Morgan et al. (2001) grew alfalfa plants for 20 days post-defoliation in growth chambers maintained at atmospheric CO2 concentrations of 355 and 700 ppm and low or high levels of soil nitrogen to see how these factors affected plant re-growth. They determined that the plants in the elevated CO2 treatment attained total dry weights over the 20-day re-growth period that were 62% greater than those reached by the plants grown in ambient air, irrespective of soil nitrogen concentration.

De Luis et al. (1999) grew alfalfa plants in controlled environment chambers in air of 400 and 700 ppm CO2 for two weeks before imposing a two-week water treatment on them, wherein the soil in which half of the plants grew was maintained at a moisture content approaching field capacity while the soil in which the other half grew was maintained at a moisture content that was only 30% of field capacity. Under these conditions, the CO2-enriched water-stressed plants displayed an average water-use efficiency that was 2.6 and 4.1 times greater than that of the water-stressed and well-watered plants, respectively, growing in ambient 400-ppm-CO2 air. In addition, under ambient CO2 conditions, the water stress treatment increased the mean plant root:shoot ratio by 108%, while in the elevated CO2 treatment it increased it by 269%. As a result, the nodule biomass on the roots of the CO2-enriched water-stressed plants was 40 and 100% greater than the nodule biomass on the roots of the well-watered and water-stressed plants, respectively, growing in ambient air. Hence, the CO2-enriched water-stressed plants acquired 31 and 97% more total plant nitrogen than the well-watered and water-stressed plants, respectively, growing in ambient air. The bottom line, in terms of productivity, was that the CO2-enriched water-stressed plants attained 2.6 and 2.3 times more total biomass than the water-stressed and well-watered plants, respectively, grown at 400 ppm CO2.

Luscher et al. (2000) grew effectively- and ineffectively-nodulating (good nitrogen-fixing vs. poor nitrogen-fixing) alfalfa plants in large FACE plots for multiple growing seasons at atmospheric CO2 concentrations of 350 and 600 ppm, while half of the plants in each treatment received a high supply of soil nitrogen and the other half received only minimal amounts of this essential nutrient. The extra CO2 increased the yield of effectively-nodulating plants by about 50%, regardless of soil nitrogen supply, while it actually caused a 25% yield reduction in ineffectively-nodulating plants subjected to low soil nitrogen, yet produced an intermediate yield stimulation of 11% for the same plants under conditions of high soil nitrogen, which suggests that the ability to symbiotically fix nitrogen is an important factor in eliciting strong positive growth responses to elevated CO2 under conditions of low soil nitrogen supply.

Sgherri et al. (1998) grew alfalfa in open-top chambers at ambient (340 ppm) and enriched (600 ppm) CO2 concentrations for twenty-five days, after which water was withheld for five additional days so they could investigate the interactive effects of elevated CO2 and water stress on plant water status, leaf soluble protein and carbohydrate content, and chloroplast thylakoid membrane composition. They found that the plants grown in elevated CO2 exhibited the best water status during the moisture deficit part of the study, as indicated by leaf water potentials that were approximately 30% higher (less negative) than those observed in plants grown in ambient CO2. This beneficial adjustment was achieved by partial closure of leaf stomata and by greater production of nonstructural carbohydrates (a CO2-induced enhancement of 50% was observed), both of which phenomena can lead to decreases in transpirational water loss, the former by guard cells physically regulating stomatal apertures to directly control the exodus of water from leaves, and the latter by nonstructural carbohydrates influencing the amount of water available for transpiration. This latter phenomenon occurs because many nonstructural carbohydrates are osmotically active solutes that chemically associate with water through the formation of hydrogen bonds, thereby effectively reducing the amount of unbound water available for bulk flow during transpiration. Under water-stressed conditions, however, the CO2-induced difference in total leaf nonstructural carbohydrates disappeared. This may have resulted from an increased mobilization of nonstructural carbohydrates to roots in the elevated CO2 treatment, which would decrease the osmotic potential in that part of the plant, thereby causing an increased influx of soil moisture into the roots. If this did indeed occur, it would also contribute to a better overall water status of CO2-enriched plants during drought conditions.

The plants grown at elevated CO2 also maintained greater leaf chlorophyll contents and lipid to protein ratios, especially under conditions of water stress. Leaf chlorophyll content, for example, decreased by a mere 6% at 600 ppm CO2, while it plummeted by approximately 30% at 340 ppm, when water was withheld. Moreover, leaf lipid contents in plants grown with atmospheric CO2 enrichment were about 22 and 83% higher than those measured in plants grown at ambient CO2 during periods of ample and insufficient soil moisture supply, respectively. Furthermore, at elevated CO2 the average amounts of unsaturation of two of the most important lipids involved in thylakoid membrane composition were approximately 20 and 37% greater than what was measured in plants grown at 340 ppm during times of adequate and inadequate soil moisture, respectively. These greater lipid contents observed at elevated CO2, and their increased amounts of unsaturation, may allow thylakoid membranes to maintain a more fluid and stable environment, which is critical during periods of water stress in enabling plants to continue photosynthetic carbon uptake. These effects are so important, in fact, that some researchers have suggested that adaptive plant responses such as these may allow plants to better cope with any altered environmental condition that produces stress.

