Manipulation of hemoglobin expression affects Arabidopsis shoot organogenesis

Over the past few years non-symbiotic plant hemoglobins have been described in a variety of plant species where they fulfill several functions ranging from detoxification processes to basic aspects of plant growth and post-embryonic development. To date no information is available on the role of hemoglobins during in vitro morphogenesis. Shoot organogenesis was induced in Arabidopsis lines constitutively expressing class 1, 2 and 3 hemoglobins (GLB1, 2 and 3) and lines in which the respective genes were either downregulated by RNAi (GLB1) or knocked out (GLB2 and GLB3). The process was executed by culturing root explants on an initial auxin-rich callus induction medium (CIM) followed by a transfer onto a cytokinin-containing shoot induction medium (SIM). While the repression of GLB2 inhibited organogenesis the over-expression of GLB1 or GLB2 enhanced the number of shoots produced in culture, and altered the transcript levels of genes participating in cytokinin perception and signalling. The up-regulation of GLB1 or GLB2 activated CKI1 and AHK3, genes encoding cytokinin receptors and affected the transcript levels of cytokinin responsive regulators (ARRs). The expression of Type-A ARRs (ARR4, 5, 7, 15, and 16), feed-back repressors of the cytokinin pathway, was repressed in both hemoglobin over-expressors whereas that of several Type-B ARRs (ARR2, 12, and 13), transcription activators of cytokinin-responsive genes, was induced. Such changes enhanced the sensitivity of the root explants to cytokinin allowing the 35S::GLB1 and 35S::GLB2 lines to produce shoots at low cytokinin concentrations which did not promote organogenesis in the WT line. These results show that manipulation of hemoglobin can modify shoot organogenesis in Arabidopsis and possibly in those systems partially or completely unresponsive to applications of exogenous cytokinins.