Gene Editing for Sugar Perception Transport and Source–Sink Optimization in Soybean

Abstract

Sugars serve as primary energy sources and key essential signaling molecules, playing pivotal roles in regulating plant growth and development. Crop yield is tightly linked to the efficient partitioning of photoassimilates from source leaves to sink organs. This process is intricately regulated by sugar sensing and transport systems, which orchestrate the dynamic source–sink balance by modulating phloem loading, long-distance translocation, and sink unloading. While substantial progress has been made in deciphering these mechanisms in model organisms, a comprehensive understanding of the regulatory network in soybean—a globally significant crop with unique plant architecture in which leaves, inflorescences, and pods are borne on nodes—remains elusive. The emergence of gene-editing tools, notably CRISPR/Cas9, offers powerful tools for dissecting redundant transporter families and targeted engineering of key regulatory nodes. This review synthesizes the current understanding of the molecular networks governing sugar perception and long-distance transport, with a specific focus on soybean. It further explores the utility of gene editing in accelerating the functional characterization of critical components and highlights potential molecular targets for manipulating source–sink dynamics to enhance soybean yield.

1. Introduction

The sessile nature of plants requires a complex internal metabolic regulatory network to adjust to changing environmental conditions [1]. Sugars play a dual role within this network, serving as both the primary energy source for cellular functions and crucial signaling molecules that regulate plant development. Sugar concentrations in plant tissues are dynamic, responding to developmental and environmental signals. This dynamic process involves reallocating carbon between source and sink tissues and adjusting the strengths of source and sink organs, as well as the competitive interactions among sinks, with the goal of enhancing overall carbon utilization efficiency [2,3]. In higher plants, carbon fixed during photosynthesis in source leaves is typically converted to sucrose, which is then transported long distances through the phloem to heterotrophic sink organs like developing seeds, roots, and nodules [4]. To maintain sugar balance, source leaves must be closely coordinated with the needs of the sinks. Generally, tissues with low sugar levels increase source activities such as photosynthesis, nutrient mobilization, and export, while tissues with sufficient sugar levels promote sink activities like growth and storage [5]. However, unlike plants in the natural environment, crops may face different selection pressures in terms of sucrose homeostasis and carbohydrate distribution [2]. This difference is particularly reflected in the formation of reproductive structures, such as the parameters of grain setting and the filling process, which may involve changes in gene expression levels, gene replication, or enhancement of specific regulatory pathways to meet the demands of high-yield breeding. The breeding goal of humans is to maximize the yield of the harvestable part, which means that more photosynthetic products such as sucrose need to be transported to reproductive or storage organs such as grains, fruits or tubers. This shift in the priority of resource allocation has led to crops being subjected to different evolutionary pressures in the mechanisms of sucrose synthesis, transportation, storage and utilization compared to wild plants.

Soybean (Glycine max (L.) Merr.), the world’s foremost oilseed and protein crop, contributes more than a quarter of the global plant protein supply, playing a crucial role in ensuring food and feed security [6,7,8]. As one of the model plants, soybean exhibits distinctive biological traits including photoperiod sensitivity, symbiotic nitrogen fixation in root nodules, and complex stem growth habits and plant architecture, all of which rely heavily on precise regulation of source–sink dynamics [9,10]. An efficient molecular network governing sugar regulation underlies these complex agronomic traits, driving both photosynthetic production and the effective utilization of sugars in various sink tissues [11]. Research suggests that HEXOKINASE (HXK), SNF1-RELATED PROTEIN KINASE1 (SnRK1), and TARGET OF RAPAMYCIN (TOR) serve as central sugar sensors and energy-sensing nodes in plants, while transmembrane transporters like SWEETs (Sugars Will Eventually be Exported Transporters) and SUTs/SUCs (Sucrose Transporter) facilitate the long-distance transport of sugars. The former modulates the expression and function of transporters by detecting cellular sugar levels, while the latter offers feedback to adjust signal output, collectively maintaining sugar homeostasis. In recent years, the allocation mechanism of carbohydrate partitioning has emerged as a prominent research focus, enhancing the comprehension of plant growth and development. Concurrently, the rapid progress in gene-editing technologies, notably the maturation of the CRISPR/Cas9 system, has opened unprecedented avenues. These tools allow for precisely dissecting the functions of redundant sugar transporter families and intervening in carbon partitioning pathways [12]. In comparison to traditional breeding, gene editing presents a more efficient approach for elucidating the molecular mechanisms underlying yield traits and expediting genetic enhancements in soybean [13]. Thus, this review centers on soybean. We delve into the two fundamental modules of sugar sensing and transport and investigate their mechanistic contributions to shaping superior source–sink relationships. We also explore the potential of using gene-editing technology to optimize these source–sink relationships and bolster high-yield characteristics in soybeans.

