Supplemental Material: Annu. Rev. Plant Biol. 2011. 62:227-250 Supplementary A Negative mycorrhizal responses As negative mycorrhizal growth responses (MGR) have received more experimental attention it has become clear that they are not invariably associated with high fungal biomass (and possibly rapid colonization) or low light and hence large C costs relative to photosynthetic supply, as previously thought (3). In his extensive experiment with 64 plant species paired with 10 AM fungi Klironomos (4) demonstrated an enormous range of MGR, from positive to negative. Although colonization was not reported, he mentioned that there was no relationship between percent colonization and magnitude or direction of MGR in a subset of the species, indicating that some negative responses occurred with low colonization. Using wheat, which frequently does not respond positively to AM fungi at least during early vegetative growth, Graham and Abbott (2) showed large negative MGR in plants that were either extensively or poorly colonized by different AM fungi. Li et al. (5) also used wheat in several experiments and again showed that negative MGR were not necessarily associated with high colonization. Gigaspora margarita induced negative MGR even though it colonized poorly (2 experiments), whereas G. intraradices, which colonized well, only caused a growth depression in one of the experiments (5). In a separate experiment growth of plants highly colonized by G. intraradices was unaffected under shade, whereas growth of NM plants was reduced; that of AM plants was unaffected (H-Y. Li, S.E. Smith and F.A. Smith unpublished data). The simplest explanation is that the AM plants were not C-limited but P-limited both at high and low light. As discussed in the main text a feasible hypothesis is that the direct pathway (DP) for P uptake is suppressed in AM plants and that the mycorrhizal pathway (MP) does not compensate for this reduction, particularly where transfer of P is low
in plants with low colonization. There are (many) other examples in the literature that cannot be reviewed here. A further complexity is that negative MGR has often been observed in relatively young plants. It has been appreciated for many years that negative MGR often disappears as plant mature (1; 3; 8), but relatively few experiments have been taken to the stage of seed production. In one such case, Li et al. (6) demonstrated negative MGR in wheat at tillering across a range of applied P. However, grain yield of one wheat variety was higher in AM plants, surprisingly at the highest levels of P application because positive MGRs are conventionally assumed to be greatest at low P. The only way of resolving the complex and interacting factors that have been suggested to induce negative MGR is to design experiments that will reveal the contributions of MP and DP in plants with both negative and positive MGRs, and to link this information not only with percent colonization and fungal biomass but also to actual C use by the fungi.
Supplemental Material: Annu. Rev. Plant Biol. 2011. 62:227-250 Supplementary B. Effects of P supply on contributions of MP and DP to P uptake by tomato Table S1 uses data extracted from Nagy et al. 2008 (7) to calculate effects of P supply (0, 20 and 60 mg P kg -1 soil) on uptake via the mycorrhizal pathway (MP) and direct pathway (DP) in tomato, which had slightly negative MGR when colonized by G. intraradices. Percent MP contribution declined as P supply increased. At P60 the specific activity of 33 P in AM plants was not significantly different from NM plants, so the 10% MP contribution is a maximum estimate. Given that MP operates only in colonized regions of the root where AM-inducible Pi transporter genes are expressed, this pattern is what would be expected from the observed decrease in percent root length colonized. A clearer picture is obtained when the contributions of the two pathways are calculated from the percentage contributions and total P uptake; these data can then be used to calculate specific uptake (mg P g -1 root dry weight). Table S1 shows that in AM plants the contribution of the MP to total P uptake was unaffected at P20 (compared with P0), and probably reduced at P60 (taking into account the low specific activity of 33 P in these plants). Specific P uptake via the MP (mg P g -1 root) decreased slightly as P increased from P0 to P20, but proportionately much less than the reduction in percent root length colonized or percent root length containing arbuscules (not shown). In consequence evidence that the operation of the MP in colonized regions of the root was repressed at P20 compare with P0 is very weak. However, expression of AM-inducible transporters was barely detectable at high P in a separate experiment, making it likely that repression did contribute to the low MP activity at P60. Total P uptake and specific P uptake via the DP both increased with increasing P supply, as also found in NM plants. This finding is to be expected from much previous work. However DP uptake in the AM plants was lower than in NM plants at all P levels, as shown in other examples of non-responsive plants (see
main text). Overall the results indicate two lines for future investigation. First, P repression of the AM pathway via reduced expression of AM-inducible Pi transporter genes requires confirmation in experiments in which gene expression and the contribution of the two uptake pathways are measured concurrently. Second, the finding that the total contribution of the DP and specific uptake via the DP were lower in AM than NM over a range of P supply, again points to subtle signaling between symbionts which deserves more research. In both cases it will be valuable to extend investigation to plants with positive MGR in which MP makes a higher contribution to total P uptake.
Table S1. The contributions of mycorrhizal (MP) and direct pathways (DP) to P uptake into Solanum lycopersicum (var Micro-Tom) colonized (AM) by Glomus intraradices or not (NM). Calculations are based on data for 6 week-old plants presented in Nagy et al. (7). AM NM P 1 Col n % P MP 3 SU (MP) 4 DP SU (DP) 4 DP SU (DP) 4 % via MP (mg P plant -1 ) (mg P g -1 root) (mg P plant -1 ) (mg g -1 root) (mg P plant -1 ) (mg g -1 root) P0 75 75 3.2 5.4 1.2 1.8 6.5 6.5 P20 35 30 3.7 3.2 8.6 7.5 14.7 12.8 P60 20 <10 2 <2.2 <1.8 18.9 16.4 25.9 21.6 1. P levels were additions of 0, 20 or 60 mg P kg soil -1 2. Specific activity of 33 P in AM plants grown at P60 was not significantly different from that in NM counterparts which had high variance; hence contribution of the MP could have been lower than values calculated here. 3. Contribution of MP (mg P plant -1 ) calculated from total plant P and % P taken up by MP 4. Specific uptake (SU) via MP and DP (mg P g -1 root) calculated from total uptake via MP or DP (mg P plant -1 ) and total root weight.
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