Regulation of Root Length and Lateral Root Number in Trifoliate Orange Applied by Peroxide Hydrogen and Arbuscular Mycorrhizal Fungi

Root system morphology (RSM) in plants plays a key role in acquiring nutrients from the soil and is also altered by abiotic or biotic factors including soil microorganisms and signal molecules. The present study was made to evaluate the effects of an arbuscular mycorrhizal fungus (AMF, Glomus versiforme) and exogenous peroxide hydrogen (H2O2, 0, 1 and 100 μM) on root length, lateral root number and activities of polyamine-metabolized enzymes in trifoliate orange (Poncirus trifoliata) seedlings. After 5 months of inoculation with AMF, root mycorrhizal colonization was significantly increased by application of 1 μM H2O2, but markedly restrained by 100 μM H2O2. Inoculation with AMF significantly increased the taproot length and the number of secondand third-order lateral roots under 1 and 100 μM H2O2 application. The AMF infection significantly increased 0-1 cm classed root length and total root length, regardless of H2O2 concentration. In general, inoculation with AMF increased arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activity of roots under 0, 1 and 100 μM H2O2, increased diamine oxidase (DAO) activity of roots under 0 μM H2O2 and decreased DAO activity of roots under 1 and 100 μM H2O2. Root polyamine oxidase (PAO) activity was similar between AMF and non-AMF seedlings, irrespectively of H2O2 concentration. Results suggest that lower concentration of H2O2 (1 μM) might be regarded as a signal to stimulate mycorrhizal and lateral root development through increase of ADC and ODC and decrease of DAO, while high concentration of H2O2 (100 μM) as a toxic compound of reactive oxygen species restricted AMF colonization.


Introduction
Root is a vital plant organ which absorbs water and nutrients from the soil.Meanwhile, root system morphology (RSM), the configuration and structure of the root system, can determine the transport capacity of root in soil nutrient and water (de Dorlodot et al., 2007).In general, rhizospheric nutritional status and microorganisms would highly impact RSM (Osmont et al., 2007;Chapman et al., 2011;Sato and Miura, 2011).
Arbuscular mycorrhizal fungi (AMF), a kind of soil inhabitant microorganism, can form a mutual symbiont with the roots of ~80% of land plants, thus enhancing the uptake of mineral nutrition and water from the soil to the host plant, as well as withstanding various adversities (Koide and Mosse, 2004).Studies showed that inoculation with AMF could alter the RSM traits and promote the formation of lateral roots of higher order for the host plant (Yao et al., 2009).The AMF-mediated RSM improvement may be related to increased plant hormone biosynthesis proposed by Kaldorf and Ludwig-Müllerb (2000) in maize colonized by Glomus intraradices.Wu et al. (2011) found that the RSM alteration by mycorrhization may be due to the AMF-induced alteration of carbohydrates and peroxidase.However, other studies also revealed that AMF colonization did not alter RSM traits.Atkinson et al. (2003) found that root branching of Trifolium repens was not significantly affected by inoculation with G. mosseae.Herdlera et al. (2008) reported that the total root length and the root diameter of rice plants were notably decreased by the G. intraradices inoculation.
Hydrogen peroxide (H2O2) as one of reactive oxygen species (ROS) generally acts a key regulator in a broad range of physiological processes, such as senescence (Peng et al., 2005), stomatal movement, cell cycle and growth and development (Deng et al., 2012).The H2O2 excess accumulation in plants could be used as ROS to induce oxidative burst in cells, thereby triggering cell death.On the other hand, H2O2 is also regarded as an important signal compound concerning the interaction of plants with pathogenic microorganisms (Apel and Hirt, 2004;Laloi et al., 2004), as well as many other processes referring to root biology, such as gravitropism, root elongation growth and root hair development (Zolla et al., 2010;Jiang et al., 2012).However, the interaction between AMF and H2O2 on RSM traits of the host plant is poorly known.
Diamine putrescine (Put), triamine spermidine (Spd) and tetraamine spermine (Spm), three types of polyamine (PAs), are formed either by the direct decarboxylation of Lornithine through ornithine decarboxylase (ODC) or by decarboxylation from L-arginine to agmatine by arginine decarboxylase (ADC).The decomposition of PAs takes place under the two enzymes, diamine oxidase (DAO) and polyamine oxidase (PAO) (Kaur-Sawhney et al., 2003).It is well documented that PAs play important roles in mycorrhizal formation, plant growth, organ development, fruit formation and leaf senescence (Kusano et al., 2008;Wu et al., 2012b).In root development, accumulation of PAs is involved in the formation of adventitious root (Couée et al., 2004).Exogenous PAs could significantly promote the spore germination and hyphal growth of G. mosseae and Gigaspora margarita, in vitro culture conditions (Zhang et al., 2003).Wu et al. (2012a) observed that AMFmediated RSM improvement was related only to ADC and ODC, without any PAs concentrations.It seems that PA synthetase and degraded enzymes, except PA levels, were associated with AMF-mediated RSM.In addition, PA degradation may also act as a pathway to generate H2O2, which is considered to be an active secondary messenger (Vandenabeele et al., 2003).However, so far, in plants, the potential relationship between RSM and PA-metabolized enzymes under H2O2 and mycorrhization conditions has not been examined in detail.
The aims of the present work were to (1) clarify the integrated effects of AMF and H2O2 on root length, lateral root number and PA-metabolized enzymes of citrus and to (2) evaluate the relationship between RSM and PAmetabolized enzymes.

