Coordination of Leaf Photosynthesis,Transpiration, and Structural Traits in Rice and Wild Relatives (Genus Oryza)

The genus Oryza, which includes rice (Oryza sativa and Oryza glaberrima) and wild relatives, is a useful genus to study leaf properties in order to identify structural features that control CO₂ access to chloroplasts, photosynthesis, water use efficiency, and drought tolerance. Traits, 26 structural and 17 functional, associated with photosynthesis and transpiration were quantified on 24 accessions (representatives of 17 species and eight genomes). Hypotheses of associations within, and between, structure, photosynthesis, and transpiration were tested. Two main clusters of positively interrelated leaf traits were identified: in the first cluster were structural features, leaf thickness (Thick_leaf), mesophyll (M) cell surface area exposed to intercellular air space per unit of leaf surface area (S_mes), and M cell size; a second group included functional traits, net photosynthetic rate, transpiration rate, M conductance to CO₂ diffusion (g_m), stomatal conductance to gas diffusion (g_s), and the g_m/g_s ratio. While net photosynthetic rate was positively correlated with g_m, neither was significantly linked with any individual structural traits. The results suggest that changes in g_m depend on covariations of multiple leaf (S_mes) and M cell (including cell wall thickness) structural traits. There was an inverse relationship between Thick_leaf and transpiration rate and a significant positive association between Thick_leaf and leaf transpiration efficiency. Interestingly, high g_m together with high g_m/g_s and a low S_mes/g_m ratio (M resistance to CO₂ diffusion per unit of cell surface area exposed to intercellular air space) appear to be ideal for supporting leaf photosynthesis while preserving water; in addition, thick M cell walls may be beneficial for plant drought tolerance.Leaves have evolved in different environments into a multitude of sizes and shapes, showing great variation in morphology and anatomy (Evans et al., 2004). However, all leaf typologies share common functions associated with chloroplasts, namely to intercept sunlight, take up CO₂ and inorganic nitrogen, and perform photosynthesis as a primary process for growth and reproduction.Investigating relationships between leaf anatomy and photosynthetic features (CO₂ fixation, which involves physical and biochemical processes and loss of water by transpiration) could lead to the identification of structural features for enhancing crop productivity and improve our understanding of plant evolution and adaptation (Evans et al., 2004).Stomata, through which CO₂ and water vapor diffuse into and out of the leaf, are involved in the regulation and control of photosynthetic and transpiration responses (Jarvis and Morison, 1981; Farquhar and Sharkey, 1982). Besides stomata distribution patterns between the abaxial and adaxial lamina surfaces (Foster and Smith, 1986), stomatal density and size are leaf anatomical traits contributing to build the leaf stomatal conductance to gas diffusion (g_s). This is calculated as the reciprocal of the stomatal resistances to gas diffusion; stomatal control results in a lower concentration of CO₂ in the leaf mesophyll (M) intercellular air space (C_i) than in the atmosphere (C_a; Nobel, 2009).Leaf M architecture greatly contributes to the pattern of light attenuation profiles within the lamina (Terashima and Saeki, 1983; Woolley, 1983; Vogelmann et al., 1989; Evans, 1999; Terashima et al., 2011) and affects CO₂ diffusion from the intercellular air space (IAS) to the chloroplast stroma. Therefore, it influences photosynthetic activity (Flexas et al., 2007, 2008) and can have effects on leaf hydrology and transpiration (Sack et al., 2003; Brodribb et al., 2010; Ocheltree et al., 2012). In addition, M architecture sets boundaries for leaf photosynthetic responses to changing environmental conditions (Nobel et al., 1975).Fortunately, several methodologies are currently available (Flexas et al., 2008; Pons et al., 2009) to determine M conductance to CO₂ diffusion (g_m), expressed per unit of leaf surface area. It is calculated as the reciprocal of the cumulated partial resistances exerted by leaf structural traits and biochemical processes from the substomatal cavities to photosynthetic sites (Evans et al., 2009; Nobel, 2009). The resistance to CO₂ diffusion in the liquid phase is 4 orders of magnitude higher than in the gaseous phase (Nobel, 2009); therefore, the changes in CO₂ concentration in the leaf gas phase are small in comparison with the changes in the liquid phase (Niinemets, 1999; Aalto and Juurola, 2002; Nobel, 2009). In the liquid phase, the resistance to CO₂ transfer is built from