Cytokinins control secondary cell wall formation in the inflorescence stem of Arabidopsis

WT arr12 arr1,10 A P E X B A S E mx px px mx mx mx ph ph ph ph px px pc pc co ccif co pi co if pi co pi pi co mx px ph pc co mx px ph c co if if pi pi pi if if pi pi ph co co pi pi if if mx ph px pc if mx ph px c co co mx ph px pc if mx ph px c arr1,12 A P E X B A S E mx px ph pc co mx px ph c co if if pi pi if arr10,12 mx px ph pc co pi if if mx px ph c co pi if if ipt3,5,7 mx px ph pc pi if if co mx px ph c pi if if co arr10arr1 Apical Medial Basal Sub-Apical A B Figure S1. Cytokinin (signaling) deficiency causes a precocious onset of SCW formation in interfascicular fibers. (A) Photograph of a Col-0 WT inflorescence at the time of sampling showing the different stem portions collected for gene expression analyses. (B) Toluidine blue - stained transverse section of living WT Col-0, arr1-3, arr10-5, arr12-1, arr1-3 arr10-5, arr1-3 arr12-1, arr10-5 arr12-1, and atipt3-2 atipt5-1 atipt7-1 inflorescence stems. Where present, premature SCW formations in the apical internodes (apex) are indicated by a red arrowhead. Green arrowheads indicate lack of SCW in apical internodes. A thicker SCW layer in the basal internodes (base) is also apparent (empty arrowheads) in the cytokinin(signaling) deficient lines. Key: c – cambium; co – cortex; pi – pith; mx – metaxylem; px – protoxylem; pc – procambium; ph – phloem; if. Interfascicular fibers. Scale bars: 50 μm. b a e c ce ce a g cf d b b b a a cd aca bdc aa ddde e dc b cc dd g e e c b ccd d c dd d b c f b cd e c e d g ee d d b cf c d f cc de dbab b b a a d aa d f aa ac aaac aa aab a b aa ab b f a d aa c bd ff Figure S2. Altered sugar and acid-soluble lignin content in the cytokinin (signaling) deficient lines. Relative fucose, arabinose, rhamnose, galactose, xylose, mannose, glucose, total sugars, acid-soluble lignin and total lignin content in apical, medial and basal parts of the inflorescence stem in WT Col-0, ahk2, ahk3, p35S:CKX2 and p35S:CKX3 lines. Data are means ± SE of two independent experiments (n≥3). Different letters indicate significant differences at P < 0.05 based on a Tukey’s HSD test. A B Row Z-Score 1 3 -1 -3 ahk23 BASE ahk23 APEX WT BASE WT APEX C D FPKM expression values of 19 genes from the secondary cell wall cascade apex base apex base apex base apex base ahk23 WT Figure S3. Cytokinins regulate the transcriptional cascade controlling the onset of SCW biosynthesis. (A) Venn diagram showing the overlap of differentially expressed genes between two experiments comparing apical versus basal internodes in WT Col-0 (AB_WT) and in ahk2,3 mutant (AB_ahk23). The significance level was set at 0.05. (B) Heatmap of 1499 DEGs revealing the significant change in apicalto-basal expression in both WT and ahk2,3. At the same time, the apical-to-basal ratio of those genes differs by a factor ≥ 2 between WT and ahk2,3; for the list of the genes in individual subsets categorized according to the direction of the apical/basal difference in both genotypes (the color key on the left-hand side of the image) see Table S2. (C) Simplified scheme of the transcriptional cascade regulating SCW formation. Green and red indicate the upregulation or downregulation of the genes, respectively, in the apex of ahk2,3 when compared to WT. (D) Comparison of the mean FPKM (Fragments Per Kilobase of exon per Million reads mapped) expression values of 19 genes from the SCW cascade in apical and basal internodes. Key: sd: standard deviation; mt: ahk2,3 mutant; wt: Col-0 wild type. Figure S4. Gene ontology analysis of genes revealing differential expression between apical and basal internodes in both WT Col-0 and ahk2-1 ahk3-1 inflorescence stems. (A) Gene ontology analysis of genes demonstrating apical versus basal expression difference as seen in WT Col-0, but revealing the loss of the difference in ahk2-1 ahk3-1(3439 DEGs). For the list of corresponding GO categories