The single-cell transcriptome program of nodule development cellular lineages in Medicago truncatula.

Legumes establish a symbiotic relationship with nitrogen-fixing rhizobia by developing nodules. Nodules are modified lateral roots that undergo changes in their cellular development in response to bacteria, but the transcriptional reprogramming that occurs in these root cells remains largely uncharacterized. Here, we describe the cell-type-specific transcriptome response of Medicago truncatula roots to rhizobia during early nodule development in the wild-type genotype Jemalong A17, complemented with a hypernodulating mutant (sunn-4) to expand the cell population responding to infection and subsequent biological inferences. The analysis identifies epidermal root hair and stele sub-cell types associated with a symbiotic response to infection and regulation of nodule proliferation. Trajectory inference shows cortex-derived cell lineages differentiating to form the nodule primordia and, posteriorly, its meristem, while modulating the regulation of phytohormone-related genes. Gene regulatory analysis of the cell transcriptomes identifies new regulators of nodulation, including STYLISH 4, for which the function is validated.

Spatiotemporal oxygen dynamics in young leaves reveal cyclic hypoxia in plants.

Oxygen is essential for plant growth and development. Hypoxia occurs in plants due to limited oxygen availability following adverse environmental conditions as well in hypoxic niches in otherwise normoxic environments. However, the existence and functional integration of spatiotemporal oxygen dynamics with plant development remains unknown. In animal systems dynamic fluctuations in oxygen availability are known as cyclic hypoxia. In this study, we demonstrate that cyclic fluctuations in internal oxygen levels occur in young emerging leaves of Arabidopsis plants. Cyclic hypoxia in plants is based on a mechanism requiring the ETHYLENE RESPONSE FACTORS type VII (ERFVII) that are central components of the oxygen-sensing machinery in plants. The ERFVII-dependent mechanism allows precise adjustment of leaf growth in response to carbon status and oxygen availability within plant cells. This study thus establishes a functional connection between internal spatiotemporal oxygen dynamics and developmental processes of plants.

Temporal change in chromatin accessibility predicts regulators of nodulation in Medicago truncatula.

BACKGROUND: Symbiotic associations between bacteria and leguminous plants lead to the formation of root nodules that fix nitrogen needed for sustainable agricultural systems. Symbiosis triggers extensive genome and transcriptome remodeling in the plant, yet an integrated understanding of the extent of chromatin changes and transcriptional networks that functionally regulate gene expression associated with symbiosis remains poorly understood. In particular, analyses of early temporal events driving this symbiosis have only captured correlative relationships between regulators and targets at mRNA level. Here, we characterize changes in transcriptome and chromatin accessibility in the model legume Medicago truncatula, in response to rhizobial signals that trigger the formation of root nodules. RESULTS: We profiled the temporal chromatin accessibility (ATAC-seq) and transcriptome (RNA-seq) dynamics of M. truncatula roots treated with bacterial small molecules called lipo-chitooligosaccharides that trigger host symbiotic pathways of nodule development. Using a novel approach, dynamic regulatory module networks, we integrated ATAC-seq and RNA-seq time courses to predict cis-regulatory elements and transcription factors that most significantly contribute to transcriptomic changes associated with symbiosis. Regulators involved in auxin (IAA4-5, SHY2), ethylene (EIN3, ERF1), and abscisic acid (ABI5) hormone response, as well as histone and DNA methylation (IBM1), emerged among those most predictive of transcriptome dynamics. RNAi-based knockdown of EIN3 and ERF1 reduced nodule number in M. truncatula validating the role of these predicted regulators in symbiosis between legumes and rhizobia. CONCLUSIONS: Our transcriptomic and chromatin accessibility datasets provide a valuable resource to understand the gene regulatory programs controlling the early stages of the dynamic process of symbiosis. The regulators identified provide potential targets for future experimental validation, and the engineering of nodulation in species is unable to establish that symbiosis naturally.

Single-nuclei transcriptome analysis of the shoot apex vascular system differentiation in Populus.

Differentiation of stem cells in the plant apex gives rise to aerial tissues and organs. Presently, we lack a lineage map of the shoot apex cells in woody perennials - a crucial gap considering their role in determining primary and secondary growth. Here, we used single-nuclei RNA-sequencing to determine cell type-specific transcriptomes of the Populus vegetative shoot apex. We identified highly heterogeneous cell populations clustered into seven broad groups represented by 18 transcriptionally distinct cell clusters. Next, we established the developmental trajectories of the epidermis, leaf mesophyll and vascular tissue. Motivated by the high similarities between Populus and Arabidopsis cell population in the vegetative apex, we applied a pipeline for interspecific single-cell gene expression data integration. We contrasted the developmental trajectories of primary phloem and xylem formation in both species, establishing the first comparison of vascular development between a model annual herbaceous and a woody perennial plant species. Our results offer a valuable resource for investigating the principles underlying cell division and differentiation conserved between herbaceous and perennial species while also allowing us to examine species-specific differences at single-cell resolution.

An LCO-responsive homolog of NODULE INCEPTION positively regulates lateral root formation in Populus sp.

