Introduction

Arsenic (As) is a non-essential toxic metalloid having ubiquitous natural occurrence. It has gained attention due to its widespread contamination in many areas of the world caused by geological activities and exasperated by human intervention. Neumann et al. [1] explained why irrigation of rice fields in Bangladesh with As-contaminated groundwater adds thousands of tons of as each year with the observation that very little As returns to the aquifer or mobilizes from subsoils. Such a load of As has exposed huge population to potential As poisoning due to consumption of contaminated drinking water and food. In combination, these factors can account for around 99 % of the total human As ingestion [2, 3]. Rice is the major entry route for As because it is particularly efficient in assimilating As [4] and is consumed worldwide in some way or other [5, 6].

Aquaporins are water channel proteins belonging to the major intrinsic protein (MIP) superfamily of membrane proteins. Plant aquaporins are divided into four classes called PIPs (plasma membrane intrinsic proteins), TIPs (tonoplast intrinsic proteins), SIPs (small basic intrinsic proteins) and NIPs (nodulin 26-like intrinsic proteins) [2]. Recent search for AsIII transporter in plants identified aquaporins of the NIP subfamily, which can transport AsIII and other elements like Si, Sb and B as neutral molecules. An involvement of NIP2;1 (Lsi1) in rice [7] and NIP1;1, NIP5;1, NIP6;1, and NIP7;1 in Arabidopsis is demonstrated in AsIII uptake and transport [8–10]. Lsi1 has also been found to mediate the uptake of undissociated methylated As in rice roots [11]. Garey et al. [12] analyzed retranslocation of As from flag leaves to grains of rice by feeding flag leaves with AsIII and other chemical forms of As. Arsenite did not display retranslocation and germanic acid, an analogue of silicic acid, did not affect grain As in AsIII-treated panicles. In another study, Mathews et al. [13] analyzed the effect of glycerol and antimonite (SbIII), AsIII analogs, and silver nitrate, an aquaporin inhibitor, on As uptake in Pteris vittata plants. They found no effect on As accumulation in presence of glycerol and SbIII but a decline upon supply of silver nitrate in AsIII-fed plants. The two studies implied the presence of an aquaporin transporter different from glycerol and SbIII transporters. Very recently, members of the rice PIP subfamily were found to be involved in As tolerance and transport. qRT-PCR analysis of PIPs in rice root and shoot tissues revealed a significant down-regulation of transcripts encoding OsPIP1;2, OsPIP1;3, OsPIP2;4, OsPIP2;6, and OsPIP2;7 in response to AsIII treatment. Heterologous expression of OsPIP2;4, OsPIP2;6, and OsPIP2;7 in Xenopus laevis and Arabidopsis suggested that these PIPs participate in bidirectional AsIII permeability in plants [14].

Although plants have well-devised mechanisms to combat the As entry [15], they do suffer from toxicity when accumulation reaches beyond an optimal limit of a plant and show disturbance to whole metabolism [16, 17]. As is not a redox-active metalloid but can cause an increase in the level of reactive oxygen species (ROS) [18]. With respect to toxicity, early seedling growth may play a determining role in tolerance at later stages of plant life. And, balancing the movement of water at seedling stage is a crucial requirement for growth. Plants need to adjust their water balance in response to challenging environmental conditions and regulate the expression of aquaporins for the purpose. Some aquaporins, are constitutively expressed while the expression of others is regulated by environmental factors such as salinity [19]. Aquaporins have also been linked to plant mineral nutrition and C and N fixation [20]. It has been shown that roots are capable of monitoring the nutrient content of the solution in the root apoplasm and of initiating responses that anticipate by hours or days any metabolic disturbances caused by nutrient deficiencies. At some point, close to the initiation of these responses, changes in water channel activity may be involved [21].

Brassica juncea has been found to be a potential As accumulator and has been analyzed for biochemical and molecular responses to As exposure [22]. However, there is lack of information regarding the response of aquaporins of PIP class upon exposure to AsIII in Brassica. This study therefore analyzed the expression of PIPs and water status in response to AsIII treatment in Brassica juncea seedlings.

