Jl

Diameter classes, um

Fig. 10.4 Total length of (a) "fine roots" (<1.2 mm) and (b) "coarse roots" (>1.2 mm) within diameter classes in maize plants as in Fig. 10.1. Treatments acronyms and statistics as in Fig. 10.1. Asterisks indicate a statistically significant difference in respect to control (CONT; Tukey's test, at p < 0.05)

10.3.2 Soil Chemical Properties and CO2 Emissions from Microcosm-Confined Plant-Soil Systems b

Compost amendment markedly affected all selected soil chemical properties. The pH and EC values in COM-amended soils were significantly larger than for unamended soil treatments (Table 10.1), without being affected by plant growth. The TN content was increased by COM, either alone or in combination with CAT

Jo 300

Jo 300

CONT

CAT COM+CAT

Fig. 10.5 Root surface area in the maize plants as in Fig. 10.1. Treatments acronyms and statistics as in Fig. 10.1

CONT

CAT COM+CAT

Fig. 10.5 Root surface area in the maize plants as in Fig. 10.1. Treatments acronyms and statistics as in Fig. 10.1

(Table 10.1), while it resulted unaffected by both plant growth and time. Finally, TOC increase in both COM and the CAT + COM treatments was still noticeable at the end of the experiment (76 days), both in unplanted and in planted microcosms (Table 10.1), though TOC was found to slightly decrease (approximately by 5%) over time in all treatments.

According to Sikora and Stott (1996), these results suggest that the whole soil organic C pool does not rapidly respond to environmental changes, unless it is subjected to large amendments or anthropogenic disturbances. Thus, rather than following C-stock variations, we monitored the changes in soil respiration to evaluate the impact of the synthetic iron-porphyrin on soil C dynamics.

Before starting to monitor CO2 release from soil microcosms, a few weeks lag time was allowed to elapse, in order to avoid any masking action due to the "tillage effect", that generally occurs after soil physical disturbance (Ellert and Janzen 1999). In particular, the intensive soil mixing occurring during set-up of enclosed model systems is likely to lead to an overestimation of heterotrophic fluxes. As assessed elsewhere for the same clay loam soil used here, approximately 40 days were needed for the tillage effect to subside (Tortorella and Gelsomino 2011).

During the early experimental period, CAT further reduced the already low (<1 mmol CO2 m~2 s_1) amount of CO2 released from unamended soils (Fig. 10.10). At later stages, however, the soil CO2 efflux from unplanted microcosms sprayed with CAT appeared to be larger than control (Fig. 10.10). The contribution of the rhizosphere-derived C markedly increased soil CO2 flux. In fact, larger values were recorded in planted microcosms added with CAT.

Even though COM amendment stimulated CO2 emission from soil (CO2 efflux > 1 mmol CO2 m~2 s_1), CAT addition strongly depressed the compost-induced CO2 release over the entire experimental period (Fig. 10.11). As observed in the

30 0

30 0

0-400 400-800 800-1200 Diameter classes, um

0-400 400-800 800-1200 Diameter classes, um ii

Diameter classes, um

Fig. 10.6 Surface area of (a) "fine roots" (<1.2 mm) and (b) "coarse roots" (>1.2 mm) within diameter classes in maize plants in Fig. 10.1. Treatments acronyms and statistics as in Fig. 10.1. Asterisks indicate a statistically significant difference in respect to control (CONT; Tukey's test, at p < 0.05)

unamended soil, such depressive effect of CAT was reversed in the presence of plants, being the largest flux rates found in the planted COM + CAT treatments (Fig. 10.11).

Before transplanting, a reduced soil CO2 flux was recorded in CAT-treated microcosms, suggesting that the biomimetic catalyst was active in favoring the stabilization of soil organic compounds against microbial degradation. Although this process could have been still operating after transplanting, the increased plant-induced soil respiration rendered the role of the iron-porphyrin less evident.

