The arbuscular mycorrhizal (AM) symbiosis enhances plant tolerance to water deficit through …

• Abstract
• Implications
• Materials and Methods
• Results
• Discussion
• Conclusion
• Acknowledgments
• References
• Figures

Mycorrhizal colonization
Uninoculated control plants did not show mycorrhizal colonization.Plants inoculated with G. intraradices showed a percentage ofroot colonization ranging from 45% to 70% of root length (Fig. 1).The application of ABA did not affect the colonization of rootby G. intraradices. The only differences in root colonizationwere due to the water regime. In fact, plants cultivated underwell-watered conditions showed 45% to 50% of mycorrhizal rootlength. In contrast, plants subjected to drought showed 62%to 70% of root colonization.

Plant growth
In order to obtain AM and non-AM plants of similar size beforestarting the drought and recovery treatments, non-AM plantsreceived an application of nutrient solution. Therefore, underwell-watered conditions AM and non-AM plants had similar shootand root dry weights either with ABA or without ABA (Figs 2,3). Under drought-stress conditions and in the absence of exogenousABA, AM and non-AM plants also exhibited similar shoot dry weight,while roots of AM plants grew more after the recovery period(Fig. 3). By contrast, in the presence of exogenous ABA anddrought stress, AM plants had 34% more shoot biomass than non-AMplants, but the difference in root dry weight was not significant.

Shoot dry weight decreased in all treatments as a consequenceof drought, except in plants inoculated with G. intraradicesand supplied with exogenous ABA that showed similar shoot dryweight to those under well-watered conditions.

The application of exogenous ABA decreased shoot dry weightin all treatments, except in AM plants subjected to drought.Exogenous ABA also decreased the root dry weight of non-AM plantssubjected to drought and of AM plants subjected to drought afterthe recovery period. By contrast, exogenous ABA did not affectthe root dry weight of plants maintained under well-wateredconditions.

In the absence of ABA the 3 d recovery period allowed furtherincrease of shoot dry weight of the AM plants kept under well-wateredconditions (Fig. 2). Drought-stressed plants also increasedplant growth during the recovery period, mainly the root dryweight of AM plants (Fig. 3). In the presence of ABA, the recoveryperiod did not significantly increase the shoot dry weight,either under well-watered conditions or under drought-stressconditions. By contrast, the root dry weight of AM plants increasedsignificantly after the recovery period.

Root hydraulic conductivity (L_o)
Plants subjected to drought stress were unable to exude afterdetachment of the shoots. Hence, L_o could not be measured inany of the treatments subjected to drought. L_o could only bemeasured in well-watered plants and in plants recovered fromdrought (Fig. 4).

Well-watered AM plants had higher L_o than non-AM plants, bothwith and without ABA application (increasing by 47% and 89%,respectively). This effect was also maintained after the recoveryperiod. AM plants subjected to drought stress and recoveredfor 3 d also showed higher L_o than non-AM plants, both withand without ABA application (increasing by 346% and 520%, respectively).

The application of ABA to plants cultivated under well-wateredconditions enhanced L_o by 81% in non-AM plants and by 41% inAM ones. The same trend was observed after the extra 3 d recoveryperiod for AM and non-AM plants maintained under well-wateredconditions.

Plants that had been subjected to drought stress and recoveredfor 3 d also showed an important increase of L_o by ABA application(260% increase for non-AM plants and 158% increase for AM plants),AM plants always exhibited higher L_o than non-AM plants. However,even after recovery from the stress, the plants that had beensubjected to drought stress always showed lower L_o than thosemaintained under well-watered conditions. Only the AM plantsthat had exogenous ABA added recovered their L_o to values similarto those of well-watered plants.

Transpiration rate
Under well-watered conditions the transpiration rate was similarin AM and non-AM plants, regardless of ABA application (Fig. 5).By contrast, under drought-stress conditions, AM plants showeda lower transpiration rate than non-AM plants, both with andwithout ABA application.

Drought stress decreased the transpiration rate both in AM andin non-AM plants, this decrease being more pronounced in AMplants. After the recovery from stress for 3 d, all the treatmentsrecovered their transpiration rate to levels similar to thoseof well-watered plants. This effect was unrelated to ABA application.

The application of ABA decreased the transpiration rate bothin AM and in non-AM plants, but only under well-watered conditions.In plant subjected to drought, the application of ABA did notreduce the transpiration rate further.

