Figure 1
Scheme indicating soybean phenology and the duration of heat stress (black line) and drought stress (red line) treatments. End of seed fill in drought-stressed plots and heat-stressed combined with drought-stressed plots = 106 days after emergence (DAE); end of seed fill in irrigated heat-stressed plots and control plots =116 DAE. Samples for leaf structure, ultrastructure, leaf area, and specific leaf weight determinations were collected 95 DAE (coincidently with the end of heat stress imposition). Indicated developmental stages (Fehr & Caviness 1977) are V1, V2, V3, VN: 1st, 2nd, 3rd, Nth node; R3: beginning pod (pods in one of the uppermost nodes is 0.5 cm). ; R5.5: mid grain filling (seeds of 6mm length in a pod at one of the four uppermost nodes); R7: beginning of physiological maturity (first pod on the main stem is matured to a brown pod color); R8: full maturity (when 95% of the pods have reached their full mature color). Illustration: Oliveira Junior et al.OLIVEIRA JUNIOR AD, CASTRO CD, PEREIRA L & DOMINGOS CDS. 2016. Estádios fenológicos e marcha de absorção de nutrientes da soja. Embrapa Soja-Fôlder/Folheto/Cartilha (INFOTECA-E). (2016).
Figure 2
Biplot displaying relations between soybean leaf structure (black circles) and physiological traits (white circles). Data correspond to the average of two soybean cultivars grown under four conditions during seed filling (grey triangles): control (non-heat-stressed and non-drought-stressed), heat-stressed (HS), drought-stressed (DS), and HS × DS plots. Measurements were performed on the central leaflet of the third trifoliate leaf from the main stem apex, 21 d after the plot reached R5.5 (coincidently with the end of HT imposition), except the canopy temperature which was measured also during the HS treatment. ᶲPSII= quantum efficiency of photosystem II photochemistry; Fv/Fm= maximum quantum yield of photosystem II; Fo/Fm= thylakoid membrane damage, SPAD= leaf chlorophyll content, AD= adaxial, AB= abaxial.
Figure 3
Light micrographs of leaf anatomy in cross-sections of soybean (Glycine max) grown under four water and temperature combinations: control (non-heat-stressed and non-drought-stressed), heat-stressed (HS), drought-stressed (DS), and HS × DS treatments. (a) Control leaf midrib with turgent parenchyma cells, vascular system, and well-organized mesophyll. (b) Control leaf dorsiventral mesophyll, typically columnar palisade cells, and spongy parenchyma cells irregularly shaped with abundant air spaces. (c) HS leaf midrib with dehydrated parenchyma cells. (d) HS leaf, detail of mesophyll with large intercellular spaces, and reduced number of chlorenchyma cells. (e) DS leaf midrib. (f) DS leaf, detail of mesophyll. (g) HS × DS leaf midrib. (h) HS × DS leaf, detail of mesophyll with decreased palisade cells diameter compactly arranged and small spongy parenchyma cells. Paraveinal mesophyll (*), bundle-sheath cells (BSc), parenchyma (P), palisade parenchyma (PP), stoma (arrow), spongy parenchyma (Sp). Scale bars: a, c, e, g= 50 µm; b, d, f, h= 20 µm.
Figure 4
Scanning electron micrographs of leaf surface adaxial and abaxial epidermis of soybean (Glycine max) grown under four water and temperature combinations. (a, b) Control (non-heat-stressed and non-drought-stressed). (a) Adaxial leaf with an irregular surface, turgid and large size epidermal cells, and stomatal apparatus, note the smooth cuticle. (b) Abaxial leaf surface, similar traits as in (a), epicuticular waxes are observed, as in (d) and (f). (c-d) Heat-stressed (HS). (c) Adaxial leaf surface (d) Abaxial leaf surface. (e-f) Drought-stressed (DS). (e) Adaxial leaf surface. (f) Abaxial leaf surface. (g-h) HS × DS. (g) Adaxial leaf surface. (h) Abaxial leaf surface. Both epidermises (c-h) under the different treatments showed dehydrated and smaller epidermal cells. Subsidiary cell (Sc). Scale bars: a-h= 20 µm.
