Phylogenetic tree and gene organization of 14-3-3 proteins.

<p>The unrooted phylogenetic tree was constructed with maize 14-3-3 proteins from sorghum (Sb14-3-3), maize (Zm14-3-3), foxtail millet (Si14-3-3), <i>Arabidopsis</i> (GRF) and rice (GF). The intron-exon positions of respective members are shown in the right.</p

A model based on phosphorylation of S122 and/or S124 in 14-3-3 for deciding the cellular location of SiRSZ21A.

<p>When phosphorylation of SiRSZ21A was prevented, 14-3-3 proteins could not bind to SiRSZ21A and therefore SiRSZ21A was localized in nucleus. Another possibility for cytoplasmic retention of SiRSZ21A may be due to the masking effect of 14-3-3 on MOS14 binding site in phosphorylated SiRSZ21A.</p

Sub-cellular localization of mutants showing nucleus localization.

<p><b>I</b><i>m1</i>, <b>II</b><i>m2</i>, <b>III</b><i>m3</i>; <b>A</b> whole cell, <b>B</b> magnified view of the nucleus. More accumulation was seen in speckle region.</p

Chromosomal distribution and duplication of <i>14-3-3</i> genes.

<p><b>a</b> Maize. <b>b</b> Sorghum. <b>c</b> Foxtail millet. Vertical bars represent chromosomes and the values at left indicate the position of genes in megabase. Genes are marked in the right and Zm, Sb and Si denotes <i>Zea mays</i>, <i>Sorghum bicolor</i> and <i>Setaria italica</i>, respectively. Lines denote segmental duplication, whereas tandemly duplicated genes are highlighted in boxes.</p

Si14-3-3 and mutant SiRSZ21A (<i>m2</i> and <i>m3</i>) co-expressed in onion epidermal cells.

<p>Localization of m2 in nucleus is evidenced. <b>A</b> whole cell, <b>B</b> magnified view of nucleus.</p

Tissue-specific expression profiles of <i>14-3-3</i> genes.

<p><b>a</b> Sorghum. <b>b</b> Maize. <b>c</b> Foxtail millet. Illumina RNA-seq data of different tissues of these crops were re-analyzed and heat map was generated. Bar at the right represents log₂ transformed values, thereby values -3.0, 0.0 and 3.0 represent low, intermediate and high expression, respectively.</p

Yeast two-hybrid assay between Si14-3-3_f and SiRSZ21A.

<p>Y2H gold was co-transformed with both the constructs. Interaction was confirmed by culturing the co-transformants on selective media. Empty-BD constructs were used against the SiRSZ21A as controls. Autoactivation was also checked for SiRSZ21A.</p

<i>In vivo</i> BiFC analysis for confirming interaction of Si14-3-3_f protein with SiRSZ21A splicing factor.

<p><b>A</b> whole cell, <b>B</b> nucleus, shows the interaction in onion epidermal cells. <b>C.</b> interaction in <i>N</i>. <i>benthamiana</i> leaf.</p

Subcellular localization of candidate Si14-3-3 proteins in onion epidermal cells.

<p>Onion epidermal cells were transiently transformed with constructs containing vector <i>pAMPAT-MCS</i>-14-3-3:YFP through particle bombardment method. Subcellular localization of respective Si14-3-3:YFP fusion proteins were viewed using fluorescent confocal microscope.</p

Comparative physical mapping of <i>14-3-3</i> genes.

<p>Orthologous relationship of foxtail millet <i>14-3-3</i> genes (Sit) with the genes of sorghum (Sbi), maize (Zma), rice (Osa) and <i>Brachypodium</i> (Bdi). The coloured blocks represent the chromosomes.</p