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).

Toward unveiling transcriptome dynamics and regulatory modules at the maternal/filial interface of developing maize kernel.

The basal region of maize (Zea mays) kernels, which includes the pedicel, placenta-chalazal, and basal endosperm transfer layers, serves as the maternal/filial interface for nutrient transfer from the mother plant to the developing seed. However, transcriptome dynamics of this maternal/filial interface remain largely unexplored. To address this gap, we conducted high-temporal-resolution RNA sequencing of the basal and upper kernel regions between 4 and 32 days after pollination and deeply analyzed transcriptome dynamics of the maternal/filial interface. Utilizing 790 specifically and highly expressed genes in the basal region, we performed the gene ontology (GO) term and weighted gene co-expression network analyses. In the early-stage basal region, we identified five MADS-box transcription factors (TFs) as hubs. Their homologs have been demonstrated as pivotal regulators at the maternal/filial interface of rice or Arabidopsis, suggesting their potential roles in maize kernel development. In the filling-stage basal region, numerous GO terms associated with transcriptional regulation and transporters are significantly enriched. Furthermore, we investigated the molecular function of three hub TFs. Through genome-wide DNA affinity purification sequencing combined with promoter transactivation assays, we suggested that these three TFs act as regulators of 10 basal-specific transporter genes involved in the transfer of sugars, amino acids, and ions. This study provides insights into transcriptomic dynamic and regulatory modules of the maternal/filial interface. In the future, genetic investigation of these hub regulators must advance our understanding of maternal/filial interface development and function.