Increasing the yield potential (Y_p) of important agronomic crops is imperative for meeting predicted future production needs. Y_p is the maximum possible regional yield for a given crop in the absence of biotic and abiotic stresses (Evans and Fischer, 1999), but as radiation, water, nutrients, etc., become limiting or there is pest/pathogen pressure, realized yields decrease, resulting in a yield gap (Lobell et al., 2009). Y_p for a given crop during a growing season is the product of several components: the incident solar radiation across the growing season (S_t), the proportion of S_t that is photosynthetically active radiation (PAR; estimated as 0.487), the radiation interception efficiency (𝜀_i), the conversion efficiency of intercepted radiation into biomass (𝜀_c), and the partition efficiency of biomass into harvestable product (𝜀_p; modified from Monteith, 1977). S_t and the proportion that is PAR vary but are largely predetermined by growing season length and location, although there can be substantial interannual variability in S_t at a given location (Monteith, 1965, 1972). Of the three efficiencies, plant breeders have already pushed 𝜀_i and 𝜀_p near their theoretical upper limits in highly productive crops in the best years (Evans, 1993; Hay, 1995; Sinclair, 1998). Breeding in soybean (Glycine max Merr.) has achieved 𝜀_i season averages of approximately 0.60–0.75 (Koester et al., 2014) with peak midseason 𝜀_i of >0.90 in modern cultivars (Dermody et al., 2008; Koester et al., 2014). 𝜀_p has reached values of 0.60 for soybean (Dermody et al., 2008; Koester et al., 2014), which is at or near the estimated theoretical maximum of ∼0.55–0.67 for major food crops (Austin et al., 1980; Bugbee and Monje, 1992; Khush, 1995; Smil, 1999; Hay and Porter, 2006; Prasad et al., 2006). These advancements in 𝜀_i and 𝜀_p leave only modest potential for further improvement of Y_p (Zhu et al., 2010). However, 𝜀_c operates substantially below the theoretical maxima for C₃ (0.094) and C₄ (0.123; Zhu et al., 2010) crop canopies and therefore limits yield potential (Zhu et al., 2008, 2010), especially in soybean where the maximum realized 𝜀_c (0.028) is estimated at less than a third of the C₃ theoretical potential (Slattery and Ort, 2015).

Reducing leaf chlorophyll (chl) content has been proposed as a strategy to improve 𝜀_c in crop canopies. At low light levels, leaf photosynthesis (A_leaf) in crops such as soybean responds linearly to light intensity, but at approximately 25% of full sunlight, or 500 μmol m^-2 s^-1 photosynthetic photon flux density (PPFD), the linear relationship between absorbed quanta and A_leaf begins to plateau (Long et al., 2006). Light in excess of photosynthetic capacity is then wasted through heat dissipation, or photoprotection, which reduces light use efficiency (Niyogi, 1999; Ort, 2001). At normal chl levels, individual soybean leaves absorb approximately 85–90% of incident PPFD, which results in the uppermost ∼25% of the canopy absorbing ∼75% of incoming light (Campbell and Norman, 1998), much of which is wasted due to light saturation of photosynthesis in these leaves. Meanwhile, at full sunlight, leaves below the uppermost 25% receive half or less of the light needed to saturate A_leaf and are therefore light limited (Long et al., 2006). By distributing light more proportionately throughout leaf layers (Zhu et al., 2010; Ort et al., 2011, 2015), absorbed PPFD could be used more efficiently by mitigating both light oversaturation at the top of canopy and light limitation within the canopy. It is likely that some crops overinvest in chl content to the detriment of light distribution in the canopy similar to the manner in which soybean overinvest in leaf area (Srinivasan et al., 2016). Therefore, decreasing leaf absorbance (leaf_abs) through reduced chl content seems a potential strategy to achieve deeper light penetration into a crop canopy; thus, sun leaves would absorb only enough photons at mid-day to sustain maximum A_leaf while allowing more light to reach the lower canopy and stimulate A_leaf in shade leaves, thereby potentially improving canopy photosynthesis (A_can), 𝜀_c, and Y_p. Experimental evidence supporting the principle of this notion has been found in similar or greater rates of A_leaf in various crops with substantial reductions in chl compared to their dark-green counterparts (Highkin et al., 1969; Benedict et al., 1972; Edwards et al., 1993; Habash et al., 1994; Li et al., 2013; Kirst et al., 2017). In soybean, greater rates of A_leaf on an absorbed photon basis were evident in light-green soybean leaves, and the increase in A_leaf correlated with a more even light distribution among chloroplasts within leaves (Slattery et al., 2016). An analogous alteration of light distribution could therefore occur among leaves within a canopy. In addition, dense mass cultures of truncated light antennae (tla) green algae mutants demonstrated increased light penetration and improved solar energy conversion efficiency (Melis, 1999; Polle et al., 2002; Mitra and Melis, 2008), which ultimately led to increased hydrogen production (Kosourov et al., 2011).

