regulatory roles of the MADS-domain transcription factor FRUITFULL (FUL) in flower development and mechanisms modulating its activity. We found that the dual role of FUL in regulating floral transition and pistil development is associated with its different in vivo patterns of DNA binding in both tissues. Characterization of FUL protein complexes by liquid chromatography-tandem mass spectrometry and SELEX-seq experiments shows that aspects of tissue-specific target site selection can be predicted by tissue-specific variation in the composition of FUL protein complexes with different DNA binding specificities, without considering the chromatin status of the target region. This suggests a role for dynamic changes in FUL TF complex composition in reshaping the regulatory functions of FUL during flower development.

Twisting development, the birth of a potential new gene.

Evolution has long been considered to be a conservative process in which new genes arise from pre-existing genes through gene duplication, domain shuffling, horizontal transfer, overprinting, retrotransposition, etc. However, this view is changing as new genes originating from non-genic sequences are discovered in different organisms. Still, rather limited functional information is available. Here, we have identified TWISTED1 (TWT1), a possible de novo-originated protein-coding gene that modifies microtubule arrangement and causes helicoidal growth in Arabidopsis thaliana when its expression is increased. Interestingly, even though TWT1 is a likely recent gene, the lack of TWT1 function affects A. thaliana development. TWT1 seems to have originated from a non-genic sequence. If so, it would be one of the few examples to date of how during evolution de novo genes are integrated into developmental cellular and organismal processes.

Experimental rice seed aging under elevated oxygen pressure: Methodology and mechanism.

Seed aging during storage results in loss of vigor and germination ability due to the accumulation of damage by oxidation reactions. Experimental aging tests, for instance to study genetic variation, aim to mimic natural aging in a shorter timeframe. As the oxidation rate is increased by elevating the temperature, moisture, and oxygen levels, this study aimed to (1) investigate the effect of experimental rice seed aging by an elevated partial pressure of oxygen (EPPO), (2) elucidate the mechanism of dry-EPPO aging and (3) compare aging under dry-EPPO conditions to aging under traditional moist-controlled deterioration (CD) conditions and to long-term ambient storage. Dry seeds from 20 diverse rice accessions were experimentally aged under EPPO (200 times higher oxygen levels), at 50% relative humidity (RH), along with storage under high-pressure nitrogen gas and ambient conditions as controls. While no decline in germination was observed with ambient storage, there was significant aging of the rice seeds under EPPO storage, with considerable variation in the aging rate among the accessions, with an average decline toward 50% survival obtained after around 21 days in EPPO storage and total loss of germination after 56 days. Storage under high-pressure nitrogen gas resulted in a small but significant decline, by an average of 5% germination after 56 days. In a second experiment, seven rice seed lots were stored under EPPO as compared to a moist-CD test and two different long-term ambient storage conditions, i.e., conditioned warehouse seed storage (CWSS) and traditional rice seed storage (TRSS). Untargeted metabolomics (with identification of lipid and volatile compounds profiles) showed a relatively high increase in levels of oxidized lipids and related volatiles under all four storage conditions. These compounds had a high negative correlation with seed viability, indicating oxidation as a main deteriorating process during seed aging. Correlation analysis indicated that EPPO storage at 50% RH is more related to aging under TRSS at 60% and CD-aging at 75% ERH rather than CWSS at 40% ERH. In conclusion, aging rice seeds under EPPO conditions is a suitable experimental aging method for analyzing variation among seed lots or genotypes for longevity under storage.

BABY BOOM regulates early embryo and endosperm development.

The BABY BOOM (BBM) AINTEGUMENTA-LIKE (AIL) AP2/ERF domain transcription factor is a major regulator of plant cell totipotency, as it induces asexual embryo formation when ectopically expressed. Surprisingly, only limited information is available on the role of BBM during zygotic embryogenesis. Here we reexamined BBM expression and function in the model plant Arabidopsis thaliana (Arabidopsis) using reporter analysis and newly developed CRISPR mutants. BBM was expressed in the embryo from the zygote stage and also in the maternal (nucellus) and filial (endosperm) seed tissues. Analysis of CRISPR mutant alleles for BBM (bbm-cr) and the redundantly acting AIL gene PLETHORA2 (PLT2) (plt2-cr) uncovered individual roles for these genes in the timing of embryo progression. We also identified redundant roles for BBM and PLT2 in endosperm proliferation and cellularization and the maintenance of zygotic embryo development. Finally, we show that ectopic BBM expression in the egg cell of Arabidopsis and the dicot crops Brassica napus and Solanum lycopersicon is sufficient to bypass the fertilization requirement for embryo development. Together these results highlight roles for BBM and PLT2 in seed development and demonstrate the utility of BBM genes for engineering asexual embryo development in dicot species.