While working with wild-type Arabidopsis thaliana and two mutants deficient in thylakoid lipid unsaturation, for example, Hugly and Somerville (1992) found that "chloroplast membrane lipid polyunsaturation contributes to the low-temperature fitness of the organism," noting there was a positive correlation "between the severity of chlorosis in the two mutants at low temperatures and the degree of reduction in polyunsaturated chloroplast lipid composition." Similarly, Kodama et al. (1994) demonstrated for tobacco that the low-temperature-induced suppression of leaf growth and concomitant induction of chlorosis observed in wild-type plants was much less evident in transgenic plants containing a gene that allowed for greater expression of unsaturation in the fatty acids of leaf lipids, which observation led them to conclude that substantially unsaturated fatty acids "are undoubtedly an important factor contributing to cold tolerance."

In a closely related study, Moon et al. (1995) found that heightened unsaturation of the membrane lipids of chloroplasts stabilized the photosynthetic machinery of transgenic tobacco plaints against low-temperature photo-inhibition "by accelerating the recovery of the photosystem II protein complex." Likewise, Kodama et al. (1995), also working with transgenic tobacco plants, showed that increased fatty acid desaturation is one of the prerequisites for normal leaf development at low, nonfreezing temperatures; and Ishizaki-Nishizawa et al. (1996) demonstrated that transgenic tobacco plants with a reduced level of saturated fatty acids in most membrane lipids "exhibited a significant increase in chilling resistance."

With respect to alfalfa itself, Bertrand et al. (2007a) grew well watered and fertilized plants inoculated with one of two strains (either A2 or NRG34) of the nitrogen-fixing symbiont Sinorhizobium meliloti in pots filled with non-sterile topsoil within controlled-environment chambers maintained at atmospheric CO2 concentrations of either 400 or 800 ppm for a period of two months under optimal light and day/night temperatures of 22/17°C and for two final weeks at a reduced light level and a cold day/night temperature regime of 5/2°C, over which period they measured a number of plant physiological functions and characteristics. In doing so, they determined that the total biomass of the plants in the elevated CO2 treatment was approximately 33% greater than that of the plants in the control treatment when infected with the A2 strain of S. meliloti, but about 36% greater when infected with the NRG34 strain. However, plants in the 800-ppm CO2 treatment were found to be less freezing tolerant than those in the 400-ppm treatment, while the plants inoculated with the NRG34 strain were determined to be less freezing tolerant than those inoculated with the A2 strain.

In providing some comparative background for their freezing tolerance results, Bertrand et al. noted that "CO2 enrichment led to more severe frost damage in leaves of Eucalyptus pauciflora (Barker et al., 2005) and Ginko biloba (Terry et al., 2000), and in a native temperate grassland (Obrist et al., 2001), whereas it increased frost resistance of Betula allaghanensis (Wayne et al., 1998) and Picea mariana (Bigras and Bertrand, 2006) but had no effect on freezing tolerance of Picea abies (Dalen et al., 2001)," which suggests there may not be a single freezing tolerance response to atmospheric CO2 enrichment that is typical of plants in general. However, because their results suggest that "it is possible to select or identify rhizobial strains to improve alfalfa performance under high CO2," Bertrand et al. concluded that the "freezing tolerance as well as the expression of key over-wintering genes of alfalfa can be altered by the strain of rhizobium," which should enable farmers to obtain the best of both worlds, as it were, by benefiting from the significant growth stimulation produced by the ongoing rise in the air's CO2 content, while selecting a strain of rhizobium capable of compensating for a possible CO2-induced reduction in freezing tolerance. But in a world where air temperature is already in a rising mode -- and where minimum temperatures in the winter appear to be rising fastest of all -- this "fine tuning" of rhizobium strain may not be needed, as the environmental transformation that is currently underway automatically insures that there will be a "greening of the earth" throughout natural and agro-ecosystems alike.

But what about temperatures at the other end of the spectrum? Would global warming -- if it were to resume -- harm alfalfa production?

