Biochemical changes in barberries during adventitious root formation : the role of indole-3-butyric acid and hydrogen peroxide

Peroxidase, polyphenol oxidase (PPO), phenolic compounds and total sugars (TS) were investigated during root formation in cuttings of Berberis vulgaris var. asperma (BVA) and Berberis thunbergii var. atropurpurea (BTA) treated with indole-3-butyric acid (IBA) and IBA + H2O2. Rooting was observed on BTA cuttings but not on BVA cuttings. The BTA cuttings treated with IBA and IBA + H2O2 showed higher rooting percentages, number of roots, and root length over the control. Those treated with IBA + H2O2 recorded the lowest peroxidase activity after planting. BTA cuttings treated with IBA + H2O2 showed the highest peroxidase activity at 50 d after planting; BVA cuttings under different treatments showed no significant difference for peroxidase activity at planting time or up to 80 d after planting. PPO activity for the BTA cuttings in the control treatment was lower than for other treatments during root formation. The cuttings in the IBA and IBA+H2O2 treatments showed increased PPO activity from 0 to 50 d after planting and a slight decrease in PPO activity from 60 to 80 d after planting. PPO activity for the BVA cuttings was significantly lower than for BTA during root formation. The BTA cuttings treated with IBA and IBA + H2O2 showed the highest phenolic compound content during root formation. The BVA cuttings displayed higher TS than BTA during the initial stage of root formation. A comparison of the anatomical structure of easy-to-root and difficult-to-root cuttings indicated that physical inhibitors did not affect the rooting capacity of BVA. Additional key words: Berberis vulgaris var. asperma; Berberis thunbergii var. atropurpurea; peroxidase; polyphenol oxidase; phenolic compounds. * Corresponding autor: tehranifar@um.ac.ir Received: 23-12-12. Accepted: 14-03-14. Abbreviations used: BTA (Berberis thunbergii var. atropurpurea); BVA (Berberis vulgaris var. asperma); IBA (indole-3-butyric acid); PPO (polyphenol oxidase); TS (total sugars). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2014 12(2): 477-485 http://dx.doi.org/10.5424/sjar/2014122-3889 ISSN: 1695-971-X eISSN: 2171-9292 RESEARCH ARTICLE OPEN ACCESS 478 A. Tehranifar et al. / Span J Agric Res (2014) 12(2): 477-485 ha under cultivation and an annual production of 9414500 t (Balandari & Kafi, 2001; Tehranifar, 2003). Adventitious rooting consists of three successive, but interdependent, physiological phases (induction, initiation and expression) with different requirements (Hartmann et al., 1989). The induction phase comprises molecular and biochemical events with no visible change. The initiation phase is characterized by cell division and root primordia organization. The expression phase consists of the intra-stem growth of rootprimordia and the emergence of roots (Ford et al., 2001; Sebastiani et al., 2002). The ability to form adventitious roots is affected by genotype, stock plants, types of cuttings, collection date, and endogenous factors related to cutting (Kibber et al., 2004; Tworkoski & Takeda, 2007). Numerous studies on adventitious root formation have shown that changes in phenolic compounds, sugar and protein content significantly affect root formation (Kaur et al., 2002; Sivaci & Yalcim, 2006; Kevresan et al., 2007; Satish et al., 2008). Carbohydrates are the direct products of photosynthesis; they serve as an energy source and provide the carbon necessary for production of new tissue (Sivaci & Yalcim, 2006). Previous studies have noted changes in enzyme activity, such as polyphenol oxidase (PPO) and peroxidase, during rooting (Gaspar et al., 1992; Gunes, 2000; Ludwig-Muller, 2003; Yilmaz et al., 2003). PPO is involved in the oxidation of phenol and contributes energy for cell division and cell differentiation in organogenesis of explants in tissue cultures (Satisha et al., 2008). Peroxidase is a marker of the inductive and initiative phases of root formation in plants (Yilmaz et al., 2003; Kevresan et al., 2007). Currently, indole-3-butyric acid (IBA) is the most widely used auxin to stimulate rooting in cuttings. It has a strong ability to promote root initiation, low toxicity and good stability in comparison to naphthalene acetic acid and indole-3-acetic acid (Strzelecka, 2007). It has been reported that hydrogen peroxide (H2O2) also effectively increases the activity of enzymes such as superoxide dismutase and catalase and is also related to morphogenesis. Li et al. (2009a) reported that exogenous hydrogen peroxide promotes the formation and development of adventitious roots in mung beans (Vigna radiate) and demonstrated that H2O2 may function as a signal molecule in the auxininduced formation of adventitious roots. The present study investigated biochemical and anatomical changes during rooting of Berberis thunbergii var. atropurpurea (easy-to-root) and Berberis vulgaris var. asperma (hard-to-root) barberry species. It also examined the influence of IBA and H2O2 on the formation and development of adventitious roots for these two species. Material and methods