Materials and methods

Plant material and treatment

Brassica juncea (L.) Czern. var. TPM-1 was used as the plant material, which is an As tolerant variety [22]. Seeds were sterilized with ethanol (30 %) for 3 min and then rinsed with distilled water to remove any traces of ethanol. The seeds were put on fine cotton bed in small plastic bottles (5.5 cm height, 3.5 cm diameter) having holes at the bottom. These bottles were fitted in a plastic box (6 bottles in each box) having 150 ml of 50 % Hoagland nutrient medium up to a level where cotton bed in the bottles just touches the solution and remains wet. Plastic boxes were incubated in dark for 1 day and then transferred to a Plant Growth Chamber (Sanyo, Japan) having a daily cycle of a 14 h photoperiod with a light intensity of 150 μE m−2s−1, day/night temperature of 25 ± 2 °C and relative humidity of 65–75 % for a week. After 7 days, seedlings were exposed to 100 μM AsIII (prepared using NaAsO2) for 8 days. Seedlings were harvested for analyzing various parameters from 0.5 h to 8 days, roots and shoots were separated, powdered in liquid nitrogen, filled in 1.5 ml pre-weighed microfuge tubes and stored till further use at −80 °C.

Measurement of root and shoot length and weight, total water content

Seedling length was measured using a metric scale. For fresh and dry weight estimation, roots and shoots were separated after harvesting, washed with double distilled water and blotted gently to remove adhering water. After taking fresh weight (FW), root and shoot were dried in a hot air oven at 80 °C for 2 days to constant weight and weighed again to note dry weight (DW). The water content (WC; mL H2O g−1 DW) was estimated using the formula: WC = (FW−DW)/DW.

Estimation of superoxide radicals, hydrogen peroxide, and malondialdehyde

The rate of superoxide radicals (O2•−) production was spectrophotometrically measured following the method of [23]. For H2O2 determination, 0.5 mL of 0.5 % (w/v) trichloroacetic acid (TCA) extracted sample was mixed with 0.5 mL 100 mM potassium phosphate buffer (pH 7.0) and 1 mL of freshly prepared 1 M potassium iodide. Reaction was allowed to develop for 1 h in dark and absorbance was measured at 390 nm [24]. Lipid peroxidation was determined by the estimation of the malondialdehyde (MDA; ε of 155 mM−1 cm−1) content following [25].

Determination of root oxidizability (RO)

RO is a measure of root’s capacity to oxidize. It was determined by red tetrazolium (TTC) reduction assay resulting in red-coloured triphenyl formazan formation [26] as given in Singh et al. [27]. Root tissue (100 mg) was treated with 5 ml of 0.4 % TTC (w/v) and 5 mL of 66.7 mM phosphate buffer (pH 7.0). Reaction mixture was incubated for 3 h at 40 °C and then 2 mL of 2 N H2SO4 was added to it. Roots were ground in 10 ml of ethyl acetate to extract red triphenyl formazan and absorbance of the extract read at 485 nm. It was expressed as A485 g−1 h−1.

Histochemical detection of hydrogen peroxide and cell death

In vivo H2O2 accumulation was analyzed using 3,3′-diaminobenzidine (DAB) according to Schraudner et al. [28] as per the procedures given in Wohlgemuth et al. [29]. The stained leaves were mounted on glass slides and scanned using a HP Scanjet G4010 photo scanner. Plant plasma membrane integrity was detected by incubating roots in triplicate in 20 mL of Evan’s Blue solution (0.025 %, w/v in 100 μM CaCl2; pH 5.6) [27]. For obtaining roots having 100 % cell death, detached leaves were boiled for 5 min and then stained. The stained leaves were washed 3 to 4 times with DW, mounted on glass slides and scanned using HP Scanjet G4010 photo scanner.