The TCP transcription factor HvTB2 heterodimerizes with VRS5 and controls spike architecture in barley.

KEY MESSAGE: Understanding the molecular network, including protein-protein interactions, of VRS5 provide new routes towards the identification of other key regulators of plant architecture in barley. The TCP transcriptional regulator TEOSINTE BRANCHED 1 (TB1) is a key regulator of plant architecture. In barley, an important cereal crop, HvTB1 (also referred to as VULGARE SIX-ROWED spike (VRS) 5), inhibits the outgrowth of side shoots, or tillers, and grains. Despite its key role in barley development, there is limited knowledge on the molecular network that is utilized by VRS5. In this work, we performed protein-protein interaction studies of VRS5. Our analysis shows that VRS5 potentially interacts with a diverse set of proteins, including other class II TCP's, NF-Y TF, but also chromatin remodelers. Zooming in on the interaction capacity of VRS5 with other TCP TFs shows that VRS5 preferably interacts with other class II TCP TFs in the TB1 clade. Induced mutagenesis through CRISPR-Cas of one of the putative VRS5 interactors, HvTB2 (also referred to as COMPOSITUM 1 and BRANCHED AND INDETERMINATE SPIKELET 1), resulted in plants that have lost their characteristic unbranched spike architecture. More specifically, hvtb2 mutants exhibited branches arising at the main spike, suggesting that HvTB2 acts as inhibitor of branching. Our protein-protein interaction studies of VRS5 resulted in the identification of HvTB2 as putative interactor of VRS5, another key regulator of spike architecture in barley. The study presented here provides a first step to underpin the protein-protein interactome of VRS5 and to identify other, yet unknown, key regulators of barley plant architecture.

Auxin biosynthesis maintains embryo identity and growth during BABY BOOM-induced somatic embryogenesis.

Somatic embryogenesis is a type of plant cell totipotency where embryos develop from nonreproductive (vegetative) cells without fertilization. Somatic embryogenesis can be induced in vitro by auxins, and by ectopic expression of embryo-expressed transcription factors like the BABY BOOM (BBM) AINTEGUMENTA-LIKE APETALA2/ETHYLENE RESPONSE FACTOR domain protein. These different pathways are thought to converge to promote auxin response and biosynthesis, but the specific roles of the endogenous auxin pathway in somatic embryogenesis induction have not been well-characterized. Here we show that BBM transcriptionally regulates the YUCCA3 (YUC3) and YUC8 auxin biosynthesis genes during BBM-mediated somatic embryogenesis in Arabidopsis (Arabidopsis thaliana) seedlings. BBM induced local and ectopic YUC3 and YUC8 expression in seedlings, which coincided with increased DR5 auxin response and indole-3-acetic acid (IAA) biosynthesis and with ectopic expression of the WOX2 embryo reporter. YUC-driven auxin biosynthesis was required for BBM-mediated somatic embryogenesis, as the number of embryogenic explants was reduced by ca. 50% in yuc3 yuc8 mutants and abolished after chemical inhibition of YUC enzyme activity. However, a detailed YUC inhibitor time-course study revealed that YUC-dependent IAA biosynthesis is not required for the re-initiation of totipotent cell identity in seedlings. Rather, YUC enzymes are required later in somatic embryo development for the maintenance of embryo identity and growth. This study resolves a long-standing question about the role of endogenous auxin biosynthesis in transcription factor-mediated somatic embryogenesis and also provides an experimental framework for understanding the role of endogenous auxin biosynthesis in other in planta and in vitro embryogenesis systems.

Passiflora organensis FT/TFL1 gene family and their putative roles in phase transition and floral initiation.

KEY MESSAGE: Comprehensive analysis of the FT/TFL1 gene family in Passiflora organensis results in understanding how these genes might be involved in the regulation of the typical plant architecture presented by Passiflora species. Passion fruit (Passiflora spp) is an economic tropical fruit crop, but there is hardly any knowledge available about the molecular control of phase transition and flower initiation in this species. The florigen agent FLOWERING LOCUS T (FT) interacts with the bZIP protein FLOWERING LOCUS D (FD) to induce flowering in the model species Arabidopsis thaliana. Current models based on research in rice suggest that this interaction is bridged by 14-3-3 proteins. We identified eight FT/TFL1 family members in Passiflora organensis and characterized them by analyzing their phylogeny, gene structure, expression patterns, protein interactions and putative biological roles by heterologous expression in Arabidopsis. PoFT was highest expressed during the adult vegetative phase and it is supposed to have an important role in flowering induction. In contrast, its paralogs PoTSFs were highest expressed in the reproductive phase. While ectopic expression of PoFT in transgenic Arabidopsis plants induced early flowering and inflorescence determinacy, the ectopic expression of PoTSFa caused a delay in flowering. PoTFL1-like genes were highest expressed during the juvenile phase and their ectopic expression caused delayed flowering in Arabidopsis. Our protein-protein interaction studies indicate that the flowering activation complexes in Passiflora might deviate from the hexameric complex found in the model system rice. Our results provide insights into the potential functions of FT/TFL1 gene family members during floral initiation and their implications in the special plant architecture of Passiflora species, contributing to more detailed studies on the regulation of passion fruit reproduction.

