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2.4.6.1 The activity of ILA in inhibiting root growth regulated by UGT76B1 expression

The flagellin peptide flg22, which strongly interferes with the defense pathway of the leaves, also inhibits root growth of Arabidopsis seedlings (Gomez-Gomez and Boller, 2000; Zipfel et al., 2004). Furthermore, flg22 confers positive regulation on plant defense against pathogens in roots apart from the well-known regulation of defense responses against pathogens in leaves (Millet et al., 2010). Similarly the hormone SA, which is the crucial player in defense, and then non-protein amino acid BABA, which can confer resistance to pathogen infection were previously reported to inhibit root growth of Arabidopsis seedlings (Zimmerli et al., 2008; Wu et al., 2010).

To investigate whether ILA can inhibit seedling growth, the seedling growth was observed on plates containing ILA. Indeed, ILA clearly showed a root growth inhibition phenotype in a concentration dependent manner (Figure 23). Since UGT76B1 is mainly expressed in the root tissue and ILA is the substrate of UGT76B1, the root growth inhibition by ILA was observed among lines with different UGT76B1 expression. Consistent with being the substrate of UGT76B1, ILA caused a much stronger root growth inhibition phenotype of ugt76b1-1, but a less pronounced root growth inhibition of UGT76B1-OE-7 as compared to Col-0 in a concentration dependent manner (Figure 23). This indicated that ILA, but not its conjugate ILA-glucoside, was active in inhibiting root growth.

Since UGT76B1 is mainly expressed in the root of Arabidopsis, the susceptibility towards the root pathogen Vertimicillum longisporum was tested by our collaborator, the laboratory of Christiane Gatz (Universität Göttingen). However, there was no difference in susceptibility among the lines with different UGT76B1 expression level and wild type (data not shown).

control 200 µM ILA 500 µM ILA 400 µM ILA ugt76b1 -1 Col-0 UGT76B1 -OE-7  

Figure 23. Direct effects of exogenously applied ILA.

Root growth inhibition phenotype of ILA inversely correlates with UGT76B1 expression. Pictures were taken 10 d after sawing the seeds on plates containing 0, 200, 400 and 500 µM ILA.

2.4.6.2. Response to ILA in roots does not require SA, JA and ET pathways

Lines deficient in SA, JA or ET pathways were grown on plates containing ILA in order to explore whether or not the inhibition of root growth required SA, JA or ET pathways. The lines NahG, npr1 and sid2 are deficient in SA response; the jar1 mutant is deficient in JA- mediated response and the etr1 mutant is deficient in ET-mediated response. The mutation of CONSTITUTIVE EXPRESSION OF PR GENES 5 leads to the cpr5 mutant with a constitutive activation of the SA pathway (Bowling et al., 1997). The root growth of seedlings of the NahG, npr1 and sid2 lines could still be inhibited by ILA indicating that ILA perception in the root was independent from the SA pathway. The root growth of seedlings of the jar1 and etr1 mutants could also be inhibited by ILA, suggesting independence of ILA perception of the JA/ ET pathways (Figure 24). The root inhibition in the cpr5 line was effective, though SA levels were very high in the cpr5 mutant (Figure 24).

jar1 Col-0 npr1

NahG Col-0 sid2 NahG Col-0 sid2

npr1 Col-0 jar1

ugt76b1 Col-0UGT76B1 etr1 cpr5

ugt76b1 Col-0UGT76B1 etr1 cpr5

Control 500 µM ILA

Figure 24. ILA perception by roots in lines deficient in SA, JA and ET pathways and the cpr5

Root growth inhibition phenotypes by 500 µM ILA were observed among lines deficient in SA pathway (NahG , sid2), JA pathway (jar1) and ET pathway (etr1) and the cpr5 mutant. Col-0, ugt76b1 and UGT76B1 were taken as the control. The pictures were taken 10 d after sawing the seeds on plates containing 500 µM ILA.

2.4.6.3. The activity of ILA in inhibiting root growth regulated by GOX3 expression

During the photo-respiratory process, glycolic acid, the simplest hydroxy acid, is produced by oxidation of RuBP and further exported to peroxisomes where it is then oxidized by glycolate oxidase (GOX) to glyoxylic acid, causing the parallel formation of H2O2. Subsequently, glyoxylic acid is transferred to the amino acid glycine by glutamate: glyoxylate aminotransferase (Igarashi et al., 2003; Igarashi et al., 2006). Since ILA (2-hydroxy-3-methyl- pentanoic acid) has a very similar structure as the amino acid Ile, ILA may be converted to Ile via an intermediator precursor, 2-keto-3-methylpentanoic acid. This process would require a dehydrogenase. There are several candidates such as LDH and GOX. GOX3 has the function to oxidize the simple hydroxy acid to the keto form glyoxylic acid in vivo. To get a hint, the mutant gox3 was placed on medium containing ILA to observe the root growth inhibition

phenotype. Shorter root length conferred by ILA in the gox3 mutant suggested the potential of GOX3 to catabolize ILA to the keto-form, the precursor of isoleucine (Figure 25). This could be linked to the amino acid precursor keto-acid forms and metabolism of ILA.

Col-0 gox3-1 gox3-1 Col-0 ILA 400 µM gox3-1 Col-0 Control  

Figure 25. Root growth inhibition by ILA inversely correlates with GOX3 expression.

The root growth of the gox3-1 mutant was observed on media containing 400 µM ILA relative to the wild type (Col-0).

2.5. Exogenous application of ILA leads to specific transformation to isoleucine