2. Core Components of the Plant Sugar Sensing Network

Sugar perception is crucial for initiating signal transduction pathways in plants [14,15]. Traditionally, sugar’s regulatory effects on plant metabolism, growth, and gene expression were regarded as direct metabolic outcomes of its role as an energy substrate; however, the discovery that non-metabolizable hexose analogs can also induce gene expression provides compelling evidence that plants possess specific sugar sensing and signal transduction mechanisms operating independently of sugar catabolism [16]. However, investigating these mechanisms in depth remains challenging, primarily because mutations in the fundamental components of sugar signaling networks often lead to lethality or severe pleiotropic defects [17,18]. The first encompasses signaling pathways directly mediated by specific sugar sensors. For instance, HXK1 sensors can bind glucose directly and initiate a signaling cascade to transduce signals. Additionally, trehalose-6-phosphate (T6P) has been identified as a signaling molecule that reflects sucrose levels. The second category includes pathways that transmit signals indirectly by sensing energy status derived from sugar metabolism. At the core of this category are two antagonistic energy-sensing nodes: the TOR kinase complex and SnRK1. TOR is activated under high-sugar, high-energy conditions, thereby stimulating anabolism and growth. In contrast, sugar deprivation and low energy levels activate SnRK1, which suppresses growth and promotes catabolism. The former relies on direct detection by sensors, while the latter synchronizes growth, metabolism, and stress response pathways according to energy and metabolic status. Together, these two systems form the fundamental framework of sugar signal transduction in plants.

2.1. The Glucose Sensor Hexokinase

HXK stands out as the first identified glucose sensor and serves as a pivotal integration point that connects nutrient, light, and hormone signaling pathways to collectively govern plant growth and development [17]. The elucidation of HXK’s signaling role primarily stemmed from genetic screenings conducted in Arabidopsis, leveraging the phenomenon of sugar-induced suppression of seedling growth to identify glucose-insensitive (gin) or hypersensitive mutants [19,20]. Notably, the loss-of-function mutant of AtHXK1, gin2-1, displays significant developmental abnormalities, such as stunted shoot and root growth, diminished leaf expansion, and delayed flowering and senescence, underscoring the disconnection between glucose signaling and its metabolic processes [20]. Furthermore, a recent study has revealed that HXK1 also promotes branch development in Arabidopsis, rose, and pea [21].

Symbiotic nitrogen fixation in legume root nodules heavily relies on shoot-derived carbohydrates. Sucrose, the main transport form, is transported to the roots and nodules, where it is hydrolyzed to provide energy and carbon skeletons to rhizobia, facilitating nitrogen fixation. Within the host cell cytoplasm, hexokinase mediates the glucose phosphorylation, indicating its involvement in a crucial rate-limiting step of nodule carbon metabolism [22]. Despite significant advancements in plant research, a comprehensive functional analysis of the soybean HXK gene family is lacking. A recent genome-wide analysis has identified 17 GmHXK genes (GmHXK1-17) [23,24]. Phylogenetic and subcellular localization predictions have clearly classified them into type A (GmHXK1-4) and type B (GmHXK5-17) [23]. These family members share similar protein structures and conserved catalytic domains, suggesting a universal potential for hexose phosphorylation. Tissue expression profiling has revealed significant tissue specificity among GmHXKs. For example, GmHXK3 is highly expressed in pods, GmHXK11 is predominantly expressed in seeds and nodules, and GmHXK5 is mainly expressed in flowers. Analysis of promoter cis-acting elements also supports their involvement in multiple processes, including growth, development, stress responses, and hormone induction [23,24]. Furthermore, preliminary functional characterization has revealed differentiation among family members. GmHXK2 expression is induced by salt or drought stress. Knockdown of its expression through virus-induced gene silencing (VIGS) technology aggravates the wilting, coiling and yellowing of plant leaves, reduces the intracellular ROS clearance ability and down-regulates the expression of salt stress-related genes, indicating that it plays an important role in maintaining Na⁺ and K⁺ homeostasis. Under salt stress, overexpression of GmHXK2 increased the root length and fresh weight of soybeans, and the growth state was better than that of the wild type [24]. An additional investigation demonstrated that the overexpression of GmHXK15 in transgenic soybean hairy roots notably stimulated root growth and improved alkali tolerance. Subsequent studies unveiled that GmHXK15 has the capability to catalyze hexose phosphorylation [23]. These research advances provide evidence for the functional diversity of the GmHXK gene family in soybean.