Decreasing chl content could also have other benefits at the canopy level. Reducing light absorption and thereby increasing albedo at the top of the canopy could decrease leaf temperature (T_leaf) in the upper canopy, similar to the manner in which paraheliotropism reduces T_leaf in other species (Gamon and Pearcy, 1989). During times of above optimal temperatures, this should increase A_leaf by mitigating negative heat stress effects (Ainsworth and Ort, 2010) and in turn also improve water use efficiency (WUE). Cooler soybean canopies lower vapor pressure deficit, resulting in higher WUE (Baldocchi et al., 1985), which was reported for alfalfa with reduced chl content compared to the full green control (Estill et al., 1991). Greater light availability with depth in the canopy could also increase WUE by facilitating greater A_leaf in deeper layers where humidity is higher and therefore vapor pressure deficit is lower (Drewry et al., 2014; Ort and Long, 2014). If monoculture crops are overinvesting in chl biosynthesis, reallocation of nitrogen from an excess of pigment-protein complexes to other nitrogen-limited photosynthesis-related molecules might also be a benefit of chl reduction. In a modeling study, reallocating nitrogen resources among Calvin cycle enzymes predicted increased potential A_leaf without any additional nitrogen (Zhu et al., 2007). If nitrogen that would otherwise be used in pigment and pigment-proteins were reinvested in increased photosynthetic capacity, a similar increased nitrogen use efficiency would be expected.

Soybean is the world’s third most economically important commodity crop (FAO, 2012). At agricultural planting densities, soybean develops a dense canopy with a leaf area index (LAI) often greater than six. This creates a situation in which the majority of leaves are experiencing light levels below the light compensation point during most daylight hours and makes it an ideal candidate crop for testing the effects of reduced chl content on 𝜀_c. A large number of chl-deficient mutants have been identified in soybean, and two chl-deficient soybean mutants with robust canopy growth, Y11y11 and y9y9, were previously reported to have greater A_can compared to the nearly isogenic “Clark” wild-type (WT) throughout the growing season (Pettigrew et al., 1989). These mutants display a disproportionately large truncation in the antennae associated with photosystem II (PSII) compared to photosystem I (PSI; Ghirardi and Melis, 1988). This leads to higher chl a/b and PSII/PSI ratios, the latter of which serves to balance light absorption between the two photosystems (Eskins et al., 1983; Ghirardi and Melis, 1988). However, comprehensive studies of reduced chl effects on soybean at both the leaf and canopy scale have not yet been conducted in the same experiment. Therefore, the same light-green soybean mutants and the WT control were grown in the field during the 2012 growing season. The following year only the Y11y11 mutant was grown with the WT so that a row spacing treatment could be added. These field experiments were used to characterize the light-green mutants and to investigate the impact that reducing chl content has on leaf and canopy photosynthesis, photosynthetic efficiency, and yield.

