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Mycorrhizal colonization represents functional equilibrium on root morphology and carbon distribution of trifoliate orange grown in a split-root system
Scientia Horticulturae 199 (2016) 95–102
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Mycorrhizal colonization represents functional equilibrium on root morphology and carbon distribution of trifoliate orange grown in a split-root system Qiang-Sheng Wu a,b,∗ , Ming-Qin Cao a , Ying-Ning Zou a,b , Chu Wu a,b , Xin-Hua He c,d,∗∗ a
College of Horticulture and Gardening, Yangtze University, Jingzhou, Hubei 434025, China Institute of Root Biology, Yangtze University, Jingzhou, Hubei 434025, China c Centre of Excellence for Soil Biology, College of Resources and Environment, Southwest University, Chongqing 400715, China d School of Plant Biology, University of Western Australia, Crawley, WA 6009, Australia b
a r t i c l e
i n f o
Article history: Received 25 August 2015 Received in revised form 9 December 2015 Accepted 18 December 2015 Keywords: Carbon partitioning Citrus Glucose Mycorrhizal fungi Split root
a b s t r a c t Trifoliate orange seedlings were grown in perspex pots of a split-root system, where one half of the split roots was inoculated with or without arbuscular mycorrhizal fungi (AMF, Acaulospora scrobiculata or Funneliformis mosseae). Five months after inoculation, the growth performance, leaf, stem and root biomass and photosynthetic rate were generally significantly higher in AM than in non-AM seedlings. Greater root morphological traits were observed in the AM root side than in the non-AM side for the same plant and in AM than in non-AM plants. AM seedlings had significantly higher leaf sucrose and glucose contents and total (leaf + root) glucose and fructose contents as compared to non-AM ones. In the split roots, the AM side displayed substantially higher sucrose, glucose and fructose contents than the non-AM side for the same plant, and in AM than in non-AM plants. These results showed greater C movement into the non-AM side from an AM-colonized plant than from a non-AM-colonized plant. These results conclude that the presence of AM in one side of the split roots benefited C acquisition and root development to another half non-AM side, suggesting functional equilibrium and resource allocation of AM within a root system. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Arbuscular mycorrhizal fungi (AMF), belonging to the phylum Glomeromycota, can form the mutualistic symbioses, arbuscular mycorrhizas (AMs), with roots of more than 80% terrestrial plants (Smith and Read, 2008). The formation of AMs provides multiple benefits to the fungal partner, including uptake of mineral nutrients and water, while consumes ∼20% of plant photosynthetically fixed carbon (C) to sustain the symbiosis life cycle (Smith and Read, 2008). AMs thus function as a metabolic C-sink causing basipetal mobilization of photosynthates to roots. Sucrose, a major photosynthate, is transported by sucrose transporters from source leaves to sink tissues such as AMs, where it is cleaved by invertases and sucrose synthases (degraded direction)
∗ Corresponding author at: College of Horticulture and Gardening, Yangtze University, Jingzhou, Hubei 434025, China. Fax.: +86 716 8066262. ∗∗ Corresponding author at: Centre of Excellence for Soil Biology, College of Resources and Environment, Southwest University, Chongqing 400715, China. E-mail address: [email protected] (W. Qiang-Sheng). http://dx.doi.org/10.1016/j.scienta.2015.12.039 0304-4238/© 2015 Elsevier B.V. All rights reserved.
into hexoses, further into trehalose and glycogen for the storage and utilization of C in AMs (Bago et al., 2003; Doidy et al., 2012b). Meanwhile, glucose, one of hexoses, is preferentially taken up by the mycobiont (Doidy et al., 2012a), though the percentage of hexoses allocated to AM versus non-AM roots is unknown. As a consequence, AM growth is a C-limited process, whilst the host plants have ultimate control over fungal activity through the regulation of carbohydrate transfer to the roots (Miller et al., 2002). Root AM colonization was generally linearly correlated with the C-sink strength of roots (Lerat et al., 2003). A higher plant C assimilation rate would hence compensate for their greater below-ground C expenditure in AM symbioses (Eissenstat et al., 1993). Foraging strategies of AMs and roots for energy are linked to plant C source (Gavito and Olsson, 2003). Even so, the cost of an AM formation involved in C partitioning between AMs and plants is poorly understood (Doidy et al., 2012a). Plant root systems exhibit highly plastic characteristics while are affected by various abiotic and biotic factors, including AMF (Hodge et al., 2009). AMF had been confirmed to improve root morphology in strawberry, rice and citrus plants (Norman et al., 1996;
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Gutjahr et al., 2009; Wu et al., 2011, 2012), which would consume more root carbohydrates for root respiration and root biomass production (Liu et al., 2006). The presence of AM would lead to an increase of carbohydrates in the non-AM root sides in the split-root citrus seedlings (Koch and Johnson, 1984). Early studies showed a 3–4% higher transfer of carbohydrates in AM root sides than in non-AM root sides of split roots (Douds et al., 1988). Citrus, one of the most planted fruit trees worldwide is strongly dependent on the AM symbiosis because of lacking of root hairs (Wu et al., 2011). Trifoliate orange [Poncirus trifoliata (L.) Raf.], a close relative to Citrus, is widely used as a rootstock of citrus plantation in Asia, including China, India, and Japan. In field, root colonization of citrus trees by native AMF is less than 10% in China (Zeng et al., 2004) and ∼20% in Japan (Ishii and Kadoya, 1996). It seems that in whole root systems, a large proportion of citrus roots in the field are not colonized by native AMF. As a result, we do not know whether these non-AM roots in whole root systems obtain some carbohydrates for better root development. Split-root system approaches can provide differential treatments for separate and independent root side of the whole root system but share a conjunct aerial part (Larrainzar et al., 2014). Split-root experiments have been extensively used to study the effects of AMF colonization on the production of carbohydrates and biomass (Koch and Johnson, 1984; Vierheilig et al., 2000; Lerat et al., 2003). Using this technique, the root system of a plant is divided into two parts, one of which is inoculated with an AM fungus, allowing a comparison of the C-sink strength between the AM and non-AM root halves of the same plant (Lerat et al., 2003). However, these studies considered C allocation only, while neglecting the root modification between AM and non-AM root sides. Here, we thus hypothesized that the non-AM root of split roots could gain a certain amount of carbohydrates from C pools of AM root side to support better root development, though how carbohydrates are distributed in roots to ensure the fungal metabolism and greater root growth is poorly understood. To confirm this hypothesis, in the present study, with one half of trifoliate orange root colonized by AMF or not, we firstly compared differences in root morphology and carbohydrate contents in two root sides, and then addressed if AM root side represent functional equilibrium and resource allocation in non-AM root side.
2.2. Experimental set-up Perspex pots (20 × 10 × 18 cm = length × width × height) were separated in the middle by a 15-cm-height perspex to form two equal sized compartments (see Fig. 1). At the top of the separated perspex, a semicircle of 1-cm diameter was designed to arrange nearly equal lateral roots of the seedlings into two halve compartments. A 5 × 3 × 2 cm (length × width × height) foam board was placed in the shoot bottom of a seedling to fix the seedlings for upright growth. Such plants were defined as the split-root plants. Each compartment was filled with 1550 g autoclaved (121 ◦ C, 0.11 Mpa, 2 h) soil. The soil (Xanthi-Udic Ferralsols, FAO system) was collected from the same Citrus Orchard on the Yangtze University campus. The soil chemical properties were pH 6.1, 9.7 g kg−1 organic carbon, 11.8 mg kg−1 available nitrogen, 15.3 mg kg−1 Oslen-P, and 21.5 mg kg−1 available potassium. The AMF inoculated split-root compartment (M) received 50 g AMF inocula, and the other compartment (NM) of the pot received the same amount of autoclaved (121 ◦ C, 0.11 Mpa, 2 h) inocula plus 2 mL filtrate (25 m) of AMF inocula to keep similar other microorganisms. The experiment consisted of a completely randomized design with three mycorrhizal treatments (A. scrobiculata, F. mosseae, and non-AMF control). Each treatment replicated four times, creating a total of 12 pots. The seedlings were grown in an environment controlled plastic greenhouse from April to September, 2013, where photosynthetic photon flux density was 965 mol m−2 s−1 , day/night temperature 28/21 ◦ C, and relative humidity 85%. The seedlings were watered in an interval of three days with distilled water to avoid waterlogging at the bottom of the compartment. A 30 mL standard Hoagland solution was biweekly supplied into each compartment.
