1 Introduction
3.5 Can we increase the growth of trees by manipulation of the
It has been calculated that the production of phenolics has high construction costs for plants (Vivin et al. 2003). According to allocation theory, plants have to allocate limited resources to different functions and to meet important needs (Caretto et al.
2015). For example, salicylates, PAs, flavonol glycosides and cinnamic acids may comprise up to one third of the dry mass of aspen leaves (Tsai et al. 2006). Major phenolics in birches are
typically PAs, which may constitute over 10% of the dry biomass (e.g. Witzell & Martin 2008). The leaves of ANRi birches contained phenolics accounted for 2–5% of DW (except for PAs) and WT birches for 1%, whereas in the stems, phenolics accounted for 11–13% and 4% of the dry biomass of ANRi birches and WT birches, respectively (II). According to GVA the concentration effect of phenolics was huge in ANRi birches, which may be due to the small size of the leaves (Figure 2b). ANRi birches used the energy for phenolics during the growth period, and there was a dramatic reduction in the number of resin glands from the optimum nitrogen (Figure 4 a-d) to the low nitrogen level (Figure 4 e-h). In addition, the overexpression of MYB134 in hybrid aspen led to increased amounts of phenolic acids and to reduced production of flavonoids and salicylates (II). These changes indicate that the plants allocate carbon differently if the synthesis of some compounds is blocked or enhanced.
The CNB and GDB hypotheses assumed that carbon is the limiting resource for phenolic synthesis (Bryant et al. 1983, Herms
& Mattson 1992). PCM argues that protein and phenol synthesis compete for the common enzyme L-phenylalanine, which is the branch point between the shikimate and phenolic pathways (Haukioja et al. 1998, Jones & Hartley 1999). However, PCM suggested that nitrogen rather than carbon is the limiting resource for both growth and synthesis of phenolics, since the biosynthesis of proteins and phenolics share the same precursor, Phe (Haukioja et al. 1998, Jones & Hartley 1999). Phe is considered to be a limiting precursor for phenylpropanoid synthesis, and at the same time it is a necessary amino acid. PAL catalyses the deamination of Phe producing a molecule of cinnamic acid and a molecule of ammonium (e.g. Craven-Bartle et al. 2013). In addition, MYB transcription factor regulates the expression of genes involved in Phe metabolism, and nitrogen recycling in conifers (Craven-Bartle et al. 2013).
If the reduction of phenolics were possible, it would be feasible to make a tree that could grow fast, as argued in the PCM hypothesis. High concentrations of PAs and salicylates have been associated with low growth in Populus species (Donaldson et al.
51 2006, Häikiö et al. 2009, Randriamanana et al. 2014). The growth of ANRi birches decreased, and the concentration of phenolics increased (II). By contrast, overexpression of the genes that increased the concentration of PAs did not cause decreased growth in aspen (III) (data not shown).
CNB suggests that low nitrogen availability limits growth more than it limits photosynthesis, and as a consequence, the accumulation of phenolics is enhanced (Bryant et al. 1983, Herms
& Mattson 1992). The GDB hypothesis suggests that situations where limited resources reduce photosynthesis, carbon supply is a factor predicted to limit both growth and defense. The birches grown at a low nitrogen level (II) had a lower content of chlorophyll than birches grown at optimum nitrogen level.
Birches also grew slower under the low nitrogen level than did birches under the optimum nitrogen level. Thus in this case, the GDB and CNB hypotheses are accepted, and a low level of nitrogen limited both growth and chlorophyll level in birches (II).
The same results have been found in several similar studies (e.g.
Keski-Saari & Julkunen-Tiitto 2003, Randriamanana et al. 2014).
According to GDB, limited resources should be shunted to growth processes rather than to differentiation (Stamp 2003). As mentioned earlier, limited resources have only minor effects for photosynthesis and this, connected with the slow growth, cause the excess of photosynthates. The limitation of growth processes modifies the concentration of phenolics, because there is found an excess pool of photosynthates that are used for phenolic formation (Herms & Mattson 1992, Stamp 2003).
