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The term heterosis refers to the superior performance of heterozygous hybrid plants over their homozygous parental inbred lines. In maize, heterosis was paramount in raising the yield ceiling seven decades ago, when the first commercial hybrids were introduced (Tollenaar & Lee, 2006). Heterosis has also been reported to help maize to better adapt under diverse stress conditions (Tollenaar et al., 2004; Tollenaar & Lee, 2006). Thus, differences in grain yield between hybrids and their parental inbred lines increased in plants grown under drought (Blum, 1997; Betrán et al., 2003a,b,c) and greater plant density (Liu & Tollenaar, 2009b).
Although heterosis in maize has been studied since the early 1900s and is of vital agronomical importance, the genetic and physiological basis of heterosis remains unsolved (Sinha & Khanna, 1975; Tollenaar et al., 2004). Understanding this phenomenon might open up oppor-tunities for increasing yield potential and stress adapta-tion. Plant physiologists have long attempted to explain the greater biomass and grain production in F1 hybrids (Sinha & Khanna, 1975; Tollenaar et al., 2004; Tollenaar & Lee, 2006; and references herein). There are reports that indicate that heterosis for growth is mediated by a single factor, such as embryo size (Ashby, 1930) or endogenous concentration of gibberellins (Rood et al., 1988). However, overall heterosis is considered to be the product of quantitative characters, which are the result of complex interactions between simpler processes throughout the crop cycle. Recent studies explain heterosis as a dynamic attribute affected by both the environment and the stage of development (Tollenaar et al., 2004; Tollenaar & Lee, 2006). Thus, Tollenaar et al. (2004) dissected the physiological mechanisms responsible for heterosis for grain yield in maize. They concluded that three main processes are involved: heterosis for DM accumulation before silking, which results mainly from greater light interception owing to increased leaf size; heterosis for DM accumulation during the grain-filling period, which results from greater light interception owing to greater maximum leaf area index and increased stay-green; and heterosis for harvest index. However, how heterosis mediates these processes remains elusive.
We postulate that heterosis confers better water use to plants during growth. The responses associated with heterosis are in some way opposed to what is considered the effect of water stress. For example, the larger total leaf area of the plant in hybrids is sustained basically through longer leaves rather than through variation in final leaf number or even leaf width (Sinha & Khanna, 1975; Tollenaar et al., 2004). When maize encounters water deficit, there is a decline in photosynthesis. This is caused by a reduction in light interception as leaf expansion is reduced or as leaves senesce, and the reduction in photosynthetic assimilation per unit leaf area as stomata close or as photoinhibition increases (Bruce et al., 2002). Overall, these symptoms may fall within what is considered inbred depression.
Alternatively, heterosis might confer better nutritional status, which would explain its effect on growth and yield. Hybrids have better tolerance to high sowing density than inbreds (Liu & Tollenaar, 2009b). In this context, lower nutrient acquisition by inbreds might affect either plant growth or maximum leaf area and its duration. Thus, leaf senescence might be accelerated as the demand for nitrogen (N) by grains exceeds capacity for uptake (Chapman & Edmeades, 1999).
Even though heterosis is most evident in adult traits such as plant biomass or yield, it is also apparent during embryo (Meyer et al., 2007) and early seedling development (Hoecker et al., 2006). Thus, differences in rooting pattern and performance of seedling between inbreds and hybrids have been reported (Hoecker et al., 2006, 2008). The effect of such differences, even if tiny at the beginning of the crop cycle, accumulate during the growth of the plant, following what is known as the ‘compound interest law of growth’ (Blackman, 1919; Sinha & Khanna, 1975).
