Regulation of in vitro morphogenesis

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How cells acquire specific characteristics as they grow towards an organ or organism is a fundamental and yet poorly understood question in animal and plant developmental biology. Plant morphogenesis relies on the ability of cells to redirect their fate, a process best exemplified in the shoot and root apical meristems (SAMs and RAMs). Meristems serve as a reservoir of pluripotent cells producing derivatives recruited for the formation of lateral organs. Balance between self-renewal and retention of pluripotent cells, and differentiation of their derivatives, is therefore key for meristem function. Phytoglobins (Pgbs), plant hemoglobins with nitric oxide (NO) scavenging properties, influence cell fate commitmentand reprogram cell fate by delaying differentiation and maintaining meristematic cells in an undifferentiated state during conditions of stress.This program of research comprises two main objectives. The first objective will examine the ontogeny of RAMs and SAMs from cultured Arabidopsis roots and the participation of Pgbs (by experimentally altering their amount in cultured cells) in the specification of the pluripotent meristematic cells analysed. Intermediates of Pgb-responses, such as nitric oxide, ethylene and changes in cellular redox state will also be investigated in relation to SAM and RAM formation. Dissection of root meristem compartments, including the quiescent cells responsible for conferring pluripotency, will also be employed to increase the resolution of these studies. Collectively, results from this objective will provide a model integrating Pgb action and response during the acquisition of pluripotency.The second objective will define the mechanisms underlying the somatic-embryogenic fate transition by inducing embryogenic cells from Arabidopsis somatic tissue. In this in vitro system, embryogenic cells are defined by the expression of WUSCHEL (WUS), and suppression of Pgb increases the number of WUS-expressing embryogenic cells. The proposed research will focus on how Pgb regulates WUS and the specification of embryogenic cells by investigating cytokinin- and auxin-mediated pathways operating during meristematic cell specification in vivo. The microdissection and separation of nascent embryogenic cells from the adjacent somatic cells will identify factors determining the somatic-embryogenic transition.The novelty of this program resides in the utilization and integration of a large number of techniques (structural, physiological and molecular) to test proposed developmental models controlling the redirection of cell fate in response to changing culture conditions. Results will be ground breaking in that they will advance not only the field of embryogenesis, but also all those processes requiring a redirection of cell behavior which are crucial for plant propagation, productivity, and survival under adverse conditions.