Plant culture
Seeds of trifoliate orange [Poncirus trifoliata (L.) Raf.] were surface-sterilized with 70% ethanol solution for 5 min, washed with distilled water and then sown into a plastic pot (18 cm upper diameter × 13 cm below diameter × 17 cm height) including 2.5 kg autoclaved (121 ºC, 0.11 Mpa, 2 h) soil.The inoculums of Glomus versiforme (40 g) including spore and extraradical hyphae were placed below 5 cm of the soil surface at the time of sowing.The non-AMF pots were also supplied with 40 g sterilized inoculums as the control.

Experimental design
The experiment consisted in a completely randomized design with two factors: inoculation with or without an AMF (G.versiforme) and application of exogenous H2O2 with 0, 1 and 100 µM.Each treatment had three replicates, reaching a total of 18 pots.Three months after mycorrhizal inoculation, different concentrations (0, 1 and 100 µM) of H2O2 were weekly applied into the designed pots, nine times during the experiment.

Measurement of plant growth and root colonization
The AM and non-AM plants were harvested after 63 days of exogenous H2O2 treatments.Before harvest, plant height, stem diameter and leaf number were directly determined.
Root mycorrhizal colonization was measured by the method of Phillips and Hayman (1970) after clearing with 10% KOH and staining with 0.05% trypan blue.

Determinations of root system morphology and lateral root number
The root systems were carefully taken out from the plastic pots and the intact root systems were scanned immediately by the Epson Perfection V700 Photo Dual Lens System (Seiko Epson Corp, Japan).The images of root systems were analyzed by a WinRHIZO version from 2007 (Regent Instruments Incorporated, Canada) and the 0-1, 1-2, 2-3, 3-4 and >4 cm classed root lengths were automatically obtained.Taproot lengths were measured by a flexible rule and the number of lateral roots was artificially quantified.

Determinations of PA-metabolized enzyme activities
Activities of PA synthetases (ADC and ODC) and PAdegrading enzymes (PAO and DAO) were determined according to the methods previously described by Wu et al. (2012a).

Statistical analyses
Data were subjected to the analysis of variance (ANOVA) with SAS v8.1, and the significant differences among treatments were used for comparison to the Duncan test at P<0.05.

