3.2 Analysis of ANAC058 knock-out and knock-down mutants
3.2.5 Physiological effect of decreased suberin amounts in anac058 mutants
3.2.5.1 Susceptibility of anac058 mutants to salt and osmotic stress
Possible effects of lower suberin amounts in anac058 on stress susceptibility were tested by applying salt stress. The plants were grown on ½ MS plates with 50 mM NaCl by letting them germinate on as well as transferring 3 d old seedlings to this high salt medium. Root growth was measured as main root length.
ANAC058 RALPH GPAT5 HORST RBOHF
fol d dif fer en ce in ex pr es sio n r el ativ e t o c on tro l
0.0 0.2 0.4 0.6 0.8 1.0 1.2
1.4 WT
anac058-1 anac058-2
anac058-3 (amiRNAi)
n.i. n.i.
Germination on control medium and 50 mM NaCl MS medium yielded no significant differences between mutants and their respective wild types as suggested by figure 3.16. Plant growth was observed over 5 weeks in total without any visible effect of the NaCl treatment. Representative seedlings of 10 d age were selected for in-depth analysis of salt-treatment effect. The measurement of root length shows that seedlings of all genotypes are affected by NaCl, causing increased root growth compared to seedlings growing on control medium (fig. 8.7, supp.). This effect is predominantly observed for Col and anac058-2. The measurement also confirms same root growth of wild type and mutants. Additionally, occasional growth arrest shortly after germination was observed under NaCl conditions. This effect does not differ between wild type and mutant plants (not shown) and is therefore not influenced by a mutation in ANAC058.
Fig. 3.16 Germination of anac058 mutants and corresponding wild type on 50 mM NaCl and control medium
Representative picture of plants observed in total over 5 weeks, here seedlings grown for 10 d on 1/2 MS and 1/2 MS supplemented with 50 mM NaCl are depicted. For root length measurements, see fig. 8.7, and table 8.20, supp.. Scale bars represent 1 cm.
In order to exclude potential early effects of NaCl as well as prior adjustment of plants to high salt concentration, seedlings were grown for 3 d on MS medium and transferred to MS with 50 mM NaCl.
Seedling growth was observed over 5 weeks in total but no significant differences between wild type and mutants due to salt treatment were observed (fig. 3.17). For representative in-depth analysis, 10 d old seedlings, grown for 3 d on MS and 7 d on NaCl medium were selected. Root growth differed between individual seedlings considerably, especially in case of anac058-1 and its respective wild type. Significant differences between mutant lines and their wild type were not observed, as shown by their similar root length (fig. 3.17). Ler seedlings had a root length of 3.9 ± 1.54 cm, anac058-1 seedlings of 3.63 ± 1.2 cm (fig. 3.17 B). Comparing Col and anac058-2 root length with respective 4.68 ± 0.75 cm and 4.42 ± 0.69 cm revealed only slightly shorter roots for the mutant. To measure only root growth on NaCl medium, the distance from the root tip until the dot marking the position of the root tip directly after transfer to NaCl medium was quantified. After 7 d growth on NaCl medium, same
Col anac058-2
Ler anac058-1
controlNaCl
trends as for total root length were observed. The slight differences were not significant and differences between ecotypes were larger than between mutant and respective wild type. Generally, a very slight trend toward shorter roots in the mutant appeared which, on the other hand, was not observed for seedlings germinating on NaCl medium (fig. 3.16 and fig. 8.7, supp.).
Fig. 3.17 Growth of anac058 seedlings on 50 mM NaCl in comparison to the corresponding wild type Of seedlings observed in total for 5 weeks, representative pictures of seedlings transferred after 3 d growth on 1/2 MS and grown for 7 d on 1/2 MS with 50 mM NaCl were selected (A). Root tip position directly after the transfer to NaCl medium was marked by a black dot and total root length of the main root as well as main root length grown only on NaCl medium (dot to root tip) was measured (B). Mean values and standard deviation represented by error bars were calculated from 23 (Col), 24 (anac058-2), 16 (Ler) and 14 (anac058-1) independent seedlings. Significant differences are represented by one (p ≤ 0.05) or two (p ≤ 0.01) asterisks as determined by student t-test. For related values see table 8.21, supp.. Scale bars represent 1 cm.
In contrast to the results from seedlings growing on 50 mM NaCl MS plates, long term salt treatment of anac058 mutants and wild types with 50 mM NaCl by growing them in hydroponics had a different effect (fig. 3.18).
anac058-1, anac058-2 and wild type plants were transferred to hydroponic solution with 50 mM NaCl for salt treatment and control plants were transferred to new hydroponic solution without changed salt concentrations. Water potential of respective solutions was measured and – 0.09 MPa was detected for the control solution, whereas water potential in the NaCl solution was distinctly lower (- 0.36 MPa).
All genotypes were affected by the salt treatment with leave margins showing necrotic spots (fig. 3.18 B). Additionally, fresh weight and dry weight of plants subjected to 50 mM NaCl is lower than for control plants (fig. 3.18 C and fig. 8.8, supp.).
whole root length root growth on NaCl
root length [cm]
0 1 2 3 4 5
6 Ler
anac058-1 Col anac058-2
Col anac058-2
Ler anac058-1
A B
Fig. 3.18 Growth of anac058 mutants and wild type in 50 mM NaCl and control solution
Plants were grown for 25 d in hydroponics (A) and were transferred to new hydroponics solution (control) or hydroponics solution supplemented with 50 mM NaCl. Plant health was investigated after 2 weeks of NaCl treatment (B - D). Root and shoot fresh weight (C) and dry weight (fig. 8.8 A, supp.) were measured and root:shoot ratio was calculated from fresh weight (fig. 8.8 B, supp.) and dry weight (D). The ratio fresh weight:dry weight is depicted in fig. 8.8 C, supp. Mean values and standard deviations represented by error bars were calculated from 5 (Col), 4 (anac058-2), 6 (Ler) and 3 (anac058-1) independent seedlings for control conditions and from 7 (Col), 9 (anac058-2), 7 (Ler) and 8 (anac058-1) independent seedlings for salt conditions.
