Osmolyte accumulation: can it really help increase crop yield under drought conditions?

Osmolyte accumulation (OA) is frequently cited as a key putative mechanism for increasing yields of crops subjected to drought conditions. The hypothesis is that OA results in a number of benefits that sustain cell and tissue activity under water-deficit conditions. It has been proposed as an effective tolerance mechanism for water deficits, which could be enhanced in crops by traditional plant breeding, marker-assisted selection or genetic engineering, to generate drought-tolerant crops. However, field studies examining the association between OA and crop yield have tended to show no consistent benefit. The few, often-cited, investigations with positive associations were obtained under severe water deficits with extremely low yields or conditions with special water-supply scenarios when much of the benefit is plant survival. Under conditions where water deficits threaten crop survival, yields are so low that even large fractional yield gains offer little practical benefit to growers. Indeed, the often-cited benefit of turgor maintenance in cells is likely to result in crop behaviour that is exactly opposite to what is beneficial to crops. The one clear mechanism identified in this review for beneficial yield responses to OA is in the maintenance of root development in order to reach water that may be available deeper in the soil profile.     

Evidence that carbon dioxide enrichment alleviates ureide-induced decline of nodule nitrogenase activity

The hypothesis that elevated [CO(2)] alleviates ureide inhibition of N(2)-fixation was tested. Short-term responses of the acetylene reduction assay (ARA), ureide accumulation and total non-structural carbohydrate (TNC) levels were measured following addition of ureide to the nutrient solution of hydroponically grown soybean. The plants were exposed to ambient (360 micromol mol(-1)) or elevated (700 micromol mol(-1)) [CO(2)]. Addition of 5 and 10 mM ureide to the nutrient solution inhibited N(2)-fixation activity under both ambient and elevated [CO(2)] conditions. However, the percentage inhibition following ureide treatment was significantly greater under ambient [CO(2)] as compared with that under elevated [CO(2)]. Under ambient [CO(2)] conditions, ARA was less than that under elevated [CO(2)] 1 d after ureide treatment. Under ambient [CO(2)], the application of ureide resulted in a significant accumulation of ureide in all plant tissues, with the highest concentration increases in the leaves. However, application of exogenous ureide to plants subjected to elevated [CO(2)] did not result in increased ureide concentration in any tissues. TNC concentrations were consistently higher under elevated [CO(2)] compared with those under ambient [CO(2)]. For both [CO(2)] treatments, the application of ureide induced a significant decrease of TNC concentrations in the leaves and nodules. For both leaves and nodules, a negative correlation was observed between TNC and ureide levels. Results indicate that product(s) of ureide catabolism rather than tissue ureide concentration itself are critical in the regulation of N(2)-fixation.     

Salt stress induces a decrease in the oxygen uptake of soybean nodules and in their permeability to oxygen diffusion

The effects of short-term NaCl-salinity on nodules of soybean ( Glycine max L. cv. Kingsoy) were studied on hydroponically-grown plants. Both acetylene reducing activity (ARA) and nodule respiration (O₂ uptake and CO₂ evolution) were immediately inhibited, and the stimulation of them by rising the external partial pressure of O₂ (pO₂) was diminished by the application of 0.1 M NaCl in the nutrient solution. The permeability of the nodule to O₂ diffusion, estimated by O₂ consumption or CO₂ evolution, was significantly lower in the stressed nodules than in the cootrol ones. The respiratory quotient of intact nodules and the ethanol production of excised nodules were increased by low pO₂ and by salt stress. These data confirm that in salt-stressed soybean nodules, O₂ availability is reduced and fermentative pathways are stimulated.     

Review article. Symbiotic N2 fixation response to drought

Symbiotic nitrogen fixation is highly sensitive to drought, which results in decreased N accumulation and yield of legume crops. The effects of drought stress on N2 fixation usually have been perceived as a consequence of straightforward physiological responses acting on nitrogenase activity and involving exclusively one of three mechanisms: carbon shortage, oxygen limitation, or feedback regulation by nitrogen accumulation. The sensitivity of the nodule water economy to the volumetric flow rate of the phloem into the nodule offers a common framework to understand each of these mechanism. As these processes are sensitive to volumetric phloem flow into the nodules, variations in phloem flow as a result of changes in turgor pressure in the leaves are likely to cause rapid changes in nodule activity. This could explain the special sensitivity of N2 fixation to drying soils. It seems likely that N feedback may be especially important in explaining the response mechanism in nodules. A number of studies have indicated that a nitrogenous signal(s), associated with N accumulation in the shoot and nodule, exists in legume plants so that N2 fixation is inhibited early in soil drying. The existence of genetic variation in N2 fixation response to water deficits among legume cultivars opens the possibility for enhancing N2 fixation tolerance to drought through selection and breeding.     