Therefore, when evaluating the kind of physiological factors responsible for heterosis, it is necessary to use traits able to integrate in time (crop cycle) and scale (whole plant) the physiological response of the plant. We showed recently that the oxygen isotope enrichment above source water (Δ18O) of plant dry matter as well as the total accumulated mineral content in transpirative tissues (Cabrera-Bosquet et al., 2009a,b), provide an indication of stomatal conductance and transpiration and thus of water use during the crop cycle. These approaches are precise enough to trace genotypic differences in yield within a given water regime. In addition, carbon isotope discrimination (Δ13C) in plant tissues, even if it is not feasible for assessing genotypic differences in C4 species such as maize (Monneveux et al., 2007; Cabrera-Bosquet et al., 2009b), may still trace differences in photosynthetic gas exchange in response to changes in water status.
Over two consecutive years we examined the physiological expression of heterosis in maize using a set of inbreds and derived hybrids of tropical maize grown in the field under three contrasting water regimes. We followed the approach proposed by Tollenaar et al. (2004), studying, together with biomass and grain yield, informative traits about plant growth, stay-green during grain filling and partitioning. The water and N status of hybrids and inbreds were also measured. Water status was assessed (mostly as water use) using instantaneous and time-integrated traits. Among the former, stomatal conductance, chlorophyll fluorescence, relative water content (RWC) and leaf temperature were measured. Integrated traits included signatures of stable isotopes 18O and 13C in leaves and kernels, as well as total mineral content in leaves. Nitrogen status was inferred by analysing the N content in leaves and kernels. The final aim was to examine whether, regardless of the water conditions during growth, heterosis gives hybrids better water use. Indeed, such differences in water use may underlie the effect of heterosis on growth and yield.
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Our range of yields for hybrids and inbreds under well-watered conditions is comparable to that reported previously by Betrán et al. (2003a,b), using a set of tropical lines and the derived single hybrids from the same LPS population grown in the absence of water stress during a winter season at the CIMMYT station, Tlaltizapán. The other two growing conditions assayed may be considered as intermediate–severe and very severe drought stress (Betrán et al., 2003a,b). In this previous study with the LPS population, hybrids gave higher yield and had taller plants, slower leaf senescence and higher leaf chlorophyll content than inbreds, under both drought stress and optimal conditions (Betrán et al., 2003a,b). Moreover, heterosis for GY increased with the intensity of drought stress (Betrán et al., 2003b). Overall, our results agree with previous findings in the LPS population.
In agreement with Tollenaar et al. (2004), we found that heterosis for biomass and grain yield was accompanied by heterosis for growth, stay-green and partitioning traits. Moreover, growth traits such as the total aerial biomass or the NDVI values around middle grain filling correlated positively with GY when all hybrids and inbreds were combined under the three growing conditions. In both cases, hybrids and inbreds followed a common relationship pattern (Fig. 2), which further indicates that heterosis for growth and stay-green traits is responsible for the higher GY attained by hybrids.
The main agronomical yield component responsible for differences in grain yield across environments combining hybrids and inbred lines was the number of kernels per ha (Fig. 2). Moreover, agronomical traits influencing kernel number showed consistent heterosis, regardless of water conditions (Table 2). Liu & Tollenaar (2009a) reported that heterosis for grain yield was closely associated with heterosis for kernel number, with this trait being associated with the amount of dry matter partitioning to the kernels during the sensitive period for kernel establishment (Echarte et al., 2004). Furthermore, Liu & Tollenaar (2009a) conclude that heterosis for kernel set was attributable, in part, to the relationship between kernel number and plant growth rate during the period bracketing flowering. In this context, our results suggest that hybrids show consistently higher partitioning to kernels than inbreds, irrespective of the water conditions during growth. Nevertheless, short anthesis-to-silking interval (ASI) in hybrids and subsequently better pollination should be not discarded as explanation of heterosis in grain number (Bolaños & Edmeades, 1996).
The number of grain-bearing ears per plant was the only agronomical yield component with negative heterosis. A similar pattern has also been reported in temperate maize (Tollenaar et al., 2004).
In summary, all the effects of heterosis on growth, stay-green, biomass, retranslocation and grain yield are consistent with the findings of Tollenaar and coworkers (Tollenaar et al., 2004; Tollenaar & Lee, 2006). Heterosis may be the result of improved N use efficiency or may result from the better water status of hybrids. We now discuss these two options.