CONT

CAT COM+CAT

Fig. 10.7 Root mass ratio in maize plants as in Fig. 10.1. Treatments acronyms and statistics as in Fig. 10.1

3 100 o

CONT

COM CAT COM+CAT

Fig. 10.8 Root fineness in maize plants as in Fig. 10.1. Treatments acronyms and statistics as in Fig. 10.1

An increased soil CO2 flux from plant-hosting microcosms was not unexpected, as plants may contribute strongly to total CO2 emission, due to root and rhizo-microbial respiration (Kuzyakov 2006). It is known that a close interaction occurs at the plant-root interface. This affects total soil CO2 flux, both directly, due to respiration by living root tissues (autotrophic flux component), and indirectly, due to microbial decomposition of dead plant residues, as well as rhizodeposits from living roots (heterotrophic flux component; Cheng and Kuzyakov 2005; Moyano et al. 2007). Here, it appears that iron-porphyrin and maize plant alone exerted an b a a c c a

0,10

JD O

0,04

ro 0,02

0,00

Fig. 10.9 Root tissue density in maize plants as in Fig. 10.1. Treatments acronyms and statistics as in Fig. 10.1

Table 10.1 Changes in pH, electrical conductivity (EC1:2), total organic C (TOC), and total N (TN) in the experimental microcosms (unplanted or planted) filled with control soil (CONT), compost-amended soil (COM), soil amended with iron-porphyrin (CAT) or compost-amended and iron-porphyrin-treated soil (COM + CAT) before and after the 76 days experimental period

Table 10.1 Changes in pH, electrical conductivity (EC1:2), total organic C (TOC), and total N (TN) in the experimental microcosms (unplanted or planted) filled with control soil (CONT), compost-amended soil (COM), soil amended with iron-porphyrin (CAT) or compost-amended and iron-porphyrin-treated soil (COM + CAT) before and after the 76 days experimental period

Treatment

pH

EC12

(dS m-1; at 25°C)

Day 0

Day 76

Day 0

Day 76

Unplanted

Planted

Unplanted

Planted

CONT

6.64 (0.08)a

6.66 (0.06)

6.68 (0.04)

0.156 (0.030)

0.221 (0.051)

0.229 (0.053)

COM

7.01 (0.07)*

6.95 (0.04)*

6.99 (0.03)*

0.384 (0.015)*

0.501 (0.087)*

0.472 (0.126)*

CAT

6.69 (0.06)ns

6.66 (0.06)ns

6.71 (0.05)ns

0.187 (0.024)ns

0.192 (0.069)ns

0.209 (0.023)ns

COM + CAT

6.98 (0.07)*

6.98 (0.03)*

6.95 (0.02)*

0.309 (0.075)*

0.415 (0.034)*

0.435 (0.074)*

Treatment

TOC (g kg-1

)

TN (g kg-1

)

Day 0

Day 76

Day 0

Day 76

Unplanted

Planted

Unplanted

Planted

CONT

19.2(0.5)

18.1 (0.6)

18.2(0.4)

1.8 (0.2)

1.7 (0.1)

1.7 (0.2)

COM

22.0 (0.2)*

20.9 (0.7)*

21.0(0.5)

*

2.1 (0.3)*

2.0 (0.1)*

2.0 (0.2)*

CAT

19.1 (0.6)ns

18.4 (0.4)ns 18.1 (0.4)ns

1.8 (0.1)ns

1.7 (0.1)ns

1.6 (0.1)ns

COM + CAT

21.5 (0.4)*

20.8 (0.5)*

20.7 (0.5)

*

2.1 (0.1)*

1.9 (0.2)*

1.8 (0.2)ns

a Values are means (n = 6) with standard deviation in brackets; for column means, asterisks denote significant differences respect to the control (CONT) at the 0.01 < p < 0.05 level (Tukey's test); ns, not significant at the chosen level of statistical significance.

a Values are means (n = 6) with standard deviation in brackets; for column means, asterisks denote significant differences respect to the control (CONT) at the 0.01 < p < 0.05 level (Tukey's test); ns, not significant at the chosen level of statistical significance.

opposite action on soil respiration. The combination of the two factors, however, unexpectedly increased soil CO2 emission.

A possible explanation of such unforeseen outcome is that the maize root system might have acted as a secondary target in the CAT treatment. This could have changed certain root morphological features, thereby determining an altered pattern of soil respiration rates. In fact, although maize roots from CAT and CONT

CONT COM CAT COM+CAT

Time (elapsing days)

Fig. 10.10 Soil CO2 efflux from compost-free microcosms. Treatments acronyms and statistics as in Fig. 10.1

Time (elapsing days)

Fig. 10.10 Soil CO2 efflux from compost-free microcosms. Treatments acronyms and statistics as in Fig. 10.1

Time (elapsing days)

Fig. 10.11 Soil CO2 efflux from compost-amended microcosms. Treatments acronyms and statistics as in Fig. 10.1

Time (elapsing days)

