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Keywords:

  • Arabidopsis thaliana ;
  • drought tolerance;
  • flowering phenology;
  • Phyllobacterium brassicacearum STM196;
  • plant growth-promoting rhizobacteria (PGPR);
  • water-use efficiency

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Understanding how biotic interactions can improve plant tolerance to drought is a challenging prospect for agronomy and ecology. Plant growth-promoting rhizobacteria (PGPR) are promising candidates but the phenotypic changes induced by PGPR under drought remain to be elucidated.
  • We investigated the effects of Phyllobacterium brassicacearum STM196 strain, a PGPR isolated from the rhizosphere of oilseed rape, on two accessions of Arabidopsis thaliana with contrasting flowering time. We measured multiple morphophysiological traits related to plant growth and development in order to quantify the added value of the bacteria to drought-response strategies of Arabidopsis in soil conditions.
  • A delay in reproductive development induced by the bacteria resulted in a gain of biomass that was independent of the accession and the watering regime. Coordinated changes in transpiration, ABA content, photosynthesis and development resulted in higher water-use efficiency and a better tolerance to drought of inoculated plants.
  • Our findings give new insights into the ecophysiological bases by which PGPR can confer stress tolerance to plants. Rhizobacteria-induced delay in flowering time could represent a valuable strategy for increasing biomass yield, whereas rhizobacteria-induced improvement of water use is of particular interest in multiple scenarios of water availability.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Drought is one of the major limitations to food production worldwide. The development of drought-resistant cultivars and water-use-efficient plants is therefore of global concern. In their habitats, plants are not single organisms but are surrounded by dense populations of diverse microorganisms with which they probably interact. Some of these plant–microorganism interactions are beneficial for plant growth and allow plants to better cope with biotic and abiotic stresses (Yang et al., 2009).

Drought periods lead to large physiological and developmental alterations in plants. Water deprivation decreases above-ground vegetative biomass accumulation and therefore reduces plant performance (Boyer & Westgate, 2004; Hummel et al., 2010; Tardieu et al., 2011; Vile et al., 2012). Vegetative growth and the total production of dry matter are closely related to key developmental switches such as reproductive transition (Jung & Muller, 2009). Specifically, flowering time can be delayed (McMaster et al., 2009; Tisne et al., 2010) or hastened (Verslues & Juenger, 2011) in response to drought, most likely depending upon plant species and the occurrence, duration and severity of the stress.

In order to minimize the negative effects of water deficit and complete their life cycle under unfavourable conditions, plants exhibit a variety of strategies (for reviews, see Farooq et al., 2009; Verslues & Juenger, 2011). Physiological changes occur rapidly after the onset of water deficit in order to maintain high tissue water potential. One of the swiftest responses is a reduction of transpiration through reduced leaf conductance following stomata closure. This response is often associated with an accumulation of ABA or enhancement of sensitivity to this hormone in the leaf cells, leading to the induction of related signalling genes (Harb et al., 2010). Rapid osmotic adjustment through active accumulation of solutes also helps in maintaining cell turgor and increases the driving force of water influx into the cell (Yoshiba et al., 1997). In the long term, increased root-to-shoot ratio, through reduced above-ground growth and/or increased root growth, participates in reducing evaporative area and increasing water absorption capacities from the soil (Boyer, 1985), together contributing to increased water-use efficiency (WUE). WUE reflects the tradeoff between CO2 acquisition for growth and water losses and is therefore an important indicator of how plants manage water stress (Blum, 2005; Tardieu, 2012).

Soil microorganisms may interact with plant-specific mechanisms related to drought resistance. Some naturally occurring free-living soil bacteria, namely plant growth-promoting rhizobacteria (PGPR), colonize the root system and maintain mutualistic interactions that lead to plant growth improvement and plant protection against multiple stresses, including drought, salt, heavy metals or pathogens (Dimkpa et al., 2009; Lugtenberg & Kamilova, 2009; Yang et al., 2009). PGPR such as Azospirillum, Azotobacter and Pseudomonas fluorescens are well known for their plant growth-promoting effects and are notably used for improving crop yields (Kloepper et al., 1989; Lucy et al., 2004). PGPR effects involve multiple changes in plant metabolism and signalling networks (Lugtenberg & Kamilova, 2009; Friesen et al., 2011). Modifications in phytohormone content and/or signalling have been reported (see, for review, Dodd et al., 2010), such as decreased ethylene production via bacterial ACC deaminase activity (Glick et al., 1998; Belimov et al., 2009), changes in cytokinin–ABA balance (Figueiredo et al., 2008; Cohen et al., 2009) or changes in auxin signalling (Persello-Cartieaux et al., 2003; Contesto et al., 2010). These effects on hormone pathways are likely to interfere with plant tolerance to drought stress. Some PGPR strains improve plant enzyme activity, such as catalase or superoxide dismutase, which alleviates the oxidative damage induced by drought (Kohler et al., 2008; Wang et al., 2012). Finally, PGPR have been shown to increase drought-response transcript abundances (Wang et al., 2005, 2012).

