Hide menu

AM symbiosis affects the morphology and the growth of M. truncatula

The M. truncatula plants were treated with Gi as a fungus root (AM),without Gi (NAM), with inoculums’ filtrate (Fil.) and 5mM KH2PO4 (Pi 5mM).   Growth was observed during the development and pictures were taken after one, two, three and four weeks. The plant appearance reveals that AM and Pi (5mM) plants look similar to each other but greater than NAM and filtrates plants. The AM plant looks more greenish than NAM (fig. 1).

Fig.1: Morphological study of M. truncatula plants that were grown under growth light ( 300 µmol m-2 s-1) with G. intraradices (AM), Without G. intraradices(NAM), without G. intraradices but AM inoculums’ filtrate were given at the time plantation ( Fil.) and without G. intraradices but KH2PO4 were given at 5mM concentration (Pi 5mM).

Root mycorrhization

To monitor the level of development and establishment of the symbiosis, the root systems of all plant groups were analyzed with black ink staining. An apparent fungal structure indicating symbiosis was seen in AM plants and it comprised of intra-radical hyphae, arbuscules and vesicles (Fig. 2A). No sign of fungal structure inside the roots were seen in the control plants including NAM , Pi (5mM) and filtrate (Fig. 2B, 2C, 2D).

Fig.2: M. truncatula root staining with blank ink solution. (2A) Arbuscular mycorrhized (M. truncatula cultured with Glomus intraradices) is showing fungal colonization and it is easily distinguishable from nonmycorrhized roots. Arrows are indicating arbuscules, double arrow is indicating intra-radical hyphae while star is showing vesicles. No colonization was found in roots of other plant groups.

Effect of AM symbiosis on plant biomass

All groups of M. truncatula plants were harvested after four weeks. Fresh leaves, stems and roots were weighed for biomass.   Our results indicate that AM plants have more biomass of roots, stems and leaves. Overall, the AM plant shows significant increase in biomass as compared to NAM and filtrate (an average increase of 2.44 fold, fig.3). We found no difference in biomass between AM and Pi (5mM) plants while NAM and filtrate also did not differ significantly in biomass.   We calculated fresh shoot to root ratio and over all, we found that AM roots has more contribution in biomass as compared to NAM .  

 

Fig.3: Plant biomass; fresh roots, stems, leaves & total weight increased and differ significantly from NAM & Filtrate (P < 0.0001) while no difference from Pi (5mM)(P > 0.01 ).

AM symbiosis increases phosphorus uptake

Our result shows that the AM plant has less remaining free Pi (mg/kg) in the pot soil as compared to NAM and filtrate. A significant decrease in soil free Pi levels (average decrease of 58%; Fig. 4)  in AM plant was observed indicating that fungal symbiosis has increased the free Pi uptake in the AM plants. We have not found large differences in the amount of remaining total P (mg/kg) in the pot soil of AM, NAM , filtrate and Pi (5mM) plants (fig.4).

Figure 4: Remaining Total P and Free Pi in the soil of 4 weeks old M. truncatula that were grown under growth light (GL=300µmolm-2 s-1) and with their respective treatments.

The amount of total phosphorus (nmole/fresh plant) and free Pi (nmole/fresh plant) were also analyzed in roots, stems and leaves of fresh plants, harvested after four weeks.   Our results shows that AM plants have increased uptake of total P and free Pi content in roots, stems and leaves. The AM plant shows significant increase in total P and free Pi uptake as compared to NAM and filtrate (fig. 5) while decreased from Pi (5mM). 

Figure 5: Shoot and root phosphorus contents of 4 week’s old M. truncatula that were grown under growth light (GL=300µmolm-2 s-1) and with their respective controls.

Levels of photosynthetic proteins

The isolated thylakoid membranes were tested to monitor the changes in protein level of PsII complex.   Initially, western blotting was performed for D1 and Lhcb1 antibodies. Both are subunit of PSII complex, namely the reaction centre D1 protein and the light harvesting Chl binding protein 1. The results shows that the overall D1 protein contents not differ significantly in AM as compared to NAM, Filtrate and Pi (5mM) (fig. 6). Initial western blot analyses with anti- Lhcb1 and Lhcb2 antibodies showed that overall Lhcb1 proteins are accumulated at about 20% higher levels (fig. 6) in AM as compared to NAM , Fil and Pi (5mM) plants.Similar pattern to Lhcb2 was obtained when Lhcb2 antibody was used.  

Fig.6: Protein western blot analysis by using antibody as a probe of thylakoids membrane, isolated from 4 weeks old M. truncatula plants groups (AM, NAM, Pi (5mM), Fil.) and assayed for the, D1 protein contents, Lhcb1 and Lhcb2 protein contents.

Oxygen evolution and chlorophyll content

The results show that the Chl a/b ratio was not significantly affected by AM symbiosis in AM plants as compared to NAM, filtrate and Pi (5mM) plants. But the total chlorophyll was increased in AM plants (an average increase of 1.8 fold from NAM , table 1) as compared to NAM . The steady state oxygen evolution activities in AM plants were also increased and highly significant differences were found (an average increase of 1.5 fold from NAM , table 1) as compared to NAM and filtrate. Taken together, these results indicate that AM plants have more active PSP machinery as compared to NAM while Pi (5mM) plants have better PSP than AM

Table 1: O2 evolution and chlorophyll content of 4 weeks old M. truncatula plants (AM, NAM, Fil and Pi (5mM)) grown under growth light (GL=300µmolm-2s-1) conditions.

Chlorophyll fluorescence as a measure of PSII activity

Our result showed that photosynthetic electron transport rate (ETR) increased in AM plants as compared to NAM plants. In the similar way the results showed that Pi (5mM) plants have greater ETR values as compared to AM (fig. 7). Our results shows that at this tested PAR intensity, the AM plants showed similar NPQ values to other plant groups. No differences were found among all respective plants group (fig. 7). Taken together, the significantly higher ETR indicates that AM plant has more efficient and responsive to solar energy while greater ETR value indicates that Pi (5mM) plants have better PS P than AM plants

Fig.7: Measurement of electron transport rate and kinetics for induction of non photochemical quenching in M. truncatula plants that were grown under growth light (GL=300µmol m-2 s-1).

Effect of AM symbiosis on reproductive fitness of M. truncatula

The results show that AM plant has better seed quality as compared to NAM , Fil and Pi (5mM) (fig. 8). Statistically the number of fruits per plants did not show any difference among plants groups while mg of seeds per plants in AM plants increased and differ significantly from NAM, fil and Pi (5mM) ( an average increase of 1.7 fold; table 2).

Fig.8: Reproductive fitness of M. truncatula that were grown under growth light (GL=300µmolm-2 s-1) for 12th weeks with AM, NAM, Fil and Pi (5mM) conditions.
Table 2: Seed quantity of M. truncatula that were grown under growth light (GL=300µmolm-2 s-1) for 12th weeks with AM, NAM, Fil and Pi (5mM) conditions.


Responsible for this page: Director of undergraduate studies Biology
Last updated: 05/25/10