Sex determination in the monoecious species cucumber is confined to specific floral whorls

Abstract

In unisexual flowers, sex is determined by the selective repression of growth or the abortion of either male or female reproductive organs. The mechanism by which this process is controlled in plants is still poorly understood. Because it is known that the identity of reproductive organs in plants is controlled by homeotic genes belonging to the MADS box gene family, we analyzed floral homeotic mutants from cucumber, a species that bears both male and female flowers on the same individual. To study the characteristics of sex determination in more detail, we produced mutants similar to class A and C homeotic mutants from well-characterized hermaphrodite species such as Arabidopsis by ectopically expressing and suppressing the cucumber gene CUCUMBER MADS1 (CUM1). The cucumber mutant green petals (gp) corresponds to the previously characterized B mutants from several species and appeared to be caused by a deletion of 15 amino acid residues in the coding region of the class B MADS box gene CUM26. These homeotic mutants reveal two important concepts that govern sex determination in cucumber. First, the arrest of either male or female organ development is dependent on their positions in the flower and is not associated with their sexual identity. Second, the data presented here strongly suggest that the class C homeotic function is required for the position-dependent arrest of reproductive organs.

Figure 1.

Flower Morphology of Wild-Type Cucumber Plants. (A) Longitudinal section through male flower buds at two developmental stages. The carpel primordia are arrested in whorl 4. Stamen primordia arise from the flanks of the petals and produce sporogenous tissue. (B) Longitudinal section through a young wild-type male cucumber flower at a later developmental stage just before opening of the flower. The anthers start to produce pollen. (C) Macroscopic view of a male flower. A pair of sepals and petals were removed to allow a view inside. The arrested carpel primordia are visible at the bottom of the flower. (D) Longitudinal section through a female flower bud. Stamen and carpel primordia develop in whorls 3 and 4, respectively. The sepals cover the flower completely. (E) Longitudinal section through a female flower bud at a later developmental stage. The stamen primordia are arrested, and in the fourth whorl an inferior ovary and superior stigmas develop. (F) Macroscopic view of a female flower at a stage just before opening of the flower. As in (C), a pair of sepals and petals have been removed to allow a view inside the flower. The whorl numbers indicate the positions of the floral organs within the flower. O, ovary. (A) (B) (D) (E) .

Figure 2.

Amino Acid Sequence of CUM26 in the Wild Type and the gp Mutant and Expression Patterns of CUM1 and CUM26. (A) The CUM26 protein sequence deduced from the longest reading frame of CUM26 cDNA. The conserved MADS box is underlined with a thick line, and the K box region is underlined with a thin line. The 15 amino acid residues that are deleted in the CUM26 protein of the gp mutant are boxed. (B) CUM1 and CUM26 expression in cucumber leaves and floral organs. Total RNA was isolated from mature leaves (L), sepals (S), petals (P), stamens (St), styles (Sl), stigmas (Sg), nectaries (N), and inferior ovaries (O).

Figure 3.

Expression Patterns of CUM1 and CUM26 in Wild-Type and gp Mutant Flowers. Longitudinal sections were hybridized with digoxigenin-labeled antisense CUM1 ([A], [B], [G], and [H]) or CUM26 ([C], [D], [E], and [F]) RNA. All sections were viewed using dark-field microscopy. The whorl numbers indicate the positions of the floral organs within the flower. (A) and (C) Young male flower buds from a wild-type plant. The carpel primordia are arrested and the anthers start to produce sporogenous tissue. The bud is completely covered by sepals. (B) and (D) Young female flower buds from a wild-type plant. The stamen primordia are arrested and in the fourth whorl an inferior ovary and superior stigmas develop. (E) and (G) Male flowers from a gp plant grown under normal temperature conditions (22°C). The red arrowheads indicate new buds as they appear in these bushy indeterminate flowers (cf. Figure 4B). (F) and (H) Male flowers from a gp plant grown under high-temperature conditions (35°C). Carpels are formed in whorl 3, and the carpel primordia in the fourth whorl are arrested. The outer two whorls are sepals. O, ovary. (A) (A) (H)

Figure 4.