Despite strong evidence that PGPR influence overall plant performance, their detailed effects on development, growth and physiology under drought have been less well explored. Therefore, integrative studies to explain how PGPR can improve drought tolerance are lacking. Among the specific PGPR-mediated mechanisms identified is the enhancement of wheat growth by Azospirillum sp. strains under various drought intensities, which was associated with better maintenance of plant water status as a result of increased cell wall elasticity (Creus et al., 2004). An increase of photosynthetic capacity has also been shown in Pinus halepensis inoculated with P. fluorescens (Rincon et al., 2008) or in Azospirillum-inoculated rice (Ruiz-Sanchez et al., 2011). Although these physiological studies have detailed measurements of plant water relations, most failed to report drought effect on the dynamics of plant development. Moreover, most studies focus on a single time point, generally at flowering or seed maturity, and reports on plant growth throughout the whole plant cycle are very scarce.

Here, we investigated the growth and physiological responses of Arabidopsis thaliana inoculated with a free-living PGPR, Phyllobacterium brassicacearum strain STM196 under long-term water deficit. A. thaliana is a useful organism to study plant interactions with PGPR (Ryu et al., 2005; Desbrosses et al., 2009), but the effects of PGPR on the development or physiology of this model species under water stress have been little investigated. STM196 belongs to the Phyllobacteriaceae family in the Rhizobiales order of α-Proteobacteria (Mantelin et al., 2006). This strain was the most efficient PGPR isolated from the rhizoplan of field-grown Brassica napus roots (Bertrand et al., 2001; Larcher et al., 2003).

Previous in vitro studies showed that STM196 enhances shoot and root growth of A. thaliana, and modifies its root architecture and hormonal signalling (Mantelin et al., 2006; Contesto et al., 2010; Galland et al., 2012; Kechid et al., 2013). However, we lack information on plant–bacteria interactions under soil conditions and no study has investigated the effects of this particular strain on plant response to drought. We used the high-throughput plant phenotyping platform PHENOPSIS (Granier et al., 2006) to decipher the effects of STM196 on multiple plant traits related to growth dynamics, development and physiology under well-defined soil water availability. First, we show that the plant growth-promoting effect of STM196 is related to a delay in reproductive transition in two A. thaliana accessions with contrasting flowering phenology. Then, we show that STM196 induces a suite of physiological and developmental changes that lead to enhanced WUE and to a better plant tolerance to water deficit.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Bacterial inoculum and soil inoculation

The strain Phyllobacterium brassicacearum STM196 was grown for 3 d in Petri dishes on a sterile (20 min at 120°C) 1.5% agar (w/v; Sigma-Aldrich) medium (E′) containing 2.87 mM K2HPO4, 0.81 mM MgSO4, 1.71 mM NaCl, 7.91 mM KNO3, 0.34 mM CaCl2, 30 μM FeCl3, 1% mannitol (w/v) and 0.3% yeast extract (w/v; Sigma-Aldrich), adjusted to pH 6.8. Next, the bacteria were grown aerobically in 750 ml liquid E′ medium on a rotary shaker (145 rpm) at 25°C for 24 h to reach the exponential phase of growth. The culture of bacteria cells was pelleted by centrifugation (3200 g, 15 min, 20°C) and resuspended in water. To obtain 3 × 107 colony-forming units (CFU) g–1 of soil, the volume was adjusted based upon a correspondence with the absorbance measured at 595 nm (WPA UV 1101; Biotech Photometer, Cambridge, UK). This inoculum was placed directly into the soil substrate, which was then manually homogenized.

Plant material, growth conditions and irrigation treatments

We selected two accessions of A. thaliana (L.) Heynh differing in flowering time: Col-0, one of the reference accessions in A. thaliana research; and An-1, an early-flowering accession (Granier et al., 2006; Tisne et al., 2010). A total of 96 individual plants per genotype were studied (see Supporting Information, Table S1, for details on replicate numbers per trait and conditions). Five seeds were sown at the soil surface in 260 ml culture pots filled with a damped mixture (1 : 1, v/v) of loamy soil and organic compost (Neuhaus N2) inoculated (or not) with STM196. Noninoculated soil was previously damped with deionized water to avoid difference in initial soil humidity between this soil and inoculated soil. Soil water content was controlled before sowing to estimate the initial amount of dry soil and water in each pot. The 192 pots were placed in the dark at 4°C for 48 h to ensure stratification and were then transferred into the PHENOPSIS growth chamber (Granier et al., 2006). Pots were kept in the dark for 2 d and were dampened with sprayed deionized water three times a day until germination. Then, plants were cultivated under conditions of 12 h day length (180 μmol m−2 s−1 photosynthetic photon flux density (PPFD) at plant height). During the germination phase (7 d), the air temperature was set to 20°C day and night, and the relative humidity of the air was adjusted to maintain a constant water vapour pressure deficit (VPD) at 0.6 kPa (Fig. S1). Plants were then grown at 20 : 17°C, day : night temperatures and a VPD of 0.8 kPa (Fig. S1). Soil relative water content was maintained at 0.35 g H2O g−1 dry soil (corresponding to −0.07 MPa soil water potential; WP4-T dewpoint meter; Decagon Devices, Pullman, WA, USA) until the emergence of the first two true leaves (stage 1.02 in Boyes et al., 2001). After stage 1.02, seedlings were thinned to one plant per pot and soil water deficit was started. Soil water content was maintained at 0.35 g H2O g−1 dry soil in the well-watered treatment (WW) and was decreased to 0.20 g H2O g−1 dry soil (corresponding to −0.28 MPa soil water potential) by stopping irrigation (Fig. S2) in the water-deficit treatment (WD). The weight of each pot was adjusted daily with a modified 1/10th-strength Hoagland solution (Hoagland & Arnon, 1950) to reach the target soil water content. These two values of soil water content were maintained until harvest.

