Integrative Seed Biology - Oregon State University

In a carrot (Daucus carota L.) seed (schizocarp) the embryo does not complete its development prior to seed desiccation, and apparently ‘mature’ carrot seeds contain immature embryos when seeds are dispersed from the maternal plants (Fig. 1). Different tissues, such as the epidermis and procambium, are already differentiated and are distinguishable in the embryo, but the cotyledons and the axis are very small compared with the size of the seed itself at maturity. The immature embryos of carrot seeds develop following imbibition and then undergo radicle protrusion, while the endosperm layers are being degraded (Fig. 2).

Figure 1. Completion of embryogenesis in imbibed carrot seeds before radical protrusion.

Figure 2. Schematic representation of embryo development in imbibed carrot seeds

Endo-b-mannanase is involved in the dissolution of carrot endosperm. Mannan is one of the major carbohydrates in the endosperm in Umbelliferae seeds. We have isolated a cDNA from imbibed carrot seeds that encodes endo-b-mannanase (termed DcMAN1).

DcMAN1 mRNA expression is not detected early in imbibition, but it is observed after 18 h from the start of imbibition and increases during 24 h to 30 h from the start of imbibition, leveling off by 36 h (Fig. 3). That is, DcMAN1 is expressed before radicle protrusion.

Figure 3. Northern blot of total RNA from carrot seeds imbibed for 6, 12, 18, 24, 30, and 36 h, probed with LeMAN2.

The expression of DcMAN1 is first detected only in the micropylar-half seed and then occurred in the lateral-half seed at later stages (Fig. 4).

Figure 4. Northern blot of total RNA from micropylar- (M) and the lateral (L) half- carrot seeds imbibed for 30, and 40 h, probed with LeMAN2.

Endo-b-mannanase activity is first detected only in the micropylar-half seed after 18 h from the start of imbibition, and it increases about five-fold from 24 h to 30 h from the start of imbibition, before leveling off. Enzyme activity appears in the lateral-half seed after 24 h from the start of imbibition, with an increase during 30 h to 36 h. The timing of the increase of total endo-b-mannanase activity in imbibed carrot seeds corresponds to that of mRNA expression (Fig. 5).

Figure 5. Endo-b-mannanase activity in carrot seeds before radicle protrusion. Enzyme activity in the micropylar-half seed (£) and the lateral-half seed (¢) was measured separately.

Most endo-b-mannanase activity is detected in the endosperm and very low activity is detected in the embryo. The activity in the embryo is approximately two-orders less than in the endosperm, suggesting that endo-b-mannanase activity is involved in endosperm degradation (Fig. 6).

Figure 6. Gel diffusion assay for endo-b-mannanase activity in the embryo and the endosperm of germinating carrot seeds. The endosperm (Endo) and the embryo (Emb) were isolated from a single seed imbibed for 20 h and directly placed on a gel plate containing the substrate. The endosperm and the embryo from the same seed were given the same number. Standards are commercial endo-b-mannanase from Asperguillus niger.

The immature embryo is located at the micropylar region of carrot seed early in imbibition and elongates into the lateral half seed at the later stages. This occurs concomitantly with enlargement of the corrosion cavity, which presumably is caused by enzymatic degradation of the endosperm, since the spatial and temporal patterns of expression of DcMAN1 mRNA and endo-b-mannanase activity in carrot seeds are consistent with the patterns of the enlargement of the corrosion cavity and embryo development in imbibed seeds. Thus, an endo-b-mannanase is likely to be associated with the developmental program in imbibed carrot seeds.

Carrot seeds complete embryogenesis during seed imbibition after the shedding of seeds. The increase in embryo size in imbibed seeds starts as early as 12 h from the start of imbibition. But most embryos excised from seeds before 24 h from the start of imbibition are not capable of germinating in water (Fig. 7) or MS medium (data not shown).

Figure 7. Developmental growth of excised embryos. Carrot embryos excised from seeds imbibed for 6 h, 18 h, 24 h and 36 h, respectively. The photographs were taken before and after 3 d incubation in water at 25 °C.

Inability of the excised embryos to complete germination at these stages is most likely due to the physiological state of the embryos. At 36 h from the start of imbibition, the embryos appear to have completed development, when they reach two-thirds the length of seed, or more, since most excised embryos can complete germination, with a normally elongated radicle. It is most likely that completion of the developmental program occurs first and then the germinative programs starts, although both occur in imbibed seeds before radicle emergence. The transition from seed development to germination must occur in carrot seeds at later stages of imbibition.

Endo-b-mannanase is involved in the dissolution of carrot endosperm. Mannan is one of the major carbohydrates in the endosperm in Umbelliferae seeds. We have isolated a cDNA from imbibed carrot seeds that encodes endo-b-mannanase (termed DcMAN1).