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Will modifying plant ethylene status improve plant productivity in water-limited environments ?

Ian C. Dodd1, Andrei A. Belimov2, Wagdy Y. Sobeih1, Vera I. Safronova2, Donald Grierson3 and William J. Davies1

1 Department of Biological Sciences, Lancaster Environment Centre, University of Lancaster LA1 4YQ, United Kingdom.
E-mail I.Dodd@lancaster.ac.uk
2
All-Russia Research Institute for Agricultural Microbiology, Podbelskogo Sh., 3, Pushkin-8, 196608, St.-Petersburg, Russia.
E-mail belimov@rambler.ru
3
Plant Science Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, United Kingdom

Abstract

The plant hormone ethylene is generally inhibitory to shoot and root growth. We tested two independent strategies to overcome ethylene-mediated growth inhibition when plants are grown in drying soil. Antisense suppression of plant ethylene synthesis was trialled to overcome the inhibition of tomato (Lycopersicon esculentum Mill.) leaf expansion. Rhizosphere bacteria containing the enzyme ACC deaminase (to decrease plant levels of the ethylene precursor ACC (1-aminocyclo-propane carboxylic acid)) were trialled to overcome the inhibition of pea (Pisum sativum L.) root growth. Two pea genotypes (cv. Sparkle and its E2(sym5) mutant) were grown at two levels of soil moisture in pots containing the plant growth-promoting rhizobacterium Variovorax paradoxus 5C-2. The bacterium stimulated root biomass by 20-25% irrespective of soil moisture regime, and whole plant biomass was stimulated also by 25% in plants grown in drying soil. Isogenic wild-type (WT) and ACO1AS genotypes of tomato were grown at two different soil moisture regimes. The ACO1AS genotype has decreased ACC oxidase activity and is thus less able to convert ACC to ethylene. Leaves of WT and ACO1AS plants elongated at the same rate under well-watered conditions. When plants were exposed to drying soil, both genotypes dried the soil to a similar extent but ACO1AS leaves had a 10% higher growth rate than WT leaves. The significance of these responses to plant yield are under investigation.

Media summary

Decreasing synthesis of the plant hormone ethylene, via a genetic modification or naturally occurring root-associated bacteria, stimulated the growth of plants in drying soil.

Key Words

soil drying, leaf expansion, root growth, rhizobacteria, ACC deaminase

Introduction

Plant biomass accumulation is linearly related to the amount of radiation intercepted by the crop (Monteith 1977). The development of the leaf canopy is critical to intercept radiation, particularly in young developing crops which have not yet covered the soil. Leaf expansion is highly sensitive to soil drying, which can limit growth even when there is still substantial water available in the soil profile. Even when shoot water status is maintained, chemical signals generated as a result of the interaction between root systems and drying soil can directly inhibit leaf growth (reviewed in Davies and Zhang 1991). If we can develop genotypes which do not produce chemical growth inhibitors as the soil dries or have leaf growth processes that are insensitive to these signals, then we can perhaps optimise biomass accumulation and yield of vegetative plant parts in dryland agriculture. This strategy is dependent on identifying the chemical signals that limit leaf expansion during drought.

Although the plant hormone ethylene is generally inhibitory to shoot and root growth (Abeles et al. 1992), an influential report indicated that water stress did not increase ethylene evolution of intact plants (Morgan et al. 1990). However soil drying increases both soil strength and decreases nitrogen availability and both these factors can increase ethylene evolution (Hussain et al. 1999; Lege et al. 1997). It therefore seems possible that some soil drying episodes can perturb ethylene evolution, thus modifying leaf growth. Hence we re-examined the possibility that ethylene might be involved in the regulation of leaf growth when the soil dries. Upon finding that mild soil drying (which didn’t decrease leaf water status) increased leaf ethylene evolution (W.Y Sobeih, I.C. Dodd, M.A. Bacon & W.J. Davies, unpublished results), we investigated whether antisense suppression of ethylene synthesis could overcome soil drying-induced limitation of leaf growth.

