Mycorrhizal Fungi Regulate Root Responses and Leaf Physiological Activities in Trifoliate Orange

Plant responses to mycorrhization are mediated through secretion of certain signal molecules deposited in mycorrhizosphere in response to environmental stimuli. Responses of four arbuscular mycorrhizal fungi (AMF), namely Claroideoglomus etunicatum, Diversispora versiformis, Funneliformis mosseae, and Rhizoglomus intraradices on root morphology, lateral root (LR) number, and leaf carbohydrates, nitric oxide (NO), and calmodulin (CaM) changes were studied using trifoliate orange. Inoculation response of D. versiformis, F. mosseae, and R. intraradices registered significantly higher plant growth performance (plant height, stem diameter, leaf number, and shoot and root biomass), root morphological traits (total length, projected area, surface area, and volume), and LR number (first-, second-, third-, and forth-order), compared to un-inoculated response. Higher concentrations of CaM, NO, glucose, and fructose and lower sucrose level in leaves were observed in AMF-seedlings than in non-AMF seedlings. Correlation studies further revealed, root morphological traits and LR numbers were significantly negatively correlated with sucrose whereas positively correlated with glucose, fructose, NO, and CaM level in leaves. These results suggested, AMF-induced root modification is routed through sucrose cleavage and partly through changes in NO and CaM.


Introduction
Arbuscular mycorrhizal fungi (AMF) are reported to establish symbiotic association, with roots of ~80% of terrestrial plants (Kiers and van der Heijden, 2006).Such symbiosis derives ~20% of photosynthetic carbohydrates from the host plant on account for mycorrhizal growth, and in return, AMF provide the host plant, a greater access to nutrients and water absorption (Smith and Read, 2008;Parniske, 2008).The essential roles of AMF in crops like citrus, litchi, strawberry, lettuce, pepper etc are well documented (Borowicz, 2010;Ortas et al., 2011).Trifoliate orange [Poncirus trifoliata (L.) Raf.] is a rootstock used in citriculture in Asia (Srivastava et al., 2008).Citrus is usually considered as a crop severely lacking in root hair (Srivastava and Singh, 2009;Wu et al., 2016), and thus depends heavily on AMF for meeting the nutrient requirement.
Root systems play a significant role in acquisition of nutrients from within a given soil volume (Yao et al., 2009).Different microbial communities play an important role in growth and developmental responses in host plants (Sorgona et al., 2007;Jung et al., 2013).Of them, AMF have shown to regulate root system architecture through enhanced mineral nutrient absorption (Smith and Read, 2008).Earlier studies showed that AMF-inoculation strong stimulated morphological modification in root features like root length, surface area, and volume to varying proportions (Schellenbaum et al., 1991;Wu et al., 2011Wu et al., , 2015a)).AMF-induced root modification is reported to have strong relations with metabolism of endogenous polyamines (Wu et al., 2010).However, such relationship is independent of signaling of common symbiotic transactions (Gutjahr et al., 2009).On the other hand, Isobe et al. (2002) reported a negative effect of AMF inoculation on the length and the number of tap roots and lateral roots (LRs) in Phaseolus vulgaris.Root morphological alteration depends on AMF species and plant genotypes (Yao et al., 2009;Wu et al., 2011;Li et al., 2013).These studies suggested number of mechanisms involved in describing AMF-induced LR development such as, excretion of AMF spore germination, phosphorus (P) nutrition Root mycorrhizal colonization was determined using clearing with 10% KOH at 90 °C for 1.5 h, then staining with 0.05% trypan blue for 5 min (Phillips and Hayman, 1970).
NO concentration in leaves was estimated with the ELISA assay with NO kit (A012, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and CaM using the Plant CaM ELISA Kit (YAD 001,Beijing Dingguochangsheng Biotechnology Co.,Ltd.,Beijing,China).Carbohydrate forms such as fructose, glucose and sucrose concentrations in leaves were assayed through the protocol as described by Wu et al. (2015b).

Statistical analysis
Data (means ± SD, n = 4) were analyzed by one-way variance (ANOVA).Significant differences between treatments were compared with the Duncan's multiple range tests at P < 0.05.Pearson correlation coefficients between variables were carried out with Proc Corr procedure.All the statistical analyses were performed using the SAS software 8.1v.