ABA content
ABA content was measured in leaves and roots. In leaves, thedrought stress considerably enhanced (by 388%) the ABA contentin plants colonized by G. intraradices, while it had no effecton non-AM plants (Fig. 6). However, after the recovery period,AM plants subjected to drought considerably decreased theirABA content reaching levels similar to those of non-inoculatedplants maintained under well-watered conditions. By contrast,when exogenous ABA was added to the plant, the non-AM plantsalso increased their ABA content considerably as a consequenceof drought. Under such conditions, the increase of ABA contentwas even higher for non-AM plants than for AM ones. In the presenceof exogenous ABA, the level of ABA in leaves of non-AM lettuceplants that had been previously subjected to drought did notsignificantly decrease after recovery, while the decrease afterrecovery in AM plants was significant (by 44%).

In roots, the picture is considerably different (Fig. 7). Infact, drought stress did not increase the ABA content in roots.Surprisingly, roots of non-AM plants cultivated under well-wateredconditions enhanced their ABA content after the 3 d recoveryperiod. The application of exogenous ABA to the plants enhancedthe ABA content of plants inoculated with G. intraradices, mainlywhen subjected to drought stress (increasing by 216% comparedwith the absence of exogenous ABA). In the presence of exogenousABA, the recovery from drought only decreased the content ofABA in the roots of AM plants, while non-AM plants even increasedtheir ABA content.

Gene expression
The expression of four stress-related plant genes was analysedby northern blot both in shoot and root tissues of lettuce plantsunder the various growing conditions tested in this work. Exceptfor the LsPIP2 gene, the pattern of gene expression was quitesimilar in shoots and roots (Figs 8, 9).

Gene Lsnced
In the absence of exogenous ABA, the expression of the Lsncedgene was only remarkable in shoots and roots of plants colonizedby G. intraradices and subjected to drought, although the expressionof this gene could also be detected in shoots of non-AM plantssubjected to drought (Figs 8, 9). The application of exogenousABA considerably enhanced the expression of this gene in shootsand roots of non-AM plants subjected to drought, while in AMplants it did not significantly change the expression patternin roots (Fig. 9) or decreased it slightly in shoots (Fig. 8).Well-watered plants and plants recovered from drought did notexpress the Lsnced gene.

Gene LsPIP2
In the absence of exogenous ABA, the expression of the LsPIP2gene was inhibited by drought stress both in the shoots androots, mainly in AM plants, that showed the lowest rate of geneexpression (Figs 8, 9). In shoots, the AM plants cultivatedunder well-watered conditions always exhibited about 45% reductionof gene expression compared with non-AM plants (Fig. 8). Inany case, in shoots, AM and non-AM plants that had been subjectedto drought, recovered the expression rate of the LsPIP2 geneafter the recovery period. By contrast, in roots, the expressionrate of the LsPIP2 gene in the plants that had been previouslysubjected to drought only partially recovered after the recoveryperiod (Fig. 9). The expression rate of this gene in roots ofAM and non-AM plants that were maintained under well-wateredconditions was considerably lower than before the recovery period.

The application of exogenous ABA considerably inhibited thegene expression in shoots of AM and non-AM plants, regardlessof water treatment (Fig. 8). By contrast, in roots, the applicationof ABA to these plants did not significantly change the rateof gene expression (Fig. 9). The plants with added exogenousABA having recovered from a previous drought also recoveredthe expression rate of the LsPIP2 gene in shoots and in roots.By contrast, roots of non-AM plants that had been maintainedunder well-watered conditions showed reduced gene expression(Fig. 9).

Gene Lsp5cs
In the absence of exogenous ABA, this gene was only expressedin plants subjected to drought, mainly in AM plants (both shootsand roots) (Figs 8, 9). A residual expression was observed afterthe recovery period in plants that had been previously drought-stressed.In any case, the application of exogenous ABA considerably inducedthe expression of this gene in shoots and roots of non-AM plantsonly (even under well-watered conditions) so, under drought-stressconditions, the expression of that gene was higher than in AMplants. Again, a residual expression of the Lsp5cs gene wasobserved after the recovery period in plants that had previouslybeen drought-stressed.

Gene Lslea
In the absence of exogenous ABA, the Lslea gene was only expressedin plants subjected to drought, mainly in AM plants (both shootsand roots) (Figs 8, 9). No expression was detected in well-wateredplants or after the recovery period. The application of exogenousABA considerably induced the expression of this gene in shootsand roots, mainly in the case of non-AM plants. In fact, afterthe addition of exogenous ABA, the expression of that gene underdrought-stress conditions was higher in non-AM plants than inAM ones. Again, no expression was detected in well-watered plantsor after the recovery period.