Measurement of plant traits

Rosette expansion, leaf production dynamics and phenology

Individual areas of the two first leaves and projected area of the rosette (RAproj) were determined every 3 d from semi-automated analysis (ImageJ 1.43C; Rasband, Bethesda, MD, USA) of zenithal images of the plants (PROSILICA AVT GC 1600C camera, ALLIED, Stradroda, Germany). The initial relative expansion rate of the rosette (RER, d−1) was estimated as the slope of the linear relationship between total leaf area and time after sowing. A sigmoid curve was fitted for each plant following RAproj = a/(1 + exp−((d−a/2)/b)), where a is the maximum area, and d is the number of days after sowing. The maximum rate of leaf expansion (Rmax, mm2 d−1) was calculated from the first derivative of this logistic model at d0 as Rmax = a/(4b). The duration (d) of rosette expansion was estimated as the time period for rosette area to increase from 5 to 95% maximum area following a/2 − bloge ((1/0.95) – 1).

The number of leaves that were visible to the naked eye was counted every 2–3 d to determine the phyllochron (d), that is, the time necessary to have a new visible leaf, until emergence of the flowering stem. Bolting and flowering time were determined as the number of d from germination until macroscopic visualization of flower buds (stage 5.01; Boyes et al., 2001) and the first flower open (stage 6.00), respectively.

Whole-plant and leaf morphology

Col-0 individuals were harvested at bolting, and An-1 individuals were harvested both at bolting and at first flower open. Rosettes were cut and immediately weighed after the removal of inflorescence stems to determine above-ground vegetative fresh mass (FM). The rosettes were wrapped in moist paper and placed into Petri dishes at 4°C in darkness overnight to achieve complete rehydration. Water-saturated fresh mass (SM) was then determined. The total leaf number was determined, and the leaf blades were separated from their petiole and scanned for measurements of leaf area, length and width (ImageJ 1.43C). Leaf blades, petioles and reproductive structures were then oven-dried separately at 65°C for 48 h, and their dry mass (DM) was determined. Rosette DM was calculated as the sum of blade DM and petiole DM. From these measurements, leaf dry matter content (LDMC = DM/SM (mg g−1)) and relative water content (RWC = (FM − DM) × 100 × (SM − DM)−1) were calculated at the rosette level. Leaf dry mass per area (LMA, g m−2) was calculated as DM divided by the projected rosette area as determined from the last zenithal image before harvest. Roots were carefully extracted from the soil, gently washed in deionized water, placed in a paper bag at 65°C for 5 d and their dry mass (DMroot) determined.

Leaf and shoot development

Postembryonic development of the shoot is characterized by distinct phases: a reproductively incompetent juvenile vegetative phase, a reproductively competent adult phase and a reproductive phase (Willmann & Poethig, 2011). In Arabidopsis, during each phase different types of leaves are produced: juvenile, adult and cauline leaves, which can be distinguished from each other by morphological characteristics (Steynen et al., 2001; Willmann & Poethig, 2011). Juvenile leaves are flat and round with a small blade and a long petiole. Generally, juvenile leaves consist of the first two leaves. Adult leaves are recognized by a larger, curled blade and a lanceolate shape, whereas cauline leaves are recognized by their small and pointed leaf blade and lack of petiole (Steynen et al., 2001). We used the leaf blade length-to-width ratio to quantify leaf shape and estimate leaf types. The length of the blade was determined as the distance from the blade-to-petiole junction to the distal leaf tip and the width was determined at the midpoint of this line. This ratio approximates to 1 (rounded leaves) in juvenile leaves and increases in adult leaves (Willmann & Poethig, 2011).

Stomata and cell density

Adaxial epidermal imprints of the sixth leaf were obtained by drying off a varnish coat spread on the surface of the leaf. The imprint was peeled off and then stuck on microscope slides with one-sided adhesive. Imprints were placed under a microscope (Leitz DM RB; Leica, Wetzlar, Germany) coupled to an image analyser (BioScan-Optimas 4.10, Edmond, WA, USA). Mean cell density and stomatal density were then determined in two 0.12 mm2 zones in the middle part of the leaf blade distributed on both sides of the midvein, halfway from the margins distributed on both sides. The stomatal index was calculated as 100 × stomatal number/(stomatal number + cell number).