contributions by the cell walls, the plasmalemma, cytoplasm, chloroplast membranes, and stroma (Tholen and Zhu, 2011; Tholen et al., 2012); in addition, it involves factors associated with the carboxylation reaction (Kiirats et al., 2002; Evans et al., 2009). Thus, the concentration of CO₂ in the chloroplasts (C_c) is lower than C_i and can limit photosynthesis.At steady state, the relationships between the leaf net photosynthetic rate (A), the concentrations of CO₂, and the stomatal conductance to CO₂ diffusion (g_{s_CO2}) and g_m are modeled based on Fick’s first law of diffusion (Nobel, 2009) as:(1)where C_a, C_i, and C_c are as defined above (Flexas et al., 2008).The magnitude of g_m has been found to correlate with certain leaf structural traits in some species, in particular with the M cell surface area exposed to IAS per (one side) unit of leaf surface area (S_mes) and its extent covered by chloroplasts (S_chl; Evans and Loreto, 2000; Slaton and Smith, 2002; Tholen et al., 2012). From a physical modeling perspective, increasing S_mes provides more pathways acting in parallel for CO₂ diffusion (to and from the chloroplasts) per unit of leaf surface area; thus, it tends to reduce the resistance to CO₂ movement into the M cells and to increase g_m (Evans et al., 2009; Nobel, 2009). A number of leaf structural traits affect S_mes, including leaf thickness, cell density, cell volume and shape, and the fraction of the M cell walls in contact with the IAS (Terashima et al., 2001, 2011), and the degree they are linked to S_mes can vary between species (Slaton and Smith, 2002; Terashima et al., 2006). In particular, the presence of lobes on M cells, which are prominent in some Oryza species, may contribute to g_m through increasing S_mes (Sage and Sage, 2009; Terashima et al., 2011; Tosens et al., 2012). The M cell wall can provide resistance in series for M CO₂ diffusion (Nobel, 2009); thicker cell walls may increase resistance to CO₂ movement into the M cells and decrease g_m (Terashima et al., 2006, 2011; Evans et al., 2009).Other leaf traits, such as M porosity (the fraction of M volume occupied by air spaces [Vol_IAS]), has been shown to have a positive correlation with g_m in some species (Peña-Rojas et al., 2005), but the association may be mediated by light availability (Slaton and Smith, 2002). Leaf thickness (Thick_leaf) tends to be negatively linked to g_m, and it may set an upper limit for the maximum g_m, according to Terashima et al. (2006), Flexas et al. (2008), and Niinemets et al. (2009).With respect to leaf structural traits and water relations, Thick_leaf may increase the apoplast path length (resistances in series; Nobel, 2009) in the extra-xylem M (Sack and Holbrook, 2006; Brodribb et al., 2007) for water to reach the evaporation sites, which could decrease the conductance of water through the M and lower the transpiration rate. Interestingly, while thicker M cell walls may reduce g_m, they can enable the development of higher water potential gradients between the soil and leaves, which can be decisive for plant survival and longevity under drought conditions (Steppe et al., 2011).The purpose of this study was to provide insight into how the diversity of leaf structure relates to photosynthesis and transpiration among representative cultivated species and wild relatives in the genus Oryza. This includes, in particular, identifying leaf structural features associated with the diffusion of CO₂ from the atmosphere to the chloroplasts, photosynthesis, transpiration efficiency (A/E), and drought tolerance. The genus consists of 10 genomic groups and is composed of approximately 24 species (the number depending on taxonomic preferences; Kellogg, 2009; Brar and Singh, 2011), including the cultivated species Oryza sativa and Oryza glaberrima. Oryza species are distributed around the world, and they exhibit a wide range of phenotypes, with annual versus perennial life cycles and sun- versus shade-adapted species (Vaughan, 1994; Vaughan et al., 2008; Brar and Singh, 2011; Jagadish et al., 2011). This diversity in the genus is an important resource, which is being studied to improve rice yield, especially under unfavorable environmental conditions. In particular, O. glaberrima, Oryza australiensis, and Oryza meridionalis are of interest as drought-tolerant species (Henry et al., 2010; Ndjiondjop et al., 2010; Scafaro et al., 2011, 2012), while Oryza coarctata is salt tolerant (Sengupta and Majumder, 2010). In this study, a total of 43 leaf functional and structural parameters were collected on 24 accessions corresponding to 17 species within eight genomes (Brar and Singh (2011). Life cycle is as follows: A = annual; B = biennial; P = poliennial. Habitat is as follows: S = shade; S-Sh = sun-shade.