Soybean nodulation and N2 fixation response to drought under carbon dioxide enrichment

The combined effects of carbon dioxide (CO₂) enrichment and water deficits on nodulation and N₂ fixation were analysed in soybean [Glycine max (L.) Merr.]. Two short-term experiments were conducted in greenhouses with plants subjected to soil drying, while exposed to CO₂ atmospheres of either 360 or 700 μmol CO₂ mol^–1. Under drought-stressed conditions, elevated [CO₂] resulted in a delay in the decrease in N₂ fixation rates associated with drying of the soil used in these experiments. The elevated [CO₂] also allowed the plants under drought to sustain significant increases in nodule number and mass relative to those under ambient [CO₂]. The total non-structural carbohydrate (TNC) concentration was lower in the shoots of the plants exposed to drought; however, plants under elevated CO₂ had much higher TNC levels than those under ambient CO₂. For both [CO₂] treatments, drought stress induced a substantial accumulation of TNC in the nodules that paralleled N₂ fixation decline, which indicates that nodule activity under drought may not be carbon limited. Under drought stress, ureide concentration increased in all plant tissues. However, exposure to elevated [CO₂] resulted in substantially less drought-induced ureide accumulation in leaf and petiole tissues. A strong negative correlation was found between ureide accumulation and TNC levels in the leaves. This relationship, together with the large effect of elevated [CO₂] on the decrease of ureide accumulation in the leaves, indicated the importance of ureide breakdown in the response of N₂ fixation to drought and of feedback inhibition by ureides on nodule activity. It is concluded that an important effect of CO₂ enrichment on soybean under drought conditions is an enhancement of photoassimilation, an increased partitioning of carbon to nodules and a decrease of leaf ureide levels, which is associated with sustained nodule growth and N₂ rates under soil water deficits. We suggest that future [CO₂] increases are likely to benefit soybean production by increasing the drought tolerance of N₂ fixation.     

Water-use efficiency and carbon isotope discrimination of <Emphasis Type="Italic">Acacia ampliceps</Emphasis> and <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis> at different soil moisture regimes under semi-arid conditions

Acacia ampliceps Maslin and Eucalyptus camaldulensis Dehnh. were grown for one year in lysimeters at three soil moisture regimes: 100 % (well-watered), 75 % (medium-watered) and 50 % (low-watered) of total plant available water. Biomass yield of both species increased with increase in soil moisture. Water-use efficiency (WUE) of E. camaldulensis decreased and that of A. ampliceps increased markedly with decrease in available soil moisture. A. ampliceps showed 4 – 5 times more biomass yield than E. camaldulensis grown at similar soil moisture. A. ampliceps showed almost 5, 9 and 12 times higher WUE than E. camaldulensis under low-, medium- and well-watered treatments, respectively. Significant negative correlation of ¹³C with WUE (r = –0.99) was observed in A. ampliceps. In contrast, ¹³C of E. camaldulensis showed a significant positive correlation with WUE (r = 0.82).     

Manganese application alleviates the water deficit-induced decline of N2 fixation

Water deficit is a very serious constraint on N₂ fixation rates and grain yield of soybean (Glycine max Merr.). Ureides are transported from the nodules and they accumulate in the leaves during soil drying. This accumulation appears responsible for a feedback mechanism on nitrogen fixation, and it is hypothesized to result from a decreased ureide degradation in the leaf. One enzyme involved in the ureide degradation, allantoate amidohydrolase, is manganese (Mn) dependent. As Mn deficiency can occur in soils where soybean is grown, this deficiency may aggravate soybean sensitivity to water deficit. In situ ureide breakdown was measured by incubating soybean leaves in a 5 mol m ^{? 3} allantoic acid solution for 9 h before sampling leaf discs in which remnant ureide was measured over time. In situ ureide breakdown was dramatically decreased in leaves from plants grown without Mn. At the plant level, allantoic acid application in the nutrient solution of hydroponically grown soybean resulted in a higher accumulation of ureide in leaves and lower acetylene reduction activity (ARA) by plants grown with 0 mol m ^{? 3} Mn than those grown with 6·6 mol m ^{? 3} Mn. Those plants grown with 6·6 mol m ^{? 3} Mn in comparison with those grown with 52·8 mol m ^{? 3} Mn had, in turn, higher accumulated ureide and lower ARA. To determine if Mn level also influenced N₂ fixation sensitivity to water deficit, a dry‐down experiment was carried out by slowly dehydrating plants that were grown in soil under four different Mn nutritions. Plants receiving no Mn had the lowest leaf Mn concentration, 11·9 mg kg ^{? 1}, and had N₂ fixation more sensitive to water deficit than plants treated with Mn in which leaf Mn concentration was in the range of 21–33 mg kg ^{? 1}. The highest Mn treatments increased leaf Mn concentration to 37·5 mg kg ^{? 1} and above but did not delay the decline of ARA with soil drying, although these plants showed a significant increase in ARA under well‐watered conditions.     

