A telomere-to-telomere genome of raintree (Samanea Saman) reveals its ecological adaptation characteristics of nyctinastic movement and symbiotic nitrogen fixation

A telomere-to-telomere genome of raintree (Samanea Saman) reveals its ecological adaptation characteristics of nyctinastic movement and symbiotic nitrogen fixation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A telomere-to-telomere genome of raintree (Samanea Saman) reveals its ecological adaptation characteristics of nyctinastic movement and symbiotic nitrogen fixation Kejing Yang, hao Wang, GUO Guilian, Haixiang Yu, Zhidong Li, Fei Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7165529/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Raintrees are the predominant species within tropical monsoon forest ecosystems, largely due to their distinctive ecological adaptations, such as nyctinastic movement and nitrogen fixation. However, the comprehensive understanding of their functions has been constrained by lack of genomic resources. Here, we present the first T2T genome of the raintree, comprising 13 pseudo-chromosomes and a contig N50 42.86 Mb, with 25,997 gene models annotated. Additionally, we sequenced the genome of the rhizobium symbiotic with the raintree, measuring 7.28 Mb and containing 6,882 annotated genes, and designated it as Bradyrhizobium saman. Through transcriptomic analysis, we identified 41 key genes that are significantly upregulated in pulvinus cells, which are exclusively involved in nyctinastic movement. These genes include basic regulatory factors, ion transporters, and aquaporins. Meanwhile, RNA-seq identified 699 core genes upregulated in root nodules, crucial for symbiotic nitrogen fixation. These include seven SsaHGs coding hemoglobin proteins that extremely high expressed to maintain anaerobic conditions in symbiosomes; 26 genes for amino acid transporters, glutamate synthetases (SsaGh) and aspartate synthetases (SsaAsn), 16 for auxin transport facilitators, and nine in cytokinin signaling. Furthermore, 17 MYB transcription factors are upregulated. These genomic resources and findings are vital for enhancing raintree genetics and investigating their ecological adaptations. Biological sciences/Plant sciences/Plant stress responses/Abiotic Biological sciences/Genetics/Genomics/Transcriptomics Raintree Rhizobium Genome nyctinastic movement symbiotic nitrogen fixation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The raintree ( Samanea saman Merr.), in the Fabaceae family, originates from the tropical arid regions of South and Central America, notably in countries such as Belize, Ecuador, and Colombia (Merrill ED, 1916 ). It has since become prevalent in tropical semi-arid regions worldwide. The species flourishes under conditions of light, heat, and humidity, exhibiting tolerance to drought and poor soil quality. It is predominantly utilized for landscaping and garden enhancement. The tree's sweet, succulent fruit and protein-rich foliage serve as suitable feed for livestock, while its durable heartwood is employed in the production of furniture and crafts. The Fabaceae family encompasses 796 confirmed genera, including the genus Samanea, which comprises 20 species. Samanea is closely related to genera such as Albizia and Calliandra , as well as Cylindrokelupha , Enterolobium , and Pithecellobium . Cytological studies reveal that the raintree is diploid (2n = 26), although comprehensive genome sequencing has yet to be conducted. Nonetheless, some gene and transcriptome data are available in GenBank. Rain trees adapt to the arid environment of the native atmosphere and form the morphological characteristics of the pinnate compound leaves that unfold during the day and close at night. Closing and drooping leaves at night reduces the leaf surface area exposed to the air, reduces transpiration water loss, and maintains plant water balance. This phenomenon is called nyctinastic movement, which is only found in the genus Phaseolus of the Fabaceae family Phaseolus, Robinia, Albizzia, Mimosa , and Samanea (Moran 2015). The nyctinastic movement of the pinnate leaves of raintree is achieved through the movement of pulvinus cells located at the base of the tertiary compound leaves. The pulvinus of raintree is composed of two parts with opposite positions and functions: one is the extensor, which extends longitudinally when the leaves are unfolded, and the other is the flexor, which contracts longitudinally simultaneously (Oikawa et al., 2018 ). Primary signal of the nyctinastic movement is the differential response of the phytochrome receptor to blue and red light in raintree (Kim et al., 1993 ; Mano, et al., 2021). Blue light promotes the opening of leaflets by increasing the activity of K + influx channels in extensor cells and K + efflux channels in flexor cells. By inhibiting the H + pump, blue light leads to depolarization of the flexor cell membrane, which may be a mechanism of blue light regulation in leaf movement (Suh et al., 2000). In darkness, red and far-red light induce the opening of K + influx channels in the flexor cells of rain tree, leading to the closing movement of the leaves. The reversible osmotic swelling of extensor cells accompanied by the contraction of flexor cells results in the opening of the pulvinus, which in turn lifts the leaves and leaflets. When potassium ions redistribute, the extensor cells contract and the flexor cells swell, closing the pulvinus. The water permeability of the membranes in both motor tissues is strictly regulated in time (Mano, et al., 2006; Zeng et al., 2024 ). The reversible osmotic swelling of extensor cells accompanied by the contraction of flexor cells causes the opening of the pulvinus, which in turn raises the leaves and leaflets. When potassium ions redistribute, the extensor cells contract and the flexor cells expand, closing the pulvinus (Zeng et al., 2024 ). The water permeability of the membrane in the two motor tissues is strictly regulated by time. Further research found that several potassium ion channel protein genes in the extensor and flexor tissues of the pulvinus of rain tree leaves, such as SPICK3, SPICK4, SPORK2, SPORK3, and SPORK4, are involved in the nyctinastic movement of pulvinus cells. Among them, only SPORK2 has been confirmed as a functional K + channel (Oikawa et al., 2018 ; Ueda, et al. 2019 ). The aquaporin gene localized in the plasma membrane has been proven to be involved in the movement of flexor and extensor cells in pulvinus. There are two types, namely SsAQP1 and SsAQP2, which belong to the two subfamilies of PIP1 and PIP2, respectively (Moshelion et al., 2002 ; Uehlein et al., 2008). The rain tree's root system shows dense nodulation, suggesting it uses symbiotic nitrogen fixation for ecological adaptation (Qadri et al., 2007 ). Rhizobia, Gram-negative soil bacteria, form nodules with leguminous plants, converting atmospheric nitrogen into plant nutrition. This legume-rhizobium symbiosis fixes about 55 million tons of nitrogen annually, representing 55% of terrestrial biological nitrogen fixation and is crucial to the nitrogen cycle (Kistner and Parniske 2002; Markmann and Parniske 2009). About 65 million years ago, plants in the Caesalpinioideae and Papilionoideae subfamilies of the Fabaceae family developed symbiotic nitrogen fixation with rhizobia (Sprent and James 2007; Zhao et al. 2021). Rhizobia, part of the Alphaproteobacteria and Betaproteobacteria classes in the Proteobacteria phylum, include 19 genera such as Rhizobium and Cupriavidus. They have large genomes (5–10 Mb) to adapt to the rhizosphere and nitrogen fixation (Remigi et al., 2016; Zhang C, 2023). Functional analysis indicates that the rhizobia genome tends to encode a set of genes responsible for transport, regulation, and stress resistance. However, the genome size and structure of the symbiotic nitrogen-fixing bacteria associated with the rain tree have not been reported yet. The symbiotic nitrogen fixation in the legume-rhizobia system is a complex biological process that is tightly regulated by both the host plant and the rhizobia (Oldroyd, 2013; Yu H et al., 2025 ). Upon sensing flavonoid compounds secreted by the host plant, the rhizobia are specifically recognized by nodulation factor receptors on the host plant's cell membrane surface. This triggers the expansion and curling of the root hair tips, enclosing the rhizobia to form an infection chamber. Subsequently, the root hair cell wall and membrane invaginate to grow into an infection thread, guided by a pre-infection thread (PIT), and enter the plant's root cortical cells (Bladergroen and Paink 1998). The formation of a microaerobic environment in nodules is a prerequisite for nitrogen fixation. The molybdenum-iron nitrogenase requires 16 ATP and 8 electrons to reduce each molecule of N2, producing 2 molecules of NH3 and 1 molecule of H2. Exposure to oxygen leads to irreversible loss of enzyme activity (Rutledge et al. 2022). Legume nodule cells specifically express large amounts of leghemoglobin to maintain the concentration of free oxygen at nanomolar levels per liter (Dixon and Kahn 2004; Ott et al. 2005; Wang et al. 2019a; Cui et al. 2021; Jiang et al. 2021 ). Meanwhile, rhizobia express the CBB3 terminal oxidase with higher oxygen affinity in nodules to perform oxidative phosphorylation, generating ATP for maintaining nitrogen fixation reactions and basic life activities (Preisig et al. 1993, 1996; Zufferey et al. 1996). Plant hormones such as auxin, cytokinin (CTK), gibberellins (GAs), and ethylene (ETH) are involved in the rhizobia infection process, nodule formation, and development, playing crucial roles in regulating the interaction between legumes and rhizobia and the formation of nodule organs. Adequate carbon sources and energy supply are necessary for efficient nitrogen fixation by bacteroids in nodules. In nodules, the host provides malate as the primary energy and carbon source to sustain the nitrogen fixation reactions of rhizobia, which are absorbed by bacteroids through the four-carbon dicarboxylate transporter Dct system (Mitsch et al. 2018). The process of plant symbiotic nitrogen fixation is co-regulated by both rhizobia and host genes. Specific rhizobia can only form symbiotic nitrogen-fixing nodules with specific groups of legume plants. In peas, host-specificity genes are determined by the carried sym2 locus, whose gene products interact with NodX encoded by specific Rhizobium leguminosarum sv. viciae rhizobia to achieve successful nodulation and symbiotic nitrogen fixation (Geurts et al., 1997; Limpens et al., 2003). The nodulation (nod) and nitrogen fixation (nif) genes mediating rhizobial symbiotic nitrogen fixation are typically located on transferable elements, including plasmids with transfer capabilities (known as symbiotic plasmids, pSym) and genomic islands (known as symbiotic islands, SI). Nodulation genes include nodA, nodB, nodC, nodI, and nodJ, while nitrogen fixation genes include nifH, nifD, nifK, etc. Host nitrogen fixation gene types include Lysin motif receptor-like kinase ( LysM-RLK ), encoding proteins such as MtAAP7 , Ammonium transporter 1 ( MtAMT1 ), and Aromatic and neutral transporter 50 ( MtANT50 ). Although the core genes and reactions of nitrogen fixation are highly conserved among rhizobia, different legume plants provide rhizobia with distinct working micro environments resulting in species-host-dependent diversity in the resource supply mechanisms required to maintain bacteroid nitrogen fixation efficiency. In summary, the genome of the rain tree and its symbiotic nitrogen-fixing rhizobia is largely unexplored. While there are some studies on the circadian movement of legumes and the rain tree, examining the gene family structure related to this process at the genomic level is a new approach that could support existing models (Bai Q et al., 2022 ). Although there has been progress in understanding plant symbiotic nitrogen fixation, research on the rain tree's symbiotic nitrogen fixation is minimal. Understanding this mechanism in tropical fast-growing trees and comparing it with legumes could reveal how legumes manage stress resistance and nitrogen fixation. This study reported the first near T2T genome of the raintree and genome of rhizobium symbiotic in raintree. And further annotated the gene resources of its symbiotic nitrogen-fixing and nyctinastic movement, the key double characteristics. Based on it, we clarified the phylogenetic tree of the specific evolutionary relationships with its closely related genera. Integrated the transcriptomic analysis, we deciphered the molecular mechanism of nyctinastic movement response to circadian and light signal, also the symbiotic nitrogen-fixing. These data and findings will establish the cornerstone for genetic research and breeding of raintree, and service for better understanding the ecological adaptability and economic utilization of the rain tree. Results Genome assembly and annotation of raintree Using a flow cytometry estimate the genome size of 530 Mb for the raintree ( Samanea saman ) (2n = 26). A total of 68 Gb of Hifi long reads sequences data was generated, resulting in a genome coverage of 130X. To achieve a high-quality, chromosome-level reference genome, we leveraged 53 Gb of Hi-C paired-end sequencing data to scaffold the contigs. Finally, the genome assembly resulted in 13 contigs equal to the chromosome numbers with a total length of 539.53Mb, with a contig N50 size of 42.86 Mb. There are 25,997 gene models have been annotated (Table 1 , DataSet 1 ). The assembly quality was evaluated using BUSCO revealing a completeness of 98.8% in genome and gene regions. The basic structural features (gene density, repeat sequence density, and GC density) of the T2T genome are shown in Fig. 1 A. We used the seven-base telomere sequence (CCCTAAA at the5’ end or TTTAGGG at the 3’ end) as a query sequence to scan the genome. We identified almost all 26 telomeres in 13 chromosomes except of Chr4. With Tandem Repeats Finder (Benson, 1999 ) software, we detected 13 putative centromeres of 13 chromosomes, with size ranging from 102kb-2.5Mb of the T2T genome (Fig. 1 B, DataSet 1). In summary, we released the T2T genome for Raintree in the first time, enabling a foundation for evolutionary studies and gene mining. Table 1 Genome assembly ann annotation of raintree ( Samanea saman ) Genome Samanea saman Ploidy 2n = 26 Estimated genome size (Mb) 530 Assembled genome size (Mb) 539.53 Genomic heterozygosity (%) 0.253 Contig N50 (Mb) 42.86 Number of contigs 13 Number of chromosomes 13 Repeat sequence content (%) 58.51 GC content (%) 32.85 Number of gene models 25,997 Genome BUSCO (%) 98.8 Gene BUSCO (%) 98.8 Utilizing the protein sequences of single-copy genes from the genomes of the rain tree and its related legume species, a phylogenetic tree was constructed to elucidate species evolution. The analysis demonstrated that the rain tree shares the closest genetic relationship with the Partridge Pea ( Chamaecrista fasciculata ), with their divergence occurring approximately 46 million years ago (MYA). In contrast, the divergence between the cultivated soybean ( Glycine max ) and its wild relative ( Glycine soja ) occurred around 6 MYA. Furthermore, the divergence of the common bean ( Phaseolus vulgaris ) and another cultivated species, the runner bean ( Phaseolus coccineus ), as well as lentils ( Lens culinaris ) and their wild relative species ( Lens ervoides ), is estimated to be much later occurred approximately 2.5 MYA (Fig. 1 C). These findings suggest that the raintree is a tropical rainforest plant that has not undergone extensive cultivation and domestication. However, its distinctive nyctinastic movement, adaptation to atmospheric drought, and ability to fix nitrogen symbiotically through nodules confer ecological advantages, contributing to its widespread distribution. The expression profiling of the specific genes involved in nyctinastic movement and the co-expression network The nyctinastic movement of the raintree is unique phenotype adapted to atmospheric drought. It achieves the closure of lobules at night and the re-opening during the day through the asymmetric movement of extensor cells and flexor motor cells in the pulvinus. This function may be jointly regulated by light and the circadian clock (Kim et al., 1993 ; Minorsky, 2019 ; Zeng F, 2024). Although it has been initially indicated that the movement of motor cells in pulvinus is driven by chloride channels, potassium channels and aquaporins (Moran et al., 1988 ; Yu et al., 2006 ; Oikawa et al., 2018 ; Ueda et al., 2019 ), the variations of related genes systematically analyzed at the genomic and transcriptome levels have not been reported yet. We designed a transcriptome sampling strategy for the circadian clock and light response of the rain tree: Taking the dark time of 5:30 (T0) in the morning as the control (Fig. 2 A right), samples of pulvinus and leaflets were taken at intervals of 2hs respectively and treated rapidly with liquid nitrogen with total of 4 time points (T0, T1, T2 and T3); Meanwhile, we further collected the samples after 2hs dark treatment at 9:30 a.m (T4) to distinguish the response to the light signal. Each sample was biologically repeated three times for transcriptome sequencing. Referring to the raintree T2T genome, we obtained high-quality transcriptome annotated genes and their expression information. For differential expression analysis, transcripts exhibiting expression differences exceeding fourfold were extracted. In the pulvinus, the number of differentially expressed genes at various time points following light exposure was 444, 1,234, and 1,136, respectively, with a total of 146 genes consistently differentially expressed across all three time points. Specifically, at the T1 time point, 322 genes were up-regulated, while 122 were down-regulated. At T2, 867 genes were up-regulated, and 387 were down-regulated. At T3, 530 genes showed increased expression, whereas 606 exhibited decreased expression (Fig. 2 B). These differentially expressed genes are predominantly associated with metabolic pathways involved in processes such as nyctinastic movement and photosynthesis. In the leaf blade, there were 1,893, 2,089, and 2,149 differentially expressed genes at three distinct time points following morning light exposure, in comparison to the early morning darkness. A total of 481 differentially expressed genes were common across these time points. Among these, the numbers of up-regulated versus down-regulated genes were 825 vs 1,068, 1,547 vs 542, and 1,200 vs 949, respectively (Fig. 2 C). These differentially expressed genes are predominantly associated with photosynthesis and the growth and development processes stimulated by enhanced light signals. How to screen out the differentially expressed genes in the pulvinus cells that are prority to nyctinastic movement? We focused on four kinds of genes that met the following conditions: 1) Genes that were upregulated above four folds in the pulvinus under light treatment, but not upregulated in the leaf blades. The statistical results showed a total of 190. This set excludes genes directly related to photosynthesis and covers positive regulation genes response to circadian clock and light signal in nyctinastic movement ( DataSet 2 ). 2) In the set, we selected that down-regulated under dark treatment (T4) Majority of them encode splicing factor 3B subunit ( SF3B ), Zinc finger A20 and AN1 domain-containing stress-associated protein ( SAP ), EIN3-binding F-box protein ( EBF ), RING-H2 finger protein ( RING-H2 ) and protein kinase superfamily ( PKs ). They are basic regulation factors, directly responded to light signals in motor cells. There are genes for ion and water transport, like sodium ion transport ( CitMHS ), aquaporins as plasm membrane intrinstic proteins ( PIP2 and PIP3 ), S-type anion channel ( SLAC1 ) and exocyst complex component ( EXOC ). However, although several genes for potassium channel proteins ( AKT1, KAT1 and SKOR3 ) were upregulated, their expression levels were relatively low (Fig. 2 D). 3) Genes were decreased over four- fold (T1) but increased under dark treatment (T4) in the pulvinus, and not down-regulated in the leaflets under light. It enabling us found 19 genes, they are transcriptional regulatory factors, coordinators of cellular basic processes (Fig. 2 E). These genes are negative regulatory genes of nyctinastic movement that only respond to light signals. The genome of the Bradyrhizobium Saman unlocks its function in symbiotic nitrogen fixation The total DNA of the root nodule samples of raintree was collected and sequenced using the second-generation sequencing technology. The bacterial metagenomic sequences were extracted using software, totaling approximately x Mb. After quality control, the metagenomic data were assembled using Megahit, resulting in 4813 contigs (total length approximately 16.94 Mb, N50 = 32.61 kb, GC content 63.33%). Five MAGs were obtained through MetaBAT2 binning. The CheckM assessment showed that only bin.2 met the quality requirements for single-bacterial analysis (completeness 97.51%, contamination 0.17%, strain consistency 0), while the remaining bins were excluded due to low completeness. This genome was further assembly and annotated with genome size 7.28 Mb, composed of 50 contigs with N50 of 309.5 kb and a GC content of 64.63% (Fig. 3 A). The genome annotation identified 6882 genes, including 51 transfer RNAs (tRNAs), three ribosomal RNAs (rRNAs) and one transfer-messenger RNA (tmRNA). The classification of raintree rhizobia was assessed utilizing both Average Nucleotide Identity (ANI) and DNA-DNA Hybridization (DDH) methods. Comprehensive genomic sequences from six rhizobial bacteria were obtained and used for comparsion with the bin.2 genome (Fig. 3 B). Sequence alignment analysis revealed that the ANI value of the raintree-associated rhizobium and the rhizobium isolated from Cajanus cajan originated in Africa, Bradyrhizobium cajani , was 95.33% (Fig. 3 C), suggesting a close genetic relationship at the species level. However, employing Model 2 of the Genome-to-Genome Distance Calculator (GGDC) network service (accessible at ggdc.dsmz.de/ggdc.php#), the calculated digital DDH (dDDH) value between these two bacterial strains was 63.1%, which falls below the 70% threshold typically required to classify them as the same species ( Dataset 3 ). Consequently, this rhizobium is named as Bradyrhizobium saman . Gene resources involved in symbiotic nitrogen fixation in raintree During the vigorous growth phase of the raintree (Fig. 4 A), we analyzed the transcriptomes of root nodules (RN), adventitious roots (R), and leaflets (L). Comparative analysis of transcript expression identified 4,805 differentially expressed genes (DEGs) between RN and R, and 7,841 DEGs between RN and L. Notably, 2,273 DEGs were common to both comparisons. In comparison to the R group, the NR group exhibited upregulation of 1,608 genes and downregulation of 3,197 genes. When comparing NR with L, the NR group demonstrated an upregulation of 4,588 genes and a downregulation of 3,253 genes relative to the L (Fig. 4 B). From the 1,608 genes upregulated in NR compared to R, we excluded 472 genes that were four folds upregulated also in the L, resulting in a subset of 669 genes (DataSet 2). This subset is considered to represent the primary gene types involved in symbiotic nitrogen fixation in raintrees. Six members in the hemoglobin gene family were extremely high expressed, specifically identified as SsaHB2A, SsaHB2B, SsaHB2C, SsaHB2D, SsaHb1 and SsaGLB3 (Fig. 4 E). Their coding proteins contribute to maintaining a low-oxygen environment within the symbiosome cells of the raintree's root nodules (Ma et al., 2024 ). Gene Ontology (GO) enrichment analysis revealed significant enrichment in 20 pathways, including those related to amino acid transport and metabolic processes, cytokinin metabolism, auxin signaling pathways, and transcription factors, encompassing a total of 227 genes (Fig. 4 D). Within the nitrogen metabolism-related pathways, 26 key components include ammonium ion and amino acid transport (involving ten genes), nitrate transporters (double NRTs), and genes encoding asparagine synthetase ( AS ) and glutamate synthase ( GS ) (Fig. 4 F). These genes, derived from the host genome, are essential for the symbiotic nitrogen fixation process in the raintree. Sixteen genes exhibited significant upregulation within the auxin signaling pathways (Fig. 4 G). Notably, ten of these genes are identified as WALLS ARE THIN1 ( WAT1 ), a vacuolar auxin transport facilitator essential for maintaining auxin homeostasis (Ranocha et al., 2013 ). Additionally, three genes belong to the Aux/IAA family, while the remaining three are related protein genes. Aux/IAA proteins play a crucial role in modulating the transcriptional response to auxin (Cancé et al., 2022 ). In the cytokinin pathway, nine genes demonstrated significant upregulation (Fig. 4 H). These include Adenine Phosphoribosyl Transferase ( APT ), three Isopentenyl Transferases ( IPTs ), three Cytokinin Dehydrogenases ( CKXs ), and Cytokinin Hydroxylase ( CYP735 ), among others. IPT functions as a rate-limiting enzyme in cytokinin biosynthesis, whereas adenine phosphoribosyl transferase is pivotal in catalyzing the conversion of cytokinin from nucleobases to nucleotides (Zhang et al., 2013 ). Cytokinin serves as a crucial hormonal signal in symbiotic nitrogen fixation, contributing to the formation of symbiosomes and influencing cellular metabolism. Furthermore, it was found that 17 transcription factors were significantly upregulated in the root nodules of raintree (Fig. 4 I). Notably, the majority these transcription factors belong to the MYB family, with 14 instances identified, alongside 2 R2R3-MYB-Like Regulatory Factors, specifically EOBI, which are involved in regulating substrate availability for volatile biosynthesis (Spitzer-Rimon et al., 2012 ). This indicates that the MYB transcription factors play a particularly vital role in the symbiotic nitrogen fixation process within the