Introduction
More than 40 cultivars of Japanese barberry (Berberis thunbergii DC) are utilized as ornamental shrubs in landscaping because they are hardy, drought tolerant, easy to grow and generally attractive.They vary in leaf colour (green, purple, yellow or variegated) and plant habitat, and are easily propagated from seeds and cuttings (Lubell et al., 2008).Berberis thunbergii var.atropurpurea (BTA) has adapted to the drought conditions of Khorasan province in Iran.It is a deciduous shrub with purple leaves and yellow aggregate blooms (Lubell et al., 2008).Berberis vulgaris var.asperma (BVA) is native to Iran and Khorasan province and is known for its drought and salt tolerance.
BVA is commonly known in Iran as seedless barberry; its fruit is consumed fresh and is used to prepare syrups, jams, beverages, jellies, flavoring, and coloring agents.BVA cuttings are generally difficult to root and they are usually propagated by suckers.The development of seedless barberry orchards is limited because of the low rooting ability of its cuttings (Balandari & Kafi, 2001).The salinity of water and soil make large areas in eastern Iran (latitude 32.5-34.5°N)unsuitable for most agricultural crops.In the last 20 years, BVA has become a major crop in the area, with about 6000 ha under cultivation and an annual production of 941-4500 t (Balandari & Kafi, 2001;Tehranifar, 2003).
Adventitious rooting consists of three successive, but interdependent, physiological phases (induction, initiation and expression) with different requirements (Hartmann et al., 1989).The induction phase comprises molecular and biochemical events with no visible change.The initiation phase is characterized by cell division and root primordia organization.The expression phase consists of the intra-stem growth of rootprimordia and the emergence of roots (Ford et al., 2001;Sebastiani et al., 2002).The ability to form adventitious roots is affected by genotype, stock plants, types of cuttings, collection date, and endogenous factors related to cutting (Kibber et al., 2004;Tworkoski & Takeda, 2007).Numerous studies on adventitious root formation have shown that changes in phenolic compounds, sugar and protein content significantly affect root formation (Kaur et al., 2002;Sivaci & Yalcim, 2006;Kevresan et al., 2007;Satish et al., 2008).Carbohydrates are the direct products of photosynthesis; they serve as an energy source and provide the carbon necessary for production of new tissue (Sivaci & Yalcim, 2006).
Previous studies have noted changes in enzyme activity, such as polyphenol oxidase (PPO) and peroxidase, during rooting (Gaspar et al., 1992;Gunes, 2000;Ludwig-Muller, 2003;Yilmaz et al., 2003).PPO is involved in the oxidation of phenol and contributes energy for cell division and cell differentiation in organogenesis of explants in tissue cultures (Satisha et al., 2008).Peroxidase is a marker of the inductive and initiative phases of root formation in plants (Yilmaz et al., 2003;Kevresan et al., 2007).
Currently, indole-3-butyric acid (IBA) is the most widely used auxin to stimulate rooting in cuttings.It has a strong ability to promote root initiation, low toxicity and good stability in comparison to naphthalene acetic acid and indole-3-acetic acid (Strzelecka, 2007).It has been reported that hydrogen peroxide (H 2 O 2 ) also effectively increases the activity of enzymes such as superoxide dismutase and catalase and is also related to morphogenesis.Li et al. (2009a) reported that exogenous hydrogen peroxide promotes the formation and development of adventitious roots in mung beans (Vigna radiate) and demonstrated that H 2 O 2 may function as a signal molecule in the auxininduced formation of adventitious roots.
The present study investigated biochemical and anatomical changes during rooting of Berberis thunbergii var.atropurpurea (easy-to-root) and Berberis vulgaris var.asperma (hard-to-root) barberry species.It also examined the influence of IBA and H 2 O 2 on the formation and development of adventitious roots for these two species.

