Fig 3. Expression analysis of H2B.S histone variants in Arabidopsis, rice and maize.

(A) RT-PCR analysis of HTB8 expression in Arabidopsis tissues. GAPC is served as an endogenous control. DAP: days after pollination. Seedling, root and rosettes were collected as described in Fig 2B. At least 150 mature anthers and 30 mature pistils were used for RNA extraction. Seeds were collected based on the days after pollination, with at least 500 seeds at 2DAP and 5DAP, and 250 seeds at 20DAP used for RNA extraction. (B) Expression of HTB2, HTB3 and HTB8 during seed maturation. Data represents relative expression levels normalized to UBQ10, error bars represent the standard deviation (SD) from three biological replicates. At least 500 seeds at 5DAP and 10DAP, and 250 seeds at 15DAP and 20DAP were used in each biological replicate. (C) Expression of HTB2, HTB3, and HTB8 during seed storage and imbibition. Data represents relative expression levels normalized to UBQ10, error bars represent the SD from three biological replicates. At least 250 seeds at each time point were used in each biological replicate. (D) H2B.8-RFP expression in cotyledon nuclei of developing embryos at 18 DAP. DAPI staining indicates the position of nuclei. (E) H2B.8-RFP expression in a mature embryo alongside a corresponding DAPI stained image. (F-G) Expression of HTB8 relatives in rice (Oryza sativa) and maize (Zea mays) in selected tissues and different stages of seed development, respectively. Data represents microarray transcriptomes deposited in Genevestigator. (H) Modelled structure of an H2B.8-containing nucleosome with a close-up view of the predicted location of highly conserved amino acids in H2B.S orthologs, which are highlighted in red. Yellow indicates histone H2A, green indicates histone H2B, pink indicates histone H3 and blue indicates histone H4. The position of the ten highly conserved H2B.S amino acids within the nucleosome are marked in red.