Maize (Zea mays), a C4 photosynthetic plant, uses C4 carbon assimilation to perform carbon fixation. Its leaf photosynthetic cells possess Kranz anatomy, which comprises two distinct photosynthetic cell types: mesophyll and bundle sheath. The mesophyll (M) cells, located beneath the leaf epidermis, surround the bundle sheath (BS) cells, while the BS cells enclose the veins (Langdale and Nelson, 1991; Braun and Slewinski, 2009; Garner et al., 2016). Transitory starch primarily accumulates in the BS cells, while sucrose is synthesized in mesophyll cells (Rhoades and Carvalho, 1944; Lunn and Furbank, 1999; Furbank and Kelly, 2021).

Carbohydrate partitioning refers to the process of carbon assimilation, transport and distribution from photosynthetic source tissues to non-photosynthetic sink tissues. Sucrose is the primary carbohydrate transported over long distances via the phloem, supporting plant growth, development and host-pathogen interactions (Braun and Slewinski, 2009; Julius et al., 2017; Chen et al., 2024). In maize leaves, phloem loading occurs through abaxial bundle sheath cells (Bezrutczyk et al., 2021). Phloem loading of sucrose includes both symplastic and apoplastic pathways. Sucrose moves symplastically through plasmodesmata (PD) from mesophyll cells into the BS cells and vascular parenchyma (VP) cells. Subsequently, sucrose is effluxed into the apoplastic space via SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERs (SWEETs) and then imported into companion cells (CC) via SUCROSE TRANSPORTER (SUT). Finally, sucrose moves through PD into the sieve elements (SE) for long-distance translocation from source to sink tissues (Braun and Slewinski, 2009; Braun et al., 2014; Braun, 2022).

Using forward genetic approaches, a number of genes controlling carbohydrate partitioning have been identified, and their mutants exhibit hyperaccumulation of carbohydrates in leaves, leading to chlorotic leaves, lower photosynthetic capacity, and premature senescence (Braun et al., 2006; Baker and Braun, 2008; Slewinski et al., 2009; Bezrutczyk et al., 2018; Tran et al., 2019; Julius et al., 2021; Qin et al., 2022). Among them, several mutants exhibit impaired PD-mediated symplastic phloem loading. The sucrose export defective1 (sxd1) mutant shows ectopic callose deposition, leading to PD blockage between BS and VP cells (Russin et al., 1996; Provencher et al., 2001; Ma et al., 2008). Carbohydrate partitioning defective1 (Cpd1) mutants accumulate callose occlusions in developing phloem (Julius et al., 2018). Tie-dyed1 (Tdy1) and Tdy2 function within the same genetic pathway. Mutants of these genes develop stable, nonclonal variegated yellow and green leaf sectors. Yellow sectors exhibit excessive accumulation of soluble sugars and starch, whereas green sectors remain unaffected. Notably, both tdy1 and tdy2 mutants show no physical blockage or structural alteration along the symplastic transport pathway (Braun et al., 2006; Baker and Braun, 2007, 2008; Ma et al., 2009). Tdy2 functions in vein development and affects symplastic trafficking within the phloem of maize leaves (Slewinski et al., 2012). The cpd33 mutant displays reduced PD between CC and SE in minor veins (Tran et al., 2019). Brittle Stalk2-Like3 (BK2L3) is required for proper cellulose deposition and cell wall formation during phloem development (Julius et al., 2021). Additionally, ZmSUT1, ZmSUT2, and ZmSWEET13a/b/c encode sucrose transporters for apoplastic phloem loading, and their mutants hyperaccumulate starch and soluble sugars in leaves (Slewinski et al., 2009; Slewinski et al., 2010b; Baker et al., 2016; Leach et al., 2017; Bezrutczyk et al., 2018). Zpu1, SBE2a and ZmPHOH encode starch-metabolizing enzymes (pullulanase-type starch-debranching enzyme, starch-branching enzyme, and starch phosphorylase, respectively), and their mutations fail to degrade starch nocturnally, causing hyperaccumulation of transitory starch (Dinges et al., 2003; Yandeau-Nelson et al., 2011; Qin et al., 2022). Collectively, these genes appear to regulate carbohydrate partitioning via symplastic/apoplastic phloem loading and starch metabolism.

Fumarylacetoacetate hydrolase, an enzyme involved in the final step of the tyrosine (Tyr) degradation, catalyzes the hydrolysis of fumarylacetoacetate into fumarate and acetoacetate (Lindblad et al., 1977; Dixon and Edwards, 2006). In humans, FAH deficiency causes accumulation of toxic metabolites (e.g., succinylacetone, maleylacetoacetate, and fumarylacetoacetate), leading to tyrosinaemia type 1, a disorder affecting the liver and kidneys (Lindblad et al., 1977; Grompe and al-Dhalimy, 1993; Grompe, 2001; de Laet et al., 2013). Compared to extensive research on FAH in microbes and animals, its function in plants remains largely unknown. Mutation of FAH in Arabidopsis leads to spontaneous cell death (Han et al., 2013), while loss-of-function of OsFAH causes sterility in rice (Hu et al., 2021). Recent studies demonstrate that wheat fumarylacetoacetate hydrolase (TaFAH) positively regulates Fusarium head blight resistance (Shang et al., 2025).

In this study, we found some recombinant inbred lines (RILs) from a maize-teosinte BC2S3 population showed chlorotic leaf spots (CLS). Quantitative trait locus (QTL) mapping and map-based cloning revealed that the CLS phenotype in this population is controlled by a single gene Chlorotic Leaf Spot1 that encodes a putative FAH. The teosinte parental allele is a weak allele of CLS1 that causes chlorotic leaf spots. We showed that CLS1 mutation leads to hyperaccumulated carbohydrates in leaves, reduced photosynthetic efficiency, disrupted tyrosine metabolism, ectopic callose accumulation and impaired plasmodesmata ultrastructure between mesophyll, bundle sheath and vascular parenchyma cells. Our findings demonstrate CLS1’s essential role in maintaining PD-mediated symplastic phloem loading and carbohydrate partitioning, highlighting an important role for FAH in plant development and metabolism.