Net photosynthetic and transpiration rates

Gas exchanges were determined at the bolting stage, that is, just before harvest, only in the Col-0 accession. The rate of CO2 assimilation was measured using a whole-plant chamber designed for Arabidopsis (Li-Cor 6400-17, Li-Cor Inc., Lincoln, NE, USA) connected to a gas analyzer system (LI-6400XT; Li-Cor). Carbon fluxes (μmol CO2 s−1 cm−2) were determined at steady state under growing conditions (180 μmol m−2 s−1 PPFD, 20°C) and at 350 ppm reference CO2.

Transpirational water loss was determined by successive weighting of the pots over 3 d and nights (every 3 h approximately). Evaporation of water from the soil was prevented by sealing the soil surface with four layers of a plastic film. Whole-plant transpiration rate (mg H2O h−1) was estimated as the slope of the linear relationship between weight and time, and then expressed per projected rosette area (mg H2O h−1 cm−2). WUE (g mg−1 H2O), the amount of dry matter synthesized per unit of water lost, was calculated as the ratio of absolute growth rate during the period of transpiration measurement to transpiration rate. Absolute growth rate was estimated from zenithal images and converted per unit dry mass using LMA.

Sucrose and leaf ABA content

Plants were harvested at the bolting stage, in the middle of the day, and immediately frozen in liquid nitrogen to determine the sucrose and ABA content. Sucrose content was determined by enzymatic assay as in Gibon et al. (2004). Leaf ABA content (ng g−1 FM) was determined by radioimmunoassay, as previously described in Barrieu & Simonneau (2000). Leaf samples were ground finely under liquid nitrogen, placed in distilled water (5 ml mg−1 FM) and immediately warmed at 70°C for 5 min before shaking at 4°C overnight. Extracts were then centrifuged at 16 000 g for 10 min at 4°C, and the supernatant was conserved at −20°C and used for radioimmunoassay.

Statistical analyses

Comparisons of mean trait values between treatments were performed using Kruskal–Wallis nonparametric tests. The effect of inoculation on the phyllochron through time was tested using repeated-measures ANOVA (time treated as random) for each watering condition. All analyses were performed using R 2.15 (R Development Core Team, 2009).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

STM196 promotes growth of Arabidopsis Col-0 and increases plant tolerance to water deficit

STM196 had a growth-promoting effect on the Arabidopsis ecotype Col-0 under both the WW and WD soil conditions. Under WW, soil inoculation induced a 25% increase of above-ground vegetative FM at emergence of the flowering buds (i.e. bolting stage) (Fig. 1a; < 0.05), but the increase in above-ground vegetative DM was not significant (Fig. 1b). Root DM of inoculated plants increased by 30% (Fig. 1c; < 0.001) under WW. Under WD, inoculation resulted in a larger relative increase in plant size at bolting. First, above- and below-ground DM of inoculated plants were doubled and increased by 67%, respectively (Fig. 1). Secondly, under WD, above-ground FM was reduced by 80% in noninoculated plants but only by 72% in inoculated plants (Fig. 1a).

image

Figure 1. Effects of Phyllobacterium brassicacearum STM196 and water deficit on above- and below-ground mass of Arabidopsis thaliana Col-0. Data indicate the mean (± SE) shoot fresh mass (a; = 21–27; < 0.05), shoot dry mass (b; = 8; < 0.05) and root dry mass (c; = 8; < 0.001) of inoculated (I) and noninoculated (NI) plants under well-watered (WW) and water deficit (WD) conditions measured at bolting. Different letters indicate significant differences following Kruskal–Wallis test.

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The PGPR strain STM196 induces changes in the developmental dynamics of Col-0 plants

The effects of soil inoculation by STM196 on the growth and development of Col-0 appeared very early after germination. A counterintuitive observation is that the growth-promoting effect of STM196 was associated with a delay in the timing of emergence of the first leaves (i.e. inoculated plants had a higher phyllochron; Fig. 2a; < 0.001 in both watering conditions). The phyllochron of inoculated plants remained higher than the phyllochron of noninoculated plants during the vegetative phase (Fig. 2a; < 0.001 in both watering conditions) and was not the result of different germination rates between inoculated and noninoculated plants (the mean (± SE) number of d to germination was 2.83 ± 0.18 (= 40) and 2.71 ± 0.16 (= 49) for noninoculated and inoculated plants, respectively; = 0.63). The first two leaves of inoculated plants were also smaller as a result of a reduced growth rate (Fig. 2c). By contrast, the initial and the maximal relative expansion rates of the rosette (RER and Rmax, respectively) were not affected by inoculation (Figs 2f, S3b,d), and therefore inoculated plants remained smaller than noninoculated plants until these latter reached the bolting stage. However, inoculated plants produced more leaves at bolting (Fig. 2b,d), which occurred, on average, 5.5 and 12.5 d later in inoculated plants than in noninoculated plants under WW and WD, respectively (arrows in Fig. 2e).

