Transcriptional Regulation of SWEET15_A01 by MYB44/bHLH3 Modulates Carbon Allocation in Cotton Ovule and Fibre to Affect Seed and Fibre Traits

Research Background

Cotton is one of the world's most important economic crops, serving not only as a primary raw material for the texte industry (cotton fibers) but also as a significant source of oil and protein feed from its seeds (cottonseeds). In cotton plants, developing seeds (ovuthe attached fibers are the two main "sink" organs that compete for carbohydrates (primarily sucrose) produced by "source" organs (leaves) through photosynthesis. Thce of carbon allocation between the ovule and fibers directly determines the yield and quality of cottonseeds (e.g., oil content) as well as the yield and quality of cott fibers (e.g., length, strength). However, a trade-off relationship often exists between seed development and fiber growth; how to synergistically improve these two important agronomic traits remains a core ge in cotton genetic breeding. Sugar transporters act as key "valves" regulating carbon allocation, with proteins of the SWEET (Sugars Will Eventually be Exported rter) family playing a central role. Although studies have shown that SWEET genes are involved in cotton development, the specific molecular mechanisms underlying their fine-tuning of carbon allocation during late particularly between the ovule and fibers, remain to be elucidated.

Introduction to Key Research Findings

1. SWEET15_A01 is a key sucrose transporter regulating the late stage of development. The research team first identified a gene named SWEET15_A01 as a key candidate gene affecting cotton seed oil content through QTL mapping and transcriptonalysis. Phylogenetic analysis indicated that this gene belongs to the third clade of the SWEET family, which is primarily responsible for sucrose transport. Functional complementation experiments in yts confirmed that SWEET15_A01 encodes a functional sucrose-specific transporter. Subcellular localization experiments showed that this protein is primarily localized to the ce, consistent with its function as a transmembrane transporter (Figure 1). Analysis of spatiotemporal expression patterns revealed that SWEET15_A01 is highessed mainly during the late stage of cotton ovule development (25-35 days after flowering), which coincides with the critical stage of massive accumulation of oil and protein in otton seeds, suggesting that it plays an important role in cotton seed filling.

2. SWEET15_A01 promoter variation is a key factor in the differentiation of agronomic traits between *Gossypium barbade* and *Gossypium hirsutum*. Researchers compared the sequence differences of SWEET15_A01 between high-oil-content *G. barbadense* and retively low-oil-content *G. hirsutum*. The results showed that the coding sequences were identical, but there were 8 variant sites in the promoter region, includinga critical 21-base-pair insertion/deletion (InDel). Haplotype analysis indicated that this 21-bp InDel could clearly separate the vast majoritf *G. barbadense* and *G. hirsutum* germplasm resources. Correlation analysis showed that varieties with the *G. barbadense*-type prmoter (21-bp deletion, Indel_D) had significantly higher seed oil content and seed index, but lower lint percentage. Further promoter activity analysis confirmed that the *G. adense*-type promoter exhibited significantly higher activity in the ovule than the *G. hirsutum*-type. This suggests that natural variation in the SWEET15_A01 promoter is a significant cause of differences in oil content and fiber yield between cotton species, and this 21-bp InDel can serve as an excellent molecular marker for improvington seed oil content (Figure 2).

3. The expression level of SWEET15_A01 finely regulates the allocation of carbon between the ovule and fibers. To directly verify the function of SWEET15_A01, the research team generated transgenic cotton overexpressing SWEET15_A01 (OE-SWEET15). Inngly, during the late stage of ovule development (25 DPA), the expression level of SWEET15_A01 was significantly downregulated in the transgenic linesexhibiting a typical "co-suppression" phenomenon. Phenotypic analysis revealed that these transgenic lines exhibited complex changes in agronomic traits: increased fiber initiation density, higher lint percentseed protein content, but shorter fibers, smaller seeds, and significantly reduced oil content (Figure 3). Sugar component analysis revealed that in the transgenic lines, sucrose accumulation eased in the ovules but increased in the fibers.

