Effect of exogenous IAA on tension wood formation by facilitating polar auxin transport and cellulose biosynthesis in hybrid poplar (Populus deltoids × Populus nigra) wood

Abbreviations:

ABCB, ATP-binding cassette transporters of the B class; CesA, cellulose synthase; G-layer, gelatinous layer; IAA, indole-3-acetic acid; MFA, microfibril angle; NAA, naphthalene acetic acid; NPA, N-1-naphthylphthalamic acid; PAT, polar auxin transport; SuSy, sucrose synthase; TW, tension wood; UDPG, uridine diphosphate glucose; UGPase, UDP-glucose pyrophosphorylase.

Plant materials:

Homogeneous 25 cm long woody stems were cut of P. deltoides×P. nigra clone grown in the Agricultural Park of Anhui Agricultural University (31°52′N, 117°14′E). During March, 1-month-old rooted cuttings were transplanted into pots containing a 50:50 (v/v) mixture of brown peat and cuttage substrate. Cuttings were grown in a greenhouse with a light/dark photoperiod of 16 h/8 h and the day/night temperature of 30°C/22°C.

Preparation and application of IAA and NPA:

IAA and NPA (Sigma, Germany) were applied in the form of lanolin pastes and prepared as described by Aloni (1979). Nine vigorous seedlings with straight stem and uniform size were selected for the experiment. The average heights and basal diameters of the new stems were 90.2 cm and 8.5 mm, respectively. Seedlings were randomly assigned to three groups (three trees per group) and subjected to the following treatments: (a) plain lanolin as control; (b) 10 mg·g⁻¹ IAA lanolin paste; (c) 10 mg·g⁻¹ NPA lanolin paste. On June, a 2 cm long band of epidermis was slightly removed from the stem circumference in each seedling with a scalpel at 18 to 20 cm (denoted application site) below the shoot apex and then 1 g lanolin paste was applied only once in each treatment. The application site was wrapped with a piece of aluminum foil promptly.

Growth measurement and sampling:

Tree height and basal diameter were measured before and after the experiment with measuring tape and vernier caliper, respectively. On July, stem samples were harvested and isolated at 0.5 to 12.5 cm below the application site in each seedling. Fresh tissues were harvested and immediately frozen in liquid nitrogen and stored at −80°C until RNA isolation. For microscopy, subsamples were fixed in a formalin-acetic-acid-alcohol (FAA) solution until processing. The bark and pith of the rest samples were removed and then these samples served for density determination. The samples were air dried at 60°C to a constant weight for determination of crystallinity, MFA and cellulose content. Analyses were done on three trees per treatment.

RNA extraction and CDNA synthesis:

The bark and stem-developing phloem tissues were removed with a sterile scalpel. About 1 g sample was placed into a 50 ml steel jar together with a steel bead and frozen by liquid nitrogen for more than 10 min firstly, and then the jar was set in a high throughput ball mill (DHS, Beijing, China) for powdering the samples for 2 min. Total RNA was isolated by the improved cetyl trimethylammonium bromide (CTAB) method (Dong et al. 2011). A 2 μg of total RNA was reverse transcribed by means of the PrimeScript RT reagent Kit (TaKaRa, Dalian, China) to generate cDNA.

Real-time quantitative PCR:

Quantitative real-time PCR reactions were carried out with GoTaq qPCR Master Mix (Promega, Fitchburg, MA, USA) in a total volume of 25 μl on the ABI 7300 Real-Time PCR System (ABI, Foster City, CA, USA). A negative control with sterile water (instead of template cDNA) was also performed. The real-time PCR conditions were as follows: denaturation by hot start at 95°C for 10 min, followed by 40 cycles of a two-step program consisting of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. Amplification of a single product for each primer set was confirmed by the dissociation curves analysis. The transcript abundance of genes involved in cellulose synthesis and intercellular transport of auxin were quantified, namely CesA, PtCesA4, PtCesA5, PtCesA7, PtCesA8, PtCesA13, PtCesA17, and PtCesA18; SuSy, PtSUS1 and PtSUS2; UGPase, UPG1 and UPG2; PIN1; AUX2; and ACBC1. The poplar actin gene (ACT2) served as an endogenous control to normalize transcript quantity. Primers used for amplification are listed in Table S1. The genes selected for transcript analysis are based on previously reported high levels or specific expression in poplar xylem (Suzuki et al. 2006; Richet et al. 2011; Carraro et al. 2012). Transcript relative abundance was calculated as the mean of three biological replicates (three trees per condition) and three analytical replicates.

