What are the prospects for reprogramming rice leaf development

This question really comes in two parts: What are the prospects for filling our gaps in knowledge about control of leaf development and then what are the prospects for using that knowledge to modify rice? Modification requires first isolating genes that control developmental processes. Because none of these genes that control the key features of C4 leaves have been isolated, we must therefore ask how they might be found.

Contemporary biology has made use of model genetic systems to identify and isolate genes whose protein product is unknown. Maize and Arabidopsis thaliana are often the systems of choice in plants and significant progress has been made in understanding the genetic control of some aspects of development, especially floral morphogenesis. One approach to understanding genetic control of tissue pattern, particularly vein spacing, would be to look for mutants with altered vein patterns. Although mutants are known that affect veins, such as those resulting in loss of midribs, mutants affecting vein patterns have proven difficult to identify.

Cell

Cell

lineage

position

Vein

Provascular

j

L

^ BSC

' C4 signal

Vein

Ground

signal

MC

r

Provascular

Fig. 3. Mechanisms responsible for the establishment of vein spacing and cell pattern in a C4 leaf. See text for details. BSC = bundle sheath cell, MC = mesophyll cell.

Fig. 3. Mechanisms responsible for the establishment of vein spacing and cell pattern in a C4 leaf. See text for details. BSC = bundle sheath cell, MC = mesophyll cell.

One aspect of the problem is that there are no good markers to identify provascular strands at their earliest developmental stages when patterns are formed.

Mutations in genes that control the formation of BSC and MC patterns would be very desirable, as would mutants affecting BSC and MC differentiation. It is not obvious, however, what sort of phenotype we should look for. This problem has plagued the efforts to identify "C, mutants." Langdale and colleagues reasoned that mutations affecting BSC and MC differentiation should be photosynthetically impaired. They screened mutagenized maize seedlings for pale green plants that showed altered levels of C4 enzymes or defective chloroplasts in one cell type. They have identified several mutations specific to BSC and have shown that these bundle sheath defective mutants affect chloroplast structure and the accumulation of BSC enzymes (Langdale and Kidner 1994). The Bsdl gene has been isolated and shown to encode a transcription factor (Hall et al 1998).

Another approach relies on the major worldwide research activities in structural and functional genomics. Both maize and rice are the subject of major gene sequencing efforts and both are using transposable elements to make large mutant collections so that the phenotype of a knockout mutation in any gene can be determined. Of particular interest would be a comparison of expressed sequence tag (EST) libraries from rice and maize leaves. Each library is a collection of all genes expressed in the leaf. Some of the differences should be due to genes related to the C4 nature of the maize leaf. A simple comparison, however, may not be adequate to identify the key genes that control C4 features. It is possible that some important differences between C, and C4 will be due to the timing of gene expression rather than to new genes for the C4. Vein spacing, for example, could be viewed as an additional set of provascular strands being established after the process would have finished in a C, leaf. The comparison between maize and rice will therefore need to rely on microarray technology to compare gene expression programs during all stages of rice and maize leaf development. In this approach, an EST library consisting of perhaps 10,000 clones is put into a microarray on a single glass slide by a robot. RNA from selected stages of leaf development is labeled with a fluorescent dye and then hybridized to the microarray. The extent of fluorescence at each EST gives a quantitative estimate of the expression level of that gene. From the vast amount of expression data generated, it should be possible to identify genes that may be related to C4 features. The maize transposable element collection can then be screened to look for mutations in these genes and determine their phenotypes. It is fortunate that one of the most widely used model genetic systems in plant research, maize, is a C4 plant.

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