Treatment with OGs triggers local and systemic hormone responses in tomato roots and leaves
The spatio-temporal regulation of local and systemic responses to OGs was analysed on tomato plants grown in a hydroponic system. An aqueous OG solution was applied to the fourth fully expanded leaf (leaf treatment; LT) or to the roots (root treatment; RT). Local responses were examined in the treated organs (local, leaves or roots). In the case of leaf treatment, systemic responses were analysed in roots and in the non-consecutive, fully developed younger leaf, attending to the vascular connection in tomato plants (Orians et al., 2000) (sixth true leaf. In the case of root treatment, the equivalent sixth true leaf was harvested to study systemic responses (suppl. Fig. S1).
First, we studied the changes in the main plant defence hormones and some precursors and derivatives [SA, abscisic acid (ABA), ABA glucoside (ABA-Gluc), jasmonate isoleucine (JA-Ile) and 12-oxo-phytodienoic acid (OPDA; JA precursor)]. The levels of these compounds were measured at 1, 6 and 24 hours post treatment (hpt) and the data are presented in suppl. Figs. S2 a,b,c). A summary of the changes, expressed as fold changes compared to the corresponding mock-treated controls is shown in Figure 1.
Local responses to OGs appear to be very different in leaves and roots (Figure 1). In treated leaves, changes were observed mainly at 1 hpt, consisting of a slight accumulation of ABA, an important accumulation of JA-Ile [3.7 fold compared to mock-treated leaves] and a decrease of SA. A decrease of OPDA at 6 h was the only significant later response. In OG-treated roots only SA showed a change at 1 hpt: a slight decrease, followed by a moderate increase at later time points. ABA transiently increased at 6 hpt, dropping below control levels at 24 h.
Systemic responses of leaves to OGs were also different depending on the type of treatment (root or leaf) (Figure 1). A decrease of SA at all time-points and an accumulation of OPDA and ABA-Gluc levels at 6 hpt were observed only upon leaf treatment, whereas a transient decrease of JA-Ile was observed exclusively in RT-SL at 1 hpt. Levels of ABA-Gluc increased in systemic leaves with both treatments, with a delay in the case of root treatment.
Notably, systemic changes in hormone levels in the roots of leaf-treated plants were more pronounced (Figure 1). A strong accumulation of JA-Ile occurred at 1 hpt that was maintained up to 6 hpt and paralleled with a reduction of the JA precursor OPDA, revealing an upregulation of the oxylipin pathway in the roots upon leaf treatment. ABA and its derivative ABA-Gluc also showed a systemic transient increase in roots, and SA levels increased at the latest time point.
These data show that tomato plants respond systemically to OG treatment in leaves, with the most conspicuous hormone changes occurring in the root. Regardless of the site of OG treatment [leaf or root], an early and transient increase of ABA and a late increase in SA occur mainly in roots. Instead, induction of the oxylipin pathway, shown as early increases in JA-Ile levels, occurs in both leaves and roots only upon leaf treatments, although the effect is more durable in roots.
This hormone analysis was complemented with the expression analysis of genes involved in the JA, ABA and ET biosynthesis pathways (Table 1). In the local response, we observed an early and transient upregulation of the JA biosynthetic gene LOXD in both leaves and roots. In the systemic response, LOXD was induced only upon leaf treatment, with a stronger and more sustained induction in the roots than in upper leaves, confirming a general activation of the oxylipin pathway upon leaf treatment. Induction of the ABA biosynthetic gene NCED was found mostly in roots as both a local and a systemic response to leaf treatment. In addition, we examined the expression of the geneACO1 , encoding the ACC oxidase 1, responsible of the limiting step in ethylene biosynthesis. ACO1 was mostly upregulated as a local response in both leaves and roots. The induction was transient in OG-treated leaves, but stronger and more sustained in OG-treated roots. Thus, gene expression analyses confirm the activation by OG treatment of JA, ABA and ET signalling, with varying patterns according to the application site. They also support the conclusion of a faster response to OGs in roots, either as a local or a systemic response (Table 1).
As a whole, the transcriptional and metabolic data reveal that OG treatment impacts hormone signalling both at a local and a systemic level with induction of the oxylipins, ABA and ET pathways. Local hormone-related responses to OGs in leaves occur early and transiently, while systemic responses are delayed compared to local ones, with the stronger effects detected at 6 hpt. Remarkably, changes in hormone levels and expression of hormone biosynthetic genes were, in general, stronger and more sustained during time in roots.