Reducing chl content in soybean was hypothesized to lead to an improved distribution of light in the canopy, resulting in benefits to leaf and canopy photosynthesis and therefore yield (Zhu et al., 2010; Ort et al., 2011; Drewry et al., 2014). This hypothesis was tested on two chl-deficient mutants that reportedly performed well in optimal field conditions in a previous study (Pettigrew et al., 1989). As the 1st year of the field study revealed severe limitations in the y9y9 mutant, the 2nd year focused on comparison of the Y11y11 mutant to the WT and by adding a narrow row spacing treatment that modeling predicted would advantage the light-green phenotype due to earlier canopy closure. Despite transient benefits to mutant leaf-level photosynthesis, no increases in canopy-level processes were evident during either growing season, both of which experienced drought conditions. While the results of this study confirmed neither our hypothesis nor the earlier published work in which the Y11y11 mutant significantly outyielded the parental Clark cultivar, they do indicate that soybean, and likely many other crop plants, significantly overinvest in chl since a >50% chl reduction had little negative impact on biomass accumulation or yield. In addition, the small negative effects reduced chl did have on yield in our study were likely due to pleiotropic effects of the mutation. This outcome suggests that more sophisticated approaches for chl reduction, such as developmentally timed transgenic technology, may promote an opportunity to reinvest nitrogen and energy resources saved in chl reduction into increasing the biochemical photosynthetic capacity, leading to increased yield.

Although the Y11y11 mutant demonstrated the potential to surpass WT in light use efficiency and A_sat, the improvements were transient. The increases in light use efficiency corresponded with a narrow range of chl reductions (Table 3 and Figures 2C,D, 6), which may support the hypothesis that an optimal chl content exists and is lower than current levels (Ort et al., 2011). Benefits to A_leaf were seen only when chl content in Y11y11 was reduced to 30–40% of WT (Figures 2C,D), which corresponded to a 22% reduction in leaf_abs (Figure 3) that is less than the reduction predicted by Beer’s law (Slattery et al., 2016). As chl content increased in the mutant throughout the growing season (Figures 2C,D) and the percent difference between mutant and WT leaf_abs decreased (Figure 3), benefits at the leaf level became too small to resolve (Figure 6). In other species, chl contents correlated with increased A_leaf ranged from 30% in tobacco (Edwards et al., 1993) to 60% in maize (Edwards et al., 1993) and cowpea (Habash et al., 1994) as compared to the dark-green WT, suggesting that optimal chl content may be species specific.

Improved leaf light use efficiency is the anticipated result of improved light distribution within the low chl leaf. In dark-green leaves, the majority of light absorption occurs in the uppermost adaxial chloroplasts, causing light limitation in lower chloroplasts (Vogelmann and Evans, 2002; Evans and Vogelmann, 2003; Slattery et al., 2016). This has been shown to create a within-leaf gradient in photosynthesis (Evans and Vogelmann, 2003) as well as photoinhibition (Oguchi et al., 2011) that decreases with depth in the leaf and limits overall photosynthetic efficiency. Light sheet microscopy analyses revealed more gradual PPFD attenuation in light-green soybean leaves compared to dark-green leaves. This resulted in significantly more red and blue light reaching the spongy mesophyll chloroplasts of the light-green leaves, which correlated with greater photosynthetic light use efficiency (Slattery et al., 2016). Thus, if chloroplasts within the leaf mimic leaves within a canopy, then decreasing chl content may have ameliorated the large disparity of light availability in the lower leaf to increase photosynthetic light use efficiency.

However, other attributes of the chl mutants may have also played roles in altering photosynthetic performance. SLW was reduced in both mutants (Figures 2A,B). Changes in SLW are due to changes in either leaf thickness or leaf density, both of which can affect photosynthesis (Niinemets, 1999). Although leaf thickness was not directly measured in this study, leaf thickness did not change in a previous study on the same soybean genotypes, even when SLW was significantly different (Slattery et al., 2016). Therefore, changes in SLW were almost certainly due to reduced leaf density in the chl mutants, which can increase the proportion of intercellular space and thus improve CO₂ diffusion throughout the leaf (Niinemets, 1999). In addition, chl deficiency in these mutants was accompanied by increased g_s, which would increase carbon supply to the inner leaf and further confound deciphering the chl reduction impact on A_leaf. A similar increase in g_s was seen in rice chl mutants that exhibited greater rates of A_leaf in non-limiting environmental conditions (Gu et al., 2017). Those authors hypothesized that the increase in g_s may have been due to changes in vein structure in the rice mutants (Gu et al., 2017). More analyses are needed to separate the effects of reduced chl, increased g_s, and altered leaf anatomy on A_leaf improvements or the lack thereof.

