Plant genetic effects on microbial hubs impact host fitness in repeated field trials.

Although complex interactions between hosts and microbial associates are increasingly well documented, we still know little about how and why hosts shape microbial communities in nature. In addition, host genetic effects on microbial communities vary widely depending on the environment, obscuring conclusions about which microbes are impacted and which plant functions are important. We characterized the leaf microbiota of 200 Arabidopsis thaliana genotypes in eight field experiments and detected consistent host effects on specific, broadly distributed microbial species (operational taxonomic unit [OTUs]). Host genetic effects disproportionately influenced central ecological hubs, with heritability of particular OTUs declining with their distance from the nearest hub within the microbial network. These host effects could reflect either OTUs preferentially associating with specific genotypes or differential microbial success within them. Host genetics associated with microbial hubs explained over 10% of the variation in lifetime seed production among host genotypes across sites and years. We successfully cultured one of these microbial hubs and demonstrated its growth-promoting effects on plants in sterile conditions. Finally, genome-wide association mapping identified many putatively causal genes with small effects on the relative abundance of microbial hubs across sites and years, and these genes were enriched for those involved in the synthesis of specialized metabolites, auxins, and the immune system. Using untargeted metabolomics, we corroborate the consistent association between variation in specialized metabolites and microbial hubs across field sites. Together, our results reveal that host genetic variation impacts the microbial communities in consistent ways across environments and that these effects contribute to fitness variation among host genotypes.

Pleiotropic and nonredundant effects of an auxin importer in Setaria and maize.

Directional transport of auxin is critical for inflorescence and floral development in flowering plants, but the role of auxin influx carriers (AUX1 proteins) has been largely overlooked. Taking advantage of available AUX1 mutants in green millet (Setaria viridis) and maize (Zea mays), we uncover previously unreported aspects of plant development that are affected by auxin influx, including higher order branches in the inflorescence, stigma branch number, glume (floral bract) development, and plant fertility. However, disruption of auxin flux does not affect all parts of the plant, with little obvious effect on inflorescence meristem size, time to flowering, and anther morphology. In double mutant studies in maize, disruptions of ZmAUX1 also affect vegetative development. A green fluorescent protein (GFP)-tagged construct of the Setaria AUX1 protein Sparse Panicle1 (SPP1) under its native promoter showed that SPP1 localizes to the plasma membrane of outer tissue layers in both roots and inflorescences, and accumulates specifically in inflorescence branch meristems, consistent with the mutant phenotype and expected auxin maxima. RNA-seq analysis indicated that most gene expression modules are conserved between mutant and wild-type plants, with only a few hundred genes differentially expressed in spp1 inflorescences. Using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, we disrupted SPP1 and the other four AUX1 homologs in S. viridis. SPP1 has a larger effect on inflorescence development than the others, although all contribute to plant height, tiller formation, and leaf and root development. The AUX1 importers are thus not fully redundant in S. viridis. Our detailed phenotypic characterization plus a stable GFP-tagged line offer tools for future dissection of the function of auxin influx proteins.

Multiple FLC haplotypes defined by independent cis-regulatory variation underpin life history diversity in Arabidopsis thaliana.

Relating molecular variation to phenotypic diversity is a central goal in evolutionary biology. In Arabidopsis thaliana, FLOWERING LOCUS C (FLC) is a major determinant of variation in vernalization--the acceleration of flowering by prolonged cold. Here, through analysis of 1307 A. thaliana accessions, we identify five predominant FLC haplotypes defined by noncoding sequence variation. Genetic and transgenic experiments show that they are functionally distinct, varying in FLC expression level and rate of epigenetic silencing. Allelic heterogeneity at this single locus accounts for a large proportion of natural variation in vernalization that contributes to adaptation of A. thaliana.

The Evening Complex Establishes Repressive Chromatin Domains Via H2A.Z Deposition.

