Symbiotic nitrogen fixation (SNF) is essential for legume productivity and sustainable agriculture, yet the genetic and regulatory bases of its natural variation remain incompletely understood. Here, we implemented an integrative multi-omics framework to dissect SNF architecture in a diversity panel of 360 soybean accessions encompassing both wild and cultivated lineages. SNF-related traits exhibited extensive variation and strong environmental sensitivity. Genome-wide association studies (GWAS) detected only modest-effect loci, consistent with a polygenic and context-dependent genetic architecture. To resolve regulatory mechanisms underlying this complexity, we analyzed population-scale mature nodule transcriptomes using independent component analysis (ICA), identifying 136 expression modules, of which 15 were significantly associated with SNF traits and enriched for circadian rhythm, lipid metabolism, and defense response pathways. Transcriptome-wide association studies (TWAS) identified 1,453,806, and 178 significant gene-trait associations for nitrogen fixation per plant (NFP), nodule weight (NW), and nitrogen fixation efficiency (NFE), respectively. Among these, 185 transcription factors were identified, 39% overlapping selective sweeps, suggesting evolutionary selection on transcriptional regulation. Expression quantitative trait locus (eQTL) mapping further uncovered 4,654 significant regulatory variants (1,241 cis-, 2,505 trans-, and 908 mixed), including 38 trans-regulatory hotspots collectively influencing ~2,400 genes, nearly half of which are located in domestication-diverged genome regions. Functional validation confirmed that the circadian regulator GmLHY acts as a negative modulator of nodulation, while Dt2, a developmental transcription factor, exerts pleiotropic effects on nodule biomass and fixation efficiency. To facilitate community access, we developed SoySNFdb, an open database integrating all information for SNF in soybean, featuring AI-assisted querying for interactive exploration of regulatory networks. Together, our results suggest that, within this population and experimental context, natural diversity in SNF is associated with regulatory and expression-level variation rather than major-effect coding variants. This integrative framework and accompanying resources establish a basis for system-level dissection and predictive improvement of nitrogen fixation efficiency in legumes.

Three-dimensional structural reconstruction revealed that localized cell wall degradation is essential for the transition from the infection thread (IT) to the infection droplet (ID). Specifically, NPL-mediated local pectin degradation drives this transition and promotes efficient bacterial release

Rhizosphere microbiota play an important role in maintaining plant root growth. However, the physiological and molecular basis of microbial regulation of root traits remains poorly understood. Here, we report that Massilia efficiently colonizes roots and promotes root elongation. In particular, the M117 strain of Massilia significantly inhibits salicylic acid (SA)-related immune signaling, which is required for M117-mediated root elongation. M117 can directly degrade SA, thereby reducing SA levels in the roots. Integrated omics reveal the presence of multiple SA hydrolytic pathways in M117. Among them, the NagGHAaAb pathway is strongly induced by SA. This pathway regulates root growth and is nonrandomly distributed across Massilia species. Finally, we show that M117 colonization enriches specific bacterial taxa within roots. Our findings reveal a specific pathway employed by rhizosphere bacteria to colonize roots and promote their growth and highlight a useful microbial strategy and information for balancing host immunity and growth.