Heterosis for N status
Heterosis in the uptake of N and other mineral nutrients was reported for many years, when data were expressed on a plant basis (Sinha & Khanna, 1975). However, as early as 1934, Smith (1934) reported that no heterosis for N accumulation existed if data were calculated on a unit weight basis. In our results, the higher N concentration in leaves and kernels of inbred lines, compared with hybrids, not only precludes a lower intrinsic capacity of inbreds for N uptake and use, but is also coherent with a somewhat lower water status of inbreds than of hybrids. Thus, it is widely reported that, under well-fertilized conditions, plants exposed to water stress usually increase their leaf N concentration on a dry matter basis, because of the limitations placed by drought on plant growth and carbon assimilation (Bänziger et al., 2000). In addition, when data from hybrids and inbreds under the three growing conditions were combined, the N concentration of grains correlated negatively with 100-kernel weight and both hybrids and inbreds followed a similar relationship pattern (Fig. 5). This kind of negative relationship is widely documented in maize and other cereals, when plants that are grown under different water conditions, but with good N fertilization, are compared (Bänziger & Lafitte, 1997; Bänziger et al., 2000). This does not support differences in N metabolism as a primary cause of heterosis. In fact, Betrán et al. (2003b), when working with inbreds and hybrids derived from the LPS population, concluded that the expression of heterosis was smaller under low N environments than under nonstress environments. In addition the higher values in the ratio total shoot N/total kernel N in inbreds than hybrids (data not shown), also precludes a limitation in N acquisition as a cause of heterosis.
Water status and heterosis for growth and stay-green
In our study, heterosis for plant growth and stay-green traits was present, irrespective of the growing conditions. However, there are some differential patterns between them. Thus, heterosis for growth traits showed the maximal differences under well-watered conditions. It has long been known that cell expansion is one of the first physiological changes caused by dehydration (Hsiao, 1973). This may occur even under conditions considered well-watered or in the transition to mild water stress (Kramer & Boyer, 1995; Passioura, 1996). Under our well-watered conditions, better water use of hybrids than inbreds, even if mild, might be responsible for these differences in growth. Moreover, the possible effect of a higher endogenous concentration of gibberellins for heterosis for shoot growth (Rood et al., 1988) may be better expressed under well-watered conditions.
Differences across water regimes for leaf chlorophyll content and Fv/Fm were already present at anthesis (Table 1), suggesting that permanent photoinhibition and photodamage were already present under drought conditions (Valentinuz & Tollenaar, 2004; Ding et al., 2006). Heterosis for stay-green traits was already evident at anthesis and steadily increased under the three growing conditions during grain filling. However, unlike during growth, heterosis for stay-green was higher under water stress than with well-watered conditions. Higher heterosis under drought may be a consequence of more severe stress causing accelerated senescence in inbreds (Hsiao, 1973; Passioura, 1996).
Water status and heterosis for partitioning and grain yield
Heterosis for grain yield as well as the number of kernels per ha and per plant were higher under drought than under WW conditions (Table 2). Betrán et al. (2003a,b) also reported increases in heterosis for GY and kernel number under drought stress, indicating that hybrids may be more tolerant to drought stress than inbred lines. However, the effects of drought (Bruce et al., 2002) and heterosis on GY are both sustained by decreases in kernel number (Fig. 2c).
Other stresses, such as increasing plant density, which may have a similar effect to increasing water stress, also increased heterosis for GY, while not affecting heterosis for dry matter at maturity (Liu & Tollenaar, 2009b). By contrast, stresses very different from drought, such as shading during the grain-filling period, did not increase heterosis for GY (Liu & Tollenaar, 2009a).
