Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing.

Major advances in crop yields are needed in the coming decades. However, plant breeding is currently limited by incremental improvements in quantitative traits that often rely on laborious selection of rare naturally occurring mutations in gene-regulatory regions. Here, we demonstrate that CRISPR/Cas9 genome editing of promoters generates diverse cis-regulatory alleles that provide beneficial quantitative variation for breeding. We devised a simple genetic scheme, which exploits trans-generational heritability of Cas9 activity in heterozygous loss-of-function mutant backgrounds, to rapidly evaluate the phenotypic impact of numerous promoter variants for genes regulating three major productivity traits in tomato: fruit size, inflorescence branching, and plant architecture. Our approach allows immediate selection and fixation of novel alleles in transgene-free plants and fine manipulation of yield components. Beyond a platform to enhance variation for diverse agricultural traits, our findings provide a foundation for dissecting complex relationships between gene-regulatory changes and control of quantitative traits.

A pair of readers of bivalent chromatin mediate formation of Polycomb-based "memory of cold" in plants.

The Polycomb-group chromatin modifiers play important roles to repress or switch off gene expression in plants and animals. How the active chromatin state is switched to a Polycomb-repressed state is unclear. In Arabidopsis, prolonged cold induces the switching of the highly active chromatin state at the potent floral repressor FLC to a Polycomb-repressed state, which is epigenetically maintained when temperature rises to confer "cold memory," enabling plants to flower in spring. We report that the cis-acting cold memory element (CME) region at FLC bears bivalent marks of active histone H3K4me3 and repressive H3K27me3 that are read and interpreted by an assembly of bivalent chromatin readers to drive cold-induced switching of the FLC chromatin state. In response to cold, the 47-bp CME and its associated bivalent chromatin feature drive the switching of active chromatin state at a recombinant gene to a Polycomb-repressed domain, conferring cold memory. We reveal a paradigm for environment-induced chromatin-state switching at bivalent loci in plants.

Noncoding SNPs influence a distinct phase of Polycomb silencing to destabilize long-term epigenetic memory at Arabidopsis FLC.

In Arabidopsis thaliana, the cold-induced epigenetic regulation of FLOWERING LOCUS C (FLC) involves distinct phases of Polycomb repressive complex 2 (PRC2) silencing. During cold, a PHD-PRC2 complex metastably and digitally nucleates H3K27me3 within FLC On return to warm, PHD-PRC2 spreads across the locus delivering H3K27me3 to maintain long-term silencing. Here, we studied natural variation in this process in Arabidopsis accessions, exploring Lov-1, which shows FLC reactivation on return to warm, a feature characteristic of FLC in perennial Brassicaceae This analysis identifies an additional phase in this Polycomb silencing mechanism downstream from H3K27me3 spreading. In this long-term silencing (perpetuated) phase, the PHD proteins are lost from the nucleation region and silencing is likely maintained by the read-write feedbacks associated with H3K27me3. A combination of noncoding SNPs in the nucleation region mediates instability in this long-term silencing phase with the result that Lov-1 FLC frequently digitally reactivates in individual cells, with a probability that diminishes with increasing cold duration. We propose that this decrease in reactivation probability is due to reduced DNA replication after flowering. Overall, this work defines an additional phase in the Polycomb mechanism instrumental in natural variation of silencing, and provides avenues to dissect broader evolutionary changes at FLC.

In vivo single-molecule RNA structural profiling.

Cellular RNAs exhibit substantial heterogeneity in structure and function. Recently, Yang et al. developed an in vivo single-molecule RNA structure profiling methodology and revealed that individual isoforms of noncoding transcripts adopt multiple diverse and functionally relevant structural conformations, which change in abundance and structure in response to temperature conditions.

LncRNA FLAIL affects alternative splicing and represses flowering in Arabidopsis.

