The cartoon in Figure 1 shows a longitudinal section through a maize kernel. The endosperm represents the storage tissue of the seed, providing nourishment to the growing seedling at germination. The endosperm is a simple tissue composed of 3 major cell types: the starchy endosperm (yellow), the basal transfer layer (blue) and the aleurone (red). The transfer layer functions in nutrient uptake from the mother plant during seed development. The starchy endosperm is the major storage site for starch and proteins. While performing some storage function, the primary role of the aleurone is as a digestive tissue. At germination, it secretes amylase into the starchy endosperm causing the breakdown of of the stored starch, providing the growing seedlings with sugars for energy and growth. The apparent simplicity is deceptive; endosperm development is a highly specialized process with many unique features. For detailed information, see one of these reviews (Becraft, 2001; Becraft et al., 2001; Berger, 1999; Olsen, 2001).
Our lab focuses on the differentiation of the aleurone layer. From an experimental perspective, this is advantageous system because in addition to the simplicity already mentioned, the aleurone layer can be conveniently marked facilitating genetic analyses. In the proper genotypes, the aleurone layer specifically accumulates purple anthocyanin pigment. Our approach is to search for mutants that have disrupted aleurone development by screening for loss of pigment. These mutants define genes that are important for normal aleurone development (Becraft and Ascuncion-Crabb, 2000). Figures 2 and 3 below show what a normal kernel looks like in microscopic section and some examples of aleurone mutants.
Although the endosperm is often considered a dead-end tissue, endosperm development shows remarkable plasticity. This can be demonstrated by using unstable mutants that disrupt the aleurone cell fate decision. As shown in Figure 4 at the right, the recessive dek1 mutant lacks an aleurone layer. The white kernel in the top panel is a dek1 mutant and because it lacks an aleurone layer, it lacks the purple anthocyanin pigment found in the aleurone layer of surrounding normal kernels. The next 2 panels show histological sections of normal and dek1 mutant kernels at 12 days after pollination, or about midway through development. At this stage, an aleurone layer is already established in the peripheral layer of the normal endosperm. The peripheral endosperm cells of the dek1 mutant kernel have attributes of starchy endosperm. This altered cell identity indicates that the normal Dek1+ gene is required for the perception and/or response to positional cues that specify aleurone cell fate in the peripheral endosperm cells (Becraft and Ascuncion-Crabb, 2000).
Figure 5 shows a gain-of-function experiment. The dek1 mutation is caused by a Mu transposon inserted into the dek1 gene disrupts its function. Occasionally Mu jumps, restoring the function of the gene and creating revertant (normal) sectors in an otherwise mutant background. The size of the sector reflects how many cycles of cell division occurred following the reversion event, therefore indicating the relative time during development. Small sectors happened late in development. Revertant aleurone sectors as small as a single cell were observed, indicating very late events. Because the mutant has starchy endosperm in the periphery midway in development and onward, these revertant cells have switched identity from starchy endosperm to aleurone. This demonstrates that the positional cues that specify aleurone identity are present throughout the later stages of development and that peripheral endosperm cells remain responsive to those cues (Becraft and Ascuncion-Crabb, 2000).
Figure 6 shows a related loss-of-function experiment. A chromosome-breaking Ds transposon was used to create dek1 mutant sectors in an otherwise normal endosperm. Again some of the sectors were small, indicating late events. Iodine staining revealed that loss of anthocyanin pigment was accompanied by accumulation of starch grains. Thus, these sectors represent cells that had previously possessed aleurone identity but which switched to starchy endosperm upon loss of Dek1+. Therefore the positional cues are also required to maintain aleurone fate (Becraft and Ascuncion-Crabb, 2000).
Mutations in the crinky4 (cr4) gene cause a switch in aleurone cell fate, similar to dek1. Mutations in cr4 also cause crinkled leaves and a short contorted plant. The gene was cloned and found to encode a receptor kinase. Thus, we hypothesize that this represents the receptor for the positional cues that signal the peripheral endosperm cells to adopt an aleurone cell fate (Becraft, et al., 1996). Analysis of the plant phenotype shows that the CR4 receptor regulates an array of processes suggesting its function is analogous to a growth factor receptor (Becraft, et al., 2001; Jin, et al., 2000). The implication of this is that CR4 is involved in a signal transduction pathway that is important for the aleurone cell fate decision. We are currently working on isolating additional genes with mutant phenotypes similar to cr4 to identity other factors necessary for endosperm cell fate decisions. One gene we think might fit into the CR4 pathway is dek1 (Becraft, et al., 2002). Dek1 was recently cloned but we don't yet know molecularly how it is related to CR4 signal transduction (Lid, et al., 2002). For more information on the research in my lab to dissect the CR4 signal transduction pathway, see the section on receptor kinases.
Becraft, P. W. (2001). Cell fate specification in the cereal endosperm. Semin Cell Dev Biol 12, 387-394.
Becraft, P. W., Li, K., Dey, N., and Asuncion-Crabb, Y. T. (2002). The maize dek1gene functions in embryonic pattern formation and in cell fate specification. Development , In press.
Becraft, P. W., Kang, S.-H., and Suh, S.-G. (2001). The maize CRINKLY4 receptor kinase controls a cell-autonomous differentiation response. Plant Physiol 127, 486-496.
Becraft, P. W., Brown, R. C., Lemmon, B. E., Opsahl-Ferstad, H. G., and Olsen, O.-A. (2001). Endosperm Development. In “Current Trends In The Embryology Of Angiosperms” (S. S. Bhojwani, Ed.), pp. 353-374. Kluwer.
Becraft, P. W., and Asuncion-Crabb, Y. T. (2000). Positional cues specify and maintain aleurone cell fate during maize endosperm development. Development 127, 4039-4048.
Becraft, P. W., Stinard, P. S., and McCarty, D. R. (1996). CRINKLY4: a receptor kinase with TNFR similarity, involved in maize epidermal differentiation. Science 273, 1406-1409.
Berger, F. (1999). Endosperm development. Curr. Opin. Plant Biol. 2, 28-32.
Jin, P., Guo, T., and Becraft, P. W. (2000). The maize CR4 receptor-like kinase mediates a growth factor-like differentiation response. Genesis 27, 104-116.
Lid, S. E., Gruis, D., Jung, R., Lorentzen, J. A., Ananiev, E., Chamberlin, M., Niu, X., Meeley, R., Nichols, S., and Olsen, O.-A. (2002). The defective kernel 1 (dek1) gene required for aleurone cell development in the endosperm of maize grains encodes a membrane protein of the calpain gene superfamily. PNAS 99, 5460-5465.
Olsen, O. A. (2001). Endosperm development: cellularization and cell fate specification. Annu Rev Plant Physiol Plant Mol Biol 52, 233-267.