image

Figure 2. Effects of Phyllobacterium brassicacearum STM196 and water deficit (WD) on growth and development dynamics of Arabidopsis thaliana Col-0. (a–e) Phyllochron (a), leaf number (b), area of the two first leaves (c), representative vegetative phenotypes under WD (d) and total projected leaf area (e) of inoculated (I) and noninoculated (NI) plants under well-watered (WW) and WD conditions. Data are means (± SE) of 11–13 plants, except for the area of the two first leaves values (= 9–14). Means of total leaf area and phyllochron are estimated from 3 d intervals around each time point. Arrows indicate bolting time. Insert (f) shows the log-linearized projected area in the exponential phase of vegetative growth.

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The delay in the transition from the vegetative to the reproductive phase of inoculated plants was in accordance with the changes in size and shape of the leaves (Fig. 3). The length-to-width ratio of the blades of the first two leaves was close to 1 regardless of the watering condition and the presence of bacteria in the soil (Fig. 3a,c). This ratio then increased rapidly in later adult leaves and decreased at the beginning of bolting stage. The length-to-width ratio was increased for a higher number of leaves in inoculated plants compared with noninoculated plants (Fig. 3a), which indicates that inoculated plants produced a higher number of vegetative adult leaves than noninoculated plants, notably under WD (Fig. 3b,c). Under WW conditions the production of larger leaves occurred beyond the 25th leaf. Under WD, a more pronounced increase in leaf area occurred beyond the 10th leaf (Fig. 3a). As bolting time and the duration of vegetative growth are correlated in Arabidopsis, it was not surprising to observe that inoculated plants exhibited a longer duration of rosette area expansion (Figs 2e, S3c). As a result, despite the developmental slowdown, inoculated Col-0 plants had a higher total leaf area at bolting (Fig. 2d,e), and the larger effect of STM196 under WD compared with WW resulted in a better tolerance to WD.

image

Figure 3. Effects of Phyllobacterium brassicacearum STM196 and water deficit (WD) on leaf growth and morphology of Arabidopsis thaliana Col-0. (a–d) Length-to-width ratio of the blades (a), number of juvenile and adult leaves (b), representative morphology of rosette leaves arranged from left to right by order of emergence under WD (c) and area of individual leaves (d) of inoculated (I) and noninoculated (NI) plants under well-watered (WW) and WD conditions. Data are means (± SE) of eight plants. Different letters indicate significant differences following the Kruskal–Wallis test at < 0.05.

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The developmental slowdown induced by inoculation also led to increased tolerance to water deficit in the early-flowering accession An-1

Next, we investigated the effects of STM196 on the response to WD of the Arabidopsis accession An-1, an early-flowering accession. In An-1, flowering stems emerged, on average, 16.4 and 10.8 d earlier than in Col-0 under WW and WD, respectively (solid arrows in Figs 2a, 4a). Inoculation by STM196 did not affect bolting time of An-1, but flowering time was delayed. Flowering time of inoculated plants was delayed by 2 and 5 d compared with noninoculated plants (dashed arrows in Fig. 4a; < 0.05) under WW and WD, respectively. In An-1, shoot FM at bolting was not affected by the presence of bacteria in the soil (Fig. S4a,c). However, STM196 induced a 65% increase of shoot FM and DM at flowering under WD (Fig. S4b,c, < 0.05). Inoculated plants also produced larger flowering stems under WD (Fig. 4d, P < 0.05). In this accession, root mass of inoculated plants was significantly increased under WD but not under WW conditions (Fig. S4d). The increase of shoot biomass at flowering was associated with an increase in leaf number and in individual leaf area that occurred beyond the 10th leaf (Fig. 4b,c). An-1 plants exhibited the same number of juvenile leaves regardless of the soil treatment, but inoculated plants exhibited more adult and cauline leaves (Fig. 4c).

image

Figure 4. Effects of Phyllobacterium brassicacearum STM196 and water deficit (WD) on growth and development of Arabidopsis thaliana An-1. (a–d) Total projected leaf area (a), area of individual leaves (b), number of juvenile, adult and cauline leaves (c) and length of reproductive stem (d) of inoculated (I) and noninoculated (NI) plants under well-watered (WW) and WD conditions. Data are means (± SE) of seven to 17 plants. In panel (a), solid arrows and dashed arrows indicate bolting and flowering time, respectively. Different letters indicate significant differences following the Kruskal–Wallis test at < 0.05.

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Hence, under drought, An-1 and Col-0 exhibited similar trends in response to soil inoculation by STM196. The developmental slowdown was characterised by a delayed bolting and flowering time in Col-0, whereas in the early-flowering accession An-1, only flowering time was delayed. In both accessions, the production and expansion of adult leaves were increased in response to soil inoculation by STM196.

