Microarray data from field grown plants.

<p>Microarrays performed on tissue sampled throughout the day from two <i>AtBBX32</i>-expressing lines (lines 1 and 2 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030717#pone-0030717-t001" target="_blank">Table 1</a>) in the field demonstrate 219 genes show 2–8 fold changes (8-fold is maximum change observed) in abundance in both transgenic events relative to the control and that the majority of these changes occur around ZT 0 (6 am). Dark bar represents genes increased in abundance and light bar represents genes decreased in abundance. All changes significant at a false discovery rate of 5 percent.</p

<i>AtBBX32</i> transgenic soybean plants increase yield components.

<p>Both field and growth chamber grown plants show increases in plant height and pod number in the transgenic lines relative to controls. Node number, flower number, seed number, and 100 seed weight were also increased in growth chamber grown plants. Growth chamber experiments were performed in a 10:14 hour photoperiod (Light∶Dark) with 900 mE of light. Data was collected from ten plant replicates that were randomized among entries in the chamber. Field grown plants were grown under standard agronomic conditions and ambient light. All differences between the transgenic lines and control are significant to p<0.05 unless otherwise indicated as (ns) not significant.</p

<i>AtBBX32</i> transgenic soybean plants demonstrate improved grain yield over non-transgenic controls.

<p>Mean yield values (kilograms per hectare) and percent improvement over controls for transgenic plots are shown for three growing seasons. The difference in the day of flowering (DOF) between the transgenic lines and control was calculated to determine delta DOF. The difference in day of final maturity (MAT) was examined in transgenic lines and compared to control to determine delta MAT (units = days). The low yielding event 4 produced no detectable transcript. N represents the number of environments tested. p-values were based on the difference between the transgenic lines and wildtype control.</p><p>*p≤0.05,</p><p>**p≤0.01.</p

<i>AtBBX32</i> expression in soybean delays leaf senescence and brown pod maturity.

<p>A) Field grown soybeans were visually assessed and scored every few days late in the season on a whole plot basis according to green leaf color. Leaf senescence was rated on a 1–9 scale based on whole plot appearance. 9 = dark green, no yellow leaves on the top canopy; 5 = 40 percent change yellow leaves, 10 percent change fallen leaves; 1 = more than 95 percent change fallen leaves. B) The same soybean plots were visually inspected for the appearance of brown pods and the percentage of the plot containing brown pods was determined. * Event 1 significantly different from control at p<0.05, † Event 2 significantly different from control at p≤0.05.</p

<i>AtBBX32</i> extends the reproductive period between R3 and R7 developmental stages in soybean resulting in a delay in final maturity compared to control.

<p>The timing of reproductive development was measured according to standard methods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030717#pone.0030717-Fehr1" target="_blank">[16]</a> in ten field plot replicates for each line. R1 is the initiation of flowering. R3 is the onset of pod development. R7 is the beginning of maturation. R8 is the stage where 95 percent of the pods are physiologically mature. The number of days to reach each developmental stage was calculated on a whole plot basis and the mean is indicated below, where units are days after planting.</p><p>*p<0.05.</p

Expression of <i>AtBBX32</i> in soybean affects the transcript abundance of central clock components near ZT 0.

<p>Levels of both central clock components <i>GmLCL2</i> (A) and <i>GmTOC1</i> (B) were assayed by quantigene RNA extraction and expression analysis from V2 leaf tissue harvested from soybean plants grown in a controlled environment. Growth chamber experiment was performed in a 14:10 hour photoperiod (Light∶Dark) with 650 mE of light. p-values based on the difference between both transgenic lines and wildtype control. * p≤0.05. Where error bars are not visible they are smaller than the data points.</p

Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

<div><p><i>ATHB17</i> (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.</p></div

Phenology of <i>ATHB17</i> events and control.

<p>Two independent <i>ATHB17</i> events in three hybrids were used in physiological studies conducted in 2011 and 2012 under standard agricultural practices (SAP) for corn production in the Central Corn Belt. The number of days to 50% silking and anthesis were measured and the number of days between anthesis and silking was calculated (ASI) each year for physiological studies conducted under standard agronomic practices conditions. Differences in phenology between <i>ATHB17</i> events and control were determined using an across year combined analysis using a mixed model ANOVA. N denotes the number of data points included per entry in the statistical analysis. Number of event data points were within ±3 of control data points. Results for individual hybrids per year are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094238#pone.0094238.s004" target="_blank">Table S2</a>.</p

Full- length ATHB17 protein functions as transcriptional repressor and ATHB17Δ113 can relieve repression caused by full-length ATHB17 protein.

<p>Maize mesophyll protoplasts were transformed (A) with 4 µg cells of reporter (Class II::GUS, Class I::GUS or No BS::GUS) and 0–5 µg cells of effector (Full-length ATHB17) or 5 µg of ATHB17Δ113 and <i>Renilla</i> luciferase (B) with 4 µg reporter (Class II::GUS, Class I::GUS or No BS::GUS), 0–5 µg ATHB17Δ113, and 0 (grey bars) or 0.2 µg (blue bars) of ATHB17 full length. DNA amounts are per 320,000 cells. After 18 h, cells were assayed for GUS and luciferase expression. GUS values were divided by luciferase internal control values for each well and normalized to respective GFP samples. Bars are means and error bars represent 1 SD.</p

List of differentially expressed genes in ear inflorescence in common between two <i>ATHB17</i> events.

<p>Table lists the fold change (Fc), raw and fdr p-values for common genes in each event.</p