Fig. 10.11 Soil CO2 efflux from compost-amended microcosms. Treatments acronyms and statistics as in Fig. 10.1

treatments showed similar length- and mass ratio values, those for the former were coarser than for the latter (lower fineness and higher length in the >1.2 mm diameter classes). Noticeably, coarse roots are able to penetrate hard soil layers (Clark et al. 2003) and reorganize soil structure by alternate formation and destruction of soil aggregates (Haynes and Beare 1997). In this respect, Helal and Sauerbeck (1984, 1986) and then Cheng and Kuzyakov (2005) suggested "a positive priming effect of living roots on SOM decomposition by soil aggregate destruction". It may thus be inferred that a coarser maize root system induced by the biomimetic catalyst favored the breakdown of soil macroaggregates, thus exposing physically protected SOM to microbial attack.

Interestingly, a stimulatory effect on soil respiration due to CAT was also observed in the CAT + COM/maize plant treatment, suggesting that compost addition enhanced the response of the CAT-maize root interaction. However, stimulation of soil respiration by compost addition became statistically significant only at a late experimental stage, when compost was a single source of variability. An increase of soil respiration following compost addition had been previously observed by Borken et al. (2002) and Bastida et al. (2008), who hypothesized that such increase may be caused by (1) microbial decomposition of applied compost, (2) increased OM decomposition rate, (3) increased root respiration. Regarding this latter aspect, we noticed that the maize root system exposed to CAT + COM treatment was longer and characterized by more numerous fine and coarse roots than for control. We may speculate that morphological root traits of CAT + COM-treated plants could be functionally related to soil CO2 flux. In fact (1) a greater root length is related to enhanced efficiency of plant root systems to explore larger soil volumes (Ryser 1998) and, consequently, enhanced rhizo-microbial respiration may be inferred, (2) a greater proportion of fine roots has the potential to increase both autotrophic and heterotrophic microbial respiration through an increased release of root-derived C inputs (Pregitzer et al. 2000, 2008), (3) the disaggregating effect of the coarse portion of the maize root systems may contribute to soil respiration, as discussed earlier. Hence, root morphological changes induced by the combined CAT + COM treatment may be responsible for an increased soil CO2 flux.

As our results appear to suggest that a direct interaction between plant root system and biomimetic catalyst overcomes, or even contrasts, the catalyst action in stabilizing soil C, further studies were undertaken to understand if and how the iron-porphyrin per se could influence seed germination, as well as seedling establishment and growth. The main results of such studies are presented as follows.

10.3.3 Direct CAT Effects on Seed Germination and Post-germinative Processes

The CAT treatment had no significant effect on the germination of both garden cress and carrot seeds (data not shown). However, CAT appeared to stimulate

Fig. 10.12 Root growth in carrot (a) or garden cress (b) plantlets, grown for 9 or 6 days in the dark, respectively, as affected by the presence of increasing concentrations of CAT iron-porphyrin

Fig. 10.12 Root growth in carrot (a) or garden cress (b) plantlets, grown for 9 or 6 days in the dark, respectively, as affected by the presence of increasing concentrations of CAT iron-porphyrin radicle elongation in carrot seedlings, but not in garden cress, depending on concentration until the 6th day of growth (Fig. 10.12).

Since CAT-treated garden cress leaflets were visibly greener than control, exploratory experiments on the photo-induction of chlorophyll (Chl) and accessory pigment were run. In seedlings grown for 6 days in the dark and then exposed to 5 h of light, CAT increased the contents of total Chl (Fig 10.13) as well as carotenoids (Fig. 10.14) as a function of concentration, except for the seedlings treated with the largest CAT concentration (243 mM), whose Chl values were similar to control (Fig 10.13).

When the above photo-induced garden cress plants were exposed to a subsequent 20-h dark period, the Chl content decreased in all treatments, even though it remained larger in CAT-treated seedlings (except for CAT = 243 mM; Fig. 10.13). Carotenoids contents, on the other hand, were not reduced by exposure to dark, both for control and CAT up to 27 mM (Fig. 10.14).

