Sukumar Poornima, Edwards Karin S, Rahman Abidur, Delong Alison, Muday Gloria K
Department of Biology, Wake Forest University, Winston-Salem, North Carolina 27109, USA.
Plant Physiol. 2009 Jun;150(2):722-35. doi: 10.1104/pp.108.131607. Epub 2009 Apr 10.
Reversible protein phosphorylation is a key regulatory mechanism governing polar auxin transport. We characterized the auxin transport and gravitropic phenotypes of the pinoid-9 (pid-9) mutant of Arabidopsis (Arabidopsis thaliana) and tested the hypothesis that phosphorylation mediated by PID kinase and dephosphorylation regulated by the ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1) protein might antagonistically regulate root auxin transport and gravity response. Basipetal indole-3-acetic acid transport and gravitropism are reduced in pid-9 seedlings, while acropetal transport and lateral root development are unchanged. Treatment of wild-type seedlings with the protein kinase inhibitor staurosporine phenocopies the reduced auxin transport and gravity response of pid-9, while pid-9 is resistant to inhibition by staurosporine. Staurosporine and the phosphatase inhibitor, cantharidin, delay the asymmetric expression of DR5revGFP (green fluorescent protein) at the root tip after gravistimulation. Gravity response defects of rcn1 and pid-9 are partially rescued by treatment with staurosporine and cantharidin, respectively. The pid-9 rcn1 double mutant has a more rapid gravitropic response than rcn1. These data are consistent with a reciprocal regulation of gravitropism by RCN1 and PID. Furthermore, the effect of staurosporine is lost in pinformed2 (pin2). Our data suggest that reduced PID kinase function inhibits gravitropism and basipetal indole-3-acetic acid transport. However, in contrast to PID overexpression studies, we observed wild-type asymmetric membrane distribution of the PIN2 protein in both pid-9 and wild-type root tips, although PIN2 accumulates in endomembrane structures in pid-9 roots. Similarly, staurosporine-treated plants expressing a PIN2GFP fusion exhibit endomembrane accumulation of PIN2GFP, but no changes in membrane asymmetries were detected. Our data suggest that PID plays a limited role in root development; loss of PID activity alters auxin transport and gravitropism without causing an obvious change in cellular polarity.
可逆性蛋白质磷酸化是一种控制生长素极性运输的关键调节机制。我们对拟南芥的类萜醇-9（pid-9）突变体的生长素运输和向重力性表型进行了表征，并检验了由PID激酶介导的磷酸化作用以及由根对萘基邻苯二甲酸卷曲1（RCN1）蛋白调节的去磷酸化作用可能对根生长素运输和重力反应起拮抗调节作用的假说。在pid-9幼苗中，向基性吲哚-3-乙酸运输和向重力性减弱，而向顶运输和侧根发育未改变。用蛋白激酶抑制剂星形孢菌素处理野生型幼苗，可模拟pid-9中生长素运输减少和重力反应减弱的现象，而pid-9对星形孢菌素的抑制具有抗性。星形孢菌素和磷酸酶抑制剂斑蝥素会延迟重力刺激后根尖处DR5rev绿色荧光蛋白（GFP）的不对称表达。分别用星形孢菌素和斑蝥素处理可部分挽救rcn1和pid-9的重力反应缺陷。pid-9 rcn1双突变体的向重力性反应比rcn1更快。这些数据与RCN1和PID对向重力性的相互调节作用一致。此外，在pin形成2（pin2）中，星形孢菌素的作用消失。我们的数据表明，PID激酶功能降低会抑制向重力性和向基性吲哚-3-乙酸运输。然而，与PID过表达研究不同，我们观察到在pid-9和野生型根尖中，PIN2蛋白均呈现野生型的不对称膜分布，尽管PIN2在pid-9根的内膜结构中积累。同样，表达PIN2GFP融合蛋白且经星形孢菌素处理的植物表现出PIN2GFP在内膜中的积累，但未检测到膜不对称性的变化。我们的数据表明，PID在根发育中起有限作用；PID活性丧失会改变生长素运输和向重力性，而不会导致细胞极性发生明显变化。