STM196 affects whole-plant physiology and carbon status of Arabidopsis Col-0

We measured whole-plant physiology and carbon status only in Col-0 plants, before the reproductive phase. These traits were affected by both the amount of water and the presence of the bacteria in the soil. Transpiration rate was significantly reduced in response to WD in both noninoculated and inoculated plants (Fig. 5a; both < 0.01). Inoculation was also associated with a large decrease in transpiration rate regardless of the watering treatment, especially during the night (Fig. 5a; both < 0.01). This difference in transpiration rate did not result from changes in stomatal density or stomatal index, which were not significantly different among inoculated and noninoculated plants (Fig. S5). LDMC was significantly increased under WD and in inoculated plants regardless of the watering treatment (Fig. 6a, P < 0.05). However, the RWC of the rosette was only weakly affected by WD and was not affected by inoculation (Fig. S6; < 0.05). Leaf ABA content per unit FM was not significantly affected by the watering treatment but increased in response to bacteria inoculation (Fig. 5b; both < 0.001).

image

Figure 5. Effects of Phyllobacterium brassicacearum STM196 and water deficit (WD) on plant physiology and carbon status of Arabidopsis thaliana Col-0. (a–d) Night-time and daytime transpiration (a), ABA content in leaves (b), photosynthesis (c) and sucrose content (d) of inoculated (I) and noninoculated (NI) plants under well-watered (WW) and WD conditions. Data are means ± SE of six to eight plants. Different letters indicate significant differences following the Kruskal–Wallis test.

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image

Figure 6. Effects of Phyllobacterium brassicacearum STM196 and water deficit (WD) on water status of Arabidopsis thaliana Col-0. (a, b) Leaf dry matter content (a) and water-use efficiency (b) of inoculated (I) and noninoculated (NI) plants under well-watered (WW) and WD conditions. Data are means ± SE of seven to eight plants. Different letters indicate significant differences following the Kruskal–Wallis test at < 0.05.

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The net photosynthetic rate was not significantly affected by the watering treatment but it was significantly reduced in the presence of bacteria in the soil (Fig. 5c, P < 0.05). The leaf carbon status was modified by the watering treatment and the bacteria. Sucrose contents were increased under WD and by the presence of the bacteria in the soil (Fig. 5d; < 0.05).

Water-use efficiency of noninoculated plants was not affected by WD (Fig. 6b). WUE was also not impacted by soil inoculation under WW conditions. However, it was significantly increased in the presence of bacteria in the soil under WD (Fig. 6b, P < 0.05).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant growth-promoting rhizobacteria can enhance plant performance and plant tolerance to environmental stresses by a large variety of mechanisms (for reviews, see Lugtenberg & Kamilova, 2009; Friesen et al., 2011). These mechanisms have to be elucidated to design strategies for PGPR application in agriculture (for review, see Lucy et al., 2004). Here, we show that Phyllobacterium brassicacearum strain STM196, a PGPR isolated from the rhizosphere of oilseed rape B. napus (Bertrand et al., 2001; Larcher et al., 2003), enhances plant tolerance to drought in two accessions of A. thaliana with contrasting flowering phenology. We highlight a new means by which bacteria can enhance plant performance under both well-watered and drought soil conditions. Specifically, our results show for the first time a PGPR-induced delay in the transition from vegetative to reproductive development. Inoculated plants accumulated more biomass before reproduction and exhibited a better WUE.

STM196 induces a delay in reproductive timing that leads to increased biomass accumulation

The switch from vegetative to reproductive development is highly critical for wild and crop species. Indeed, the timing of flowering is a key event that determines the production of plant biomass and therefore yield (Jung & Muller, 2009). Floral transition can be influenced by abiotic changes in the environment, such as day length, ambient temperature and water availability (Bernier & Perilleux, 2005), and by endogenous stimuli (Huijser & Schmid, 2011). Multiple pieces of evidence support the fact that plant growth rate and the duration of the growth phases depend on flowering time. Alteration of flowering time by genetic modifications or photoperiod resulted in accelerated or decelerated progress towards the vegetative phases of Arabidopsis (Steynen et al., 2001) and others species (Salehi et al., 2005). In addition, close relationships among leaf production, individual leaf growth and flowering time have also been reported (Cookson et al., 2007). Here, we showed that Arabidopsis plants grown in soil inoculated by STM196 exhibit contrasting growth dynamics and phenology. Developmental changes induced by STM196 appeared very early during plant development. For instance, the phyllochron was increased in inoculated plants as early as the emergence of the two first leaves until the reproductive phase. The most noticeable phenological change was a significant delay in flowering time in inoculated plants. This delay coincided with a prolonged adult vegetative phase, as indicated by the postponed morphological transition between adult and cauline leaves. Moreover, inoculated plants exhibited a prolonged production of adult vegetative and cauline leaves that resulted in a higher number of both types of leaves. Previous studies reported that delaying flowering time as a result of shortening photoperiod coincided with a longer duration of leaf production and growth (Koornneef et al., 1998; Cookson et al., 2007). Here, the prolonged vegetative growth and the delayed flowering of PGPR-inoculated plants led to a greater production of vegetative and reproductive biomass. Strikingly, the direction of changes was similar in the two watering regimes, but the intensity of changes was more pronounced under drought and led to better plant tolerance to drought. PGPR effects on the timing of flowering are not common and we found no study reporting a PGPR-induced delay in flowering time. Growth promotion by rhizobacteria is often shown at a given date after germination or inoculation (Ryu et al., 2003; Jaleel et al., 2007; Zahir et al., 2008) and we lack a precise study of their effects on growth dynamics and development. Recently, it has been shown that A. thaliana plants inoculated with a naturally associated rhizobacterium, Pseudomonas sp., exhibited a faster rate of development – plants reached the floral transition earlier – and were bigger (Schwachtje et al., 2011), indicating that various PGPR strains mediate different plant responses.