Introduction12-oxophytodienoate reductases (OPRs) are vital enzymes for jasmonic acid (JA) biosynthesis, which mediate plant growth, development and stress defense. Characterizing the soybean OPR gene family is essential for breeding soybean cultivars with strong seed storage stability.MethodsGenome-wide identification and systematic analysis of soybean OPR genes were performed in this study. Twelve OPR members were identified and renamed according to their chromosomal positions, and their chromosomal distribution patterns were analyzed. A multi-species phylogenetic tree of OPR proteins was constructed, along with analyses of conserved motifs and gene structures. Cis-acting elements were predicted to explore hormone-responsive regulatory sequences, and the expression correlation between GmOPR11 and seed deterioration resistance was analyzed under different sowing dates.ResultsA total of 12 GmOPR genes were identified and unevenly scattered on soybean chromosomes. Phylogenetic tree, conserved motif and gene structure analyses clarified the evolutionary features of the OPR family. Cis-element prediction showed that abscisic acid (ABA)- and methyl jasmonate (MeJA)-responsive cis-elements occupied the largest proportion. The expression of GmOPR11 was significantly negatively correlated with soybean seed deterioration resistance across different sowing treatments.DiscussionThis negative correlation may be caused by excessive accumulation of jasmonic acid (JA). Excess JA could disrupt endogenous hormone homeostasis and trigger reactive oxygen species production, which in turn accelerates soybean seed deterioration. This study systematically characterizes the evolutionary patterns and biological functions of the soybean OPR family, laying a theoretical foundation for breeding soybean varieties with enhanced seed storage stability.

Amino acids are the primary assimilated form of nitrogen (N) in plants, and their transport and distribution between source and sink organs determine the yield and quality of tea plants (Camellia sinensis L.). Amino acid permeases (AAPs) play critical roles in amino acid uptake, transport, and distribution, yet the specific functions of most CsAAPs remain largely unelucidated. Here, we aimed to identify the functional role of CsAAP3.1, in amino acid transport and N allocation dynamics. Based on bioinformatics analysis and the expression pattern profiling, we identified CsAAP3.1 as a candidate gene. Its expression patterns across tissues and N conditions were characterized and its function using heterologous overexpression in Arabidopsis and virus-induced gene silencing (VIGS) in tea plants. CsAAP3.1 showed distinct transcript levels in leaves, veins, and roots. Under low nitrogen (LN, 0.25 mmol · L-1 N) conditions, its expression was repressed in roots but induced in shoots. Functional assays revealed that CsAAP3.1 is a broad-specificity amino acid transporter, capable of transporting theanine (Thea) and twelve other amino acids. Compared to wild-type (WT), CsAAP3.1 overexpressing Arabidopsis lines exhibited lower amino acid levels and enhanced N accumulation under low-N conditions, and higher amino acid levels with reduced N allocation in young leaves under normal nitrogen (NN, 5 mmol · L-1 N) conditions at maturity. Moreover, in tea plants, VIGS-mediated silencing of CsAAP3.1 reduced free amino acids and N in young leaves, with 15N tracing showing inhibited amino acid transport from mature to young leaves and downregulated key nitrogen assimilation genes in young leaves, indicating CsAAP3.1 mediated source-to-sink amino acid transport and regulates nitrogen accumulation in young leaves. Taken together, these findings suggest that CsAAP3.1 optimizes N allocation and partitioning to acclimate to low-N, which may have applications for tea quality improvement by upregulating amino acid level.

Sugar content is a key determinant of fruit quality, and sugars also act as signalling molecules that regulate ripening processes, including anthocyanin accumulation. However, the molecular mechanisms underlying sugar accumulation and sugar signal-mediated ripening remain incompletely understood. In this study, we identify FvMAPK6 as an important phosphorylation hub that coordinates both sugar and anthocyanin accumulation in strawberry fruit. FvMAPK6 forms a phosphorylation cascade with FvMAPKK4, which directly phosphorylates the transcription factors FvMYB44.1 and FvMYB44.2. This phosphorylation reduces the stability and transcriptional activity of these proteins, attenuates their repression of downstream target genes such as FvCHI, FvSPS3 and FvSWEET1, thereby coordinating anthocyanin and sugar accumulation. Furthermore, FvMAPK6 increases the protein abundance of the hexose transporter FvSWEET1 in strawberry fruits and alters its transport activity through phosphorylation. We demonstrate that sucrose treatment activates FvMAPK6, reinforcing its regulation of FvMYB44s and FvSWEET1 and thus amplifying sugar and anthocyanin accumulation. These findings establish FvMAPK6 as a key regulator that integrates both sugar accumulation and signalling at both transcriptional and post-transcriptional levels. Although FvMAPK6 promotes sugar accumulation, it significantly reduces fruit yield and vegetative growth. To overcome this limitation, we screen for downstream targets of FvMAPK6 and identify FvSPS3 as a promising breeding target: modulating FvSPS3 improves fruit quality without compromising vegetative growth or yield. Collectively, our findings reveal novel regulatory pathways modulating sugar accumulation and signalling in strawberry while providing a valuable molecular target for the simultaneous improvement of fruit quality and agricultural productivity.

