Abstract
Biochemical changes of CaMsrB2 expressing transgenic rice seed during germination in heavy metal stress condition were studied. Transgenic lines, L-8 (single copy) and L-23 (two copy), along with WT were evaluated under metal stress conditions. All the plants were treated with different metals and their two selected concentration. Final germination rate, changes in amylase activity, total protein, reducing and total sugar was observed in all treated and control samples. Metal stress showed considerable impact on final germination rate in CaMsrB2 expressing transgenic rice seed. Application of lead salt showed 100% germination in L-23 compared to Zn and Cu. However, maximum germination rate was recorded in L-23 seed when it was treated with 4 mM PbCl2 and 0.5 mM CuCl2 compared to WT. Amylase activity and total reducing sugar was increased in transgenic rice seed treated with 2 mM and 4 mM PbCl2 as compared to WT. L-23 showed substantial increase in amylase activity and total reducing sugar compared to L-8 and WT. However, transgenic seeds treated with Zn and Cu showed substantial decreased in amylase activity and total reducing sugar with few exceptions. L-23 performed well regarding amylase activity and total reducing sugars in metal stress condition particularly in Pb as compared to Cu and Zn. CaMsrB2 expressing transgenic seed germination and their carbohydrate metabolism under metal stress condition were discussed. It was evident from the data that PbCl2 showed better germination rate due to enhance amylase activity and carbohydrate mobilization of CaMsrB2 expressing transgenic seed as compared to Cu and Zn.
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Key words: CaMsrB2, Rice, Seed, Amylase activity, Carbohydrate
INTRODUCTION
Abiotic stress like drought, salinity, temperature, chemical toxicity and metal stress is the major constraint in crop productivity. Germination is the most critical stage in crop plant life and often inhibited by several stress factors. Abiotic stress like heavy metal stress causes substantial reduction in germination ability and plant growth. It was observed that heavy metals decrease germination, reduced root and shoot elongation, dry weight, total soluble protein of several plant species (
Katiya-Agarwal et al. 1999;
Siddiqui et al. 2015). Many literatures are available for the effects of heavy metal stress caused by copper, zinc toxicity on plants which not only affects germination but also reduces crop yields, soil biomass, and fertility (
Shety and Ghosh 2013). However, literature regarding the effect of heavy metal such as Cu, Zn and Pb on germination of transgenic plant seeds is rather scarce.
As we reported in the previous work (
Kim et al. 2014;
Siddiqui et al. 2015), transgenic rice expressing the
Capsicum annuum methionine sulfoxide reductase B2 (
CaMsrB2) gene has shown drought tolerance at growth and reproductive stages. However, the effect of heavy metal like Cu, Zn and Pb on
CaMsrB2 expressing transgenic rice during germination has not been studied so far. It was reported that
CaMsrB2 gene expressing rice works well against abiotic and biotic stresses, therefore, it is presumed that
CaMsrB2 expressing transgenic rice seeds may work well in heavy metal stress during germination. For the germination study we selected amylase activity and their subsequent roles in carbohydrate metabolism. It was observed that amylase is a key enzyme of carbohydrate metabolism which is essential for the completion of germination, often regulated by several stresses (
Siddiqui and Khan 2011). Endo-1,4-D-glucan glucohydrolase (amylase) is a class of hydrolases which are universally found in living organisms. They break ortho-glycosidic bonds in starch (amylose) which is a principal storage of polysaccharides present in seeds of various plants along with other related oligo and polysaccharides playing key role in carbohydrate metabolism of developing and germinating seeds (
Muralikrishna and Nirmala 2005). There are few studies on the regulation of carbohydrate in seeds during germination and their early developmental stages under stress conditions (
Muralikrishna and Nirmala 2005;
Mei and Song 2008).
MATERIALS AND METHODS
Plant materials and growth
Seeds of wild-type Ilmi and two CaMsrB2 transgenic rice plants, L-8 and L-23, were provided by the National Center for GM Crops, National Institute of Agricultural Sciences, Rural Development Administration, Korea and were washed with distilled water several times before sowing.
Real-time PCR analysis
Entire RNA was extracted from seed samples of each wild-type (WT) Ilmi, and two
CaMsrB2 transgenic rice plants by the RNeasy Plant Mini Kit and RNase-Free DNase Set (Qiagen), and reverse-transcribed by the amfiRivert Platinum cDNA Synthesis Master Mix (Gendepot). The gene-specific primers used for quantitative real-time PCR for
CaMsrB2 were 5′-AGTTTACACCGGCAAATTCCTA-3′ and 5′-GAAAGCGCAAGGCTTAAAAGTA-3′ and for
OsActin1 were 5′-ACAGGTATTGTGTTGGACTCTGG-3′ and 5′-AGTAACCACGCTCCGTCAGG-3′ (
Siddiqui et al. 2015).
