Mycorrhiza stimulates root-hair growth and IAA synthesis and transport in trifoliate orange under drought stress

Root-hair growth and development regulated by soil microbes is associated with auxin. In this background, we hypothesized that mycorrhizal fungal inoculation induces greater root-hair growth through stimulated auxin synthesis and transport under water stress conditions. Trifoliate orange (Poncirus trifoliata) was inoculated with an arbuscular mycorrhizal (AM) fungus (Funneliformis mosseae) under well-watered (WW) and drought stress (DS) for 9 weeks. Compared with non-AM seedlings, AM seedlings displayed significantly higher density, length, and diameter of root hairs and root indoleacetic acid (IAA) level, whereas lower total root IAA efflux, regardless of soil moisture status. Root PtYUC3 and PtYUC8 involved in IAA biosynthesis were up-regulated by mycorrhization under WW and DS, whereas AM-modulated expression in PtTAA1, PtTAR2, PtYUC4, and PtYUC6 depended on status of soil moisture. Mycorrhizal inoculation down-regulated the transcript level of root auxin efflux carriers like PtPIN1 and PtPIN3, whereas significantly up-regulated the expression of root auxin-species influx carriers like PtABCB19 and PtLAX2 under DS. These results indicated that AMF-stimulated greater root-hair growth of trifoliate orange under DS that is independent on AMF species is related with mycorrhiza-modulated auxin synthesis and transport, which benefits the host plant to enhance drought tolerance.

P-uptake in promoting the plant growth, plant water relations or photosynthetic capacity under DS, thereby, providing an evidence for enhancing drought tolerance of host plant under DS. The magnitude of plant responses to mycorrhization is considered highly dependent on the compatibility between AMF species and host plant species 15 . It is still not clear whether mycorrhizas could play a role in root hair growth, with an exception of D. versiformis 14 .
The mechanisms regarding mycorrhizal effects on root-hair growth of host plants are unknown. It is well documented that root-hair initiation is manipulated by two different pathways, viz., developmental pathway and the environmental/hormonal pathway 16 . In roots, a variety of of phytohormones, like auxin, ethylene, jasmonic acid, brassinosteroid, and strigolactone participate in root-hair growth and development, but auxin is most extensively studied [17][18][19] . Auxin is primarily synthesized at the shoot apex, transported to the root tip by vascular tissues of the stem, finally moves in a basipetal orientation towards the elongation zone through root peripheral tissues 20 . Such auxin efflux at the root apex is mainly controlled by various Pin-formed (PIN) auxin efflux carriers, besides AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) auxin influx carriers and some members of the ATP-BINGING CASSETTE B (ABCB) transporters (auxin efflux proteins) 21,22 . Besides auxin transport, auxin synthesis is controlled by a number of genes, such as tryptophan aminotransferase (TAA), tryptophan aminotransferase related (TAR), flavin monooxygenase-like enzyme (YUC), etc. 23 .
Trifoliate orange (Poncirus trifoliata L. Raf.) is a widely used rootstock in citriculture in Southeast Asia, the root configuration of which is characterized by distinctively few and short root hairs which, accompanied with highly drought-sensitive nature 24 . Based on our previous results 13 , we hypothesized that mycorrhizal inoculation with Funneliformis mosseae could induce greater root-hair growth of trifoliate orange through enhanced auxin synthesis and transport under DS for enhanced drought tolerance. To confirm this hypothesis, trifoliate orange seedlings were inoculated with Funneliformis mosseae and subsequently exposed to well-watered (WW) and DS conditions. The responses were evaluated through root-hair morphology, root auxin concentration, root auxin effluxes, and relative expression of root auxin relevant genes.

