Central Roles of ZmNAC128 and ZmNAC130 in Nutrient Uptake and Storage during Maize Grain Filling.

Grain filling is critical for determining yield and quality, raising the question of whether central coordinators exist to facilitate the uptake and storage of various substances from maternal to filial tissues. The duplicate NAC transcription factors ZmNAC128 and ZmNAC130 could potentially serve as central coordinators. By analyzing differentially expressed genes from zmnac128 zmnac130 mutants across different genetic backgrounds and growing years, we identified 243 highly and differentially expressed genes (hdEGs) as the core target genes. These 243 hdEGs were associated with storage metabolism and transporters. ZmNAC128 and ZmNAC130 play vital roles in storage metabolism, and this study revealed two additional starch metabolism-related genes, sugary enhancer1 and hexokinase1, as their direct targets. A key finding of this study was the inclusion of 17 transporter genes within the 243 hdEGs, with significant alterations in the levels of more than 10 elements/substances in mutant kernels. Among them, six out of the nine upregulated transporter genes were linked to the transport of heavy metals and metalloids (HMMs), which was consistent with the enrichment of cadmium, lead, and arsenic observed in mutant kernels. Interestingly, the levels of Mg and Zn, minerals important to biofortification efforts, were reduced in mutant kernels. In addition to their direct involvement in sugar transport, ZmNAC128 and ZmNAC130 also activate the expression of the endosperm-preferential nitrogen and phosphate transporters ZmNPF1.1 and ZmPHO1;2. This coordinated regulation limits the intake of HMMs, enhances biofortification, and facilitates the uptake and storage of essential nutrients.

The transcription factors ZmNAC128 and ZmNAC130 coordinate with Opaque2 to promote endosperm filling in maize.

Endosperm filling in maize (Zea mays), which involves nutrient uptake and biosynthesis of storage reserves, largely determines grain yield and quality. However, much remains unclear about the synchronization of these processes. Here, we comprehensively investigated the functions of duplicate NAM, ATAF1/2, and CUC2 (NAC)-type transcription factors, namely, ZmNAC128 and ZmNAC130, in endosperm filling. The gene-edited double mutant zmnac128 zmnac130 exhibits a poorly filled kernel phenotype such that the kernels have an inner cavity. RNA sequencing and protein abundance analysis revealed that the expression of many genes involved in the biosynthesis of zein and starch is reduced in the filling endosperm of zmnac128 zmnac130. Further, DNA affinity purification and sequencing combined with chromatin-immunoprecipitation quantitative PCR and promoter transactivation assays demonstrated that ZmNAC128 and ZmNAC130 are direct regulators of 3 (16-, 27-, and 50-kD) γ-zein genes and 6 important starch metabolism genes (Brittle2 [Bt2], pullulanase-type starch debranching enzyme [Zpu1], granule-bound starch synthase 1 [GBSS1], starch synthase 1 [SS1], starch synthase IIa [SSIIa], and sucrose synthase 1 [Sus1]). ZmNAC128 and ZmNAC130 recognize an additional cis-element in the Opaque2 (O2) promoter to regulate its expression. The triple mutant zmnac128 zmnac130 o2 exhibits extremely poor endosperm filling, which results in more than 70% of kernel weight loss. ZmNAC128 and ZmNAC130 regulate the expression of the transporter genes sugars that will eventually be exported transporter 4c (ZmSWEET4c), sucrose and glucose carrier 1 (ZmSUGCAR1), and yellow stripe-like2 (ZmYSL2) and in turn facilitate nutrient uptake, while O2 plays a supporting role. In conclusion, ZmNAC128 and ZmNAC130 cooperate with O2 to facilitate endosperm filling, which involves nutrient uptake in the basal endosperm transfer layer (BETL) and the synthesis of zeins and starch in the starchy endosperm (SE).