Plants
The barberries species selected were Berberis thunbergii var.atropurpurea (easy-to-root) and Berberis vulgaris var.asperma (diff icult-to-root) cultivars.Hardwood cuttings 6-9 mm in thickness and 180-200 mm in length with four buds were prepared from the basal parts of 1-year-old shoots collected from 8-yearold barberry shrubs.The shrubs were maintained in the experimental orchard of Ferdowsi University in the city of Mashhad.The cuttings were treated with cold water (tap water) for 24 h to leach out rooting inhibitors.
Hardwood cuttings of the two species were treated with IBA and H 2 O 2 solutions by wetting 2.5 cm of their basal ends.The cuttings were planted in a nursery bed with bottom heat in a sand medium without nutrients for 80 d.The following rooting treatments were used: (i) control, dipped in distilled water for 90 s; (ii) dipped in 3 g L -1 IBA for 60 s; and (iii) dipped in 3.5% (w/v) H 2 O 2 30 s + dipped in 3 g L -1 IBA 60 s.

Chemical composition
Fresh stem tissue was prepared for the enzyme assays according to the method proposed by Rout (2006).Protein concentrations were determined as recommended by Bradford (1976) and bovine albumin (BSA) was used as the standard.Peroxidase enzyme was determined spectrophotometrically at 470 nm after 10 min incubation at 30 ± 0.5 °C using 3,3-dimethyl-glutaric acid (3,3-DGA)-NaOH and guaiacol (Beffa et al., 1990).One unit of peroxidase activity corresponds to ΔA 470 of 1.0 for 1 mg of protein in 10 min.
Polyphenol-oxidase enzyme assay was carried out using pyrogallol as the substrate (Kar & Mishra, 1976).The activity was determined as mg -1 protein min -1 .Each value was the mean of f ive replicates.Total phenol was extracted and estimated using Folin-Ciocalteu reagent (Jayaprakasha et al., 2001) and expressed as mg g -1 of fresh weight.Total sugar (TS) was estimated using anthrone agent according to the method described by Irigoyen et al. (1992) and expressed as mg g -1 of fresh weight.

Anatomical studies
Basal sections of cuttings were fixed in formalin/ methanol/acetic acid (1:18:1 v/v/v) for 2 wk.Section were then mounted by hand on slides, stained with saffranin for 30 min and fast green for 3 min and rinsed in distilled water.The samples were photographed using a camera (U-TVO-5XC-2, Olympus Optical Co., Tokyo) attached to an optical microscope (BX41TF, Olympus Optical Co., Tokyo) under the control of a computer.

Statistical analysis
The hardwood cuttings were sampled at 0, 10, 20, 30, 40, 50, 60, 70, and 80 d after planting.Cuttings of each species and treatment were used for biochemical analysis; 30 cuttings were used for root scoring.The treatments were arranged in a completely randomized design with four replications of 30 cuttings each.Analysis of the data was conducted using SAS software (version 9.1) and the mean values and standard error (SE) were calculated for all tests.

Rooting percentage, number of roots and root length
The rooting percentage differed between species and was observed only on cuttings of BTA (Fig. 1).The highest rooting percentage (73.33%),number of roots and root lengths were observed on cuttings treated with IBA + H 2 O 2 .The control cuttings of B. thumbergii var.atropurpurea showed the lowest rooting percentage (43.33%),number of roots and root length (Table 1).

BTA
The cuttings treated with IBA + H 2 O 2 showed the lowest peroxidase activity after planting.The peroxidase activity of the cuttings decreased slightly up to 40 d after planting and the lowest peroxidase activity was observed in cuttings treated with IBA + H 2 O 2 .A sharp increase was observed in peroxidase activity in each treatment at 40 d after planting (Fig. 2A).The cuttings treated with IBA + H 2 O 2 showed the highest peroxidase activity at 50 d after planting.The highest peroxidase activity in the IBA and control treatments were observed at 60 d after planting.Peroxidase activity was highest in the IBA treatment.Peroxidase activity decreased rapidly for all treatments from 60 to 80 d after planting.

BVA
There were no signif icant differences between treatments for peroxidase activity at planting time.All treatments showed constant levels of peroxidase activity up to 40 d after planting and increasing activity from 40 to 80 d after planting (Fig. 2B).

