Model-based inference of a plant-specific dual role for HOPS in regulating guard cell vacuole fusion.

Stomata are the pores on a leaf surface that regulate gas exchange. Each stoma consists of two guard cells whose movements regulate pore opening and thereby control CO2 fixation and water loss. Guard cell movements depend in part on the remodeling of vacuoles, which have been observed to change from a highly fragmented state to a fused morphology during stomata opening. This change in morphology requires a membrane fusion mechanism that responds rapidly to environmental signals, allowing plants to respond to diurnal and stress cues. With guard cell vacuoles being both large and responsive to external signals, stomata represent a unique system in which to delineate mechanisms of membrane fusion. Fusion of vacuole membranes is a highly conserved process in eukaryotes, with key roles played by two multi-subunit complexes: HOPS (homotypic fusion and vacuolar protein sorting) and SNARE (soluble NSF attachment protein receptor). HOPS is a vacuole tethering factor that is thought to chaperone SNAREs from apposing vacuole membranes into a fusion-competent complex capable of rearranging membranes. To resolve a counter-intuitive observation regarding the role of HOPS in regulating plant vacuole morphology, we derived a quantitative model of vacuole fusion dynamics and used it to generate testable predictions about HOPS-SNARE interactions. We derived our model by applying simulation-based inference to integrate prior knowledge about molecular interactions with limited, qualitative observations of emergent vacuole phenotypes. By constraining the model parameters to yield the emergent outcomes observed for stoma opening - as induced by two distinct chemical treatments - we predicted a dual role for HOPS and identified a stalled form of the SNARE complex that differs from phenomena reported in yeast. We predict that HOPS has contradictory actions at different points in the fusion signaling pathway, promoting the formation of SNARE complexes, but limiting their activity.

Solving the puzzle of Fe homeostasis by integrating molecular, mathematical, and societal models.

To ensure optimal utilization and bioavailability, iron uptake, transport, subcellular localization, and assimilation are tightly regulated in plants. Herein, we examine recent advances in our understanding of cellular responses to Fe deficiency. We then use intracellular mechanisms of Fe homeostasis to discuss how formalizing cell biology knowledge via a mathematical model can advance discovery even when quantitative data is limited. Using simulation-based inference to identify plausible systems mechanisms that conform to known emergent phenotypes can yield novel, testable hypotheses to guide targeted experiments. However, this approach relies on the accurate encoding of domain-expert knowledge in exploratory mathematical models. We argue that this would be facilitated by fostering more "systems thinking" life scientists and that diversifying your research team may be a practical path to achieve that goal.

The HK5 and HK6 cytokinin receptors mediate diverse developmental pathways in rice.

The phytohormone cytokinin regulates diverse aspects of plant growth and development. Our understanding of the metabolism and perception of cytokinin has made great strides in recent years, mostly from studies of the model dicot Arabidopsis Here, we employed a CRISPR/Cas9-based approach to disrupt a subset of cytokinin histidine kinase (HK) receptors in rice (Oryza sativa) in order to explore the role of cytokinin in a monocot species. In hk5 and hk6 single mutants, the root growth, leaf width, inflorescence architecture and/or floral development were affected. The double hk5 hk6 mutant showed more substantial defects, including severely reduced root and shoot growth, a smaller shoot apical meristem, and an enlarged root cap. Flowering was delayed in the hk5 hk6 mutant and the panicle was significantly reduced in size and infertile due to multiple defects in floral development. The hk5 hk6 mutant also exhibited a severely reduced cytokinin response, consistent with the developmental phenotypes arising from a defect in cytokinin signaling. These results indicate that HK5 and HK6 act as cytokinin receptors, with overlapping functions to regulate diverse aspects of rice growth and development.

Mutagenomics: A Rapid, High-Throughput Method to Identify Causative Mutations from a Genetic Screen.

Genetic screens are powerful tools to dissect complex biological processes, but a rate-limiting step is often the cloning of targeted genes. Here, we present a strategy, "mutagenomics," to identify causal mutations from a screen in a high throughput fashion in the absence of backcrossing. Mutagenomics is initiated by sequencing the genomes of the mutants identified, which are then subjected to a three-stage pipeline. The first stage identifies sequence changes in genes previously linked to the targeted pathway. The second stage uses heuristics derived from a simulation strategy to identify genes that are represented by multiple independent alleles more often than expected by chance. The third stage identifies candidate genes for the remaining lines by sequencing multiple lines of common descent. Our simulations indicate that sequencing as few as three to four sibling lines generally results in fewer than five candidate genes. We applied mutagenomics to a screen for Arabidopsis (Arabidopsis thaliana) mutants involved in the response to the phytohormone cytokinin. Mutagenomics identified likely causative genes for many of the mutant lines analyzed from this screen, including 13 alleles of the gene encoding the ARABIDOPSIS HIS KINASE4 cytokinin receptor. The screen also identified 1-AMINOCYCLOPROPANE-1-CARBOXYLATE (ACC) SYNTHASE7, an ACC synthase homolog involved in ethylene biosynthesis, and ELONGATED HYPOCOTYL5 (HY5), a master transcriptional regulator of photomorphogenesis. HY5 was found to mediate a subset of the transcriptional response to cytokinin. Mutagenomics has the potential to accelerate the pace and utility of genetic screens in Arabidopsis.

Using indCAPS to Detect CRISPR/Cas9 Induced Mutations.

Cleaved amplified polymorphic sequences (CAPS) assays are useful tools for detecting small mutations such as single nucleotide polymorphisms (SNPs) or insertion/deletions (indels) present in an amplified DNA fragment. A mutation that disrupts or creates a restriction site will prevent cleavage by a restriction enzyme, allowing discrimination of wild-type and mutant alleles. In cases where no convenient restriction site is present, a derived Cleaved Amplified Polymorphic Sequence (dCAPS) assay can be used, where mismatches in the primer are used to create a diagnostic restriction site. No special design constraints are present for a CAPS assay, but cases where CAPS assays can be used are infrequent. A dCAPS assay can be burdensome to design by hand, but it is more broadly applicable. This protocol will describe the use of the indCAPS tool for the design of CAPS and dCAPS primers. The indCAPS tool was designed to be compatible with indel alleles, which prior tools struggled with but have increased importance since the rise of CRISPR/Cas9 mutagenesis methods.

Three Auxin Response Factors Promote Hypocotyl Elongation.

The hormone auxin regulates growth largely by affecting gene expression. By studying Arabidopsis (Arabidopsis thaliana) mutants deficient in AUXIN RESPONSE FACTORS (ARFs), we have identified three ARF proteins that are required for auxin-responsive hypocotyl elongation. Plants deficient in these factors have reduced responses to environmental conditions that increase auxin levels, including far-red-enriched light and high temperature. Despite having decreased auxin responses, the ARF-deficient plants responded to brassinosteroid and gibberellin, indicating that different hormones can act partially independently. Aux/IAA proteins, encoded by IAA genes, interact with ARF proteins to repress auxin response. Silencing expression of multiple IAA genes increased hypocotyl elongation, suggesting that Aux/IAA proteins modulate ARF activity in hypocotyls in a potential negative feedback loop.

indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events.

Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.

A regulatory network for coordinated flower maturation.

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs.