Carbohydrate partitioning from source to sink tissues is essential for plant growth and development. However, in maize (Zea mays L.), the molecular mechanisms by which callose synthase genes regulate this process remain largely unexplored. This study demonstrates that mutation of maize callose synthase12 (ZmCals12) results in increased carbohydrate accumulation in photosynthetic leaves but decreased carbohydrate content in sink tissues, leading to plant dwarfing and male sterility. Histochemical β-glucuronidase (GUS) activity assay and mRNA in situ hybridization (ISH) revealed that ZmCals12 expression mainly occurs in the vascular transport system. ZmCals12 loss-of-function decreased callose synthase activity and callose deposition in plasmodesmatas (PDs) and surrounding phloem cells (PCs) of the vascular bundle. The drop-and-see (DANS) assay indicated reduced PD permeability in photosynthetic cells and diminished transport competence of leaf veins in Zmcals12 mutants, resulting in decreased symplastic transport. Paraffin section analysis revealed that less-developed vascular cells (VCs) in Zmcals12 mutants likely disrupted sugar transport, contributing to the pleiotropic phenotype. Furthermore, impaired sugar transport inhibited internode development by suppressing auxin (IAA) biosynthesis and signaling in Zmcals12 mutant. These findings elucidate the mechanism by which ZmCals12-mediated callose deposition and symplastic transport regulate maize growth and development.

Maize plant architecture influences planting density and, in turn, grain yield. Most of the plant architecture-related traits can be described as organ size. We describe a miniature maize mutant, Tiny plant 4 (Tip4), which exhibits reduced size of multiple organs and exhibits a semi-dominant monofactorial inheritance characteristic. Positional cloning confirmed that a 4-bp deletion in the NAC TF with transmembrane motif 1-Like (NTL) gene ZmNTL2, denoted as ZmNTL2^Δ, confers the Tip4 mutation. qRT-PCR showed that ZmNTL2 was expressed in all tested tissues. ZmNTL2 functions as a transcriptional activator and is located in both the nucleus and biomembranes. The mutation does not affect the mRNA abundance of ZmNTL2 locus, but it does result in the loss of transmembrane domain and confines the ZmNTL2^Δ protein to the nucleus. Knocking out ZmNTL2 has no effect on maize organ size development, indicating that the 4-bp deletion might be a gain-of-function mutation in organ size regulation. Combining transcriptome sequencing with cytokinin and auxin content determination suggests that the decreased organ size may be possibly mediated by changes in hormone homeostasis.

Callose contributes to many biological processes of higher plants including pollen development, cell plate and vascular tissue formation, as well as regulating the transport function of plasmodesmata. The functions of callose synthase genes in maize have been little studied. We describe a maize male-sterile mutant 39 (ms39) characterized by reduced plant height. In this study, we confirmed using CRISPR/Cas9 technology that a mutation in Zm00001d043909 (ZmCals12), encoding a callose synthase, is responsible for the male sterility of the ms39 mutant. Compared with male-fertile plants, callose deposition around the dyads and tetrads in ms39 anthers was significantly reduced. Increased cell autophagy observed in ms39 anthers may have been due to the premature programmed cell death of tapetal cells, leading to collapse of the anther wall structure. Disordered glucose metabolism in ms39 may have intensified autophagy in anthers. Evaluation of the ms39 gene on maize heterosis by paired-crossed experiment with 11 maize inbred lines indicated that ms39 can be used for maize hybrid seed production.

A nuclear-encoded sigma (σ) factor is essential for the transcriptional regulation of plant chloroplast-encoded genes. Five putative maize σ factors have been identified by database searches, but their functions are unknown. We report a maize leaf color mutant etiolated/albino leaf 1 (eal1) that was derived from space mutation breeding. The eal1 mutant displays etiolated or albino leaves that then gradually turn to normal green at the seedling stage. The changes in eal1 leaf color are associated with changes in photosynthetic pigment content and chloroplast development. Map-based cloning revealed that a single amino-acid deletion changing Val₄₈₀-Val₄₈₁-Val₄₈₂ to Val₄₈₀-Val₄₈₁, in the C-terminal domain σ₄ of the putative σ factor ZmSig2A, is responsible for the eal1 mutation. In comparison with the expression level of the wild-type (WT) allele ZmSig2A⁺ in WT plants, much higher expression of the mutant allele ZmSig2A^ΔV in eal1 plants was detected before the eal1 plants turned to normal green. ZmSig2A shows the highest similarity to rice OsSig2A and Arabidopsis SIG2. Ectopic expression of ZmSig2A⁺ or ZmSig2A^ΔV driven by the cauliflower mosaic virus 35S promoter rescued the pale green leaf of the sig2 mutant, but ectopic expression of ZmSig2A^ΔV driven by the SIG2 promoter did not. We propose that the Val deletion generated a new weak allele of ZmSig2A that cannot completely abolish the ZmSig2A function. Some genes involved in chloroplast development and photosynthesis-associated nuclear genes showed significant expression differences between eal1 and WT plants. We conclude that ZmSig2A encoding a σ factor is essential for maize chloroplast development. The eal1 mutant with a weak allele of ZmSig2A represents a valuable genetic resource for investigating the regulation of ZmSig2A-mediated chloroplast development in maize. The eal1 mutation may be useful as a marker for early identification and elimination of false hybrids or transgene transmission in the application of genetic male sterility to commercial hybrid seed production.