Sex expression and its variation in cucumber plants
Plants were grown until anthesis of flowers on node 25, andthe sex of the flowers on each node of the main stem was recorded.Figure 1 shows the sex expression of RS-G and RS-M cucumberplants in Experiment I. In this experiment, the first flowerappeared on nodes 3–4 in both cucumber lines, and thepercentage of flowered nodes with female flowers was 100% inRS-G and 89.1% in RS-M cucumbers. RS-M plants produced maleflowers on nodes 10–12, although female flowers developedon other nodes. Figure 2 shows the sex expression of RS-G andRS-M cucumber plants grown for Experiment II. The first flowerappeared on node 5 in RS-G and on node 3 in RS-M cucumbers.The percentage of flowered nodes with female flowers in RS-Gand RS-M cucumber plants was 100% and 21.9%, respectively, underthe growth conditions of this experiment. RS-M cucumber plantsdeveloped female flowers on nodes 6–7, 12–13, 19–20,and 24–25, and male flowers were produced at the othernodes. Numbers of nodes with female flower on the main stemsin RS-M cucumber in Experiment I and Experiment II were 20.7and 5.6, respectively, and the difference was statisticallysignificant by Student t test (P <0.01). Thus, sex expressionof the gynoecious RS-G cucumber was relatively stable, but thatof the monoecious RS-M cucumber varied dramatically in the twoexperiments.
Expression of the CS-ACS2 gene in different organs of cucumber plants
An ACC synthase gene, CS-ACS2, was isolated from cucumber plantsand shown to be expressed in shoots and female flowers (Kamachi et al., 1997,2000; Yamasaki et al., 2003b). However, no detailed analysisof the expression of the CS-ACS2 gene in other organs of cucumberplants has been conducted. Therefore, RT-PCR was used to analyseCS-ACS2 expression in different organs of the monoecious cucumberRS-M and the gynoecious cucumber RS-G (Fig. 3). CS-ACS2 expressionwas specific to the shoot apex in both monoecious and gynoeciouscucumber plants. Moreover, the spatial expression analysis usedhere revealed that accumulation of CS-ACS2 mRNA was not detectedin leaves, stems, or roots. CS-ACS2 mRNA accumulated more abundantlyin the shoot apices of gynoecious cucumber than that of themonoecious line, as reported previously. Accumulation of CS-ACS2mRNA in roots was detected when the number of PCR cycles wasincreased to 30, but there was no difference in the signal intensitybetween monoecious and gynoecious cucumber plants (data notshown). To identify the specific site of CS-ACS2 expression,the shoot apices were analysed by in situ hybridization. Inboth RS-G and RS-M cucumber plants, CS-ACS2 mRNA accumulatedbeneath the pistil primordia or in the region of the flowerbud locules, but not in other organs such as the immature leavesand stems (Fig. 4A, B). When a CS-ACS2 sense probe was used,no signals were detected in any organs including the flowerbuds (data not shown).
Expression of the CS-ACS2 gene in flower buds at different developmental stages in gynoecious and monoecious cucumber plants
Since CS-ACS2 was expressed predominantly in the flower budsof cucumber shoots, its mRNA accumulation was examined in eachflower bud in the shoot apices of cucumber plants. Because theproportion of male and female flowers in monoecious cucumbersvaries in response to environmental cues, shoot apices for insitu hybridization were obtained from cucumber plants grownin different seasons for Experiment I and Experiment II.
