3.1 Protein and expression analysis
3.1.3 Activity of the putative ANAC058 promoter
In order to test the activity of the putative ANAC058 promoter regarding root tissue-specificity and in response to treatments, a promoter reporter construct was generated. It contains the putative promoter of ANAC058 upstream of the green fluorescence protein gene (GFP) and the β-glucuronidase gene (GUS) (fig. 8.24, supp.). This construct was used to stably transform wild type
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plants (genotyping of transformants in fig. 8.14, supp.). To test the promoter activity, PromANAC058::GFP-GUS seedlings were grown on 1/2 MS plates for 5 d.
Seedlings were submerged in X-Gluc (cyclohexylammonium salt) staining solution (8.1, supp.) which turns blue in the presence of the GUS protein activity. Putative ANAC058 promoter activity was detected in the root but not in the shoot (fig. 3.3 A). Specifically, it is active in the endodermis and causes GUS activity as well as GFP fluorescence in a specific pattern. According to this pattern, promoter activity changes along the root from root tip towards root-shoot junction, also known as the root base (fig. 3.3 F - K). No activity was found at the root meristem, whereas single blue and fluorescing endodermal cells (patchy) appeared later, finally leading to a fully stained and fluorescing endodermis. This activity is similar to the pattern of suberin deposition (see model in fig. 2.7) which was published for example by Naseer and colleagues in 2012. Similar promoter activity was observed in the same study for several suberin-associated genes.
Fig. 3.3 Putative ANAC058 promoter activity in roots of PromANAC058::GFP-GUS plants
GUS activity and GFP fluorescence was detected in roots of 5 d old seedlings (A), specifically in the root cap cells (B - E, fig. 3.4 C) and the endodermis (F - K). Promoter activity via GPF fluorescence was investigated using confocal microscopy. Pictures were taken with bright field (C, J) employing a standard GFP filter (B, G, I).
Depicted are Z-stacks of 6 - 7 individual pictures (G - K). Overlays of bright field and GFP fluorescence pictures were generated with FluoView program (D, H, K). Scale bars represent 200 µm (A) and 10 µm (B - K). ep epidermis, co cortex, en endodermis, cc central cylinder
Accordingly, no staining or fluorescence is visible in the meristematic zone and the only GUS staining and GFP fluorescence in the root tip was observed in root cap cells (fig. 3.3 A - E). GUS activity and GFP fluorescence starts being visible in single endodermal cells (fig. 3.3 F - H) in the differentiation zone. Measuring the distance from the root tip to the first stained cell shows that promoter activity starts at approx. 20 % of the whole root length (19.74 ± 5.22 %, fig. 8.2, supp.). The putative promoter of the suberin biosynthesis gene HORST in PromHORST::GUS (Höfer et al., 2008) becomes active at a very similar distance from the root tip (fig. 3.4 H and fig. 8.2, supp.). The distance corresponds well to the start of suberin deposition (fig. 8.2, supp.). Closer to the root base all endodermis cells with only
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few exceptions show staining (fig. 3.3 A, fig. 3.4 F) and fluorescence (Fig. 3.3 I - K). No staining or fluorescence is observed in the outermost cell layer, the epidermis (fig. 3.3 F, G and I, labeled ep) or the cell layer below, the cortex (fig. 3.3 F, G and I, labeled co). Neither was GFP detected in the central cylinder (fig. 3.3, I - K, labeled cc). Single cells without staining in an otherwise fully stained endodermis (not depicted) indicate passage cells. Those cells are unsuberized and are suspected to allow passage of ions and water into the central cylinder (Peterson and Enstone, 1996).
While the root of PromANAC058::GFP-GUS plants in fig. 3.3 A and fig. 3.4 F shows continuous GUS staining until root-shoot junction, roots that are developmentally slightly older indicate that promoter activity in the endodermis decreases again towards the root base (fig. 3.4 H, labeled A). At the root base, on the other hand, staining intensity was often observed to increase again. It appears in sub-epidermal root cells (fig. 3.4 D and F), observed in three of four independent transformed lines.
Blue staining in the endodermis and root cap cells was observed in all four independent transformed lines.
Specific promoter activity of the putative ANAC058 promoter was also observed during lateral root development. In root sections in which the promoter is already strongly active, no additional promoter induction was present in cells surrounding the lateral root base (fig. 3.4 A). If promoter activity on the other hand is weak at the site of lateral root development, the activity increases in endodermal cells of the main root in the vicinity (fig. 3.4 B and C). In later developmental stages of the lateral root, cells at its root base which seem to be morphologically part of the lateral root but in contact with the main root epidermis occasionally also show staining (fig. 3.4 B and C). In case the emergence of the lateral root disrupts cells of the epidermal cell layer the promoter is activated locally in those epidermal cells (fig. 3.4 A).
Additionally, the putative ANAC058 promoter becomes active in the root tip of lateral roots at a certain developmental stage. Only when the meristematic and elongation zone are distinct in lateral roots, was promoter activity observed in the root tip (fig. 3.4 C). This staining seems to be localized to the root cap cells in the same manner as for the main root (fig. 3.3 B - E). The activity of the promoter was also observed occasionally in the radicle of mature embryos, extracted from green seeds. Whereas promoter activity in lateral roots seemed to be localized to single root cap cells, localization of activity in the embryo was less certain. Staining was observed in root cap cells but might also be present in other cell types in the radicle (fig. 3.4 E).
