Lignin and lignans in the defense response of Arabidopsis

In this thesis, lignan glucosides have been detected to be strongly enriched in Arabidopsis leaves in response to infection with V. longisporum in undirected measurements and in quantification by RP-HPLC (Fig. 14). In cross sections of hypocotyls and petioles it was additionally detected that lignin accumulated (Fig. 16).

Semiquantitative RT-PCR studies revealed that two CAD genes are induced upon infection, CAD5 and CAD8 (Fig. 17). This is in line with the accumulation of lignans and lignin because these enzymes catalyze the synthesis of coniferyl alcohol and sinapyl

85 alcohol – the precursors of lignin and lignans. Out of nine putative CADs in Arabidopsis these both genes are also described to be induced in P. syringae infection in Arabidopsis (Tronchet et al, 2010). The authors demonstrated by infecting knock out mutants of CAD5 and CAD8, that both genes are involved in the basal resistance of Arabidopsis against P. syringae. Based on this study a cad5 SALK-line was tested in this thesis for its susceptibility against V. longisporum, but no difference compared to the wild type could be determined (Fig. S2). This may be due to a redundant function to CAD4, as Tronchet et al (2010) also showed that in their infection system the double mutant of CAD4 and CAD5 was more susceptible than both single mutants. Another possibility of the unaltered susceptibility of cad5 might be based on the fact that the mutant is not a total knock out of the gene but has only reduced levels of cad5 mRNA.

Lignification is a common reaction of plants in response to infection with different pathogens. In the work of Michael Reusche (Rg Dr. Teichmann, Göttingen; personal communication) and in this thesis it could be shown that more lignified cells in V. longisporum infected Arabidopsis petioles and hypocotyls are present (Fig. 16). Also Floerl (2007) detected that lignin is enriched in V. longisporum infected Arabidopsis leaves.

In different studies it was shown that upon infection with different pathogens plants synthesize ‘defense lignin’, which can differ in the composition from the normal lignin (Zhang et al, 2007; Lloyd et al, 2011; Wuyts et al, 2006; Pomar et al, 2004). These defense lignins can not only have the function to reinforce the cell wall but also an antimicrobial one (De Ascensano et al, 2003).

The higher susceptibility of the fah1-2 mutant in the V. longisporum–Arabidopsis interaction shown in this thesis (Fig. 18), could therefore also be due to the different lignin composition of the mutant, which is due to a defect in the syringyl lignin synthesis (Meyer et al, 1998). There are several examples where syringyl lignin is highly enriched at the infection site, for example in Puccinia graminis infected wheat (Menden et al, 2007), in F. oxysporum elicited flax cells (Hano et al, 2006) and in B. cinerea infected Arabidopsis plants (Lloyd et al, 2011). Also in the B. cinerea interaction it was shown that the fah1-2 mutant was stronger infected than the wild type (Lloyd et al, 2011). The authors suggested that this is due to less defense related lignin locally deposed on the infection site. Lignin composition cannot only have an impact on the stability of the polymer but also on the degradability for pathogens. Syringyl lignin is a more linear polymer than coniferyl lignin and therefore better protects large areas of secondary cell walls from degradation (Jung & Deetz, 1993). Degradability of the cell wall in fah1-2 mutants was tested in different systems. For rumen microorganisms it was not shown to be higher degradable

86 (Jung et al, 1999), but in tobacco the cell wall of mutants with lower amounts of syringyl units was better degradable for fungal enzymes (Vailhé et al, 1996). So it is conceivable that the type of lignin found in the fah1-2 mutant results in a higher susceptibility.

In addition to the fah1-2 mutant also an overexpressor mutant (C4H:F5H) of the gene was analyzed in infections (Fig. 19). This mutant was described in the literature to harbor especially syringyl lignin instead of guaiacyl lignin (Meyer et al, 1998). Consistent with the theory of the importance of defense lignin in the interaction, fungal growth was reduced in the C4H:F5H mutants (Fig. 19). The VL-DNA amounts in infected plants were lower compared to wild type plants, although the reduction of the leaf area was comparable.

This leaf area reduction might be a secondary effect due to water deficiency symptoms in infected C4H:F5H plants at the end of the light period (Fig. S10). The reason for this symptom could not be elucidated so far.

Next to lignin, also lignans were shown in this thesis to be affected in Arabidopsis upon infection with V. longisporum. Lignans are widely distributed in different plant species. In Arabidopsis they were so far only described in the roots (Nakatsubo et al, 2008). But in this thesis, it could be shown that lignans like pinoresinol and lariciresinol are also abundant in the leaves of Arabidopsis and that they accumulate as glucosides in high amounts upon infection with V. longisporum (Fig. 14). In line with this, an induction of DIR6, which is involved in lignan synthesis, could be shown by semiquantitative RT-PCR analysis (Fig. 17). The induction of dirigent proteins was described to be active in conifer resistance against insects (Ralph et al, 2007) and may also imply a possible function in fungal defense of plants.

Next to the identified lignans pinoresinol and lariciresinol, also masses of related metabolites were found by the HPLC analyses of V. longisporum infected Arabidopsis leaves, but their identity could not be proven so far (Fig. 15). Due to the high variety between different plant species it may well be that Arabidopsis possess other lignans apart from pinoresinol and lariciresinol that are not yet identified.

Due to the lack of mutants the impact of lignans on the infection could not be analyzed in this thesis. The pinoresinol-reductase double mutant (Nakatsubo et al, 2008) was examined but in this mutant only the ratio of lariciresinol to pinoresinol is changed (Fig S12). Therefore it was not surprising that no differences compared to wild type could be detected (Fig. S2). In wheat plants it was described that overexpression of the pinoresinol reductase led to an increase in total amount of secoisolariciresinol diglucoside which is the final lignan in wheat (Ayella et al, 2007). Maybe also in Arabidopsis an overexpression of the gene could lead to higher amounts of lignans and therefore, to a valuable tool to test the importance of these compounds for infections.

87 Different functions of lignans in defense are conceivable, one is the function as an antifungal compound. Carpinella et al (2003) showed that pinoresinol has antifungal activity against different phytopathogenic fungi, for example F. verticillioides. The minimal inhibitory concentration in the study was determined to be 1 mg ml^-1 for pinoresinol. In this thesis, an inhibition of lariciresinol or pinoresinol on V. longisporum growth could not be shown, but this may be due to lower concentrations (up to 100 µM, approximately 0.035 mg ml^-1) used in the analysis (Fig. S13). Because the concentrations of the lignans at infection sites could not be estimated, the possibility of fungal growth inhibition upon higher concentrations of lignans cannot be ruled out. Additionally, the synergistic effects of different lignans, lignans with coniferin or other upregulated phenylpropanoids were not tested so far. Synergistic effects can lead to higher toxicity compared to single compounds and are widely described (Osbourn 1999, Carpinella 2005).

Another function of lignans in defense could be the generation of vascular obstructions to block the fungal spread. In woody plants lignans are described to be highly abundant in heartwood as antioxidant and to shut of non-productive water and nutrient transport to protect the sapwood and to tighten the longevity against wood rotting fungi (Naoumkina et al, 2010). Eynck et al (2009) could also show that phenolic compounds play a role in V. longisporum infected B. napus to build up occlusion in the vessels against the fungus.

This function could also be related to lignan accumulation. But the fact that the lignan accumulation is equally distributed in petiole and lamina (Fig. 23), does not favor this explanation.

4.2.3 Monolignols and its glucosides in the defense response of