Cytokinin signaling regulates cambial development in poplar.

Although a substantial proportion of plant biomass originates from the activity of vascular cambium, the molecular basis of radial plant growth is still largely unknown. To address whether cytokinins are required for cambial activity, we studied cytokinin signaling across the cambial zones of 2 tree species, poplar (Populus trichocarpa) and birch (Betula pendula). We observed an expression peak for genes encoding cytokinin receptors in the dividing cambial cells. We reduced cytokinin levels endogenously by engineering transgenic poplar trees (P. tremula x tremuloides) to express a cytokinin catabolic gene, Arabidopsis CYTOKININ OXIDASE 2, under the promoter of a birch CYTOKININ RECEPTOR 1 gene. Transgenic trees showed reduced concentration of a biologically active cytokinin, correlating with impaired cytokinin responsiveness. In these trees, both apical and radial growth was compromised. However, radial growth was more affected, as illustrated by a thinner stem diameter than in WT at same height. To dissect radial from apical growth inhibition, we performed a reciprocal grafting experiment. WT scion outgrew the diameter of transgenic stock, implicating cytokinin activity as a direct determinant of radial growth. The reduced radial growth correlated with a reduced number of cambial cell layers. Moreover, expression of a cytokinin primary response gene was dramatically reduced in the thin-stemmed transgenic trees. Thus, a reduced level of cytokinin signaling is the primary basis for the impaired cambial growth observed. Together, our results show that cytokinins are major hormonal regulators required for cambial development.

The effects of drugs inhibiting protein secretion in the filamentous fungus Trichoderma reesei. Evidence for down-regulation of genes that encode secreted proteins in the stressed cells.

To study the mechanisms of protein secretion as well as the cellular responses to impaired protein folding and transport in filamentous fungi, we have analyzed Trichoderma reesei cultures treated with chemical agents that interfere with these processes, dithiothreitol, brefeldin A, and the Ca(2+)-ionophore A23187. The effects of the drugs on the kinetics of protein synthesis and transport were characterized using metabolic labeling of synthesized proteins. Cellobiohydrolase I (CBHI, Cel7A), the major secreted cellulase, was analyzed as a model protein. Northern analysis showed that under conditions where protein transport was inhibited (treatments with dithiothreitol or brefeldin A) the unfolded protein response pathway was activated. The active form of the hac1 mRNA that mediates unfolded protein response signaling was induced, followed by induction of the foldase and chaperone genes pdi1 and bip1. Concomitant with the activation of the unfolded protein response pathway, the transcript levels of genes encoding secreted proteins, like cellulases and xylanases, were drastically decreased, suggesting a novel type of feedback mechanism activated in response to impairment in protein folding or transport (repression under secretion stress (RESS)). By studying expression of the reporter gene lacZ under cbh1 promoters of different length, it was shown that the feedback response was mediated through the cellulase promoter.