2. Materials and methods 2.1. Biological materials Two AMF strains, Acaulospora scrobiculata Trappe and Funneliformis mosseae (Nicol. and Gerd.) Schüßler and Walker, respectively isolated from the rhizosphere of Bauhinia blakeana in Hongkong and of Incarvillea younghusbandii in Tibet in China, were used. The inocula were propagated from the identified fungal spores and cultivated with host plant of Sorghum vulgare (A. scrobiculata) or Trifolium repens (F. mosseae) for 16 weeks. After harvesting, the growth substrate contained 21 or 23 spores g−1 for A. scrobiculata or F. mosseae, respectively. Seeds of trifoliate orange were collected from a Citrus Orchard on the Yangtze University campus, surface-sterilized with 70% ethanol for 10 min, and germinated in a plastic box containing autoclaved sands in a controlled growth chamber at 28/20 ◦ C and 16/8 photoperiod hours (day/night) for 3 weeks under 80% relative humidity and 1200 mol m−2 s−1 photosynthetic photon flux density. Fresh taproots of four-leaf-old (under sterilization) trifoliate orange seedlings were excised and kept 3-cm long. Subsequently, these treated seedlings were re-planted into the plastic box for 7 weeks for inducing the formation of lateral roots.
Fig. 1. Schematic diagram of a two-chambered split-root system to grow trifoliate orange seedlings with or without Acaulospora scrobiculata or Funneliformis mosseae colonization. Abbreviations: NM, non-AMF inoculation; M, AMF inoculation.
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2.3. Plant harvest, AM colonization, and root system scanning After 5 months of AMF inoculation, the seedlings had shown significant differences in growth performance, and thus were harvested. The plant height, stem diameter, and leaf number per plant were determined. Leaf, stem and root dry biomass was recorded after over-drying at 75 ◦ C for 48 h. The two sides of split root of one plant were separately collected. The root systems were scanned with an Epson Perfection V700 Photo (Seiko Epson Corp, Japan), and then the root images were analyzed using a WinRHIZO professional 2007b (Regent Instruments Incorporated, Canada). Morphological traits, including total length, projected area, surface area and volume were obtained. For the determination of AM colonization, 1-cm-long root segments were stained with the protocol described by Phillips and Hayman (1970). Root AM colonization was expressed as the percentage of colonized root lengths against total root lengths. 2.4. Determinations of photosynthetic rate and carbohydrate concentrations On a sunny day (September 13, 2013) between 9:00 to 10:00 am, the photosynthetic rate (Pn) of AM and non-AM seedlings was determined with an infrared gas analyzer (Li-6400 Portable Photosynthesis System, Li-Cor, Lincoln, USA) using the fourth and fifth fully expanded leaf from the apices of these seedlings. Pn values were recorded when the total coefficient of variation was less than 0.5%. The reference [CO2 ] was 400 mol mol−1 . For extraction of fructose, glucose, and sucrose, 100 mg of ground dry tissues of leaves and roots was incubated with 8 mL 80% ethanol for 40 min at 80 ◦ C and centrifuged at 2500 × g for 5 min. The centrifugal residues were re-extracted using the above procedure, and the two supernatants were combined for the analysis. Determination of fructose, glucose, and sucrose concentrations was followed by the procedure described by Wu et al. (2015). These sugar contents (mg DW plant−1 ) were the amounts of corresponding tissue biomass × sugar concentration (mg DW g−1 ). Sugar contents of total (M + NM sides) were the sum of sugar contents in M and NM root sides from a treated plant. 2.5. Statistical analysis Data (means ± SE, n = 4) were subjected to one-way analysis of variance (ANOVA) using the SAS software (v8.1, SAS Institute Inc., Cary, NC 27513-2414, USA). The data about percentage were arcsine transformed before variance analysis. The significant differences in means between treatments were tested with the Duncan’s multiple range test at 5% level. The Pearson’s correlation coefficients between variables were performed using the Proc Corr’s procedure. 3. Results 3.1. Root AM colonization After five months of AMF inoculation, in the M chamber, the root AMF colonization was significantly higher under F. mosseae than under A. scrobiculata (Table 1). No root colonization was found in the NM root side. 3.2. Plant growth performance Except leaf numbers, M/NM seedlings represented significantly higher plant height, stem diameter, leaf, stem and root biomass than those in NM/NM seedlings, irrespective of AMF strains
Fig. 2. Effects of AMF on photosynthetic rates of trifoliate orange seedlings colonized with or without Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system. Data (means ± SE, n = 4) followed by different letters among treatments indicate significant differences at P < 0.05. Abbreviations: NM, non-AMF inoculation; MAs, Acaulospora scrobiculata inoculation; MFm, Funneliformis mosseae inoculation.
(Table 1). Moreover, all these growth traits were significantly greater in MFm/NM than in MAs/NM seedlings. 3.3. Photosynthetic rate (Pn) Pn was 65% and 101% significantly higher in MAs/NM and MFm/NM seedlings than in NM/NM seedlings, respectively (Fig. 2). Meanwhile, MFm/NM seedlings had significantly higher Pn than MAs/NM seedlings. 3.4. Root morphology AM seedlings recorded considerably greater root morphology than non-AM seedlings (Fig. 3a). In a same treated plant, total length, projected area, surface area and volume were similar in the two split root compartments under NM/NM treatment, whereas significantly higher in the M root side than in the NM root side, no matter whether the plants were colonized by A. scrobiculata or F. mosseae (Fig. 3b–e). Amongst the three treatments, significantly higher root morphological traits were ranked as in MFm/NM seedlings > MAs/NM colonized seedlings > NM/NM seedlings, no matter whether the roots were from the M root side, NM root side or total (M + NM root sides). 3.5. Carbohydrate contents in leaf and split roots For the same split root side (i.e. the M or NM root side) and the total (M + NM root side) root, significantly higher root sucrose contents ranked in the order: F. mosseae ≥ A. scrobiculata ≈ nonAMF, whilst significantly higher root fructose and glucose contents ranked as F. mosseae > A. scrobiculata ≈ non-AMF (Fig. 4a). For the same AMF treatment, root fructose, glucose and sucrose contents were significantly higher in the split M side than in the split NM side under A. scrobiculata and F. mosseae inoculation, whilst were similar between the two split NM sides of the non-AMF seedlings. Contents of leaf sucrose or glucose were 116–169% or 18–20% significantly higher in the AM seedlings than in the non-AM seedlings, but similar between MAs/NM and MFm/NM seedlings (Fig. 4b). Compared to the non-AMF inoculated seedlings, leaf fructose contents were significantly increased by 43% under MFm/NM whereas markedly decreased by 69% under MAs/NM. Compared to the NM/NM seedlings, the total (leaf + root) contents of fructose, glucose and sucrose were increased by 130, 259 and 120% or 19, 41 and 13% under MFm/NM or MAs/NM (Fig. 4c).
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Table 1 Effects of AMF on root mycorrhization, soil hyphal length and plant growth of trifoliate orange (Poncirus trifoliata) seedlings inoculated with Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system. Inoculated treatments
Root AM colonization (%)
Plant height (cm)
Stem diameter (mm)
Leaf number
Biomass (g/plant DW) Leaf
NM/NM MAs/NM MFm/NM a
0 ± 0c 15.8 ± 1.2b 38.9 ± 3.5a
19.7 ± 4.5c 25.6 ± 3.1b 43.4 ± 2.5a
2.47 ± 0.24c 2.85 ± 0.18b 4.52 ± 0.15a
24 ± 6b 26 ± 6b 44 ± 7a
0.12 ± 0.04c 0.18 ± 0.04b 0.31 ± 0.03a
Stem 0.31 ± 0.11c 0.50 ± 0.10b 1.23 ± 0.08a
Roota 0.25 ± 0.02c 0.31 ± 0.02b 0.70 ± 0.03a
Total production of both root sides. Abbreviations: NM, non-AMF inoculation; MAs, Acaulospora scrobiculata inoculation; MFm, Funneliformis mosseae inoculation.