How the noncoding genome affects cellular functions is a key biological question. A particular challenge is to distinguish the effects of noncoding DNA elements from long noncoding RNAs (lncRNAs) that coincide at the same loci. Here, we identified the flowering-associated intergenic lncRNA (FLAIL) in Arabidopsis through early flowering flail mutants. Expression of FLAIL RNA from a different chromosomal location in combination with strand-specific RNA knockdown characterized FLAIL as a trans-acting RNA molecule. FLAIL directly binds to differentially expressed target genes that control flowering via RNA-DNA interactions through conserved sequence motifs. FLAIL interacts with protein and RNA components of the spliceosome to affect target mRNA expression through co-transcriptional alternative splicing (AS) and linked chromatin regulation. In the absence of FLAIL, splicing defects at the direct FLAIL target flowering gene LACCASE 8 (LAC8) correlated with reduced mRNA expression. Double mutant analyses support a model where FLAIL-mediated splicing of LAC8 promotes its mRNA expression and represses flowering. Our study suggests lncRNAs as accessory components of the spliceosome that regulate AS and gene expression to impact organismal development.

Artificial selection of two antagonistic E3 ubiquitin ligases finetunes soybean photoperiod adaptation and grain yield.

The precise control of flowering time is of utmost importance for crop adaptation to varying environmental conditions and consequently determines grain yield and plant fitness. Soybean E2, the homolog of Arabidopsis GIGANTEA, is a major locus contributing to high-latitude adaptation and is involved in photoperiod sensitivity. However, due to major effects of E2, additional genetic loci controlling soybean flowering and adaptation have historically been masked and difficult to identify. Here, by eliminating the effect of E2, we identified a Tof9 locus controlling flowering in which ZEITLUPE 2 (ZTL2) is the causal gene. ZTL2 encodes an F-box E3 ubiquitin ligase with homology to Arabidopsis ZEITLUPE and is shown to play a key role in the soybean photoperiodic flowering pathway. ZTL2 physically interacts with E2 to mediate its degradation. Intriguingly, ZTL2 and FKF1, both belong to the F-box-type E3 ubiquitin-ligase family, exhibit opposite roles in regulating soybean flowering. ZTL2 degrades E2, leading to early flowering, while FKF1 stabilizes E2, resulting in delayed flowering. The balance between ZTL2-mediated degradation and FKF1-mediated stabilization enables soybeans to finetune flowering time and maximize grain yield. Field-grown ztl2 mutants are taller, flower late, and have increased yield parameters. ZTL2 and FKF1b bear contrasting artificial-selection patterns to adapt to different latitudes. This antagonistic regulation is crucial for soybean adaptation to diverse ecological settings and allows plants to fine-tune their flowering time in response to photoperiod and latitudinal changes.

Strigolactones promote flowering by inducing the miR319-LA-SFT module in tomato.

Strigolactones are a class of phytohormones with various functions in plant development, stress responses, and in the interaction with (micro)organisms in the rhizosphere. While their effects on vegetative development are well studied, little is known about their role in reproduction. We investigated the effects of genetic and chemical modification of strigolactone levels on the timing and intensity of flowering in tomato (Solanum lycopersicum L.) and the molecular mechanisms underlying such effects. Results showed that strigolactone levels in the shoot, whether endogenous or exogenous, correlate inversely with the time of anthesis and directly with the number of flowers and the transcript levels of the florigen-encoding gene SINGLE FLOWER TRUSS (SFT) in the leaves. Transcript quantifications coupled with metabolite analyses demonstrated that strigolactones promote flowering in tomato by inducing the activation of the microRNA319-LANCEOLATE module in leaves. This, in turn, decreases gibberellin content and increases the transcription of SFT. Several other floral markers and morpho-anatomical features of developmental progression are induced in the apical meristems upon treatment with strigolactones, affecting floral transition and, more markedly, flower development. Thus, strigolactones promote meristem maturation and flower development via the induction of SFT both before and after floral transition, and their effects are blocked in plants expressing a miR319-resistant version of LANCEOLATE. Our study positions strigolactones in the context of the flowering regulation network in a model crop species.

Analysis of pea mutants reveals the conserved role of FRUITFULL controlling the end of flowering and its potential to boost yield.