Root mycorrhizal colonization and plant growth performance
Mycorrhizal colonization is associated with different plant growth parameters through an increase in root absorptive surface area (Aguín et al., 2004).Root AMF colonization was observed to vary from 17.64% to 29.69%, following the order of F. mosseae ≈ D. versiformis > R. intraradices > C. etunicatum in the decreasing order (Table 1).With the exception of C. etunicatum, other three AMF species showed a significant increase in different growth attributing parameters viz., plant height, stem diameter, leaf number, and shoot and root biomass, compared to non-AMF treatment (Table 1).Studies in the past showed strong positive effect of AMF colonization on growth of the host plant (Aguín et al., 2004).Our studies showed, except C. etunicatum, inoculation with F. mosseae, D. versiformis, and R. intraradices produced significantly better plant growth performance, which is possible due to improved nutrients and the compatibility between AMF and host plants (Yao et al., 2009).
Response on root traits AMF-inoculation was observed to alter different root traits of trifoliate orange seedlings (Fig. 1; Table 2).Out of four AMF species, three species viz., F. mosseae, D. versiformis and R. intraradices significantly increased root total length, projected area, surface area, and volume; however, C. etunicatum increased root total length only.Root average diameter also, remained unaffected with all the four AMF species.Likewise higher LR number was recorded in different order than non-AMF-improvement, changes in hormone levels, sugar signals, synthesis of nitric oxide (NO) as a signaling molecule, and active involvement of calmodulin (CaM) as Ca 2+ receptors (Zhao et al., 2007;Yang et al., 2010;Chen and Kao, 2012;Zhang et al., 2013;Fusconi, 2014).
In this background, the present study evaluated the responses of different AMF species on root colonization, root morphology, LR number, and leaf carbohydrates, NO, and CaM changes to understand the underlying mechanistic insights involved.

Experimental setup
The experiment was carried out during March-August, 2013 at Yangtze University, Jingzhou, China.On March 30, 2013, five-leaf-old seedlings (~6 cm height) without mycorrhization were transplanted into a 4.8-L pot filled with 4.5 kg autoclaved (121 °C, 2h)  At the same time, approx.1000 spores of each AM fungus used were mixed along with sands for AMF treatment.The non-AMF control received the same amount of autoclaved inoculums plus 2 mL filtrate (25 μm filter) of mycorrhizal inoculums to minimize the differences in other microbial communities.Seedlings were grown in a controlled environment characterized by 27/20 °C (day/night) temperature, 982 μmol/m 2 /s photonflux density, and 80% relative air humidity.A 150 mL Hoagland solution (1/10 P strength) was applied into each pot at an alternate day.The AMF-and non-AMF seedlings were harvested on August 17, 2013.

Observations and analysis
Plants following their harvest, were divided into shoots and roots, and recorded the fresh biomass.The root system from each pot was scanned with the Epson Perfection V700 Photo Dual Lens System (Seiko Epson Corp, Japan).Root images were then analyzed through the WinRHIZO software (Regent Instruments Incorporated, Canada), to obtain different root traits viz., length, surface area, volume, and diameter.The root of seedlings was divided into taproot and LRs, to count the number of LRs artificially on a test-bed.68±2.61bNote: Data (means ± SD, n = 4) followed by different letters indicate significant differences (P < 0.05) between treatments colonized seedlings (Table 2).F. mosseae showed significantly superior response on these root-related traits than other three AMF species.These observations are in agreement with our previous study in trifoliate orange (Wu et al., 2015a).

