PhysiologyNitrogen metabolism and gas exchange parameters associated with zinc stress in tobacco expressing an ipt gene for cytokinin synthesis
Introduction
Zinc is an important component of a large number of plant enzymes. It is associated with carbohydrate metabolism, protein synthesis, and gene expression and regulation (Broadley et al., 2007). Zinc plays critical roles in the defense system of cells against oxidative stress, and thus represents a protective agent against the oxidation of several vital cell components, such as membrane lipid and chlorophyll (Cakmak, 2000). According to Puzina (2004), Zn application to plants shifted the hormonal balance toward a substantial increase in the cytokinin (CK) content and the CK/ABA ratio, as well as a decrease in the IAA/CK ratio. Depending on the plant species and plant part, Zn proportions in the range of 58–91% of total Zn content may be soluble (Brown et al., 1993, Pavlíková et al., 2001). This soluble Zn part is considered to be the physiologically active fraction. A high Zn concentration plays a negative toxic role in plant metabolism. At the organism level, excess Zn inhibits seed germination, plant growth (Mrozek and Funicelli, 1982) and root development (Lingua et al., 2008), and causes leaf chlorosis (Ebbs and Kochian, 1997, Wang et al., 2009). It is likely that trace element stress induces senescence through enhancement of catabolism of key metabolites such as chlorophyll, protein and RNA (Khudsar et al., 2004). At the cellular level, excess Zn can significantly alter mitotic activity (Rout and Das, 2003), affect membrane integrity and permeability (Stoyanova and Doncheva, 2002), and even kill cells (Chang et al., 2005, Wang et al., 2009). At the cellular level, excess Zn can significantly alter gene expression. Many products of these genes are involved in various biological processes (e.g. lignin biosynthesis), among which are many genes encoding proteins that are associated with defense against oxidative stress (Van de Mortel et al., 2006, Wang et al., 2009). Several studies have demonstrated the effects of Zn stress on the activity of many antioxidative enzymes and low-molecular antioxidants in plants (Wójcik et al., 2006, Tewari et al., 2008). These data suggest there is cross-talk between Zn-induced differentially expressed genes and antioxidant defensive genes, and represents a complex mechanism developed to cope with Zn toxicity (Van de Mortel et al., 2006).
Zn also causes the decline in protein content and the corresponding increase in the activity of hydrolytic enzymes such as protease due to trace element stress, and this strongly suggests the induction of catabolic activities. A decline in the protein level may be a consequence of a decrease in nitrate reductase activity. Solanki and Dhankhar (2011) reported that when trace element toxicity crosses the threshold limit, the protein level decreases, and this might be due to the breakdown of the protein synthesis mechanism at toxic concentration levels of trace elements or due to reduced incorporation of free amino acids into proteins. According to Atici et al. (2005), high Zn concentrations can affect CK metabolism and decrease CK content in plants, while optimum Zn concentrations increased CK content.
CK levels have been found to change significantly in plants under a variety of stress conditions. The role of CK in plant during stress is relatively inconsistent (Ha et al., 2012). Their endogenous level decreases in response to various stress conditions (Hare et al., 1997). Alvarez et al. (2008) found that isoprene-type CK declined, but benzylaminopurine was concurrently elevated in drought-stressed maize. The effect of CK on the plant stress response was investigated mainly by exogenous CK application and in plants over-expressing the isopentyl transferase (ipt) gene under different promoters. A positive role of CK application in abiotic stresses has been shown in many studies. For example, CK alleviated drought (Hu et al., 2013), heat (Barciszewski et al., 2000, Wang et al., 2012) and salinity (Barciszewski et al., 2000). CKs reverse ABA-induced stomatal closure (Pospíšilová, 2003), thus promoting stomatal reopening following drought, leading to enhanced stomatal conductance (gs) (Hu et al., 2013). Plants with the introduced SAG12:ipt gene construct, which increases CK biosynthesis in response to senescence initiation. Thus, these plants have a longer life span and show better tolerance against abiotic stresses compared to nontransformed plants (Merewitz et al., 2010, Procházková et al., 2012, Pavlíková et al., 2014). Their resistance has been associated with the maintenance of greater antioxidant enzyme activities (Merewitz et al., 2011a, Merewitz et al., 2011b). According to Thomas et al. (2005), enhanced CK production in transgenic tobacco also led to lower lipid peroxidation compared to controls under non-stressed and copper-stressed conditions.
The objectives of the study were to evaluate metabolite changes differentially exhibited between ipt and non-transgenic tobacco plants under zinc stress conditions in order to identify potential tolerance mechanisms related to the maintenance of CK content under trace element stress in tobacco. We hypothesized that the impact of Zn on photosynthesis and nitrogen utilization by plants can result in changes of free amino acid concentrations, and that these changes can differ in transformed and non-transformed plants.
Section snippets
Plant material and cultivation conditions
For the pot experiment, tobacco plants (Nicotiana tabacum L., cv. Wisconsin 38) transformed with a construct consisting of the SAG12 promoter fused with ipt gene for CK synthesis (SAG) were planted. The seeds were a gift from Prof. R. Amasino at the University of Wisconsin, USA. As the control, its wild type (WT plants) was used. After 30 days of in vitro pre-cultivation, plants (three plants per pot) were cultivated for 90 days in pots. The pots were filled with the soil from the non-polluted
Results
The results of the pot experiment revealed the different toxic effect of Zn for WT and SAG tobacco plants. The plant response to Zn contamination was assessed on the basis of reduced tobacco dry matter and increased concentrations of this element in the above-ground biomass (Table 1). The yields of both WT and SAG plants were reduced only by the highest Zn concentration (18% and 17% decrease of yield for Zn3 in WT and SAG plants, respectively, compared to the control). In comparison with the
Discussion
The yields of both WT and SAG tobacco plants were reduced only by higher Zn concentrations. A relatively small decline of biomass yield caused by Zn treatment was affirmed during the entire plant growth period by Pavlíková et al. (2008). Compared to the untreated control, the lowest Zn rate (Zn1) stimulated the dry matter yield. According to Puzina (2004), Zn treatment shifted the hormonal balance toward a substantial increase in the CK content and in the CK/ABA ratio and increased the yield of
Acknowledgement
This research was supported by Grant Agency of the Czech Republic Grant No. P501/11/1239.
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