Growth inducing effects of AHLs

The influence of AHLs on the morphology of barley was investigated using a newly developed glass bowl system (fig. 2.2), providing axenic conditions, easy handling, and good observation of growth. The growth in agar-agar supplemented with 10 µM AHL resulted in increased biomass for fresh and dry matter of shoots and roots (fig. 3.1). Interestingly, not only the short-chain AHL was able to induce biomass gains, but also, to a lesser extent, the long-chain AHL, which is contradicting to recent publications. In Götz-Rösch et al. (2015) no effects on the root and leaf fresh weight could be determined when barley and yam bean plants were treated with C6-, C8, and C10-HSL, whereas A. thaliana showed significantly

DISCUSSION

65 increased root fresh weights after C6-HSL and shoot fresh weights after C6-, C8- and C10-HSL treatments (Schenk et al., 2012). Further, the long-chain AHLs C12- and oxo-C14-HSL did not lead to any biomass gain (Schenk et al., 2012), which could be refuted here with the biomass gaining effect of C12-HSL. Beside the fresh weights, the dry weights were also determined, as fresh weights tend to show biomass variations, caused by moisture differences of the plant, by the growth system, and by the experimental environment (Bashan and De-Bashan, 2005). These effectors could possibly explain the wide range of deviation of the fresh weights compared to the dry weights in the present investigations (fig. 3.1).

Additionally, the determination of the dry weights provides proof that the significant biomass gain in fresh weight is achieved by higher dry matter and not by increased water storage of the plant, as it has been discussed by Bashan and De-Bashan (2005). In any case, the application of short-chain AHL led to slight root elongation and a 19 % increase in the root tip number, whereas the long-chain AHL increased the number of root tips by 28 %, compared to the control (fig. 3.2 and 3.3). Concluding that every root tip stands for a single root, it could be assumed that, in fact, AHLs have the ability to influence post-embryonic root development by the stimulation of lateral root formation. Our results clearly demonstrate that the alterations in the root system architecture are AHL acyl-side chain length dependent.

Accordingly, previous studies displayed the relationship between various AHL derivatives and the magnitude in changes of growth and morphology of plants (reviewed in Hartmann et al., 2014). Thus, short-chain AHLs are related to root elongation or inhibiting effects and long-chain AHLs are involved in lateral root and root hair formation (Ortíz-Castro et al., 2008;

Teplitski et al., 2011). Furthermore, the observed root weight increase after AHL treatment correlates well with the stimulation of lateral roots. Besides gains in plant biomass and root hair growth (Dobbelaere et al., 1999), PGPRs induce the promotion of lateral roots (Verbon and Liberman, 2016). Inoculation experiments with the PGPR strain Serratia marcescens 90-166 and the application of different fractions of the cell culture and the cell lysate led to lateral root formation in A. thaliana (Shi et al., 2010). The authors discuss that besides the auxin production of the strain, which could induce lateral root formation in plants (Vacheron et al., 2013), additional compounds are possibly involved, because they could determine an AHL production of this strain (Shi et al., 2010). Three different short-chain AHLs are produced by Serratia marcescens 90-166: C4-HSL, oxo-C6-HSL and C8-HSL (Ryu et al., 2013). It is likely that these AHLs might also be involved in the generation of lateral roots because the different fractions of the cell culture and the cell lysate were tested positive (Huang et al., 2016). In the present study, the single application of C8-and C12-HSL led to the formation of lateral roots in barley (see fig. 3.3). Accordingly, C10-HSL also induced lateral roots in the model plant A. thaliana (Ortíz-Castro et al., 2008). The present data confirm lateral root induction after AHL treatments and show that purified AHLs are sufficient

DISCUSSION

66 for lateral root promotion. This gives rise to the assumption that these quorum sensing molecules could be an additional acting part in PGPR induced lateral root formation.

It has been demonstrated that AHLs are systemically transported. So, short-chain AHLs were detectable in roots and shoots, long-chain AHLs only in the root using UPLC and FT-ICR/MS, whereas application of tritium-labeled AHLs led to the confirmation of C10-HSL in leaves (Götz et al., 2007). Furthermore, reportedly long-chain AHLs are faster degraded in plant tissue compared to short-chain AHLs and therefore it might be possible that their metabolites are partly transported into the leaves (Götz-Rösch et al., 2015). The systemic AHL-transport leads to the assumption that further signaling pathways are activated, implying the induction of phytohormones. Auxin and cytokinin possess a distinct role in root and leaf morphological development, which was demonstrated by Skoog and Miller (1957). It seems that higher cytokinin concentrations lead to leaf development and increased auxin levels to better root formation. Von Rad et al. (2008) demonstrated increased cytokinin concentrations in leaves and 4-fold induced auxin levels in roots after AHL application, changing the auxin-cytokinin ratio towards higher auxin concentrations, compared to the control. Similar reactions can be assumed for barley, leading to the demonstrated biomass gain (see chapter 3.1.1 and summarizing figure 4.1).

We further hypothesize that a stronger branched root system and a larger root surface lead to better and higher nutrient uptake, which then results in enhanced biomass. The augmentation of the root system improves nutrient availability and enables the plant to access new, so far unrooted soil regions. Also rhizobacteria profit and obtain more nutrients (rhizodeposits) from colonizing new root tissue. This effect results in a positive feedback loop, where the AHL producing bacteria improve plant growth, fitness, and nutrient supply while the host plant provides the bacteria with more nutrients and habitat. Therefore, rhizobacteria might secrete AHLs to create a better living space for themselves, whereas plants are able to interfere and to direct the microbial signal production, so that no bacterial overgrowth will occur (Zarkani et al., 2013). Accordingly, a positive impact of microbial derived molecules on the plant nutrient supply has been demonstrated by Joseph and Phillips (2003). Here, Phaseolus vulgaris was treated with AHL breakdown products (L-homoserine), resulting in enhanced stomatal conductance and transpiration. In this context Palmer et al. (2014) suggest that the growth stimulation of AHLs is dependent on the activity of a fatty acid amide hydrolase, which cleaves the AHL by obtaining the L-homoserine, the active compound that demonstrably stimulates transpiration. This enhanced transpiration implies higher water and nutrient uptake and may lead to growth stimulation (see fig. 4.1, Palmer et al., 2014).

DISCUSSION

67 Figure 4.1 AHL application leads to root and leaf growth induction and root system augmentation. Possibly, increased cytokinin levels in leaves and increased auxin levels in roots (von Rad et al., 2008) are involved in the total biomass gain and root architecture change. Augmentation of root system may recruit more PGPRs due to more rhizodeposits. AHL-degradation products reportedly increase transpiration and therefore positively influence nutrient uptake (N=nitrogen, P=phosphorus and K⁺).