Mycorrhizal colonization
After inoculation with G. versiforme, root colonization of the trifoliate orange seedlings ranged from 55.28 to 75.91% (Fig. 1).Compared to 0 μM H2O2 control, 1 μM H2O2 significantly increased AMF colonization by 16.6%, whereas 100 μM H2O2 level notably decreased mycorrhizal infection by 15.1%.There is increasing evidence indicating that during the early establishment of a symbiotic relationship, many plant signals and fungal perception are involved in the process (Requena et al., 2007).It seems that the low concentration of H2O2 (1 μM) might be regarded as a signal molecule to stimulate mycorrhizal development, while high concentration of H2O2 (100 μM) as a toxic compound of ROS restricted AMF colonization.

Growth performance
The present study showed that exogenous H2O2 and AMF colonization did not significantly alter the stem diameter of the seedlings (Tab.1).Leaf number per plant was significantly higher in AMF than in non-AMF seedlings under 0 and 100 µM H2O2, except treatments under 1 µM H2O2 (Tab.1).Compared to non-AMF seedlings, AMF seedlings showed notably higher plant height under 0 µM H2O2 conditions, but lower under 100 µM H2O2 conditions.In addition, compared to 0 µM H2O2, 1 and 100 µM H2O2 generally increased plant height and leaf number, suggesting that the low or high concentration of H2O2 did not absolutely interfere with the plant growth performance.Previous studies had shown that AMF could facilitate the growth of the host plants, attributed to extraradical hyphal absorption of water and mineral nutrients (Sharif and Claassen, 2011).Although low concentrations of H2O2 promoted root AMF colonization, the H2O2-induced increase of root colonization, they did not show significant effects on plant height, stem diameter and leaf number (Tab.1).There was a significantly interacted effect of AMF and H2O2 on leaf was no significant influence on the number of first-order lateral roots between AM and non-AM trifoliate orange seedlings, but the number of the second-order lateral roots were markedly increased by mycorrhization.

Different classed root lengths
In the present study, the trifoliate orange seedlings in total root lengths depended on 0-1 cm classed root lengths (Tab.3).Compared to non-AMF control, G. versiforme significantly increased 0-1 cm classed root lengths by 12.6, 10.6 and 7.3% and 3-4 cm classed root lengths by 100, 200 and 83.3% under 0, 1 and 100 µM H2O2, respectively.In addition, the AMF seedlings showed higher total root lengths than the non-AMF controls, regardless of exogenous H2O2 concentration.The results imply that AMF highly induced elongation of lateral roots, which is not dependent on exogenous H2O2 level.Yao et al. (2009) reported that the fine roots lengths in trifoliate orange seedlings colonized by Gigaspora margarita, G. mosseae, G. versiforme and G. caledonium were with 35.0, 35.1, 30.5 and 45.5% higher than in non-AMF seedlings.In addition, AMF and exogenous H2O2 generally did not alter 1-2, 2-3, and >4 cm classed root lengths, suggesting that the AMF and exogenous H2O2 mainly regulated the fine roots lengths

Number of lateral roots
Our results showed that the root systems of the five months old trifoliate orange seedlings were primarily dominated by first-order and second-order lateral roots (Tab.2).Mycorrhizal inoculation significantly increased taproot length compared to non-mycorrhizal seedlings, irrespectively of exogenous H2O2 levels.
A study showed that inoculation with Paraglomus occultum significantly increased number of the lateral roots in Citrus tangerina plants (Wu et al., 2012b).The present study indicated that the AMF and exogenous H2O2 significantly influenced the number of the second-order and third-order lateral roots, but did not alter the number of the first-order lateral roots.This result is in compliance with the previous reports by Zai et al. (2007), who found that there Tab. 2. Effects of Glomus versiforme and exogenous H2O2 on taproot length and number of lateral roots in trifoliate orange (Poncirus trifoliata) seedlings (e.g., 0-1 cm classed root lengths).