BTA
Polyphenol oxidase activity of the control cuttings was lower than for the other treatments during root formation.It gradually increased for all treatments from 0 to 60 d after planting and then decreased slightly from 60 to 80 d after planting.The IBA and IBA + H 2 O 2 treatments showed an increase in PPO activity from 0 to 50 d after planting; the highest PPO activity was observed for the IBA + H 2 O 2 treatment.In both treatments, PPO activity decreased slightly by 80 d after planting (Fig. 3A).

BVA
Polyphenol oxidase activity of the BVA cuttings was signif icantly lower than for BTA during root formation.The cuttings treated with IBA, and IBA + H 2 O 2 and the control showed no signif icant differences in PPO activity from 0 to 80 d after planting.Polyphenol oxidase activity of some  treatments increased gradually, but not significantly, during root formation (Fig. 3B).

BTA
The IBA and IBA + H 2 O 2 treatments showed higher phenolic compound concentrations over the control during root formation.Phenolic compounds of all treatments gradually increased up to 40 d after planting.It was observed that phenolic compound concentrations in the IBA and IBA + H 2 O 2 treatments increased rapidly from 40 to 50 d after planting and then decreased slightly, while for the control treatment, it increased rapidly from 40 to 60 d after planting and then gradually decreased from 60 to 80 d after planting (Fig. 4A).

BVA
The phenolic compound content in BVA was significantly lower than for BTA during the initial stage of root formation.The cuttings treated with IBA and IBA + H 2 O 2 and the control showed no signif icant differences in phenolic compounds between 0 to 80 d after planting.The phenolic compounds increased gradually, but not significantly, from 0 to 50 d after planting and then decreased slightly from 60 to 80 d after planting (Fig. 4B).

BTA
The cuttings treated with IBA and IBA + H 2 O 2 and the control showed no significant differences in TS during the initial stage of root formation.IBA and IBA + H 2 O 2 treatments showed a slight decrease in TS from 0 to 40 d after planting and a strong decrease from 40 to 50 d after planting; from 60 to 80 d, TS increased.The control showed a gradual decrease in TS from 0 to 50 d after planting.The rate of TS depletion increased from 50 to 60 d after planting and continued to increase gradually from 60 to 80 d after planting (Fig. 5A).

BVA
The Berberis vulgaris var.asperma showed higher TS than the Berberis thunbergii var.atropurpurea during the initial stage of root formation.The cuttings treated with IBA and IBA + H 2 O 2 and the control showed no significant differences in TS from 0 to 60 d after planting; TS decreased gradually from 60 to 80 d after planting (Fig. 5B).

Anatomical studies
Transverse sections of the BVA and BTA cuttings were compared before transfer to the rooting medium (Fig. 6).The cortex tissue of BVA contained more cell layers than did that of BTA.The dye density of the cell wall indicated lignif ication of the cell wall.This indicates that the cell wall of the cortex layer of BVA was thicker than that of BTA.The sclerenchymatic tissue of BVA consisted of more cell layers than that of BTA sclerenchyma, while the cell wall of the sclerenchymatic tissue of BVA was thinner than that of BTA.Other BVA and BTA tissues showed no signif icant differences.

