Ethylene modulates root cortical senescence in barley

Abstract

Background and aims: Root cortical senescence (RCS) is a poorly understood phenomenon with implications for adaptation to edaphic stress. It was hypothesized that RCS in barley (Hordeum vulgare L.) is (1) accelerated by exogenous ethylene exposure; (2) accompanied by differential expression of ethylene synthesis and signalling genes; and (3) associated with differential expression of programmed cell death (PCD) genes.

Methods: Gene expression of root segments from four barley genotypes with and without RCS was evaluated using quantitative real-time PCR (qRT-PCR). The progression of RCS was manipulated with root zone ethylene and ethylene inhibitor applications.

Key results: The results demonstrate that ethylene modulates RCS. Four genes related to ethylene synthesis and signalling were upregulated during RCS in optimal, low nitrogen and low phosphorus nutrient regimes. RCS was accelerated by root zone ethylene treatment, and this effect was reversed by an ethylene action inhibitor. Roots treated with exogenous ethylene had 35 and 46 % more cortical senescence compared with the control aeration treatment in seminal and nodal roots, respectively. RCS was correlated with expression of two genes related to programmed cell death (PCD).

Conclusions: The development of RCS is similar to root cortical aerenchyma formation with respect to ethylene modulation of the PCD process.

Fig. 1.

The progression of RCS in plants grown with optimal nutrient availability in control (air), ethylene and 1-MCP aeration treatments in barley seminal and nodal roots of modern cultivars and landraces. Progression of RCS in plants grown in low nitrogen and low phosphorus treatments in control (air), ethylene and 1-MCP aeration treatments in barley seminal and nodal roots of modern cultivars and landraces. No differences in RCS were observed in low nitrogen and phosphorus treatments, so data were combined for analysis. Points show the means of four replicates for each of two genotypes ± s.e. RCS was quantified by disappearance of the cortex compared with a non-senesced root segment on the same root.

Fig. 2.

Expression of ethylene-related genes in optimal, low nitrogen and low phosphorus conditions in root segments with different levels of RCS. Distal root segments had no RCS, mid-root segments had partial RCS, and basal root segments had complete cortical senescence. (A) HvACS6, (B) HvRAF, (C) HvACO7 and (D) BERF1 were significantly upregulated in mid-root sections compared with distal and basal root segments. In mid-root segments, HvACO7 and HvRAF were significantly upregulated in low nitrogen compared with optimal nutrient conditions. In low phosphorus, expression of HvACO7 was significantly upregulated in mid-root segments compared with optimal nutrient conditions. Points show the means of four replicates for each of four genotypes ± s.e. since no significant differences among genotypes were observed. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). Relative expression is the ratio of expression of the target gene compared with the reference genes.

Fig. 3.

Cross-sections of barley seminal root stained with acridine orange. (A) Root cross-section from a plant grown in optimal nutrient conditions with no RCS from the distal portion of the root (3 cm from the apex). (B) Root cross-section from a plant grown in optimal nutrient conditions with partial RCS from the mid-root section (8 cm from the apex). (C) Root cross-section from a plant grown in optimal nutrient conditions with maximal RCS from the basal portion of the root (34 cm from the apex). (D) Root cross-section from an ethylene-treated plant with partial RCS from the mid-root section. (E) Root cross-section from a 1-MCP-treated plant with partial RCS from the mid-root section. Scale bar =100 µm.

Fig. 4.

RCS in optimal, low nitrogen and low phosphorus treatments in (A) seminal roots of modern cultivars, (B) nodal roots of modern cultivars, (C) seminal roots of landraces and (D) nodal roots of landraces. In seminal and nodal roots of modern cultivars and landraces, mid-root segments in low nitrogen and phosphorus conditions had significantly greater RCS compared with optimal nutrient conditions. Distal segments (no RCS) are 3–4 cm from the root apex, mid-root segments (RCS forming) are 8–9 cm from the apex and basal segments (complete RCS) are 34–46 cm from the apex. Points show the means of four replicates for each of two genotypes ± s.e. since no significant differences within the landraces or modern cultivar classes were observed. RCS was quantified by disappearance of the cortex compared with a non-senesced root segment on the same root. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05).

Fig. 5.

Expression of PCD genes in optimal, low nitrogen and low phosphorus conditions in root segments with different levels of RCS. Distal root segments (3–4 cm from root apex) had no RCS, mid-root segments (8–9 cm from the root apex) had partial RCS and basal root segments (34–36 cm from the root apex) had complete cortical senescence. (A) Relative expression of DAD1. Relative expression is the ratio of expression of the target gene compared with the reference genes. DAD1 was significantly downregulated in mid- and basal root segments compared with distal root segments. DAD1 was significantly upregulated in low nitrogen and low phosphorus conditions compared with optimal nutrient conditions. (B) Relative expression of DAD2. DAD2 was significantly upregulated in mid-root segments compared with distal root segments. DAD2 was significantly upregulated in low phosphorus conditions compared with optimal nutrient conditions. Points show the means of four replicates for each of four genotypes ± s.e. since no significant differences among genotypes were observed. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05).