Lily Cdc42/Rac-interactive binding motif-containing protein, a Rop target, involves calcium influx and phosphoproteins during pollen germination and tube growth

Abstract

We report unique desiccation-associated ABA signaling transduction through which the Rop (Rho GTPase of plants) and its target LLP12-2 are regulated during the stage of pollen maturation and tube growth. Overexpression of LLP12-2 drastically inhibited pollen germination and tube growth. Studies on the germination inhibitors, Ca (2+) influx blocking agents LaCl 3 and EGTA and an actin-depolymerizing drug, latrunculin B (LatB), revealed that the LLP12-2-induced inhibition of germination and tube growth is significantly suppressed by LaCl 3 and EGTA in the LLP12-2-overexpressing pollen but not by LatB. These results suggested that LLP12-2 is associated with Ca (2+) influx in the cytoplasm and may be not with actin assembly. With the addition of LaCl 3 and EGTA, LLP12-2-overexpressing pollen increased germination and tube growth compared with the one without addition, whereas pollen expressing GFP decreased germination and tube growth. Thus, an optimum level of [Ca (2+) ]cyt influx is crucial for normal germination and tube growth. Studies on the inhibitors, staurosporine and okadaic acid in the LLP12-2-overexpressing pollen, showed no appreciable increase in germination when compared with the one without addition, suggesting that staurosporine-sensitive protein kinases and dephosphorylation of phosphoproteins may be not involved in the LLP12-2 mediated germination. However, the LLP12-2-induced inhibition of tube length was slightly but significantly suppressed by staurosporine, suggesting that staurosporine-sensitive protein kinases involve in the LLP12-2-induced inhibition of tube growth.

Figure 1

LLP12-2 inhibits pollen germination by regulating the calcium influx channels. Germination percentages were determined 9 h after particle bombardment and subsequent in vitro germination. Equal amounts of GFP and LLP12-2 DNA (7.5 µg) were transiently expressed in lily pollen after which pollen was treated without (control) or with either LaCl₃ (1 µM), EGTA (0.5 mM), LatB (0.05 nM), okadaic acid (5 nM) or staurosporine (1 µM) during germination.

Figure 2

Schematic diagram of the LLP-Rop1 signaling during pollen germination and tube growth. During germination and tube growth, LLP-Rop1 is activated at the tip and activates LLP12-2, which affects calcium influx in the cytoplasm that in turn promotes germination and tube elongation. In addition, staurosporine-sensitive protein kinases are involved in the LLP12-2-induced inhibition of pollen tube elongation.