Rhizobacteria often induce modifications in phytohormone signalling (for a review, see Yang et al., 2009), which may mediate effects on meristem activity and identity (Hayat et al., 2010). Our results showed that ABA was increased in STM196-inoculated plants. By contrast with other PGPR strains, STM196 is not a high auxin producer (Contesto et al., 2010) and, thus, cannot supply plant roots with extra auxin. However, it has been shown that inoculation with STM196 changed auxin distribution within Arabidopsis roots towards apices, which probably explains the positive effect of STM196 on lateral root development (Contesto et al., 2010). It is worth mentioning these effects of STM196 on auxin distribution because this hormone also plays a role in the regulation of leaf and floral initiation and of the position of lateral organs (Reinhardt et al., 2000). In addition, other hormonal pathways are modified by STM196, including ethylene, which participates in root hair elongation in vitro (Contesto et al., 2008; Galland et al., 2012). Further investigations are required to disentangle the interactions between signalling pathways that might explain the developmental changes following STM196 inoculation (Achard et al., 2006).

These results are novel in the context of plant–microorganism interactions and are promising for agronomy. Reducing or eliminating flowering by altering the endogenous mechanisms involved in the flowering pathway is one of the strategies to increase the yield of biomass crops (Jung & Muller, 2009). For instance, overexpression of the Arabidopsis floral repressor gene, FLOWERING LOCUS C (FLC), in tobacco resulted in a significant delay in flowering time and a concomitant increase in the biomass yield (Salehi et al., 2005). In vegetative crops such as cabbage (Brassica oleracea), early bolting and flowering limit the potential for yield increases (Jung & Muller, 2009). Therefore, manipulation of flowering time through rhizospheric flora can have important applications in stressed conditions, but underlying regulatory genes remain to be investigated.

The growth slowdown of STM196-inoculated plants, superimposed on that of water deficit, contributes to lifetime water economy and to increased drought resistance

Multiple combinations of traits can participate in plant strategies for dealing with drought, including those that allow drought escape or drought resistance (Verslues & Juenger, 2011). In addition, several soil microorganisms, including PGPR, can represent an added value to these strategies. For instance, some rhizobacteria help plants to maintain a favourable water status under water deficit (Creus et al., 2004), by enhancing the development of the root system (Marulanda et al., 2009). Here, we quantified numerous morphophysiological traits related to plant growth and development in order to decipher the added value of the PGPR STM196 to the drought response strategies of Arabidopsis. The automated phenotyping platform PHENOPSIS allowed the water limitation in the soil to be precisely controlled and maintained from as early as germination and up to the reproductive phase. Steady-state drought as applied here during the whole-plant cycle is highly relevant for the study of plant acclimation to drought (Verslues & Juenger, 2011). Acclimation processes during steady-state drought may reinforce plant resistance to this stress. Here, the soil water deficit was strong enough to cause an 80% decrease in above-ground FM of noninoculated plants at bolting. This biomass reduction is comparable to previous reports using a similar experimental procedure (daily irrigation to a steady-state soil water content) and similar intensities of drought applied to Col-0 (Hummel et al., 2010; Vile et al., 2012) and other accessions (Tisne et al., 2010).

Reduced plant size and total leaf area are common plant strategies to reduce water consumption and therefore drought injury (Tardieu et al., 2011). Indeed, we recently showed that the inherent size of various Arabidopsis ecotypes was negatively related to drought resistance (Vile et al., 2012), and that mutants that cope better with extreme stress often display a dwarfed stature (see references in Skirycz & Inze, 2010). Our results suggest that the growth slowdown of inoculated plants, superimposed on that of water deficit, has contributed to lifetime water economy and to increased drought resistance. In addition, as found in a previous study performed under similar drought scenarios, the reproductive timing under drought tended to occur earlier in Col-0 and later in An-1 (Vile et al., 2012). This illustrates the variability in drought response strategies in terms of reproductive phenology, and contrasts with the generally held view that drought escape is a common strategy of Arabidopsis (Verslues & Juenger, 2011). However, as in well-watered conditions, inoculated plants of both accessions exhibited delayed reproductive timing under drought. Inoculated plants accumulated twice as much biomass and produced more leaves of a larger area before flowering, had bigger reproductive stems and therefore higher expected reproductive yield. Several lines of evidence point to a higher survival and seed production of later-flowering Arabidopsis accessions (Korves et al., 2007). The timing of flowering often correlates with abiotic and biotic stress avoidance, which is frequently scored as a component of yield, for example in maize (Chardon et al., 2004). In addition, quantitative trait loci for adaptation to drought are often related to flowering time loci (Ducrocq et al., 2008). The advantage of a delay is that there is more time to accumulate more mass that can be invested towards seeds (Metcalf & Mitchell-Olds, 2009). Among the drawbacks of such a strategy, plants have to maintain a favourable use of water during a longer period, especially under drought conditions.