rhizobium-nodule system of the raintree. Samanea saman Database: An Open-Access Platform for the Genomics of Raintree Currently, the absence of a centralized web server for sharing rain tree genome information impedes progress in rain tree breeding and molecular breeding research. To address this issue, we established a Samanea saman genome database (SSDB) available at https://bioinformatics.hainanu.edu.cn/SSDB/ . This repository offers functionalities for data browsing, searching, analysis, and downloading, serving as a comprehensive resource for rain tree genome information. SSDB provides a user-friendly interface to facilitate the retrieval of information (Fig. 5 A). The SSDB structure consists of six main modules: Home, Tools, Download, Search, Breeding, and About. To enhance the utility of rain tree genomic resources, we have developed several data retrieval and analysis interfaces: KEGG enrichment tool (Fig. 5 B); GO enrichment tool (Fig. 5 C); Basic Local Alignment Search Tool (BLAST+) tool for sequence alignments (Fig. 5 D); Furthermore, SSDB offers search functionalities for 63 transcription factor families (Fig. 5 E), alongside a gene search tool (Fig. 5 F) and a sequence extraction tool (Fig. 5 G). The database also incorporates a primer design tool (Fig. 5 H) and an SSR identification tool. SSDB facilitates data download, granting access to publicly available sequences, such as the rain tree genome, transcriptomes, coding sequences, and other related sequences. Discussion The genome of raintree, symbiotic rhizobium and their database The raintree represents a predominant vegetation type within the tropical seasonal rainforests of Central and South America and is extensively cultivated across global tropical regions for use as street trees and in landscaping (Durr, 2001 ). Despite its ecological significance, research concerning the genetics and species diversity of raintrees remains scarce. In this study, we present the first telomere-to-telomere (T2T) genomic sequence of the raintree and provide a comprehensive annotation. Additionally, we elucidate the evolutionary relationships between the raintree and its phylogenetically related genera. Concurrently, we have obtained a high-quality genome of the raintree's symbiotic nitrogen-fixing bacteria through a metagenomic approach. Comparative genomic analysis confirmed its classification within the Bradyrhizobium genus, symbiotically associated with raintree species, and it has been designated as Bradyrhizobium saman . These are new findings in the taxonomic family (Durr PA, 2001 ; Jia et al., 2024 ). To facilitate the utilization of these genetic resources, we have developed a raintree genome database, accessible online at https://bioinformatics.hainanu.edu.cn/SSDB/ . The gene resources in nyctinastic movement of raintree The nyctinastic movement is an adaptation to atmospheric drought observed in rain trees and certain leguminous plants. This movement aids in minimizing water loss through transpiration and respiration, also facilitates the collection of surface water. In this study, we performed a comparative transcriptomic analysis of nyctinastic movement involving both the pulvinus and leaflets under pod cultivation conditions. Using 5:30 a.m. as the control time point, samples were collected every two hours. To differentiate the effects of the circadian clock from light influences, additional samples were collected following a two-hour dark treatment at 9:30 a.m., with each sample replicated three times for RNA sequencing. We identified 41 genes closely associated with circadian rhythm regulation and discovered 19 negative regulatory factors. The co-expression network analysis highlighted two hub genes, SsaMYB4 and SsaAMPK1 . While the mechanisms underlying nyctinastic movements in the raintree have been extensively studied, it has been established that the movements of the two types of cells in the pulvinus are regulated by potassium ions, chloride ions, and aquaporins (Kim et al., 1993 ; Moshelion et al., 2002 ; Yu et al., 2006 ; Uehlein et al., 2008; Ueda et al., 2019 ; Wang et al., 2023 ). Nevertheless, the comprehensive regulatory mechanism remains inadequately understood. Our study identified that fundamental regulatory factors, including kinases, splicing factors, and transcription factors, exhibited notably increased expression levels. For instance, SsaAMPK1 , which encodes a kinase, functions as a nutrient and energy sensor to maintain energy homeostasis. Additionally, SsaMYB4 may play a crucial role in regulating cell movement within the pulvinus of the raintree. We also observed a significant upregulation of sodium ion and aquaporin gene expression, aligning with previous findings (Moshelion et al., 2002 ; Oikawa et al., 2018 ). However, contrary to existing reports (Yu et al., 2006 ), we did not detect a significant upregulation of the potassium ion channel protein gene. The gene resources in symbiotic nitrogen fixation of raintree The importance of symbiotic nitrogen fixation within the root nodules of leguminous plants is well-established. The raintree, as the predominant tree species in tropical seasonal rainforests, exhibits a vibrant and consistent green foliage, potentially attributable to its symbiotic nitrogen-fixing capabilities. Through genomic sequencing of both the raintree and its symbiotic rhizobium, complemented by transcriptomic analysis, we have identified that the genetic resources from both the raintree and the rhizobium offer essential insights into the nitrogen fixation processes in the raintree. These enduring nitrogen-fixing mechanisms in their native ecological context may hold substantial potential for enhancing nitrogen fixation in cultivated crops. Our study identified that six members of the hemoglobin gene family were expressed at the highest levels within the nodules, suggesting their pivotal role in establishing the anaerobic environment of the nodules and in coordinating oxygen balance (Larrainzar et al., 2020 ; Jiang et al., 2021 ). Gene Ontology (GO) term enrichment analysis revealed genes across 20 categories. Notably, 17 MYB transcription factors were exclusively and highly expressed in the pulvinus, indicating their significant, yet previously unreported, role in symbiotic nitrogen fixation. Additionally, genes associated with ammonium ion and amino acid transport, nitrate transporters ( NRTs ), asparagine synthetase ( AS ), and glutamate synthase ( GS ) exhibited elevated expression levels solely in the pulvinus. These genes, related to nitrogen metabolism from the raintree genome, are implicated in nodule formation and the conversion and transport of ammonia synthesized by rhizobium (Stokstad, 2016 ; Xu et al., 2023; Bu et al., 2020 ). Furthermore, we observed that multiple genes involved in auxin and cytokinin signaling pathways were highly expressed in the pulvinus, including WALLS ARE THIN1 ( WAT1 ), a vacuolar auxin transport facilitator crucial for maintaining auxin homeostasis, as well as genes related to the IAA/AUX response to auxin and Adenine Phosphoribosyl Transferase ( APT ), along with three Isopentenyl Transferases ( IPTs ) associated with the cytokinin pathway. Partial reports of these findings have been documented in previous studies (Cancé et al., 2022 ; Zhang et al., 2013 ; Spitzer-Rimon et al., 2012 ). Materials and Methods Materials and sampling design Rain tree seedlings were procured from Yazhou Bay, Hainan, and cultivated in pots until they reached a height of 2 meters during their vigorous growth phase. Young leaves and fresh root nodules were immediately frozen in liquid nitrogen for subsequent DNA extraction and genome sequencing of both the rain tree and its symbiotic rhizobium. A sampling strategy was devised in a natural environment to assess nyctinastic movement and symbiotic nitrogen fixation in the rain tree using RNA sequencing (RNA-seq). The control point was established at 5:30 a.m., with pulvinus and leaflet samples collected every two hours until 11:30 a.m., resulting in a total of four sampling intervals. To distinguish between the effects of the circadian clock and light, additional samples were collected following a 2-hour dark treatment at 9:30 a.m. Root nodules were collected at 7:30 a.m. All samples, with three biological replicates, were rapidly frozen in liquid nitrogen and stored at − 80°C until required for RNA-seq analysis. Estimation of genome size by k-mer analysis The k-mer analysis was performed as part of a comprehensive genome survey. Initially, raw sequencing data underwent a stringent filtering process to obtain high-quality reads, which involved the removal of low-quality reads, short reads, adapter sequences, and polyG tails. Following this, Jellyfish version 2.2.10 (Marçais et al., 2011) was employed to conduct a frequency distribution analysis with k-mer size set to 21. Subsequently, GenomeScope version 2.0 (Vurture et al., 2017 ) was used to estimate the genome size, heterozygosity, and duplication rate. Genome sequencing, assembly and quality assessment For the assembly of the genome using PacBio HiFi data, Hifiasm version 0.16.1 was utilized (Feng et al., 2022 ). Subsequently, the Hi-C data were aligned to the final assembled contigs using Juicer version 1.6 (Durand et al., 2016 ) to generate the interaction matrix. Both the HiFi and Hi-C sequencing methodologies produced 50 Gb of data, corresponding to approximately 50X coverage. The contigs were subsequently ordered and anchored employing 3D de novo assembly version 180114 (Dudchenko et al., 2017 ). Manual inspection of the Hi-C contact map of the final assembly was conducted using Juicebox version 1.11.08 (Robinson et al., 2018 ). The completeness of the rain tree genome was evaluated using BUSCO with the embryophyte_odb10 database of single-copy genes (Simão et al., 2015 ). Telomere and centromere detection For chromosomes devoid of telomeric regions, telomere-associated reads were identified from HiFi sequencing data, and the de novo assembled telomeric repeats were incorporated into the chromosomal termini utilizing TGS-GapCloser (Xu et al., 2020 ). Employing quarTeT (Lin et al., 2023 ), we identified telomeric repeat sequences (CCCTAAA) in 23 out of the 26 anticipated chromosomal termini and detected potential centromeric repeat sequences. The approximate boundary of the centromeric region was inferred from the frequency distribution of all candidate repeat sequences. Gene prediction and annotation RepeatModeler v2.0.3 (Flynn et al., 2020 ) was utilized to cluster repeats through the construction of a de novo repeat library. Subsequently, RepeatMasker v4.1.2 (Tarailo-Graovac et al., 2009) was employed to identify repetitive sequences. HISAT2 v2.1.0 (Kim et al., 2015 ) was then used to align all transcriptome data to the genome. Following this, BRAKER v3.0.3 (Gabriel et al., 2024 ) was applied to automatically train species-specific parameter models and annotate gene structures, integrating both transcriptome alignment results and protein evidence. For functional annotation, the predicted protein-coding genes were queried against the eggNOG database (Hernández-Plaza et al., 2023 ). Phylogenetic and phylogenomic analysis The alignment of individual gene sequences was performed utilizing MUSCLE (Edgar et al., 2004), as implemented in MEGA12 (Kumar et al., 2024 ). This analysis included homologous sequences from related Bradyrhizobium species and Rhodopseudomonas palustris, which were retrieved from the NCBI database. Phylogenetic trees were reconstructed employing the Neighbor-Joining (NJ) method (Saitou and Nei, 1987 ) under the Maximum Composite Likelihood model specifically for 16S rRNA gene sequences. The robustness of the tree branches was evaluated through 1000 bootstrap replicates. For whole-genome comparisons, the Average Nucleotide Identity (ANI) was calculated using FastANI (Jain et al., 2018 ). Additionally, digital DNA-DNA hybridization (dDDH) values were estimated using the formula 2 model available on the Genome-to-Genome Distance Calculator (GGDC) web server ( https://ggdc.dsmz.de/ggdc.php ), as described by Meier-Kolthoff et al. ( 2013 ). Metagenome assembly and annotation of Rhizobium DNA was extracted using the CTAB method (Porebski et al., 1997 ) and subsequently sequenced on the NovaSeq 6000 platform, employing the paired-end sequencing protocol (PE150), which produced sequence reads of 150 base pairs. The sequencing data were aligned to the rain tree genome using Bowtie2 version 2.5.4 (Langmead et al., 2012). The host genome was then removed using Samtools version 1.21 (Danecek et al., 2021 ) to isolate the metagenomic data for further assembly. The metagenomic data were assembled using MEGAHIT version 1.2.9 (Li et al., 2016 ), resulting in the metagenomic assembly. The contigs derived from this assembly were assigned to distinct genomes using MetaBAT2 version 2.17 (Kang et al., 2019 ), facilitating the identification of individual genomes within the metagenome, referred to as metagenome-assembled genomes (MAGs). The quality of the MAGs was evaluated using CheckM (Parks et al., 2015 ), with high-quality genomes selected based on predefined completeness and contamination thresholds. Subsequently, dRep (Olm et al., 2017 ) was utilized to eliminate redundancies and select representative species genome clusters.Taxonomic annotation was conducted utilizing GTDB-Tk version 2.4.0 (Chaumeil et al., 2022 ) in conjunction with the GTDB_r2 14 reference library. Annotation of the individual bacterial genome was carried out using Prokka version 1.14.6 (Seemann, 2014 ). Additionally, the protein sequences of the bacterial genome were annotated employing the eggNOG database. Transcriptome analysis The high-quality clean RNA reads were aligned to the rain tree T2T reference genome using HISAT2 version 2.1.0 with default parameters. Read counts mapped to each gene were quantified using FeatureCounts (Liao et al., 2014 ). Gene expression levels were subsequently estimated as transcripts per million (TPM). Differentially expressed genes (DEGs) across various tissues and developmental stages were identified using DESeq2 (Love et al., 2014 ). A threshold of |log2FoldChange| ≥ 2 and an adjusted p-value (padj) ≤ 0.05 was set as the criterion for identifying DEGs between samples. These DEGs were further utilized to construct gene regulatory networks. For the acetylene reduction assay, nodules at 14 days post-inoculation (dpi) were harvested and placed in 20-mL vials, followed by the injection of 2 mL of acetylene. The vials were then incubated for 1 hour at 25°C. The amount of ethylene produced was quantified using gas chromatography (GC9310-VI) through an acetylene reduction assay (ARA). Weighted gene co-expression network analysis Utilizing the "WGCNA" R package (Langfelder et al., 2008), we developed a gene co-expression network to investigate pulvinus movement. Subsequently, we assessed the correlation between various modules and the mechanism of pulvinus movement, identifying the most pertinent module as the central gene derived from the WGCNA analysis. This key module was then visualized using Cytoscape software (Shannon et al., 2003 ). Declarations Acknowledgements This work was supported by the National Natural Science Foundation of China (32172614) and supported by the Project of National Key Laboratory for Tropical Crop Breeding (NO. NKLTCB202337), Hainan Province Science and Technology Special Fund (ZDYF2023XDNY050), Hainan Provincial Natural Science Foundation of China (324RC452). Author contributions K. Yang designed the sampling strategy of RNA seq for nyctinastic movement, and proposal ideas of the paper. S Wang response for genome assembly and annotation. Z Li Participated in the analysis of repetitive sequences and the construction of co-expression networks of the transcriptome, constructed the website. G Guo participated the collection of the samples. H Yu separated and purified the rhizobium and identified its function of nitrogen fixation. K Yang and S Wang prepared the materials and analyzed the data and drew the figures. Y Xu provided good guidance for experimental design. K Yang wrote the paper. F Chen and Y Xu edited the paper. All authors read and approved the final manuscript. Data Availability The genome sequences described in this article have been deposized in The National Genomics Data Center (NGDC, https://ngdc. cncb.ac.cn) under accession number PRJCA042275 and PRJCA042279 (whole genome and assembly data). 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Supplementary Files DataSet1Assemblyandannotationofraintree.xlsx DataSet 1 DataSet2Predominantexpressionofgenesfornyctinasticmovement.xlsx DataSet 2 DataSet3GenomeassemblyandannotationofrhizobiumB.Saman.xlsx DataSet 3 DataSet4Genesexpresseddominantlyinrootnoduleofraintree.xlsx DataSet 4 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7165529","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":497761253,"identity":"caf1d6c9-32b3-41d1-b30b-c0b89f9f07f5","order_by":0,"name":"Kejing Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIie3RMUvEMBTA8YTAm8Ld+uTg+hXeUSgn4vlVWrKL4HKDaKTQ0a4KfoiCi2NCoV2EWwsudnJxOBcXb7AGRBxaOwrmvyW835AXxny+Pxgwe/m0JZxPv86f4RCZ8DJdXJ8swz0tRpK5qLOZ3K6Twojv20ECYDVKQn632VTI7ycBGWEfJVsd9xJpNSGhiBoFyB9gURhQB5Kp016CVsdECFEjOpIBL4yMZpKZRPeRoNUmJpRhXjpyVJjp2zDplqwNIRJTjnR7kPAbSbl7TaPCZZKBuikh3L8l1UsCXT+/73bnF3lu2+Y1qw6v6rRtXtarXvKzmFWMuf+hUfOus/GjPp/P92/6ANhBUx/oiNV/AAAAAElFTkSuQmCC","orcid":"","institution":"Nanjing Agriculture University","correspondingAuthor":true,"prefix":"","firstName":"Kejing","middleName":"","lastName":"Yang","suffix":""},{"id":497761254,"identity":"8f2493cd-e782-4a8e-a278-3f3dcd52ab2d","order_by":1,"name":"hao Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"hao","middleName":"","lastName":"Wang","suffix":""},{"id":497761255,"identity":"987ea526-6952-4f34-8428-e67dad7d33e6","order_by":2,"name":"GUO Guilian","email":"","orcid":"https://orcid.org/0009-0000-5356-2769","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"GUO","middleName":"","lastName":"Guilian","suffix":""},{"id":497761256,"identity":"e7c1cf27-b28d-4658-a3d8-f12c8b25ad92","order_by":3,"name":"Haixiang Yu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Haixiang","middleName":"","lastName":"Yu","suffix":""},{"id":497761257,"identity":"87ba9c7d-5d5f-4397-b96f-9345f9897a96","order_by":4,"name":"Zhidong Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhidong","middleName":"","lastName":"Li","suffix":""},{"id":497761258,"identity":"b0ec2d96-77c8-4cb3-adf8-7d2b3ebcccd4","order_by":5,"name":"Fei Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Chen","suffix":""},{"id":497761259,"identity":"67a7127a-f548-4ad5-afce-f4d0b1cac750","order_by":6,"name":"Yingchun Xu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yingchun","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2025-07-19 16:00:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7165529/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7165529/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88977360,"identity":"9bb749f8-7b87-4cc3-9636-2e87f83ab7a0","added_by":"auto","created_at":"2025-08-13 10:43:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6622130,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe T2T genome assembly and evolutionary tree of raintree. \u003c/strong\u003eA: Circos plot showing the genome details. (a) 13 chromosomes of \u003cem\u003eSamanea saman; \u003c/em\u003e(b) gene density; (c) repeat sequence content; (d) GC content density; (e) density of Copia LTR-RTs; (f) density of Gypsy LTR-RTs; (g) syntenic blocks (all window sizes = 50 kb). B: Telomere and centromere detection map. Red square and purple half-circles represent centromere and telomeres within the assembled genome; C: \u0026nbsp;Evolutionary tree between raintree and its legume relative species. Specially, the rain tree is estimated to have differentiated from the \u003cem\u003eChamaecrista fasciculata \u003c/em\u003earound 46 MYAs. The dots represent the number of gene expansions and contractions between adjacent species; The abscissa represents the age of species differentiation (MYA -million years).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7165529/v1/0b222f1e6a2a3a2b3a84617f.jpg"},{"id":88977362,"identity":"0b3b5eb3-218a-4b73-8a6e-cbebcf63689b","added_by":"auto","created_at":"2025-08-13 10:43:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5515813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA-Seq revealed genes involved in nyctinastic movement of the pulvinus cells in raintree.\u003c/strong\u003e A. The opening and closing phenotypes of leaves in day and night. B. The number of differentially expressed genes in the pulvinus at different time points of light exposure. T0: AM5:30 dark check; T1: AM7:30 light; T2: AM9:30 light; T3: AM11:30 light. C. The number of differentially expressed genes in the leaflets at different time points of light exposure. D. The expression heat map of genes significantly upregulated only in the pulvinus cells that directly response to nyctinastic movement. E. The heatmap of down-regulated genes only in pulvinus those are negative regulatory factors in nyctinastic movement. F. The expression trend analysis yielded 32 different expression modules. G. The correlation analysis revealed that only the blue module had the highest correlation with the nyctinastic movement. H. Co-expression network in the blue module.\u003c/p\u003e\n\u003cp\u003eresulting 41 genes.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7165529/v1/5394cf973effc575ec7a1a64.jpg"},{"id":88977361,"identity":"5ec7373c-9197-4d6e-9840-23053647d49f","added_by":"auto","created_at":"2025-08-13 10:43:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4424455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome of \u003c/strong\u003e\u003cem\u003eBradyrhizobium saman\u003c/em\u003e and genes for symbiotic nitrogen fixation in raintree A. genome assembly of 50 contigs with 7.28Mb in length. B. the evolutionary tree of raintree- associated rhizobia using 16S RNA. C. Average Nucleotide Identity (ANI) among 6 relatives of rhizobium from raintree. D. Determination of nitrogenase activity of RN vs R of raintree by acetylene reduction analysis (ARA).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7165529/v1/24b9e9692072fab8c316d1eb.jpg"},{"id":88978525,"identity":"acadc5e4-4aea-4bd1-88fb-54678792c15a","added_by":"auto","created_at":"2025-08-13 10:59:25","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7948735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression profiling of genes involved in symbiotic nitrogen fixation in the root nodules of raintree \u003c/strong\u003eA. Phenotypes of root and root nodules in vigor growth plants of raintree. B. Differentially expressed genes between root nodules to roots and between root nodules to leaflets of raintrees. C. GO enrichment of the genes specific expressed in root nodule. D. The extremely high expression of \u003cem\u003eleghemoglobins\u003c/em\u003e. E. Extremely expression of the 6 members in Hemoglobin family. F. Predominant expression of genes in amino transport and metabolism. G. Prefer expression of genes in auxin signaling pathways. H. Specific expressed genes in cytokinin pathway. I. The advantageous expression of the \u003cem\u003eMYB\u003c/em\u003e transcription factors.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7165529/v1/6984c9fd1865264f99e70492.jpg"},{"id":88978526,"identity":"39b1a582-4f1c-43d6-9e8a-314066de324b","added_by":"auto","created_at":"2025-08-13 10:59:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8601343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the Genome Database for Raintree A. \u003c/strong\u003eWeb interface designed. B. KEGG enrichment tool. C. GO enrichment tool. D. tool for sequence alignments. E.\u003c/p\u003e\n\u003cp\u003eBroser of transcription factor family. F. Broser of genes. G. Tool for sequence extraction H. Window of primer design.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7165529/v1/f12fa11d93bd7034de759a67.jpg"},{"id":88979551,"identity":"f3a7ddc4-0731-4494-9871-2ff3a8f85ac4","added_by":"auto","created_at":"2025-08-13 11:15:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":34369049,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7165529/v1/9d1e671f-1c80-406e-9899-3d47c1ad2047.pdf"},{"id":88977363,"identity":"1422c9f0-1ebf-4f88-b262-338a8d00d589","added_by":"auto","created_at":"2025-08-13 10:43:25","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5418644,"visible":true,"origin":"","legend":"DataSet 1","description":"","filename":"DataSet1Assemblyandannotationofraintree.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7165529/v1/3336a17b2083c311869e1b7e.xlsx"},{"id":88977373,"identity":"ba996694-5fca-4684-9571-dbf6afde0c50","added_by":"auto","created_at":"2025-08-13 10:43:26","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12700040,"visible":true,"origin":"","legend":"\u003cp\u003eDataSet 2\u003c/p\u003e","description":"","filename":"DataSet2Predominantexpressionofgenesfornyctinasticmovement.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7165529/v1/255956faba581b830a1a2f07.xlsx"},{"id":88977810,"identity":"436edeb9-605a-4023-8f54-ebe74f391e15","added_by":"auto","created_at":"2025-08-13 10:51:25","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1154602,"visible":true,"origin":"","legend":"DataSet 3","description":"","filename":"DataSet3GenomeassemblyandannotationofrhizobiumB.Saman.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7165529/v1/34d91cdb6d0dc4f14691ed08.xlsx"},{"id":88977370,"identity":"7b0a0081-9b66-43c7-82f6-bc194dd88d59","added_by":"auto","created_at":"2025-08-13 10:43:25","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":6622484,"visible":true,"origin":"","legend":"DataSet 4","description":"","filename":"DataSet4Genesexpresseddominantlyinrootnoduleofraintree.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7165529/v1/5223571f32d88a40160d2280.