The rhizobia-legume symbiosis, essential for nitrogen fixation, is initiated through Nod Factor (NF) perception by the NF receptors (NFRs). However, the mechanisms regulating NFR activity remain poorly defined. Here, we identify a NODULE INCEPTION 1 (GmNIN1)- Subtilisin-like Protease 1.2 (GmSBT1.2) - Phytosulfokine 4 (GmPSK4) - Phytosulfokine Receptor 1 (GmPSKR1) signaling module that directly activates the GmNFR1ɑ to promote nodulation in soybean. We demonstrate that GmNIN1a specifically activates GmSBT1.2b/1.2d, which are important for nodulation. GmSBT1.2b cleaves the proprotein of the phytosulfokine peptide GmPSK4a into mature form. Knockout of GmPSK4a and its homolog GmPSK4b severely impairs nodulation. Genetic analyses confirm the functionally interdependency of GmSBT1.2b/1.2d and GmPSK4a/4b. GmPSKR1a/1b/1c, identified as GmPSK4 receptors, are beneficial to nodulation. Notably, GmPSKR1a physically interacts with GmNFR1ɑ and enhances its kinase activity. Thus, the activation of GmNFR1ɑ by GmPSKR1a is important to its role in initiating NF signaling. Our findings uncover a previously unknown regulatory mechanism that controls the core NF signaling, providing fundamental insights into the governance of symbiotic nitrogen fixation.

Dark septate endophytes (DSEs) are a type of endophytic fungi colonizing plant roots with a broad distribution, especially in extreme habitats such as deserts. Various studies have demonstrated that DSEs can enhance plant growth under stress conditions. In this study, wheat seedlings were inoculated with two desert-derived dark septate endophytes, Alternaria alstroemeriae (Aa), Paraphoma chrysanthemicola (Pc), or left uninoculated as control (CK), and exposed to either adequate watering (WW) or drought (DD) conditions. Six treatment groups, Aa_WW, Aa_DD, Pc_WW, Pc_DD, CK_WW and CK_DD, were established. The results suggested that inoculation with either Aa or Pc significantly promoted wheat performance under WW and DD conditions. Under two water conditions, inoculation with Aa and Pc significantly promoted the shoot and root growth of wheat. Notably, Aa exhibited a stronger promoting effect on root system under DD treatment, increasing total root length by 17.75%. Under DD treatment, inoculation with Pc significantly enhanced wheat photosynthesis, increasing photosynthesis rate, stomatal conductance, transpiration rate, and chlorophyll content by 30.00%, 33.33%, 47.22%, and 5.61%, respectively. Both DSE strains enhanced osmotic adjustment under drought stress. Aa increased soluble sugar, soluble protein and glutathione contents, while Pc elevated proline and glutathione levels, leading to significant reductions in malondialdehyde accumulation by 24.24% and 19.09%, respectively. In addition, both Aa and Pc significantly increased auxin content of wheat leaves. Consistent with the improved physiological and growth performance, transcriptomic analysis revealed that DSE inoculation induced differential expression of genes primarily enriched in pathways related to photosynthesis, carbohydrate metabolism, signal transduction, antioxidants defense, collectively contributing to enhanced wheat drought tolerance. Consequently, these findings demonstrate the great potential of desert-derived DSE strains for improving wheat growth and drought resilience in arid and semi-arid agricultural ecosystems.