FRUITFULL-like genes regulate flowering time and inflorescence architecture in tomato.

The timing of flowering and the inflorescence architecture are critical for the reproductive success of tomato (Solanum lycopersicum), but the gene regulatory networks underlying these traits have not been fully explored. Here, we show that the tomato FRUITFULL-like (FUL-like) genes FUL2 and MADS-BOX PROTEIN 20 (MBP20) promote the vegetative-to-reproductive transition and repress inflorescence branching by inducing floral meristem (FM) maturation. FUL1 fulfils a less prominent role and appears to depend on FUL2 and MBP20 for its upregulation in the inflorescence- and floral meristems. MBP10, the fourth tomato FUL-like gene, has probably lost its function. The tomato FUL-like proteins cannot homodimerize in in vitro assays, but heterodimerize with various other MADS-domain proteins, potentially forming distinct complexes in the transition meristem and FM. Transcriptome analysis of the primary shoot meristems revealed various interesting downstream targets, including four repressors of cytokinin signaling that are upregulated during the floral transition in ful1 ful2 mbp10 mbp20 mutants. FUL2 and MBP20 can also bind in vitro to the upstream regions of these genes, thereby probably directly stimulating cell division in the meristem upon the transition to flowering. The control of inflorescence branching does not occur via the cytokinin oxidase/dehydrogenases (CKXs) but may be regulated by repression of transcription factors such as TOMATO MADS-box gene 3 (TM3) and APETALA 2b (AP2b).

ABA signalling promotes cell totipotency in the shoot apex of germinating embryos.

Somatic embryogenesis (SE) is a type of induced cell totipotency where embryos develop from vegetative tissues of the plant instead of from gamete fusion after fertilization. SE can be induced in vitro by exposing explants to growth regulators, such as the auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The plant hormone abscisic acid (ABA) has been proposed to be a downstream signalling component at the intersection between 2,4-D- and stress-induced SE, but it is not known how these pathways interact to induce cell totipotency. Here we show that 2,4-D-induced SE from the shoot apex of germinating Arabidopsis thaliana seeds is characterized by transcriptional maintenance of an ABA-dependent seed maturation pathway. Molecular-genetic analysis of Arabidopsis mutants revealed a role for ABA in promoting SE at three different levels: ABA biosynthesis, ABA receptor complex signalling, and ABA-mediated transcription, with essential roles for the ABSCISIC ACID INSENSITIVE 3 (ABI3) and ABI4 transcription factors. Our data suggest that the ability of mature Arabidopsis embryos to maintain the ABA seed maturation environment is an important first step in establishing competence for auxin-induced cell totipotency. This finding provides further support for the role of ABA in directing processes other than abiotic stress response.

Live Imaging of embryogenic structures in Brassica napus microspore embryo cultures highlights the developmental plasticity of induced totipotent cells.

KEY MESSAGE: In vitro embryo development is highly plastic; embryo cell fate can be re-established in tissue culture through different pathways. In most angiosperms, embryo development from the single-celled zygote follows a defined pattern of cell divisions in which apical (embryo proper) and basal (root and suspensor) cell fates are established within the first cell divisions. By contrast, embryos that are induced in vitro in the absence of fertilization show a less regular initial cell division pattern yet develop into histodifferentiated embryos that can be converted into seedlings. We used the Brassica napus microspore embryogenesis system, in which the male gametophyte is reprogrammed in vitro to form haploid embryos, to identify the developmental fates of the different types of embryogenic structures found in culture. Using time-lapse imaging of LEAFY COTYLEDON1-expressing cells, we show that embryogenic cell clusters with very different morphologies are able to form haploid embryos. The timing of surrounding pollen wall (exine) rupture is a major determinant of cell fate in these clusters, with early exine rupture leading to the formation of suspensor-bearing embryos and late rupture to suspensorless embryos. In addition, we show that embryogenic callus, which develops into suspensor-bearing embryos, initially expresses transcripts associated with both basal- and apical-embryo cell fates, suggesting that these two cell fates are fixed later in development. This study reveals the inherent plasticity of in vitro embryo development and identifies new pathways by which embryo cell fate can be established.