2.2. Trehalose-6-Phosphate: A Key Signal of Sucrose Status

T6P serves as a key signaling molecule in plants, acting as a crucial regulator of sucrose sensing and optimizing crop growth and yield by modulating sucrose utilization and distribution, thus linking sucrose status to agronomic performance [25]. The Sucrose-T6P nexus model posits that T6P acts as a dual-role molecule: reporting sucrose levels in plants, and as a negative regulator that inhibits excessive sucrose accumulation—ultimately keeping sucrose concentrations within an optimal physiological range [26]. The regulatory role of T6P is intricate and accurate, impacting the allocation of photoassimilates between sucrose and organic/amino acids in source leaves through the post-translational control of phosphoenolpyruvate carboxylase (PEPC) and nitrate reductase (NR). During the night, T6P facilitates the mobilization of transiently stored starch in leaves, effectively connecting a carbon supply from the source to the developmental needs of remote sinks. In sink tissues, T6P governs sucrose utilization and growth. Furthermore, research indicates that the T6P metabolic pathway plays a role in modulating stomatal regulation in guard cells [27]. In Arabidopsis, the identification and functional analysis of genes encoding trehalose-6-phosphate synthase (TPS) and phosphatase (TPP) have demonstrated that modifying T6P levels can notably impact leaf structure, delay senescence, and improve photosynthetic efficiency, providing compelling evidence that T6P serves as a crucial signaling metabolite connecting carbon status to plant development [28,29]. Research on the T6P signaling pathway in soybean remains in its nascent stage but has already revealed its critical roles in stress response and morphogenesis. For instance, the oomycete pathogen Phytophthora sojae can trigger the expression of the host’s GmTPS6 via secreted effectors to promote infection—indicating that modulating trehalose biosynthesis could be a viable strategy for managing soybean root and stem rot [30]. Additionally, studies have indicated that soybean can adjust T6P metabolism in response to varying light qualities, thereby modifying carbon distribution patterns and reshaping plant morphology. This underscores the substantial impact of the T6P pathway on mediating plant adaptability to environmental conditions [31]. Nevertheless, the mechanisms underlying the perception and signal transduction of T6P in soybean necessitate further clarification.

2.3. SnRK1: The Central Energy Sensor

Organisms must precisely regulate carbon intake and energy expenditure to maintain essential life processes [17]. SnRK1 serves as a crucial energy sensor in this metabolic balance [32]. Comprising a catalytic α-subunit and regulatory β and βγ subunits, SnRK1 forms a complex structure that can detect cellular energy variations and regulate metabolic and growth pathways globally [33,34]. In Arabidopsis, the overexpression of its catalytic subunit KIN10 significantly boosts the plant’s resilience to energy deprivation, adjusts transcriptional networks to modulate developmental processes, and ensures energy equilibrium and stress resilience [35]. Recent studies have also demonstrated that SnRK1, specifically through its catalytic subunit KIN10, interacts with NODULE INCEPTION PROTEIN-LIKE PROTEIN (NLP7) to suppress nitrate signaling, establishing a connection between carbon availability and nitrate signals to coordinate carbon-nitrogen metabolism [36]. In legumes, SnRK1 plays a crucial role, particularly in the energy-demanding process of symbiotic nitrogen fixation. Previous studies have demonstrated that GsSnRK1 from wild soybean interacts with essential nodulation components like the nodulation factor receptor GsNFR5α, indicating its involvement in regulating the symbiotic nodulation network [37]. Knocking out GmSnRK1.1 and GmSnRK1.2 has been shown to impede root growth, induce stomatal closure, and decrease tolerance to various abiotic stresses, underscoring their significance in stress adaptation [38,39]. A recent groundbreaking study has elucidated the core regulatory mechanism of a legume-specific SnRK1 subunit in symbiotic nitrogen fixation. This study revealed that enhancing the expression of the novel legume-specific SnRK1α4 led to increased nodule size and nitrogenase activity, whereas mutants lacking snrk1α4 exhibited the opposite effect. The upstream kinase DMI2 activates SnRK1α4 through phosphorylation at Thr175. Subsequently, SnRK1α4 phosphorylates malate dehydrogenase, stimulating the production of cytoplasmic malate, which serves as a carbon source for bacteroids, thereby supporting their efficient symbiotic nitrogen fixation. This investigation highlights the pivotal role of SnRK1α4 in mediating the carbon-nitrogen interchange between the host plant and bacteroids [40].