Results
Mycorrhizal colonization of roots. No mycorrhizal colonization was observed in non-AM roots. AMFinoculated seedlings showed 55.6-61.4% of root mycorrhizal colonization ( Table 1). As much as 9.4% reduction in root mycorrhizal colonization was observed under DS than under WW. Plant growth. Plant growth traits, including plant height, stem diameter, leaf number, and shoot and root biomass were adversely affected by DS treatment, as compared with WW treatment, regardless of AM or non-AM seedlings (Table 1). On the other hand, AM seedlings showed significantly higher these plant growth-related traits than non-AM seedlings, irrespective of WW or DS condition.
Root-hair features. Length, diameter and density of root hairs were significantly increased by DS treatment, in comparison with WW treatment (Fig. 1). AM seedlings displayed better root hair features than non-AM seedlings under both WW and DS, in a range of 41% and 15% higher for root hair length, 50% and 40% higher for root hair density, and 16% and 25% higher for root hair diameter, respectively. Root IAA concentration. Concentration of root indole-3-acetic acid (IAA) was significantly reduced by DS treatment, as compared to WW treatment (Fig. 2). AM seedlings exhibited significantly higher root IAA concentration than non-AM seedlings by 36% and 37% under WW and DS condition, respectively.
Total root IAA efflux. An IAA efflux was observed in trifoliate orange from the root to the rhizosphere, regardless of WW or DS treatment. The DS treatment produced a significant reduction in total root IAA efflux by 3% in non-AM seedlings and an increase by 35% in AM seedlings (Fig. 3). AMF inoculation conferred a significant reduction in total root IAA efflux by 58% and 41% under WW and DS, respectively, in relative to non-AMF treatment.
Root IAAO activity. Compared with WW treatment, root IAA oxidase (IAAO) activity was significantly increased by DS treatment in AM or non-AM seedlings (Fig. 4). There were no significant difference in root IAAO activity between AM and non-AM seedlings exposed to both WW and DS.  Table 1. Effects of an arbuscular mycorrhizal fungus (AMF), Funneliformis mosseae, on plant growth performance of trifoliate orange (Poncirus trifoliata) seedlings exposed to well-watered (WW) and drought stress (DS). Data (means ± SD, n = 4) followed by different letters in the column indicate significant differences (P < 0.05) between treatments.

Figure 1.
Effects of an arbuscular mycorrhizal fungus (AMF), Funneliformis mosseae, on average density, length, and diameter of root hairs of trifoliate orange (Poncirus trifoliata) seedlings exposed to well-watered (WW) and drought stress (DS). Data (means ± SD, n = 4) followed by different letters above the bars indicate significant differences (P < 0.05) between treatments.

Figure 2.
Effects of an arbuscular mycorrhizal fungus (AMF), Funneliformis mosseae, on root IAA concentration of trifoliate orange (Poncirus trifoliata) seedlings exposed to well-watered (WW) and drought stress (DS). Data (means ± SD, n = 4) followed by different letters above the bars indicate significant differences (P < 0.05) between treatments. Effects of an arbuscular mycorrhizal fungus (AMF), Funneliformis mosseae, on root IAA effluxes of trifoliate orange (Poncirus trifoliata) seedlings exposed to well-watered (WW) and drought stress (DS). Data (means ± SD, n = 4) followed by different letters above the bars indicate significant differences (P < 0.05) between treatments. Transcript levels of root EXPAs. Root EXPAs such as PtEXPA4, PtEXPA5, and PtEXPA7 genes were significantly up-regulated by DS, compared with WW, irrespective of AM or non-AM-seedlings ( Fig. 7a-c).