Activities of PA synthetases ADC and ODC
Studies demonstrated that PAs are an important regulatory factor involved in the process of mycorrhizal formation (Wu et al., 2012b) and that the formation of PAs derives either from L-ornithine by ODC or from Larginine by ADC, being disintegrated by PAO or DAO (EI Meskaoui and Trembaly, 2009).In the present study, ADC activity of both leaves and roots was generally significantly higher under AMF than under non-AMF treatment under 0 and 100 µM H2O2 levels, while under 1 µM H2O2 concentration AMF seedlings recorded higher ADC activity of roots and lower ADC activity of leaves than non-AMF seedlings (Fig. 2a, 2b).ODC activity of leaves was similar between AM and non-AM seedlings under 1 and 100 µM and was significantly higher in AM than in non-AM seedlings grown in 0 µM H2O2 level (Fig. 2c).The G. versiforme increased the ODC activity of roots by 135.9, 3.8 and 7.8% under 0, 1 and 100 µM H2O2, respectively (Fig. 2d).It seems that AMF plants presented higher ADC and ODC activity of roots, irrespectively of exogenous H2O2 concentrations.It is well known that ADC is generally associated with cell expansion and ODC with cell division (Paschalidis and Roubelakis-Angelakis, 2005).Wu et al. (2012a) revealed that AMF-mediated RSM was related to the change of ADC and ODC activity.Therefore, in AMF seedlings, greater activity of ADC and ODC would benefit the development of lateral roots.

Activities of PA-degrading enzyme DAO and PAO
As compared to that of the non-mycorrhizal seedlings, DAO and PAO activities in leaves of the mycorrhizal seedlings decreased by 31.0 and 19.4% (Fig. 3a, 3c) and DAO and PAO in roots by 19.6 and 7.0% under 100 µM H2O2 treatment, respectively (Fig. 3b, 3d).Under 1 µM H2O2 treatment, DAO and PAO activities in leaves and roots were similar between AMF and non-AMF seedlings.Under 0 µM H2O2 concentration conditions, PAO activity in leaves and roots of AMF and non-AMF seedlings was not significantly different, but lower leaf DAO and higher root DAO were found in AMF seedlings.DAO and PAO are not only involved in the degradation of PAs, but also in the production of H2O2, during root cell growth and differentiation (Wisniewski et al., 2000).Since no significant differences in PAO of roots were observed between AMF and non-AMF trifoliate orange seedlings under 1 and 100 µM H2O2 application, higher total root length and number of second-order and third-order lateral roots in AMF seedlings were not related to PAO.

Conclusions
Low concentration (1 µM) of exogenous H2O2 may act as an important regulating factor of mycorrhizal development in trifoliate orange seedlings inoculated with Glomus versiforme, but high concentration (100 µM) of exogenous H2O2 may confer oxidative damage and thus restrict root mycorrhizal colonization.In addition, under application of a low H2O2 concentration (1 µM) conditions, AMF colonization significantly increased the number of second-order later root and total roots length (mainly 0-1 cm classed roots length), which were related to the increase of ADC and ODC and the decrease of DAO in roots.

Fig. 1 .
Fig. 1.Effect of exogenous H 2 O 2 on AMF colonization of Glomus versiforme-inoculated trifoliate orange (Poncirus trifoliata) seedlings.Data (means±SE, n=3) followed by the different letter above the bars are significantly different at P<0.05.

Tab. 1 .
Effect of Glomus versiforme alone or in combination with exogenous H2O2 on growth performance of trifoliate orange (Poncirus trifoliata) seedlings number.

Fig. 2 .
Fig. 2. Influence of Glomus versiforme and exogenous H 2 O 2 on activity of PA synthetic enzymes (ADC and ODC) in leaves and roots of trifoliate orange (Poncirus trifoliata) seedlings.Data (means±SE, n=3) followed by the same letter above the bars are not significantly different at P<0.05 Fig. 3. Effects of Glomus versiforme and exogenous H 2 O 2 on activity of PA-metabolic enzymes (DAO and PAO) in leaves and roots of trifoliate orange (Poncirus trifoliata) seedlings.Data (means±SE, n=3) followed by the same letter above the bars are not significantly different at P<0.05 Note: The data (means±SE, n=3) in a column followed by different letters are significantly different at 5% level.Tab.3. Effect of Glomus versiforme and exogenous H2O2 on the different classed root lengths of trifoliate orange (Poncirus trifoliata) seedlings