Imaging and quantifying carbohydrate transport to the developing ovaries of maize

Abstract

Background and aims: Shade or inadequate water can inhibit photosynthesis and limit the development of maize (Zea mays) ovaries around the time of pollination, potentially reducing the number of kernels at harvest. This study investigated whether the decreased photosynthesis diminished only the sugar supply or also altered the transport path to the ovaries.

Methods: Photosynthesis and water potentials (Psiw) were measured in the leaves while dry matter delivery was monitored in the ovaries. Ovary glucose, starch and acid invertase activities were measured in situ. Stems were fed xylem-mobile safranin or phloem-mobile carboxyfluorescein (CF), and the dye transport to the ovaries was determined.

Key results: Under normal conditions, the ovaries gained in dry mass, and starch accumulated in the pedicel and ovary wall. Glucose accumulated in the pedicel, apparently in the apoplast where insoluble (cell-wall-bound) acid invertase acted on the arriving sucrose. A glucose gradient developed from pedicel to nucellus. Safranin moved in the xylem and did not reach the ovary, but CF moved in the phloem and arrived at the ovary. CF also spread into the pedicel but unlike glucose it did not enter the nucellus. Low Psiw or shade decreased leaf photosynthesis, ovary dry mass accumulation, invertase activities, pedicel glucose, starch accumulation and CF delivery. Removal of these treatments reversed the effects.

Conclusions: The success of CF in tracing the general path and rate of carbohydrate transport gave visual evidence that phloem transport to the ovary decreased at low Psiw or in the shade but otherwise remained functional. The decreases indicated that losses in carbohydrate delivery are central features of failed ovary development under these conditions. The selectivity of transport into the nucellus resembled the situation later when embryo and endosperm are present and selective uptake occurs from the apoplast.

Fig. 1.

(A) Leaf water potential (Ψ_w), (B) net photosynthesis, (C) water use, and (D) ovary dry mass in maize deprived of water or shaded around the time of pollination. Open circles: control at high Ψ_w; closed circles: low Ψ_w; closed squares: shade. The black bar on the abscissa indicates when water was withheld from the soil and the white bar indicates the time of shading. The arrow marks the beginning of silking. Plants were hand-pollinated on day 0. Data are means ± s.e. of 4–6 plants.

Fig. 2.

Structure of maize ovary on the day of pollination. Phloem terminates in or immediately below cup-shaped tissue below the nucellus. Stigma and style have been removed. N, nucellus; T, phloem termini; V, vascular structures; PD, pedicel. Scale bar = 1 mm.

Fig. 3.

Starch in maize ovaries around the time of pollination. Control: (A–D) display starch (black) as ovaries develop. Starch was located around the vascular tissue in the pedicel and as part of the cup below the nucellus. No starch was detected in the nucellus. Low Ψ_w: starch was present in (E) but was depleted at low Ψ_w (F), and began to reappear (G) after rewatering. Shade: starch was present in (H) but depleted in shade (J) and remained depleted for 1 d after shade was removed (K). The black bar on the abscissa indicates when water was withheld from the soil and the white bar indicates the time of shading. Scale bar = 1 mm.

Fig. 4.

(A) Localization of carboxyfluorescein (green) and safranin (red) in horizontally cut node (cross-section) below ear on maize ear shoot. (B) Localization of carboxyfluorescein (green) and safranin (red) in vertically cut node (axial-section) below ear of maize ear shoot. Scale bars: (A) 100 mm, (B) 1 mm.

Fig. 5.

Localization and content of carboxyfluorescein (green) in maize ovaries 24 h after stem infusion around the time of pollination. Control: (A–D) as ovaries developed, carboxyfluorescein was increasingly evident (green) in and around the vascular tissues in the pedicel. Low Ψ_w: carboxyfluorescein was nearly absent (E, F) but reappeared 1 d after rewatering (G). Shade: (H) carboxyfluorescein was less abundant than in controls and disappeared (J). It began to reappear 1 d after shade was removed (K). Shade was imposed to match photosynthesis at low Ψ_w (see Fig. 1). Background: (L–O) autofluorescence in the absence of carboxyfluorescein. Scale bar = 1 mm. (P) Spectofluorophotometer measurement of fluorescence from carboxyfluorescein in extracts from ovaries in images. Open circles: control at high Ψ_w; closed circles: low Ψ_w; closed squares: shade. The black bar on the abscissa indicates when water was withheld from the soil and the white bar indicates the time of shading. Data are means ± s.e. of 3–5 plants.

Fig. 6.

In vivo localization of glucose in maize ovaries on the day of pollination (day 0). Epifluorescence micrographs of glucose are paired with respective UV autofluorescence (below) showing ovary anatomy. High, moderate and low concentrations of glucose are shown as red, yellow and blue, respectively. Control: (A, B), glucose (A) was concentrated in the upper pedicel but was low in the nucellus shown in (B). Low Ψ_w: (C, D) glucose concentration was low in (C) compared with control in (A). Shade: (E, F) glucose concentration was low in (E) compared with control in (A). Scale bar = 1 mm. (G) Spectrofluorophotometer quantification of glucose concentrations in extracts from cryosections like those in (A–F). Data are means ± s.e. of three plants.

Fig. 7.

Insoluble (A–C) and soluble (D–F) acid invertase activities in maize ovaries on the day of pollination (day 0). (A) Insoluble activity in vivo was localized in upper pedicel tissue. (B, C) Insoluble activity was not detected at low Ψ_w (B) or in shade (C) but was present if the assay was extended to 24 h. This indicated that insoluble activity was present but very low at low Ψ_w or in shade. (D) Soluble activity in situ was localized in the nucellus. Although there is some overlap of upper pedicel tissues and nucellus tissue in this section, other sections showed the soluble activity to be restricted to the nucellus (McLaughlin and Boyer, 2004a). (E, F) No activity was detected for soluble invertase at low Ψ_w (E) or in shade (F). Scale bar = 1 mm. Images are representative of eight control plants, eight plants at low Ψ_w and three plants in shade.

Fig. 8.

Comparison of mean rates of dry mass (DM) and carboxyfluorescein (CF) accumulation per day during the experiments. (A) Control, (B) low Ψ_w, and (C) shade. Rates of DM accumulation were calculated from the slope of the means shown in Fig. 1D. Rates of CF accumulation are means from Fig. 5P. The black bar on the abscissa indicates when water was withheld from the soil and the white bar indicates the time of shading.