Drought serves as an important abiotic factor influencing plant growth and constraining agricultural progress. Ammopiptanthus mongolicus (Maxim. ex Kom.) Cheng f., a vital plant for afforestation and medicine in the deserts of northwestern China, frequently exhibits extensive root colonization by dark septate endophytes (DSE), while their precise role of DSEs in drought mitigation for A. mongolicus remains ambiguous. This study investigated the effects of DSE symbiosis (Gaeumannomyces hyphopodioides, Paraphoma radicina, and Magnaporthiopsis agrostidis) on the physiological and morphological changes in A. mongolicus leaves through a simulated water limitation experiment. Drought decreased shoot biomass by 20.94 %; however, all DSE strains mitigated this growth inhibition, particularly G. hyphopodioides, which enhanced biomass by 39.41 %. DSE inoculation further improved photosynthetic efficiency, leading to increases in chlorophyll content, net photosynthetic rate (Pn), and stomatal conductance (Gs). Additionally, DSE symbiosis elevated antioxidant enzyme activity and osmotic compound levels, thereby mitigating drought damage. Anatomical analysis showed that G. hyphopodioides-inoculated plants maintained superior leaf structure under drought, with ultrastructural studies confirming the stability of chloroplasts and mitochondria. Variation partitioning further confirmed that DSE symbiosis is a primary factor influencing plant growth and morphological changes. Overall, our study concludes that DSE inoculation, particularly with G. hyphopodioides, could enhance the growth of A.mongolicus under conditions of drought stress through the modulation of leaf morphology and physiology characteristics. These findings confirm that DSE symbiosis can enhance the yield and cultivation of A. mongolicus in arid regions, which is crucial for the its community restoration and resource utilization.

Legumes form root nodules with symbiotic nitrogen-fixing rhizobacteria, which require ample iron to ensure symbiosis establishment and efficient nitrogen fixation. The functions and mechanisms of iron in nitrogen-fixing nodules are well established. However, the role of iron and the mechanisms by which legumes sense iron and incorporate this cue into nodulation signalling pathways remain unclear. Here we show that iron is a key driver of nodulation because symbiotic nodules cannot form without iron, even under conditions of sufficient light and low nitrogen. We further identify an iron optimum for soybean nodulation and the iron sensor BRUTUS A (BTSa) which acts as a hub for integrating iron and nodulation cues. BTSa is induced by rhizobia, binds to and is stabilized by iron. In turn, BTSa stabilizes and enhances the transcriptional activation activity of pro-nodulation transcription factor NSP1a by monoubiquitination from its RING domain and consequently activates nodulation signalling. Monoubiquitination of NSP1 by BTS is conserved in legumes to trigger nodulation under iron sufficiency. Thus, iron status is an essential cue to trigger nodulation and BTSa integrates cues from rhizobial infection and iron status to orchestrate host responses towards establishing symbiotic nitrogen fixation. The authors report that iron binds to and stabilizes the iron sensor BRUTUS A (BTSa), which monoubiquitinates the pro-nodulation transcription factor NSP1a to increase its stability and transcriptional activity, thereby regulating nodulation in legumes.

Soybean domestication has led to a remarkable transformation from the prostrate, vining growth pattern of wild soybeans to the erect growth habit characteristic of modern cultivars. Despite this significant morphological shift, the intricate molecular mechanisms governing the alteration in shoot architecture during domestication remain elusive. Here, we integrated transcriptomic, biochemical, and genetic approaches to dissect the regulatory mechanism underlying basal node branching during the transition of stem growth habit in soybean domestication. We found that the erect growth habit of cultivated soybean restricts basal node branching and reduces cytokinin levels. Exogenous cytokinin application to cultivated soybean enhances basal branching. GmBRC1b is specifically expressed in cotyledonary buds and node 1 buds, and its expression is suppressed by cytokinin. Knocking out GmBRC1a and GmBRC1b increases basal branching and yield, in part, by activating SPL (SQUAMOSA promoter-binding protein-like) transcription factors. Evolutionary analysis reveals that GmBRC1a/b genes underwent artificial selection during domestication, causing their expression to rise over time. Our study reveals that the erect growth habit in cultivated soybeans restricts basal branching via the cytokinin-GmBRC1 module, clarifying the genetic basis of shoot architectural evolution during domestication and highlighting the role of basal branching regulation in crop improvement.