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A telomere-to-telomere genome of raintree (Samanea Saman) reveals its ecological adaptation characteristics of nyctinastic movement and symbiotic nitrogen fixation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe raintree (\u003cem\u003eSamanea saman\u003c/em\u003e Merr.), in the Fabaceae family, originates from the tropical arid regions of South and Central America, notably in countries such as Belize, Ecuador, and Colombia (Merrill ED, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1916\u003c/span\u003e). It has since become prevalent in tropical semi-arid regions worldwide. The species flourishes under conditions of light, heat, and humidity, exhibiting tolerance to drought and poor soil quality. It is predominantly utilized for landscaping and garden enhancement. The tree's sweet, succulent fruit and protein-rich foliage serve as suitable feed for livestock, while its durable heartwood is employed in the production of furniture and crafts. The Fabaceae family encompasses 796 confirmed genera, including the genus Samanea, which comprises 20 species. \u003cem\u003eSamanea\u003c/em\u003e is closely related to genera such as \u003cem\u003eAlbizia\u003c/em\u003e and \u003cem\u003eCalliandra\u003c/em\u003e, as well as \u003cem\u003eCylindrokelupha\u003c/em\u003e, \u003cem\u003eEnterolobium\u003c/em\u003e, and \u003cem\u003ePithecellobium\u003c/em\u003e. Cytological studies reveal that the raintree is diploid (2n\u0026thinsp;=\u0026thinsp;26), although comprehensive genome sequencing has yet to be conducted. Nonetheless, some gene and transcriptome data are available in GenBank.\u003c/p\u003e\u003cp\u003eRain trees adapt to the arid environment of the native atmosphere and form the morphological characteristics of the pinnate compound leaves that unfold during the day and close at night. Closing and drooping leaves at night reduces the leaf surface area exposed to the air, reduces transpiration water loss, and maintains plant water balance. This phenomenon is called nyctinastic movement, which is only found in the genus Phaseolus of the Fabaceae family \u003cem\u003ePhaseolus, Robinia, Albizzia, Mimosa\u003c/em\u003e, and \u003cem\u003eSamanea\u003c/em\u003e (Moran 2015). The nyctinastic movement of the pinnate leaves of raintree is achieved through the movement of pulvinus cells located at the base of the tertiary compound leaves. The pulvinus of raintree is composed of two parts with opposite positions and functions: one is the extensor, which extends longitudinally when the leaves are unfolded, and the other is the flexor, which contracts longitudinally simultaneously (Oikawa et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Primary signal of the nyctinastic movement is the differential response of the phytochrome receptor to blue and red light in raintree (Kim et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Mano, et al., 2021). Blue light promotes the opening of leaflets by increasing the activity of K\u003csup\u003e+\u003c/sup\u003e influx channels in extensor cells and K\u003csup\u003e+\u003c/sup\u003e efflux channels in flexor cells. By inhibiting the H\u003csup\u003e+\u003c/sup\u003e pump, blue light leads to depolarization of the flexor cell membrane, which may be a mechanism of blue light regulation in leaf movement (Suh et al., 2000). In darkness, red and far-red light induce the opening of K\u003csup\u003e+\u003c/sup\u003e influx channels in the flexor cells of rain tree, leading to the closing movement of the leaves. The reversible osmotic swelling of extensor cells accompanied by the contraction of flexor cells results in the opening of the pulvinus, which in turn lifts the leaves and leaflets. When potassium ions redistribute, the extensor cells contract and the flexor cells swell, closing the pulvinus. The water permeability of the membranes in both motor tissues is strictly regulated in time (Mano, et al., 2006; Zeng et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The reversible osmotic swelling of extensor cells accompanied by the contraction of flexor cells causes the opening of the pulvinus, which in turn raises the leaves and leaflets. When potassium ions redistribute, the extensor cells contract and the flexor cells expand, closing the pulvinus (Zeng et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The water permeability of the membrane in the two motor tissues is strictly regulated by time. Further research found that several potassium ion channel protein genes in the extensor and flexor tissues of the pulvinus of rain tree leaves, such as SPICK3, SPICK4, SPORK2, SPORK3, and SPORK4, are involved in the nyctinastic movement of pulvinus cells. Among them, only SPORK2 has been confirmed as a functional K\u0026thinsp;+\u0026thinsp;channel (Oikawa et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ueda, et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The aquaporin gene localized in the plasma membrane has been proven to be involved in the movement of flexor and extensor cells in pulvinus. There are two types, namely SsAQP1 and SsAQP2, which belong to the two subfamilies of PIP1 and PIP2, respectively (Moshelion et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Uehlein et al., 2008).\u003c/p\u003e\u003cp\u003eThe rain tree's root system shows dense nodulation, suggesting it uses symbiotic nitrogen fixation for ecological adaptation (Qadri et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Rhizobia, Gram-negative soil bacteria, form nodules with leguminous plants, converting atmospheric nitrogen into plant nutrition. This legume-rhizobium symbiosis fixes about 55\u0026nbsp;million tons of nitrogen annually, representing 55% of terrestrial biological nitrogen fixation and is crucial to the nitrogen cycle (Kistner and Parniske 2002; Markmann and Parniske 2009).\u003c/p\u003e\u003cp\u003eAbout 65\u0026nbsp;million years ago, plants in the Caesalpinioideae and Papilionoideae subfamilies of the Fabaceae family developed symbiotic nitrogen fixation with rhizobia (Sprent and James 2007; Zhao et al. 2021). Rhizobia, part of the Alphaproteobacteria and Betaproteobacteria classes in the Proteobacteria phylum, include 19 genera such as Rhizobium and Cupriavidus. They have large genomes (5\u0026ndash;10 Mb) to adapt to the rhizosphere and nitrogen fixation (Remigi et al., 2016; Zhang C, 2023). Functional analysis indicates that the rhizobia genome tends to encode a set of genes responsible for transport, regulation, and stress resistance. However, the genome size and structure of the symbiotic nitrogen-fixing bacteria associated with the rain tree have not been reported yet.\u003c/p\u003e\u003cp\u003eThe symbiotic nitrogen fixation in the legume-rhizobia system is a complex biological process that is tightly regulated by both the host plant and the rhizobia (Oldroyd, 2013; Yu H et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Upon sensing flavonoid compounds secreted by the host plant, the rhizobia are specifically recognized by nodulation factor receptors on the host plant's cell membrane surface. This triggers the expansion and curling of the root hair tips, enclosing the rhizobia to form an infection chamber. Subsequently, the root hair cell wall and membrane invaginate to grow into an infection thread, guided by a pre-infection thread (PIT), and enter the plant's root cortical cells (Bladergroen and Paink 1998).\u003c/p\u003e\u003cp\u003eThe formation of a microaerobic environment in nodules is a prerequisite for nitrogen fixation. The molybdenum-iron nitrogenase requires 16 ATP and 8 electrons to reduce each molecule of N2, producing 2 molecules of NH3 and 1 molecule of H2. Exposure to oxygen leads to irreversible loss of enzyme activity (Rutledge et al. 2022). Legume nodule cells specifically express large amounts of leghemoglobin to maintain the concentration of free oxygen at nanomolar levels per liter (Dixon and Kahn 2004; Ott et al. 2005; Wang et al. 2019a; Cui et al. 2021; Jiang et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Meanwhile, rhizobia express the CBB3 terminal oxidase with higher oxygen affinity in nodules to perform oxidative phosphorylation, generating ATP for maintaining nitrogen fixation reactions and basic life activities (Preisig et al. 1993, 1996; Zufferey et al. 1996). Plant hormones such as auxin, cytokinin (CTK), gibberellins (GAs), and ethylene (ETH) are involved in the rhizobia infection process, nodule formation, and development, playing crucial roles in regulating the interaction between legumes and rhizobia and the formation of nodule organs. Adequate carbon sources and energy supply are necessary for efficient nitrogen fixation by bacteroids in nodules. In nodules, the host provides malate as the primary energy and carbon source to sustain the nitrogen fixation reactions of rhizobia, which are absorbed by bacteroids through the four-carbon dicarboxylate transporter Dct system (Mitsch et al. 2018).\u003c/p\u003e\u003cp\u003eThe process of plant symbiotic nitrogen fixation is co-regulated by both rhizobia and host genes. Specific rhizobia can only form symbiotic nitrogen-fixing nodules with specific groups of legume plants. In peas, host-specificity genes are determined by the carried sym2 locus, whose gene products interact with NodX encoded by specific \u003cem\u003eRhizobium leguminosarum\u003c/em\u003e sv. viciae rhizobia to achieve successful nodulation and symbiotic nitrogen fixation (Geurts et al., 1997; Limpens et al., 2003). The nodulation (nod) and nitrogen fixation (nif) genes mediating rhizobial symbiotic nitrogen fixation are typically located on transferable elements, including plasmids with transfer capabilities (known as symbiotic plasmids, pSym) and genomic islands (known as symbiotic islands, SI). Nodulation genes include nodA, nodB, nodC, nodI, and nodJ, while nitrogen fixation genes include nifH, nifD, nifK, etc. Host nitrogen fixation gene types include Lysin motif receptor-like kinase (\u003cem\u003eLysM-RLK\u003c/em\u003e), encoding proteins such as \u003cem\u003eMtAAP7\u003c/em\u003e, Ammonium transporter 1 (\u003cem\u003eMtAMT1\u003c/em\u003e), and Aromatic and neutral transporter 50 (\u003cem\u003eMtANT50\u003c/em\u003e).\u003c/p\u003e\u003cp\u003eAlthough the core genes and reactions of nitrogen fixation are highly conserved among rhizobia, different legume plants provide rhizobia with distinct working micro environments resulting in species-host-dependent diversity in the resource supply mechanisms required to maintain bacteroid nitrogen fixation efficiency.\u003c/p\u003e\u003cp\u003eIn summary, the genome of the rain tree and its symbiotic nitrogen-fixing rhizobia is largely unexplored. While there are some studies on the circadian movement of legumes and the rain tree, examining the gene family structure related to this process at the genomic level is a new approach that could support existing models (Bai Q et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although there has been progress in understanding plant symbiotic nitrogen fixation, research on the rain tree's symbiotic nitrogen fixation is minimal. Understanding this mechanism in tropical fast-growing trees and comparing it with legumes could reveal how legumes manage stress resistance and nitrogen fixation.\u003c/p\u003e\u003cp\u003eThis study reported the first near T2T genome of the raintree and genome of rhizobium symbiotic in raintree. And further annotated the gene resources of its symbiotic nitrogen-fixing and nyctinastic movement, the key double characteristics. Based on it, we clarified the phylogenetic tree of the specific evolutionary relationships with its closely related genera. Integrated the transcriptomic analysis, we deciphered the molecular mechanism of nyctinastic movement response to circadian and light signal, also the symbiotic nitrogen-fixing. These data and findings will establish the cornerstone for genetic research and breeding of raintree, and service for better understanding the ecological adaptability and economic utilization of the rain tree.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eGenome assembly and annotation of raintree\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUsing a flow cytometry estimate the genome size of 530 Mb for the raintree (\u003cem\u003eSamanea saman\u003c/em\u003e) (2n\u0026thinsp;=\u0026thinsp;26). A total of 68 Gb of Hifi long reads sequences data was generated, resulting in a genome coverage of 130X. To achieve a high-quality, chromosome-level reference genome, we leveraged 53 Gb of Hi-C paired-end sequencing data to scaffold the contigs. Finally, the genome assembly resulted in 13 contigs equal to the chromosome numbers with a total length of 539.53Mb, with a contig N50 size of 42.86 Mb. There are 25,997 gene models have been annotated (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eDataSet 1\u003c/b\u003e). The assembly quality was evaluated using BUSCO revealing a completeness of 98.8% in genome and gene regions. The basic structural features (gene density, repeat sequence density, and GC density) of the T2T genome are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. We used the seven-base telomere sequence (CCCTAAA at the5\u0026rsquo; end or TTTAGGG at the 3\u0026rsquo; end) as a query sequence to scan the genome. We identified almost all 26 telomeres in 13 chromosomes except of Chr4. With Tandem Repeats Finder (Benson, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) software, we detected 13 putative centromeres of 13 chromosomes, with size ranging from 102kb-2.5Mb of the T2T genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, DataSet 1). In summary, we released the T2T genome for Raintree in the first time, enabling a foundation for evolutionary studies and gene mining.