Nucleosome positioning influences the access of transcription factors (TFs) to their binding sites and gene expression. Studies in plant, animal, and fungal models demonstrate similar nucleosome positioning patterns along genes and correlations between occupancy and expression. However, the relationships among nucleosome positioning, cis-regulatory element accessibility, and gene expression in plants remain undefined. Here, we showed that plant nucleosome depletion occurs on specific 6-mer motifs and this sequence-specific nucleosome depletion is predictive of expression levels. Nucleosome depleted regions in Arabidopsis thaliana tend to have higher G/C content, unlike yeast, and are centered on specific G/C-rich 6-mers, suggesting that intrinsic sequence properties, such as G/C content, cannot fully explain plant nucleosome positioning. These 6-mer motif sites showed higher DNase I hypersensitivity and are flanked by strongly phased nucleosomes, consistent with known TF binding sites. Intriguingly, this 6-mer specific nucleosome depletion pattern occurs not only in promoter but also in genic regions and is significantly correlated with higher gene expression, a phenomenon also found in rice but not in yeast. Among 6-mer motifs enriched in genes responsive to treatment with the defense hormone jasmonate, there are no significant changes in nucleosome occupancy, suggesting that these sites are potentially pre-conditioned to enable rapid response without changing chromatin state significantly. Our study provides the first global assessment of the joint contribution of nucleosome occupancy and 6-mer motifs that are likely cis-elements for control of gene expression. Our findings pave the way for further understanding the impact of chromatin state on plant transcriptional regulatory circuits.