Sugar transporter modulates nitrogen-determined tillering and yield formation in rice

Nitrogen (N) is crucial for crop yield, but excessive N fertilizer use is environmentally damaging. Boosting shoot branching, or tillering, can improve yield under high N conditions. Previous research highlighted the role of *OsTCP19* in N-responsive tillering, but a significant tillering response persists in modern rice varieties lacking the high NUE *OsTCP19-H* allele, indicating other regulatory pathways. This study investigates the genetic basis of this N-regulated tillering, focusing on identifying genes that control tillering in response to varying nitrogen levels and exploring the underlying mechanisms. Understanding these mechanisms can lead to improved nitrogen use efficiency (NUE), crucial for sustainable agriculture and global food security. Rice yield is primarily determined by tiller number (panicle number), grain number per panicle, and grain weight. Tillering is complex, influenced by environmental factors such as N availability. Increased N promotes tiller outgrowth, improving effective panicle number and yield. This process involves phytohormones like auxin, strigolactone (SL), and gibberellin (GA), with GA acting as a tiller development inhibitor. The GA-NGR5 module is known to be involved, but the integration of N availability into this module remains unclear. This research aims to identify and characterize a key gene mediating the N-tillering relationship, focusing on potential interactions with the GA signaling pathway.

Extensive research has been conducted on the role of nitrogen in regulating plant growth and development, particularly in rice. Studies have shown that nitrogen is a crucial macronutrient that significantly impacts crop yield. However, excessive nitrogen fertilizer application can lead to environmental issues such as soil acidification, water eutrophication, and greenhouse gas emissions. Therefore, improving nitrogen use efficiency (NUE) is a major goal in sustainable agriculture. Previous studies have investigated the genetic basis of nitrogen-responsive tillering in rice, with *OsTCP19*, a transcription factor, being identified as a key player. Interestingly, an elite allele, *OsTCP19-H*, exhibiting higher NUE, is largely absent in modern cultivated rice. Research also highlighted the complex interplay of phytohormones, such as auxin, strigolactone, and gibberellin (GA), in regulating tillering. GA typically acts as an inhibitor of tiller development, and studies have explored the role of the GA signaling pathway and its downstream component, NGR5, in mediating nitrogen-induced tillering. However, the precise mechanisms by which rice integrates nitrogen availability into the GA-NGR5 module remain largely unknown. Existing literature provides a strong foundation for this study, highlighting the gaps in our understanding of N-responsive tillering and prompting the search for novel regulators.

This study employed a multiparent advanced generation intercross (MAGIC) population of rice, developed from four elite varieties with diverse tillering responses to N. The researchers first evaluated tiller number in these parental lines under low N (LN) and high N (HN) conditions. Subsequently, they measured tillering in the MAGIC population under both LN and HN conditions to assess phenotypic variation. Genome-wide association studies (GWAS) were performed to identify genetic loci associated with tiller number under HN conditions. Candidate genes within the significant locus were identified, and their expression patterns under different N levels were analyzed. Haplotype analysis was conducted to pinpoint the causal gene and its allelic variation. Further investigations included cis-acting element analysis of the promoter regions, transient dual-luciferase assays, and analysis of *OsSTP28* haplotype distribution in a large collection of rice accessions. Functional characterization of *OsSTP28* involved generating CRISPR/Cas9 knockout mutants and performing complementation experiments using different *OsSTP28* alleles. Subcellular localization of OsSTP28 was determined using rice protoplasts and its sugar transport activity was characterized through heterologous expression in yeast and Xenopus laevis oocytes. Rhythmic expression patterns of *OsSTP28* and sugar levels in shoot bases were analyzed under different N conditions using qRT-PCR and HPLC. The impact of *OsSTP28* on sugar distribution was assessed using 14C-glucose feeding experiments. Apoplastic and intracellular sugar levels were measured using a centrifugal method and UPLC. To investigate the molecular mechanism, RNA sequencing (RNA-seq) was performed on shoot bases from WT and *stp28* mutants under LN and HN conditions. Gene ontology (GO) enrichment analysis and gene set enrichment analysis (GSEA) were used to identify significantly enriched biological processes and pathways. The interactions between *OsSTP28*, gibberellin metabolism, and the transcription factor *OSH15* were further explored through CRISPR/Cas9 mutagenesis, electrophoretic mobility shift assays (EMSA), transactivation assays, and analysis of GA2-oxidase activity. Epigenetic regulation by H3K27me3 modification was investigated using ChIP-PCR. Field experiments were conducted to evaluate the contribution of *OsSTP28* to yield and NUE. Statistical analysis methods included two-tailed Student's t-test, Dunnett's multiple test, and Tukey's HSD test.