Response on leaf carbohydrates
It is a known fact that root growth is strongly dependent on carbon import, and the major loss of root carbon is due to root respiration and AM growth (Walter and Nagel, 2006).AMFseedlings showed significantly lower leaf sucrose concentration, but significantly higher leaf glucose concentration, as compared with non-AMF seedlings (Fig. 2), regardless of AMF species.Such responses were observed highly variable.Maximum reduction in leaf glucose concentration was observed with D. versiformis (45.97%), followed by F. mosseae (42.25%),R. intraradices (34.84%) and C. etunicatum (14.41%) over uninoculated control.While, mycorrhization induced significantly higher glucose concentration in leaves with D. versiformis and F. mosseae (30.00-30.70%)followed by R. intraradices (21.44%) and C. etunicatum (5.22%) over control.While, the response of AMF on fructose concentration was entirely different.Only F. mosseae produced an increase in leaf fructose concentration by 13.45% over non-AMF control (Fig. 2).AMF as a symbiotic fungi, rely on the host plant to provide carbohydrates for its own growth (Bago et al., 2003), and glucose would be preferentially absorbed and transformed in terms of sucrose cleaving (Schubert et al., 2004) to support the uninterrupted growth.Root mycorrhizal colonization was significantly and positively correlated with leaf glucose and fructose, and negatively with leaf sucrose (Table 3), due to sucrose cleavage (Wu et al., 2015a).AMF-induced root modification was significantly negatively correlated with leaf sucrose but positively with leaf fructose and glucose, indicating that mycorrhiza-induced changes in carbohydrates are associated with changes in root morphology and numbers of LR formation (Table 3).A relatively greater hexose level in AMF plants would provide greater substrates for the growth of both AMF and roots.Glucose stimulates the accumulation of a transcription factor or Auxin-mediated signalling for root initiation (Mishra et al., 2009;Singh et al., 2014).As a result, the AMF-induced root modification is so closely related to the AMF-stimulated sucrose cleavage.

Response on leaf NO and CaM
Compared with non-AMF treatment, leaf NO and CaM levels were significantly higher with mycorrhization conditions, irrespective of AMF species (Fig. 3).As much as 46.35%, 74.92%, 103.14%, and 8.75% significantly higher leaf NO concentration was observed upon inoculation with C. etunicatum, D. versiformis, F. mosseae, and R. intraradices, respectively, over non-AMFseedlings.Likewise 28.65%, 35.15%, 27.94% and 15.01%significantly higher leaf CaM concentration was observed in C. etunicatum, D. versiformis, F. mosseae, and R. intraradices, respectively, over non-AMF-seedlings.These results are in agreement with the results of Huang et al. (2014) in trifoliate orange.Strong correlation of NO and CaM with root AMF colonization suggested that, root AMF colonization could be driven through NO and CaM (Huang et al., 2014).Leaf NO and CaM also correlated positively with root total length and number of LR in first order, indicating that AMF-induced NO and CaM as a signalling molecule are partly involved in the root development.Both NO and CaM interacted synergistically to stimulate root development and LR formation with the cross-talk of auxins (Liao et al., 2012).The interaction between NO and CaM/Ca 2+ under mycorrhizaiton would further decode the underlying mechanisms involved.3).Amongst LR numbers, first-order, second-order, third-order, and fourth-order were negatively correlated with leaf sucrose concentration (r = -0.67 ~ -0.93, P < 0.01) and positively correlated with leaf glucose concentration (r = 0.61 ~ 0.93, P < 0.01), corresponding to pattern of similar response on root volume viz., root volume versus sucrose (r = -0.73,P < 0.01) and root volume cooperate with leaf glucose concentration (r = 0.76, P < 0.01).The other root related parameters like root AMF-colonization, total root length, root projected area, and root surface area were positively correlated with fructose as well as glucose concentration in leaves.With exception of root projected area, leaf NO and CaM concentration showed a positive correlation with root AMF-colonization (r = 0.74 and 0.79, P < 0.01) and total root length (r = 0.63 and 0.45, P < 0.01 and P < respectively), and with root diameter remaining unaffected with any of the three forms of carbohydrates, NO, and CaM in leaves.These observations suggested, mycorrhizal response of trifoliate orange is strongly dependent upon leaf carbohydrate metabolism, NO, and CaM activity, as underlying mechanisms to explain the physiological responses of AMF.20

Conclusions
Responses of trifoliate orange to mycorrhization are regulated through carbon metabolism coupled with root morphological changes.These changes were further partitioned, partly into sucrose cleavage and partly as NO-and CaM-induced changes.