Heterosis for HKW was only important under fully irrigated conditions, which suggests that, compared with hybrids, inbreds suffered a shortage of photoassimilates during grain filling owing some degree of water stress, together with less stay-green behavior, (i.e. shorter grain filling) while the number of kernels to fill was still relatively high. Deeper roots of hybrids under well-watered conditions may offer an advantage in plant water status, particularly when plants are subjected to high vapour pressure deficit (VPD) such in our growing conditions (Fig. 1, Table S2), by preventing atmospheric water deficit.
Heterosis for water-use traits
Stomatal conductance has been proposed as a key trait indicative of the effect of water stress on photosynthetic leaf performance (Medrano et al., 2002; Jiang et al., 2006). However, in our first-year results, heterosis for gs measured at different times during the plant cycle did not show a coherent pattern throughout any of the growing conditions (Table 2). Results illustrate the inherent heterogeneity associated with the characterization of water status by use of an instantaneous trait such as gs measured in single leaves and affected by daily patterns. The available literature does not conclusively support or discard the effect of heterosis on gas exchange of leaves. Thus, the recent studies do not provide values of gs or transpiration that might reflect water status during growth (Ahmadzadeh et al., 2004; Tollenaar et al., 2004; Tollenaar & Lee, 2006).
Importantly, most of the integrative water-use traits measured in our study showed consistent differences between hybrids and inbreds across the different growing conditions. Thus, mineral accumulation in transpirative tissues was lower and Δ18O values in leaves and kernels and Δ13C values in leaves were higher in inbreds. The Δ13C of grains also increased in inbreds, but only under well-watered conditions. Ash content in leaves has been reported in maize (Cabrera-Bosquet et al., 2009b) and other cereals (Araus et al., 1998, 2002a) to be positively associated with higher transpiration because of better water status and in turn with higher grain yield. The Δ18O from plant tissues may also be considered a proxy for time-integrated stomatal conductance and transpiration (Barbour et al., 2000; Barbour, 2007; Farquhar et al., 2007). An increase in Δ18O of both leaves and kernels as a result of water stress has been reported recently in maize hybrids (Cabrera-Bosquet et al., 2009a). Previous results with other cereals also support an increase in Δ18O in response to increased soil water stress (Ferrio et al., 2007; Cabrera-Bosquet et al., 2009c). The small, yet significant, increase in Δ13C in inbreds compared with hybrids also reflects poorer water status, in this case affecting photosynthetic carbon assimilation and water-use efficiency (Dercon et al., 2006; Monneveux et al., 2007; Cabrera-Bosquet et al., 2009b). While leaf ash content and Δ18O for both leaves and kernels are traits precise enough to show genotypic differences in maize, Δ13C seems less precise and to be only affected by growing conditions (Cabrera-Bosquet et al., 2009b). Even so, the differences in water status between hybrids and inbred lines are strong enough to be reflected by the Δ13C in leaves.
The strength of the integrated traits used in our study is that they may provide an overall indication of the transpirative and photosynthetic record experienced by the plant. Moreover, as these traits have a different mechanistic basis, they provide independent lines of evidence on the poorer water status of inbreds. In addition, the Δ18O and Δ13C in kernels suggests that differences in water status during grain filling between hybrids and inbreds were maximal under well-watered conditions.
Nevertheless, even instantaneous traits measured in individual leaves reflected different water statuses of hybrids and inbreds during flowering – a critical stage for GY. Thus, during the first year Fs measured at anthesis was higher in hybrids than in inbreds, regardless of the water treatment. Fs it is a faster and less intrusive parameter to measure than gs (Flexas et al., 1999, 2002) which allowed for more replications per plot than for gs. During the second year, gs (this time greatly increasing the measurements per plot compared with the first year) together with leaf temperature and RWC of individual leaves also indicated a better water status for hybrids at anthesis. Moreover, these traits were strongly correlated with GY when inbreds and hybrids under three growing conditions were combined (Fig. S1). Kernel number (and thus grain yield) in maize is greatly affected by stress in the period between 2-wk-before and 3-wk-after silking (Andrade et al., 2000 and references therein).
In conclusion, the traits discussed clearly show that hybrids experienced better water use and status than inbreds, regardless of the water conditions during growth.