Ten seeds of each wild type, Ilmi, and CaMsrB2 transgenic lines L-8 (single copy) and L-23 (two copy line), were surface sterilized with methyl thiophenate (7 mM) for 10 minutes and washed with distilled water several times before sowing seeds were allowed to germinate in 90 mm diameter glass petri-plates. Filter paper in each petri plate soaked with respective concentration of each metals like CuCl2 (0.5 mM, 1 mM), PbCl2 (2 mM, 4 mM), ZnCl2 (0.007, 0.6 mM). Seeds treated with distilled water only served as control. The whole set up was kept at Hotpack germinator and allowed to germinate at 25–30°C night/day temperature. Final germination rate was examined after seven days. Biochemical changes were measured at 24, 48 and 72 hours during germination.
Extraction procedure and biochemical analysis
Five germinating seeds of each type, i.e. L-8, L-23 and WT were randomly collected from each treatment and control and homogenized separately in chilled 10 mL Tris-HCl buffer pH 6.8, centrifuged at 14,000 rpm for 15 minutes at 4°C. Supernatant was collected, and total protein (
Bradford 1976), total sugar (
Hassid and Abraham 1957), reducing sugar (
Nelson 1944) and amylase activity (
Bernfeld 1955) were determined. Total non-reducing sugars (polysaccharides) were calculated by subtracting the values of total reducing sugar form the total sugar. Change in total protein, total sugar, reducing sugar and amylase activity was measured after 24, 48, and 72-hour time intervals. Amylase kinetics of transgenic rice seed were calculated using substrate and pH maxima data by the method of
Lineweaver and Burk (1934). The substrate saturation kinetic curve was calculated using GraphPad Prism 5.00 software. Each treatment and control was replicated four times.
RESULTS
Two transgenic lines, L-8 (single copy) and L-23 (two copy), along with WT were evaluated under metal stress condition. All the plants were treated with different metals and their two selected concentrations. For instance, lead chloride (PbCl2) 2 or 4 mM, zinc chloride (ZnCl2) 0.007 or 0.6 mM, copper chloride (CuCl2) 0.5 mM or 1 mM were treated at different time interval such as 24, 48 and 72 hours, respectively. Final germination rate, changes in amylase activity, total protein, reducing and total sugar were observed in all treated and control samples.
Metal stress showed considerable impact on final percent germination in
CaMsrB2 expressing transgenic rice (
Fig. 1). Application of lead salt showed 100% germination in L-23 compared to Zn and Cu. However, maximum final percent germination was recorded in L-23 seed when it was treated with 4 mM PbCl
2 and 0.5 mM CuCl
2 compared to WT. It was observed that in L-8 significant reduction in final percent germination were recorded when seeds were treated Cu and Zn salts.
Amylase activity was increased in transgenic rice seeds treated with 2 mM and 4 mM PbCl
2 as compared to WT after 24 hours (
Fig. 2). Later, enzyme activity decreased in both transgenic lines compared to the control. L-23 showed substantial increase in amylase activity compared to L-8 and WT. However, transgenic seeds treated with Zn and Cu showed a substantial decrease except 48 hour 0.6 mM, ZnCl
2 and 24 hour, 1 mM CuCl
2 treated samples. L-23 showed significant increase in unstressed condition at 72 hours. It was observed L-23 performed well regarding amylase activity in metal stress condition particularly in Pb as compared to Cu and Zn.
It was observed that reducing sugar increased in L-8, L-23 including WT in the late hours of the treatment as compared to early hours (
Fig. 3). Transgenic seed L-8 treated with Pb showed substantial increase in 48 and 72 hours of the imbibition. However, L-8 and L-23 showed almost similar pattern of increased as compared to WT. It was observed that an increase in reducing sugar took a little longer in higher concentrations of Pb as compared to lower concentrations (2 mM).
CaMsrB2 expressing transgenic seeds showed greater decrease in reducing when seeds were treated with ZnCl
2. However, a considerable increase in reducing was found when transgenic seeds were treated with Zn salt at 72 hours of the imbibition. L-8 showed a greater increase compared to L-23 and WT.
CaMsrB2 expressing transgenic seeds treated with CuCl
2 showed similar patterns in total reducing sugar content except 48 hours of the imbibition where L-8 showed greater increased when it was treated with 1 mM CuCl
2.
Total sugar in
CaMsrB2 expressing transgenic rice seeds increased in 24 hours of the treatment compared to 72 hours (
Fig. 4) later it was decreased with time of imbibition. Maximum reduction was found when transgenic seeds were treated with Zn after 72 hours.
Total protein content was higher in
CaMsrB2 expressing transgenic rice seeds compared to WT (
Fig. 5). Maximum increase was found when L-8 and L23 were treated with Zn and Cu salt. Substantial increases in total protein were observed at 72 hours of the imbibition when transgenic seed treated with ZnCl
2 at 0.007 or 0.5 mM concentration.
DISCUSSION
Two transgenic lines, L-8 and L-23 along with WT were evaluated under metal stress conditions. It was observed that metal stress showed considerable impact on final germination rate in
CaMsrB2 expressing transgenic rice. Maximum germination rate was recorded in L-23 seed when it was treated with 4 mM PbCl
2 and 0.5 mM CuCl
2 compared to WT.
