NKD Transcription Factors Are Central Regulators of Maize Endosperm Development

Abstract

NAKED ENDOSPERM1 (NKD1) and NKD2 are duplicate INDETERMINATE DOMAIN (IDD) transcription factors important for maize (Zea mays) endosperm development. RNA-seq analysis of the nkd1 nkd2 mutant endosperm revealed that NKD1 and NKD2 influence 6.4% of the transcriptome in developing aleurone and 6.7% in starchy endosperm. Processes regulated by NKD1 and NKD2 include gene expression, epigenetic functions, cell growth and division, hormone pathways, and resource reserve deposition. The NKD1 and NKD2 proteins bind a consensus DNA sequence of TTGTCGT with slightly different properties. This motif was enriched in the promoters of gene transcripts differentially expressed (DE) in mutant endosperm. DE genes with a NKD binding motif in the 5' promoter region were considered as likely direct targets of NKD1 and NKD2 regulation, and these putative direct target genes were notably enriched for storage proteins. Transcription assays demonstrate that NKD1 and NKD2 can directly regulate gene transcription, including activation of opaque2 and viviparous1 promoters. NKD2 functions as a negative regulator of nkd1 transcription, consistent with previously reported feedback regulation. NKD1 and NKD2 can homo- and heterodimerize through their ID domains. These analyses implicate NKD1 and NKD2 as central regulators of gene expression in developing maize endosperm.

Figure 1.

Summary of Transcriptomic Analysis (A) Shared and unique differentially expressed genes in nkd1-R nkd2-R mutant AL and SE. (B) Proportion of genes with ≥1 read counts detected in RNA-seq (expressed) to number of DE genes in AL and SE. (C) Scatterplot of RNA-seq transcript abundance fold change values in nkd1-R nkd2-R mutant AL relative to wild-type AL (on the x axis) against RT-PCR expression values (on y axis) for the15 RNA-seq DE genes in nkd1 nkd2 mutant AL tested by qRT-PCR.

Figure 2.

Pathway Analysis of Differentially Expressed Gene Transcripts Log₂ fold change heat maps of differentially expressed genes functioning in selected disrupted pathways in nkd1 nkd2 mutants. Aleurone (A) and starchy endosperm (B).

Figure 3.

Disrupted Nutrient Reserve Deposition in nkd1 nkd2 Mutant Endosperm (A) Opaque endosperm phenotype in wild-type and nkd1-Ds nkd2-Ds0297 mutant kernels from an F2 segregating ear (B) Difference plots of nkd1-Ds nkd2-Ds0297 mutant minus wild-type mean starch branch chain length abundance. Asterisk denotes statistically significant differences (P < 0.05) determined by Student’s t test. (C) to (F) Scanning electron microscopy of starch grains of mature segregating nkd1-Ds nkd2-Ds0297 mutant ([C] and [E]) and wild type ([D] and [F]) kernels. Arrows indicate hollow core in bisected starch granules.

Figure 4.

Marker Transgene Expression in Wild-Type and nkd1 nkd2 Mutant Endosperm. Florescence microscopy using narrow violet-broad range (NV) filter to visualize autofluorescence and an mCherry filter to visualize FL2-RFP (A) and GL3B-RFP (B) or a YFP florescence filter for RAB17-YFP (C). Bars = 100 μm.

Figure 5.

NKD1 and NKD2 DNA Binding. (A) and (B) SAAB-MEME derived BCSs for NKD1ID (A) and NKD2ID (B). (C) Oligonucleotide sequences used as probes for EMSAs. The wild type contains the consensus binding sequence, while M1 to M8 contain base substitutions indicated by magenta lettering. Binding, relative to the wild type for a given protein is indicated by +++ (>66%), ++ (33 to 66%), + (<33%), and – (no detected shift). (D) to (G) EMSAs. (D) and (F) EMSA of consensus (wild type) and mutant probes for NKD1-ID (D) and NKD2-ID (F) proteins. (E) and (G) Competition assays using labeled wild-type probe, and 50-, 100-, or 500-fold excess of unlabeled wild type, M2, or M8 oligonucleotide, incubated with NKD1ID (E) or NKD2ID (G) protein. The + indicates reaction with no competitor, and – indicates negative control with GST protein instead of NKD. Overexposed images, additional EMSAs, and negative controls using purified GST are shown in Supplemental Figures 8 and 9.

Figure 6.

NKD1 and NKD2 Protein BiFC and Co-Pull-Down. (A) Transient BiFC assays in onion epidermal cells for full-length NKD1 and NKD2 proteins. Vectors used for each BiFC assay are listed to the left of each row. Arrows designate nuclei viewed under differential interference contrast (DIC). mCherry florescence marks transient cells transformation and YFP florescence indicates a positive protein-protein interaction. Control bombardments containing NYFP + NKD1-CYFP or NKD2-NYFP + CYFP did not produce YFP fluorescence. Bars = 100 μm. Additional controls are shown in Supplemental Figure 12. (B) and (C) Co-pull-down immunoblots for full-length NKD1 and NKD2 proteins (B) and NKD1-ID and NKD2-ID proteins (C). Affinity column and antibody (Ab) used for each assay are indicated at the top of each panel. Total soluble bacterial lysates (inp) were immunoblotted as a positive control for protein expression. The first set of control lanes show no cross-reaction between NKD-GST protein with anti-6xHis antibody or NKD-6xHis protein with anti-GST antibody.

Figure 7.

NKD1 and NKD2 Transcription Assays. (A) Schematic of constructs transfected into aleurone protoplasts for transient reporter assays of NKD1 and NKD2 transregulatory activity on selected direct target promoters. Effector, reporter, and normalization constructs were cotransfected. The null construct was substituted for the effector construct as a negative control. See Supplemental Table 14 for details on promoters used for each reporter construct. (B) Relative luciferase activities. Reporter activity (firefly LUC) is shown in proportion to the normalization standard (Renilla LUC). Error bars represent standard errors of the means for three biological replicates. Reporter and effector constructs used for each assay are listed. Asterisk designates statistically significant difference (P = 0.05, Student’s t test) relative to control (35S_pro:null) or between NKD1 (35S_pro:NKD1) and NKD2 (35S_pro:NKD2) effectors.

Figure 8.

A NKD Binding Motif Is Required for Transcriptional Activation. (A) Reporter constructs used to test the requirement of a NKD binding site for transcriptional activation of the vp1 promoter by NKD1 and NKD2. The mutant vp1 promoter construct (vp1-mut_pro:LUC) was cloned with the thymine in the 7th position of the second NKDcore BCS in the vp1_pro:LUC construct substituted with a cytosine [TTGTCG(T) to TTGTCGC]. (B) Activities of NKD1 and NKD2 on expression of wild-type and mutant vp1 promoter constructs. Reporter and effector constructs are listed. Error bars represent standard errors of the means. Asterisk designates statistically significant (P = 0.05, Student’s t test) difference relative to control (35S_pro:null) or between NKD1 and NKD2 effectors.