As the only evergreen relict species in the desert environment of western China, Ammopiptanthus mongolicus (Leguminosae) roots is colonized with dark septate endophytes (DSE), but the potential of DSE to alleviate the adverse effects of drought on seedling roots remains uncertain. This study examined the effects of DSE on root growth, physiology and transcriptome of A. mongolicus under drought stress. Drought drastically reduced root biomass by 47.7%, while all DSE strains established positive symbiosis with A.mongolicus, with G.hyphopodioides having the most pronounced promoting effect. Inoculation with G. hyphopodioides alleviated drought stress injury by increasing CAT activity, AsA content and soluble sugar content in the roots, with a significant reduction in MDA accumulation by 97.7%. G. hyphopodioides also significantly increased zeatin and brassinosteroid contents, which in turn regulated the root structure and increased root activity, resulting in a 208.6% increase in root biomass. Transcriptome analysis screened 1246 differentially expressed genes (542 up-regulated and 704 down-regulated) between G. hyphopodioides inoculation under drought treatment, mainly associated with phenylpropanoid biosynthesis, ascorbic acid and aldehyde metabolism, hormone synthesis and signalling, sucrose and starch metabolism, and vitamin B6 metabolism, and further investigated and identified key potential genes and transcription factors (DREB, ERF, NAC, MYB, C2H2). These findings reveal the physiological and molecular mechanisms by which DSE symbiosis improves the drought resistance of A. mongolicus seedlings, providing valuable guidance on the use of DSE resources to promote ecological construction and production of desert plants.

Symbiotic nitrogen fixation (SNF) is a key trait in legume productivity, yet the genetic and regulatory basis underlying its natural variation remains poorly understood. Here, we integrated genome, transcriptome, and chromatin accessibility data from a soybean diversity panel comprising 380 accessions, including 108 wild and 272 cultivated lines. Genome-wide association studies (GWAS) detected multiple loci for SNF traits but with limited resolution due to polygenic architecture and environmental influences. Independent component analysis (ICA) identified 136 co-expression modules; ten ICs were strongly correlated with SNF phenotypes and enriched in circadian clock components (e.g., GmLHY1a/b), lipid metabolism, or defense signaling pathways. Transcriptome-wide association studies (TWAS) linked 1,453, 806, and 178 genes to NFP, NW, and NFE traits, respectively. Among TWAS hits, 185 transcription factors were identified, with 39.0% overlapping selective sweeps, suggesting regulatory evolution under domestication. To further dissect expression regulation, we performed eQTL mapping and detected 4,654 significant eQTLs, including 1,241 local (cis), 2,505 distal (trans), and 908 mixed. By integrating ATAC-seq data from sorted nodule nuclei, we found that eQTLs, particularly local eQTLs, are significantly enriched within open chromatin regions, indicating their regulatory potential. Notably, we validated the circadian clock gene GmLHY1b as a negative regulator of nodulation using CRISPR mutagenesis and CUT&Tag. Our integrative study provides comprehensive genomic and transcriptomic resources from a diverse soybean population, offering novel insights into SNF regulatory networks and a valuable foundation for future SNF research and soybean improvement. ### Competing Interest Statement The authors have declared no competing interest. the National Natural Science Foundation of China, 32272064, 32330078 the National Key Research and Development Program of China, 2022YFD1201502, 2022YFD201400