CaMsrB2 expressing L-8 line seems to be more sensitive to metal stress and showed significant reduction in final percent germination when seeds were treated with Cu and Zn salts. It was observed that transgenic lines respond differently to different metals and concentrations. In plant life, germination is a critical stage, and it is often regulated by several ecological biotic and abiotic factors. Earlier, it was reported that metal stress caused significant reduction in final percent germination which varied from species to species, depending on the type of metal and concentration (
Wierzbicka and Obidzinska 1998;
Moise et al. 2005;
Vogel-Mikuš et al. 2008;
Klatte et al. 2009;
Siddiqui and Khan 2011). In the present study, L-23 showed greater germination percent compared to WT and L-8. Lead salt produced good effects compared to Zn and Cu.
Amylase activity was increased in CaMsrB2 expressing transgenic rice seed when it was treated with 2 mM and 4 mM PbCl2 as compared to WT after 24 hours. However, L-23 showed substantial increase in amylase activity compared to L-8 and WT. Contrary, transgenic seeds treated with Zn and Cu showed substantial decreased with few exceptions. In early hours of imbibition, reducing sugar increased substantially in L-8 and L-23. Transgenic seed showed substantial increase in Pb treated sample as compared to Zn and Cu. Compared to WT, CaMsrB2 expressing transgenic seeds showed greater increase in metal stress condition. Total sugar in CaMsrB2 expressing transgenic rice seeds increased in 24 hours of the treatment compared to 72 hours. Maximum reduction was found when transgenic seeds treated with Zn after 72 hours.
Total protein content was higher in CaMsrB2 expressing transgenic rice seeds compared to WT. Maximum increase were found when L-8 and L23 were treated with Zn and Cu salt. Substantial increases in total protein were observed at 72 hours of imbibition when transgenic seed treated with ZnCl2 at 0.007, and 0.5 mM concentrations.
In germination, plasma membrane protein played a significant role for controlling negative impact of heavy metal. A common transmembrane transporter protein was found for some heavy metal like Cd, Cu, and Ni (
Clarkson and Luttge 1989) and their uptake was competitively inhibited by K, Ca, and Mg in seeds during germination (
Clarkson and Luttge 1989). It is presumed that
CaMsrB2 expressing transgenic seeds may reduce the uptake of heavy metal compared to WT through selective and effective plasma membrane transporter protein which does not only promote amylase activity under metal stress condition but also improved seed storage carbohydrate mobilization under stress condition. Increase in total protein of
CaMsrB2 expressing transgenic seed under stress condition also strengthens the idea. Further, it was reported that heavy metal like Zn, Ni, Cu, V, Co, W, and Cr are toxic elements for germination with high or low concentration as compared to Pb (
Godbold and Huttermann 1985;
Nies 1999).
It was clearly indicated that PbCl2 showed better effects rather than negative effect on germination due to enhance amylase activity and carbohydrate mobilization of CaMsrB2 expressing transgenic seeds as compared to Cu and Zn.
ACKNOWLEDGEMENTS
This work was supported by a grant from the Next-Generation Bio-Green 21 Program (No. PJ01367503, PJ01365802 & PJ01131902), Rural Development Administration, Republic of Korea.
Fig. 1Effect of heavy metals on final germination percent of CaMsrB2 expressing transgenic rice seed. Symbol stand for A = Control, B1 = PbCl2 2 mM, B2 = PbCl2 4 mM, C1 = CuCl2 0.5 mM, C2 = CuCl2 1 mM, D1 = ZnCl2 0.007 mM, D2 = ZnCl2 0.6 mM.
Fig. 2Amylase activity of CaMsrB2 expressing transgenic rice seed in heavy metal stress environment. Vertical line on the bar graph represents mean S.E (±). WT: Wild type, L-8: transgenic line carrying single-copy T-DNA insertion, L-23: transgenic line carrying two-copy T-DNA insertions, (0): untreated, (1): treated.
Fig. 3Effect of heavy metals on total reducing sugar of CaMsrB2 expressing transgenic rice seed. Vertical line on the bar graph represents mean S.E (±). WT: Wild type, L-8: transgenic line carrying single-copy T-DNA insertion, L-23: transgenic line carrying two-copy T-DNA insertions, (0): untreated, (1): treated.
Fig. 4Effect of heavy metals on total sugar content of CaMsrB2 expressing transgenic rice seed. Vertical line on the bar graph represents mean S.E (±). WT: Wild type, L-8: transgenic line carrying single-copy T-DNA insertion, L-23: transgenic line carrying two-copy T-DNA insertions, (0): untreated, (1): treated.
Fig. 5Effect of heavy metals on total protein content of CaMsrB2 expressing transgenic rice seeds. Vertical line on the bar graph represents mean S.E (±). WT: Wild type, L-8: transgenic line carrying single-copy T-DNA insertion, L-23: transgenic line carrying two-copy T-DNA insertions, (0): untreated, (1): treated.
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