Potential mechanisms and implications for breeding
There are potential mechanisms responsible for better water use in hybrids, such as the ability of the plant to capture water from a drying soil through deeper or more thorough soil exploration or by osmotic adjustment. Some reports link osmotic adjustment to stable GY of hybrids under conditions of transient stresses (Lemcoff et al., 1998). However, osmotic adjustment seems to not be as relevant in maize (Tardieu, 2006; but see Chimenti et al., 2006). Moreover, heterosis for biomass and GY is always expressed regardless of the water conditions during growth.
Heterosis in root growth was being investigated in the 1930s (Ashby, 1932). Apparently, relative root size is not the target trait, thus no heterosis (Sinha & Khanna, 1975) and positive heterosis (Ahmadzadeh et al., 2004) have been reported for the shoot : root ratio. Therefore, compared with inbreds, hybrids seem to allocate a smaller proportion of their total dry matter to the root, which may be interpreted as an indirect indication that hybrids maintain a constitutively better water use than inbreds.
Hoecker et al. (2006) demonstrated in maize that heterosis can already be seen during the very early stages of root development, a few days after germination. In that study lateral root density showed the highest degree of heterosis. Chun et al. (2005) reported heterosis for total length of lateral roots together with a higher ratio of lateral to axial root length in 20-d maize seedlings growing under different N conditions. Later in development, lateral roots become dominant and are responsible, together with the post-embryonic shoot-borne root system, for the major portion of water and nutrient uptake (Hochholdinger et al., 2004). In addition Li et al. (2008) concluded that seedlings of hybrids develop a greater number of fine roots than their parents. Furthermore heterosis exists for water uptake ability at the root cell level under well-watered conditions (Liu et al., 2009). Moreover, Hoecker et al. (2008) identified nonadditive gene expression in primary roots of maize hybrids, compared with the average expression levels of their parental inbred lines. Among those genes, a superoxide dismutase 2 was expressed significantly above the mid-parent value in all hybrids studied. This may play a protective role in heterosis-related anti-oxidative defense in the primary root of maize hybrids. Paschold et al. (2010) state that a subtle regulation of particular biochemical pathways such as the phenylpropanoid pathway in hybrids might contribute to the manifestation of heterosis in maize primary roots.
Our study may also provide some clues for breeding. It is widely accepted that, while yield potential is important in determining yield under moderate water stress (Araus et al., 2002b, 2008), it becomes much less so if yields fall below 50–60% of the potential (Bänziger & Lafitte, 1997). This is when stress-adaptive traits acquire real significance. Thus, constitutive whole-plant traits play a major role in affecting plant water use and plant dehydration avoidance under stress. These largely determine some of the negative relations between yield potential and the ability to sustain yield under severe water shortage (Blum, 2005). However, heterosis, even if differentially affected by water conditions during growth, may be considered an example of nonexistence of significant interactions between the genotype and the environment. Thus heterosis, while associated with higher yield potential, also confers adaptation under a full range of stress conditions. Therefore, there is the possibility of a common constitutive physiological trait (responsible for heterosis) which confers both higher yield potential and stress adaptation. To introduce some degree of heterosis in maize open-pollinated varieties may confer better performance under drought stress (Betrán et al., 2004).
The data of the present study on physiological mechanisms of heterosis also illustrate that the effective use of water rather than water-use efficiency is the target for crop improvement under drought stress (Blum, 2009). However, the strength of the relationships between lines and derived hybrids for water-use traits (Fig. 6) and GY is low and comparable to that previously reported for GY (Betrán et al., 2003b). This illustrates the potential difficulty in breeding for a quantitative trait such as heterosis.
We conclude that, in our study, regardless of the growing conditions, hybrids have better water use than inbreds. Heterosis for growth and yield in maize may (at least in part) be mediated by such accumulated differences in water use and status. Further studies comparing root development and soil moisture extraction of inbreds and hybrids are necessary.