Enhanced waterlogging tolerance in barley by manipulation of expression of the N‐end rule pathway E3 ligase PROTEOLYSIS6

Abstract

SummaryIncreased tolerance of crops to low oxygen (hypoxia) during flooding is a key target for food security. In Arabidopsis thaliana (L.) Heynh., the N‐end rule pathway of targeted proteolysis controls plant responses to hypoxia by regulating the stability of group VII ethylene response factor (ERFVII) transcription factors, controlled by the oxidation status of amino terminal (Nt)‐cysteine (Cys). Here, we show that the barley (Hordeum vulgare L.) ERFVII BERF1 is a substrate of the N‐end rule pathway in vitro. Furthermore, we show that Nt‐Cys acts as a sensor for hypoxia in vivo, as the stability of the oxygen‐sensor reporter protein MCGGAIL‐GUS increased in waterlogged transgenic plants. Transgenic RNAi barley plants, with reduced expression of the N‐end rule pathway N‐recognin E3 ligase PROTEOLYSIS6 (HvPRT6), showed increased expression of hypoxia‐associated genes and altered seed germination phenotypes. In addition, in response to waterlogging, transgenic plants showed sustained biomass, enhanced yield, retention of chlorophyll, and enhanced induction of hypoxia‐related genes. HvPRT6 RNAi plants also showed reduced chlorophyll degradation in response to continued darkness, often associated with waterlogged conditions. Barley Targeting Induced Local Lesions IN Genomes (TILLING) lines, containing mutant alleles of HvPRT6, also showed increased expression of hypoxia‐related genes and phenotypes similar to RNAi lines. We conclude that the N‐end rule pathway represents an important target for plant breeding to enhance tolerance to waterlogging in barley and other cereals.

Group VII Ethylene Response Factors Coordinate Oxygen and Nitric Oxide Signal Transduction and Stress Responses in Plants

“…Barley PRT6 (HvPRT6) RNA interference lines with reduced HvPRT6 expression had increased levels of anaerobic response gene transcripts (including ADHs), similar to what is observed in Arabidopsis N-end rule mutants (Gibbs et al, 2011;Licausi et al, 2011). Furthermore, RNA interference lines performed better than null controls under waterlogging stress, as evidenced by retention of chlorophyll, increased biomass, and sustained yield poststress (Mendiondo et al, 2015). This translational study highlights the value of targeting ERFVIIs and the N-end rule pathway for engineering flooding tolerance in agronomically important species.…”

“…Interestingly, analysis of rice SUB1A-1 demonstrated that it is not an N-end rule substrate in vitro, in contrast to all five of the Arabidopsis ERFVIIs and at least one barley (Hordeum vulgare) ERFVII (Gibbs et al, 2011;Mendiondo et al, 2015). Enhanced tolerance of rice varieties carrying the SUB1A-1 gene might therefore be due to the increased stability of the protein, although more research is needed to fully support this hypothesis.…”

“…This indicates that the N-end rule pathway is a promising target for manipulating flooding tolerance in crops. In barley, the ERFVII BARLEY ERF1 was shown to be a putative N-end rule substrate in vitro, and posttranscriptional accumulation of an artificial N-end rule reporter protein consisting of the ERFVII Nt domain fused to GUS (MCGGAIL-GUS) was observed under waterlogged conditions, indicating that ERFVIIs are also stabilized by low oxygen in monocots (Mendiondo et al, 2015). Barley PRT6 (HvPRT6) RNA interference lines with reduced HvPRT6 expression had increased levels of anaerobic response gene transcripts (including ADHs), similar to what is observed in Arabidopsis N-end rule mutants (Gibbs et al, 2011;Licausi et al, 2011).…”

Creation of a TILLING Population in Barley After Chemical Mutagenesis with Sodium Azide and MNU

“…There are many successful examples of using TILLING for crop improvement, for example, in Triticum aestivum and Triticum turgidum subsp. durum for obtaining a better starch quality (Slade and Knauf 2005); in Hordeum vulgare for the manipulation of spike morphology (Gottwald et al 2009), starch content (Sparla et al 2014), and waterlogging tolerance (Mendiondo et al 2016); in Cucumis melo (Dahmani-Mardas et al 2010) and Solanum lycopersicum (Okabe et al 2011) for enhanced shelf life; and in Sorghum bicolor (Xin et al 2008) and Avena sativa (Chawade et al 2010) for increased digestibility.…”

“…The application of a double mutagenic treatment with a period of inter-incubation germination of seeds between treatments gave a high frequency of point mutations in barley (Szarejko and Maluszynski 1999) and rice (Till et al 2007). The TILLING population that was developed in barley after the combined treatment with sodium azide and MNU has been successfully used for the functional analysis of the genes involved in the control of DNA repair (Stolarek 2015a, b), waterlogging (Mendiondo et al 2016), and strigolactone perception (Marzec et al 2016 1. Before a large-scale mutagenic treatment, evaluation of a critical dose of mutagen is recommended.…”