Type-A Arabidopsis Response Regulators Are Partially Redundant Negative Regulators of Cytokinin Signaling

AbstractThe phytohormone cytokinin influences many aspects of plant growth and development, several of which also involve the cellular process of autophagy, including leaf senescence, nutrient re-mobilization, and developmental transitions. The Arabidopsis type-A Response Regulators (type-A ARR) are negative regulators of cytokinin signaling that are transcriptionally induced in response to cytokinin. Here, we describe a mechanistic link between cytokinin signaling and autophagy, demonstrating that plants modulate cytokinin sensitivity through autophagic regulation of type-A ARR proteins. Type-A ARR proteins were degraded by autophagy in an AUTOPHAGY-RELATED (ATG)5-dependent manner. EXO70D family members interacted with Type-A ARR proteins, likely in a phosphorylation-dependent manner, and recruited them to autophagosomes via interaction with the core autophagy protein, ATG8. Consistently, loss-of-function exo70D1,2,3 mutants compromised targeting of type-A ARRs to autophagic vesicles, have elevated levels of type-A ARR proteins, and are hyposensitive to cytokinin. Disruption of both type-A ARRs and EXO70D1,2,3 compromised survival in carbon-deficient conditions, suggesting interaction between autophagy and cytokinin responsiveness in response to stress. These results indicate that the EXO70D proteins act as selective autophagy receptors to target type-A ARR cargos for autophagic degradation, demonstrating that cytokinin signaling can be modulated by selective autophagy.

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Response Regulators 9 and 10 Negatively Regulate Salinity Tolerance in Rice

Plant and Cell Physiology ◽

10.1093/pcp/pcz149 ◽

2019 ◽

Vol 60 (11) ◽

pp. 2549-2563 ◽

Cited By ~ 4

Author(s):

Wei-Chen Wang ◽

Te-Che Lin ◽

Joseph Kieber ◽

Yu-Chang Tsai

Keyword(s):

Salinity Tolerance ◽

Type A ◽

Gene Families ◽

Loss Of Function ◽

Wild Type ◽

Response Regulators ◽

Cytokinin Signaling ◽

Signaling Systems ◽

Identical Type ◽

And Function

Abstract Cytokinins are involved in the regulation of many plant growth and development processes, and function in response to abiotic stress. Cytokinin signaling is similar to the prokaryotic two-component signaling systems and includes the transcriptional upregulation of type-A response regulators (RRs), which in turn act to inhibit cytokinin signal response via negative feedback. Cytokinin signaling consists of several gene families and only a handful full of genes is studied. In this study, we demonstrated the function of two highly identical type-A RR genes from rice, OsRR9 and OsRR10, which are induced by cytokinin and only OsRR10 repressed by salinity stress in rice. Loss-of-function mutations give rise to mutant genes, osrr9/osrr10, which have higher salinity tolerance than wild type rice seedlings. The transcriptomic analysis uncovered several ion transporter genes, which were upregulated in response to salt stress in the osrr9/osrr10 mutants relative to the wild type seedlings. These include high-affinity potassium transporters, such as OsHKT1;1, OsHKT1;3 and OsHKT2;1, which play an important role in sodium and potassium homeostasis. In addition, disruption of the genes OsRR9 and OsRR10 also affects the expression of multiple genes related to photosynthesis, transcription and phytohormone signaling. Taken together, these results suggest that the genes OsRR9 and OsRR10 function as negative regulators in response to salinity in rice.

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EXO70D isoforms mediate selective autophagic degradation of type-A ARR proteins to regulate cytokinin sensitivity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2013161117 ◽

2020 ◽

Vol 117 (43) ◽

pp. 27034-27043

Author(s):

Atiako Kwame Acheampong ◽

Carly Shanks ◽

Chia-Yi Cheng ◽

G. Eric Schaller ◽

Yasin Dagdas ◽

...

Keyword(s):

Type A ◽

Dependent Manner ◽

Loss Of Function ◽

Response Regulators ◽

Cytokinin Signaling ◽

Developmental Transitions ◽

Selective Autophagy ◽

Receiver Domain ◽

Autophagic Degradation ◽

Response To Stress

The phytohormone cytokinin influences many aspects of plant growth and development, several of which also involve the cellular process of autophagy, including leaf senescence, nutrient remobilization, and developmental transitions. The Arabidopsis type-A response regulators (type-A ARR) are negative regulators of cytokinin signaling that are transcriptionally induced in response to cytokinin. Here, we describe a mechanistic link between cytokinin signaling and autophagy, demonstrating that plants modulate cytokinin sensitivity through autophagic regulation of type-A ARR proteins. Type-A ARR proteins were degraded by autophagy in an AUTOPHAGY-RELATED (ATG)5-dependent manner, and this degradation is promoted by phosphorylation on a conserved aspartate in the receiver domain of the type-A ARRs. EXO70D family members interacted with type-A ARR proteins, likely in a phosphorylation-dependent manner, and recruited them to autophagosomes via interaction of the EXO70D AIM with the core autophagy protein, ATG8. Consistently, loss-of-function exo70D1,2,3 mutants exhibited compromised targeting of type-A ARRs to autophagic vesicles, have elevated levels of type-A ARR proteins, and are hyposensitive to cytokinin. Disruption of both type-A ARRs and EXO70D1,2,3 compromised survival in carbon-deficient conditions, suggesting interaction between autophagy and cytokinin responsiveness in response to stress. These results indicate that the EXO70D proteins act as selective autophagy receptors to target type-A ARR cargos for autophagic degradation, demonstrating modulation of cytokinin signaling by selective autophagy.