Fig. 3.4 GUS activity during lateral root emergence, in root base and embryo of PromANAC058::GFP-GUS plants and in roots of PromHORST::GUS plants
During lateral root development in PromANAC058::GFP-GUS plants, the promoter is active in adjacent endodermal cells of the main root, the basal-most endodermal cells of the developing lateral root and occasionally in main-root epidermal cells at the site of lateral root emergence (A - C). At the main root base, GUS activity was detected in sub-epidermal cell in roots (D). In mature embryos extracted from green seeds, promoter activity was occasionally observed in the root tip (E). The distance from root tip to the start of GUS activity was the same for PromANAC058::GFP-GUS (G and H, seedling labeled A) and PromHORST::GUS (G and H, seedling labeled H). G shows the root section encircled in H in higher magnification, the arrow marks the first stained cells. Scale bars represent 200 µm (G, H), 50 µm (D, E, F) and 10 µm (A - C).
In PromANAC058::GFP-GUS plants grown for 3 weeks on 1/2 MS, activity of the putative ANAC058 promoter was clearly present in young sections of the main root as well as in young parts of lateral roots (fig. 3.5, black arrows). Close to the root tip of main root or side roots, GUS staining was absent.
Staining in root cap cells was detected in main and side roots using a higher magnification than in fig.
3.5 (not shown). Promoter activity at the age of 3 weeks was investigated for 1 transgenic line.
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Fig. 3.5 Putative ANAC058 promoter activity in 3 weeks old PromANAC058::GFP-GUS plants
The putative ANAC058 promoter is active in young root sections (black arrows) of plants grown on 1/2 MS plates. Promoter activity was visualized via GUS activity and observed under the binocular microscope. The scale bar represents 2 mm.
3.1.3.1 Induction of putative ANAC058 promoter activity by wounding
Suberin-associated genes are known to respond to environmental stress with increased expression (Domergue et al., 2010; Franke et al., 2009). The ANAC058 promoter is induced in leaves by wounding after 3 d and 7 d (fig. 3.6 C and D, G and H) and shortly after wounding in seeds (fig. 3.6 I, black arrow). Wounding of leaves for 3 h and 6 h did not result in GUS staining (fig. 3.6 A and B, E and F) and promoter activity in 3 d and 7 d wounded leaves is restricted to wounding sites.
Fig. 3.6 Putative ANAC058 promoter activity in wounded leaves and seeds of PromANAC058::GFP-GUS plants
Leaves were pierced with forceps and tested for GUS activity 3 h (A and E), 6 h (B and F), 3 d (C and G) and 7 d (D and H) after wounding. Seeds show GUS activity in seed coats at sites where siliques were opened with a cut perpendicular to silique length (I). Localization of incision is marked with an arrow. Scale bars signify 5 mm (A - D), 0.5 mm (E - H) and 1 mm (I).
Seeds not in the vicinity of the silique incision site were absent of GUS activity as well (fig. 3.6 I), as was also observed for intact seeds at various developmental stages (data not shown). When
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separating seed coats from embryos, wounding response was observed in both, not necessarily together (data not shown). In order to cause the wounding response, leaves were pierced with a forceps to generated small holes and siliques were cut perpendicular to their lengths shortly before covering them with X-Gluc solution. Wounding response was observed in four of four investigated transgenic lines.
3.1.3.2 Induction of putative ANAC058 promoter activity by ABA application
Abscisic acid (ABA), a known mediator of stress signals has been shown to induce several genes related to suberin assembly (Barberon et al., 2016; Boher et al., 2013; Yadav et al., 2014).The same applying to ANAC058 is possible, supported by high sensitivity of ANAC058 overexpressing plants to ABA (Coego et al., 2014).
Accordingly, testing for ABA susceptibility of ANAC058 by transferring PromANAC058::GFP-GUS seedlings to MS medium with 1 µM and 30 µM ABA resulted in promoter activity closer to the root tip in ABA treated plants in comparison to mock-treated plants (fig. 3.7). Distance from the root tip at which putative promoter activity decreases again in basal root sections varied in ABA-treated seedlings around the distance observed in the control. Staining intensity also did not seem to differ significantly. The effect was observed in two of two independent transformed lines and appeared to be the same for both ABA concentrations.
Fig. 3.7 Promoter activity in PromANAC058::GFP-GUS seedlings exposed to ABA
3 d old seedlings were transferred to 1/2 MS with 1 µM and 30 µM ABA, mock-treated seedlings were transferred to ½ MS without ABA (seedlings labeled m). Incubation in X-Gluc solution started 13 h after transfer to ABA.
Depicted are representative seedlings for 1 µM and 30 µM ABA treatment (seedling labeled ABA) since both treatments had the same effect. Overview (A) was taken with a binocular microscope; detailed picture (B) of encircled area in A was generated with bright field microscopy. Scale bars signify 1 mm (A) and 0.1 mm (B).