Physiological and growth responses to water deficit in the bioenergy crop Miscanthus x giganteus

Abstract

High yielding perennial biomass crops of the species Miscanthus are widely recognized as one of the most promising lignocellulosic feedstocks for the production of bioenergy and bioproducts. Miscanthus is a C4 grass and thus has relatively high water use efficiency. Cultivated Miscanthus comprises primarily of a single clone, Miscanthus x giganteus, a sterile hybrid between M. sacchariflorus and M. sinensis. M. x giganteus is high yielding and expresses desirable combinations of many traits present in the two parental species types; however, it responds poorly to low water availability. To identify the physiological basis of the response to water stress in M. x giganteus and to identify potential targets for breeding improvements we characterized the physiological responses to water-deficit stress in a pot experiment. The experiment has provided valuable insights into the temporal aspects of drought-induced responses of M. x giganteus. Withholding water resulted in marked changes in plant physiology with growth-associated traits among the first affected, the most rapid response being a decline in the rate of stem elongation. A reduction in photosynthetic performance was among the second set of changes observed; indicated by a decrease in stomatal conductance followed by decreases in chlorophyll fluorescence and chlorophyll content. Measures reflecting the plant water status were among the last affected by the drought treatment. Metabolite analysis indicated that proline was a drought stress marker in M. x giganteus, metabolites in the proline synthesis pathway were more abundant when stomatal conductance decreased and dry weight accumulation ceased. The outcomes of this study in terms of drought-induced physiological changes, accompanied by a proof-of-concept metabolomics investigation, provide a platform for identifying targets for improved drought-tolerance of the Miscanthus bioenergy crop.

Keywords: Miscanthus; bioenergy; drought; metabolite profiling; physiology; stress; water deficit.

FIGURE 1

Soil moisture. Soil moisture content was measured every 2 days over a period of 32 days. The last watering of the drought stressed plants was on day 12. The control plants continued to be watered every 2 days for the duration of the experiment. A significant decline in soil moisture occurred on day 16, 4 days after final watering, with a steady decline over the remaining period to levels similar to drought in grassland ecosystems.

FIGURE 2

Leaf relative water content and above ground biomass moisture content. The control plants maintained a constant leaf RWC of between 72 and 80% throughout the duration of the investigation (A). In water-stressed plants leaf RWC declined from 80% at the start of the investigation to <20% by day 32. Total above ground biomass moisture content followed a very similar pattern to the leaf RWC (B).

FIGURE 3

Stem elongation. Control plants showed a fairly constant rate of stem elongation throughout the investigation (A). Water-stressed plants showed a significant decrease in elongation rate from day 20 (A) when the soil moisture decreased to <0.2 m³ m^-3 (B).

FIGURE 4

Leaf area and expansion of leaf 0. Leaf area showed a linear increase in control plants throughout the experiment while no significant increase after day 26 was observed in water-stressed plants (A). Rate of leaf expansion remained constant in control plants and decreased gradually in drought treatment plants (B). The leaf expansion rate became significantly different between the two treatments at day 28.

FIGURE 5

Above ground biomass. Fresh (A) and dry weights (B) of the sampled plants were measured at each harvesting point (n = 7). Fresh and dry biomass of control plants increased throughout the experiment. Water-stressed plants only showed a small increase in fresh weight between day 12 and 24 and a significant decrease at day 32. Dry weight accumulation was similar between the two treatments up to day 24 but dry biomass did not increase further in stressed plants between day 24 and 32.

FIGURE 6

Chlorophyll content. Chlorophyll content was measured in five leaves per plant using a SPAD meter. Leaf 0 is shown because no significant differences were seen between the different leaves. Chlorophyll content was maintained in the well-watered plants for the duration of the experiment and decreased from day 28 under water-stressed conditions.

FIGURE 7

Chlorophyll fluorescence. The maximum quantum yield (F_v/F_m) (A) was maintained in well-watered plants for the duration of the experimentanddecreased significantly in water-stressed plants from day 28. F_v/F_m was measured in three leaves per plant and no significant differences were seen between the different leaves. Shown here is leaf 0. Performance index (B), which incorporates more parameters than (F_v/F_m), was compared across the two treatments and was slightly more sensitive to drought differing significantly between the two treatments at day 26.

FIGURE 8

Stomatal conductance. Under controlled well-watered conditions stomatal conductance remained constant throughout the experiment. Water stress caused increased stomatal resistance and therefore a decrease in stomatal conductance, this became significantly different between the two treatments at day 24.

FIGURE 9

Principal components analysis (PCA) of metabolite profiles from well-watered and water-stressed Miscanthus plants. Polar and non-polar extracts from leaf samples of Miscanthus plants at the start of the experiment day 12 (T0) and from well-watered control plants at day 24 (C1) and day 32 (C2) and also from water-stressed plants at day 24 (D1) and day 32 (D2) were analyzed using direct infusion electrospray ionization mass-spectrometry (DI-ESI-MS). Derived spectra were analyzed by PCA both including C2, D2 (A, D) and excluding C2, D2 (E, F). PCA plots (A) and (D) are based on data for all metabolites in the spectrum. PCA plots (E) and (F) are based on m/z within the spectra which were tentatively linked to metabolites forming the proline biosynthetic pathway namely L-glutamate (147 m/z);L-ornithine (132 m/z); L-glutamate-semi-aldehyde (131 m/z), (S)-pyrroline-5-carboxylate (113 m/z), and L-proline (115 m/z). (B) The percentage contribution by each m/z to the variation explained by PC1 in (A). (C) Percentage to total ion count contributed by each of the five metabolites tentatively associated with proline biosynthesis.

FIGURE 10

Time-course of physiological changes observed after drought treatment. Soil moisture content decreased significantly 4 days after drought treatment (DADT). The earliest physiological change observed was a decrease in stem elongation (8 DADT) followed by a significant decrease of stomatal conductance and dry weight accumulation (12 DADT). The multi-parametric performance index which incorporates the main photochemical processes was significantly affected at 14 DADT. Leaf expansion rate decreased significantly 16 DADT, although a declining trend could be observed from 8 DADT onward. A significant decline in chlorophyll fluorescence at 16 DADT was accompanied by a decrease of chlorophyll content. Finally, above ground biomass moisture content, fresh weight and RWC all declined significantly at 20 DADT. Numbers in parenthesis indicate days after start of drought treatment. The DADT indicated after each physiological event indicates the day that a significant effect was first observed. Gray vertical bars indicate days for destructive harvests.