In Experiment I, flower buds on nodes 3–14 were obtainedfrom cucumber plants at the 4-leaf stage. In cucumber plantsat this stage, flower buds had already initiated and developedby node 17–18, and the flower sex had been determinedby node 10–11. In the gynoecious RS-G cucumber, flowerbuds on nodes 13 and 14 were at the stage of primordia formationof the sepals and stamens (Fig. 5A, B). Flower buds on nodes11 and 12 were at a bisexual stage (Fig. 5C, D), but the sexof flower buds on the lower nodes had been determined, and theseflower buds possessed pistils that had begun to form a cavityfor ovary development (Fig. 5E–L). Accumulation of CS-ACS2mRNA was seen in flower buds obtained from node 5 through 12(Fig. 5C–J). No accumulation of CS-ACS2 was observed inflower buds on nodes 13 and 14, in which pistil primordia hadnot yet been formed (Fig. 5A, B). Flower buds on nodes 11 and12, which were at the bisexual stage, as judged by the presenceof primordia of both pistils and stamens, showed CS-ACS2 mRNAaccumulation only beneath the pistil primordial (Fig. 5C, D).In flower buds, on nodes 5–10, ovary development startedafter sex determination, and CS-ACS2 mRNA accumulated in thelocule of the ovary, the cavity where the ovule will develop(Fig. 5E–J). Female flower buds on nodes 3 and 4 developedto approximately 4 mm long and did not show CS-ACS2 mRNA accumulation(Fig. 5K, L).
Flower buds from nodes 3 through 13 were examined in monoeciousRS-M cucumber plants (Fig. 6A–K). As in the RS-G gynoeciouscucumber, flower buds on nodes 10–12 were at the bisexualstage (Fig. 6B–D). CS-ACS2 mRNA accumulation was seenin flower buds on nodes 4–9 and node 12 (Fig. 6B, E–J).The accumulation site of CS-ACS2 mRNA in flower buds of monoeciouscucumber was similar to that of gynoecious cucumber. That is,flower buds on node 12 belonged to the bisexual stage, and CS-ACS2mRNA accumulated just beneath the pistil primordial (Fig. 6B).Flower buds on nodes 4–9 had begun to form a cavity forlocule development in the ovary, and CS-ACS2 mRNA was detectedsurrounding the cavity (Fig. 6E–J). Flower buds on nodes3, 10, 11, and 13 did not show CS-ACS2 expression (Fig. 6A, C, D, K).
In Experiment II, flower buds were obtained from nodes 5 through18 of RS-G and from nodes 5 through 17 of RS-M cucumber plantsat the 6-leaf stage (Figs 7A–N, 8A–M). In cucumberplants at this stage, flower buds had developed up to node 21,and the flower sex had been determined by node 13–14.In RS-G cucumber plants, flower buds on nodes 17 and 18 wereforming stamen primordia (Fig. 7A, B). Sex determination hadoccurred in flower buds on nodes 5 through 13, which had begunto form a cavity beneath the pistil or to develop ovules (Fig. 7F-N).CS-ACS2 expression was strongly observed in flower buds on nodes5–16 in RS-G cucumber plants (Fig. 7C–N). Flowerbuds on nodes 14–16 were at the bisexual stage and showedCS-ACS2 mRNA accumulation (Fig. 7C–E). However, flowerbuds on nodes 17 and 18, which had not developed to the bisexualstage, showed no accumulation of CS-ACS2 mRNA (Fig. 7A, B).In RS-M cucumber plants, flower buds on nodes 14 through 17were still at the bisexual stage or before the stage of formationof pistil primordia (Fig. 8A–D), whereas flower buds onnodes 5 through 13 had already undergone sex differentiation(Fig. 8E–M). The flower buds on nodes 7 and 13 appearedto be developing as female flowers, and CS-ACS2 mRNA accumulationoccurred only in the flower buds on nodes 7 and 13 in the RS-Mcucumber plants (Fig. 8E, K).
Thus, the nodes that produced female flowers well coincidedwith those expressed CS-ACS2 gene in flower buds in both ExperimentI and Experiment II.Location of the CS-ACS2 expression in developing ovary
To clarify further the tissue that accumulates CS-ACS2 mRNA,flower buds were obtained from node 9 of RS-G cucumber plantsgrown for Experiment I and cross-sections were prepared forin situ hybridization. CS-ACS2 expression occurred specificallysurrounding the tissues destined to develop as ovules and placentain the female flowers of cucumber plants (Fig. 9A–C).