Transient reconstitution of the plant cell cycle regulatory machinery in leaf mesophyll protoplasts to identify direct SnRK1 growth targets

As sessile and autotrophic organisms, plants are significantly challenged by the changing environment and continuously need to adjust their growth and metabolism to resource supplies. The cellular carbon and energy status functions as an important point of integration of both developmental and environmental signals. The SnRK1 (SNF1-related kinase 1) kinase acts as a cellular fuel gauge, that is activated in response to diverse energy-depleting stress conditions to maintain energy homeostasis for optimal growth and survival. Although alteration of SnRK1 activity can have dramatic effects on plant development, whether and how SnRK1 directly affects growth-controlling processes, such as cell division and cell expansion, is still unclear. We started exploring Arabidopsis leaf mesophyll protoplasts as a tool to study possible molecular interactions with the cell cycle regulatory machinery. Interestingly, both application of sugars and hormones and reconstitution of the upstream regulatory pathways by transient over- expression effectively induced S- and M-phase specific gene expression in differentiated leaf cells. Co-expression with the SnRK1 catalytic α subunit (KIN10) now enables identification of direct targets. One reported link between energy availability and cell division is the transcriptional regulation of the G1/S phase transition-specific D-type cyclins by sugar availability. The use of PrCYCD3;1::LUC reporter constructs and transient expression of tagged proteins in protoplasts reveals both repression of CYCD3;1 expression and enhanced proteasome-mediated degradation of the CYCD3;1 protein in response to increased SnRK1 activity. In conclusion, leaf mesophyll protoplasts can be used to identify and dissect molecular mechanisms linking metabolic status to growth and development, although interactions need to be confirmed in meristematic tissue in planta and by genetics.status: publishe