Discussion
In this study, inoculation with F. mosseae showed a significant increase in length, density, and diameter of root hairs in trifoliate orange under DS. These observations are in agreement with the study carried out by Zou et al. 13 in trifoliate orange colonized by Diversispora versiformis under DS. It also suggested that mycorrhiza-stimulated root hair growth in trifoliate orange is independent of AMF species. Better root hair growth in mycorrhizal trifoliate orange plants under DS provides a much higher nutrient foraging ability for the inoculated plants to absorb more water and nutrients from mycorrhizosphere, eventually alleviating negative effects of drought on plants 25 . The present study showed that mycorrhizal inoculation produced a remarkably increased root IAA level in trifoliate orange seedlings exposed to either WW or DS, in accordance with our previous study in trifoliate orange seedlings colonized by Claroideoglomus etunicatum, D. versiformis, F. mosseae, and Rhizoglomus intraradices 12 . Auxin is now considered as a key regulator in the whole process of root-hair initiation, growth and development 18,19,26 . Any increase in the root IAA accumulation under mycorrhization, thereby, would benefit root-hair growth and plant growth performance.
Auxin is transported from one cell to another cell, following a strict directionality in uptake and efflux of carrier proteins involved 23 . Auxin efflux is regulated through members of PIN family, the sequence of which encodes a family of auxin efflux carriers 22 . Our study revealed that mycorrhizal seedlings displayed lower root IAA efflux than non-mycorrhizal seedlings under either WW or DS treatment. Under DS, mycorrhizal treatment down-regulated the transcript level of root PtPIN1 and PtPIN3, but not PtPIN4, indicating a potential reduction in the amount of auxin efflux and finally resulting in an elevated accumulation of root IAA in AM plants over non-AM plants.  reported that PIN1, PIN3, and PIN4 were located in stele cells, collectively responsible for auxin flow towards the quiescent center (QC), close to root tip for auxin reflux. Therefore, the lower expression level of PtPIN1 and PtPIN3 in AM versus non-AM plants exposed to DS is speculated to decrease the auxin flow towards the QC, thereby, reducing the auxin reflux 27 and inducing greater auxin accumulation in the root-hair zones to stimulate root-hair growth. In fact, auxin reflux is associated with the five PIN proteins (PIN1, PIN2, PIN3, PIN4 and PIN7) 27,28 . Further studies are needed to decode the functioning of the PIN family on AMF-induced root hair modification, especially under DS condition. It is well known that YUC (YUCCA encoding a flavin monooxygenase) and TAA/TAR (Tryptophan Aminotransferase of Arabidopsis) are two families of genes associated with auxin biosynthesis in plants 29 . In our work, the expression of root PtTAA1, PtYUC3, and PtYUC8 under WW and root PtTAR2, PtYUC3, PtYUC4, and PtYUC8 under DS was up-regulated by AMF inoculation, relative to non-AMF treatment. In the indole-3-pyruvic acid (IpyA) pathway, TAA1 and its close homologue, PtTAR2 convert L-Trp into IpyA, and YUC enzymes synthesize IAA from IpyA 30 . Our present study showed a different capacity in conversation of L-Trp into IpyA by mycorrhization from WW to DS. And, PtYUC3 and PtYUC8 were jointly activated by mycorrhization, regardless of WW or DS, implying the high responsiveness of the two genes to mycorrhization.
Initiation and growth of root hairs require loosening of cell-wall components, mediated by cell wall-loosening expansin proteins (EXPs), represented by two major EXP subgroups, viz., EXPA and EXPB 31 . In our study, the transcript level of root PtEXPA4 and PtEXPA7 genes was not affected by AMF inoculation under WW, but the relative expression of root PtEXPA5 was down-regulated. Under DS, AM seedlings were characterized by relatively higher transcript level of root PtEXPA5 and lower transcript level of root PtEXPA4 and PtEXPA7 genes, indicating that AMF-mediated expression of root PtEXPAs is strongly dependent on soil moisture status. Inactivation or down-regulation in the expression of root PtEXPAs upon mycorrhization (except an up-regulated expression of root PtEXPA5 under DS) further suggested that the member of root EXPAs is not stimulated by AMF to initiate growth elongation of root hairs.
Cell-to-cell auxin transport is dependent on two families of auxin-species carrier proteins, viz., ABCB family and AUX1/LAX family of influx carriers 32 , besides PIN carriers. ABCB auxin transporters are involved in the polar transport of IAA in plants, whilst ABCB1 and ABCB19 operate long-distance IAA-transport 33 . The AUX1/LAX family of PM permeases possess H + -symport activity to transport auxin into the cells 34 . Our work showed no changes in the expression of root PtABCB1 and PtABCB19 genes in response to mycorrihization under WW. Nevertheless, the root PtABCB1 transcript level was decreased and transcript level of root PtABCB19 was increased under DS in response to AMF inoculation. These observations warranted that mycorrhizal inoculation only induced the up-expression of root PtABCB19 under DS to accelerate long-distance auxin-transport. A considerably higher transcript level of root PtAUX1, PtLAX2, and PtLAX3 genes under WW was observed in AM than in non-AM seedlings. And, a higher expression level of root PtLAX2 and lower expression level of root PtLAX1 were observed in AM seedlings than in non-AM seedlings under DS, suggesting that these auxin carrier proteins, especially PtLAX2 responded well to mycorrhization for cell-to-cell auxin transport under DS.
IAAO is usually involved in auxin catabolism and negatively correlated with IAA levels, thereby, regulating the concentration of IAA 35 . In our work, DS treatment induced a higher root IAAO activity in both AM and non-AM seedlings, thereby, leading to the lower root IAA level in DS-treated seedlings. AMF-inoculation did not alter the In short, the present study confirmed our proposed hypothesis that mycorrhizal inoculation induced greater root-hair growth of trifoliate orange, closely associated with auxin pathway under DS, where mycorrhiza activated the auxin relevant genes (PtYUC3 and PtYUC8), up-regulated the auxin-species influx carrier genes (PtABCB19 and PtLAX2), and down-regulated the auxin-species efflux carrier genes (PtPIN1 and PtPIN3).  , PtLAX3 (f), PtPIN1 (g), PtPIN3 (h), and PtPIN4 (i) in trifoliate orange (Poncirus trifoliata) seedlings exposed to wellwatered (WW) and drought stress (DS). Data (means ± SD, n = 4) followed by different letters above the bars indicate significant differences (P < 0.05) between treatments. density is 880 μmol/m 2 /s, day/night temperature 28/21 °C, and relative humidity 85%) in the campus of Yangtze University, Hubei, China.