At present, wheat production is increasingly failing to meet the demands of a growing population. Studies have shown that dark septate endophytes (DSE) can promote host growth, providing new insights for promoting wheat growth and improving crop yields. In this study, we screened the growth-promoting DSE strain Paraphoma pye (Pp) and inoculated it into wheat seedlings under conditions of no fertilizer, organic nitrogen fertilizer, and inorganic nitrogen fertilizer (0, Y, and W). In combination with transcriptome sequencing, the growth, physiology, and soil indicators of wheat were studied. The results indicated that Pp generally had a positive effect on wheat growth, and this promoting effect was more pronounced under the fertilized conditions. Under different fertilizer treatments, Pp inoculation showed altered impacts on host growth and transcription analysis, particularly in the expression of genes associated with growth and secondary metabolite synthesis. In addition, the combined application of Pp and organic nitrogen fertilizer significantly increased soil enzyme activity, whereas the combined application of Pp and inorganic nitrogen fertilizer increased the host photosynthetic rate, chlorophyll content and nitrogen accumulation. Furthermore, weighted gene co-expression network analysis revealed that the “MAPK signaling pathway” and “glycosphingolipid biosynthesis pathway” may be the key pathways affecting plant phenotypes under Pp-inorganic fertilizer treatment. Overall, this study contributes to the understanding of the effects of DSE combined with fertilizer on plant growth and provides a feasible approach to better promote wheat growth.

The combination of mutation-based genetics and functional genomics has allowed a detailed dissection of the nodulation-induction and autoregulation of nodulation(AON) pathways of soybean. Applicable to all legumes, nodulation is induced by Rhizobium/Bradyrhizobium-produced lipopolysaccharides(Nod factors), perceived by Nod factor receptors(NFR1/NFR5 dimers), leading to cortical and pericycle cell divisions. These induce the production of CLAVATA3-like(CLE) peptides, which travel in the xylem to the shoot, where they are perceived by a receptor complex including a leucine-rich repeat(LRR) receptor kinase, encoded by Gm NARK, Lj HAR1, Mt SUNN and closely related receptors in other legumes like Phaseolus vulgaris(common bean), Pisum sativum(pea), and Glycine soja. The activated receptor complex negatively regulates by phosphorylation of the constitutive synthesis of mi R2111 in the shoot. This is normally is translocated via the phloem to the entire plant body, initiating suppression of a rootexpressed Kelch repeat-containing F-box protein“Too Much Love(TML),” which in turn suppresses the nodule initiation cascade. Nodulation is therefore permitted during a developmental window between the induction and progress of the nodulation/cell division/infection cascade during the first few days after inoculation and the functional“readiness” of the AON cascade, delayed by the root–shoot–root loop. Loss-of-function mutations in Gm NARK and Lj TML result in excessive nodulation(supernodulation/hypernodulation/supernummary nodulation) as well as localized tolerance to externally applied nitrate. Recent analyses have indicated an interaction of the AON with lateral root formation as well as with the autoregulation of mycorrhization(AOM). Further details of the parallel functions of key points in this regulatory loop remain to be elucidated.

The domestication process and origin of soybeans remain a topic of debate. In a recent study, Zhu et al. establish black soybeans as a pivotal evolutionary intermediate. They reveal the dual origins of domestication and how regional haplotype diversity was shaped. Their discoveries provide a genomic roadmap for the intelligent design of future soybeans.