Another strategy to overcome the limitation of leaf growth by soil drying is to promote root growth to allow water uptake from deeper parts of the soil profile to maintain leaf water status, a feed-forward response to soil drying (Reid and Renquist 1997). Convenient systems to assay root growth have shown that ethylene is inhibitory to roots grown in agar (Locke et al. 2000), in root growth pouches (eg. Penrose et al. 2001) and on saturated filter paper in petri dishes (Belimov et al. 2002). The extent to which ethylene depresses root growth of plants in the field is less certain. Since chemical inhibitors of ethylene synthesis and/or action are both expensive and toxic to the environment, there has been considerable interest in the use of naturally occurring rhizosphere bacteria which contain the enzyme ACC deaminase that breaks down the precursor of ethylene, ACC. Since a dynamic equilibrium of ACC concentration exists between root, rhizosphere and bacterium, bacterial uptake of rhizospheric ACC (for use as a carbon and nitrogen source) decreases root ACC concentration and root ethylene evolution and can increase root growth (Glick et al. 1998, Penrose et al. 2001). We compared the effects of modifying root ethylene status with a chemical inhibitor of ethylene biosynthesis (AVG) and a rhizosphere bacterium containing the enzyme ACC deaminase on biomass accumulation at two soil water contents.

Methods

Effects of rhizosphere bacteria on pea root and shoot growth during soil drying

Two pea (Pisum sativum L.) genotypes (cv. Sparkle and its E2(sym5) mutant) were grown at two levels of soil moisture (achieved by daily watering of pots to 75% and 50% of field capacity) in pots containing the plant growth-promoting rhizobacterium Variovorax paradoxus 5C-2, to determine whether soil moisture status affected growth response to the bacterium. Since V. paradoxus contains the enzyme ACC deaminase, which is expected to decrease plant ethylene evolution, other pots were irrigated with the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) at 10-5M. Both treatments were compared to control pots irrigated with distilled water. Transpiration was measured gravimetrically daily in vegetative plants, and biomass and leaf area determined at harvest.

Antisense suppression of tomato ethylene synthesis during soil drying

Isogenic wild-type (cv. Ailsa Craig) and ACO1AS (Hamilton et al. 1990) genotypes of tomato (Lycopersicon esculentum Mill.) were used. The ACO1AS genotype has decreased ACC oxidase activity and is thus less able to convert the ethylene precursor ACC to ethylene. Both genotypes were grown in a controlled environment cabinet with the root systems split between two columns of peat-based compost. This medium was chosen to minimise the effects of increasing soil strength as the soil dried. After plant establishment, water was applied daily to one (partial root-zone drying – PRD) or both (control well watered - WW) columns. Water was withheld from the other column in the PRD treatment to expose a proportion of the root system to drying soil. Soil and plant water status was monitored using a theta probe (ML2, Delta-T, Cambridge, UK) and pressure chamber (Plant Moisture Systems, Santa Barbara, CA, USA) respectively. Leaf growth was measured daily with a ruler and at the end of an experiment using a leaf area meter (Model 3100, Li-Cor, Lincoln, Nebraska, USA). Ethylene evolution of entire detached leaflets was measured as described previously (Hussain et al. 1999) using a gas chromatograph. Transpiration was measured gravimetrically.

Results

Effects of rhizosphere bacteria on pea root and shoot growth during drought

Both pea genotypes behaved similarly thus data were pooled. Although both AVG and bacterial treatments promoted root growth at both soil moisture regimes (Figure 1c), shoot (and whole plant) biomass was only increased (relative to control plants irrigated with distilled water) in plants grown in drying soil with bacteria (Figure 1b, d). Plant responses to bacteria were qualitatively similar to treatment with AVG, suggesting that bacterial ACC deaminase was involved in the plant-bacterium interaction and the observed effects of V.paradoxus 5C-2 on pea plants were mediated by ethylene.

Antisense suppression of tomato ethylene synthesis during soil drying

Ethylene evolution of wild-type (WT) plants increased as the soil dried (Table 1) but could be suppressed using transgenic (ACO1AS) plants containing an antisense gene for one isoenzyme of ACC oxidase. Both ACO1AS and WT plants used similar amounts of water after the initiation of PRD (Table 2) and had similar soil water contents at the end of the experiment (Table 1). Stomatal closure of both genotypes (data not shown) maintained leaf water potential throughout the experiments (Tables 1, 2). When elongation of tagged leaves was monitored during the experiment, ACO1AS plants showed no significant inhibition of leaf elongation when exposed to PRD (Tables 1, 2). This was reflected in an increased (compared to WT) area of the tagged individual leaves at harvest (Table 2). However, total leaf area of both ACO1AS and WT plants was decreased by PRD (Table 2), indicating the existence of other chemical inhibitors of leaf growth.