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DEG9, a serine protease, modulates cytokinin and light signaling by regulating the level of ARABIDOPSIS RESPONSE REGULATOR 4

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1601724113 ◽

2016 ◽

Vol 113 (25) ◽

pp. E3568-E3576 ◽

Cited By ~ 15

Author(s):

Wei Chi ◽

Jing Li ◽

Baoye He ◽

Xin Chai ◽

Xiumei Xu ◽

...

Keyword(s):

Serine Protease ◽

Response Regulator ◽

Light Signaling ◽

Plant Signaling ◽

Response Regulators ◽

Cytokinin Signaling ◽

Periplasmic Proteins ◽

The Stability ◽

Protease Degradation ◽

Signaling Components

Cytokinin is an essential phytohormone that controls various biological processes in plants. A number of response regulators are known to be important for cytokinin signal transduction. ARABIDOPSIS RESPONSE REGULATOR 4 (ARR4) mediates the cross-talk between light and cytokinin signaling through modulation of the activity of phytochrome B. However, the mechanism that regulates the activity and stability of ARR4 is unknown. Here we identify an ATP-independent serine protease, degradation of periplasmic proteins 9 (DEG9), which localizes to the nucleus and regulates the stability of ARR4. Biochemical evidence shows that DEG9 interacts with ARR4, thereby targeting ARR4 for degradation, which suggests that DEG9 regulates the stability of ARR4. Moreover, genetic evidence shows that DEG9 acts upstream of ARR4 and regulates the activity of ARR4 in cytokinin and light-signaling pathways. This study thus identifies a role for a ubiquitin-independent selective protein proteolysis in the regulation of the stability of plant signaling components.

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SCFKMD controls cytokinin signaling by regulating the degradation of type-B response regulators

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1300403110 ◽

2013 ◽

Vol 110 (24) ◽

pp. 10028-10033 ◽

Cited By ~ 65

Author(s):

H. J. Kim ◽

Y.-H. Chiang ◽

J. J. Kieber ◽

G. E. Schaller

Keyword(s):

Type B ◽

Response Regulators ◽

Cytokinin Signaling

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Type-B response regulators of rice play key roles in growth, development and cytokinin signaling

Development ◽

10.1242/dev.174870 ◽

2019 ◽

Vol 146 (13) ◽

pp. dev174870 ◽

Cited By ~ 5

Author(s):

Jennifer M. Worthen ◽

Maria V. Yamburenko ◽

Jeewoo Lim ◽

Zachary L. Nimchuk ◽

Joseph J. Kieber ◽

...

Keyword(s):

Type B ◽

Response Regulators ◽

Cytokinin Signaling ◽

Growth Development

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The Role of Cytokinin During Infection of Arabidopsis thaliana by the Cyst Nematode Heterodera schachtii

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-15-0156-r ◽

2016 ◽

Vol 29 (1) ◽

pp. 57-68 ◽

Cited By ~ 18

Author(s):

Carly M. Shanks ◽

J. Hollis Rice ◽

Yan Zubo ◽

G. Eric Schaller ◽

Tarek Hewezi ◽

...

Keyword(s):

Type A ◽

Sole Source ◽

Nematode Infection ◽

Heterodera Schachtii ◽

Cytokinin Signaling ◽

Cyst Nematodes ◽

Nematode Parasitism ◽

Arabidopsis Mutant ◽

Nematode Development

Plant-parasitic cyst nematodes induce the formation of hypermetabolic feeding sites, termed syncytia, as their sole source of nutrients. The formation of the syncytium is orchestrated by the nematode, in part, by modulation of phytohormone responses, including cytokinin. In response to infection by the nematode Heterodera schachtii, cytokinin signaling is transiently induced at the site of infection and in the developing syncytium. Arabidopsis lines with reduced cytokinin sensitivity show reduced susceptibility to nematode infection, indicating that cytokinin signaling is required for optimal nematode development. Furthermore, lines with increased cytokinin sensitivity also exhibit reduced nematode susceptibility. To ascertain why cytokinin hypersensitivity reduces nematode parasitism, we examined the transcriptomes in wild type and a cytokinin-hypersensitive type-A arr Arabidopsis mutant in response to H. schachtii infection. Genes involved in the response to biotic stress and defense response were elevated in the type-A arr mutant in the absence of nematodes and were hyperinduced following H. schachtii infection, which suggests that the Arabidopsis type-A arr mutants impede nematode development because they are primed to respond to pathogen infection. These results suggest that cytokinin signaling is required for optimal H. schachtii parasitism of Arabidopsis but that elevated cytokinin signaling triggers a heightened immune response to nematode infection.

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Reconstitution of Cytokinin Signaling in Rice Protoplasts

International Journal of Molecular Sciences ◽

10.3390/ijms22073647 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3647

Author(s):

Eunji Ga ◽

Jaeeun Song ◽

Myung Ki Min ◽

Jihee Ha ◽

Sangkyu Park ◽

...

Keyword(s):

Expression System ◽

Transient Gene Expression ◽

Firefly Luciferase ◽

Signaling Proteins ◽

Type B ◽

Response Regulators ◽

Reporter Construct ◽

Cytokinin Signaling ◽

Firefly Luciferase Gene ◽

Signaling Components

The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.

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