The rin, nor and Cnr spontaneous mutations inhibit tomato fruit ripening in additive and epistatic manners.

Tomato fruit ripening is regulated by transcription factors (TFs), their downstream effector genes, and the ethylene biosynthesis and signalling pathway. Spontaneous non-ripening mutants ripening inhibitor (rin), non-ripening (nor) and Colorless non-ripening (Cnr) correspond with mutations in or near the TF-encoding genes MADS-RIN, NAC-NOR and SPL-CNR, respectively. Here, we produced heterozygous single and double mutants of rin, nor and Cnr and evaluated their functions and genetic interactions in the same genetic background. We showed how these mutations interact at the level of phenotype, individual effector gene expression, and sensory and quality aspects, in a dose-dependent manner. Rin and nor have broadly similar quantitative effects on all aspects, demonstrating their additivity in fruit ripening regulation. We also found that the Cnr allele is epistatic to rin and nor and that its pleiotropic effects on fruit size and volatile production, in contrast to the well-known dominant effect on ripening, are incompletely dominant, or recessive.

Revisiting the Role of Master Regulators in Tomato Ripening.

The study of transcriptional regulation of tomato ripening has been led by spontaneous mutations in transcription factor (TF) genes that completely inhibit normal ripening, suggesting that they are 'master regulators'. Studies using CRISPR/Cas9 mutagenesis to produce knockouts of the underlying genes indicate a different picture, suggesting that the regulation is more robust than previously thought. This requires us to revisit our model of the regulation of ripening and replace it with one involving a network of partially redundant components. At the same time, the fast rise of CRISPR/Cas mutagenesis, resulting in unexpectedly weak phenotypes, compared with knockdown technology, suggests that compensatory mechanisms may obscure protein functions. This emphasises the need for assessment of these mechanisms in plants and for the careful design of mutagenesis experiments.

Comprehensive phenotyping reveals interactions and functions of Arabidopsis thaliana TCP genes in yield determination.

Members of the Arabidopsis thaliana TCP transcription factor (TF) family affect plant growth and development. We systematically quantified the effect of mutagenizing single or multiple TCP TFs and how altered vegetative growth or branching influences final seed yield. We monitored rosette growth over time and branching patterns and seed yield characteristics at the end of the lifecycle. Subsequently, an approach was developed to disentangle vegetative growth and to determine possible effects on seed yield. Analysis of growth parameters showed all investigated tcp mutants to be affected in certain growth aspects compared with wild-type plants, highlighting the importance of TCP TFs in plant development. Furthermore, we found evidence that all class II TCPs are involved in axillary branch outgrowth, either as inhibitors (BRANCHED-like genes) or enhancers (JAW- and TCP5-like genes). Comprehensive phenotyping of plants mutant for single or multiple TCP TFs reveals that the proposed opposite functions of class I and class II TCPs in plant growth needs revision and shows complex interactions between closely related TCP genes instead of full genetic redundancy. In various instances, the alterations in vegetative growth or in branching patterns result into negative trade-off effects on seed yield that were missed in previous studies, showing the importance of comprehensive and quantitative phenotyping.

Re-evaluation of transcription factor function in tomato fruit development and ripening with CRISPR/Cas9-mutagenesis.

Tomato (Solanum lycopersicum) is a model for climacteric fleshy fruit ripening studies. Tomato ripening is regulated by multiple transcription factors together with the plant hormone ethylene and their downstream effector genes. Transcription Factors APETALA2a (AP2a), NON-RIPENING (NOR) and FRUITFULL (FUL1/TDR4 and FUL2/MBP7) were reported as master regulators controlling tomato fruit ripening. Their proposed functions were derived from studies of the phenotype of spontaneous mutants or RNAi knock-down lines rather than, as it appears now, actual null mutants. To study TF function in tomato fruit ripening in more detail, we used CRISPR/Cas9-mediated mutagenesis to knock out the encoding genes, and phenotypes of these mutants are reported for the first time. While the earlier ripening, orange-ripe phenotype of ap2a mutants was confirmed, the nor null mutant exhibited a much milder phenotype than the spontaneous nor mutant. Additional analyses revealed that the severe phenotype in the spontaneous mutant is caused by a dominant-negative allele. Our approach also provides new insight into the independent and overlapping functions of FUL1 and FUL2. Single and combined null alleles of FUL1 and FUL2 illustrate that these two genes have partially redundant functions in fruit ripening, but also unveil an additional role for FUL2 in early fruit development.