This question was broached by Aranjuelo et al. (2005), who grew alfalfa for three consecutive June-July periods (2001-2003) out-of-doors within pots in polyethylene-covered temperature gradient tunnels maintained at atmospheric CO2 concentrations that averaged 405 and 730 ppm at ambient (AT) and elevated (ET = AT + 4°C) temperatures and at high (HW) and low (LW = 0.5 x HW) soil water contents, where all plants were fed adequate nutrients except for nitrogen, in order to insure that the only source of nitrogen for the plants was that fixed by the nodules, which were induced to form in response to inoculation with Sinorhizobium meliloti strain 102F78. Following this protocol revealed, in their words, that "the effect of elevated CO2 on plant growth interacted positively with temperature," and that the "higher dry mass production of plants grown under elevated CO2 and temperature was a consequence of enhanced photosynthetic rates," which conclusion derives directly from their data, where mean CO2-induced increases in leaf net photosynthesis over the entire experiment were found to be: +5% (HW, AT), +50% (HW, ET), +17% (LW, AT) and +42% (LW, ET), as best we can determine from the bar graphs in the researchers' paper. Likewise, mean CO2-induced increases in leaf biomass were approximately +4% (HW, AT), +54% (HW, ET), +23% (LW, AT) and +58% (LW, ET). For both leaf net photosynthesis and biomass production, these results indicate that the stimulatory effect of the elevated CO2 of this study was about 2.5 times greater in the warmer of the two temperature treatments in the low soil water regime, and that it was ten times greater in the warmer of the two temperature treatments in the high soil water regime. In addition, we note that plant water loss via transpiration was also benefited by the extra CO2 of this study, declining by 25% (HW, AT), 41% (HW, ET), 31% (LW, AT) and 31% (LW, ET). Hence, if the planet were to warm as predicted by most climate models, the CO2-induced stimulation of photosynthesis and biomass production of alfalfa would likely be even greater than it is currently.

In another study that comes to bear on the subject of high-temperature stress in alfalfa, Erice et al. (2007) grew 33-day-old nodulated alfalfa plants in two temperature-gradient greenhouses (one maintained at an atmospheric CO2 concentration of 350 ppm and the other at a concentration of 700 ppm) in pots recessed into the ground in an alfalfa field under conditions of ambient temperature (TA) and elevated temperature (TE = TA + 4°C) and well-watered (to field capacity) and water-stressed (50% field capacity) conditions for one month (after which a first cutting took place), plus an additional month (after which a second cutting took place). After each of these cuttings, plant dry matter production was determined, while taproots were analyzed for vegetative storage protein (VSP) contents.

At the time of the first cutting, it was determined that the alfalfa plants had had their dry matter production boosted by about 30% due to the experimental doubling of the air's CO2 content in the well-watered treatment (averaged across both temperature treatments), but by only about 10% in the water-stressed treatment. At the time of the second cutting, however, the well-watered plants had experienced an average dry matter production increase on the order of 20%, while plants in the water-stressed treatment had experienced a mean increase of fully 40%. In addition, Erice et al. report that over the first growth period "taproot VSP content increased in response to drought and elevated CO2."

With respect to the latter finding, the researchers say "it has been demonstrated that nitrogen pools in alfalfa taproots, especially vegetative storage proteins, condition new re-growing shoots," which appears to have been what happened in their study. At the end of the first growth period, for example, the enhanced taproot VSP content in the water-stressed and CO2-enriched treatment may have been the reason why the elevated CO2 was so effective in stimulating biomass production in the water-stressed treatment over the second growth period.

This finding is somewhat analogous to the observations of Idso et al. (2001), who found that nitrogen reabsorbed from second-year leaves of sour orange trees during the process of senescence in the fall was stored over winter in much greater quantities in putative VSPs in first-year leaves of CO2-enriched trees than in first-year leaves of trees growing in ambient air, so that when the stored nitrogen was released in the spring to produce flushes of new leaves on the trees, leaf production on the CO2-enriched trees vastly outpaced the production of new leaves on the trees growing in ambient air.

In one final study, Bertrand et al. (2007b) grew well-watered and adequately-fertilized alfalfa plants inoculated with two different strains (A2 and NRG34) of rhizobia (Sinorhizobium meliloti) in controlled-environment growth chambers, wherein they studied the effects of a 400- to 800-ppm doubling of the atmosphere's CO2 concentration on a number of plant physiological parameters, as well as on plant nutritive value and digestibility, which they assessed at the end of their 56-day experiment. This study revealed that plant shoot dry weight was increased by approximately 70% and 50% in the 800-ppm CO2-enriched air compared to the 400-ppm ambient air in the plants harboring the A2 and NRG34 strains of rhizobia, respectively, while corresponding root dry weight enhancements were 60% and 0%. In addition, nitrogenase activity (indicative of the degree of symbiotic nitrogen fixation}, was stimulated by about 55% and 25%, respectively, in the plants harboring the A2 and NRG34 rhizobia strains. Last of all, the CO2-enriched air resulted in what the researchers describe as "a slight increase" in the in vitro true digestibility of the A2 strain, while it resulted in the opposite in the NRG34 strain. In commenting on their findings, Bertrand et al. say their results "show that it is possible to identify rhizobial strains to improve plant performance under predicted future CO2 concentrations with no negative effect on nutritive value," which ability -- when utilized -- should enable mankind to greatly benefit from the ongoing rise in the air's CO2 content.

Consequently, and in view of the many positive effects of atmospheric CO2 enrichment on the growth and development of alfalfa plants, one can only hope that the air's CO2 content continues to rise, as we will need all the help we can get from it in our attempt to feed the world's growing number of people in the years and decades ahead.

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