The transition from vegetative to reproductive development is one of the most important phase changes in the plant life cycle. This step is controlled by various environmental signals that are ...integrated at the molecular level by so-called floral integrators. One such floral integrator in Arabidopsis (Arabidopsis thaliana) is the MADS domain transcription factor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1). Despite extensive genetic studies, little is known about the transcriptional control of SOC1, and we are just starting to explore the network of genes under the direct control of SOC1 transcription factor complexes. Here, we show that several MADS domain proteins, including SOC1 heterodimers, are able to bind SOC1 regulatory sequences. Genome-wide target gene analysis by ChIP-seq confirmed the binding of SOC1 to its own locus and shows that it also binds to a plethora of flowering-time regulatory and floral homeotic genes. In turn, the encoded floral homeotic MADS domain proteins appear to bind SOC1 regulatory sequences. Subsequent in planta analyses revealed SOC1 repression by several floral homeotic MADS domain proteins, and we show that, mechanistically, this depends on the presence of the SOC1 protein. Together, our data show that SOC1 constitutes a major hub in the regulatory networks underlying floral timing and flower development and that these networks are composed of many positive and negative autoregulatory and feedback loops. The latter seems to be crucial for the generation of a robust flower-inducing signal, followed shortly after by repression of the SOC1 floral integrator.
Pepper (
Capsicum
L.) is a nutritionally and economically important crop that is cultivated throughout the world as a vegetable, condiment, and food additive. Genetic transformation using
...Agrobacterium tumefaciens
(agrobacterium) is a powerful biotechnology tool that could be used in pepper to develop community-based functional genomics resources and to introduce important agronomic traits. However, pepper is considered to be highly recalcitrant for agrobacterium-mediated transformation, and current transformation protocols are either inefficient, cumbersome or highly genotype dependent. The main bottleneck in pepper transformation is the inability to generate cells that are competent for both regeneration and transformation. Here, we report that ectopic expression of the
Brassica napus
BABY BOOM AP2/ERF transcription factor overcomes this bottleneck and can be used to efficiently regenerate transgenic plants from otherwise recalcitrant sweet pepper (
C. annuum
) varieties. Transient activation of BABY BOOM in the progeny plants induced prolific cell regeneration and was used to produce a large number of somatic embryos that could be converted readily to seedlings. The data highlight the utility of combining biotechnology and classical plant tissue culture approaches to develop an efficient transformation and regeneration system for a highly recalcitrant vegetable crop.
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.
Members of the plant type I MADS domain subfamily have been reported to be involved in reproductive development in Arabidopsis (Arabidopsis thaliana). However, from the 61 type I genes in the ...Arabidopsis genome, only PHERES1, AGAMOUS-LIKE80 (AGL80), DIANA, AGL62, and AGL23 have been functionally characterized, which revealed important roles for these genes during female gametophyte and early seed development. The functions of the other genes are still unknown, despite the fact that the available single T-DNA insertion mutants have been largely investigated. The lack of mutant phenotypes is likely due to a considerable number of recent intrachromosomal duplications in the type I subfamily, resulting in nonfunctional genes in addition to a high level of redundancy. To enable a breakthrough in type I MADS box gene characterization, a framework needs to be established that allows the prediction of the functionality and redundancy of the type I genes. Here, we present a complete atlas of their expression patterns during female gametophyte and seed development in Arabidopsis, deduced from reporter lines containing translational fusions of the genes to green fluorescent protein and β-glucuronidase. All the expressed genes were revealed to be active in the female gametophyte or developing seed, indicating that the entire type I subfamily is involved in reproductive development in Arabidopsis. Interestingly, expression was predominantly observed in the central cell, antipodal cells, and chalazal endosperm. The combination of our expression results with phylogenetic and protein interaction data allows a better identification of putative redundantly acting genes and provides a useful tool for the functional characterization of the type I MADS box genes in Arabidopsis.
Successful plant reproduction relies on the perfect orchestration of singular processes that culminate in the product of reproduction: the seed. The floral transition, floral organ development, and ...fertilization are well-studied processes and the genetic regulation of the various steps is being increasingly unveiled. Initially, based predominantly on genetic studies, the regulatory pathways were considered to be linear, but recent genome-wide analyses, using high-throughput technologies, have begun to reveal a different scenario. Complex gene regulatory networks underlie these processes, including transcription factors, microRNAs, movable factors, hormones, and chromatin-modifying proteins. Here we review recent progress in understanding the networks that control the major steps in plant reproduction, showing how new advances in experimental and computational technologies have been instrumental. As these recent discoveries were obtained using the model species Arabidopsis thaliana, we will restrict this review to regulatory networks in this important model species. However, more fragmentary information obtained from other species reveals that both the developmental processes and the underlying regulatory networks are largely conserved, making this review also of interest to those studying other plant species.
