Fig 1.
Root length and meristem size of the barley cv. Morex upon cytokinin treatment for 10 days.
(A) Root length after 10 day-treatment with cytokinin; experiment was performed twice; values normalized to mock-treated plants; n = 7–18 plants per data point. (B)-(B”) Representative pictures of meristem phenotypes upon cytokinin treatment according to the captions; arrowheads mark the transition zones in the outer cortex layer; insets show magnifications of the transition zones; scale bars 200 μm (overviews) and 100 μm (magnified insets). (C) Meristem size after 10-day cytokinin treatment, measured by meristem length; experiment was performed twice; n = 11–16 roots per data point; significance was determined using the two-tailed Student’s t test, * = p<0.05, ** = p<0.001.
Fig 2.
Expression of the cytokinin reporter TCSn:VENUS-H2B in the root meristem of the barley cv. Golden Promise 8 DAG.
(A), (A’) TCSn:VENUS-H2B expression in untreated roots; transmitted light and VENUS emission (A)) and VENUS emission only (A’)); white arrow head in A’: metaxylem, gray arrow head in A’: QC; seven independent transgenic lines were examined and exhibit a similar expression pattern. (B) Quantification of TCSn:VENUS-H2B expression by the mean gray value of the region marked by red box in C); mean gray value normalized to the PBS control; significance was determined using the two-tailed Student’s t test, ** = p<0.001. (C) Representative pictures of TCSn:VENUS-H2B expression in root meristems upon 24 h of cytokinin treatment according to the captions; PBS without hormone was used as control; three independent transgenic lines were examined; the experiment was performed three times; n = 8–31 per treatment. (D) Magnification of the stem cell niche and root cap of roots upon treatments indicated by the captions; expression in the cortex/ endodermis initials, the DSCs, the QC layer adjacent to the root cap and the epidermis initials (PBS: 0/21 roots, 1 μM 6-BA: 1/9 roots, 10 μM 6-BA 8/18 roots, 1 μM t-Z 2/9 roots, 10 μM t-Z 5/8 roots); the root cap border is marked with a white frame; for a better comparison between samples, roots were cleared before microscopy (C), D), E)); scale bars 100 μm.
Fig 3.
Root length, meristem size and DSC phenotype of the cv. Morex upon auxin treatment for 10 days.
(A) Root length after 10 day-treatment with auxin; experiment was performed twice; for a better comparison between the experiments, all values were normalized to the mock-treated plants; n = 4–18 plants per data point. (B)–(B”) Representative pictures of the root meristem phenotype at 10 DAG upon hormone treatment according to the captions; arrow heads mark the transition zones; insets show magnifications of the transition zones; scale bars 200 μm and 100 μm in the magnification. (C) Meristem length upon hormone treatment, measured by meristem length; experiment was performed twice; all values are normalized to the mock-treated control; n = 7–17 roots per data point; significance was determined using the two-tailed Student’s t test, * = p<0.05, ** = p<0.001.
Fig 4.
PLT phylogenetic tree, HvPLT1 gene structure, promoter activity and protein localization in the root meristem of the barley cv. Golden Promise 8 DAG.
(A) Phylogenetic tree of PLT homologue proteins. (B) Genomic structure of the HvPLT1 coding sequence; boxes represent exons, black horizontal lines represent introns; dark gray boxes indicate coding sequence for AP2 domains, light gray boxes indicate coding sequence for the linkers between AP2 domains. (C) Representative picture of RNA in situ hybridizations with a probe for HvPLT1 (purple staining, C)) or the respective sense probe (C’)). (D) Representative picture of the HvpPLT1:HvPLT1-mVENUS emission in the root meristem; transmitted light and mVENUS emission (D)), mVENUS emission only (D’)); arrow head in D’) points to the QC; hand sections; seven independent transgenic lines were examined and exhibited similar expression patterns; scale bars 100 μm.
Fig 5.
Phylogenetic tree of maize, Arabidopsis, rice and barley PINs.
Protein subfamilies are framed with the same colour; gray frame marks HvPIN1a.
Fig 6.
HvPIN1a expression in the root meristem of the barley cv. Golden Promise 8 DAG.
(A) Representative picture of HvpPIN1a:HvPIN1a-mVENUS expression; transmitted light and mVENUS emission (A)), mVENUS emission only (A’)); six independent transgenic lines were examined which vary only in expression level but not in localisation or pattern; white box in A’) marks magnification in B); gray box in A’) marks magnification in D). (B) Magnification of the epidermal, cortical and endodermal cell layers depicted with white frame in A’). (C) Schematic illustration of HvPIN1a expression in the root meristem, high = dark gray, low = gray; red arrows indicate possible auxin flow created by localisation of PIN1 auxin transporters; En = endodermis, Co = cortex, Ep = epidermis, LRC = lateral root cap, RC = root cap. (D) Magnification of the stem cell niche depicted with gray frame in A’); transmitted light and mVENUS emission (D)), mVENUS emission only (D’)); white arrow heads mark apically localised PIN1, gray arrow heads mark basally localised PIN1; scale bars 100 μm in A), D); 50 μm in B).
Fig 7.
HvPIN1a expression in the shoot meristem of the barley cv. Golden Promise.
(A), (A’) Representative picture of HvpPIN1a:HvPIN1a-mVENUS expression in the barley SAM in Waddington stage I; longitudinal view (A’)) and top view (A’)) of the same SAM. (B) Surface projection of HvpPIN1a:HvPIN1a-mVENUS of the same SAM as in A), created with MorphoGraphX [56]; (C), (C’) Representative picture of HvpPIN1a:HvPIN1a-mVENUS expression in the barley SAM in Waddington stage II; transmitted light and mVENUS emission (C)), mVENUS emission only (C’)); four independent transgenic lines were examined and vary only in expression strength but not in localisation and pattern; scale bars 50 μm.
Fig 8.
HvPIN1a localisation is influenced by BFA and its expression is decreased by cytokinin.
(A) Representative pictures of the HvpPIN1a:HvPIN1a-mVENUS expression in the outer cortex cell layer of the root meristem or in the epidermis of the SAM immediately after (0 h) or 2 h after mock (PBS) or 50 μM BFA treatment; gray arrow heads point to vesicles; scale bars 20 μm; three independent transgenic lines were examined; experiments were performed twice; n = 4–6; one transgenic line was used (in case of the root meristem); experiments were performed twice; n = 3–5; two transgenic lines were used (in case of shoot meristem). (B) Representative pictures of HvpPIN1a:HvPIN1a-mVENUS expression upon either mock (PBS, B)) or cytokinin (B’)) treatment as indicated in the captions; scale bar 200 μm. (C) Quantitative analysis of the HvpPIN1a:HvPIN1a-mVENUS expression upon cytokinin expression in B), measured by the mean gray value of the whole root meristem and the root cap; values are normalized to the PBS-control; five different independent transgenic lines were used; experiment was performed twice; n = 24 per treatment; significance was determined using the two-tailed Student’s t test, * = p<0.05.