2.4. TOR: The Master Regulator of Growth

The evolutionarily conserved serine/threonine protein kinase, TOR serves as a central growth regulatory hub in plants, integrating nutrient signals from the environment with internal developmental and metabolic programs to maintain cellular and organismal homeostasis [17,41]. However, studying plant TOR function has encountered two significant challenges. First, unlike yeast and animals, plants lack a protein facilitating the binding of rapamycin to TOR, rendering them generally insensitive to this inhibitor and restricting the use of pharmacological approaches. Second, TOR, being a single-copy gene in Arabidopsis, results in complete loss-of-function mutants being embryo-lethal, further complicating classical genetic analysis [42]. Despite technical challenges, evidence supporting TOR as an energy/nutrient sensor has progressively emerged. Nicotinamide treatment in Arabidopsis alters cytoplasmic ATP levels, affecting the regulation of circadian period length, meristem activation, and root growth by the glucose-TOR energy signal [43]. Moreover, during the transition from seed to photoautotrophic seedling, glucose generated through photosynthesis drives TOR signaling via sugar metabolism and energy, regulating the activation of root meristems and leaf primordia [44]. TOR plays a pivotal role at the intersection of the “light-sugar-energy” signaling axis, orchestrating processes like cell growth, translation, ribosome biogenesis, and autophagy to align with environmental resources. In soybean, the TOR pathway is directly involved in symbiotic nitrogen-fixing nodule development. Upon nodule formation, both GmTOR and its downstream effector GmS6K1 are transcriptionally activated. Notably, RNA interference (RNAi)-mediated knockdown of GmS6K1 significantly impairs nodule development—reducing nodule number and weight, simultaneously suppressing nodulation-related genes, and nearly halving nitrogen fixation capacity [45]. This underscores the essential role of the TOR-S6K module in establishing and sustaining functional symbiotic nitrogen-fixing nodules in soybean, serving as a promising target for enhancing soybean nodulation and source–sink relationships—thereby boosting nitrogen fixation efficiency.

3. Sugar Transport for Plant Growth and Development

3.1. Phloem Loading in Source Leaves

Sucrose, the primary product of photosynthesis, must be transported from source leaves to sink organs such as seeds and roots to support overall plant growth and development. In most crops, this transport primarily takes place via an apoplastic loading pathway [46]. Sucrose, produced in mesophyll cells, is transported into phloem parenchyma cells via plasmodesmata. Subsequently, SWEET—a class of efflux transporters—mediate the export of sucrose from the cytoplasm of phloem parenchyma cells into the apoplastic space [47]. Following this, sucrose/H⁺ symporters belonging to the SUT/SUC family actively uptake sucrose from the apoplast, loading it into the sieve element-companion cell complex for long-distance transport to sink tissues [48,49] (Figure 1). SUC2 and its homologs in crops are specifically active in companion cells, regulating the pace of carbon export from source leaves to sink tissues (Figure 1). In Arabidopsis, the simultaneous disruption of two highly expressed paralogs in the phloem, AtSWEET11 and AtSWEET12, results in impaired sucrose export, excessive sucrose accumulation in leaves, and diminished transfer of photoassimilates to the roots, consequently hindering root growth [50]. The sweet11/sweet12 double knockout mutant also displays drought sensitivity [51]. Sucrose induction reduces the efficiency of the stomatal closure feedback mechanism. In the sweet16/sweet17 double knockout mutant, fructose partitioning in mesophyll and vascular cells is altered, positively impacting gas exchange [52]. Impaired sugar partitioning, rather than the total sugar amount, is the primary reason for the enhanced drought sensitivity in the quadruple knockout mutant [52]. In maize, the expression of ZmSWEET13a/b/c is concentrated in the bundle sheath cells and vascular regions of the leaves, making them essential for apoplastic phloem loading. Knocking out ZmSWEET13a/b/c using gene editing results in severely stunted growth and impaired photosynthesis in the triple knockout mutants. The leaves accumulate high levels of soluble sugars and starch, leading to compromised phloem loading [53]. Further RNA-seq analysis revealed significant transcriptional dysregulation of genes associated with photosynthesis and carbohydrate metabolism [53]. In summary, the coordinated action of SWEETs and SUTs in source leaves is pivotal for sucrose homeostasis at both the local and whole-plant levels, acting as a precise valve for carbon flux—accurately regulating the direction and rate of sugar flow. Nevertheless, the mechanisms of phloem transport in soybean remain to be further elucidated.