Fig. 3. Effects of AMF on root morphology (a) and root total length (b), projected area (c), surface area (d) and volume (e) of trifoliate orange seedlings colonized with (M) or without (NM) Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system. Data (means ± SE, n = 4) followed by different letters among AMF treatments for the same split root compartment or the total (M + NM) (a–c) or between the M and NM split root compartment for the same AMF treatment (x, y) indicate significant differences at P < 0.05. Abbreviations: NM, non-AMF inoculation; MAs, Acaulospora scrobiculata inoculation; MFm, Funneliformis mosseae inoculation. In ‘b–e’ subfigures, M side column in x-axis orientation represented NM root side data only in NM/NM treated split-root seedlings.
3.6. Carbohydrate distribution Distributions (%) of total (leaves + roots) tested carbohydrates were significantly decreased in leaf fructose and glucose whilst
increased in leaf sucrose under AMF than under non-AMF inoculation (Fig. 5a–c). Compared to the NM/NM treatment, percentages of root fructose, glucose and sucrose in the M side were 19, 31 and 6% or 45, 92 and 29% higher under the A. scrobiculata or F.
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Fig. 4. Effects of AMF on fructose, glucose and sucrose contents in root (a), leaf (b) and total (leaf + root) (c) of trifoliate orange seedlings colonized with (M) or without (NM) Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system. Data (means ± SE, n = 4) followed by different letters among AMF treatments for the same split root compartment or the total (M + NM) (a–c) or between the M and NM split root compartment for the same AMF treatment (x, y) indicate significant differences at P < 0.05. Abbreviations: NM, non-AMF inoculation; MAs, Acaulospora scrobiculata inoculation; MFm, Funneliformis mosseae inoculation. In ‘a’ subfigure, M side column in x-axis orientation represented NM root side data only in NM/NM treated split-root seedlings.
mosseae inoculation, respectively. Interestingly, compared to the NM root side in NM/NM seedlings, percentages of root fructose in the NM root side were increased by 7% under the A. scrobiculata but decreased by 33% under the F. mosseae inoculation (Fig. 5a). Meanwhile, percentages of root glucose and sucrose in the NM root side of M/NM seedlings were 27 and 31% or 38 and 36% lower under the A. scrobiculata or F. mosseae inoculation than under the non-AMF inoculation, respectively (Fig. 5b and c). 3.7. Relationships between root traits or photosynthesis and sugar production Total root (the split M + NM side) fructose, glucose and sucrose contents were positively (P < 0.01) correlated with root total length (Fig. 6a–c), root projected area (Fig. 6d–f), root surface area (Fig. 6d–f), and root volume (Figure 6g–i), respectively. The contents of total plant (leaf + root) fructose, glucose and sucrose were positively (P < 0.01) correlated with leaf Pn (Fig. 7). 4. Discussion 4.1. Greater plant growth performance in the AMF colonized half side of split roots Root production was significantly lower in the half non-AM side than in the half AM side of the split root colonized by either A. scrobiculata or F. mosseae, which was in agreement with studies in non-split-root citrus (Wu et al., 2011; Ortas and Ustuner, 2014) and apricot (Dutt et al., 2013). A greater plant biomass production in AM seedlings might reflect an AM-enhanced C partitioning belowground (Eissenstat et al., 1993). Under higher C expenditure by AMs
and greater root biomass conditions, root growth performance was still enhanced by AMF inoculation (Table 1). Moreover, AM-induced positive growth responses were significantly higher with F. mosseae than with A. scrobiculata, indicating that trifoliate orange seedlings have a certain degree of specificity in AM symbiosis (Ortas and Ustuner, 2014). 4.2. AMF inoculation enhanced photosynthetic rate and plant carbohydrate accumulation In plants, sucrose is synthesized in the photosynthetic leaves as a principal form of carbohydrates for long-distance transport from source leaves to plant and non-plant C sinks including AMs (Doidy et al., 2012b). Sucrose generally is cleaved into hexoses (e.g., fructose and glucose) through sucrose synthase (degraded direction) or invertase, which is then transformed into typical fungal carbohydrates, trehalose and glycogen (Bago et al., 2003; Wu et al., 2013). Studies showed that AMF inoculation with F. mosseae increased leaf glucose and fructose in tomato plants (Boldt et al., 2011). These are true in the present study that M/NM seedlings had significantly higher leaf fructose, glucose and sucrose than NM/NM seedlings (Fig. 4b), except a decrease of leaf fructose in MAs/NM than in NM/NM control seedlings. These carbohydrate accumulations are dependent on the activity of sucrose cleaving enzymes (Wu et al., 2013). In addition, leaf Pn was significantly higher in AM than in non-AM seedlings (Fig. 2) and was notably positively correlated with total (leaf + root) fructose, glucose, and sucrose (Fig. 7). It suggests that a greater AMF-induced Pn would produce more photosynthates for a greater C expenditure below-ground by AM than by non-AM plants, in addition to a higher chlorophyll and RuBPCase activity (Nemec and Vu, 1990; Eissenstat et al., 1993; Wu
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Fig. 5. Effects of AMF colonization on the distribution of total (leaves + roots) fructose, glucose or sucrose in leaf, the split M and NM root side in the trifoliate orange seedlings colonized with or without Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system. Abbreviations: NM, non-AMF inoculation; MAs, Acaulospora scrobiculata inoculation; MFm, Funneliformis mosseae inoculation.
et al., 2011; Birhane et al., 2012). Furthermore, more plant photosynthates had been transferred to the C sink of AM roots (Douds et al., 1988), thereby, inducing a significantly higher total (M + NM sides) root carbohydrate accumulation (Fig. 4a). The additional C gain derived from mycorrhization would promote root growth and maintenance of AM symbioses (Wright et al., 1998). 4.3. Higher carbohydrate allocation in M root side modulated greater root morphology in M root side In the present study, AM split-roots recorded relatively higher total (M + NM sides) sucrose content than non-AM control (Fig. 4a), implying that more sucrose was translocated to the AM split-roots to sustain the extra cost of AMs. The higher total (M+NM sides) fructose and glucose contents in roots were found in AM roots than in non-AM roots, which is not consistent with the findings of García-Rodriguez et al. (2007), who reported the lower fructose and glucose concentration in AM roots in F. mosseae- and R. intraradices-colonized tomato plants. Our study further showed that significantly higher root fructose, glucose, and sucrose contents were found in the NM root side of the MFm/NM or MAs/NM treated plants than in the NM root side of NM/NM counterparts, except a similar sucrose no matter whether roots were colonized by A. scrobiculata or not (Fig. 4a). It implied that the NM splitroot side of the M/NM plants had obtained extra photosynthetically fixed C, showing a functional equilibrium on C distribution for NM and M root sides. Gavito and Olsson (2003) pointed out that the energy foraging strategies between M and NM root sides in a split
root system might be more collaborative, rather than competitive. Our results demonstrated that part of mycorrhizal-promoted carbohydrates in the M colonized side could be moved into the NM root side, indicating a collaborative behaviour and functional equilibrium for C request in split roots. Furthermore, stronger C-sink capacity by F. mosseae than by A. scrobiculata indicated a fungal species dependent, which was in agreement with studies in split roots of barley and sugar maple (Lerat et al., 2003). Since sucrose can be cleaved by invertases and sucrose synthases into hexoses (Bago et al., 2003; Doidy et al., 2012b), greater fructose and glucose content in AM split-roots indicates that more sucrose was cleaved into hexoses, which is taken up and transformed by the symbiont (Doidy et al., 2012a). In the process of sucrose cleavage in AM plants, various sucrose cleaved enzyme genes are up-regulated (GarcíaRodriguez et al., 2007), which will be further highlighted in AM trifoliate orange seedlings. In addition, considerably higher sucrose, glucose, and fructose in the M root side than in the NM root side of split roots (Fig. 4a) further indicated that in whole root, roots in the mycorrhizal side acted as a C sink, which might be independent of enhanced nutrient effects, especially P (Koch and Johnson, 1984; Miller et al., 2002). Greater carbohydrates in the M root side (Figs. 4a; 5) would benefit the increase of root biomass production, root respiration, and mycorhizal hyphal biomass production and respiration (Miller et al., 2002; Mishra et al., 2009). Eissenstat et al. (1993) also reported that a higher percentage of below-ground C was in below-ground respiration in AM than in non-AM sour orange plants under the equivalent P status. The present study further confirmed that root
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Fig. 6. Relationships between root total length, projected area, surface area or volume and fructose, glucose or sucrose content in two sides of split-root in trifoliate orange seedlings colonized with Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system (n = 24).