Transcript expression profiling

All the primers used for SyBr green real-time RT-PCR were obtained from the Arabidopsis thaliana RT-PCR primer pair database [30]. The details of the primers used are mentioned in Supplementary Table 1. Specificity of all the primers was confirmed by sequence analysis of RT-PCR amplicons derived from Brassica juncea as detailed earlier [19]. The DNA free total RNA was isolated from root samples (100 mg) and then quantitative real-time PCR was performed as described previously [22] using a Corbett rotor gene 3000 (Corbett Life Science; www. corbettlifescience.com). The PCR cycling conditions comprised of 94 °C for 5 min and 40 cycles each comprising of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s and final extension at 72 °C for 10 min. For each sample, reactions were set up in triplicate to ensure the reproducibility of the results. At the end of each PCR run, a melting curve was generated and analyzed with the dissociation curve software built into the Corbett rotor gene 3000. A relative expression ratio plot was generated using the software REST-MCS [31].

Results

Arsenite exposure to plants hampered seedling growth and beyond the point of stress imposition, there was very little increase in root and shoot length. Beyond 16 h, the difference in seedling length of control and AsIII-exposed became significant. At 8 days, root and shoot lengths were 39 and 31 %, respectively lower than control (Fig. 1a, b). However, the ratio of shoot to root length did not differ significantly between control and AsIII-exposed seedlings (Fig. 1c). Total water content of both root (Fig. 2a) and shoot (Fig. 2b) of AsIII-exposed seedlings was found to be significantly lower than control beyond 2 h with the decline at 8 days being 46 and 25 %, respectively.

Fig. 1
figure 1

Growth analysis of Brassica juncea exposed to 100 μM arsenite for 8 d. Root (A) and shoot (B) lengths (cm) and shoot length/root length ratio of seedlings were analyzed. All values are means of triplicates ± SD

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Fig. 2
figure 2

Effect of arsenite treatment on water content of roots (A) and shoots (B) of Brassica juncea. All values are means of triplicates ± SD

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Biochemical assays and expression analysis of aquaporins were performed only in the roots of control and AsIII-exposed seedlings. The rate of superoxide radical production in seedlings exposed to AsIII was found to be higher than control at 0.5 h; then it came at par with control until 16 h and again increased from 48 h onwards (Fig. 3a). The maximum increase of 38 % in superoxide radical production was noticed at 5 days. The level of root oxidizability also increased in AsIII-exposed plants beyond 6 h and was 42 % higher than control at 8 days (Fig. 3b). The level of MDA increased in AsIII-exposed plants in comparison to control from the very start of 0.5 h to 8 days with the maximum increase being 49 % at 8 days (Fig. 3c). The level of H2O2 and cell death was found to be significantly higher than control in AsIII-exposed seedlings beyond 16 h and 6 h time point, respectively (Fig. 4a, b). The maximum increase in H2O2 and cell death was 47 and 121 % at 8 days. Similar to superoxide radical production and MDA, H2O2 also showed increase upon AsIII exposure at the early time point of 0.5 h. Histochemical staining was also performed to visualize H2O2 and cell death in vivo and representative pictures of 5 and 8 days show significant difference in their levels between control and AsIII-exposed seedling roots (Fig. 4c–f).

Fig. 3
figure 3

Effect of arsenite treatment on superoxide radical production (A), root oxidizability (B) and malondialdehyde level (C) in roots of Brassica juncea. All values are means of triplicates ± SD

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Fig. 4
figure 4

Effect of arsenite treatment on the level of hydrogen peroxide (A) and  % cell death (B) in roots of Brassica juncea. In vivo visualization of hydrogen peroxide (C & D) and cell death (E & F) was done using roots exposed to control and arsenite for 5 and 8 d. Line graphs represent the mean of triplicates ± SD

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The expression analysis of PIPs revealed that most of the PIP genes were down-regulated in response to AsIII exposure from 0.5 h to 8 days duration (Figs. 5, 6). At 0.5 h, PIP1;1, PIP1;3, PIP1;5, PIP2;2, PIP2;3 and PIP2;7 showed slight change (less than 0.5-fold), PIP1;2 and PIP2;1, were down-regulated (0.74- and 0.65-fold, respectively), while others showed up-regulation (PIP1;4-1.62, PIP2;4-1.37, PIP2;5-2.69, PIP2;6-0.91 and PIP2;8-1.99). From 2 to 16 h, all PIPs were slightly or significantly down-regulated except PIP2;6 at 6 h (1.61-fold up-regulated) and PIP2;5 at 16 h (0.50-fold up-regulated). At 48 h, PIPs like PIP1;3, PIP1;4, PIP1;5, PIP2;3, PIP2;5, PIP2;8 showed an up-regulation, while others remained down-regulated. At longer time points of 5 and 8 days again, all PIPs became down-regulated except PIP2;3 at 8 days (1.51-fold up-regulated).