3.2. Phloem Unloading in Sink Tissues

Sucrose unloading from the phloem into heterotrophic sink tissues plays a crucial role in determining both crop yield and quality [8,54]. The formation of crop yield is significantly influenced by the distribution of photoassimilates to the seeds [49]. In globally significant grain and oilseed crops like rice, maize, and soybean, SWEETs regulate the distribution of sugar to the seeds, thereby impacting their carbohydrate, protein, and oil content, as well as seed size. This characteristic has been subjected to selective pressure throughout the extensive domestication history of crops [55,56]. In soybean, GmSWEET10a and GmSWEET10b are specifically expressed in the seed coat, facilitating the transport of sucrose and fructose to the embryo, thereby influencing seed size, oil content, and protein content [57]. Knockout mutants of GmSWEET10a and GmSWEET10b exhibit reduced seed size, lower oil content, and higher protein content, indicating functional redundancy between the two genes [57]. A novel member of the sugar transporter family gene, GmSWEET30a, has been recently identified, exhibiting high expression in the seed coat. Through CRISPR/Cas-mediated knockout of GmSWEET30a and its homolog GmSWEET30b, researchers observed a reduction in total protein and oil content in the homozygous double knockout mutant compared to the wild type. Despite sharing substantial homology, GmSWEET30b, when edited, only exhibits a minor deletion of two amino acids without inducing frameshift mutations. Given its significant implication in soybean quality, GmSWEET30a emerges as a crucial target for enhancing soybean quality in future endeavors [56]. Similarly, in rice, OsSWEET14 and OsSWEET11 play crucial roles in grain filling, with strong expression in the caryopsis, ovular vascular trace, nucellar epidermis, and cross cells [58,59]. Disruption of OsSWEET11 results in decreased sucrose levels in the embryo sac, leading to impaired grain filling and abnormal seed development [59]. While single knockout of OsSWEET14 shows no phenotypic differences from the wild type, the OsSWEET14/OsSWEET11 double knockout mutant displays more severe phenotypes than the OsSWEET11 single mutant—relative to the wild type—including reduced grain weight, grain filling rate, and yield, along with increased starch accumulation in the pericarp [58] (Figure 1).

Symbiotic nitrogen fixation in legumes, facilitated by the interaction between rhizobia and host plants, is a highly energy-intensive process akin to seed development, where sucrose plays a crucial role. Nevertheless, the mechanism of sucrose allocation post-rhizobia inoculation remains obscure. Recent studies have identified GmSUT1 and GmSWEET3c as key players influencing nodule formation [60,61]. GmSUT1 exhibits high expression levels in root nodules, predominantly localized in the peripheral fixed zone and vascular bundles. Overexpression of GmSUT1 led to a notable rise in nodules numbers and dry weight compared to the wild type, consequently enhancing total plant dry weight and nitrogen content. Conversely, RNAi plants exhibited sucrose accumulation in the shoot due to disrupted sucrose transport. In overexpressing plants, sucrose concentration decreased while hexose concentration increased in root nodules. Conversely, RNAi plants displayed reduced glucose concentration. These findings suggest that GmSUT1 facilitates soybean root nodule formation by modulating sucrose transport to root nodules. Particularly, GmSWEET3c exhibits high induction and expression during the initial phase of rhizobia infection. Precision targeting of this gene through CRISPR-Cas9 technology leads to a notable decrease in infection threads and root nodules, impeding sucrose accumulation in rhizobia-induced susceptible regions. Conversely, overexpression in plants elicits contrasting effects, underscoring the essential role of GmSWEET3c in facilitating sucrose redistribution by rhizobia [61]. Subsequent molecular studies revealed that the nodulation transcription factor GmNSP1 can directly interact with the promoter region of GmSWEET3c, promoting its expression. Knockout mutants of Gmnsp1 exhibit significantly downregulated GmSWEET3c expression, thus establishing a GmNSP1-GmSWEET3c regulatory module that precisely governs sucrose distribution in rhizobium-inoculated plants in response to symbiotic cues [61].