sucrose, glucose, or fructose significantly positively correlated with root total length, surface area, projected area or volume (Fig. 6). It is hence reasonable that AM-induced greater C allocation into the mycorrhizal side of split roots would benefit AMF colonization and root growth, resulting in greater root morphological traits in M root side (Fig. 3a–e). 4.4. Significantly higher carbohydrates in NM root side of M/NM plants than in NM side of NM/NM plants indicated a functional equilibrium between M and NM root sides of split-roots to affect root morphology Our previous studies had confirmed greater root morphology in AM than in non-AM trifoliate orange seedlings (Wu et al., 2011, 2012). In the present study, an AM-enhanced root morphology also occurred in the split roots, showing higher root total length, projected area, surface area and volume in the split M roots than in the split NM roots (Fig. 3a–e). Although greater C expenditure below-ground (e.g., AMs presence and greater root biomass and respiration) occurred by AM than by non-AM seedlings, the NM root side of split roots in the M/NM seedlings still exhibited significantly greater root morphological traits and higher C contents than in the NM root side in the NM/NM seedlings (Fig. 4). We speculated that the NM root side of split roots in the M/NM seedlings might gain a certain amount of carbohydrates (such as glucose)
Fig. 7. Relationships between photosynthesis rate and total (leaf + root) fructose, glucose or sucrose content of trifoliate orange seedlings colonized with Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system (n = 12).
from the whole root C pool to support root development. Moreover, such AM-enhanced effects were firstly observed in the split roots of trifoliate orange seedlings. It suggests the functional equilibrium and resource allocation of AMs in enhancing belowground
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root morphological development. So, the present study confirmed our foregoing hypothesis, namely, the non-AM root of split roots could gain a certain amount of carbohydrates from C pools of AM root side to support better root development. Certainly, using 13 C or 14 C isotope labelling will be able to further confirm the translocation process. 5. Conclusion In short, the whole root system in the split-root trifoliate orange had significantly greater carbohydrates in M/NM than in NM/NM plants, which resulted in better root morphological characters in the NM colonized side of split roots under mycorrhization. Meanwhile, significantly greater carbohydrates in the NM root side of split roots in the M/NM seedlings than in NM/NM seedlings might derive from a better root morphology under AMF colonization. It seemed that mycorrhizal effects on root morphology and carbon distribution in split-root trifoliate orange exhibited the characteristic of a functional equilibrium. Our results could have implications for AM functioning on root morphological development and C distribution within a root system belowground in citrus trees under low root AMF colonization condition. Acknowledgements This study was supported by the National Natural Science Foundation of China (31372017), the Key Project of Chinese Ministry of Education (211107), and the Open Fund of Institute of Root Biology, Yangtze University (R201401). References Bago, B., Pfeffer, P.E., Abubaker, J., Jun, J., Allen, J.W., Brouillette, J., Douds, D.D., Lammers, P.J., Shachar-Hill, Y., 2003. Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid. Plant Physiol. 131, 1496–1507. Birhane, E., Sterck, F.J., Fetene, M., Bongers, F., Kuyper, T.W., 2012. Arbuscular mycorrhizal fungi enhance photosynthesis, water use efficiency, and growth of frankincense seedlings under pulsed water availability conditions. Oecologia 169, 895–904. Boldt, K., Pors, Y., Haupt, B., Bitterlich, M., Kuhn, C., Grimm, B., Franken, P., 2011. Photochemical processes, carbon assimilation and RNA accumulation of sucrose transporter genes in tomato arbuscular mycorrhiza. J. Plant Physiol. 168, 1256–1263. Doidy, J., Grace, E., Kuhn, C., Simon-Plas, F., Casieri, L., Wipf, D., 2012a. Sugar transporters in plants and in their interactions with fungi. Trends Plant Sci. 17, 413–422. Doidy, J., Tuinen, D., Lamotte, O., Corneillat, M., Alcaraz, G., Wipf, D., 2012b. The Medicago truncatula sucrose transporter family: characterization and implication of key members in carbon partitioning towards arbuscular mycorrhizal fungi. Mol. Plant 5, 1346–1358. Douds, D.D., Johnson, C.R., Koch, K.E., 1988. Carbon cost of the fungal symbiont relative to net leaf P accumulation in a split-root VA mycorrhizal symbiosis. Plant Physiol. 86, 491–496. Dutt, S., Sharma, S.D., Kumar, P., 2013. Arbuscular mycorrhizas and Zn fertilization modify growth and physiological behavior of apricot (Prunus armeniaca L.). Sci. Hortic. 155, 97–104.
Eissenstat, D.M., Graham, J.H., Syvertsen, J.P., Drouillard, D.L., 1993. Carbon economy of sour orange in relation to mycorrhizal colonation and phosphorus status. Ann. Bot. 71, 1–10. García-Rodriguez, S., Azcón-Aguilar, C., Ferrol, N., 2007. Transcriptional regulation of host enzymes involved in the cleavage of sucrose during arbuscular mycorrhizal symbiosis. Physiol. Plant. 139, 737–746. Gavito, M.E., Olsson, P.A., 2003. Allocation of plant carbon to foraging and storage in arbuscualr mycorrhizal fungi. FEMS Microbiol. Ecol. 45, 181–187. Gutjahr, C., Casieri, L., Paszkowski, U., 2009. Glomus intraradices induces changes in root system architecture of rice independently of common symbiosis signaling. New Phytol. 182, 829–837. Hodge, A., Berta, G., Doussan, C., Merchan, F., Crespi, M., 2009. Plant root growth, architecture and function. Plant Soil 321, 153–187. Ishii, T., Kadoya, K., 1996. Utilisation of vesicular–arbuscular mycorrhizal fungi in citrus orchards. Proc. Int. Soc. Citric. 2, 777–780. Koch, K.E., Johnson, C.R., 1984. Photosynthate partitioning in split-root citrus seedlings with mycorrhizal and nonmycorrhizal root systems. Plant Physiol. 75, 26–30. Larrainzar, E., Gil-Quintana, E., Arrese-lgor, C., González, E.M., Marino, D., 2014. Split-root systems applied to the study of the legume-rhizobial symbiosis: what have we learned? J. Integr. Plant Biol. 56, 1118–1124. Lerat, S., Lapointe, L., Gutjahr, S., Piche, Y., Vierheilig, H., 2003. Carbon partitioning in a split-root system of arbuscular mycorrhizal plants is fungal and plant species dependent. New Phytol. 157, 589–595. Liu, H.S., Li, F.M., Jia, Y., 2006. Effects of shoot removal and soil water content on root respiration of spring wheat and soybean. Environ. Exp. Bot. 56, 28–35. Miller, R.M., Miller, S.P., Jastrow, J.D., Rivetta, C.B., 2002. Mycorrhizal mediated feedbacks influence net carbon gain and nutrient uptake in Andropogon gerardii. New Phytol. 155, 149–162. Mishra, B.S., Singh, M., Laxmi, A., 2009. Glucose and auxin signaling interaction in controlling Arabidopsis thaliana seedlings root growth and development. PLoS One 4, e4502. Nemec, S., Vu, J.C.V., 1990. Effects of soil phosphorous and Glomus intraradices on growth, nonstructural carbohydrates, and photosynthetic activity of Citrus aurantium. Plant Soil 128, 257–263. Norman, J.R., Atkinson, D., Hooker, J.E., 1996. Arbuscular mycorrhizal fungal-induced alteration to root architecture in strawberry and induced resistance to the root pathogen Phytophthora fragariae. Plant Soil 185, 191–198. Ortas, I., Ustuner, O., 2014. Determination of different growth media and various mycorrhizae species on citrus growth and nutrient uptake. Sci. Hortic. 166, 84–90. Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55, 158–161. Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis, 3rd ed. Academic Press, London. Vierheilig, H., Garcia-Garrido, J.M., Wyss, U., Piche, Y., 2000. Systemic suppression of mycorrhizal colonization of barley root already colonized by AM fungi. Soil Biol. Biochem. 32, 589–595. Wright, D.P., Read, D.J., Scholes, J.D., 1998. Mycorrhizal sink strength influences whole plant carbon balance of Trifolium repens L. Plant Cell Environ. 21, 881–891. Wu, Q.S., He, X.H., Zou, Y.N., Liu, C.Y., Xiao, J., Li, Y., 2012. Arbuscular mycorrhizas alter root system architecture of Citrus tangerine through regulating metabolism of endogenous polyamines. Plant Growth Regul. 68, 27–35. Wu, Q.S., Srivastava, A.K., Li, Y., 2015. Effects of mycorrhizal symbiosis on growth behavior and carbohydrate metabolism of trifoliate orange under different substrate P levels. J. Plant Growth Regul. 34, 495–508. Wu, Q.S., Zou, Y.N., He, X.H., Luo, P., 2011. Arbuscular mycorrhizal fungi can alter some root characters and physiological status in trifoliate orange (Poncirus trifoliata L. Raf.) seedlings. Plant Growth Regul. 65, 273–278. Wu, Q.S., Zou, Y.N., Huang, Y.M., Li, Y., He, X.H., 2013. Arbuscular mycorrhizal fungi induce sucrose cleavage for carbon supply of arbuscular mycorrhizas in citrus genotypes. Sci. Hortic. 160, 320–325. Zeng, M., Li, D.G., Yuan, J., 2004. Effect of the pesticide on the arbuscular mycorrhizal fungi in the soil of citrus orchard. Mycosystema 23, 429–433.