Determination of cellulose crystallinity and microfibril angle (MFA):

MFA and cell wall crystallinity were determined by X-ray diffractometry (Persee, Beijing, China), with CuKα radiation (λ=1.54 Å) at 36 kV and 20 mA. Diffractograms were obtained from tangential sections of radial wood strip samples with precisely cut (3×1×25 mm³, T×R×L). Both the X-ray source and the detector were set to θ=0° for wood crystallinity determination, whereas the 2θ (source) was set to 22.4° for MFA determination. The MFA was calculated according to Cave (1966), i.e. MFA=0.6 T, where T is the half width of the (002) diffraction peak. Crystallinity was calculated by the method of Segal et al. (1959).

Determination of cellulose content:

The determination was done according to Updegraff (1969). After grinding, wood powders (50 mg) were added into an acetic acid/nitric acid mixture. Then, the acid insoluble material (cellulose) was collected by centrifugation at 5000 g for 10 min. The dried pellets were resuspended in 67% H₂SO₄ aqueous solution. Cellulose content was determined on a glucose basis by anthrone colorimetry at A₆₂₀ (U-2900 spectrophotometer, Hitachi, Tokyo, Japan).

Wood density and anatomy:

Wood density was determined by water displacement and calculated as minimum dry mass vs. fresh volume (Richet et al. 2011). Samples were fixed for 1 week and then embedded in polyethylene glycol (Aladdin, Shanghai, China) according to Barbosa et al. (2010). Transverse sections (10 μm thick) were obtained with a sliding microtome (Leica, Wetzlar, Germany). The sections were stained in a saturated solution of phloroglucinol in 20% HCl and mounted in glycerol for measuring the radial widths of xylem and phloem as well as for counting layers of undifferentiated cells in the cambium zone. The measurements of vessel lumen diameter, cell wall percentage and G-layer area were conducted on the images of a stained cross section (safranin O-fast green). For each tree, images of nine random scanning zones were recorded under a light microscope with a digital camera (Nikon, Tokyo, Japan). The proportion of cell walls and G-layers in the total image area are presented as cell wall percentage and G-layer area, respectively. All image analyses were performed by ImageJ software (Version 1.43u, USA). The significance of IAA and NPA effects compared with controls was assessed in a one-way analysis of variance followed by Tukey’s test.

Effects of IAA and NPA

The cambial cells of trees with IAA treatment contained two more layers than that of the control, while one layer less was observed for trees with NPA treatment (Table 1 and Figure 1a–c). Accordingly, the cambial activity was severely affected by these treatments. The height and radial growth increased by 9 and 13% in seedlings subjected to 10 mg·g⁻¹ IAA compared with the controls, while the xylem width increased by 13% (Figure 2a–c). However, the height growth, the so-called primary growth that mainly proceeds by cell division at the tips of stems, was significantly decreased in seedlings treated with 10 mg·g⁻¹ NPA, which were 41% shorter than the control (Figure 2a). Simultaneously, NPA treatment resulted in the slight reduction of xylem and phloem width, so that the radial growth was reduced, but the differences are not statistically relevant (Figure 2b–d). The higher dosage NPA has resulted in dramatic reduction of shoot growth of Populus tremuloides (Schuetz et al. 2007) and suppressed wood formation in P. deltoides (Meicenheimer and Larson 1985). As stated by Hellgren et al. (2004), NPA application inhibits cambial growth. NPA inhibits not only the macro-growth of the trees (mainly primary growth), but also the development of xylem and phloem. There was TW formation in trees treated with exogenous IAA (Figure 5b) and this observation is consistent with the report of Morey and Cronshaw (1968b). Du et al. (2004) found that NPA inhibits TW formation if the cellular auxin transport is perturbed by NPA.

Figure 1:

Micrographs of cross section stained with phloroglucinol-HCl showing differential cambial zone in stems of erect hybrid poplars cultivated for one month after treatments of control (a), exogenous IAA (b) and NPA (c), respectively.

Xy, Xylem; Ca, Cambium; Ph, phloem. Scale bar=25 μm.