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGenome assembly ann annotation of raintree (\u003cem\u003eSamanea saman\u003c/em\u003e)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGenome\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eSamanea saman\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePloidy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2n\u0026thinsp;=\u0026thinsp;26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEstimated genome size (Mb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e530\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAssembled genome size (Mb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e539.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGenomic heterozygosity (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.253\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eContig N50 (Mb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e42.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of contigs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of chromosomes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRepeat sequence content (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGC content (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e32.85\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of gene models\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25,997\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGenome BUSCO (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e98.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene BUSCO (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e98.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eUtilizing the protein sequences of single-copy genes from the genomes of the rain tree and its related legume species, a phylogenetic tree was constructed to elucidate species evolution. The analysis demonstrated that the rain tree shares the closest genetic relationship with the Partridge Pea (\u003cem\u003eChamaecrista fasciculata\u003c/em\u003e), with their divergence occurring approximately 46\u0026nbsp;million years ago (MYA). In contrast, the divergence between the cultivated soybean (\u003cem\u003eGlycine max\u003c/em\u003e) and its wild relative (\u003cem\u003eGlycine soja\u003c/em\u003e) occurred around 6 MYA. Furthermore, the divergence of the common bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e) and another cultivated species, the runner bean (\u003cem\u003ePhaseolus coccineus\u003c/em\u003e), as well as lentils (\u003cem\u003eLens culinaris\u003c/em\u003e) and their wild relative species (\u003cem\u003eLens ervoides\u003c/em\u003e), is estimated to be much later occurred approximately 2.5 MYA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These findings suggest that the raintree is a tropical rainforest plant that has not undergone extensive cultivation and domestication. However, its distinctive nyctinastic movement, adaptation to atmospheric drought, and ability to fix nitrogen symbiotically through nodules confer ecological advantages, contributing to its widespread distribution.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe expression profiling of the specific genes involved in nyctinastic movement and the co-expression network\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe nyctinastic movement of the raintree is unique phenotype adapted to atmospheric drought. It achieves the closure of lobules at night and the re-opening during the day through the asymmetric movement of extensor cells and flexor motor cells in the pulvinus. This function may be jointly regulated by light and the circadian clock (Kim et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Minorsky, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zeng F, 2024). Although it has been initially indicated that the movement of motor cells in pulvinus is driven by chloride channels, potassium channels and aquaporins (Moran et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Oikawa et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ueda et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the variations of related genes systematically analyzed at the genomic and transcriptome levels have not been reported yet.\u003c/p\u003e\u003cp\u003eWe designed a transcriptome sampling strategy for the circadian clock and light response of the rain tree: Taking the dark time of 5:30 (T0) in the morning as the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA right), samples of pulvinus and leaflets were taken at intervals of 2hs respectively and treated rapidly with liquid nitrogen with total of 4 time points (T0, T1, T2 and T3); Meanwhile, we further collected the samples after 2hs dark treatment at 9:30 a.m (T4) to distinguish the response to the light signal. Each sample was biologically repeated three times for transcriptome sequencing. Referring to the raintree T2T genome, we obtained high-quality transcriptome annotated genes and their expression information.\u003c/p\u003e\u003cp\u003eFor differential expression analysis, transcripts exhibiting expression differences exceeding fourfold were extracted. In the pulvinus, the number of differentially expressed genes at various time points following light exposure was 444, 1,234, and 1,136, respectively, with a total of 146 genes consistently differentially expressed across all three time points. Specifically, at the T1 time point, 322 genes were up-regulated, while 122 were down-regulated. At T2, 867 genes were up-regulated, and 387 were down-regulated. At T3, 530 genes showed increased expression, whereas 606 exhibited decreased expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These differentially expressed genes are predominantly associated with metabolic pathways involved in processes such as nyctinastic movement and photosynthesis.\u003c/p\u003e\u003cp\u003eIn the leaf blade, there were 1,893, 2,089, and 2,149 differentially expressed genes at three distinct time points following morning light exposure, in comparison to the early morning darkness. A total of 481 differentially expressed genes were common across these time points. Among these, the numbers of up-regulated versus down-regulated genes were 825 vs 1,068, 1,547 vs 542, and 1,200 vs 949, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These differentially expressed genes are predominantly associated with photosynthesis and the growth and development processes stimulated by enhanced light signals.\u003c/p\u003e\u003cp\u003eHow to screen out the differentially expressed genes in the pulvinus cells that are prority to nyctinastic movement? We focused on four kinds of genes that met the following conditions: 1) Genes that were upregulated above four folds in the pulvinus under light treatment, but not upregulated in the leaf blades. The statistical results showed a total of 190. This set excludes genes directly related to photosynthesis and covers positive regulation genes response to circadian clock and light signal in nyctinastic movement (\u003cb\u003eDataSet 2\u003c/b\u003e). 2) In the set, we selected that down-regulated under dark treatment (T4)\u003c/p\u003e\u003cp\u003eMajority of them encode splicing factor 3B subunit (\u003cem\u003eSF3B\u003c/em\u003e), Zinc finger A20 and AN1 domain-containing stress-associated protein (\u003cem\u003eSAP\u003c/em\u003e), EIN3-binding F-box protein (\u003cem\u003eEBF\u003c/em\u003e), RING-H2 finger protein (\u003cem\u003eRING-H2\u003c/em\u003e) and protein kinase superfamily (\u003cem\u003ePKs\u003c/em\u003e). They are basic regulation factors, directly responded to light signals in motor cells. There are genes for ion and water transport, like sodium ion transport (\u003cem\u003eCitMHS\u003c/em\u003e), aquaporins as plasm membrane intrinstic proteins (\u003cem\u003ePIP2\u003c/em\u003e and \u003cem\u003ePIP3\u003c/em\u003e), S-type anion channel (\u003cem\u003eSLAC1\u003c/em\u003e) and exocyst complex component (\u003cem\u003eEXOC\u003c/em\u003e). However, although several genes for potassium channel proteins (\u003cem\u003eAKT1, KAT1 and SKOR3\u003c/em\u003e) were upregulated, their expression levels were relatively low (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). 3) Genes were decreased over four- fold (T1) but increased under dark treatment (T4) in the pulvinus, and not down-regulated in the leaflets under light. It enabling us found 19 genes, they are transcriptional regulatory factors, coordinators of cellular basic processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These genes are negative regulatory genes of nyctinastic movement that only respond to light signals.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe genome of the\u003c/b\u003e \u003cb\u003eBradyrhizobium Saman\u003c/b\u003e \u003cb\u003eunlocks its function in symbiotic nitrogen fixation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe total DNA of the root nodule samples of raintree was collected and sequenced using the second-generation sequencing technology. The bacterial metagenomic sequences were extracted using software, totaling approximately x Mb. After quality control, the metagenomic data were assembled using Megahit, resulting in 4813 contigs (total length approximately 16.94 Mb, N50\u0026thinsp;=\u0026thinsp;32.61 kb, GC content 63.33%). Five MAGs were obtained through MetaBAT2 binning. The CheckM assessment showed that only bin.2 met the quality requirements for single-bacterial analysis (completeness 97.51%, contamination 0.17%, strain consistency 0), while the remaining bins were excluded due to low completeness. This genome was further assembly and annotated with genome size 7.28 Mb, composed of 50 contigs with N50 of 309.5 kb and a GC content of 64.63% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The genome annotation identified 6882 genes, including 51 transfer RNAs (tRNAs), three ribosomal RNAs (rRNAs) and one transfer-messenger RNA (tmRNA).\u003c/p\u003e\u003cp\u003eThe classification of raintree rhizobia was assessed utilizing both Average Nucleotide Identity (ANI) and DNA-DNA Hybridization (DDH) methods. Comprehensive genomic sequences from six rhizobial bacteria were obtained and used for comparsion with the bin.2 genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Sequence alignment analysis revealed that the ANI value of the raintree-associated rhizobium and the rhizobium isolated from \u003cem\u003eCajanus cajan\u003c/em\u003e originated in Africa, \u003cem\u003eBradyrhizobium cajani\u003c/em\u003e, was 95.33% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), suggesting a close genetic relationship at the species level. However, employing Model 2 of the Genome-to-Genome Distance Calculator (GGDC) network service (accessible at ggdc.dsmz.de/ggdc.php#), the calculated digital DDH (dDDH) value between these two bacterial strains was 63.1%, which falls below the 70% threshold typically required to classify them as the same species (\u003cb\u003eDataset 3\u003c/b\u003e). Consequently, this rhizobium is named as \u003cem\u003eBradyrhizobium saman\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene resources involved in symbiotic nitrogen fixation in raintree\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDuring the vigorous growth phase of the raintree (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), we analyzed the transcriptomes of root nodules (RN), adventitious roots (R), and leaflets (L). Comparative analysis of transcript expression identified 4,805 differentially expressed genes (DEGs) between RN and R, and 7,841 DEGs between RN and L. Notably, 2,273 DEGs were common to both comparisons. In comparison to the R group, the NR group exhibited upregulation of 1,608 genes and downregulation of 3,197 genes. When comparing NR with L, the NR group demonstrated an upregulation of 4,588 genes and a downregulation of 3,253 genes relative to the L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). From the 1,608 genes upregulated in NR compared to R, we excluded 472 genes that were four folds upregulated also in the L, resulting in a subset of 669 genes (DataSet 2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis subset is considered to represent the primary gene types involved in symbiotic nitrogen fixation in raintrees. Six members in the hemoglobin gene family were extremely high expressed, specifically identified as SsaHB2A, SsaHB2B, SsaHB2C, SsaHB2D, SsaHb1 and SsaGLB3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Their coding proteins contribute to maintaining a low-oxygen environment within the symbiosome cells of the raintree's root nodules (Ma et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Gene Ontology (GO) enrichment analysis revealed significant enrichment in 20 pathways, including those related to amino acid transport and metabolic processes, cytokinin metabolism, auxin signaling pathways, and transcription factors, encompassing a total of 227 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Within the nitrogen metabolism-related pathways, 26 key components include ammonium ion and amino acid transport (involving ten genes), nitrate transporters (double NRTs), and genes encoding asparagine synthetase (\u003cem\u003eAS\u003c/em\u003e) and glutamate synthase (\u003cem\u003eGS\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These genes, derived from the host genome, are essential for the symbiotic nitrogen fixation process in the raintree.