The growth of ANRi birches decreased when compared with WT, and the total phenolic concentration (except for PAs) was significantly higher in ANRi birches than in WT birches (II). Even at the low nitrogen level the concentration of phenolics was higher in ANRi than in WT in both leaves and stems. Moreover, in ANRi birches, and likewise in well-grown WT, differentiated resin glands have been found. ANRi birches shunted the resources to differentiation, and not to growth processes. Thus, assumptions of growth over differentiation, as GDB suggests, did not apply to the ANRi birches. However, the production of trichomes is genetically controlled, and it occurs at the same time
with vegetative growth (Szymanski et al. 2000.) In addition, very young birch leaves contain both glandular and non-glandular trichomes (Valkama et al. 2003). Growth and differentiation are simultaneous processes, and differentiation may occur early in the juvenile phase (Lapinjoki et al. 1991). However, it was impossible to defend or reject the GDB hypothesis on the basis of this data set, since the GDB hypothesis makes assumptions about resource allocation by plants across a gradient. Two levels of nitrogen (II) are insufficient to give enough data to draw further conclusions according to the GDB hypothesis.
On the basis of my results, it would not be possible to achieve increased growth of birch trees by reducing the defense agents via genetic modification techniques, such as RNAi. Theoretically, recent developments in biotechnology allow the defense enhancement of plants, which could help to reduce risks posed by pathogens or herbivory. However, there is still a lack of extensive understanding of the advantages and trade-offs involved with genetically modified trees (Hjältén & Axelssons 2015). We do not know whether complete silencing of the ANR gene is a lethal factor for birches. mRNA levels of ANR at its best was reduced by about 90% in ANRi birches and had strong effects on the phenotype. Moreover, about 20–25% (depending on the line) of ANRi birches died after acclimation time. These findings draw attention to the real importance of phenolics in regulating the growth of the plant, particularly in the early juvenile phases.
Traditionally, metabolism has been divided into discrete pathways. Generally, the phenolic pathways appear to be plastic.
Studies conducted so far suggest that it may be hard to alter the specific phenolic constituents of trees without changes in other phenolics, since metabolism operates as a highly integrated network (e.g. Boeckler et al. 2011, Sweetlove et al. 2008).
Modifications in the phenolic pathways are extremely prone to unexpected effects. As suggested by Koricheva et al. (1998), Keinänen et al. (1999a), and Keski-Saari et al. (2007), the phenolic pathway may be a network with trade-offs between different branches (II): When concentrations of some phenolics in different
53 branches are reduced (PAs, II; PGs, III), others are increased (phenolic acids, flavonols, II; PAs, phenolic acids, III).
Unintended effects resulting from GM can have both negative and positive ecological impacts on plant resistance to herbivores (Hjältén et al. 2007, Hjältén et al. 2012, Klocko et al. 2014, Vihervuori et al. 2013). Boeckler et al. (2014) have reported that upregulation of PA synthesis in Populus increases performance and leaf consumption by generalist herbivores; tent caterpillar (Malacosoma disstria) and gypsy moth (Lymantria dispar), which was explained by decreased levels of PGs in GM lines (Boeckler et al. 2014). Similarly, overexpression of MYB134 caused changes in aspen that increased the preference of specialist P. vitellinae (III) for these lines where the concentrations of salicylates were the highest.
Foliar PAs have been found to have functional importance in Populus (Harding et al. 2014). Differences have been found in some metabolites in the phenolic pathway when comparing the fast-growth Populus line with slow-growth Populus lines (Harding et al. 2014). PA accumulation can affect both chemical defense and growth rate in Populus (Harding et al. 2014). PA accumulation impacts nitrogen and carbon utilization in developing slow-growth Populus leaves (Harding et al. 2014).
Nitrogen or climate conditions can modulate the phenylpropanoid metabolism, and perhaps also growth in Populus (Harding et al. 2009). Findings in ANRi birches versus WT birches support these results. Nitrogen and carbon might be important factors for formation in phenolics, especially in the juvenile phase of ontogeny.
3.6 MAIN FINDINGS OF EACH ORIGINAL PUBLICATION
The main findings for each article are summarized below:
Article I
The early-flowering birch (B. pendula) has a similar phenolic composition to that of the northern, native B. pendula.