Monocarpic plants have a single reproductive phase in their life. Therefore, flower and fruit production are restricted to the length of this period. This reproductive strategy involves the regulation of flowering cessation by a coordinated arrest of the growth of the inflorescence meristems, optimizing resource allocation to ensure seed filling. Flowering cessation appears to be a regulated phenomenon in all monocarpic plants. Early studies in several species identified seed production as a major factor triggering inflorescence proliferative arrest. Recently, genetic factors controlling inflorescence arrest, in parallel to the putative signals elicited by seed production, have started to be uncovered in Arabidopsis, with the MADS-box gene FRUITFULL (FUL) playing a central role in the process. However, whether the genetic network regulating arrest is also at play in other species is completely unknown. Here, we show that this role of FUL is not restricted to Arabidopsis but is conserved in another monocarpic species with a different inflorescence structure, field pea, strongly suggesting that the network controlling the end of flowering is common to other plants. Moreover, field trials with lines carrying mutations in pea FUL genes show that they could be used to boost crop yield.

Needs for a conceptual bridge between biological domestication and early food globalization.

The past 15 y has seen much development in documentation of domestication of plants and animals as gradual traditions spanning millennia. There has also been considerable momentum in understanding the dispersals of major domesticated taxa across continents spanning thousands of miles. The two processes are often considered within different theoretical strains. What is missing from our repertoire of explanations is a conceptual bridge between the protracted process over millennia and the multiregional, globally dispersed nature of domestication. The evidence reviewed in this paper bears upon how we conceptualize domestication as an episode or a process. By bringing together the topics of crop domestication and crop movement, those complex, protracted, and continuous outcomes come more clearly into view.

Genome evolution of the ancient hexaploid Platanus × acerifolia (London planetree).

Whole-genome duplication (WGD; i.e., polyploidy) and chromosomal rearrangement (i.e., genome shuffling) significantly influence genome structure and organization. Many polyploids show extensive genome shuffling relative to their pre-WGD ancestors. No reference genome is currently available for Platanaceae (Proteales), one of the sister groups to the core eudicots. Moreover, Platanus × acerifolia (London planetree; Platanaceae) is a widely used street tree. Given the pivotal phylogenetic position of Platanus and its 2-y flowering transition, understanding its flowering-time regulatory mechanism has significant evolutionary implications; however, the impact of Platanus genome evolution on flowering-time genes remains unknown. Here, we assembled a high-quality, chromosome-level reference genome for P. × acerifolia using a phylogeny-based subgenome phasing method. Comparative genomic analyses revealed that P. × acerifolia (2n = 42) is an ancient hexaploid with three subgenomes resulting from two sequential WGD events; Platanus does not seem to share any WGD with other Proteales or with core eudicots. Each P. × acerifolia subgenome is highly similar in structure and content to the reconstructed pre-WGD ancestral eudicot genome without chromosomal rearrangements. The P. × acerifolia genome exhibits karyotypic stasis and gene sub-/neo-functionalization and lacks subgenome dominance. The copy number of flowering-time genes in P. × acerifolia has undergone an expansion compared to other noncore eudicots, mainly via the WGD events. Sub-/neo-functionalization of duplicated genes provided the genetic basis underlying the unique flowering-time regulation in P. × acerifolia. The P. × acerifolia reference genome will greatly expand understanding of the evolution of genome organization, genetic diversity, and flowering-time regulation in angiosperms.

The Angiosperm Terrestrial Revolution buffered ants against extinction.

