Genome-wide comparative analysis of type-A Arabidopsis response regulator genes by overexpression studies reveals their diverse roles and regulatory mechanisms in cytokinin signaling

Abstract

Cytokinin is a critical growth regulator for various aspects of plant growth and development. In Arabidopsis, cytokinin signaling is mediated by a two-component system-based phosphorelay that transmits a signal from the receptors, through histidine phosphotransfer proteins, to the downstream response regulators (ARRs). Of these ARRs, type-A ARR genes, whose transcription can be rapidly induced by cytokinin, act as negative regulators of cytokinin signaling. However, because of functional redundancy, the function of type-A ARR genes in plant growth and development is not well understood by analyzing loss-of-function mutants. In this study, we performed a comparative functional study on all ten type-A ARR genes by analyzing transgenic plants overexpressing these ARR genes fused to a MYC epitope tag. Overexpression of ARR genes results in a variety of cytokinin-associated phenotypes. Notably, overexpression of different ARR transgenes causes diverse phenotypes, even between phylogenetically closely-related gene pairs, such as within the ARR3-ARR4 and ARR5-ARR6 pairs. We found that the accumulation of a subset of ARR proteins (ARR3, ARR5, ARR7, ARR16 and ARR17; possibly ARR8 and ARR15) is increased by MG132, a specific proteasomal inhibitor, indicating that stability of these proteins is regulated by proteasomal degradation. Moreover, similar to that of previously characterized ARR5, ARR6 and ARR7, stability of ARR16 and ARR17, possibly including ARR8 and ARR15, is regulated by cytokinin. These results suggest that type-A ARR proteins are regulated by a combinatorial mechanism involving both the cytokinin and proteasome pathways, thereby executing distinctive functions in plant growth and development.

Cytokinin signaling regulates pavement cell morphogenesis in Arabidopsis

“…Because type-B, positive regulators of cytokinin signaling also controls the transcription of the type-A ARRs [23], we observed PC interdigitation in various type-A ARR over-expression lines. Among various type-A ARRs that negatively regulate cytokinin signaling [34], only over-expression of ARR7 or its paralog ARR15 enhanced PC interdigitation ( Figure 4A www.cell-research.com | Cell Research Hongjiang Li et al 295…”

“…TAIL-PCR analysis revealed that each group contained an ARR gene that participates in cytokinin signaling [9,22,34]. Group A includes ARR7 (Supplementary information, Figure S2), a type-A ARR, that negatively regulates cytokinin signaling through repression of type-B ARRs.…”

“…The majority of the mutant or transgenic lines used in this work were previously described, including ARR7OX (35S::ARR7) and ARR7OX M (D85N) [24], ahk3 cre1 (ahk3-7 cre1-2) [2], ahp12345 (ahp1,2-2,3,4,5 quintuple mutant) [43], various type-A ARR over-expression lines (ARR3OX, ARR4OX, ARR5OX, ARR6OX, ARR7OX, ARR8OX, ARR9OX, ARR15OX, ARR16OX, ARR17OX) [34], and cre1-12, ahk2-2, ahk3-3, ahk2-2 ahk3-3, ahk2-2 cre1-12, ahk3-3 cre1-12 [37]. ARR20OX (At3g62670) was obtained from the Ohio State University Arabidopsis Biological Resource Center.…”

Cytokinin Interplay with Ethylene, Auxin, and Glucose Signaling Controls Arabidopsis Seedling Root Directional Growth

“…The steady-state levels of type A ARRs are increased by CK; these ARRs negatively regulate CK signal transduction (To and Kieber, 2008). CK signaling is regulated by a mechanism involving proteasomal degradation of certain type A ARRs, which may act to derepress type B ARRs (Ren et al, 2009). Combined loss of type A ARR genes increased CK sensitivity.…”

“…Type A ARRs are negative regulators of this response; mutants exhibited enhanced CK-induced root growth, with ARR3, -4, -5, -6, -8, and -9 having prominent but redundant functions. Some type A ARRs are degraded by CK through the proteasomal pathway, which is an integrator of a number of signaling pathways such as light and circadian rhythm (To and Kieber, 2008;Ren et al, 2009;Vierstra, 2009). Hence, it would be interesting to determine if CKinduced growth is also affected by other environmental or endogenous cues via the control of proteasomal degradation of type A ARRs.…”