Discussion
The ability to form adventitious roots has been associated with the physiologic and ontogenetic age of plant material, genotype and the characteristics of the cuttings, such as growth conditions of the stock plant (Kibber et al., 2004).There was a significant difference in rooting ability between BVA and BTA (Table 1, Fig. 1).The rooting ability of BTA was strongly affected by IBA and IBA + H 2 O 2 , while the cuttings of BVA showed no response to the treatments.Previous studies have shown that IBA promotes root formation (Kaur et al., 2002;Ludwig-Muller, 2003;Rout, 2006).Li et al. (2009b) reported that the application of exogenous H 2 O 2 promoted the formation and growth of adventitious roots in mung bean seedlings and that its effects were dose and time dependent.H 2 O 2 treatment of olive cuttings at the beginning of rooting promoted early rooting, increased the root percentage and number of roots, but the application of H 2 O 2 alone and with IBA promoted rooting ability in both easyand difficult-to-root cuttings (Sebastiani & Tognetti, 2004).Plant peroxidases are known to affect auxin metabolism and lignification in the cell wall in the presence of phenol (Rout, 2006).Peroxidase activity increased during cell division and primordium formation.Peroxidase helps form the cofactors necessary for root initiation (Gunes, 2000).Rout (2006) showed that IBA-treated cuttings of Camellia sinensis L. recorded higher peroxidase activity than the control and that increased peroxidase activity at the early stages of rooting was a good marker of later rooting success.Seedlings of mung beans treated with either IBA or H 2 O 2 showed decreased peroxidase activity during the root induction phase and increased activity during the initiation and expression phases.These treatments increased root formation (Li et al., 2009b).
Changes in peroxidase activity in BTA delineate the root formation phases.It appears that the induction phase of root formation occurred from 0 to 40 d after planting and the initiation and expression phases from 40 to 70 d after planting.Cuttings treated with IBA and IBA + H 2 O 2 showed decreased peroxidase activity during the induction phase and increased peroxidase activity during the initiation and expression phases of root formation.Cuttings of BVA showed no obvious changes in peroxidase activity (Fig. 2).
Polyphenol oxidase is a key element of cell division, differentiation and primordium development (Huystee & Cairns, 1982).Rout (2006) reported that PPO activity increased in Camellia sinensis L. cuttings during the induction and initiation phases and then decreased during the expression phase.IBA treatment increased PPO activity over the control during root formation.It has been shown that cuttings of grape rootstock with low rooting ability showed decreased PPO activity during the induction and initiation phases and increased PPO during the expression phase (Satish et al., 2008).Yilaz et al. (2003) reported that there was a relationship between PPO activity and root formation in grape cuttings with different rooting abilities.Difficult-to-root cuttings showed lower PPO activity than easy-to-root cuttings during root formation.
In the present study, PPO activity of BVA was significantly lower than BTA during the root formation period.IBA and IBA + H 2 O 2 treatments increased PPO activity in cuttings of BTA.PPO activity increased from 0 to 50 d after planting (induction and initiation phase) and then decreased from 50 to 80 d after planting (expression phase) (Fig. 3).
A negative correlation has been observed for phenolic compounds and seed germination (White, 1994) and a positive correlation has been observed between phenolics and totipotency (Tomas & Ravindra, 1999).Phenolic compounds have inhibitory or stimulatory effects on plant growth depending upon the species (Ozyuigit, 2008).
Phenolic compounds of BVA were signif icantly lower than for BTA during root formation and showed no response to the stages of root formation (Fig. 4B).For BTA, phenolic compounds increased during the induction and initiation phases and decreased during the expression phase.IBA and IBA + H 2 O 2 treatments increased the phenolic compounds during root formation of BTA (Fig. 4A).Satish et al. (2008) have shown that phenolic compounds play a key role in the rooting of cuttings and that those with higher phenolic compounds in the initial stage of rooting formed roots rapidly.
Although cuttings of Berberis vulgaris var.asperma showed higher TS concentration than those of Berberis thunbergii var.atropurpurea during the initial stage of root formation, no stimulation of root formation was observed.The BTA treatment showed depletion of TS during the expression phase of rooting, which could be related to carbohydrates being a source of energy.Comparison of the anatomical structure of easy-to-root (BVA) and difficult-to-root (BTA) cuttings showed that physical inhibitors did not affect rooting of BVA.This indicates that the low rooting ability of BVA is related to its biochemical properties.

Figure 2 .
Figure 2. Peroxidase activity in the cuttings of Berberis thunbergii var.atropurpurea (A) and Berberis vulgaris var.asperma (B) with different treatments during root formation.One unit of peroxidase activity is equivalent to a ΔA470 of 1.0 for 1 mg of protein in 10 min.Values are means ± SE.

Figure 3 .
Figure 3. Polyphenol oxidase activity in cuttings of Berberis thunbergii var.atropurpurea (A) and Berberis vulgaris var.asperma (B) with different treatments during root formation.The activity was determined in terms of enzyme activity mg -1 protein min -1 .Values are means ± SE.

Figure 4 .
Figure 4. Phenolic compounds in cuttings of Berberis thunbergii var.atropurpurea (A) and Berberis vulgaris var.asperma (B) with different treatments during root formation.The phenol content was expressed as mg g -1 fresh weight.Values are means ± SE.

Figure 5 .
Figure 5.Total sugars in cuttings of Berberis thunbergii var.atropurpurea (A) and Berberis vulgaris var.asperma (B) with different treatments during root formation.Total sugars were expressed as mg g -1 fresh weight.Values are means ± SE.

Table 1 .
Root formation in the cuttings of Berberis thunbergii var.atropurpurea (BTA) and Berberis vulgaris var.asperma (BVA) with different treatments