The Evening Complex (EC) is a core component of the Arabidopsis (Arabidopsis thaliana) circadian clock, which represses target gene expression at the end of the day and integrates temperature information to coordinate environmental and endogenous signals. Here we show that the EC induces repressive chromatin structure to regulate the evening transcriptome. The EC component ELF3 directly interacts with a protein from the SWI2/SNF2-RELATED (SWR1) complex to control deposition of H2A.Z-nucleosomes at the EC target genes. SWR1 components display circadian oscillation in gene expression with a peak at dusk. In turn, SWR1 is required for the circadian clockwork, as defects in SWR1 activity alter morning-expressed genes. The EC-SWR1 complex binds to the loci of the core clock genes PSEUDO-RESPONSE REGULATOR7 (PRR7) and PRR9 and catalyzes deposition of nucleosomes containing the histone variant H2A.Z coincident with the repression of these genes at dusk. This provides a mechanism by which the circadian clock temporally establishes repressive chromatin domains to shape oscillatory gene expression around dusk.

The G-Box Transcriptional Regulatory Code in Arabidopsis.

Plants have significantly more transcription factor (TF) families than animals and fungi, and plant TF families tend to contain more genes; these expansions are linked to adaptation to environmental stressors. Many TF family members bind to similar or identical sequence motifs, such as G-boxes (CACGTG), so it is difficult to predict regulatory relationships. We determined that the flanking sequences near G-boxes help determine in vitro specificity but that this is insufficient to predict the transcription pattern of genes near G-boxes. Therefore, we constructed a gene regulatory network that identifies the set of bZIPs and bHLHs that are most predictive of the expression of genes downstream of perfect G-boxes. This network accurately predicts transcriptional patterns and reconstructs known regulatory subnetworks. Finally, we present Ara-BOX-cis (araboxcis.org), a Web site that provides interactive visualizations of the G-box regulatory network, a useful resource for generating predictions for gene regulatory relations.

Characterization of Linaria KNOX genes suggests a role in petal-spur development.

Spurs are tubular outgrowths of perianth organs that have evolved iteratively among angiosperms. They typically contain nectar and often strongly influence pollinator specificity, potentially mediating reproductive isolation. The identification of Antirrhinum majus mutants with ectopic petal spurs suggested that petal-spur development is dependent on the expression of KNOTTED 1-like homeobox (KNOX) genes, which are better known for their role in maintaining the shoot apical meristem. Here, we tested the role of KNOX genes in petal-spur development by isolating orthologs of the A. majus KNOX genes Hirzina (AmHirz) and Invaginata (AmIna) from Linaria vulgaris, a related species that differs from A. majus in possessing long, narrow petal spurs. We name these genes LvHirz and LvIna, respectively. Using quantitative reverse-transcription PCR, we show that LvHirz is expressed at high levels in the developing petals and demonstrate that the expression of petal-associated KNOX genes is sufficient to induce sac-like outgrowths on petals in a heterologous host. We propose a model in which KNOX gene expression during early petal-spur development promotes and maintains further morphogenetic potential of the petal, as previously described for KNOX gene function in compound leaf development. These data indicate that petal spurs could have evolved by changes in regulatory gene expression that cause rapid and potentially saltational phenotypic modifications. Given the morphological similarity of spur ontogeny in distantly related taxa, changes in KNOX gene expression patterns could be a shared feature of spur development in angiosperms.

Flower-specific KNOX phenotype in the orchid Dactylorhiza fuchsii.

