DEG10 but not CAT3 is needed for optimal root growth, especially

The GUS staining results hypothesize that DEG10 seems to play an important role in roots under elevated temperatures. Additionally, publicly accessible microarray data revealed that the highest DEG10 transcript levels are present in imbibed seeds (eFP Browser (Winter et al., 2007)). In order to evaluate, if DEG10 functions during germination and early seedling development, seeds of WT plants, Δdeg10-1 and Δcat3-1 mutants were incubated in axenic culture. Since Deg proteases are often associated with responses to protein folding stress, the seedlings were grown under four different conditions: absence or presence of sucrose (to promote mitochondrial respiration) and room temperature (RT;

22°C : 20°C, light : dark) or elevated temperature (ET; 30°C : 25°C, light : dark) (Figure 18). To score the seedling development, four measurements were performed: seedling establishment, continuous development until day 18, number of true leaves at day 10 and primary root length at day 18.

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At first glance it got already obvious that sucrose and especially the combination of sucrose and ET promoted development and growth of aboveground organs and roots in all seedlings (Figure 18).

Figure 18: Phenotypic analyses on growth and development of A. thaliana wildtype (WT) plants, Δdeg10-1 and Δcat3-1 mutants in axenic culture. Influence of elevated temperature and addition of sucrose on growth and development: 18-day-old WT, Δdeg10-1 and Δcat3-1 seedlings grown in random order under axenic conditions on agar plates, half supplemented with 2% (w v^-1) sucrose. The seedlings were either incubated at 22°C or 30°C; color code: blue: WT, red: Δdeg10-1, green: Δcat3-1.

Germination in the strict sense (i.e. radicle emergence) did not differ significantly between WT, Δdeg10-1 and Δcat3-1 seeds and temperature and medium had no effect (data not shown). The presence of sucrose promoted the seedling establishment by up to 10% and more than 99% of the seedlings of all three genotypes established. Overall, there was no difference between WT, Δdeg10-1 and Δcat3-1 seeds (no significant effect of genotype;

data not shown).

In medium without sucrose, most seedlings of all three genotypes had formed two rosette leaves (subsequent leaves after the cotyledons, as defined in Boyes et al. (2001)) by day 10, regardless of the temperature regime (Figure 19a). Both adding sucrose to the medium and elevated temperature had a positive effect on the number of leaves (Figure 19a;

significant effect of medium and temperature, Table 2), and the effects were additive, so that seedlings developed fastest on a medium with sucrose at ET (Figure 19a; significant T*M interaction, Table 2). However, there were no significant differences between the WT

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and mutant plants (no significant effect of genotype or interactions with genotype, Table 2).

Overall, regardless of the treatments the seedlings of the analyzed genotypes differed in root length (significant effect of genotype, Table 2): Δdeg10-1 plants had significantly shorter roots than the WT plants, whereas the Δcat3-1 seedlings did not differ from WT plants (Figure 19b; Tukey post-hoc comparison Δdeg10 vs. WT and WT vs. Δcat3 see Table 3). The reduced root growth of the Δdeg10-1 seedlings was more pronounced at ET (Figure 19b; significant effect of temperature in Table 2). Temperature had an overall positive effect on root length in all genotypes (Figure 19b; significant effect of temperature in Table 2), but compared to the WT and Δcat3-1 seedlings, the increase in root length of the Δdeg10-1 seedlings in response to ET was not as strong (Figure 19b; significant G*T interaction in Table 2) and only present on medium with sucrose. Also adding sucrose had an overall positive effect on root length (Figure 19b; significant effect of medium in Table 2), but the response depended on the temperature regime (significant T*M interaction, Table 2). Adding sucrose promoted root growth only at ET, but not at 22°C (Figure 19b).

Moreover, all three analyzed genotypes responded differently to the addition of sucrose (significant G*M interaction, Table 2): at elevated temperature the Δdeg10-1 seedlings benefited much more from added sucrose than the WT and Δcat3-1 seedlings. However, the addition of sucrose did not fully compensate the reduced root length of the Δdeg10-1 seedlings at ET (Figure 19b). Although the difference between the Δdeg10-1 mutant and the WT plant and Δcat3-1 mutant was more pronounced without sucrose, plants of the Δdeg10-1 mutant still had significantly shorter roots when sucrose was added (Tukey post-hoc comparison at ET with sucrose in Table 3). The root lengths of WT and Δcat3 plants were not significantly different from each other in any of the treatments (Table 3), which indicated that CAT3 does not influence the elongation of the primary root, whereas expression of DEG10 is required for normal root growth.

In absence of sucrose, the continuous seedling development (seedlings that germinated and did not arrest growth until day 18) was lower than with sucrose (Figure 19c; significant effect of medium, Table 2) and the effect was more pronounced in the ET treatment (Figure 19c; significant T*M interaction, Table 2). However, there were no significant differences between the WT and mutant plants (no significant effect of genotype or interactions with genotype, Table 2).

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Since no significant differences in germination potential between WT, Δdeg10-1 and Δcat3-1 seeds were found, the negative effects of absence of sucrose in interaction with ET are due to problems in seedling development after germination.

Table 2: Statistical analyses of the effects of temperature and addition of sucrose on seedling development of wildtype (WT), Δdeg10-1 and Δcat3-1 seedlings presented in Figure 19. Chi-square (χ2) tests were performed to compare the different groups:

Summary of linear mixed model analysis to test for effects of genotype (G), temperature (T), medium (M) and their interactions on leaf number (Leaf nr.), primary root length (R.

length) and continuous seedling development (C. dev.),. Values in bold are significant at the specified level, *: p<0.05; **: p<0.01; ***: p<0.001

aPoisson error distribution

bGaussian error distribution, square root data transformation

cBinomial error distribution

ddegree of freedom

eRemoval of the main effects genotype (G), temperature (T) or medium (M) compared to:

random part + G + T + M

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Figure 19: Effect of temperature and sucrose on development of wildtype (WT), Δdeg10-1 and Δcat3-1 seedlings: Per genotype and condition, at least 350 seeds from three parental plants were analyzed in axenic culture at the indicated temperatures and in the presence or absence of 2% (w v^-1) sucrose in the medium. (a) Leaf number at day 10: Dot size is proportional to the number of seedlings with the given number of true leaves. (b) Primary root length at day 18: The median and interquartile range are indicated by thick black lines and boxes, respectively. Whiskers extend to the most extreme data point within 1.5 times the interquartile range. Significance of differences (Table 3) is indicated as follows:

n.s.: not significant; *: p<0.05; **: p<0.001. (c) Percentage of seedlings which showed continuous development until day 18; horizontal lines indicate means, error bars indicate

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95% confidence intervals. In (a) and (c), no significant differences between the genotypes were detected.

Table 3: Statistical analyses of the effects of temperature and addition of sucrose on primary root length of wildtype (WT), Δdeg10-1 and Δcat3-1 seedlings presented in Figure 19. Differences were analyzed for significance in multiple pairwise comparisons with Tukey Post-hoc tests (z-tests): Asterisks indicate statistical significance, *: p<0.05; **:

p<0.001

aTreatment: RT: room temperature (22°C), ET: elevated temperature (30°C), with (w) and without (w/o) sucrose

bEstimated difference; the bigger the value the bigger the root length difference, positive or negative values indicate a longer or shorter root length, respectively, for line 1

cStandard error

2.3.4 Loss of DEG10 did not severely affect total respiration, but