We showed that WUE of inoculated Col-0 plants was significantly improved under water deficit. We did not find any significant change in WUE in response to drought in noninoculated plants, in contrast to previous Arabidopsis studies that reported an increase in WUE (Juenger et al., 2005; Aubert et al., 2010). Interestingly, McKay et al. (2003) reported that higher WUE was genetically correlated with delayed flowering in Arabidopsis. Here, higher WUE of inoculated plants was mainly a result of a significantly lower water loss through daytime and night-time transpiration, which may reflect a better drought avoidance strategy. However, as reported by Westgate & Boyer (1985), a decrease in transpiration (by stomatal closure) can be followed on a longer timescale by a reduced plant growth rate, as was observed here in inoculated plants. We also reported a decline in photosynthesis in inoculated plants, but the sucrose content in leaves was increased regardless of the soil condition. This is in accordance with the literature reporting an increase in sucrose content in leaves even if CO2 diffusion is lowered under water deficit (Quick et al., 1992; Hummel et al., 2010). This could be a result of the uncoupling between photosynthesis and growth under water deficit (Muller et al., 2011). Higher concentrations of ABA in the leaves of inoculated plants can explain the lower transpiration rate resulting from stomatal closure. Some bacteria have the capacity to modulate gas exchanges and ABA metabolism. In A. thaliana, Zhang et al. (2008) interpreted the augmentation of photosynthetic rate in plants inoculated with Bacillus subtilis as being the result of decreased ABA concentrations in planta. In the common bean, Paenibacillus polymyxa and Rhizobium tropici coinoculation has been shown to decrease the ABA content in response to WD (Figueiredo et al., 2008). In addition to acting on the biosynthesis of ABA, some pathogenic bacteria can also modify stomatal opening by acting downstream of ABA biosynthesis. This is the case with Pseudomonas syringae, which exude coronatine, a substance that inhibits ABA signalling and prevents stomatal closure (Melotto et al., 2006). By contrast, Cohen et al. (2009) found a twofold increase in ABA concentrations in Azospirillum brasilense-inoculated Arabidopsis. Bacteria-induced increase of ABA content has been proposed to play a role in alleviation of the drought effect in maize (Cohen et al., 2009). Such an observation would be consistent with our results.

Roots also play a key role in WUE and adaptation to drought. Root biomass was higher in STM196-inoculated plants, and modifications of the root architecture as a result of the presence of the bacteria may have enhanced the water absorption capacity. Indeed, studies performed in gnotobiotic conditions showed that STM196 increased lateral root length (Mantelin et al., 2006; Kechid et al., 2013), and the density and length of root hairs (Galland et al., 2012). Both effects must lead to a greater exchange surface with soil and consequently higher water flux through the whole root system up to the leaves.

Conclusion

Overall, our results show that the PGPR P. brassicacearum STM196 induces a suite of developmental and physiological changes that represent a significant added value to the drought response strategy of Arabidopsis. Developmental and early growth slowed down, but prolonged vegetative growth and reduced transpiration contributed to increasing drought resistance and WUE. Prolonged vegetative growth and delayed flowering induced by PGPR are new in the context of plant–microorganism interactions and may be promising for agronomy. Reducing or eliminating flowering by altering the endogenous mechanisms involved in flowering is one of the strategies for increasing crop yield. Delaying flowering time by rhizobacteria inoculation could represent a valuable strategy for increasing biomass yield.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Christine Granier, Bertrand Muller and François Vasseur for comments on previous versions of the present paper; and Myriam Dauzat, Gaëlle Rolland, Alexis Bediee, Frederic Bouvery, Jessica Kok and Crispulo Balsera for help during the experiments. J.B. was funded by the French Ministry of Higher Education and Research.

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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Fig. S1 Time courses of meteorological parameters.

Fig. S2 Time course of mean relative soil water content during plant growth.

Fig. S3 Duration and rate of rosette expansion of A. thaliana Col-0.

Fig. S4 Effects of P. brassicacearum STM196 and water deficit on above- and below-ground mass of A. thaliana An-1.

Fig. S5 Effects of P. brassicacearum STM196 and water deficit on anatomical leaf traits of A. thaliana Col-0.

Fig. S6 Effects of P. brassicacearum STM196 and water deficit on relative water content of A. thaliana Col-0.

Table S1 Replicate numbers per trait and conditions