Figure 2:

Stem growth of erect hybrid poplars cultivated for one month after different treatments.

Height (a) and radial (b) growth of hybrid poplars were the height and diameter differences between the end and beginning of treatment. Xylem (c) and phloem (d) width were obtained at the end of treatment. CK, SI and SN represent plant samples from control, IAA and NPA treatments, respectively. Data are mean values±SD (n=9). **Indicate significant differences at 0.01 level by comparing with control.

Effects on PAT and cellulose biosynthesis

The exogenous IAA increased significantly the level of transcript abundance of the three selected genes involved in PAT (Figure 3a), which has an effect on the signal transduction of auxin. Similar finding are described in the literature. The expression of one or more PIN and AUX/LAX genes are up-regulated following exogenous application of IAA and/or gibberellins (Schrader et al. 2003). Carraro et al. (2012) reported that the expression of PtaPIN1 is strongly up-regulated in the developing internodes of P. tremula×alba by exogenous IAA. In contrast, the mRNA expression of PIN1 gene was significantly decreased in trees after NPA treatment, while a moderate increase in the expression of ABCB1 and AUX2 genes was observed (Figure 3a). According to Petrášek et al. (2006), PIN proteins perform a rate-limiting function in cellular auxin efflux. For example, PIN1 overexpression resulted in the stimulation of auxin efflux in Arabidopsis, and NPA treatment suppressed this effect leading to an [³H]NAA accumulation inside the cells. Additionally, the sensitivity to exogenous IAA and NPA varies from these genes associated with auxin perception, transduction, and transport (Petrášek et al. 2006; Carraro et al. 2012), which brings about different expression patterns. Probably, the same effect occurs in hybrid poplar stems.

Figure 3:

Effects of exogenous IAA and NPA on the mRNA level of some PAT (a) and cellulose biosynthesis genes (b) in hybrid poplars.

After 1 month treatment, mRNA levels in stem were determined by qRT-PCR and normalized to ACT2. Values were expressed as mean (% control)±SD (n=3 biological replicates). *^,**Indicate significant differences at 0.05 and 0.01 level by comparing with control, respectively.

The expression of SUS1 and SUS2 were all down-regulated (Figure 3b) in trees treated with IAA and NPA compared to control plants. Gerber et al. (2014) also observed a strong reduction of SuSy activity followed by the reduced transcript abundance of SUS1 and SUS2 in developing wood of transgenic hybrid aspen. These have only marginal effects on tree growth and the composition of wood. This is because the SuSy isoforms possess some synergetic effects (Baroja-Fernández et al. 2012). In Populus, both UGPase1 and UGPase2 appeared to be involved in cellulose biosynthesis during TW formation (Joshi et al. 2004). We observed that the mRNA expression of UGP1 and UGP2 were significantly up-regulated with exogenous IAA treatment, increasing by 350 and 420% in comparison with control, respectively. NPA treatment caused 160 and 150% higher increments at transcript level than that of control (Figure 3b). Although the 96% reduction of UGPase activity by antisense suppression show minor change in plant biomass in potato (Zrenner et al. 1993), the overexpression of UGPs generated by transgenetic method has proved to be effective in increasing the overall cellulose yield in tobacco (Coleman et al. 2006), and cellulose contents in hybrid poplar (Coleman et al. 2007), Arabidopsis (Wang et al. 2011), and jute (Zhang et al. 2013). Accordingly, the UGPs play an important role in the allocation of carbon, especially in cellulose biosynthesis. A previous study focused on native UGP expression patterns in poplar and provided evidence that the UGPs are up-regulated during late cell expansion and secondary cell wall formation (Hertzberg et al. 2012). This supports the hypothesis that UGPases contribute to substrate transport for cellulose synthesis. Similarly, our results show that the UGP1 and UGP2 are critical not only for cellulose biosynthesis but also for the formation of TW in erect hybrid poplars induced by exogenous IAA. The roles of SuSy and UGPase are certainly combined with the cellulose synthase complex in the procedure of cellulose biosynthesis, whether or not their transcript abundances and activities change coordinately. In the present study, seven genes specific to or highly expressed in xylem were selected among the 18 CesA genes identified in P. trichocarpa by Suzuki et al. (2006). The CesA5 and CesA13 involved in cellulose synthesis in the primary cell wall were up-regulated by IAA treatment, whereas the CesA4, CesA7, CesA8, CesA17, and CesA18 genes considered to be related to cellulose production in the secondary cell wall were down-regulated to varying degrees (Figure 3b). It was observed that the transcript abundances of all selected CesAs decreased by exogenous NPA treatment, but the percentage cellulose content was not changed in comparison to control (Figures 3b, 4a). This might be due to the redundant xylem-specific expression of paired PtCesA7/17 and PtCesA8/18 together with PtCesA4, which results in the massive production of cellulose in xylem secondary cell walls for wood formation (Suzuki et al. 2006), albeit concurrent reduction occurs at the transcriptional level. As diverged expression patterns of CesAs have been observed, it is possible that the CesA5 and CesA13 are highly expressed in TW of hybrid poplar induced by exogenous IAA, which may contribute to the formation of the primary cell wall. All these genes involved in cellulose biosynthesis show different expression patterns for the stems treated with exogenous IAA and NPA. Nevertheless, it can be concluded that UGPases as well as the corresponding genes are essential for substrate transport for cellulose biosynthesis. On the other hand, SuSy affects more the total carbon allocation into wood cell walls (Gerber et al. 2014). As expected, the expression of all selected CesA genes is inhibited by NPA in the erect hybrid poplar, which detracts the plant’s primary growth. Our findings support the theory that auxin functions as a morphogen, which conveys positional information via genes related to PAT during the xylem development (Uggla et al. 1996, 1998).

