Fluorescent reporter lines for auxin and cytokinin signalling in barley (<i>Hordeum vulgare</i>)

<div><p>The phytohormones auxin and cytokinin control development and maintenance of plant meristems and stem cell systems. Fluorescent protein reporter lines that monitor phytohormone controlled gene expression programmes have been widely used to study development and differentiation in the model species Arabidopsis, but equivalent tools are still missing for the majority of crop species. Barley (<i>Hordeum vulgare</i>) is the fourth most abundant cereal crop plant, but knowledge on these important phytohormones in regard to the barley root and shoot stem cell niches is still negligible. We have now analysed the role of auxin and cytokinin in barley root meristem development, and present fluorescent protein reporter lines that allow to dissect auxin and cytokinin signalling outputs in vivo. We found that application of either auxin or cytokinin to barley seedlings negatively impacts root meristem growth. We further established a barley cytokinin reporter, <i>TCSnew</i>, which revealed significant cytokinin signalling in the stele cells proximal to the QC, and in the differentiated root cap cells. Application of exogenous cytokinin activated signalling in the root stem cell niche. Commonly employed auxin reporters DR5 or DR5v2 failed to respond to auxin in barley. However, analysis of putative auxin signalling targets barley PLETHORA1 (HvPLT1) is expressed in a similar pattern as its orthologue AtPLT1 from <i>Arabidopsis</i>, i.e. in the QC and the surrounding cells. Furthermore, the PINFORMED1 (HvPIN1) auxin efflux carrier was found to be expressed in root and shoot meristems, where it polarly localized to the plasma membrane. HvPIN1 expression is negatively regulated by cytokinin and its intracellular localisation is sensitive to brefeldinA (BFA). With this study, we provide the first fluorescent reporter lines as a tool to study auxin and cytokinin signalling and response pathways in barley.</p></div

HvPIN1a expression in the shoot meristem of the barley cv. Golden Promise.

<p><b>(A), (A’)</b> Representative picture of <i>HvpPIN1a</i>:<i>HvPIN1a-mVENUS</i> expression in the barley SAM in Waddington stage I; longitudinal view (A’)) and top view (A’)) of the same SAM. <b>(B)</b> Surface projection of <i>HvpPIN1a</i>:<i>HvPIN1a-mVENUS</i> of the same SAM as in A), created with MorphoGraphX [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196086#pone.0196086.ref056" target="_blank">56</a>]; <b>(C)</b>, <b>(C’)</b> Representative picture of <i>HvpPIN1a</i>:<i>HvPIN1a-mVENUS</i> 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.</p

Expression of the cytokinin reporter <i>TCSn</i>:<i>VENUS-H2B</i> in the root meristem of the barley cv. Golden Promise 8 DAG.

<p><b>(A), (A’)</b><i>TCSn</i>:<i>VENUS-H2B</i> 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>(B)</b> Quantification of <i>TCSn</i>:<i>VENUS-H2B</i> 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. <b>(C)</b> Representative pictures of <i>TCSn</i>:<i>VENUS-H2B</i> 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. <b>(D)</b> 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.</p

Root length, meristem size and DSC phenotype of the cv. Morex upon auxin treatment for 10 days.

<p><b> (A)</b> 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)–(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. <b>(C)</b> 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.</p

PLT phylogenetic tree, HvPLT1 gene structure, promoter activity and protein localization in the root meristem of the barley cv. Golden Promise 8 DAG.

<p><b> (A) Phylogenetic tree of PLT homologue proteins. (B)</b> Genomic structure of the <i>HvPLT1</i> 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. <b>(C)</b> Representative picture of RNA <i>in situ</i> hybridizations with a probe for <i>HvPLT1</i> (purple staining, C)) or the respective sense probe (C’)). <b>(D)</b> Representative picture of the <i>HvpPLT1</i>:<i>HvPLT1-mVENUS</i> 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.</p

HvPIN1a localisation is influenced by BFA and its expression is decreased by cytokinin.

<p><b>(A)</b> Representative pictures of the <i>HvpPIN1a</i>:<i>HvPIN1a-mVENUS</i> 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>(B)</b> Representative pictures of <i>HvpPIN1a</i>:<i>HvPIN1a-mVENUS</i> expression upon either mock (PBS, B)) or cytokinin (B’)) treatment as indicated in the captions; scale bar 200 μm. <b>(C)</b> Quantitative analysis of the <i>HvpPIN1a</i>:<i>HvPIN1a-mVENUS</i> 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.</p

Root length and meristem size of the barley cv. Morex upon cytokinin treatment for 10 days.

<p><b> (A)</b> 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)-(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). <b>(C)</b> 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.</p

HvPIN1a expression in the root meristem of the barley cv. Golden Promise 8 DAG.

<p><b>(A)</b> Representative picture of <i>HvpPIN1a</i>:<i>HvPIN1a-mVENUS</i> 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>(B)</b> Magnification of the epidermal, cortical and endodermal cell layers depicted with white frame in A’). <b>(C)</b> 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. <b>(D)</b> 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).</p

Structure and subcellular localization of At2-MMP.

<p>(<b>A</b>) Domain structure of an At2-MMP-GFP fusion construct. Numbers denote the position of amino acids (aa). SP: signal peptide; Pro: propeptide domain; CYS: cysteine switch; F: predicted furin-cleavage site; CAT: catalytic domain; ZBD: zinc-binding domain; G: putative GPI-anchor modification site; TM: transmembrane domain. The <i>Sal</i>I site (GTCGAC, aa 337–338) is downstream of the zinc-binding domain and upstream of the GPI-anchor modification site. The GFP coding sequence was integrated at the <i>Sal</i>I site. (<b>B</b>) Subcellular localization of At2-MMP upon transiently expression of 35S::At2-MMP2-GFP together with 35S::mCherry in Arabidopsis WT leaves. At2-MMP-GFP was detected 24 h after bombardment. Left panel: without plasmolysis; middle and right panel: five min after plasmolysis induced with 50% glycerol. Bar = 20 μm.</p

Expression profiles of <i>At2-MMP</i> and <i>At3-MMP</i> in hormone signaling mutants after <i>B</i>. <i>cinerea</i> infection.

<p>Five-week-old Arabidopsis WT plants, SA pathway mutants (<i>NahG</i>, <i>ics1</i>, <i>npr1-3</i>, JA pathway mutants (<i>jar1-1</i>, <i>jin1</i>, <i>npr1-1</i>) and ethylene pathway mutant (<i>ein2-1</i>) were inoculated with <i>B</i>. <i>cinerea</i> by placing 5 μl spore suspension (2x10⁵ spores/ml in PDB) or PDB (mock) on the center of the rosette leaves. Total RNA was extracted from leaves at the indicated time points and used for RT-PCR. UBQ5 was used as an internal control. Experiments were independently repeated three times with similar results. hat: hours after treatment, hai: hours after inoculation.</p