The transcription factor NODULE INCEPTION (NIN) has been studied extensively for its multiple roles in root nodule symbiosis within plants of the nitrogen-fixing clade (NFC) that associate with soil bacteria, such as rhizobia and Frankia. However, NIN homologs are present in plants outside the NFC, suggesting a role in other developmental processes. Here, we show that the biofuel crop Populus sp., which is not part of the NFC, contains eight copies of NIN with diversified protein sequence and expression patterns. Lipo-chitooligosaccharides (LCOs) are produced by rhizobia and a wide range of fungi, including mycorrhizal ones, and act as symbiotic signals that promote lateral root formation. RNAseq analysis of Populus sp. treated with purified LCO showed induction of the PtNIN2 subfamily. Moreover, the expression of PtNIN2b correlated with the formation of lateral roots and was suppressed by cytokinin treatment. Constitutive expression of PtNIN2b overcame the inhibition of lateral root development by cytokinin under high nitrate conditions. Lateral root induction in response to LCOs likely represents an ancestral function of NIN retained and repurposed in nodulating plants, as we demonstrate that the role of NIN in LCO-induced root branching is conserved in both Populus sp. and legumes. We further established a visual marker of LCO perception in Populus sp. roots, the putative sulfotransferase PtSS1 that can be used to study symbiotic interactions with the bacterial and fungal symbionts of Populus sp.

Functional and comparative genomics reveals conserved noncoding sequences in the nitrogen-fixing clade.

Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root-nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical, but undetermined, evolutionary event at the base of this clade. To identify putative regulatory sequences implicated in the evolution of RNS, we evaluated the genomes of 25 species capable of nodulation and identified 3091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade (NFC). We show that the chromatin accessibility of 452 CNS correlates significantly with the regulation of genes responding to lipochitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of an Mtcre1 mutant showed a significant decrease of nodulation in the absence of the five CNS, when they are driving the expression of a functional copy of MtCRE1. CNS identified in the NFC may harbor elements required for the regulation of genes controlling RNS in M. truncatula.

Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development.

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

Overexpression of a SOC1-Related Gene Promotes Bud Break in Ecodormant Poplars.

Perennial species in the boreal and temperate regions are subject to extreme annual variations in light and temperature. They precisely adapt to seasonal changes by synchronizing cycles of growth and dormancy with external cues. Annual dormancy-growth transitions and flowering involve factors that integrate environmental and endogenous signals. MADS-box transcription factors have been extensively described in the regulation of Arabidopsis flowering. However, their participation in annual dormancy-growth transitions in trees is minimal. In this study, we investigate the function of MADS12, a Populus tremula × alba SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1)-related gene. Our gene expression analysis reveals that MADS12 displays lower mRNA levels during the winter than during early spring and mid-spring. Moreover, MADS12 activation depends on the fulfillment of the chilling requirement. Hybrid poplars overexpressing MADS12 show no differences in growth cessation and bud set, while ecodormant plants display an early bud break, indicating that MADS12 overexpression promotes bud growth reactivation. Comparative expression analysis of available bud break-promoting genes reveals that MADS12 overexpression downregulates the GIBBERELLINS 2 OXIDASE 4 (GA2ox4), a gene involved in gibberellin catabolism. Moreover, the mid-winter to mid-spring RNAseq profiling indicates that MADS12 and GA2ox4 show antagonistic expression during bud dormancy release. Our results support MADS12 participation in the reactivation of shoot meristem growth during ecodormancy and link MADS12 activation and GA2ox4 downregulation within the temporal events that lead to poplar bud break.

Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba.

Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype's characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.

A robust method of nuclei isolation for single-cell RNA sequencing of solid tissues from the plant genus Populus.

Single-cell transcriptome analysis has been extensively applied in humans and animal models to uncover gene expression heterogeneity between the different cell types of a tissue or an organ. It demonstrated its capability to discover key regulatory elements that determine cell fate during developmental programs. Single-cell analysis requires the isolation and labeling of the messenger RNA (mRNA) derived from each cell. These challenges were primarily addressed in mammals by developing microfluidic-based approaches. For plant species whose cells contain cell walls, these approaches have generally required the generation of isolated protoplasts. Many plant tissues' secondary cell wall hinders enzymatic digestion required for individual protoplast isolation, resulting in an unequal representation of cell types in a protoplast population. This limitation is especially critical for cell types located in the inner layers of a tissue or the inner tissues of an organ. Consequently, single-cell RNA sequencing (scRNA-seq) studies using microfluidic approaches in plants have mainly been restricted to Arabidopsis roots, for which well-established procedures of protoplast isolation are available. Here we present a simple alternative approach to generating high-quality protoplasts from plant tissue by characterizing the mRNA extracted from individual nuclei instead of whole cells. We developed the protocol using two different plant materials with varying cellular complexity levels and cell wall structure, Populus shoot apices, and more lignified stems. Using the 10× Genomics Chromium technology, we show that this procedure results in intact mRNA isolation and limited leakage, with a broad representation of individual cell transcriptomes.