With ~14,000 extant species, ants are ubiquitous and of tremendous ecological importance. They have undergone remarkable diversification throughout their evolutionary history. However, the drivers of their diversity dynamics are not well quantified or understood. Previous phylogenetic analyses have suggested patterns of diversity dynamics associated with the Angiosperm Terrestrial Revolution (ATR), but these studies have overlooked valuable information from the fossil record. To address this gap, we conducted a comprehensive analysis using a large dataset that includes both the ant fossil record (~24,000 individual occurrences) and neontological data (~14,000 occurrences), and tested four hypotheses proposed for ant diversification: co-diversification, competitive extinction, hyper-specialization, and buffered extinction. Taking into account biases in the fossil record, we found three distinct diversification periods (the latest Cretaceous, Eocene, and Oligo-Miocene) and one extinction period (Late Cretaceous). The competitive extinction hypothesis between stem and crown ants is not supported. Instead, we found support for the co-diversification, buffered extinction, and hyper-specialization hypotheses. The environmental changes of the ATR, mediated by the angiosperm radiation, likely played a critical role in buffering ants against extinction and favoring their diversification by providing new ecological niches, such as forest litter and arboreal nesting sites, and additional resources. We also hypothesize that the decline and extinction of stem ants during the Late Cretaceous was due to their hyper-specialized morphology, which limited their ability to expand their dietary niche in changing environments. This study highlights the importance of a holistic approach when studying the interplay between past environments and the evolutionary trajectories of organisms.

GmEID1 modulates light signaling through the Evening Complex to control flowering time and yield in soybean.

Soybean (Glycine max) morphogenesis and flowering time are accurately regulated by photoperiod, which determine the yield potential and limit soybean cultivars to a narrow latitudinal range. The E3 and E4 genes, which encode phytochrome A photoreceptors in soybean, promote the expression of the legume-specific flowering repressor E1 to delay floral transition under long-day (LD) conditions. However, the underlying molecular mechanism remains unclear. Here, we show that the diurnal expression pattern of GmEID1 is opposite to that of E1 and targeted mutations in the GmEID1 gene delay soybean flowering regardless of daylength. GmEID1 interacts with J, a key component of circadian Evening Complex (EC), to inhibit E1 transcription. Photoactivated E3/E4 interacts with GmEID1 to inhibit GmEID1-J interaction, promoting J degradation resulting in a negative correlation between daylength and the level of J protein. Notably, targeted mutations in GmEID1 improved soybean adaptability by enhancing yield per plant up to 55.3% compared to WT in field trials performed in a broad latitudinal span of more than 24°. Together, this study reveals a unique mechanism in which E3/E4-GmEID1-EC module controls flowering time and provides an effective strategy to improve soybean adaptability and production for molecular breeding.

INDETERMINATE1-mediated expression of FT family genes is required for proper timing of flowering in Brachypodium distachyon.

The transition to flowering is a major developmental switch in plants. In many temperate grasses, perception of indicators of seasonal change, such as changing day-length and temperature, leads to expression of FLOWERING LOCUS T1 (FT1) and FT-Like (FTL) genes that are essential for promoting the transition to flowering. However, little is known about the upstream regulators of FT1 and FTL genes in temperate grasses. Here, we characterize the monocot-specific gene INDETERMINATE1 (BdID1) in Brachypodium distachyon and demonstrate that BdID1 is a regulator of FT family genes. Mutations in ID1 impact the ability of the short-day (SD) vernalization, cold vernalization, and long-day (LD) photoperiod pathways to induce certain FTL genes. BdID1 is required for upregulation of FTL9 (FT-LIKE9) expression by the SD vernalization pathway, and overexpression of FTL9 in an id1 background can partially restore the delayed flowering phenotype of id1. We show that BdID1 binds in vitro to the promoter region of FTL genes suggesting that ID1 directly activates FTL expression. Transcriptome analysis shows that BdID1 is required for FT1, FT2, FTL12, and FTL13 expression under inductive LD photoperiods, indicating that BdID1 is a regulator of the FT gene family. Moreover, overexpression of FT1 in the id1 background results in rapid flowering similar to overexpressing FT1 in the wild type, demonstrating that BdID1 is upstream of FT family genes. Interestingly, ID1 negatively regulates a previously uncharacterized FTL gene, FTL4, and we show that FTL4 is a repressor of flowering. Thus, BdID1 is critical for proper timing of flowering in temperate grasses.

Phase separation and molecular ordering of the prion-like domain of the Arabidopsis thermosensory protein EARLY FLOWERING 3.

Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalization of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature-sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical, and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher-order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH- and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semiordered structure as determined by small-angle X-ray scattering, electron microscopy, and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

Sex-dependent phenological responses to climate vary across species' ranges.