Flower Morphology of the gp Mutant. (A) Young male flower of the gp mutant grown at 22°C. The flower is composed of two whorls of sepals only. (B) Older indeterminate male flower grown at 22°C. New buds develop inside the primary flower, which results in a bushy appearance. (C) Longitudinal section through young male flower buds grown at 22°C. Initially, the whorl 3 and 4 primordia develop as in the wild type (left bud). At a later developmental stage (right bud), new meristems develop in the third whorl, as indicated by the red arrowhead. The carpel primordia in whorl 4 remain arrested. (D) Longitudinal section through an older indeterminate male flower grown at 22°C. A new flower bud originating from the third whorl in this bushy flower is indicated by the red arrowhead. (E) Male flower of the gp mutant grown under high-temperature conditions (35°C) with stamens homeotically transformed into carpels. (F) Older male flower of the gp mutant grown under high-temperature conditions (35°C) with fruit developing in whorl 3. The whorl 1 and 2 organs are senesced. (G) Female flower of the gp mutant grown at 22°C. The inferior ovary is not affected, and the outer two whorl organs are sepals. No changes in flower phenotype were observed when the plants were grown under high-temperature conditions (35°C). (H) Longitudinal section through a young male flower of the gp mutant grown under high-temperature conditions (35°C). Carpelloid structures develop in whorl 3, and carpel primordia are arrested in whorl 4. (I) Older bud as in (H). Fruit-like bodies develop in the third whorl on positions normally occupied by stamens (cf. Figures 4E and 4F). (J) Longitudinal section through a female flower bud of the gp mutant. The two inner whorls are like those in wild-type flowers (cf. Figure 1E). The whorl numbers indicate the positions of the floral organs within the flower. O, ovary. (C) (D) (H) (I) (J) .

Figure 5.

Flower Morphology of Transgenic Cucumber Plants in Which CUM1 Was Ectopically Expressed or Cosuppressed. (A) Male flower of a CUM1 ectopically expressing plant (T340-1). Five complete superior carpels develop in whorl 1, which are partly fused at the basis forming an ovary-like structure. (B) Longitudinal section through a male flower of T340-1. The whorl 1 organs are carpelloid, and the organs in the second whorl are chimeric with petaloid and antheroid tissue. The carpel primordia in whorl 4 are arrested. (C) Detail of (B) as indicated by the box. Antheroid tissue developing on top of the second whorl organs. Sporogenous tissue with developing pollen is indicated by the arrow. (D) Female flower of T340-1 with superior carpels in whorl 1 and a malformed ovary in the fourth whorl. In the second whorl, remnants of petal tissue is indicated by an arrow. (E) Longitudinal section through a female flower of T340-1. Antheroid tissue developing on top of the second whorl organs is indicated in the box. The inner part of the flower is highly malformed and organs are not recognizable. (F) Detail of (E) as indicated by the box. An arrow shows the developing pollen in the antheroid tissue. (G) Longitudinal view of a male flower bud of transformant T340-3 in which CUM1 was cosuppressed. Indeterminate floral buds are visible in whorls 3 and 4. (H) Male flower of transformant T340-3 showing indeterminate floral bud formation in positions normally occupied by stamens. The flower is indeterminate in the center. (I) Female flower of T340-3, with petal formation in the third whorl. The inferior ovary is not affected in this homeotic mutant. (J) Detail of (I) showing the petals in whorl 3 and the indeterminacy in the fourth whorl. (K) Longitudinal view of a female flower bud of T340-3. The small fruit growing inside the primary ovary is indicated by an arrow. The line indicates the plane of the cross-section shown in (L). (L) Cross-section through a female flower bud of T340-3. The position of the section is indicated in (H). Petals develop in whorl 3 on positions normally occupied by stamens. The whorl 4 structure is the upper part of the small fruit that is growing inside the primary ovary. The whorl numbers indicate the positions of the floral organs within the flower. S, stigmatic tissue; O, ovary. (B) (E) (L) .

Figure 6.

Scheme of the ABC Model of Floral Organ Identity Determination (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994). (A) The identity of the floral organs is defined by the actions of three distinct classes of floral homeotic genes, each of which is active in two adjacent whorls in wild-type flowers. In the first whorl, only class A genes are active and lead to the formation of sepals. In whorl 2, the formation of petals is defined by the combinatorial action of class A and B genes. The combinatorial expression of class B and C homeotic genes in whorl 3 determines stamen identity. In whorl 4, in which only the class C function is active, carpels develop. In addition, the class A and C functions are mutually antagonistic, as indicated by the two-headed arrow. (B) Ectopic expression of a class C gene leads to the suppression of the class A function, resulting in a phenotype similar to that of the class A mutant: homeotic transformations of sepals into carpels and petals into stamens. (C) In the class B mutant, only class A and C genes are active, resulting in sepals in the two outer floral whorls and carpels in the two inner whorls. (D) In the class C mutant, the class A function is active in all four floral whorls, resulting in petals in whorl 3. Class C genes are also essential to specify meristem determinacy in the most inner whorl. Loss of class C function therefore results in the development of indeterminate flowers in the fourth floral whorl.

Figure 7.

Summary of Organ Identities in the Four Floral Whorls of Male and Female Flowers of Wild-Type and Class A, B, and C Cucumber Mutants. Indeterminacy is indicated by “flower.” WT, wild type.