Figure 4:

Cellulose content (a), crystallinity (b) and MFA (c) in wood of erect hybrid poplars cultivated for one month after various treatments, respectively.

**Indicate significant differences at 0.01 level by comparing with control.

Changes of cellulose content, crystallinity and MFA in hybrid poplar

Cellulose contents remained unchanged in trees treated with NPA, but it was markedly elevated by 32% in IAA treated trees compared with that of control trees (Figure 4a). The major anatomical characteristic of TW in hybrid poplar is the occurrence of xylem fibers with a thick G-layer at the inner side of the secondary wall (Chang et al. 2015). The highly crystalline MF of G-layer is oriented nearly parallel to the longitudinal axis (Jourez et al. 2001; Hillis et al. 2005). As a rule, TW contains more cellulose and less lignin than normal wood because of the high cellulose content of the G-fibers. Cellulose crystallinity, which appeared to be affected by exogenous IAA, was 8% higher in IAA treated woods (Figure 4b). Harris et al. (2012) have reported that cellulose MF crystallinity could be reduced by mutating C-terminal transmembrane region residues of cellulose synthase within the primary cell wall. Inversely, the increased expression of CesA5 and CesA13 involved in primary cell wall might modify the MF structure through the increment of crystallinity. The significantly lower MFA (4%) was observed in IAA treated trees. NPA treatment did not affect the MFA (Figure 4c). All these changes are related to the cellulose-rich G-layer.

Modifications of wood density and anatomy

Only a slight increase of wood density was found in IAA treated trees (Table 1). This might be explained by a looser structure of fiber cell walls as a result of better growth conditions (Gerber et al. 2014). Vessel frequency was dramatically reduced by the treatment of IAA, and also vessel lumen diameter was decreased slightly. Nevertheless, the cell wall, which contained 16.6% G-layer area, was increased by 10% relative to the control samples (Table 1). The appearance of G-layer marked the formation of TW in plants and the decrease of vessel frequency was one of the typical TW traits (Jourez et al. 2001). Based on these findings it is obvious that IAA induced the formation of TW (Figure 5b–c). The NPA treatment had no effects on wood density or anatomy compared (Table 1) as NPA inhibits TW formation (Figure 5d).

Figure 5:

Micrographs of stem cross section stained with safranin O-fast green in erect hybrid poplars cultivated for1 month after treatments of control (a), exogenous IAA (b and c) and NPA (d), respectively.

Pink areas showed normal fibers, light green areas in developing xylem (Dx) represented newly formed fibers, blue-green areas in mature xylem (Mx) displayed typical fibers with G-layers encountered in tension wood. R, Ray; V, vessel; Nf, normal fiber; Gf, gelatinous fiber. Scale bars=150 μm apply to a, b and d; scale bar in c=10 μm.