A decline in photosynthetic efficiency and capacity in y9y9 was correlated with severe reductions in chl. Despite greater g_s and lower SLW, which was evident in Y11y11 as well, the y9y9 mutant displayed reduced A_sat and light use efficiency compared to the WT (Figures 4A, 5A, 6, 7A and Table 3) when chl was reduced by more than 85% compared to the WT (Figure 2C). Similar responses to severe reductions in chl content (>80%) seen in y9y9 have been reported in Y11y11 when grown in controlled environment chambers at lower than field light levels (Xu et al., 1993; Slattery et al., 2016). These two mutants are characterized by higher chl a/b ratios due to greatly reduced PSII-associated light harvesting complexes (LHCII) and to some extent PSI-associated light harvesting complexes (LHCI; Ghirardi and Melis, 1988). Theoretically, severe deficiencies in LHCII could have negative effects at the leaf level, including greater levels of photoinhibition (Leverenz et al., 1992; Xu et al., 1993), reduced photoprotective capacity (Ort, 2001), and lowered connectivity among PSII centers and therefore lower quantum yield at low light (Allen and Forsberg, 2001). In this study, extreme reductions in chl coupled with large reductions in carotenoids (>40%; Table 2) were correlated with impaired photoprotective mechanisms such as NPQ (Figure 6). ϕCO₂ was also significantly reduced in y9y9 (Tables 2, 3). Thus, these data suggest improving leaf photosynthetic efficiency through lowering chl a and chl b concentrations proportionally, but not to an extent that impairs photosynthetic and photoprotective capacity.

Despite transient leaf-level improvements in photosynthesis and photosynthetic efficiency, chl reductions only significantly affected the canopy parameter of 𝜀_i. Although no improvements in A_can or 𝜀_c were evident in the chl-deficient soybean, the fact that there was also no decline in these parameters with a >50% reduction in chl suggests that soybean significantly overinvest in chl. This is consistent with a recent study that showed soybean also overinvest in LAI to the detriment of canopy productivity (Srinivasan et al., 2016). However, less pigment led to significant depressions in 𝜀_i in y9y9 in 2012 and Y11y11 in 2013 narrow row widths (Tables 4, 5), mostly due to reduced light interception by the mutants early in the season, even when LAI was similar (Figure 8). The mutant chl content in this study was the lowest early in the season (Figures 2C,D), which would be expected to both increase transmission to the soil and increase the proportion of reflectance not reabsorbed by upper canopy leaves during early growth. As LAI increased, chl content also increased in the mutants, which in turn would be expected to limit light penetration to deeper layers in the canopy. An ideal situation for maximizing light absorption early in the season would be normal chl content until the canopy has closed (Melis, 2009). This would suppress weed vigor through increased competitiveness, which is reasoned to be why plants evolved with much higher chl content than is needed to maximize photosynthesis (Donald, 1968). As LAI increases, decreasing chl biosynthesis in new leaves would alleviate oversaturation in times of high light and allow greater transmittance to the lower canopy. However, light reflection and thus loss of canopy absorbance will also increase at the top of the canopy; thus, a more light-use efficient canopy may not necessarily lead to an increase in A_can from chl reductions alone.