Colletotrichum spp., hemibiotrophic fungal pathogens, threaten global strawberry production. Jasmonate (JA) regulates plant-Colletotrichum interactions, but its mechanisms remain unclear. Here we demonstrate that both exogenous methyl jasmonate (MeJA) treatment and elevated endogenous MeJA levels increase strawberry susceptibility to anthracnose. Two key JA biosynthesis genes, FveAOS2 and FveAOC3, were identified as contributors to Colletotrichum-induced susceptibility. Further analysis revealed that the FveSnRK2.1-FveWRKY50 phosphorylation module functions as an important molecular switch in regulating disease susceptibility. Specifically, Colletotrichum infection or MeJA application activates FveSnRK2.1, which phosphorylates FveWRKY50 at serine residue 88 (S88). This phosphorylation enhances the stability and transcriptional activity of FveWRKY50, leading to increased expression of FveAOS2 and FveAOC3, higher MeJA accumulation and enhanced susceptibility. Notably, the strawberry JASMONATE-ZIM DOMAIN (JAZ) protein FveJAZ5 suppresses susceptibility by directly interacting with FveWRKY50, thereby preventing its interaction with FveSnRK2.1 and inhibiting the activation of FveAOS2 and FveAOC3. Upon pathogen attack or MeJA signalling, FveJAZ5 is degraded, thereby releasing FveWRKY50 from suppression. The study elucidates a Colletotrichum-induced 'JA signaling - JA biosynthesis' positive feedback loop that drives strawberry susceptibility. Knocking out FveWRKY50 and overexpressing FveJAZ5 generated anthracnose-resistant germplasms. These findings deepen understanding of plant-Colletotrichum interactions and provide genes for resistant strawberry breeding.

Central to the legume–rhizobium symbiosis is the formation of organelle-like symbiosomes where nitrogen-fixing bacteroids are enclosed by a host-derived symbiosome membrane. This creates the symbiosome space, which topologically resembles an apoplastic compartment within the cell. While the apoplast of plant cells is largely occupied by the cell wall, symbiosomes are devoid of cell wall polymers. Here, we describe a mechanism that functions to protect and maintain effective nitrogen fixation through the action of cell-wall-degrading enzymes that prevent accumulation of un-esterified pectin within symbiosomes. We identify two symbiotically-induced polygalacturonase (PG) genes in Medicago truncatula, SyPG1 and SyPG2, that are secreted into the symbiosome space. Silencing the expression of SyPG1/2 or editing SyPG1/2 via CRISPR-Cas9 both lead to nodule senescence and trigger excessive accumulation of un-esterified pectin in symbiosome containing cells. Additionally, we show that un-esterified pectins inhibit rhizobial growth both in vivo and in vitro. Together, our results provide evidence for a host-controlled cell wall clearance mechanism that is essential for symbiosome maintenance. Here a mechanism for nitrogen fixation maintenance is identified in Medicago truncatula where two polygalacturoneases are expressed in nodules and secreted into the symbiosome space for clearing of excessive un-esterified pectins.

BACKGROUND:Transcription initiation is a key checkpoint in plant gene regulation, yet the DNA features that determine where and the frequency of the genes start transcription remain unclear. RESULTS:We develop GenoRetriever, an interpretable deep learning model trained on base pair resolution STRIPE-seq data from multiple crop genomes, to systematically reveal and quantify the sequence code that governs transcription start sites (TSSs). Using TSS profiles from 16 soybean tissues and six additional crops, GenoRetriever identifies 27 core promoter motifs, including canonical TATA box and initiator elements, that together dictate TSS choice and activity. Model interpretation shows how each motif modulates both initiation frequency and precise start site position; these effects are confirmed by in silico motif edits, saturation mutagenesis, and targeted promoter assays. A new telomere-to-telomere assembly of wild soybean, Glycine soja, reveals that 31.85% of natural promoter variants shift dominant motifs relative to cultivated soybean, explaining domestication-driven changes in transcriptional regulation. Cross-species comparisons further indicate that, although many motif functions are conserved, monocots and dicots display distinct motif frequencies and positional preferences. CONCLUSIONS:GenoRetriever provides an interpretable, cross species framework for decoding transcription initiation in plants. By linking specific sequence motifs to quantitative transcriptional outcomes and validating these links experimentally, our study advances fundamental knowledge of promoter architecture and supplies a practical platform for rational engineering of gene expression in crop improvement and functional genomics.