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Mycorrhizal colonization represents functional equilibrium on root morphology and carbon distribution of trifoliate orange grown in a split-root system Qiang-Sheng Wu a,b,∗ , Ming-Qin Cao a , Ying-Ning Zou a,b , Chu Wu a,b , Xin-Hua He c,d,∗∗ a
College of Horticulture and Gardening, Yangtze University, Jingzhou, Hubei 434025, China Institute of Root Biology, Yangtze University, Jingzhou, Hubei 434025, China c Centre of Excellence for Soil Biology, College of Resources and Environment, Southwest University, Chongqing 400715, China d School of Plant Biology, University of Western Australia, Crawley, WA 6009, Australia b
a r t i c l e
i n f o
Article history: Received 25 August 2015 Received in revised form 9 December 2015 Accepted 18 December 2015 Keywords: Carbon partitioning Citrus Glucose Mycorrhizal fungi Split root
a b s t r a c t Trifoliate orange seedlings were grown in perspex pots of a split-root system, where one half of the split roots was inoculated with or without arbuscular mycorrhizal fungi (AMF, Acaulospora scrobiculata or Funneliformis mosseae). Five months after inoculation, the growth performance, leaf, stem and root biomass and photosynthetic rate were generally significantly higher in AM than in non-AM seedlings. Greater root morphological traits were observed in the AM root side than in the non-AM side for the same plant and in AM than in non-AM plants. AM seedlings had significantly higher leaf sucrose and glucose contents and total (leaf + root) glucose and fructose contents as compared to non-AM ones. In the split roots, the AM side displayed substantially higher sucrose, glucose and fructose contents than the non-AM side for the same plant, and in AM than in non-AM plants. These results showed greater C movement into the non-AM side from an AM-colonized plant than from a non-AM-colonized plant. These results conclude that the presence of AM in one side of the split roots benefited C acquisition and root development to another half non-AM side, suggesting functional equilibrium and resource allocation of AM within a root system. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Arbuscular mycorrhizal fungi (AMF), belonging to the phylum Glomeromycota, can form the mutualistic symbioses, arbuscular mycorrhizas (AMs), with roots of more than 80% terrestrial plants (Smith and Read, 2008). The formation of AMs provides multiple benefits to the fungal partner, including uptake of mineral nutrients and water, while consumes ∼20% of plant photosynthetically fixed carbon (C) to sustain the symbiosis life cycle (Smith and Read, 2008). AMs thus function as a metabolic C-sink causing basipetal mobilization of photosynthates to roots. Sucrose, a major photosynthate, is transported by sucrose transporters from source leaves to sink tissues such as AMs, where it is cleaved by invertases and sucrose synthases (degraded direction)
∗ Corresponding author at: College of Horticulture and Gardening, Yangtze University, Jingzhou, Hubei 434025, China. Fax.: +86 716 8066262. ∗∗ Corresponding author at: Centre of Excellence for Soil Biology, College of Resources and Environment, Southwest University, Chongqing 400715, China. E-mail address: [email protected] (W. Qiang-Sheng). http://dx.doi.org/10.1016/j.scienta.2015.12.039 0304-4238/© 2015 Elsevier B.V. All rights reserved.
into hexoses, further into trehalose and glycogen for the storage and utilization of C in AMs (Bago et al., 2003; Doidy et al., 2012b). Meanwhile, glucose, one of hexoses, is preferentially taken up by the mycobiont (Doidy et al., 2012a), though the percentage of hexoses allocated to AM versus non-AM roots is unknown. As a consequence, AM growth is a C-limited process, whilst the host plants have ultimate control over fungal activity through the regulation of carbohydrate transfer to the roots (Miller et al., 2002). Root AM colonization was generally linearly correlated with the C-sink strength of roots (Lerat et al., 2003). A higher plant C assimilation rate would hence compensate for their greater below-ground C expenditure in AM symbioses (Eissenstat et al., 1993). Foraging strategies of AMs and roots for energy are linked to plant C source (Gavito and Olsson, 2003). Even so, the cost of an AM formation involved in C partitioning between AMs and plants is poorly understood (Doidy et al., 2012a). Plant root systems exhibit highly plastic characteristics while are affected by various abiotic and biotic factors, including AMF (Hodge et al., 2009). AMF had been confirmed to improve root morphology in strawberry, rice and citrus plants (Norman et al., 1996;
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Gutjahr et al., 2009; Wu et al., 2011, 2012), which would consume more root carbohydrates for root respiration and root biomass production (Liu et al., 2006). The presence of AM would lead to an increase of carbohydrates in the non-AM root sides in the split-root citrus seedlings (Koch and Johnson, 1984). Early studies showed a 3–4% higher transfer of carbohydrates in AM root sides than in non-AM root sides of split roots (Douds et al., 1988). Citrus, one of the most planted fruit trees worldwide is strongly dependent on the AM symbiosis because of lacking of root hairs (Wu et al., 2011). Trifoliate orange [Poncirus trifoliata (L.) Raf.], a close relative to Citrus, is widely used as a rootstock of citrus plantation in Asia, including China, India, and Japan. In field, root colonization of citrus trees by native AMF is less than 10% in China (Zeng et al., 2004) and ∼20% in Japan (Ishii and Kadoya, 1996). It seems that in whole root systems, a large proportion of citrus roots in the field are not colonized by native AMF. As a result, we do not know whether these non-AM roots in whole root systems obtain some carbohydrates for better root development. Split-root system approaches can provide differential treatments for separate and independent root side of the whole root system but share a conjunct aerial part (Larrainzar et al., 2014). Split-root experiments have been extensively used to study the effects of AMF colonization on the production of carbohydrates and biomass (Koch and Johnson, 1984; Vierheilig et al., 2000; Lerat et al., 2003). Using this technique, the root system of a plant is divided into two parts, one of which is inoculated with an AM fungus, allowing a comparison of the C-sink strength between the AM and non-AM root halves of the same plant (Lerat et al., 2003). However, these studies considered C allocation only, while neglecting the root modification between AM and non-AM root sides. Here, we thus hypothesized that the non-AM root of split roots could gain a certain amount of carbohydrates from C pools of AM root side to support better root development, though how carbohydrates are distributed in roots to ensure the fungal metabolism and greater root growth is poorly understood. To confirm this hypothesis, in the present study, with one half of trifoliate orange root colonized by AMF or not, we firstly compared differences in root morphology and carbohydrate contents in two root sides, and then addressed if AM root side represent functional equilibrium and resource allocation in non-AM root side.
