Elsevier

Journal of Plant Physiology

Volume 165, Issue 16, 1 November 2008, Pages 1745-1755
Journal of Plant Physiology

Drought tolerance through overexpression of monoubiquitin in transgenic tobacco

https://doi.org/10.1016/j.jplph.2007.10.002Get rights and content

Summary

Ubiquitin (Ub) is present in all eukaryotic species examined. It is a multifunctional protein and one of its main known functions is to tag proteins for selective degradation by the 26S proteasome. In this study, Ta-Ub2, a cDNA sequence containing a single Ub repeat and a 3′ non-coding region of a polyubiquitin gene, was isolated from wheat (Triticum aestivum) by reverse transcription-polymerase chain reaction (RT-PCR). A PBI sense vector with Ta-Ub2 was constructed and transformed into tobacco plants. Ub expression in wheat leaves, monitored by semi-quantitative RT-PCR, responded to drought stress. In transgenic tobacco, determined by protein gel blot analysis, we found higher amounts of Ub–protein conjugates than in control (tobacco carrying a PBI GUS vector without Ta-Ub2) and wild-type (WT) lines. However, free Ub levels did not significantly differ in the 3 genotypes. Seeds from transgenic, Ub-overexpressing tobacco germinated faster and seedlings grew more vigorously than control and WT samples, both under drought and non-drought conditions. Furthermore, CO2 assimilation of transgenic plants was significantly higher under drought stress. Our results indicate that Ub may be involved in the response of plants to drought stress and that overexpression of monoubiquitin might be an effective strategy for enhancing drought tolerance.

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).

References (38)

  • F. Becker et al.

    Altered response to viral infection by tobacco plants perturbed in ubiquitin system

    Plant J

    (1993)
  • M.M. Bradford

    A rapid, sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding

    Anal Biochem

    (1976)
  • J. Callis et al.

    Structure and evolution of genes encoding polyubiquitin and ubiquitin-like proteins in Arabidopsis thaliana Ecotype Columbia

    Genetics

    (1995)
  • A. Christensen et al.

    Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation

    Plant Mol Biol

    (1992)
  • U. Conrath et al.

    Tobacco plants perturbed in the ubiquitin-dependent protein degradation system accumulate callose, salicylic acid, and pathogenesis-related protein

    Plant Cell Rep

    (1998)
  • D. Ferguson et al.

    Ubiquitin pool modulation and protein degradation in wheat roots during high temperature stress

    Plant Physiol

    (1990)
  • A.J. Fornace et al.

    Ubiquitin mRNA is a major stress induced transcript in mammalian cells

    Nucleic Acids Res

    (1989)
  • J.E. Garbarino et al.

    Isolation of a polyubiquitin promoter and its expression in transgenic potato plants

    Plant Physiol

    (1995)
  • P. Genschik et al.

    Ubiquitin genes are differentially regulated in protoplast-derived cultures of Nicotiana sylvestris and in response to various stresses

    Plant Mol Biol

    (1992)
  • Cited by (64)

    • Shift of calcium-induced Microcystis aeruginosa colony formation mechanism: From cell adhesion to cell division

      2022, Environmental Pollution
      Citation Excerpt :

      Higher calcium concentrations do not inhibit algal cell growth but delays algal cells from entering into the rapid growth phase. The change in total protein (T-Pro) content could reflect the influence of external factors on cyanobacterial activity to a certain extent (Guo et al., 2008). The T-Pro content was also measured in EPS on day 15 to assess the adaptation of algal cells to different ionic strengths (Fig. 2).

    • Proteomics in relation to abiotic stress tolerance in plants

      2020, Plant Life under Changing Environment: Responses and Management
    • Protein Modification in Plants in Response to Abiotic Stress

      2019, Protein Modificomics: From Modifications to Clinical Perspectives
    View all citing articles on Scopus
    1

    These authors contributed equally to this paper.

    View full text