LHY2 Integrates Night-Length Information to Determine Timing of Poplar Photoperiodic Growth.

Day length is a key indicator of seasonal information that determines major patterns of behavior in plants and animals. Photoperiodism has been described in plants for about 100 years, but the underlying molecular mechanisms of day length perception and signal transduction in many systems are not well understood. In trees, photoperiod perception plays a major role in growth cessation during the autumn as well as activating the resumption of shoot growth in the spring, both processes controlled by FLOWERING LOCUS T2 (FT2) expression levels and critical for the survival of perennial plants over winter [1-4]. It has been shown that the conserved role of poplar orthologs to Arabidopsis CONSTANS (CO) directly activates FT2 expression [1, 5]. Overexpression of poplar CO is, however, not sufficient to sustain FT2 expression under short days [5], pointing to the presence of an additional short-day-dependent FT2 repression pathway in poplar. We find that night length information is transmitted via the expression level of a poplar clock gene, LATE ELONGATED HYPOCOTYL 2 (LHY2), which controls FT2 expression. Repression of FT2 is a function of the night extension and LHY2 expression level. We show that LHY2 is necessary and sufficient to activate night length repressive signaling. We propose that the photoperiodic control of shoot growth in poplar involves a balance between FT2 activating and repressing pathways. Our results show that poplar relies on night length measurement to determine photoperiodism through interaction between light signaling pathways and the circadian clock.

Photoperiodic Regulation of Shoot Apical Growth in Poplar.

Woody perennials adapt their genetic traits to local climate conditions. Day length plays an essential role in the seasonal growth of poplar trees. When photoperiod falls below a given critical day length, poplars undergo growth cessation and bud set. A leaf-localized mechanism of photoperiod measurement triggers the transcriptional modulation of a long distance signaling molecule, FLOWERING LOCUS T (FT). This molecule targets meristem function giving rise to these seasonal responses. Studies over the past decade have identified conserved orthologous genes involved in photoperiodic flowering in Arabidopsis that regulate poplar vegetative growth. However, phenological and molecular examination of key photoperiod signaling molecules reveals functional differences between these two plant model systems suggesting alternative components and/or regulatory mechanisms operating during poplar vegetative growth. Here, we review current knowledge and provide new data regarding the molecular components of the photoperiod measuring mechanism that regulates annual growth in poplar focusing on main achievements and new perspectives.

Environmentally Sensitive Molecular Switches Drive Poplar Phenology.

Boreal and temperate woody perennials are highly adapted to their local climate, which delimits the length of the growing period. Moreover, seasonal control of growth-dormancy cycles impacts tree productivity and geographical distribution. Therefore, traits related to phenology are of great interest to tree breeders and particularly relevant in the context of global warming. The recent application of transcriptional profiling and genetic association studies to poplar species has provided a robust molecular framework for investigating molecules with potential links to phenology. The environment dictates phenology by modulating the expression of endogenous molecular switches, the identities of which are currently under investigation. This review outlines the current knowledge of these molecular switches in poplar and covers several perspectives concerning the environmental control of growth-dormancy cycles. In the process, we highlight certain genetic pathways which are affected by short days, low temperatures and cold-induced signaling.

Real-time monitoring of PtaHMGB activity in poplar transactivation assays.

BACKGROUND: Precise control of gene expression is essential to synchronize plant development with the environment. In perennial plants, transcriptional regulation remains poorly understood, mainly due to the long time required to perform functional studies. Transcriptional reporters based on luciferase have been useful to study circadian and diurnal regulation of gene expression, both by transcription factors and chromatin remodelers. The high mobility group proteins are considered transcriptional chaperones that also modify the chromatin architecture. They have been found in several species, presenting in some cases a circadian expression of their mRNA or protein. RESULTS: Transactivation experiments have been shown as a powerful and fast method to obtain information about the potential role of transcription factors upon a certain reporter. We designed and validated a luciferase transcriptional reporter using the 5' sequence upstream ATG of Populus tremula × alba LHY2 gene. We showed the robustness of this reporter line under long day and continuous light conditions. Moreover, we confirmed that pPtaLHY2::LUC activity reproduces the accumulation of PtaLHY2 mRNA. We performed transactivation studies by transient expression, using the reporter line as a genetic background, unraveling a new function of a high mobility group protein in poplar, which can activate the PtaLHY2 promoter in a gate-dependent manner. We also showed PtaHMGB2/3 needs darkness to produce that activation and exhibits an active degradation after dawn, mediated by the 26S proteasome. CONCLUSIONS: We generated a stable luciferase reporter poplar line based on the circadian clock gene PtaLHY2, which can be used to investigate transcriptional regulation and signal transduction pathway. Using this reporter line as a genetic background, we established a methodology to rapidly assess potential regulators of diurnal and circadian rhythms. This tool allowed us to demonstrate that PtaHMGB2/3 promotes the transcriptional activation of our reporter in a gate-dependent manner. Moreover, we added new information about the PtaHMGB2/3 protein regulation along the day. This methodology can be easily adapted to other transcription factors and reporters.