Abstract
Kernel size is an important factor determining grain yield. Although a number of genes affecting kernel development in maize (Zea mays) have been identified by analyzing kernel mutants, most of the corresponding mutants cannot be used in maize breeding programs due to low germination or incomplete seed development. Here, we characterized small kernel7, a recessive small-kernel mutant with a mutation in the gene encoding the second-largest subunit of RNA polymerase III (RNAPΙΙΙ; NRPC2). A frame shift in ZmNRPC2 leads to a premature stop codon, resulting in significantly reduced levels of transfer RNAs and 5S ribosomal RNA, which are transcribed by RNAPΙΙΙ. Loss-of-function nrpc2 mutants created by CRISPR/CAS9 showed significantly reduced kernel size due to altered endosperm cell size and number. ZmNRPC2 affects RNAPIII activity and the expression of genes involved in cell proliferation and endoreduplication to control kernel development via physically interacting with RNAPIII subunits RPC53 and AC40, transcription factor class C1 and Floury3. Notably, unlike the semidominant negative mutant floury3, which has defects in starchy endosperm, small kernel7 only affects kernel size but not the composition of kernel storage proteins. Our findings provide novel insights into the molecular network underlying maize kernel size, which could facilitate the genetic improvement of maize in the future.
Highlights
State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, One sentence summary: ZmNRPC2 affects RNA polymerase (RNAP) activity and regulates the expression of genes involved in cell proliferation and endoreduplication to control maize kernel development
The conducting zone (CZ) cells were compacted lengthways (Fig. 1E, F) and endosperm cells located in the area immediately internal to the aleurone layer (AL) were more similar to AL cells than endosperm cells, because they were smaller and accumulated less starch (Fig. 1I, Fig. 2K). These results indicate that smk7 is a small-kernel mutant with normal embryonic structures, but the cell shape in basal endosperm transfer layer (BETL), CZ and peripheral of endosperm are different from WT
We have described a recessive mutant that represses endosperm cell proliferation, causes defects in the endosperm peripheral regions and BETL cell-wall ingrowths, and compacts the length of the CZ cells (Fig. 1E-1L)
Summary
ZmNRPC2 and dNRPC2 were localized to the cytoplasm and the nucleus (Supplemental Fig. S5), and the G/T mutation in smk did not affect their subcellular localization These results indicated that the peptide that was absent from the mutant. Transmission electron microscopy revealed that the number and size of starch granules and protein bodies in the endosperm of the three mutants were drastically smaller than that in WT (Fig. 6G, H) This indicates that the reduced kernel size of the three mutants is associated with the repression of cell proliferation and storage reserve filling. Scanning electron microscopy revealed that the starch granules in the endosperm were tightly embedded within the storage protein body matrix in WT and smk kernels, but were naked in fl and smk7;fl (Fig. 7B, C) These results demonstrate that the ratio of zeins/non-zeins was altered in fl kernels but not in smk. These results indicated that FL3 plays a major role in zein synthesis to control seed quality, and might affect cell proliferation
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