Anthropogenic climate change has significantly altered the flowering times (i.e., phenology) of plants worldwide, affecting their reproduction, survival, and interactions. Recent studies utilizing herbarium specimens have uncovered significant intra- and inter-specific variation in flowering phenology and its response to changes in climate but have mostly been limited to animal-pollinated species. Thus, despite their economic and ecological importance, variation in phenological responses to climate remain largely unexplored among and within wind-pollinated dioecious species and across their sexes. Using both herbarium specimens and volunteer observations of cottonwood (Populus) species, we examined how phenological sensitivity to climate varies across species, their ranges, sexes, and phenophases. The timing of flowering varied significantly across and within species, as did their sensitivity to spring temperature. In particular, male flowering generally happened earlier in the season and was more sensitive to warming than female flowering. Further, the onset of flowering was more sensitive to changes in temperature than leaf out. Increased temporal gaps between male and female flowering time and between the first open flower date and leaf out date were predicted for the future under two climate change scenarios. These shifts will impact the efficacy of sexual reproduction and gene flow among species. Our study demonstrates significant inter- and intra-specific variation in phenology and its responses to environmental cues, across species' ranges, phenophases, and sex, in wind-pollinated species. These variations need to be considered to predict accurately the effects of climate change and assess their ecological and evolutionary consequences.

Arabidopsis RUP2 represses UVR8-mediated flowering in noninductive photoperiods.

Plants have evolved complex photoreceptor-controlled mechanisms to sense and respond to seasonal changes in day length. This ability allows plants to optimally time the transition from vegetative growth to flowering. UV-B is an important part intrinsic to sunlight; however, whether and how it affects photoperiodic flowering has remained elusive. Here, we report that, in the presence of UV-B, genetic mutation of REPRESSOR OF UV-B PHOTOMORPHOGENESIS 2 (RUP2) renders the facultative long day plant Arabidopsis thaliana a day-neutral plant and that this phenotype is dependent on the UV RESISTANCE LOCUS 8 (UVR8) UV-B photoreceptor. We provide evidence that the floral repression activity of RUP2 involves direct interaction with CONSTANS, repression of this key activator of flowering, and suppression of FLOWERING LOCUS T transcription. RUP2 therefore functions as an essential repressor of UVR8-mediated induction of flowering under noninductive short day conditions and thus provides a crucial mechanism of photoperiodic flowering control.

THYLAKOID FORMATION 1 interacts with FLOWERING LOCUS T and modulates temperature-responsive flowering in Arabidopsis.

The intracellular localization of the florigen FLOWERING LOCUS T (FT) is important for its long-distance transport toward the shoot apical meristem. However, the mechanisms regulating the FT localization remain poorly understood. Here, we discovered that in Arabidopsis thaliana, the chloroplast-localized protein THYLAKOID FORMATION 1 (THF1) physically interacts with FT, sequestering FT in the outer chloroplast envelope. Loss of THF1 function led to temperature-insensitive flowering, resulting in early flowering, especially under low ambient temperatures. THF1 mainly acts in the leaf vasculature and shoot apex to prevent flowering. Mutation of CONSTANS or FT completely suppressed the early flowering of thf1-1 mutants. FT and THF1 interact via their anion binding pocket and coiled-coil domain (CCD), respectively. Deletion of the CCD in THF1 by gene editing caused temperature-insensitive early flowering similar to that observed in the thf1-1 mutant. FT levels in the outer chloroplast envelope decreased in the thf1-1 mutant, suggesting that THF1 is important for sequestering FT. Furthermore, THF1 protein levels decreased in seedlings grown at high ambient temperature, suggesting an explanation for its role in plant responses to ambient temperature. A thf1-1 phosphatidylglycerolphosphate synthase 1 (pgp1) double mutant exhibited additive acceleration of flowering at 23 and 16°C, compared to the single mutants, indicating that THF1 and phosphatidylglycerol (PG) act as independent but synergistic regulators of temperature-responsive flowering. Collectively, our results provide an understanding of the genetic pathway involving THF1 and its role in temperature-responsive flowering and reveal a previously unappreciated additive interplay between THF1 and PG in temperature-responsive flowering.

