Genome-wide identification and expression profiling of auxin response factor (ARF) gene family in maize

<p>Abstract</p> <p>Background</p> <p>Auxin signaling is vital for plant growth and development, and plays important role in apical dominance, tropic response, lateral root formation, vascular differentiation, embryo patterning and shoot elongation. Auxin Response Factors (ARFs) are the transcription factors that regulate the expression of auxin responsive genes. The <it>ARF </it>genes are represented by a large multigene family in plants. The first draft of full maize genome assembly has recently been released, however, to our knowledge, the <it>ARF </it>gene family from maize (<it>ZmARF </it>genes) has not been characterized in detail.</p> <p>Results</p> <p>In this study, 31 maize (<it>Zea mays </it>L.) genes that encode ARF proteins were identified in maize genome. It was shown that maize <it>ARF </it>genes fall into related sister pairs and chromosomal mapping revealed that duplication of <it>ZmARFs </it>was associated with the chromosomal block duplications. As expected, duplication of some <it>ZmARFs </it>showed a conserved intron/exon structure, whereas some others were more divergent, suggesting the possibility of functional diversification for these genes. Out of these 31 <it>ZmARF </it>genes, 14 possess auxin-responsive element in their promoter region, among which 7 appear to show small or negligible response to exogenous auxin. The 18 <it>ZmARF </it>genes were predicted to be the potential targets of small RNAs. Transgenic analysis revealed that increased miR167 level could cause degradation of transcripts of six potential targets (<it>ZmARF3</it>, <it>9</it>, <it>16</it>, <it>18</it>, <it>22 </it>and <it>30</it>). The expressions of maize <it>ARF </it>genes are responsive to exogenous auxin treatment. Dynamic expression patterns of <it>ZmARF </it>genes were observed in different stages of embryo development.</p> <p>Conclusions</p> <p>Maize <it>ARF </it>gene family is expanded (31 genes) as compared to <it>Arabidopsis </it>(23 genes) and rice (25 genes). The expression of these genes in maize is regulated by auxin and small RNAs. Dynamic expression patterns of <it>ZmARF </it>genes in embryo at different stages were detected which suggest that maize <it>ARF </it>genes may be involved in seed development and germination.</p

Transposon Tagging of a Male-Sterility, Female-Sterility Gene, St8, Revealed that the Meiotic MER3 DNA Helicase Activity Is Essential for Fertility in Soybean.

The W4 locus in soybean encodes a dihydroflavonol-4-reductase (DFR2) that regulates pigmentation patterns in flowers and hypocotyl. The mutable w4-m allele that governs variegated flowers has arisen through insertion of a CACTA-type transposable element, Tgm9, in DFR2. In the w4-m line, reversion from variegated to purple flower indicates excision of Tgm9, and its insertion at a new locus. Previously, we have identified a male-sterile, female-sterile mutant among the selfed progenies of a revertant plant carrying only purple flowers. Co-segregation between Tgm9 and the sterility phenotype suggested that the mutant was generated by insertion of Tgm9 at the St8 locus. The transposon was localized to exon 10 of Glyma.16G072300 that shows high identity to the MER3 DNA helicase involved in crossing over. Molecular analysis of fertile branches from two independent revertant plants confirmed precise excision of Tgm9 from the st8 allele, which restored fertility. In soybean, the gene is expressed in flower-buds, trifoliate leaves and stem. Phylogenetic analysis placed St8 in a clade with the Arabidopsis and rice MER3 suggesting that St8 is most likely the orthologous MER3 soybean gene. This study established the utility of Tgm9 in gene identification as well as in forward and reverse genetics studies

A revertant plant showing a fertile branch.

<p>The transposon excised out of the <i>st8</i> allele in the bud that resulted in the fertile branch with multiple pods bearing viable seeds. All other branches on the MSFS plant remained sterile.</p

PCR amplification of the four genome walking libraries generated from the homozygous sterile bulk and the homozygous fertile bulk.

<p><b>A)</b> Results of the first PCR (PCR1) reaction with adaptor primer (AP1) and transposon specific primer (Trans R1). <b>B)</b> Results of the second PCR (PCR2) with nested adaptor primer (AP2) and nested transposon specific primer (Trans R2). M, 200 bp DNA Ladder; S, Sterile bulk (library generated from DNA of pooled 10 homozygous sterile plants); F, Fertile Bulk (library generated from DNA of pooled 10 homozygous fertile plants). Arrows indicate the bands specific to sterile plants. a, <i>Dra</i>1-sterile-AP2; b, <i>Eco</i>RV-sterile-AP2; c, <i>Stu</i>I-sterile-AP2-1; d, <i>Stu</i>I-sterile-AP2-2.</p

The multiple sequence alignment of soybean, rice, Arabidopsis and yeast MER3 helicases highlighting four conserved motifs commonly found in the MER3 helicases.

<p>There is 100% conservation of all the amino acid residues in four motifs among the four genes showing that the newly cloned <i>Glyma</i>.<i>16G072300 (St8)</i> gene is a MER3 DNA helicase. Only a part of the proteins is shown.</p

Semi-quantitative RT-PCR showing expression of <i>St8</i> in different soybean tissues.

<p><i>Glyma</i>.<i>16G072300</i> showed an expression pattern distinct from the other DEAH/D-box helicase genes (<i>Glyma</i>.<i>08g102300</i>, <i>Glyma</i>.<i>06g202500</i>, and <i>Glyma</i>.<i>01g038100</i>) in soybean. The <i>Elf1B</i> gene was used to confirm equal loading of RNA for each sample.</p

PCR analysis of two revertant plants.

<p><b>A)</b> Graphical representation showing locations of two PCR primers (Rev1 and Rev2) flanking the insertion site in <i>Glyma</i>.<i>16G072300</i> (the <i>St8</i> gene) and a third primer (Trans R1) located in <i>Tgm9</i>. With no transposon present, the primers Rev1 and Rev2 should amplify a 491 bp fragment. With <i>Tgm9</i> present, the primers Rev1 and Trans R1 should amplify a 601 bp fragment. The <i>Tgm9</i> insertion is not drawn to scale. <b>B)</b> PCR amplification of fragments from sterile and fertile branches of two revertant plants (A11-180-1 and A11-1036) using primers Rev1, Rev2 and Trans R1. In the Williams 82 sample the transposon is not present, so only 491 bp fragment is amplified. The fertile branches of A11-180-1 and A11-1036 are heterozygous, therefore both the fragments representing absence of <i>Tgm9</i> (491 bp fragment) and presence of <i>Tgm9</i> (601 bp fragment) were amplified. The sterile branches are homozygous for the insertion of <i>Tgm9</i>, so only 601 bp fragment is amplified. F, fertile branch; S, sterile branch.</p

The graphical representation of <i>Glyma</i>.<i>16G072300</i>.

<p>The gene has 28 predicted exons and the transposon <i>Tgm9</i> is inserted into exon 10. The <i>Tgm9</i> insertion is not drawn to scale.</p

Phylogenetic tree of homologs of Glyma.16G072300 (St8).

<p>The unrooted radiated phylogenetic tree shows the relationships among the soybean helicase, St8 and homologous proteins in soybean and other taxa. The tree was generated using the Neighbor-Joining method in the MEGA6 software. Numbers on the nodes represent percent bootstrap support in a 10,000 replicate bootstrap test, and the sum of branch lengths is 9.42170767. The evolutionary distance represented by the scale bar shows 0.2 amino acid substitutions per site.</p