These results collectively indicate that SWEET15_A01 acts as a "master switch" regulating the allocation of carbon sourcestween the ovule and fibers. Downregulating its expression in the ovule hinders sucrose entry for oil synthesis (resulting in small seeds with low oil content), thereby rediing more sucrose to the fibers. The excessive accumulation of sucrose in fibers promotes fiber initiation on one hand, and may prematurely trigger secondary wall synthesis on the other, leading to the prematucessation of fiber elongation (resulting in shorter and thicker fibers). This finding profoundly reveals the central hub role of SWEET15_A01 in balancing seed and fiber deelopment.

4. The transcription factor MYB44 is a direct transcriptional repressor of SWEET15_A01. To investigate the upstream regulatory mechanim of SWEET15_A01, the research team identified an R2R3-MYB transcription factor, MYB44, through yeast one-hybrid screening. A series of molec experiments (dual-luciferase reporter assays, yeast one-hybrid, and electrophoretic mobility shift assays [EMSA]) confirmed that MYB44 direcbinds to the MBS cis-element in the SWEET15_A01 promoter region and significantly inhibits its transcriptional activity. Expression pattern analysis showed that MYB44 es negatively correlated with SWEET15_A01 during late ovule development, further supporting the conclusion that MYB44 acts as its transcriptional repress (Figure 5).

5. bHLH3 interacts with MYB44 to form a fine-tuning regulatory module. Interestingly, researchers identified a conserved bHLH protein interactmotif on the MYB44 protein. Through screening, they identified a bHLH-type transcription factor, bHLH3, which interacts strongly with MYB44 in . Functional experiments showed that bHLH3 alone cannot directly regulate SWEET15_A01, but when co-expressed with MYB44, it significantly alleviate inhibitory effect of MYB44 on the SWEET15_A01 promoter. This finding reveals a more refined regulatory pattern: MYB44 a as a "brake," while bHLH3 serves as a regulator that "releases the brake." Through their interaction, they form a dynamic transcriptional regulatory complex to jointly fine-tune thession level of SWEET15_A01 (Figure 6).

6. Functional Validation of MYB44 and Its Feedback Mechanism in Response to Sucrose Signaling

To ultimately verify the function of this regultory module, the research team generated transgenic cotton overexpressing MYB44 (OE-MYB44). Phenotypic analysis revealed that fiber length was significantly increased e transgenic lines, while cotton seeds became smaller and oil content decreased (Figure 7). This phenotype was entirely consistent with the expected outcome of SWEET15_A01 sussion in the ovule: overexpressed MYB44 inhibited the expression of SWEET15_A01 in the ovule, reducing carbon sources flowing to the ceds, thereby allocating more carbon sources to fibers and promoting fiber elongation. Furthermore, the study found that high sucrose concentrations could, in turn, inhibit the expression e MYB44 gene, forming an elegant negative feedback regulatory loop that allows cells to dynamically adjust carbon allocation strategies based on their sugar status (Figure 8).

Summary and Outlook

This study systematically elucidates a key molecular module regulating carbon partitioning and trait trade-offs in cotton. A comprehensive working model osed: during the late stages of cotton ovule development, the sucrose transporter SWEET15_A01 is responsible for pumping carbon sources into the ovule for lipid syranscription factor MYB44 acts as a repressor, directly binding to and shutting down the expression of SWEET15_A01 to restrict sucrose flto the ovule. Another transcription factor, bHLH3, interacts with MYB44 to weaken its repressive function, thereby "opening" the channel for sucrose flowto the ovule. The complex formed by MYB44 and bHLH3 functions like a precise "rheostat," dynamically regulating the activity o SWEET15_A01 and ultimately determining the flow and allocation balance of carbon sources between the competing "sinks" of fibers and cotton seeds (Figure 9).

This work not only profoundly reveals the molecular basis of the trade-off between cotton yield and quality at the theoretical level but also provides highly valuable targets and tooltton genetic improvement in practice. For instance, through gene editing technology, the *SWEET15_A01* promoter in upland cotton can be precisely modified inigh-activity Gossypium barbadense type, which is expected to directly increase seed oil content; alternatively, by finely regulating the expression of MY44 or bHLH3, the negative correlation between fiber development and seed oil content can be broken, opening up a new avenue for breeding new cotton varieties with high yield, superior quality, and excellent fd oil traits.