Loss of daylength sensitivity by splice site mutation in Cannabis pseudo-response regulator.

Photoperiod insensitivity (auto-flowering) in drug-type Cannabis sativa circumvents the need for short day (SD) flowering requirements making outdoor cultivation in high latitudes possible. However, the benefits of photoperiod insensitivity are counterbalanced by low cannabinoid content and poor flower quality in auto-flowering genotypes. Despite recent studies in cannabis flowering, a mechanistic understanding of photoperiod insensitivity is still lacking. We used a combination of genome-wide association study and genetic fine-mapping to identify the genetic cause of auto-flowering in cannabis. We then used gene expression analyses and transient transformation assays to characterize flowering time control. Herein, we identify a splice site mutation within circadian clock gene PSEUDO-RESPONSE REGULATOR 37 (CsPRR37) in auto-flowering cannabis. We show that CsPRR37 represses FT expression and its circadian oscillations transition to a less repressive state during SD as compared to long days (LD). We identify several key circadian clock genes whose expression is altered in auto-flowering cannabis, particularly under non-inductive LD. Research into the pervasiveness of this mutation and others affecting flowering time will help elucidate cannabis domestication history and advance cannabis breeding toward a more sustainable outdoor cultivation system.

Short day promotes gall swelling by a CONSTANS-FLOWERING LOCUS T pathway in Zizania latifolia.

"Jiaobai" is a symbiont of Zizania latifolia and Ustilago esculenta, producing fleshy galls as a popular vegetable in South and East Asia. Current "Jiaobai" cultivars exhibit abundant variation in their gall formation date; however, the underlying mechanism is not clear. In this study, a strict short-day (SD) "Jiaobai" line "YD-3" was used. Plants were treated with two day-length regimes [14 h/10 h (day/night) (control) and 8 h/16 h (day/night) (SD)] from 100 to 130 days after planting. The gall swelling rate of the two treatments and another early SD treatment (from 60 to 90 days after planting), together with the contingent flowering plants in the experiment population, revealed that SD can improve both gall enlargement and flowering of "Jiaobai" plants. Comparison of RNA sequencing data among control, SD swelling, and SD flowering treatments of leaves and meristems indicated that SD promotion of "Jiaobai" swelling is conducted by the CONSTANS (CO)-FLOWERING LOCUS T (FT) pathway, similar but not identical to the SD-induced flowering pathway in Z latifolia and rice. "Virus-induced gene silencing", "Yeast one-hybrid assay" and "Dual-luciferase assay" showed that a FT gene, ZlGsd1, is critical in SD promotion of gall formation and is positively regulated by a CO gene, ZlCOL1. Our study elucidated how photoperiod affects the formation of a unique organ produced by plant-fungus symbiosis. The difference in SD response between "Jiaobai" and rice, as well as their potential applications in breeding of "Jiaobai" and rice, were also discussed.

Population-level gene expression can repeatedly link genes to functions in maize.

Transcriptome-wide association studies (TWAS) can provide single gene resolution for candidate genes in plants, complementing genome-wide association studies (GWAS) but efforts in plants have been met with, at best, mixed success. We generated expression data from 693 maize genotypes, measured in a common field experiment, sampled over a 2-h period to minimize diurnal and environmental effects, using full-length RNA-seq to maximize the accurate estimation of transcript abundance. TWAS could identify roughly 10 times as many genes likely to play a role in flowering time regulation as GWAS conducted data from the same experiment. TWAS using mature leaf tissue identified known true-positive flowering time genes known to act in the shoot apical meristem, and trait data from a new environment enabled the identification of additional flowering time genes without the need for new expression data. eQTL analysis of TWAS-tagged genes identified at least one additional known maize flowering time gene through trans-eQTL interactions. Collectively these results suggest the gene expression resource described here can link genes to functions across different plant phenotypes expressed in a range of tissues and scored in different experiments.