A KNAT3-like homeobox gene from Juglans nigra L., JnKNAT3-like, highly expressed during heartwood formation.

The value of black walnut (Juglans nigra L.) is affected by the quality and quantity of darkly colored heartwood in its stem. We are exploring the regulation of heartwood production by identifying genes associated with the transition from sapwood to heartwood. Previous microarray data indicated that heartwood formation may be related to programmed cell death (PCD). To test this hypothesis, we analyzed the region of heartwood formation in walnut stems (i.e., the transition zone, TZ) for the expression of 80 ESTs putatively associated with PCD. Semi-quantitative RT-PCR and real-time PCR was performed to detect the expression changes in candidate genes in the TZ and sapwood of trees harvested in summer and fall. The results revealed that the transcript of a clone that encodes a presumed homeobox protein knotted-1-like 3 (KNAT3) was highly expressed in the TZ when compared with other tissues. Analysis of the full-length coding sequence revealed that the black walnut gene contains regions with 67% similarity to Knox1 and Knox2 domains from the Arabidopsis thaliana KNAT3, as well as a putative homeodomain known to be a transcription factor in other plants. JnKNAT3-like transcript was detected in the pith meristem, roots, embryogenic callus, vascular cambium, female flowers, male flowers, green leaves, and partially and fully senescent leaves of black walnut, although transcript abundance varied considerably among tissues. These analyses may provide insight into the mechanism regulating heartwood formation in walnut and other hardwood trees.