Methods
AM and non-AM seedlings were kept at 75% of maximum water holding capacity of the soil (soil WW status) for 11 weeks. Afterwards, half of the seedlings were still maintained under WW status for 9 weeks, and the other half seedlings were exposed to 55% of maximum water holding capacity (soil DS status) of the soil for 9 weeks. Soil water levels in the pots were measured daily by weighing, and the amount of water lost was supplied to maintain the designated soil water levels.
Experimental design. The experiment consisted of four treatments with a completely randomized block arrangement: i. the seedlings inoculated with F. mosseae under WW (WW + AMF), ii. the seedlings inoculated without F. mosseae under WW (WW-AMF), iii. the seedlings inoculated with F. mosseae under DS (DS + AMF), and iv. the seedlings inoculated without F. mosseae under DS (DS-AMF). Each treatment had four replicates, for a total of 16 pots, each pot having 3 seedlings.
Variable determination. Root mycorrhizal colonization. As many fifteen 1-cm-long root segments per seedlings were cleared by 10% KOH solution at 95 °C for 1.5 h and then stained with 0.05% trypan blue in lactophenol for 5 min 36 . Root mycorrhizal colonization was calculated as the percentage of mycorrhizal infected root lengths against total observed root lengths.
Plant growth. Plant growth-related parameters like plant height, stem diameter, and leaf number per plant were determined in all the seedlings. After harvested, the seedlings were divided into shoots and roots, to measure their fresh weight.
Root hairs. Eight 1.5-cm-long root hair zones at 3 cm away from the root tip in tap root and 1 st -, 2 nd -, and 3 rd -order lateral roots were selected, fixed by 2.5% glutaraldehyde solution with 0.1 mM sodium cacodylate buffer (pH 7.4), dehydrated step by step with alcohol using increasing concentration, dried with critical-point drying, and finally sprayed with by metals 12  Root IAAO activity. Root IAAO activity was measured using the ELISA assay (BYE97073, Shanghai Bangyi Biotechnology Co. Ltd, China) according to the user's guide.
Quantitative RT-PCR. Freezed root sample was ground in liquid nitrogen. Root total RNA was extracted using an EASY spin Plus plant RNA kit (RN 38, Aidlab Biotecnolohies Co. Ltd, China). After DNase treatment, total RNA was reversely transcribed to cDNA using the PrimeScript TM RT reagent kit (PK02006, Takara Bio. Inc, Japan). Quantitative real-time PCR (qRT-PCR) were performed using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, CA, USA) on a 7900HT Fast Real-time PCR System (Applied Biosystems, CA, USA). The amplification protocol consists of one cycle of 95 °C for 10 min, followed by 40 amplification cycles of 95 °C for 15 s, 56 °C for 30 s, and 72 °C for 30 s. The primers for selected auxin efflux carriers (PIN1, PIN3, and PIN4), auxin influx carriers (AUX1, LAX1, LAX2, LAX3, ABCB1, and ABCB19), auxin synthesized genes (TAA1, TAR2, YUC3, YUC4, YUC6, and YUC8), and root hair-specific expansin genes (EXPA4, EXPA5, and EXPA7) in the qRT-PCR were shown in Table 2, as per the design from Citrus sinensis cDNA sequences (http://citrus.hzau.edu. cn/orange). The relative fold change in gene expression was calculated by the 2 −△△Ct method 39 , where the reference gene β-actin was acted as the control. Statistical analysis. The data were statistically analyzed using one-way ANOVA (SAS, version 8.1). Data of root AM colonization were arcsine transformed prior to ANOVA analyses. The Duncan's multiple range tests were used to compare significant differences among treatments at P < 0.05.  Table 2. Gene-specific primer sequences used in this work for qRT-PCR.