Sink unloading is intricately regulated by developmental and environmental cues, not functioning in isolation. For example, during the floral transition in Arabidopsis, the FLOWERING LOCUS T (FT) signaling pathway triggers the transcription of SWEET10, altering the transcription of floral regulatory factors at the shoot apex and impacting the floral transition [62]. In soybean, photoperiod signals play a role in seed filling regulation. Under long-day conditions, GmSWEET10a interacts with the Arabidopsis TFL1 (TERMINAL FLOWER 1) ortholog Dt1, inhibiting the sugar transport function of GmSWEET10a from the seed coat to the embryo, resulting in compromised sucrose transport and reduced seed sizes [8]. These findings suggest that sucrose unloading in sink tissues likely entails interactions among sugar transporters, plant hormones, protein kinases, and photoperiod signals, collectively governing sucrose distribution to uphold sugar homeostasis in response to environmental changes.

4. The Source–Sink Regulatory Network: An Integrated View

Nutrient signaling is a fundamental mechanism that regulates cellular activities and organismal development in multicellular plants, in conjunction with intrinsic regulatory factors and environmental inputs [17]. The regulation of nutrient signals is intricate, with individual molecules rarely acting alone but rather interacting with key nutrient, hormone, and environmental signals [63]. In maintaining sugar homeostasis and facilitating plant growth and development, HXK1, the T6P-SnRK1 axis, TOR, and sugar transporters play pivotal roles, although some molecular mechanisms remain unclear [64]. In Arabidopsis, the hexokinase HXK1 not only senses glucose levels but also actively participates in the ethylene signaling pathway, acting as an upstream negative regulator that influences the stability of the core ethylene pathway transcription factor EIN3. The regulated EIN3, in turn, directly binds to the promoter of the sucrose transporter SUC2, inhibiting its expression and thereby regulating glucose signaling associated with root sink growth [65]. The HXK1-EIN3-SUC2 regulatory module enhances sucrose phloem loading in source tissues, leading to increased sucrose levels in sink roots [65] (Figure 1).

Figure 1. Regulatory model of source–sink carbon allocation. In source leaves, photosynthetically derived sucrose generates a Trehalose-6-Phosphate (T6P) signal that inhibits the energy-sensing kinase SnRK1, thereby promoting anabolic pathways [33]. Sucrose is exported to the apoplast by SWEETs for phloem loading via the SUC2 transporter [65]. This process is regulated transcriptionally (EIN3 represses SUC2) and post-translationally (ABA-activated SnRK2 phosphorylates SWEETs) [51]. In sink tissues, imported sucrose fuels storage synthesis (e.g., grain filling), a process modulated by a local T6P-SnRK1 feedback loop [66]. SUS is the key enzyme for sucrose hydrolysis, which is negatively regulated by T6P. This network connects sugar status with transport machinery to control whole-plant carbon partitioning. UDPG: Uridine Diphosphate Glucose, G1P: Glucose-1-Phosphate, G6P: Glucose-6-Phosphate, F6P: Fructose-6-Phosphate, SUC2/SUT: Sucrose Transporter, SWEET: Sugars Will Eventually be Exported Transporters, SUS: Sucrose Synthase, bZIP: Basic leucine zipper. The upward black arrow represents an increase in ABA content; red lines indicate activation; blue lines indicate inhibition, and the dotted lines represent the omitted metabolic steps. This figure was created with BioRender.com.

Figure 1. Regulatory model of source–sink carbon allocation. In source leaves, photosynthetically derived sucrose generates a Trehalose-6-Phosphate (T6P) signal that inhibits the energy-sensing kinase SnRK1, thereby promoting anabolic pathways [33]. Sucrose is exported to the apoplast by SWEETs for phloem loading via the SUC2 transporter [65]. This process is regulated transcriptionally (EIN3 represses SUC2) and post-translationally (ABA-activated SnRK2 phosphorylates SWEETs) [51]. In sink tissues, imported sucrose fuels storage synthesis (e.g., grain filling), a process modulated by a local T6P-SnRK1 feedback loop [66]. SUS is the key enzyme for sucrose hydrolysis, which is negatively regulated by T6P. This network connects sugar status with transport machinery to control whole-plant carbon partitioning. UDPG: Uridine Diphosphate Glucose, G1P: Glucose-1-Phosphate, G6P: Glucose-6-Phosphate, F6P: Fructose-6-Phosphate, SUC2/SUT: Sucrose Transporter, SWEET: Sugars Will Eventually be Exported Transporters, SUS: Sucrose Synthase, bZIP: Basic leucine zipper. The upward black arrow represents an increase in ABA content; red lines indicate activation; blue lines indicate inhibition, and the dotted lines represent the omitted metabolic steps. This figure was created with BioRender.com.