Concentrations of the main phenolics decreased during the experimental time, especially in the leaves.
Concentrations of PAs and procyanidins was highest in stems after planting to peat. In roots, concentrations of the the main phenolics, hydrolysable tannins and their precursors, and proanthocyanidins, either remained stable or increased towards maturation.
The relative concentration of most of the phenolics remained fairly stable in the leaves and stems despite vigorous growth.
Only the ANS gene in the PA pathway was expressed differently in young parts of the plant when compared to mature parts in the leaves and stems.
Article II
Inhibition of ANR gene expression reduced the growth of ANRi birches, and the concentration of PAs was reduced in comparison to WT birches.
The accumulation of other than PA-phenolics, increased strongly in ANRi birches as compared to WT birches.
Compared to low nitrogen level, optimum nitrogen level reduced concentrations of phenolics, while, as expected, increased growth.
In response to low nitrogen level concentrations of certain phenolic compounds increased in the stems of WT birches and decreased in stems of ANRi birches.
PAs may play an important role for the normal growth of birches.
Article III
Two out of three MYB134 overexpression lines accumulated high levels of PAs, phenolic acids and (+)-catechin, with a concomitant decrease in the levels of salicylates and flavonoids.
Concentrations of phenolic compounds were generally lower in aspen leaves grown under elevated temperature than in those grown under ambient temperature, regardless of the line.
55
A specialist leaf beetle, Phratora vitellinae preferred leaves grown at ambient over those grown at elevated temperature.
P. vitellinae preferred the leaves that have the highest content of their feeding cues, salicylates.
The growth of aspen was increased under elevated temperature, but MYB overexpression did not affect the growth.
57
4 Conclusions
Many chemical compounds have several concomitant functions and therefore allocation to different processes, such as growth, defense or reproduction is not straightforward. PAs are multifunctional and may affect plant vitality in multiple ways (e.g. Mellway & Constabel 2009). This thesis shows that PAs and other phenolics are important for plant development in all plant tissues. The phenolic pathway is a network based on an apparent trade-offs between biosynthetic pathway, such as between PGs and PAs in aspens, and flavonols and PAs in birches. In addition, many metabolites were found in birches where PA production was downregulated, that were not detected in WT birches. When the concentration of PAs was high the concentrations of PGs and flavonols were at a lower level in aspen, whereas low concentrations of PAs increased the concentrations of flavonols in birches. These results indicate carbon reallocation between the different branches of the phenolic pathway.
Environmental stimuli induce the concentration of phenolics.
The studied temperature and nitrogen effects were clear; both environmental factors affected the concentrations of phenolics and the growth of the plants. Nonetheless, the interaction between elevated temperature and food quality may affect the intensity of herbivory and the consumption rate.
The common carbon allocation hypotheses (CNB, GDB and PCM) were partly accepted for the studied species. The generally known increase of phenolics under low nitrogen fertilization and at ambient (relatively low) temperature was also found in birches and aspens. This supports the GDB hypothesis. One question that has been a subject of debate for a long time still remains open:
How do the plants distribute carbon between primary metabolism and from stress-induced phenolics? The evidence for PCM remained incomplete as the concentration of Phe was not analyzed in this thesis.
Despite the new knowledge gathered in this study, further studies are still needed in order to understand the role of these complicated branches of flavonoid and phenylpropanoid biosynthesis in whole plant growth. It would be necessary to approach the problem taking into account the connection between the genes of PA biosynthesis, plant hormones (auxin) and, in particular, mRNA levels within different experimental levels, such as different temperatures. Phenolic pathway studies need to be integrated with studies on plant hormones related to growth, flowering and senescence. The response of different genes of the the PA pathway and individual enzymes in this pathway at different nitrogen levels should be taken into consideration. It would be interesting to offer the leaves of the ANRi birches to different herbivores and study the palatability of the leaves. It is most obvious that transgenic plants are powerful tools for these studies, while keeping in mind that the enhancement or inhibition of one gene may lead to unexpected effects on other chemical pathways.
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