see Supplemental Table 2, sheet “I GO”. (B) Gene ontology analysis for the intersecting genes (1499 DEGs) demonstrating significant change in apical versus basal expression in both WT Col-0 and ahk2-1 ahk3-1 and revealing the difference of the respective log2 fold changes ≥ 1 (representing at least two-fold change in the apical-to-basal expression ratio in WT Col-0 vs ahk2-1 ahk31; Fig. 2B). For the list of genes enriched in corresponding GO categories see Supplemental Table 2, sheet “II GO”. (C) Gene ontology analysis for subset of intersecting genes as shown in (B) (747 out of 1499 DEGs), revealing higher level at the base when compared with apex in both WT Col-0 and ahk2-1 ahk3-1. For the list of genes enriched in corresponding GO categories see Supplemental Table 2, sheet “III GO”. (D) Gene ontology analysis for subset of intersecting genes as shown in (B) (477 out of 1499 DEGs), revealing lower level at the base when compared with apex in both WT Col-0 and ahk2-1 ahk31. For the list of genes enriched in corresponding GO categories see Supplemental Table 2, sheet “IV GO”. (E) Gene ontology analysis for subset of intersecting genes as shown in (B) (182 out of 1499 DEGs), revealing higher level at the base when compared with apex in WT Col-0, while lower level at the base when compared with apex in ahk2-1 ahk3-1. For the list of genes enriched in corresponding GO categories see Supplemental Table 2, sheet “V GO”. No statistically significant enrichment in any of the GO category was found for the subset of DEGs revealing lower level at the base when compared with apex in WT Col0 while higher level at the base when compared with apex in ahk2-1 ahk3-1 (93 out of 1499 DEGs). Figure S5. Expression of SCW reporter genes VND6 and VND7 after 3 hours of cytokinin treatment. R T-qPCR analysis of VND6 and VND7 expression in the apical, sub-apical, medial, and basal portions o f s tems collected from WT Col-0 after 3 hours of control (DMSO) and cytokinin (BAP) treatment. Transcrip t l evels were normalized to UBQ10. The relative target gene/UBQ10 expression ratios are shown. Data ar e m eans ± SE from a representative experiment (n≥3). Different letters indicate significant differences a t P < 0.05 based on Tukey’s HSD test. DMSO 10μM BAP DMSO 10μM BAP 0 10 20 30 40 50 Apical Sub-Apical Medial Basal Relative expression DMSO DMSO 10μM BAP 0 5 10 15 Stem length (cm) DMSO DMSO 10μM BAP 20 30 40 50 60 First silique (day) WT ipt1,3,5,7WT ipt1,3,5,7 A ipt1,3,5,7 DMSO ipt1,3,5,7 10 μM BAP B C a a b b b c DMSO 10μM BAP DMSO 10μM BAP 0 10 20 30 40 50 60 Apical Sub-Apical Medial Basal Relative expression ARR15 ccc b ab a WT ipt1,3,5,7 a bb aa a b cc ab ARR5 b a cc ab d b WT ipt1,3,5,7 DMSO 10μM BAP DMSO 10μM BAP 0 0.5 1 Apical Sub-Apical Medial Basal Relative expression ARR7 cccc WT ipt1,3,5,7 c a aaaa aa ab a c aab bb a a ab a a c DMSO 10μM BAP DMSO 10μM BAP 0 100 200 300 400 500 Apical Sub-Apical Medial Basal Relative expression IRX8 b f f cd WT ipt1,3,5,7 a a a d a bbb cce b DMSO 10μM BAP DMSO 10μM BAP 0 500 1000 1500 2000 Apical Sub-Apical Medial Basal Relative expression IRX3 b e ff WT ipt1,3,5,7 a a dd a a bc b c b ccd D E F Figure S6. Exogenous cytokinin partially rescue the growth defect of ipt1,3,5,7mutant and the expression of SCW and cytokinin signaling reporter genes. (A) Phenotype of representative control (DMSO)-treated and cytokinin (10 μM BAP)-treated ipt1,3,5,7 plants at the time of collection. Plants were grown on soil in long day conditions for 7 weeks before harvest. Time (in days) of appearance of the first differentiated silique on the inflorescence (B) and stem length (C) of control (DMSO)-treated WT Col-0 and controland cytokinin (10 μM BAP)-treated ipt1,3,5,7 at the time of collection. In (B and C), Error bars indicate SE (n=12). Different letters indicate significant differences at P < 0.05 based on a Tukey’s HSD test. (D-F) Expression of SCW and cytokinin signaling