A pivotal homeostatic feedback loop operates within the regulatory network, with the T6P-SnRK1 axis assuming a critical role. T6P, serving as an indicator of sucrose abundance, directly hinders the activity of the energy-sensing “brake” SnRK1 through allosteric regulation, thereby stimulating anabolism in times of sufficient energy. Conversely, SnRK1 can reciprocally modulate the gene expression of T6P synthase (TPS) and phosphatase (TPP) via transcription factors, establishing a closed feedback loop [66]. Comprehending the mechanism by which T6P acts through the SnRK1 protein kinase regulatory system lays the groundwork for a cohesive mechanism that governs overall plant resource allocation and source–sink interactions. Specifically, T6P indicates the sucrose status and suppresses SnRK1 by modifying its phosphorylation, consequently enhancing biosynthetic pathways. SnRK1 governs TPS through transcriptional regulation and phosphorylation, as well as TPP transcription via bZIP transcription factors (Figure 1). Notably, SWEET sugar transporters are probable crucial downstream targets of its regulation of sucrose partitioning [66,67]. Another significant study supports this perspective [51]. Under drought stress, accumulation of the plant hormone ABA activates SnRK2 protein kinases, which subsequently phosphorylate SWEET11 and SWEET12 to enhance their sucrose transport activity. This process accelerates sucrose transport to the roots, thereby improving drought stress resistance [51]. In rice, the growth-regulating factor GRF4 activates the sucrose metabolism gene TPS1 and the sugar transporter gene SWEET11. Conversely, the DELLA protein SLR1 inhibits the activation of these genes. The physical antagonism between GRF4 and SLR1 plays a crucial role in the homeostatic regulation of growth and carbon-nitrogen metabolism [63]. These pivotal regulatory factors have been deliberately selected during the breeding process. Therefore, in the current era of advanced biotechnology, the potential of utilizing tools such as gene editing to precisely reprogram key nodes of the regulatory network and optimize source–sink relationships for increased crop yield holds great promise.

5. Targeted Editing to Optimize Source–Sink Relationships for Soybean Yield Enhancement

The CRISPR/Cas9 system represents one of the most efficient genome editing technologies available today. Its core mechanism relies on the Cas9 endonuclease, guided by a guide RNA (gRNA), to specifically identify and bind to a target DNA sequence (the protospacer) within the genome. Once the gRNA pairs with the target sequence through complementary base pairing, the Cas9 protein cleaves the DNA double helix at this precise location, generating a double-strand break (DSB). The formation of a DSB immediately triggers the cell’s endogenous DNA repair mechanisms [68]. In most eukaryotes, including plants, Non-Homologous End Joining (NHEJ) is the primary pathway for repairing such damage. The NHEJ pathway is an inherently rapid but “error-prone” process. It attempts to re-ligate the broken DNA ends; however, this process often introduces random insertions or deletions (InDels) of a few nucleotides at the cleavage site. The occurrence of these small InDels often disrupts the open reading frame (ORF) of the gene’s coding region, leading to a frameshift mutation [68].

This technical strategy—studying gene function by inactivating a specific gene—is a core application of reverse genetics. By generating gene-knockout mutants in plants, researchers can systematically observe the resulting phenotypic changes, such as growth rate, organ morphology, flowering time, and tolerance to biotic and abiotic stresses including drought, salinity, and disease. A rigorous comparison of the phenotypic differences between the mutant and its wild-type (WT) counterpart enables precise inference of the gene’s critical role within a specific biological pathway or life process [69,70]. Given this, this review comprehensively outlines the recent advancements in understanding the physiological and morphological regulation by sugar molecules in soybean (

Table 1. Summary of Key Genes Affecting Sugar Perception and Transport in Soybeans.