Six days of germination under continuous light did not change the content of pigments in garden cress leaflets (Fig.10.15), even though Chl tended to decrease as

120 100 80 60 40 20 0

120 100 80 60 40 20 0

Fig. 10.13 Effects of increasing concentrations of CAT iron-porphyrin on total chlorophyll of garden cress plantlets grown for 6 days in the dark and then exposed to two different light/dark regimes

Fig. 10.13 Effects of increasing concentrations of CAT iron-porphyrin on total chlorophyll of garden cress plantlets grown for 6 days in the dark and then exposed to two different light/dark regimes

CD 20

CD 20

Fig. 10.14 Effects of increasing concentrations of CAT iron-porphyrin on total carotenoids of garden cress plantlets grown for 6 days in the dark and then exposed to two different light/dark regimes

Fig. 10.14 Effects of increasing concentrations of CAT iron-porphyrin on total carotenoids of garden cress plantlets grown for 6 days in the dark and then exposed to two different light/dark regimes the CAT concentration increased. Under continuous light, the smallest CAT concentrations, but not the largest, promoted both radicle elongation (Fig. 10.16) and seedlings' fresh weight in respect to control (+10%; Fig. 10.17).

Light is directly required for Chl synthesis at the level of enzymatic reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a direct precursor of Chls a and b. In angiosperms, such light- and NADPH-dependent reduction of the double

350 300

JB it ra 30

20 10 0

Total chlorophyll Total carotenoids

Fig. 10.15 Effects of increasing concentrations of CAT iron-porphyrin on photosynthetic pigments of garden cress plantlets grown for 6 days under continuous light

Fig. 10.15 Effects of increasing concentrations of CAT iron-porphyrin on photosynthetic pigments of garden cress plantlets grown for 6 days under continuous light

9 27 [CAT], pM

Fig. 10.16 Effects of increasing concentrations of CAT iron-porphyrin on the root growth of garden cress plantlets grown for 6 days under continuous light bond is carried by the NADPH-protochlorophyllide oxidoreductase (POR; EC 1.3.1.33). The binding of Pchlide and NADPH to the POR polypeptide leads to an organized ternary complex. Because of its light dependency, POR is not considered simply as an enzyme, but rather as a plastid-specific photon sensor, triggering pigment biosynthesis and membrane reorganization during the transformation of etioplasts to chloroplasts, and leading to conversion of cotyledons from storage organs to photosynthetically competent structures (Heyes et al. 2007).

J 10

J 10

Fig. 10.17 Effects of increasing concentrations of CAT iron-porphyrin on the fresh weight of garden cress plantlets grown for 6 days under continuous light

Fig. 10.17 Effects of increasing concentrations of CAT iron-porphyrin on the fresh weight of garden cress plantlets grown for 6 days under continuous light

Two different species of Pchlide have been identified. One of these is aggregated to the POR ternary complex and is termed photoactive because of its immediate reduction to Chlide a following a single millisecond flash illumination. It has a fluorescence emission maximum at 655 nm (Pchlide-F655). A second Pchlide form is non-photoactive, with an emission maximum at 632 nm (Pchlide-F632). It was supposed that Pchlide-F632 is non-photoconvertible because it forms Pchlide aggregates, which are not bound to the POR complex (Armstrong et al. 2000).

It has been reported that high levels of the photoactive Pchlide may protect plants from photodamage under conditions of high irradiance. In fact, the aggregated structure of PChlide-F655 and its association to the POR polypeptide might ensure an effective NADPH supply, thus channeling excess excitation energy towards photoreduction of Pchlide to Chlide, rather than to photo-oxidative processes (Skribanek et al. 2000).

Our observations lead us to speculate that the iron-porphyrin based catalyst, structurally similar to the tetrapyrrole intermediates in the chlorophyll biosynthesis, might be integrated in the POR ternary complex, thus acting as a photoactive Pchlide and promoting the formation of Chlide. At the largest CAT concentration (243 mM), however, an excessive accumulation of tetrapyrrolic molecules might occur, thus increasing the risk of electron abstraction and radical formation upon excitation by light. Such light-induced radical character could have been transferred to molecular oxygen, leading to a hyper-production of reactive oxygen species (e.g., singlet oxygen) with a consequent enhanced risk of oxidative damage. This may explain the depressive effect of large CAT concentrations on the content of photosynthetic pigments.

All together, the above observations suggest not only a direct "contact effect" of CAT on radicle growth, but also an uptake of the molecule by roots and its translocation to shoots with an influence on Chl metabolism. Should ad hoc biochemical and physiological studies might substantiate the preliminary evidence reported here, it is tempting to consider synthetic water-soluble iron-porphyrins as potentially useful molecules under typical stressful conditions in Mediterranean environments, due to their potential in severely affecting structure and function of light-intercepting machinery in plants, such as photo-oxidative stress and lime-induced chlorosis.

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