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

Abstract

Increased 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.

Keywords: ERFVIIs; N-end rule; PRT6; targeted proteolysis; waterlogging.

Figure 1

Amino terminal cysteine controls protein stability in barley. (a) The Cys‐Arg/N‐end rule pathway. Amino acids are indicated by single letters; C*, oxidized cysteine; MAP, methionine aminopeptidase; NO, nitric oxide; ATE, arginyl tRNA transferase; PRT6 E3, ligase PROTEOLYSIS6; and PCO, plant cysteine oxidase. Circled S indicates substrate proteins. Tertiary, secondary and primary Nt‐destabilizing residues are indicated. (b)  α‐HA Western blot analysis of the in vitro stability of HA‐tagged wild‐type and Ala² variants of barley ERF1 (BERF1) in the absence or presence of MG132 following treatment with 100 μm cycloheximide at time point 0. Equal loading was confirmed by ponceau staining (data not shown). (c) Diagrammatical representation of the M(C/A)GGAIL‐GUS reporter construct used to analyse substrate stability in vivo in response to waterlogging. (d) Histochemical staining for GUS activity in transgenic barley leaf and root tissue following growth under well‐drained or waterlogged conditions for 15 days. In waterlogged conditions, the analysed tissue was submerged. (e) α‐GUS Western blot analysis of M(C/A)GGAIL‐GUS reporter and tubulin protein stability in response to waterlogging. Expression of RNA via semi‐quantitative rtPCR showing no change in GUS RNA expression in MC‐ or MA‐ constructs in response to waterlogging.

Figure 2

qRT‐PCR analysis of the influence of the HvPRT6 RNAi construct on gene expression in independent barley homozygous RNAi lines compared to un‐transformed null segregant controls. Relative expression of HvPRT6, HvADH1, HvHB , HvPDC1. Transcript levels are shown relative to respective null segregants. In each case, error bars represent standard deviation of the mean. *P < 0.05.

Figure 3

Reduced HvPRT6 expression alters barley seed germination phenotypes. (a) Germination of two independent barley homozygous RNAi lines compared to un‐transformed null segregants under dark (top) or white light (bottom) conditions. (b) The influence of the NO scavenger cPTIO on germination in the light. (c) Germination of un‐chilled and chilled arabidopsis WT (Col‐0), prt6 and ate1ate2 seeds at 22 °C in continuous light and under reduced oxygen availability. (d) Germination of barley seeds at 21 °C under reduced oxygen availability following moist chilling. Final germination percentage at each oxygen concentration (top) and germination rate over 7 days at 3% oxygen (bottom) is shown. In each case, error bars represent standard deviation of the mean of three independent experiments.

Figure 4

Reduced HvPRT6 expression alters barley response to waterlogging. (a) Photograph of 25‐day‐old plants following 20 days of waterlogging, showing enhanced green leaf material in RNAi line 55 compared to WT. (b) Relative expression of hypoxia‐related genes measured by qRT‐PCR from healthy leaves of null and RNAi lines grown under well‐drained or waterlogged (WL) conditions. Transcript levels and significance are shown relative to well‐drained null segregant controls. (c) Total chlorophyll derived from leaves of plants grown under well‐drained or waterlogged conditions. (d) Total above‐ground plant weight following growth under well‐drained (control) or waterlogged conditions. (e) Total yield (dry weight of 250 grains) following growth under well‐drained (control) or waterlogged conditions. In each case, error bars represent standard deviation of the mean. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Reduced HvPRT6 expression delays leaf senescence. (a) Photographs of leaf sections from RNAi lines and nontransgenic controls following treatment with darkness on liquid media for 6 days. (b) Quantification of colour change in leaf sections following transformation of images of leaf segments from RGB to HSV colour space. The 50% confidence regions are shown as ellipses (thick for null lines and thin for RNAi lines).

Figure 6

Phenotypes of two barley TILLING lines containing mutations in PRT6. (a) Positions of amino acid sequence changes in prt6i and prt6k TILLING lines. (b) Photograph of 20‐day‐old plants following 20 days of waterlogging, showing enhanced growth of TILLING lines compared to WT (Sebastian). (c) Total chlorophyll derived from leaves of plants grown under well‐drained or waterlogged conditions. (d) Expression of hypoxia‐related genes measured by qRT‐PCR from leaves of plants grown under well‐drained (control) or waterlogged conditions. (e) Photographs of leaf sections from RNAi lines and nontransgenic controls following treatment with darkness in 1/2MS liquid media for 6 days. (f) Quantification of colour change in leaf sections following transformation of images of leaf segments from RGB to HSV colour space. The 50% confidence regions are shown as ellipses (thick for Sebastian and thin for TILLING lines). *P < 0.05, **P < 0.01, ***P < 0.001.