Involvement of Ureides in Nitrogen Fixation Inhibition in Soybean

The sensitivity of N₂ fixation to drought stress in soybean (Glycine max Merr.) has been shown to be associated with high ureide accumulation in the shoots, which has led to the hypothesis that N₂ fixation during drought is decreased by a feedback mechanism. The ureide feedback hypothesis was tested directly by measuring the effect of 10 mm ureide applied by stem infusion or in the nutrient solution of hydroponically grown plants on acetylene reduction activity (ARA). An almost complete inhibition of ARA was observed within 4 to 7 d after treatment, accompanied by an increase in ureide concentration in the shoot but not in the nodules. The inhibition of ARA resulting from ureide treatments was dependent on the concentration of applied ureide. Urea also inhibited ARA but asparagine resulted in the greatest inhibition of nodule activity. Because ureides did not accumulate in the nodule upon ureide treatment, it was concluded that they were not directly inhibitory to the nodules but that their influence mediated through a derivative compound, with asparagine being a potential candidate. Ureide treatment resulted in a continual decrease in nodule permeability to O₂ simultaneous with the inhibition of nitrogenase activity during a 5-d treatment period, although it was not clear whether the latter phenomenon was a consequence or a cause of the decrease in the nodule permeability to O₂.The physiological basis of N₂ fixation inhibition by water deficits in legume nodules is not clearly understood. A potential physiological basis for this water-deficit sensitivity may be that drought stress decreases the P_o (Weisz et al., 1985), as has been shown with other stresses such as temperature, salinity, or nitrate (Hunt and Layzell, 1993; Serraj et al., 1994; Denison and Harter, 1995). The role of O₂ limitation in the response of nitrogenase activity to drought stress has been discussed extensively (Diaz del Castillo and Layzell, 1995; Purcell and Sinclair, 1995; Serraj and Sinclair, 1996b; Serraj et al., 1999). However, the mechanisms by which drought affects P_o have not been elucidated. It is not clear whether drought stress has a direct effect on P_o, or whether the decrease in P_o is a consequence of a decrease in nodule activity.An alternative explanation for the decrease in nitrogenase activity under drought could be a feedback mechanism involving the accumulation of N compounds. Pate et al. (1969) proposed that lower rates of water movement out of the nodule during drought stress may restrict export of products of N₂ fixation, and the accumulation of these products would inhibit nitrogenase activity. Others have suggested that N₂ fixation in legumes might be regulated by a feedback mechanism involving N metabolism and the pool of reduced N in the plant (Silsbury et al., 1986; Parsons et al., 1993; Hartwig et al., 1994). Oti-Boateng and Silsbury (1993) reported an inhibition of nitrogenase activity in fava bean after plant uptake of Asn or Gln.Soybean (Glycine max Merr.) usually exports more than 80% of the N compounds out of the nodules in the form of the ureides Aln and Alac. They are transported in the xylem to the shoots, where they are catabolized (Winkler et al., 1987). High accumulation of petiole ureides has been measured during the inhibition of N₂ fixation by drought in both controlled (de Silva et al., 1996; Serraj and Sinclair, 1996a) and field (Purcell et al., 1998) environments. Furthermore, in a comparison of grain legume species, Sinclair and Serraj (1995) reported that those species exporting ureides from the nodules had N₂ fixation that was drought sensitive. Those species that exported little or no ureide had N₂ fixation that was relatively drought tolerant.An important possibility is that the accumulation of ureides in soybean nodules under soil-water deficits might trigger a feedback mechanism that results in decreased N₂ fixation activity (Sinclair and Serraj, 1995; Serraj et al., 1999). This paper reports a series of experiments to investigate the hypothesis of a ureide feedback inhibition of N₂ fixation in soybean. First, ureide levels were measured in plant tissue (nodules, roots, and shoots) upon the imposition of water deficits to confirm that ureide levels increased in the nodules themselves, and not just in the shoot. Second, the influence of ureides on nodule activity was examined by introducing ureides, along with other compounds, into soybean plants. These experiments were designed to examine the time course of the response and to determine the concentration response. Third, data were collected to determine if P_o and the response of N₂ fixation to pO₂ were also sensitive to introduced ureides.     

Soybean leaf growth and gas exchange response to drought under carbon dioxide enrichment

This study was conducted to determine the response in leaf growth and gas exchange of soybean (Glycine max Merr.) to the combined effects of water deficits and carbon dioxide (CO₂) enrichment. Plants grown in pots were allowed to develop initially in a glasshouse under ambient CO₂ and well-watered conditions. Four-week old plants were transferred into two different glasshouses with either ambient (360 μmol mol^-1) or elevated (700 μmol mol^-1) CO₂. Following a 2-day acclimation period, the soil of the drought-stressed pots was allowed to dry slowly over a 2-week period. The stressed pots were watered daily so that the soil dried at an equivalent rate under the two CO₂ levels. Elevated [CO₂] decreased water loss rate and increased leaf area development and photosynthetic rate under both well-watered and drought-stressed conditions. There was, however, no significant effect of [CO₂] in the response relative to soil water content of normalized leaf gas exchange and leaf area. The drought response based on soil water content for transpiration, leaf area, and photosynthesis provide an effective method for describing the responses of soybean physiological processes to the available soil water, independent of [CO₂].