The KNOTTED1-like homeobox (KNOX) genes are best known for maintaining a pluripotent stem-cell population in the shoot apical meristem that underlies indeterminate vegetative growth, allowing plants to adapt their development to suit the prevailing environmental conditions. More recently, the function of the KNOX gene family has been expanded to include additional roles in lateral organ development such as complex leaf morphogenesis, which has come to dominate the KNOX literature. Despite several reports implicating KNOX genes in the development of carpels and floral elaborations such as petal spurs, few authors have investigated the role of KNOX genes in flower development. Evidence is presented here of a flower-specific KNOX function in the development of the elaborate flowers of the orchid Dactylorhiza fuchsii, which have a three-lobed labellum petal with a prominent spur. Using degenerate PCR, four Class I KNOX genes (DfKN1-4) have been isolated, one from each of the four major Class I KNOX subclades and by reverse transcription PCR (RT-PCR), it is demonstrated that DfKNOX transcripts are detectable in developing floral organs such as the spur-bearing labellum and inferior ovary. Although constitutive expression of the DfKN2 transcript in tobacco produces a wide range of floral abnormalities, including serrated petal margins, extra petal tissue, and fused organs, none of the vegetative phenotypes typical of constitutive KNOX expression were produced. These data are highly suggestive of a role for KNOX expression in floral development that may be especially important in taxa with elaborate flowers.

A Dynamic Co-expression Map of Early Inflorescence Development in Setaria viridis Provides a Resource for Gene Discovery and Comparative Genomics.

The morphological and functional diversity of plant form is governed by dynamic gene regulatory networks. In cereal crops, grain and/or pollen-bearing inflorescences exhibit vast architectural diversity and developmental complexity, yet the underlying genetic framework is only partly known. Setaria viridis is a small, rapidly growing grass species in the subfamily Panicoideae, a group that includes economically important cereal crops such as maize and sorghum. The S. viridis inflorescence displays complex branching patterns, but its early development is similar to that of other panicoid grasses, and thus is an ideal model for studying inflorescence architecture. Here we report a detailed transcriptional resource that captures dynamic transitions across six sequential stages of S. viridis inflorescence development, from reproductive onset to floral organ differentiation. Co-expression analyses identified stage-specific signatures of development, which include homologs of previously known developmental genes from maize and rice, suites of transcription factors and gene family members, and genes of unknown function. This spatiotemporal co-expression map and associated analyses provide a foundation for gene discovery in S. viridis inflorescence development, and a comparative model for exploring related architectural features in agronomically important cereals.

Sparse panicle1 is required for inflorescence development in Setaria viridis and maize.

Setaria viridis is a rapid-life-cycle model panicoid grass. To identify genes that may contribute to inflorescence architecture and thus have the potential to influence grain yield in related crops such as maize, we conducted an N-nitroso-N-methylurea (NMU) mutagenesis of S. viridis and screened for visible inflorescence mutant phenotypes. Of the approximately 2,700 M2 families screened, we identified four recessive sparse panicle mutants (spp1-spp4) characterized by reduced and uneven branching of the inflorescence. To identify the gene underlying the sparse panicle1 (spp1) phenotype, we performed bulked segregant analysis and deep sequencing to fine map it to an approximately 1 Mb interval. Within this interval, we identified disruptive mutations in two genes. Complementation tests between spp1 and spp3 revealed they were allelic, and deep sequencing of spp3 identified an independent disruptive mutation in SvAUX1 (AUXIN1), one of the two genes in the ∼1 Mb interval and the only gene disruption shared between spp1 and spp3. SvAUX1 was found to affect both inflorescence development and root gravitropism in S. viridis. A search for orthologous mutant alleles in maize confirmed a very similar role of ZmAUX1 in maize, which highlights the utility of S. viridis in accelerating functional genomic studies in maize.

The evening complex coordinates environmental and endogenous signals in Arabidopsis.

Plants maximize their fitness by adjusting their growth and development in response to signals such as light and temperature. The circadian clock provides a mechanism for plants to anticipate events such as sunrise and adjust their transcriptional programmes. However, the underlying mechanisms by which plants coordinate environmental signals with endogenous pathways are not fully understood. Using RNA-sequencing and chromatin immunoprecipitation sequencing experiments, we show that the evening complex (EC) of the circadian clock plays a major role in directly coordinating the expression of hundreds of key regulators of photosynthesis, the circadian clock, phytohormone signalling, growth and response to the environment. We find that the ability of the EC to bind targets genome-wide depends on temperature. In addition, co-occurrence of phytochrome B (phyB) at multiple sites where the EC is bound provides a mechanism for integrating environmental information. Hence, our results show that the EC plays a central role in coordinating endogenous and environmental signals in Arabidopsis.