2.2. Experimental set-up Perspex pots (20 × 10 × 18 cm = length × width × height) were separated in the middle by a 15-cm-height perspex to form two equal sized compartments (see Fig. 1). At the top of the separated perspex, a semicircle of 1-cm diameter was designed to arrange nearly equal lateral roots of the seedlings into two halve compartments. A 5 × 3 × 2 cm (length × width × height) foam board was placed in the shoot bottom of a seedling to fix the seedlings for upright growth. Such plants were defined as the split-root plants. Each compartment was filled with 1550 g autoclaved (121 ◦ C, 0.11 Mpa, 2 h) soil. The soil (Xanthi-Udic Ferralsols, FAO system) was collected from the same Citrus Orchard on the Yangtze University campus. The soil chemical properties were pH 6.1, 9.7 g kg−1 organic carbon, 11.8 mg kg−1 available nitrogen, 15.3 mg kg−1 Oslen-P, and 21.5 mg kg−1 available potassium. The AMF inoculated split-root compartment (M) received 50 g AMF inocula, and the other compartment (NM) of the pot received the same amount of autoclaved (121 ◦ C, 0.11 Mpa, 2 h) inocula plus 2 mL filtrate (25 m) of AMF inocula to keep similar other microorganisms. The experiment consisted of a completely randomized design with three mycorrhizal treatments (A. scrobiculata, F. mosseae, and non-AMF control). Each treatment replicated four times, creating a total of 12 pots. The seedlings were grown in an environment controlled plastic greenhouse from April to September, 2013, where photosynthetic photon flux density was 965 mol m−2 s−1 , day/night temperature 28/21 ◦ C, and relative humidity 85%. The seedlings were watered in an interval of three days with distilled water to avoid waterlogging at the bottom of the compartment. A 30 mL standard Hoagland solution was biweekly supplied into each compartment.
2. Materials and methods 2.1. Biological materials Two AMF strains, Acaulospora scrobiculata Trappe and Funneliformis mosseae (Nicol. and Gerd.) Schüßler and Walker, respectively isolated from the rhizosphere of Bauhinia blakeana in Hongkong and of Incarvillea younghusbandii in Tibet in China, were used. The inocula were propagated from the identified fungal spores and cultivated with host plant of Sorghum vulgare (A. scrobiculata) or Trifolium repens (F. mosseae) for 16 weeks. After harvesting, the growth substrate contained 21 or 23 spores g−1 for A. scrobiculata or F. mosseae, respectively. Seeds of trifoliate orange were collected from a Citrus Orchard on the Yangtze University campus, surface-sterilized with 70% ethanol for 10 min, and germinated in a plastic box containing autoclaved sands in a controlled growth chamber at 28/20 ◦ C and 16/8 photoperiod hours (day/night) for 3 weeks under 80% relative humidity and 1200 mol m−2 s−1 photosynthetic photon flux density. Fresh taproots of four-leaf-old (under sterilization) trifoliate orange seedlings were excised and kept 3-cm long. Subsequently, these treated seedlings were re-planted into the plastic box for 7 weeks for inducing the formation of lateral roots.
Fig. 1. Schematic diagram of a two-chambered split-root system to grow trifoliate orange seedlings with or without Acaulospora scrobiculata or Funneliformis mosseae colonization. Abbreviations: NM, non-AMF inoculation; M, AMF inoculation.
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2.3. Plant harvest, AM colonization, and root system scanning After 5 months of AMF inoculation, the seedlings had shown significant differences in growth performance, and thus were harvested. The plant height, stem diameter, and leaf number per plant were determined. Leaf, stem and root dry biomass was recorded after over-drying at 75 ◦ C for 48 h. The two sides of split root of one plant were separately collected. The root systems were scanned with an Epson Perfection V700 Photo (Seiko Epson Corp, Japan), and then the root images were analyzed using a WinRHIZO professional 2007b (Regent Instruments Incorporated, Canada). Morphological traits, including total length, projected area, surface area and volume were obtained. For the determination of AM colonization, 1-cm-long root segments were stained with the protocol described by Phillips and Hayman (1970). Root AM colonization was expressed as the percentage of colonized root lengths against total root lengths. 2.4. Determinations of photosynthetic rate and carbohydrate concentrations On a sunny day (September 13, 2013) between 9:00 to 10:00 am, the photosynthetic rate (Pn) of AM and non-AM seedlings was determined with an infrared gas analyzer (Li-6400 Portable Photosynthesis System, Li-Cor, Lincoln, USA) using the fourth and fifth fully expanded leaf from the apices of these seedlings. Pn values were recorded when the total coefficient of variation was less than 0.5%. The reference [CO2 ] was 400 mol mol−1 . For extraction of fructose, glucose, and sucrose, 100 mg of ground dry tissues of leaves and roots was incubated with 8 mL 80% ethanol for 40 min at 80 ◦ C and centrifuged at 2500 × g for 5 min. The centrifugal residues were re-extracted using the above procedure, and the two supernatants were combined for the analysis. Determination of fructose, glucose, and sucrose concentrations was followed by the procedure described by Wu et al. (2015). These sugar contents (mg DW plant−1 ) were the amounts of corresponding tissue biomass × sugar concentration (mg DW g−1 ). Sugar contents of total (M + NM sides) were the sum of sugar contents in M and NM root sides from a treated plant. 2.5. Statistical analysis Data (means ± SE, n = 4) were subjected to one-way analysis of variance (ANOVA) using the SAS software (v8.1, SAS Institute Inc., Cary, NC 27513-2414, USA). The data about percentage were arcsine transformed before variance analysis. The significant differences in means between treatments were tested with the Duncan’s multiple range test at 5% level. The Pearson’s correlation coefficients between variables were performed using the Proc Corr’s procedure. 3. Results 3.1. Root AM colonization After five months of AMF inoculation, in the M chamber, the root AMF colonization was significantly higher under F. mosseae than under A. scrobiculata (Table 1). No root colonization was found in the NM root side. 3.2. Plant growth performance Except leaf numbers, M/NM seedlings represented significantly higher plant height, stem diameter, leaf, stem and root biomass than those in NM/NM seedlings, irrespective of AMF strains
Fig. 2. Effects of AMF on photosynthetic rates of trifoliate orange seedlings colonized with or without Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system. Data (means ± SE, n = 4) followed by different letters among treatments indicate significant differences at P < 0.05. Abbreviations: NM, non-AMF inoculation; MAs, Acaulospora scrobiculata inoculation; MFm, Funneliformis mosseae inoculation.