Fruit ripening in tomato (Solanum lycopersicum) requires the coordination of both developmental cues as well as the plant hormone ethylene. Although the role of ethylene in mediating climacteric ...ripening has been established, knowledge regarding the developmental regulators that modulate the involvement of ethylene in tomato fruit ripening is still lacking. Here, we show that the tomato APETALA2a (AP2a) transcription factor regulates fruit ripening via regulation of ethylene biosynthesis and signaling. RNA interference (RNAi)-mediated repression of AP2a resulted in alterations in fruit shape, orange ripe fruits, and altered carotenoid accumulation. Microarray expression analyses of the ripe AP2 RNAi fruits showed altered expression of genes involved in various metabolic pathways, such as the phenylpropanoid and carotenoid pathways, as well as in hormone synthesis and perception. Genes involved in chromoplast differentiation and other ripening-associated processes were also differentially expressed, but softening and ethylene biosynthesis occurred in the transgenic plants. Ripening regulators RIPENING-INHIBITOR, NON-RIPENING, and COLORLESS NON-RIPENING (CNR) function upstream of AP2a and positively regulate its expression. In the pericarp of AP2 RNAi fruits, mRNA levels of CNR were elevated, indicating that AP2a and CNR are part of a negative feedback loop in the regulation of ripening. Moreover, we demonstrated that CNR binds to the promoter of AP2a in vitro.
Correct flower formation requires highly specific temporal and spatial regulation of gene expression. In Arabidopsis thaliana the majority of the master regulators that determine flower organ ...identity belong to the MADS-domain transcription factor family. The canonical DNA binding motif for this transcription factor family is the CArG-box, which has the consensus CC(A/T)
GG. However, so far, a comprehensive analysis of MADS-domain binding patterns has not yet been performed.
Eight publicly available ChIP-seq datasets of MADS-domain proteins that regulate the floral transition and flower formation were analyzed. Surprisingly, the preferred DNA binding motif of each protein was a CArG-box with an NAA extension. Furthermore, motifs of other transcription factors were found in the vicinity of binding sites of MADS-domain transcription factors, suggesting that interaction of MADS-domain proteins with other transcription factors is important for target gene regulation. Finally, conservation of CArG-boxes between Arabidopsis ecotypes was assessed to obtain information about their evolutionary importance. CArG-boxes that fully matched the consensus were more conserved than other CArG-boxes, suggesting that the perfect CArG-box is evolutionary more important than other CArG-box variants.
Our analysis provides detailed insight into MADS-domain protein binding patterns. The results underline the importance of an extended version of the CArG-box and provide a first view on evolutionary conservation of MADS-domain protein binding sites in Arabidopsis ecotypes.
Floral organs, whose identity is determined by specific combinations of homeotic genes, originate from a group of undifferentiated cells called the floral meristem. In Arabidopsis, the homeotic gene ...AGAMOUS (AG) terminates meristem activity and promotes development of stamens and carpels. To understand the program of gene expression activated by AG, we followed genome-wide expression during early stamen and carpel development. The AG target genes included most genes for which mutant screens revealed a function downstream of AG. Novel targets were validated by in situ hybridisation and binding to AG in vitro and in vivo. Transcription factors formed a large fraction of AG targets, suggesting that during early organogenesis, much of the genetic program is concerned with elaborating gene expression patterns. The results also suggest that AG and other homeotic proteins with which it interacts (SEPALLATA3, APETALA3, PISTILLATA) are coordinately regulated in a positive-feedback loop to maintain their own expression, and that AG activates biosynthesis of gibberellin, which has been proposed to promote the shift from meristem identity to differentiation.
Molecular–genetic analysis of Arabidopsis mutants revealed a novel role for abscisic acid in promoting auxin-induced somatic embryogenesis at three different levels: biosynthesis, signalling, and ...downstream transcription.
Abstract
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.
•Several mechanisms underlying ambient temperature regulation of floral timing have been uncovered.•Chromatin accessibility, alternative splicing, protein stability, and miRNAs are involved in ...thermosensory floral timing.•Two pathways containing repressing factors or activating factors of ambient temperature-dependent flowering time exist next to each other.
The floral transition is an essential process in the life cycle of flower-bearing plants, because their reproductive success depends on it. To determine the right moment of flowering, plants respond to many environmental signals, including day length, light quality, and temperature. Small changes in ambient temperature also affect the flowering process, although our knowledge of the genetic and molecular mechanisms underlying this flowering pathway is limited. However, recent advances in Arabidopsis (Arabidopsis thaliana) have uncovered multiple molecular mechanisms controlling ambient temperature regulation of flowering, which modulate both repressing and activating factors of flowering time. At a time when temperatures are rising worldwide, understanding how plants integrate ambient temperature signals can be crucial for crop production.
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