GWAS analysis identified *OsSTP28* (a sugar transporter) on chromosome 11 as strongly associated with tillering response to N. *OsSTP28* expression was negatively correlated with tillering; higher expression under low N and lower under high N. Two haplotypes, H (high response to N) and C (common response to N), were identified in the *OsSTP28* promoter region, with H associated with greater tillering response to N. *OsSTP28* knockout mutants (*stp28*) showed significantly increased tillering under both LN and HN conditions, confirming its role as a negative regulator. Complementation experiments using *OsSTP28* alleles showed that the H allele restored N-responsive tillering more effectively than the C allele. OsSTP28 was shown to be a plasma membrane-localized hexose transporter responsible for glucose influx. *OsSTP28* expression in shoot bases displayed a diurnal rhythm, peaking at night and declining during the day, mirroring a similar rhythmic pattern for sugars. *stp28* mutants showed increased glucose accumulation, particularly in the apoplast of shoot bases, at night. High N supply also increased apoplastic glucose. RNA-seq revealed that *OsSTP28* knockout activated GA catabolism by upregulating the expression of *GA2ox* genes. This, in turn, decreased levels of bioactive GA, contributing to enhanced tillering. The transcription factor *OSH15* was identified as a key regulator downstream of *OsSTP28*. *OSH15* negatively regulates *GA2ox* expression, and its expression was reduced in *stp28* mutants and under HN conditions, leading to increased *GA2ox* expression and GA catabolism. *OSH15* silencing was associated with increased H3K27me3 modification. Field experiments demonstrated that *stp28* mutants exhibited increased panicle number and yield, particularly under LN conditions. The elite *OsSTP28^H* allele contributed to improved N-responsive yield and NUE. Importantly, *OsSTP28* functions independently of *OsTCP19* in regulating N-responsive tillering.

This study successfully identifies *OsSTP28* as a crucial regulator of N-responsive tillering and yield in rice, acting independently of the previously known *OsTCP19* pathway. The findings highlight the intricate interplay between sugar transport, gibberellin metabolism, and epigenetic regulation in mediating plant responses to N availability. The identification of an elite *OsSTP28* allele with enhanced N sensitivity presents a promising target for crop improvement. The mechanistic insights provided by this research offer significant opportunities for targeted breeding strategies aimed at enhancing NUE and improving rice yield under diverse N conditions. The study's findings underscore the importance of considering multiple regulatory pathways in addressing the complex issue of optimizing N use in crops. Future research should focus on exploring the precise mechanisms of glucose sensing and signal transduction in this pathway, as well as investigating the potential for gene editing approaches to precisely manipulate *OsSTP28* expression for enhanced N use efficiency and yield in a wider range of rice cultivars.

This research demonstrates that *OsSTP28*, a sugar transporter, plays a critical role in regulating nitrogen-responsive tillering and yield in rice. The study reveals a novel regulatory pathway involving *OsSTP28*, glucose accumulation, *OSH15* silencing via H3K27me3 modification, and GA catabolism. An elite *OsSTP28* allele enhances these effects, providing a valuable target for crop improvement. These findings advance our understanding of N use efficiency and offer practical implications for breeding superior rice varieties. Future work should investigate the molecular mechanisms of glucose signaling and the potential for further optimization of *OsSTP28* function.

The study primarily focuses on a specific MAGIC population and a limited set of rice cultivars. While the findings are strongly supported by multiple lines of evidence, the generalizability of these results to other rice varieties and growing conditions requires further investigation. The precise mechanisms of glucose sensing and the role of apoplastic glucose in activating downstream signaling pathways warrant further exploration. The study also focuses on the role of *OsSTP28* in regulating tillering and yield but may not fully capture its other potential roles in rice physiology.