reporter genes. RT-qPCR quantification of the expression of the marker of cellulose IRX3 and hemicellulose synthesis IRX8 (D) and the type-A ARRs ARR5 and ARR7 (E) and ARR15 (F) in apical, sub-apical, medial and basal internodes of inflorescence stems in control (DMSO)-treated and cytokinin (10 μM BAP)-treated WT Col-0 and ipt1,3,5,7 mutant. Transcript levels were normalized to UBQ10. The relative target gene/UBQ10 expression ratios are shown. In (D-F), data are means ± SE from a representative experiment (n≥3). Different letters indicate significant differences at P < 0.05 based on a Tukey’s HSD test. ARR1 ARR2 ARR10 ARR11 ARR12 0 2 4 6 8 10 Apical Sub-Apical Medial Basal Relative expression a a a ab a a ab a a a ab b a bb b a a a a Figure S12. The expression of ARR10 and ARR11 increases during secondary growth in stems. Analysis of type-B ARR expression by RT-qPCR in Col-0 WT inflorescence stems. RT-qPCR analysis of ARR1, ARR2, ARR10, ARR11 and ARR12 expression in the apical, sub-apical, medial and basal portions of stems collected from WT (Col-0) plants. Transcript levels were normalized to UBQ10. The relative ARRs/UBQ10 expression ratios are shown. Error bars indicate SE (n≥3). For each gene, different letters indicate significant differences at P < 0.05 based on a Tukey’s HSD test. The experiments were repeated 3 times with similar results. c abaab d cb d b c ab a e a bc b cc dd a ab bb c b c a d b b a a a a b d c A B Figure S13. Analysis of the basic anatomical parameters of WT and cytokinin (signaling) deficient l ines and a model for the contribution of TE number and diameter to the hydraulic conductivity. ( A) Protoand metaxylem diameter, protoand metaxylem count and total number of vessels i n i nflorescence stems of WT Col-0, and cytokinin (signaling) deficient lines. Data are means ± SE from a r epresentative experiment (n≥6). Different letters indicate significant differences at P < 0.05 based on a T ukey’s HSD test. (B) The simple model shows dominant contribution of the TE diameter (fourth power ) to the total hydraulic conductivity in comparison to the TE number (linear). Figure S14. A hypothetical model of the cytokinin-mediated regulation of SCW formation during T Es and interfascicular fiber differentiation program. ( A) In the apical portion of the inflorescence stem, a thin layer of procambial cells (yellow) is surrounde d b y a few phloem cells (pink) and xylem cells (dark blue). At the base of the inflorescence stem, full y d ifferentiated (SCW-containing) xylem cells are present in the vascular bundles and interfascicular arc s ( light blue); cambium (red) is differentiated. Attenuated cytokinin (CK) signaling or decreased endogenou s c ytokinin levels results into an altered developmental gradient, characterized by premature SC W f ormation in both vascular bundles and the interfascicular regions, while the cambium does no t d ifferentiate. (B) In WT plants, high activity of cytokinin signaling (CK) in the apex of the inflorescenc e s tem inhibits the premature formations of secondary cell wall (SCW) via the suppression of NSTs. Thi s m ay directly or indirectly permit cell expansion and tracheary elements (TEs) to attain their prope r d iameter before the onset of SCW. In the medial portion of the stem, the upregulation of NACs take s p lace, possibly as a result of (inter)action among both positive and/or negative regulators of SC W t hickening (see in the text), permitting the initiation of SCW formation (blue line). Following programme d c ell death, the fully functional TEs are formed. A decrease in endogenous cytokinin content and/o r a ttenuation of cytokinin signaling in the apical portion of the inflorescence stem results in a precociou s u pregulation of NST expression, motivating the premature onset of SCW before the cell expansion i s c omplete. Consequently, TEs are of smaller diameter and show dramatically impaired hydrauli c conductivity. AB