Fig. 5
figure 5

Quantitative real-time RT-PCR of various PIPs in roots of Brassica juncea exposed to 100 μM arsenite for 0.5 h to 8 d. Expression change in genes is given in terms of Log2-fold. Y-axis represents the level of all genes in control roots. All values are means of triplicates ± SD

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Fig. 6
figure 6

Log2-fold expression level of various PIPs in comparison to control values of Brassica juncea exposed to 100 μM arsenite for 0.5 h to 8 d. All values are means of triplicates ± SD. The green and red color designates the up- and down-regulated genes, respectively, and the color intensity is directly proportional to the fold difference

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Discussion

Aquaporins of PIP class are known to play a major role in transmembrane water transport in plants [32]. The movement of water through PIPs occurs in a bidirectional manner that depends on the concentration gradient. In our earlier study, the changes in the expression levels of PIPs were monitored in presence of NaCl with/without thiourea (ROS scavenger and redox modulator) in Brassica juncea roots. The results demonstrated an early down-regulation of most of the PIPs as an efficient tolerance strategy to conserve water under salt stress [19]. In the present study too, PIPs were mostly down-regulated during the whole treatment duration upon AsIII exposure. Such a down-regulation of aquaporins has also been observed in a recent transcriptome analysis of transporters in rice under As stress [33]. The down-regulation of PIPs may affect uptake and/or efflux of AsIII as proposed by Mosa et al. [14]. If down-regulation of PIPs leads to a decrease in the rate of AsIII uptake, it may or may not lead to a decrease in total As accumulation. However, this would give time to plants to detoxify As using glutathione and peptides derived from glutathione, phytochelatins. Such a mechanism of lower uptake rate is known to exist for AsV tolerance in Holcus lanatus and other AsV-hypertolerance plants [34]. Here, aquaporins are not constitutively suppressed but are down-regulated at the onset of stress suggesting towards adaptive mechanism of tolerance. However, another possibility with aquaporin down-regulation is that it would decrease the efflux of accumulated AsIII and this would affect plants’ ability to regulate As levels. PIPs seem to play critical roles in regulating the As levels and hence plant’s tolerance to As stress. There exact function needs to be investigated in further studies.

The down-regulation of PIPs was associated with the compromised water balance in plants as evidenced by decrease in total water content. Water balance is extremely crucial for plants to live and sustain growth. Disturbance to water balance may affect every aspect of plant physiology. Plant water homeostasis and regulation of aquaporins are suggested to be closely linked to the production of ROS [35]. Therefore, we assessed the level of ROS, RO, MDA and cell death in roots in response to AsIII exposure. We observed a significant increase in the rate of superoxide radical production, the level of H2O2, RO and MDA, which probably lead to an increased cell death. The present observation of oxidative stress and cell death could be attributed to As toxicity [18] because cell death under As stress is demonstrated to extend from an increase in ROS to damage to lipids, proteins and DNA.

High oxidizing activity of roots has been shown to protect roots by avoiding uptake of toxic materials from soil [36]. Al tolerance/resistance of some rye cultivars has been related to their higher RO [37]. Enhanced RO relates to greater ROS (superoxide radical) generation as TTC salt used to measure RO traps electrons from mitochondrial electron respiratory chain (ETC) [38]. Thus, although high RO is related to higher oxidative stress as observed but this too may be a tolerance response. It is important to note that ROS signals are important in regulating plants’ normal growth as well as stress tolerance [39].

In conclusion, during AsIII stress imposition for 8 days, PIPs were mostly down-regulated starting at an early time point of 0.5 h. We hypothesize that this occurred to regulate the rate of AsIII uptake/efflux so as to tackle the load of As more effectively. Plants were able to combat the stress and live though at the cost of hampered growth due to disturbance to water status and subsequently induced oxidative stress.