An alternative transcription start site in the third exon of ATHB2 gives rise to a shade sensitive microProtein.

(A) 5’PEAT-seq reads map to 12,398 unique TSSs, 43% representing novel TSSs and 1.5% being candidates for alternative microProteins. (B) 5’PEAT-seq reads mapping to ATHB2. (C) Iso-seq reads mapping to ATHB2. (D) Design of constructs with GUS fused to either the promoter of ATHB2 (pATHB2::GUS), the sequence upstream of an ATG in the second intron of ATHB2 (pATHB2control::GUS; negative control), or the sequence upstream of a near-cognate start codon TTG in the third exon of ATHB2 (pATHB2miP::GUS). (E) GUS staining in pATHB2::GUS accumulates in the hypocotyl, SAM and cotyledon vasculature. (F) GUS staining in pATHB2control::GUS does not produce any GUS signal. (G) GUS staining in pATHB2miP::GUS plants accumulates in the SAM and hypocotyl. Scale bars = 200 µM.

ATHB2 and ATHB2miP localise to the nucleus and interact in vitro and in planta.

(A) Graphic representation of eGFP fused to ATHB2 variants: ATHB2, ATHB2miP, and ATHB2HD; panels show subcellular localisation of ATHB2 variants when fused to eGFP in tobacco leaves, with nuclear localisation shown in square and enlarged in lower panel. Arrows point to nuclear speckles that form when eGFP-ATHB2miP is expressed. Scale bars: upper panel = 50 µM; lower panel = 5 µM. (B) Yeast-2-hybrid with growth on histidine drop-out media when ATHB2 or ATHB2miP are fused to the activation domain and binding domain of GAL4. (C) FRET-FLIM assay. Co-expression of eGFP-ATHB2 with mCherry-ATHB2 or mCherry-ATHB2miP in tobacco leaves shows co-localization in the nucleus and causes significant reduction in fluorescent lifetime of eGFP-ATHB2. Scale bar = 5 µM. Significance levels: * = p<0.05, ** = p<0.01, *** = p<0.001, ns = not significant.

Gene expression profiling of the t-athb2 and 35S::miP lines.

(A) Heatmap showing expression changes in 95 genes whose expression depends on both the genotype and the shade treatment. Lowly-expressed genes are in blue, highly-expressed genes are in yellow. (B) 3 of the 17 clusters generated with MFuzz. Expression changes of the genes included in each cluster are on the y-axis. The 3 genotypes and shade conditions are on the x-axis. (C, D, E) Venn diagrams showing the number of genes that are upregulated and downregulated in each of the three genotypes included in the RNA-Seq and the overlap between them in WL, and 45 and 90 mins of WL+FR.

ATHB2 and ATHB2mip mediate hypocotyl elongation and root growth.

(A) Graphic representation of the gene structure of ATHB2. Location of T-DNA insertion (SALK_106790) is shown and below that the predicted protein structure of WT, transgenic and mutant isoforms of ATHB2. Blue and red triangles indicate Cas9 targeted sites. (B) RT-qPCR of ATHB2 in mutant and transgenic plants with and without WL+FR treatment. Coloured regions correspond to those in part A. (C) Plots of hypocotyl length in plants after 5 days growth in three different shade regimes: deep shade, canopy shade, and proximity shade. (D) Plot of the number of lateral roots in plants after 11 days of growth in WL or WL+FR (proximity shade). Letters in all signify results of pairwise comparisons by two-way ANOVA.

ATHB2 and ATHB2miP have a role in iron transport regulation and homeostasis.

(A) Heatmap showing the expression levels in white light of genes upregulated in both the t-athb2 mutant and the 35S::miP lines that are involved in iron transport, iron homeostasis and iron deficiency response. Lowly expressed genes are coloured blue, highly expressed genes are coloured red. Additionally, the expression level for all three replicates per genotype is displayed. (B) Heatmap showing the gene expression levels in shade conditions. (C) Concentration of Fe (mg/g) measured in the roots (grey) and the shoots of seedlings of the three genotypes (green). (D) Plots of hypocotyl length (red) and root length (blue) of plants grown on low or high iron supplemented ½ MS media in WL and WL+FR. Hypocotyls were measured after five days in either condition whilst roots were measured after nine days.

Identification of an alternative ORF in the coding sequence of ATHB2.

(A) 5’RACE carried out on 10 day old seedlings that were exposed to 0, 45 and 90 mins of WL+FR light. Red asterisk signifies the small transcript arising from ATHB2 that when purified and sequenced corresponds to the underlined portion of ATHB2 in part B. (B) Genomic sequence of ATHB2. The exonic sequence with protein sequence below is shown in bold. Double-sided blue arrows denote sites corresponding to 5’PEAT-seq read clusters. Blue highlighted sequence denotes the leucine zipper domain. Red circles indicate two alternative non-canonical start codons identified by TIS predictor. Underlined sequence corresponds to the transcript purified from 5’RACE in part A.

RNA-seq reads corresponding to ATHB2 at 0, 45 and 90 mins of WL+FR.

RNA-seq reads peak in the WT at 45 mins of WL+FR and then decrease (see y axis for maximum reads) at 90 mins. No reads are detected in t-athb2 mutants whereas 35S::miP mutant plants are highly overexpressing exon 3 and 4 of ATHB2.

(A) GO enrichment analysis performed on the 95 DEGs whose expression is shown in Figure 3 yielded several significant GO terms (FDR < 0.05). These GO terms are related to shade avoidance, hormone/auxin responses, and growth. (B) The 17 clusters generated by MFuzz. The expression changes of the genes included in each cluster are on the y-axis. The three genotypes and shade conditions are on the x-axis.

ATH2B and ATHB2miP slightly effect root elongation.

Main root length of 11-day old plants grown in either WL or WL+FR in three different shade regimes: deep shade, canopy shade and proximity shade.