Genome	Species	Life Cycle	Habitat	Accession	No.
AA	O. barthii	A	S	PI 590400*	1
AA	O. glaberrima	A	S	PI 450430*	2
AA	O. glumaepatula	P	S	PI 527362*	3
AA	O. longistaminata	P	S	IRGC 101207*	4
AA	O. longistaminata	P	S	IRGC 101754	5
AA	O. meridionalis	A/P	S	IRGC 93265*	6
AA	O. nivara	A/B	S	PI 590405*	7
AA	O. rufipogon	P	S	PI 104640	8
AA	O. rufipogon		S	PI 590421*	9
AA	O. sativa	A	S	IR64*	10
AA	O. sativa	A	S	IR72	11
BB	O. punctata	A	S-Sh	IRGC 105690*	12
BBCC	O. minuta	P	S-Sh	IRGC 101141*	13
CC	O. officinalis	P	S-Sh	PI 59412*	14
CC	O. rhizomatis	P	S	IRGC 101609	15
CC	O. rhizomatis	P	S	IRGC 105950*	16
CCDD	O. alta	P	S-Sh	PI 590398*	17
CCDD	O. latifolia	P	S-Sh	IRGC 100959*	18
CCDD	O. latifolia	P	S-Sh	IRGC 105173	19
EE	O. australiensis	P	S	IRGC 101397*	20
EE	O. australiensis	P	S	IRGC 105277*	21
EE	O. australiensis	P	S	IRGC 86527	22
FF	O. brachyantha	B	S	IRGC 101232*	23
HHKK	O. coarctata	P	S	IRGC 104502*	24

Open in a separate windowFor evaluating aspects of photosynthesis, the model in Equation 1 was considered, and all the listed functional variables, A, g_{s_CO2}, (C_a − C_i), g_m, and (C_i − C_c), were determined. In addition, among the leaf functional traits, the M resistance to CO₂ diffusion per unit of cell surface area exposed to IAS (reciprocal of g_m/S_mes) was calculated as described by Evans et al. (2009): it represents the resistance to CO₂ diffusion from IAS to chloroplasts in a liquid solution through cell wall and membranes (Nobel, 2009). Leaf transpiration rate (E), A/E, the intrinsic A/E (ratio between A and stomatal conductance to water vapor diffusion [g_{s_H2O}]), g_m/g_{s_CO2} (representing the coordination between g_m and g_s), and the carbon isotope composition of leaf biomass (δ13C; calculated as ¹³C/¹²C) were determined. The value of δ13C has been recognized as a potential indicator of leaf A/E: increased limitations on photosynthesis by decreased g_s can lead to higher A/g_{s_H2O} ratios and less discrimination against assimilation of ¹³CO₂ (for review, see Farquhar et al., 1989); the leaf A/E may also be positively linked to the g_m/g_s ratio (Flexas et al., 2008, 2013; Barbour et al., 2010). With respect to leaf structure, the stomatal density, stomatal pore length, and indices of stomatal pore area on both lamina sides (according to Sack et al., 2003), the Thick_leaf, Vol_IAS, S_mes, S_chl, area of M cell section (a_cell) in leaf cross sections, cell wall thickness (Thick_cw), and M cell surface lobing (Lob_cell) were the principal traits estimated. A statistical multivariate analysis (Child, 2006) was employed to identify clusters of highly interrelated leaf traits; trait-to-trait correlation analysis was carried out to further examine leaf structural, functional, and structural-functional relationships.The following are the main hypotheses examined in this study. (1) Leaf thickness will be associated with certain M structural features. (2) g_m will be coordinated with M structural traits. (3) A will be correlated with g_s, g_m, and E. (4) Leaf structural traits will be involved in the relationship between A and E, which will affect leaf A/E. (5) The g_m/g_s ratio will be positively correlated with leaf A/E; associations with high Thick_cw could have implications for plant drought tolerance.