Figure 1. Promotion of plant biomass in well watered (WW) and drying soil for pea plants irrigated with a chemical inhibitor of ethylene synthesis (AVG) and a suspension of Variovorax paradoxus (PGPR). Data are means of 12 plants per treatment, expressed as a percentage of plants irrigated with distilled water.

Table 1: Responses of wild-type (WT) and transgenic (ACO1AS) plants to partial rootzone drying (PRD) and well-watered (WW) conditions. Measurements of leaf water potential and ethylene evolution were made on Day 12 on the same leaves for which the 12 day leaf length increment is given. Soil water content of one side of the split-root plants is given after 12 days of treatment. Data are means S.E. of 4 replicates. Values followed by different letters are significantly different at the 0.05 level according to Tukey’s HSD test.

Parameter

WW-WT

PRD-WT

WW- ACO1AS

PRD- ACO1AS

         

Increment of entire leaf (cm)

22.3 0.9 a

19.5 0.3 b

21.0 1.0 a

21.5 0.5 a

Increment of terminal leaflet (cm)

6.45 0.10 a

5.32 0.25 b

5.92 0.37 a

6.05 0.33 a

Soil water content (g H20 g-1 soil)

2.04 0.01 a

0.57 0.02 b

2.03 0.02 a

0.61 0.03 b

Leaf water potential (MPa)

-0.61 0.05 a

-0.61 0.03 a

-0.62 0.04 a

-0.62 0.03 a

Leaf ethylene evolution (nLg-1 FW h-1)

4.5 0.3 b

6.9 0.3 c

2.1 0.1 a

2.2 0.2 a

Table 2: Responses of wild-type (WT) and transgenic (ACO1AS) plants to partial rootzone drying (PRD) and well-watered (WW) conditions. Leaf elongation was assessed during a 5 day period between Days 3-8 after PRD was applied and leaf water potential of a fully expanded leaf was assessed on Day 8. The area of this leaf, and total plant leaf area, was determined after 12 days of PRD. Data are means S.E. of 6 replicates. Values followed by different letters are significantly different at the 0.05 level according to Tukey’s HSD test.

Parameter

WW-WT

PRD-WT

WW- ACO1AS

PRD- ACO1AS

         

Leaf elongation rate (mm day-1)

31.3 1.0 a

26.3 0.5 b

29.5 1.0 a

28.7 0.6 ab

Terminal leaflet elongation rate (mm day-1)

7.8 0.5 a

6.1 0.4 b

7.3 0.4 ab

6.6 0.4 b

Leaf area at harvest (cm2)

164 7 a

131 7 b

163 7 a

147 10 ab

Total plant leaf area at harvest (cm2)

920 34 a

821 22 b

954 55 a

806 42 b

Total water use (L)

2.02 0.08 a

1.60 0.04 b

1.95 0.12 a

1.58 0.05 b

Leaf water potential (MPa)

-0.44 0.01 a

-0.48 0.04 a

-0.46 0.01 a

-0.46 0.02 a

Conclusion

Pea root growth was promoted by both chemical and bacterial decreases in root ethylene evolution, irrespective of soil moisture status. The increase in root growth was not at the expense of shoot growth, and stimulated shoot growth of plants in drying soil. It is not yet known whether the positive effects of the bacteria were due to systemic effects on shoot hormone physiology, or an improvement in shoot water relations. To determine possible effects of ethylene on leaf growth independently of shoot water status, WT and ACO1AS plants were grown using a soil drying regime that maintained leaf water status. Although individual leaves of plants producing less ethylene elongated at a greater rate in partially dry soil, suppression of ethylene biosynthesis had minimal effects on whole plant leaf area after soil drying, presumably due to the action of other chemical growth inhibitors. Since ACO1AS plants had no deleterious responses to soil drying, this genotype might be worth trialling in other agronomic systems where stress-induced ethylene may be produced. The agronomic consequences of both strategies are under further evaluation.

Acknowledgements

We thank BBSRC for funding the work on antisense suppression of ethylene synthesis, and the Royal Society for funding the work on rhizobacterial mediation of plant drought stress responses.

References

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