Gene Family	Key Gene	Expression Tissue	Phenotype	Research Approach	Reference
HXK	GmHXK2	Roots	Reduce expression exacerbates salt stress phenotype.	Homology inference	[24]
HXK	GmHXK3	Pods	Unknown	Homology inference	[23]
HXK	GmHXK5	Flowers	Unknown	Homology inference	[23]
HXK	GmHXK11	Seeds, Root nodules	Unknown	Homology inference	[23]
HXK	GmHXK15	Flowers	Overexpression promotes root growth and alkali tolerance.	Homology inference	[23]
T6P	GmTPS6	Broadly expressed	Triggered by Phytophthora sojae effector to promote infection.	Experimental identification	[30]
SnRK1	GmSnRK1.1/GmSnRK1.2	Roots	Double knockout impairs root growth and reduces abiotic stress tolerance.	Homology inference	[38]
SnRK1	SnRK1a4	Roots	Enhance expression increases nodule size and nitrogenase activity.	Homology inference	[40]
TOR	GmTOR/GmS6K1	Root nodules	Phenotype not studied	Experimental identification	[45]
SUT	GmSUT1	Root nodules	Overexpression of GmSUT1 increases nodule number and dry weight and improves plant dry weight and nitrogen content. RNAi plants impaired sucrose transport and increased shoot sucrose concentration.	Homology inference	[60]
SWEET	GmSWEET3c	Root nodules	CRISPR knockdown significantly reduces infection threads and nodules.	Experimental identification	[61]
SWEET	GmSWEET10a/b	Seed coat	Knockout mutants have smaller seeds and reduced oil content.	Experimental identification	[57]
SWEET	GmSWEET30a/b	Seed coat	Double knockout mutants have reduced oil and protein content.	Experimental identification	[56]

). It identifies pivotal genes and pathways that impact source–sink relationships, crucial for governing soybean floral transition, seed development, plant architecture, source leaf photosynthetic performance, and symbiotic nitrogen fixation. These elements serve as promising targets for potential enhancement of soybean yield and quality through CRISPR/Cas9-mediated approaches. By employing precise single-point editing, multiplex combinatorial editing, or promoter editing on these targets, it is envisioned that prevailing yield constraints can be overcome, facilitating precise reprogramming of photoassimilate carbon partitioning patterns (Figure 2).

6. Conclusions and Perspectives

This review focuses on sugar sensing and transport as core modules. We systematically analyze the functional characteristics and molecular mechanisms of their key components, and their pivotal role in optimizing soybean source–sink relationships. Studies indicate that soybean yield formation is finely regulated by a complex regulatory network. Within this system, sugar signal sensors perceive the plant’s energy status, subsequently directing sugar transporters to precisely manage carbohydrate flow and allocation. This complex process is closely associated with internal and external signals such as photoperiod, hormones, and abiotic stress. While progress has been made in deciphering this network in model plants and major cereal crops, the understanding of this network in soybean remains limited. This limitation stems mainly from the biological complexity of soybean, as its genome has undergone paleo-polyploidization, resulting in multiple copies of many genes, such as those encoding sugar transporters. The resulting functional redundancy often means that a single-gene mutation is insufficient to produce a clear phenotype, which greatly increases the difficulty of functional validation.

To overcome this challenge, gene-editing technologies represented by the CRISPR/Cas system offer powerful tools. In particular, their capability for multiplex gene editing—achieved by designing multiple sgRNAs—provides a feasible path to dissecting functionally redundant genes. However, the application of this technology still faces challenges. Firstly, regarding specificity, the varying levels of specificity and promiscuity among different gRNAs directly impact mutation efficiency. Although optimizing gRNA design based on genomic data can minimize off-target effects, it cannot entirely eliminate their possibility. Secondly, regarding predictability, even expected on-target edits can lead to unintended negative effects. For example, the product of a gene “knocked out” via base editing or a small deletion might play other, unknown, critical roles within the cell. Such unexpected functional alterations could pose unpredictable risks to the environment, food, and feed. Despite the aforementioned challenges, gene editing remains an essential tool for elucidating gene functional redundancy and analyzing complex traits in soybean. However, a balance must be struck between efficiency and safety [71]. In the future, precise editing of sugar transporters and metabolic enzymes holds promise for fine-tuning sucrose balance to increase yield. Nevertheless, given the polygenic control of yield traits, it might be imperative to modify multiple regulatory elements to achieve more substantial and coordinated optimization of both source and sink.