Reduced pigmentation was hypothesized to benefit leaf water use along with canopy 𝜀_c, but the greater g_s that accompanied chl reductions in these mutants likely prevented these effects. Increased albedo was associated with lower T_leaf in the mutants as expected (Figures 5G,H), but cooler T_leaf may have been, at least in part, an effect of greater g_s and therefore cooling via transpiration in the mutants (Figures 5E,F). Greater g_s was most likely a result of the mutations causing chl deficiency. A recent study identified a mutation in the magnesium chelatase subunit-I gene (CHLI) as the cause of the light-green phenotype of Y11y11 (Campbell et al., 2015). In A. thaliana, both CHLI and CHLH mutants have demonstrated ABA-insensitivity (Tsuzuki et al., 2011; Du et al., 2012) that is independent of chl biosynthesis (Du et al., 2012). Whether the greater g_s in y9y9 that occurred in this study is also due to ABA-insensitivity is unknown since the mutation has not yet been identified, but greater g_s has been reported in y9y9 compared to WT regardless of water stress (Luquez et al., 1997). The increase in g_s resulted in lower mutant iWUE (Figures 5C,D) and integrated canopy WUE (Supporting Information Figure S4). Lower canopy WUE in the mutants would likely result in greater soil moisture depletion (Hussain et al., 2013) and therefore greater susceptibility to drought, which occurred during both of the growing seasons (Figures 1C,D). Drought stress significantly reduces 𝜀_c (Slattery et al., 2013), and the greater susceptibility to drought in the mutants may have dampened even small benefits of reduced chl on 𝜀_c and yield. In the previous study where A_can and yields were greater in Y11y11 compared to WT, water was not limiting due to irrigation (Pettigrew et al., 1989), suggesting that these specific mutations causing chl deficiency may limit productivity in times of even moderate water stress. Although rice chl mutants with greater g_s resulted in higher yields when grown at a higher planting density, the authors do not report any water limitations. Further assessments of chl-deficient crops may need to incorporate tests of efficiency and productivity in non-optimal field conditions while maintaining or improving WUE.

The results of this study suggest that soybean overinvest in chl; thus, chl reduction represents an opportunity to reinvest nitrogen from pigment-proteins into rate limiting photosynthetic enzymes that could increase photosynthetic capacity (i.e., V_c,max and J_max). However, nitrogen reallocation was not realized to any significant extent in these mutants. For example, a study by Evans and Poorter (2001) showed that 12.9% of leaf organic nitrogen was associated with pigment-protein complexes and 21.6% was associated with Rubisco in plants grown at high light. Therefore, if pigment-proteins were reduced by 50% and all of the nitrogen associated with pigment-proteins was reallocated to Rubisco, carboxylation capacity could potentially increase by up to 30%. However, across both seasons, Y11y11 V_c,max increased <5% compared to WT (Table 2). J_max only increased by 3% in 2012 and 7% in 2013 in Y11y11 compared to WT (Table 2). Therefore, further intervention would be required to redirect nitrogen savings from reduced investment in pigment-proteins in low-chl plants to the most beneficial targets for increasing photosynthesis (Zhu et al., 2007).

This study demonstrated early season benefits of reduced chl content on leaf photosynthetic efficiency and capacity. However, the effects of reduced chl were confounded with leaf properties and greater g_s in this study and a similar study in rice. Further analyses will be needed to parse out the impacts of chl reductions on leaf structure, g_s, and A_leaf. The mutants used in this study, although relatively robust in optimal field conditions, captured less light early in the growing season and used water less efficiently, which may have impaired the effects of reduced chl during the drought conditions experienced in both growing seasons. Despite the pleiotropic effects of the mutations, limitations to biomass accumulation and yield were minimal, signifying an overinvestment in chl in dark-green soybean.

As evident from these results, the methods of obtaining and maintaining optimal chl concentrations require further consideration. Optimizing chl concentration within the canopy and throughout the season is required to reduce wasted light early in the season via transmission to the soil and later in the season as reflected light from the top of the canopy while maintaining a more even light distribution within the canopy. Additionally, it is crucial to identify and utilize targets that eliminate the pleiotropic effects, such as greater water loss and photooxidative effects, that can accompany many chl biosynthesis mutations. Lastly, nitrogen reinvestment will need optimization to maximize nitrogen use efficiency in low-chl plants. This suggests that directed approaches, such as transgenic technology, are required for greater benefits of reduced chl on A_can, 𝜀_c, and yield.

All authors participated conceiving and designing the research. RS, AV, and CB performed the research. RS and AV analyzed the data. RS and DO wrote the article, which was reviewed by all other authors.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.