(Table 1). Moreover, all these growth traits were significantly greater in MFm/NM than in MAs/NM seedlings. 3.3. Photosynthetic rate (Pn) Pn was 65% and 101% significantly higher in MAs/NM and MFm/NM seedlings than in NM/NM seedlings, respectively (Fig. 2). Meanwhile, MFm/NM seedlings had significantly higher Pn than MAs/NM seedlings. 3.4. Root morphology AM seedlings recorded considerably greater root morphology than non-AM seedlings (Fig. 3a). In a same treated plant, total length, projected area, surface area and volume were similar in the two split root compartments under NM/NM treatment, whereas significantly higher in the M root side than in the NM root side, no matter whether the plants were colonized by A. scrobiculata or F. mosseae (Fig. 3b–e). Amongst the three treatments, significantly higher root morphological traits were ranked as in MFm/NM seedlings > MAs/NM colonized seedlings > NM/NM seedlings, no matter whether the roots were from the M root side, NM root side or total (M + NM root sides). 3.5. Carbohydrate contents in leaf and split roots For the same split root side (i.e. the M or NM root side) and the total (M + NM root side) root, significantly higher root sucrose contents ranked in the order: F. mosseae ≥ A. scrobiculata ≈ nonAMF, whilst significantly higher root fructose and glucose contents ranked as F. mosseae > A. scrobiculata ≈ non-AMF (Fig. 4a). For the same AMF treatment, root fructose, glucose and sucrose contents were significantly higher in the split M side than in the split NM side under A. scrobiculata and F. mosseae inoculation, whilst were similar between the two split NM sides of the non-AMF seedlings. Contents of leaf sucrose or glucose were 116–169% or 18–20% significantly higher in the AM seedlings than in the non-AM seedlings, but similar between MAs/NM and MFm/NM seedlings (Fig. 4b). Compared to the non-AMF inoculated seedlings, leaf fructose contents were significantly increased by 43% under MFm/NM whereas markedly decreased by 69% under MAs/NM. Compared to the NM/NM seedlings, the total (leaf + root) contents of fructose, glucose and sucrose were increased by 130, 259 and 120% or 19, 41 and 13% under MFm/NM or MAs/NM (Fig. 4c).
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Table 1 Effects of AMF on root mycorrhization, soil hyphal length and plant growth of trifoliate orange (Poncirus trifoliata) seedlings inoculated with Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system. Inoculated treatments
Root AM colonization (%)
Plant height (cm)
Stem diameter (mm)
Leaf number
Biomass (g/plant DW) Leaf
NM/NM MAs/NM MFm/NM a
0 ± 0c 15.8 ± 1.2b 38.9 ± 3.5a
19.7 ± 4.5c 25.6 ± 3.1b 43.4 ± 2.5a
2.47 ± 0.24c 2.85 ± 0.18b 4.52 ± 0.15a
24 ± 6b 26 ± 6b 44 ± 7a
0.12 ± 0.04c 0.18 ± 0.04b 0.31 ± 0.03a
Stem 0.31 ± 0.11c 0.50 ± 0.10b 1.23 ± 0.08a
Roota 0.25 ± 0.02c 0.31 ± 0.02b 0.70 ± 0.03a
Total production of both root sides. Abbreviations: NM, non-AMF inoculation; MAs, Acaulospora scrobiculata inoculation; MFm, Funneliformis mosseae inoculation.
Fig. 3. Effects of AMF on root morphology (a) and root total length (b), projected area (c), surface area (d) and volume (e) of trifoliate orange seedlings colonized with (M) or without (NM) Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system. Data (means ± SE, n = 4) followed by different letters among AMF treatments for the same split root compartment or the total (M + NM) (a–c) or between the M and NM split root compartment for the same AMF treatment (x, y) indicate significant differences at P < 0.05. Abbreviations: NM, non-AMF inoculation; MAs, Acaulospora scrobiculata inoculation; MFm, Funneliformis mosseae inoculation. In ‘b–e’ subfigures, M side column in x-axis orientation represented NM root side data only in NM/NM treated split-root seedlings.
3.6. Carbohydrate distribution Distributions (%) of total (leaves + roots) tested carbohydrates were significantly decreased in leaf fructose and glucose whilst
increased in leaf sucrose under AMF than under non-AMF inoculation (Fig. 5a–c). Compared to the NM/NM treatment, percentages of root fructose, glucose and sucrose in the M side were 19, 31 and 6% or 45, 92 and 29% higher under the A. scrobiculata or F.
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Fig. 4. Effects of AMF on fructose, glucose and sucrose contents in root (a), leaf (b) and total (leaf + root) (c) of trifoliate orange seedlings colonized with (M) or without (NM) Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system. Data (means ± SE, n = 4) followed by different letters among AMF treatments for the same split root compartment or the total (M + NM) (a–c) or between the M and NM split root compartment for the same AMF treatment (x, y) indicate significant differences at P < 0.05. Abbreviations: NM, non-AMF inoculation; MAs, Acaulospora scrobiculata inoculation; MFm, Funneliformis mosseae inoculation. In ‘a’ subfigure, M side column in x-axis orientation represented NM root side data only in NM/NM treated split-root seedlings.
mosseae inoculation, respectively. Interestingly, compared to the NM root side in NM/NM seedlings, percentages of root fructose in the NM root side were increased by 7% under the A. scrobiculata but decreased by 33% under the F. mosseae inoculation (Fig. 5a). Meanwhile, percentages of root glucose and sucrose in the NM root side of M/NM seedlings were 27 and 31% or 38 and 36% lower under the A. scrobiculata or F. mosseae inoculation than under the non-AMF inoculation, respectively (Fig. 5b and c). 3.7. Relationships between root traits or photosynthesis and sugar production Total root (the split M + NM side) fructose, glucose and sucrose contents were positively (P < 0.01) correlated with root total length (Fig. 6a–c), root projected area (Fig. 6d–f), root surface area (Fig. 6d–f), and root volume (Figure 6g–i), respectively. The contents of total plant (leaf + root) fructose, glucose and sucrose were positively (P < 0.01) correlated with leaf Pn (Fig. 7). 4. Discussion 4.1. Greater plant growth performance in the AMF colonized half side of split roots Root production was significantly lower in the half non-AM side than in the half AM side of the split root colonized by either A. scrobiculata or F. mosseae, which was in agreement with studies in non-split-root citrus (Wu et al., 2011; Ortas and Ustuner, 2014) and apricot (Dutt et al., 2013). A greater plant biomass production in AM seedlings might reflect an AM-enhanced C partitioning belowground (Eissenstat et al., 1993). Under higher C expenditure by AMs
and greater root biomass conditions, root growth performance was still enhanced by AMF inoculation (Table 1). Moreover, AM-induced positive growth responses were significantly higher with F. mosseae than with A. scrobiculata, indicating that trifoliate orange seedlings have a certain degree of specificity in AM symbiosis (Ortas and Ustuner, 2014). 4.2. AMF inoculation enhanced photosynthetic rate and plant carbohydrate accumulation In plants, sucrose is synthesized in the photosynthetic leaves as a principal form of carbohydrates for long-distance transport from source leaves to plant and non-plant C sinks including AMs (Doidy et al., 2012b). Sucrose generally is cleaved into hexoses (e.g., fructose and glucose) through sucrose synthase (degraded direction) or invertase, which is then transformed into typical fungal carbohydrates, trehalose and glycogen (Bago et al., 2003; Wu et al., 2013). Studies showed that AMF inoculation with F. mosseae increased leaf glucose and fructose in tomato plants (Boldt et al., 2011). These are true in the present study that M/NM seedlings had significantly higher leaf fructose, glucose and sucrose than NM/NM seedlings (Fig. 4b), except a decrease of leaf fructose in MAs/NM than in NM/NM control seedlings. These carbohydrate accumulations are dependent on the activity of sucrose cleaving enzymes (Wu et al., 2013). In addition, leaf Pn was significantly higher in AM than in non-AM seedlings (Fig. 2) and was notably positively correlated with total (leaf + root) fructose, glucose, and sucrose (Fig. 7). It suggests that a greater AMF-induced Pn would produce more photosynthates for a greater C expenditure below-ground by AM than by non-AM plants, in addition to a higher chlorophyll and RuBPCase activity (Nemec and Vu, 1990; Eissenstat et al., 1993; Wu
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Fig. 5. Effects of AMF colonization on the distribution of total (leaves + roots) fructose, glucose or sucrose in leaf, the split M and NM root side in the trifoliate orange seedlings colonized with or without Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system. Abbreviations: NM, non-AMF inoculation; MAs, Acaulospora scrobiculata inoculation; MFm, Funneliformis mosseae inoculation.