\u003c/p\u003e\u003cp\u003eSixteen genes exhibited significant upregulation within the auxin signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Notably, ten of these genes are identified as WALLS ARE THIN1 (\u003cem\u003eWAT1\u003c/em\u003e), a vacuolar auxin transport facilitator essential for maintaining auxin homeostasis (Ranocha et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Additionally, three genes belong to the \u003cem\u003eAux/IAA\u003c/em\u003e family, while the remaining three are related protein genes. Aux/IAA proteins play a crucial role in modulating the transcriptional response to auxin (Canc\u0026eacute; et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the cytokinin pathway, nine genes demonstrated significant upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). These include Adenine Phosphoribosyl Transferase (\u003cem\u003eAPT\u003c/em\u003e), three Isopentenyl Transferases (\u003cem\u003eIPTs\u003c/em\u003e), three Cytokinin Dehydrogenases (\u003cem\u003eCKXs\u003c/em\u003e), and Cytokinin Hydroxylase (\u003cem\u003eCYP735\u003c/em\u003e), among others. IPT functions as a rate-limiting enzyme in cytokinin biosynthesis, whereas adenine phosphoribosyl transferase is pivotal in catalyzing the conversion of cytokinin from nucleobases to nucleotides (Zhang et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Cytokinin serves as a crucial hormonal signal in symbiotic nitrogen fixation, contributing to the formation of symbiosomes and influencing cellular metabolism.\u003c/p\u003e\u003cp\u003eFurthermore, it was found that 17 transcription factors were significantly upregulated in the root nodules of raintree (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Notably, the majority these transcription factors belong to the MYB family, with 14 instances identified, alongside 2 R2R3-MYB-Like Regulatory Factors, specifically EOBI, which are involved in regulating substrate availability for volatile biosynthesis (Spitzer-Rimon et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This indicates that the MYB transcription factors play a particularly vital role in the symbiotic nitrogen fixation process within the rhizobium-nodule system of the raintree.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSamanea saman\u003c/b\u003e \u003cb\u003eDatabase: An Open-Access Platform for the Genomics of Raintree\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCurrently, the absence of a centralized web server for sharing rain tree genome information impedes progress in rain tree breeding and molecular breeding research. To address this issue, we established a \u003cem\u003eSamanea saman\u003c/em\u003e genome database (SSDB) available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.hainanu.edu.cn/SSDB/\u003c/span\u003e\u003cspan address=\"https://bioinformatics.hainanu.edu.cn/SSDB/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. This repository offers functionalities for data browsing, searching, analysis, and downloading, serving as a comprehensive resource for rain tree genome information. SSDB provides a user-friendly interface to facilitate the retrieval of information (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The SSDB structure consists of six main modules: Home, Tools, Download, Search, Breeding, and About. To enhance the utility of rain tree genomic resources, we have developed several data retrieval and analysis interfaces: KEGG enrichment tool (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB); GO enrichment tool (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC); Basic Local Alignment Search Tool (BLAST+) tool for sequence alignments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD); Furthermore, SSDB offers search functionalities for 63 transcription factor families (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), alongside a gene search tool (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) and a sequence extraction tool (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). The database also incorporates a primer design tool (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH) and an SSR identification tool. SSDB facilitates data download, granting access to publicly available sequences, such as the rain tree genome, transcriptomes, coding sequences, and other related sequences.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eThe genome of raintree, symbiotic rhizobium and their database\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe raintree represents a predominant vegetation type within the tropical seasonal rainforests of Central and South America and is extensively cultivated across global tropical regions for use as street trees and in landscaping (Durr, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Despite its ecological significance, research concerning the genetics and species diversity of raintrees remains scarce. In this study, we present the first telomere-to-telomere (T2T) genomic sequence of the raintree and provide a comprehensive annotation. Additionally, we elucidate the evolutionary relationships between the raintree and its phylogenetically related genera. Concurrently, we have obtained a high-quality genome of the raintree's symbiotic nitrogen-fixing bacteria through a metagenomic approach. Comparative genomic analysis confirmed its classification within the Bradyrhizobium genus, symbiotically associated with raintree species, and it has been designated as \u003cem\u003eBradyrhizobium saman\u003c/em\u003e. These are new findings in the taxonomic family (Durr PA, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Jia et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). To facilitate the utilization of these genetic resources, we have developed a raintree genome database, accessible online at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.hainanu.edu.cn/SSDB/\u003c/span\u003e\u003cspan address=\"https://bioinformatics.hainanu.edu.cn/SSDB/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e .\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe gene resources in nyctinastic movement of raintree\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe nyctinastic movement is an adaptation to atmospheric drought observed in rain trees and certain leguminous plants. This movement aids in minimizing water loss through transpiration and respiration, also facilitates the collection of surface water. In this study, we performed a comparative transcriptomic analysis of nyctinastic movement involving both the pulvinus and leaflets under pod cultivation conditions. Using 5:30 a.m. as the control time point, samples were collected every two hours. To differentiate the effects of the circadian clock from light influences, additional samples were collected following a two-hour dark treatment at 9:30 a.m., with each sample replicated three times for RNA sequencing. We identified 41 genes closely associated with circadian rhythm regulation and discovered 19 negative regulatory factors. The co-expression network analysis highlighted two hub genes, \u003cem\u003eSsaMYB4\u003c/em\u003e and \u003cem\u003eSsaAMPK1\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eWhile the mechanisms underlying nyctinastic movements in the raintree have been extensively studied, it has been established that the movements of the two types of cells in the pulvinus are regulated by potassium ions, chloride ions, and aquaporins (Kim et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Moshelion et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Uehlein et al., 2008; Ueda et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Nevertheless, the comprehensive regulatory mechanism remains inadequately understood. Our study identified that fundamental regulatory factors, including kinases, splicing factors, and transcription factors, exhibited notably increased expression levels. For instance, \u003cem\u003eSsaAMPK1\u003c/em\u003e, which encodes a kinase, functions as a nutrient and energy sensor to maintain energy homeostasis. Additionally, \u003cem\u003eSsaMYB4\u003c/em\u003e may play a crucial role in regulating cell movement within the pulvinus of the raintree. We also observed a significant upregulation of sodium ion and aquaporin gene expression, aligning with previous findings (Moshelion et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Oikawa et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, contrary to existing reports (Yu et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), we did not detect a significant upregulation of the potassium ion channel protein gene.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe gene resources in symbiotic nitrogen fixation of raintree\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe importance of symbiotic nitrogen fixation within the root nodules of leguminous plants is well-established. The raintree, as the predominant tree species in tropical seasonal rainforests, exhibits a vibrant and consistent green foliage, potentially attributable to its symbiotic nitrogen-fixing capabilities. Through genomic sequencing of both the raintree and its symbiotic rhizobium, complemented by transcriptomic analysis, we have identified that the genetic resources from both the raintree and the rhizobium offer essential insights into the nitrogen fixation processes in the raintree. These enduring nitrogen-fixing mechanisms in their native ecological context may hold substantial potential for enhancing nitrogen fixation in cultivated crops.\u003c/p\u003e\u003cp\u003eOur study identified that six members of the hemoglobin gene family were expressed at the highest levels within the nodules, suggesting their pivotal role in establishing the anaerobic environment of the nodules and in coordinating oxygen balance (Larrainzar et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jiang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Gene Ontology (GO) term enrichment analysis revealed genes across 20 categories. Notably, 17 \u003cem\u003eMYB\u003c/em\u003e transcription factors were exclusively and highly expressed in the pulvinus, indicating their significant, yet previously unreported, role in symbiotic nitrogen fixation. Additionally, genes associated with ammonium ion and amino acid transport, nitrate transporters (\u003cem\u003eNRTs\u003c/em\u003e), asparagine synthetase (\u003cem\u003eAS\u003c/em\u003e), and glutamate synthase (\u003cem\u003eGS\u003c/em\u003e) exhibited elevated expression levels solely in the pulvinus. These genes, related to nitrogen metabolism from the raintree genome, are implicated in nodule formation and the conversion and transport of ammonia synthesized by rhizobium (Stokstad, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Xu et al., 2023; Bu et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, we observed that multiple genes involved in auxin and cytokinin signaling pathways were highly expressed in the pulvinus, including WALLS ARE THIN1 (\u003cem\u003eWAT1\u003c/em\u003e), a vacuolar auxin transport facilitator crucial for maintaining auxin homeostasis, as well as genes related to the \u003cem\u003eIAA/AUX\u003c/em\u003e response to auxin and Adenine Phosphoribosyl Transferase (\u003cem\u003eAPT\u003c/em\u003e), along with three Isopentenyl Transferases (\u003cem\u003eIPTs\u003c/em\u003e) associated with the cytokinin pathway. Partial reports of these findings have been documented in previous studies (Canc\u0026eacute; et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Spitzer-Rimon et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eMaterials and sampling design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRain tree seedlings were procured from Yazhou Bay, Hainan, and cultivated in pots until they reached a height of 2 meters during their vigorous growth phase. Young leaves and fresh root nodules were immediately frozen in liquid nitrogen for subsequent DNA extraction and genome sequencing of both the rain tree and its symbiotic rhizobium. A sampling strategy was devised in a natural environment to assess nyctinastic movement and symbiotic nitrogen fixation in the rain tree using RNA sequencing (RNA-seq). The control point was established at 5:30 a.m., with pulvinus and leaflet samples collected every two hours until 11:30 a.m., resulting in a total of four sampling intervals. To distinguish between the effects of the circadian clock and light, additional samples were collected following a 2-hour dark treatment at 9:30 a.m. Root nodules were collected at 7:30 a.m. All samples, with three biological replicates, were rapidly frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until required for RNA-seq analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEstimation of genome size by k-mer analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe k-mer analysis was performed as part of a comprehensive genome survey. Initially, raw sequencing data underwent a stringent filtering process to obtain high-quality reads, which involved the removal of low-quality reads, short reads, adapter sequences, and polyG tails. Following this, Jellyfish version 2.2.10 (Mar\u0026ccedil;ais et al., 2011) was employed to conduct a frequency distribution analysis with k-mer size set to 21. Subsequently, GenomeScope version 2.0 (Vurture et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) was used to estimate the genome size, heterozygosity, and duplication rate.