Significant differences are represented by one (p ≤ 0.05) or two (p ≤ 0.01) asterisks as determined by student t-test. All related values are present in table 8.22, supp.. Scale bars represent 2 cm.
Mutants seemed more susceptible to 2 weeks of salt treatment than control plants. As a consequence, they generated significantly less biomass (fig. 8.8, supp.). Plant health deterioration is visible (fig. 3.18 B) since the shoots of the mutants show more necrosis than the respective wild type. anac058 fresh weight is significantly reduced for the shoot as well as root under high salt conditions. The only exceptions are anac058-1 roots for which fresh weight is decreased but insignificantly. Dry weight is lower as well for mutants compared to the respective wild type but only significantly in case of anac058-2 (fig. 8.8, supp.). The fresh weight:dry weight ratio indicates the impact of salt stress on water content and biomass. The ratio generally decreases for the mutants (fig. 8.8, supp.), indicating that water content decreases in mutants more than biomass. The root:shoot ratio derived from dry weight values is significantly lower in anac058-1 and ana058-2 with 0.33 ± 0.05 mg and 0.18 ± 0.03 mg, respectively, compared to their wild types with 0.4 ± 0.06 mg (Ler) and 0.21 ± 0.03 mg
control NaCl
root : shoot ratio (dry weight)
0.0 0.1 0.2 0.3 0.4 0.5
Ler anac058-1 Col anac058-2
control NaCl control NaCl
mg fresh weight
0 200 400 600 800 1000 1200 1400
Ler anac058-1 Col anac058-2
shoot root
before transfer 50 mM NaCl
anac058-2Col anac058-1Ler anac058-2Col anac058-1Ler
A B
C D
*
** *
* **
(Col). Since biomass of root and shoot decreases, the lower ratio indicates that roots are more strongly affected than shoots in mutants compared to the respective wild types. Occasionally, differences were observed between wild type and anac058-2 already under control conditions (fig.
8.8, supp.) although effects were stronger after salt stress treatment. Additionally, differences between the wild types which belong to disparate ecotypes were observed in reaction to salt stress. For Col, root:shoot ratio decreases whereas for Ler the ratio increases with regard to dry weight (fig. 3.18) and fresh weight (fig. 8.8, supp.). Consequently, Col roots are more affected by salt stress, whereas for Ler a strong effect on shoots is observed.
In order to generate osmotic stress, anac058-1, anac058-2 and wild types were transferred to a nutrient solution with 1.73 % PEG, whereas control plants were transferred to a new hydroponic control solution.
Fig. 3.19 Growth of anac058 mutants and wild type in 1.73 % PEG and control solution
Plants were grown for 25 d in hydroponics (A) and were transferred to new hydroponics solution (control) or hydroponics solution supplemented with 1.73 % PEG (173 g/l H20, water potential Δ - 0.4 MPa). Plant health was investigated after 4 d of PEG treatment (B - D). Root and shoot fresh weight (C) and dry weight (fig. 8.9 A, supp.) was measured and root:shoot ratio was calculated from fresh weight (fig. 8.9 B, supp.) and dry weight (D). The ratio fresh weight:dry weight is depicted in fig. 8.9 C, supp.. Mean values and standard deviations represented by error bars were calculated from 3 (Col, anac058-2, Ler, anac058-1) independent seedlings for control conditions and from 6 (Col), 7 (anac058-2 and Ler) and 8 (anac058-1) independent seedlings for PEG conditions. Significant differences are represented by one (p ≤ 0.05) or two (p ≤ 0.01) asterisks as determined by student t-test. All related values are present in table 8.23, supp.. Scale bars represent 2 cm.
control PEG
root : shoot ratio (dry weight)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Ler anac058-1 Col anac058-2
control PEG control PEG
mg fresh weight
0 200 400 600 800 1000 1200
Ler anac058-1 Col anac058-2
before transfer PEG (- 0.4 MPa)
anac058-2Col anac058-1Ler anac058-2Col anac058-1Ler
A B
shoot root
C D
*
4 d of PEG treatment affected health of all genotypes in a severe manner (fig. 3.19 B). 1.73 % PEG changed the water potential of the hydroponics solution to - 0.49 MPa compared to - 0.09 MPa of the control nutrient solution as measured with a potentiometer. This change in water potential resulted in plant health deterioration visible as curling and red coloration of leaves. Specifically Ler and anac058-1 also displayed necrosis at the leaf margins, indicating the Ler ecotype to be more severely affected. When measuring root and shoot biomass, dry weight and fresh weight for plants subjected to PEG treatment was lower than for control plants. No significant differences between mutants and respective wild types appeared except for the shoot fresh weight amounts of anac058-2. Those were significantly increased compared to Col. This effect was not observed in the other mutant line or for root fresh weight and was therefore deemed unrelated to suberin amounts. The root:shoot ratio increased for all genotypes upon PEG treatment and was slightly lower in mutants than in the wild type but significant differences were absent.