et al., 2011; Birhane et al., 2012). Furthermore, more plant photosynthates had been transferred to the C sink of AM roots (Douds et al., 1988), thereby, inducing a significantly higher total (M + NM sides) root carbohydrate accumulation (Fig. 4a). The additional C gain derived from mycorrhization would promote root growth and maintenance of AM symbioses (Wright et al., 1998). 4.3. Higher carbohydrate allocation in M root side modulated greater root morphology in M root side In the present study, AM split-roots recorded relatively higher total (M + NM sides) sucrose content than non-AM control (Fig. 4a), implying that more sucrose was translocated to the AM split-roots to sustain the extra cost of AMs. The higher total (M+NM sides) fructose and glucose contents in roots were found in AM roots than in non-AM roots, which is not consistent with the findings of García-Rodriguez et al. (2007), who reported the lower fructose and glucose concentration in AM roots in F. mosseae- and R. intraradices-colonized tomato plants. Our study further showed that significantly higher root fructose, glucose, and sucrose contents were found in the NM root side of the MFm/NM or MAs/NM treated plants than in the NM root side of NM/NM counterparts, except a similar sucrose no matter whether roots were colonized by A. scrobiculata or not (Fig. 4a). It implied that the NM splitroot side of the M/NM plants had obtained extra photosynthetically fixed C, showing a functional equilibrium on C distribution for NM and M root sides. Gavito and Olsson (2003) pointed out that the energy foraging strategies between M and NM root sides in a split
root system might be more collaborative, rather than competitive. Our results demonstrated that part of mycorrhizal-promoted carbohydrates in the M colonized side could be moved into the NM root side, indicating a collaborative behaviour and functional equilibrium for C request in split roots. Furthermore, stronger C-sink capacity by F. mosseae than by A. scrobiculata indicated a fungal species dependent, which was in agreement with studies in split roots of barley and sugar maple (Lerat et al., 2003). Since sucrose can be cleaved by invertases and sucrose synthases into hexoses (Bago et al., 2003; Doidy et al., 2012b), greater fructose and glucose content in AM split-roots indicates that more sucrose was cleaved into hexoses, which is taken up and transformed by the symbiont (Doidy et al., 2012a). In the process of sucrose cleavage in AM plants, various sucrose cleaved enzyme genes are up-regulated (GarcíaRodriguez et al., 2007), which will be further highlighted in AM trifoliate orange seedlings. In addition, considerably higher sucrose, glucose, and fructose in the M root side than in the NM root side of split roots (Fig. 4a) further indicated that in whole root, roots in the mycorrhizal side acted as a C sink, which might be independent of enhanced nutrient effects, especially P (Koch and Johnson, 1984; Miller et al., 2002). Greater carbohydrates in the M root side (Figs. 4a; 5) would benefit the increase of root biomass production, root respiration, and mycorhizal hyphal biomass production and respiration (Miller et al., 2002; Mishra et al., 2009). Eissenstat et al. (1993) also reported that a higher percentage of below-ground C was in below-ground respiration in AM than in non-AM sour orange plants under the equivalent P status. The present study further confirmed that root
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Fig. 6. Relationships between root total length, projected area, surface area or volume and fructose, glucose or sucrose content in two sides of split-root in trifoliate orange seedlings colonized with Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system (n = 24).
sucrose, glucose, or fructose significantly positively correlated with root total length, surface area, projected area or volume (Fig. 6). It is hence reasonable that AM-induced greater C allocation into the mycorrhizal side of split roots would benefit AMF colonization and root growth, resulting in greater root morphological traits in M root side (Fig. 3a–e). 4.4. Significantly higher carbohydrates in NM root side of M/NM plants than in NM side of NM/NM plants indicated a functional equilibrium between M and NM root sides of split-roots to affect root morphology Our previous studies had confirmed greater root morphology in AM than in non-AM trifoliate orange seedlings (Wu et al., 2011, 2012). In the present study, an AM-enhanced root morphology also occurred in the split roots, showing higher root total length, projected area, surface area and volume in the split M roots than in the split NM roots (Fig. 3a–e). Although greater C expenditure below-ground (e.g., AMs presence and greater root biomass and respiration) occurred by AM than by non-AM seedlings, the NM root side of split roots in the M/NM seedlings still exhibited significantly greater root morphological traits and higher C contents than in the NM root side in the NM/NM seedlings (Fig. 4). We speculated that the NM root side of split roots in the M/NM seedlings might gain a certain amount of carbohydrates (such as glucose)
Fig. 7. Relationships between photosynthesis rate and total (leaf + root) fructose, glucose or sucrose content of trifoliate orange seedlings colonized with Acaulospora scrobiculata or Funneliformis mosseae and grown in a two-chambered split-root system (n = 12).
from the whole root C pool to support root development. Moreover, such AM-enhanced effects were firstly observed in the split roots of trifoliate orange seedlings. It suggests the functional equilibrium and resource allocation of AMs in enhancing belowground
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root morphological development. So, the present study confirmed our foregoing hypothesis, namely, the non-AM root of split roots could gain a certain amount of carbohydrates from C pools of AM root side to support better root development. Certainly, using 13 C or 14 C isotope labelling will be able to further confirm the translocation process. 5. Conclusion In short, the whole root system in the split-root trifoliate orange had significantly greater carbohydrates in M/NM than in NM/NM plants, which resulted in better root morphological characters in the NM colonized side of split roots under mycorrhization. Meanwhile, significantly greater carbohydrates in the NM root side of split roots in the M/NM seedlings than in NM/NM seedlings might derive from a better root morphology under AMF colonization. It seemed that mycorrhizal effects on root morphology and carbon distribution in split-root trifoliate orange exhibited the characteristic of a functional equilibrium. Our results could have implications for AM functioning on root morphological development and C distribution within a root system belowground in citrus trees under low root AMF colonization condition. Acknowledgements This study was supported by the National Natural Science Foundation of China (31372017), the Key Project of Chinese Ministry of Education (211107), and the Open Fund of Institute of Root Biology, Yangtze University (R201401). References Bago, B., Pfeffer, P.E., Abubaker, J., Jun, J., Allen, J.W., Brouillette, J., Douds, D.D., Lammers, P.J., Shachar-Hill, Y., 2003. Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid. Plant Physiol. 131, 1496–1507. Birhane, E., Sterck, F.J., Fetene, M., Bongers, F., Kuyper, T.W., 2012. Arbuscular mycorrhizal fungi enhance photosynthesis, water use efficiency, and growth of frankincense seedlings under pulsed water availability conditions. Oecologia 169, 895–904. Boldt, K., Pors, Y., Haupt, B., Bitterlich, M., Kuhn, C., Grimm, B., Franken, P., 2011. Photochemical processes, carbon assimilation and RNA accumulation of sucrose transporter genes in tomato arbuscular mycorrhiza. J. Plant Physiol. 168, 1256–1263. Doidy, J., Grace, E., Kuhn, C., Simon-Plas, F., Casieri, L., Wipf, D., 2012a. Sugar transporters in plants and in their interactions with fungi. Trends Plant Sci. 17, 413–422. Doidy, J., Tuinen, D., Lamotte, O., Corneillat, M., Alcaraz, G., Wipf, D., 2012b. The Medicago truncatula sucrose transporter family: characterization and implication of key members in carbon partitioning towards arbuscular mycorrhizal fungi. Mol. Plant 5, 1346–1358. Douds, D.D., Johnson, C.R., Koch, K.E., 1988. Carbon cost of the fungal symbiont relative to net leaf P accumulation in a split-root VA mycorrhizal symbiosis. Plant Physiol. 86, 491–496. Dutt, S., Sharma, S.D., Kumar, P., 2013. Arbuscular mycorrhizas and Zn fertilization modify growth and physiological behavior of apricot (Prunus armeniaca L.). Sci. Hortic. 155, 97–104.
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