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenome sequencing, assembly and quality assessment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor the assembly of the genome using PacBio HiFi data, Hifiasm version 0.16.1 was utilized (Feng et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Subsequently, the Hi-C data were aligned to the final assembled contigs using Juicer version 1.6 (Durand et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) to generate the interaction matrix. Both the HiFi and Hi-C sequencing methodologies produced 50 Gb of data, corresponding to approximately 50X coverage. The contigs were subsequently ordered and anchored employing 3D de novo assembly version 180114 (Dudchenko et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Manual inspection of the Hi-C contact map of the final assembly was conducted using Juicebox version 1.11.08 (Robinson et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The completeness of the rain tree genome was evaluated using BUSCO with the embryophyte_odb10 database of single-copy genes (Sim\u0026atilde;o et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTelomere and centromere detection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor chromosomes devoid of telomeric regions, telomere-associated reads were identified from HiFi sequencing data, and the de novo assembled telomeric repeats were incorporated into the chromosomal termini utilizing TGS-GapCloser (Xu et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Employing quarTeT (Lin et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we identified telomeric repeat sequences (CCCTAAA) in 23 out of the 26 anticipated chromosomal termini and detected potential centromeric repeat sequences. The approximate boundary of the centromeric region was inferred from the frequency distribution of all candidate repeat sequences.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene prediction and annotation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRepeatModeler v2.0.3 (Flynn et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) was utilized to cluster repeats through the construction of a de novo repeat library. Subsequently, RepeatMasker v4.1.2 (Tarailo-Graovac et al., 2009) was employed to identify repetitive sequences. HISAT2 v2.1.0 (Kim et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) was then used to align all transcriptome data to the genome. Following this, BRAKER v3.0.3 (Gabriel et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) was applied to automatically train species-specific parameter models and annotate gene structures, integrating both transcriptome alignment results and protein evidence. For functional annotation, the predicted protein-coding genes were queried against the eggNOG database (Hern\u0026aacute;ndez-Plaza et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic and phylogenomic analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe alignment of individual gene sequences was performed utilizing MUSCLE (Edgar et al., 2004), as implemented in MEGA12 (Kumar et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This analysis included homologous sequences from related Bradyrhizobium species and Rhodopseudomonas palustris, which were retrieved from the NCBI database. Phylogenetic trees were reconstructed employing the Neighbor-Joining (NJ) method (Saitou and Nei, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) under the Maximum Composite Likelihood model specifically for 16S rRNA gene sequences. The robustness of the tree branches was evaluated through 1000 bootstrap replicates. For whole-genome comparisons, the Average Nucleotide Identity (ANI) was calculated using FastANI (Jain et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, digital DNA-DNA hybridization (dDDH) values were estimated using the formula 2 model available on the Genome-to-Genome Distance Calculator (GGDC) web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ggdc.dsmz.de/ggdc.php\u003c/span\u003e\u003cspan address=\"https://ggdc.dsmz.de/ggdc.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), as described by Meier-Kolthoff et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetagenome assembly and annotation of Rhizobium\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDNA was extracted using the CTAB method (Porebski et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) and subsequently sequenced on the NovaSeq 6000 platform, employing the paired-end sequencing protocol (PE150), which produced sequence reads of 150 base pairs. The sequencing data were aligned to the rain tree genome using Bowtie2 version 2.5.4 (Langmead et al., 2012). The host genome was then removed using Samtools version 1.21 (Danecek et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) to isolate the metagenomic data for further assembly. The metagenomic data were assembled using MEGAHIT version 1.2.9 (Li et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), resulting in the metagenomic assembly. The contigs derived from this assembly were assigned to distinct genomes using MetaBAT2 version 2.17 (Kang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), facilitating the identification of individual genomes within the metagenome, referred to as metagenome-assembled genomes (MAGs). The quality of the MAGs was evaluated using CheckM (Parks et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), with high-quality genomes selected based on predefined completeness and contamination thresholds. Subsequently, dRep (Olm et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) was utilized to eliminate redundancies and select representative species genome clusters.Taxonomic annotation was conducted utilizing GTDB-Tk version 2.4.0 (Chaumeil et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) in conjunction with the GTDB_r2 14 reference library. Annotation of the individual bacterial genome was carried out using Prokka version 1.14.6 (Seemann, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, the protein sequences of the bacterial genome were annotated employing the eggNOG database.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptome analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe high-quality clean RNA reads were aligned to the rain tree T2T reference genome using HISAT2 version 2.1.0 with default parameters. Read counts mapped to each gene were quantified using FeatureCounts (Liao et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Gene expression levels were subsequently estimated as transcripts per million (TPM). Differentially expressed genes (DEGs) across various tissues and developmental stages were identified using DESeq2 (Love et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). A threshold of |log2FoldChange| \u0026ge; 2 and an adjusted p-value (padj)\u0026thinsp;\u0026le;\u0026thinsp;0.05 was set as the criterion for identifying DEGs between samples. These DEGs were further utilized to construct gene regulatory networks. For the acetylene reduction assay, nodules at 14 days post-inoculation (dpi) were harvested and placed in 20-mL vials, followed by the injection of 2 mL of acetylene. The vials were then incubated for 1 hour at 25\u0026deg;C. The amount of ethylene produced was quantified using gas chromatography (GC9310-VI) through an acetylene reduction assay (ARA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWeighted gene co-expression network analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUtilizing the \"WGCNA\" R package (Langfelder et al., 2008), we developed a gene co-expression network to investigate pulvinus movement. Subsequently, we assessed the correlation between various modules and the mechanism of pulvinus movement, identifying the most pertinent module as the central gene derived from the WGCNA analysis. This key module was then visualized using Cytoscape software (Shannon et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32172614) and supported by the Project of National Key Laboratory for Tropical Crop Breeding (NO. NKLTCB202337), Hainan Province Science and Technology Special Fund (ZDYF2023XDNY050), Hainan Provincial Natural Science Foundation of China (324RC452).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK. Yang designed the sampling strategy of RNA seq for nyctinastic movement, and proposal ideas of the paper. S Wang response for genome assembly and annotation. Z Li Participated in the analysis of repetitive sequences and the construction of co-expression networks of the transcriptome, constructed the website. G Guo participated the collection of the samples. H Yu separated and purified the rhizobium and identified its function of nitrogen fixation. K Yang and S Wang prepared the materials and analyzed the data and drew the figures. Y Xu provided good guidance for experimental design. K Yang wrote the paper. F Chen and Y Xu edited the paper. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome sequences described in this article have been deposized in The National Genomics Data Center (NGDC, https://ngdc. cncb.ac.cn) under accession number PRJCA042275 and PRJCA042279 (whole genome and assembly data).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSupplementary Data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(DataSet 1; DataSet 2; DataSet 3; DataSet 4.)\u003c/p\u003e\n\u003cp\u003eSupplementary data\u0026nbsp;is available at Horticulture Research online.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBai Q, Yang W, Qin G.et al. 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However, the comprehensive understanding of their functions has been constrained by lack of genomic resources. Here, we present the first T2T genome of the raintree, comprising 13 pseudo-chromosomes and a contig N50 42.86 Mb, with 25,997 gene models annotated. Additionally, we sequenced the genome of the rhizobium symbiotic with the raintree, measuring 7.28 Mb and containing 6,882 annotated genes, and designated it as Bradyrhizobium saman. Through transcriptomic analysis, we identified 41 key genes that are significantly upregulated in pulvinus cells, which are exclusively involved in nyctinastic movement. These genes include basic regulatory factors, ion transporters, and aquaporins. Meanwhile, RNA-seq identified 699 core genes upregulated in root nodules, crucial for symbiotic nitrogen fixation. These include seven SsaHGs coding hemoglobin proteins that extremely high expressed to maintain anaerobic conditions in symbiosomes; 26 genes for amino acid transporters, glutamate synthetases (SsaGh) and aspartate synthetases (SsaAsn), 16 for auxin transport facilitators, and nine in cytokinin signaling. Furthermore, 17 MYB transcription factors are upregulated. These genomic resources and findings are vital for enhancing raintree genetics and investigating their ecological adaptations.\u003c/p\u003e","manuscriptTitle":"A telomere-to-telomere genome of raintree (Samanea Saman) reveals its ecological adaptation characteristics of nyctinastic movement and symbiotic nitrogen fixation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-13 10:43:20","doi":"10.21203/rs.3.rs-7165529/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"76f70639-a8af-4d89-9da0-a9c00eb646f4","owner":[],"postedDate":"August 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52879864,"name":"Biological sciences/Plant sciences/Plant stress responses/Abiotic"},{"id":52879865,"name":"Biological sciences/Genetics/Genomics/Transcriptomics"}],"tags":[],"updatedAt":"2026-04-16T07:16:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-13 10:43:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7165529","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7165529","identity":"rs-7165529","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}