Figure 5
Transmission electron micrographs showing leaf ultrastructure of stomatal apparatus in soybean (Glycine max) grown under four water and temperature combinations: control (non-heat-stressed and non-drought-stressed), heat-stressed (HS), drought-stressed (DS), and HS × DS treatments. (a) Guard cells of control leaf with intact outer chloroplast membrane and undamaged inner membrane system consisting of grana and stroma thylakoids. (b-d) Guard cells of heat-stressed (HS), drought-stressed (DS), and HS × DS leaves, respectively, with damage to chloroplast membrane, disassemble of grana and stroma thylakoid (arrow) and increased vacuolization of the cytoplasm. Cuticle (C), chloroplast (Cl), mitochondria (Mt), starch grain (Sg), vacuole (V). Scale bars: a-d= 2 µm.
Figure 6
Transmission electron micrographs showing leaf ultrastructure of chloroplast palisade mesophyll cells in soybean (Glycine max) grown under four water and temperature combinations: control (non-heat-stressed and non-drought-stressed), heat-stressed (HS), drought-stressed (DS), and HS × DS treatments. (a) Chloroplast of control leaf with an undamaged membrane (arrow) and well-developed grana and stroma thylakoids. (b-d) Chloroplast of heat-stressed (HS), drought-stressed (DS), and HS × DS leaves, respectively, with severe unstacking and damage to membranes, abundant starch grains, and plastoglobules. Chloroplast (*), grana thylakoids (Gt) (stack of thylakoids), mitochondria (Mt), peroxisome (Po), plastoglobule (Pg), starch grain (Sg), stroma thylakoids (St). Scale bars: a-d= 1 µm.
Figure 7
Transmission electron micrographs showing leaf ultrastructure of chloroplast bundle sheath cells in soybean (Glycine max) grown under four water and temperature combinations: control (non-heat-stressed and non-drought-stressed), heat-stressed (HS), drought-stressed (DS), and HS × DS treatments. (a) Chloroplast of control leaf with normal size, intact membrane, and well-formed grana and stroma thylakoids. (b-d) Chloroplast of heat-stressed (HS), drought-stressed (DS), and HS × DS leaves, respectively, with notably increased size, damage to thylakoids membranes, and unstacking of grana. Chloroplast (*), cell wall (arrow), damaged vacuolar membrane (arrowhead), grana thylakoid (Gt), mitochondria (Mt), nucleus (N), plastoglobule (Pg), peroxisome (Po), starch grain (Sg), stroma thylakoid (St), vacuole (V). Scale bars: a-d= 1 µm.
Figure 8
Mesophyll chloroplast size (a), and bundle sheath chloroplast size (b) of the third trifoliate leaves from the main stem apex of soybean plants grown under four water and temperature treatments: control (non-heat-stressed and non-drought-stressed), heat-stressed (HS), drought-stressed (DS) and HS × DS plots, expressed as mean and standard error. Different letters indicate significant differences among means (p ≤ 0.05). Measurements were performed 21 d after the plot reached R5.5 (coincidently with the end of HT imposition).
Figure 9
Thylakoid membrane damage (Fo/Fm ratio) (a), and chlorophyll content (SPAD value) (b) of the third trifoliate leaves from the main stem apex of soybean plants during seed filling exposed to four water and temperature treatments: control (non-heat-stressed and non-drought-stressed), heat-stressed, drought-stressed and heat-stressed × drought-stressed plots. Vertical bars denote ± SE of means. Black arrow = onset of heat treatment, white arrow = end of heat treatment.
Table I
Leaf area, specific leaf weight, and thickness of foliar traits of fully expanded leaves at 21 d after the plot reached R5.5 (coincidently with the end of HT imposition) for soybean crops grown under four conditions during seed filling: control (non-heat-stressed and non-drought-stressed), heat-stressed (HS), drought-stressed (DS) and HS × DS plots. Values are mean ± SEM. Values in columns followed by the same letter are not significantly different at p = 0.05.
Table II
Leaf epidermal anatomy traits of fully expanded leaves at 21 d after the plot reached R5.5 (coincidently with the end of HT imposition) for soybean crops grown under four conditions during seed filling: control (non-heat-stressed and non-drought-stressed), heat-stressed (HS), drought-stressed (DS) and HS × DS plots. Values are mean ± SEM. Values in columns followed by the same letter are not significantly different at p = 0.05.