Phytochromes function as thermosensors in Arabidopsis.

Plants are responsive to temperature, and some species can distinguish differences of 1°C. In Arabidopsis, warmer temperature accelerates flowering and increases elongation growth (thermomorphogenesis). However, the mechanisms of temperature perception are largely unknown. We describe a major thermosensory role for the phytochromes (red light receptors) during the night. Phytochrome null plants display a constitutive warm-temperature response, and consistent with this, we show in this background that the warm-temperature transcriptome becomes derepressed at low temperatures. We found that phytochrome B (phyB) directly associates with the promoters of key target genes in a temperature-dependent manner. The rate of phyB inactivation is proportional to temperature in the dark, enabling phytochromes to function as thermal timers that integrate temperature information over the course of the night.

ELF3 controls thermoresponsive growth in Arabidopsis.

Plant development is highly responsive to ambient temperature, and this trait has been linked to the ability of plants to adapt to climate change. The mechanisms by which natural populations modulate their thermoresponsiveness are not known. To address this, we surveyed Arabidopsis accessions for variation in thermal responsiveness of elongation growth and mapped the corresponding loci. We find that the transcriptional regulator EARLY FLOWERING3 (ELF3) controls elongation growth in response to temperature. Through a combination of modeling and experiments, we show that high temperature relieves the gating of growth at night, highlighting the importance of temperature-dependent repressors of growth. ELF3 gating of transcriptional targets responds rapidly and reversibly to changes in temperature. We show that the binding of ELF3 to target promoters is temperature dependent, suggesting a mechanism where temperature directly controls ELF3 activity.

Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis.

BACKGROUND: Many experiments in modern plant molecular biology require the processing of large numbers of samples for a variety of applications from mutant screens to the analysis of natural variants. A severe bottleneck to many such analyses is the acquisition of good yields of high quality RNA suitable for use in sensitive downstream applications such as real time quantitative reverse-transcription-polymerase chain reaction (real time qRT-PCR). Although several commercial kits are available for high-throughput RNA extraction in 96-well format, only one non-kit method has been described in the literature using the commercial reagent TRIZOL. RESULTS: We describe an unusual phenomenon when using TRIZOL reagent with young Arabidopsis seedlings. This prompted us to develop a high-throughput RNA extraction protocol (HTP96) adapted from a well established phenol:chloroform-LiCl method (P:C-L) that is cheap, reliable and requires no specialist equipment. With this protocol 192 high quality RNA samples can be prepared in 96-well format in three hours (less than 1 minute per sample) with less than 1% loss of samples. We demonstrate that the RNA derived from this protocol is of high quality and suitable for use in real time qRT-PCR assays. CONCLUSION: The development of the HTP96 protocol has vastly increased our sample throughput, allowing us to fully exploit the large sample capacity of modern real time qRT-PCR thermocyclers, now commonplace in many labs, and develop an effective high-throughput gene expression platform. We propose that the HTP96 protocol will significantly benefit any plant scientist with the task of obtaining hundreds of high quality RNA extractions.

A plant developmentalist's guide to paedomorphosis: reintroducing a classic concept to a new generation.

Paedomorphosis is an evolutionary change in morphology through an alteration in the rate or timing of a developmental pathway, resulting in more derived species appearing juvenilised in comparison to their ancestors. This concept has long been the subject of extensive, often intimidating, discussion in the animal literature, but is less frequently considered by only a few plant developmentalists. We define the main types of paedomorphosis and explain how they can be applied to plants, considering the differences between plant and animal lifecycles. We provide examples of plant paedomorphoses and point out their likely significance in the evolution of floral diversity.