Drought tolerance through overexpression of monoubiquitin in transgenic tobacco
Introduction
Drought is the primary limitation to wheat production worldwide (Sio-Se Mardeh et al., 2006), which is also one of the most severe environmental stresses that affects almost all plant functions (Yamaguchi-Shinozaki et al., 2002). Increasing evidence has indicated that the molecular tailoring of genes has the potential to overcome a number of limitations in creating drought-tolerant transgenic plants (Umezawa et al., 2006).
Ubiquitin (Ub) is a 76-amino acid globular protein. As the name implies, Ub is nearly ubiquitous, being present in all eukaryotic species examined. It is also one of the most structurally conserved proteins yet identified; its amino acid sequence is invariant in all higher plants. Ub is also unique among plant proteins because it is synthesized from fusion-protein precursors. Members of the Ub family express either Ub polymers (polyubiquitin genes), in which multiples of the 228-bp coding region are concatenated head-to-tail, or ubiquitin extension protein (UbEP) genes, in which a single Ub-coding region is attached to the 5′ end of another coding region (Callis et al., 1995; Smalle and Vierstra, 2004). These polypeptides become functional after deubiquitination enzymes (DUBs) release them. Free Ubs are attached to appropriate intracellular targets by an adenosine triphosphate (ATP)-dependent E1→E2→E3 conjugation cascade (Sullivan et al., 2003; Vierstra, 2003). The subsequent addition of Ub moieties through the lysine 48 (K48) residue in Ub results in the formation of polyubiquitin chains on the target protein. The resulting Ub–protein conjugates are then recognized and degraded by the multisubunit 26S proteasome with the concomitant release of the Ub moieties for reuse. Through this cycle, the Ub/26S proteasome pathway helps remove abnormal proteins and thus performs an essential housekeeping function. Ub can also target certain normal proteins for breakdown; this pathway provides an important control point by eliminating rate-limiting enzymes and key regulatory factors and by dismantling crucial signaling networks (Vierstra, 2003; Smalle and Vierstra, 2004). The inhibition of Ub-dependent protein degradation can induce cell death program(s) in plants as in animals (Yang and Yu, 2003; Schlögelhofer et al., 2005; Vaux and Silke, 2005). Data from yeast and animal studies indicate that in addition to their more traditional roles, the components of the Ub/26S proteasome pathway may also have other functions, some of which may be used by plants. Many of these functions arise from their ability to attach a single Ub or assemble polyubiquitin chains using lysines other than K48 (Smalle and Vierstra, 2004). Monoubiquitination can direct proteolytic targets to the lysosome/vacuole for turnover (Hicke, 2001) or modify transcription (Bach and Ostendorff, 2003). A number of monoubiquitinated proteins have been identified, including the H2A and H2B subunits of the core nucleosome (Bach and Ostendorff, 2003), and numerous receptors and transporters at the plasma membrane (Hicke, 2001).
Ub is multifunctional (von Kampen et al., 1996), and one of its main known functions is to tag proteins for selective degradation by the 26S proteasome (O’Mahony and Oliver, 1999; Smalle and Vierstra, 2004). Ub is induced by various stresses in plants and animals (Fornace et al., 1989; Christensen et al., 1992; Genschik et al., 1992; Sun and Callis, 1997; O’Mahony and Oliver, 1999; Guo et al., 2004). Protein degradation is a normal cellular activity, but an increase in degradation in response to stresses can be interpreted as the result of excessive protein damage and an attempt to remove damaged proteins from the cell in order to maintain cellular function (Ferguson et al., 1990; O’Mahony and Oliver, 1999; Smalle and Vierstra, 2004). In previous experiments (Bachmair et al., 1990; Becker et al., 1993; Conrath et al., 1998; Schlögelhofer et al., 2005), the Ub variant (K48 replaced by arginine (R)) was used as an inhibitor of Ub-dependent protein degradation. The expression of UbR48 can cause changes similar to the inhibition of the proteasome that results in the induction of various forms of cell death. The additional stress causes aggravation of the phenotype with regard to both the severity and kinetics of symptom appearance (Schlögelhofer et al., 2005). However, there have been very few studies thus far on the genetic engineering of transgenic plants overexpressing Ub.
In this study, Ta-Ub2 was isolated from Triticum aestivum using reverse transcription-polymerase chain reaction (RT-PCR). Transgenic tobacco plants constitutively expressing the sense RNA of monoubiquitin were obtained. The drought resistance of transgenic plants was investigated. This research suggests that Ub may play an important role in drought resistance, and overexpressing monoubiquitin is an effective strategy to improve drought tolerance in plants.
Section snippets
Plant materials and drought treatment
Wheat (T. aestivum) and tobacco (Nicotiana tabacum) seedlings were grown in a chamber at 25 °C with a 16/8 h light/dark cycle (300–400 μmol photons m−2 s−1) and a relative humidity of 75–80%.
Wheat seeds that had been soaked for 4–5 h in tap water were germinated between moistened filter paper for 24 h and were then arrayed in 10-cm diameter Petri dishes (30 seedlings/dish) containing 2 layers of filter paper wetted with distilled water. After another 24 h of growth, the seedlings were treated with 20%
Characterization of Ta-Ub2 gene
Using RT-PCR, 2 cDNAs of polyubiquitin genes were isolated from wheat, namely, Ta-Ub1 (GenBank accession AY862401) and Ta-Ub2 (GenBank accession AY297059, Figure 1). Ta-Ub2 consists of 432 bp nucleotides and a 234-bp open reading frame at positions 1–234, encodes an intact Ub monomer (76 amino acids) and an extra amino acid sequence at its carboxyl terminus. The extra amino acid is a glutamine residue same as the terminal amino acid sequence reported for a maize polyubiquitin (Christensen et
Ub expression involved in the response of wheat plants to drought stress
Ub is considered as a stress protein and thus its response to water loss in plants may be a general stress response (O’Mahony and Oliver, 1999). The induction of Ub gene expression under various stresses was considered necessary to tag the damaged proteins for selective degradation by the 26S proteasome (Ferguson et al., 1990; Garbarino et al., 1995; O’Mahony and Oliver, 1999). In this study, the expression of Ta-Ub2 increased slightly under moderate drought stress (20% PEG, −0.64 MPa) but
Acknowledgments
This research was supported by National Natural Science Fundation of China (no. 30671259) and Natural Science Foundation of Shandong Province, China (no. Y2003D03).
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These authors contributed equally to this paper.