Membrane Regulation of Cytokinin-Mediated Cell Division in Arabidopsis

Abstract

Controlled proteolytic activation of membrane-bound transcription factors (MTFs) is an efficient adaptation strategy that ensures prompt transcriptional responses to intrinsic and environmental changes in eukaryotes. The proteolytic processing liberates active transcription factors from the membranes, which subsequently enter the nucleus and turn on downstream target genes. In the December issue of Plant Cell, we have demonstrated that an Arabidopsis membrane-bound NAC transcription factor, designated NTM1, is activated by proteolytic cleavage through regulated intramembrane proteolysis (RIP). The transcriptionally active NTM1 form induces a subset of CDK inhibitor genes (KRPs), resulting in reduced cell division. We have also shown that cytokinins regulate NTM1 activity by modulating its protein stability via an ubiquitin (Ub)-mediated protein degradation pathway, defining a unique molecular scheme by which cytokinins regulate cell division. It is thus envisioned that both positive and negative signaling components would be required for tight control of cell cycling by cytokinins. In this addendum, we propose a working hypothesis in which environmental stresses affect cell division by regulating NTM1 expression or NTM1 processing step.

Cell cycling in plants is tightly controlled through coordinate interactions of a battery of cell cycle regulators, such as cyclins, cyclin-dependent kinases (CDKs), and a group of CDK inhibitors that have been collectively termed ICKs or KIP-related protein (KRPs).¹ Cell division plays a critical role in virtually all aspects of plant growth and developmental processes^1–3 and stress responses.^4–5 Accordingly, plant growth hormones greatly affect the cell cycle progression. The regulatory roles of environmental stress-related growth hormones, including abscisic acid (ABA), jasmonic acid, and ethylene, as well as cytokinins and auxins have been confirmed in cell cycling.² Among these, cytokinins are directly related to cell division and most extensively studied. Cytokinins regulate the G₁/S and G₂/M transitions and the progression through the S phase.¹ It has been known that cytokinins stimulate cell division, at least in part, by inducing CYCD3-1 expression, and CYCD3-1-overexpressing transgenic plants are cytokinin-autotrophic, as analyzed with leaf callus culture.⁶ The ubiquitin (Ub)/26S proteasome-mediated protein degradation pathway also play an important role in cytokinin signaling. It has been shown that an Arabidopsis mutant with defective 26S proteasome activity exhibits disturbed cytokinin responses.⁷

It is notable that proteolytic cleavage is intimately associated with activation of dormant signaling mediators. Particularly, it has been demonstrated that a group of MTFs are proteolytically activated in animals and yeast.⁸ The proteolytic activation of MTFs occurs through either one of two distinct ways. In the so-called regulated intramembrane proteolysis (RIP), active transcription factors are liberated by specific membrane-integrated proteases.^8,9 In the regulated ubiquitin (Ub)/26S proteasome-dependent processing (RUP), ubiquitinated MTFs are degraded by the 26S proteasome, releasing transcriptionally active forms.

Recent genome-wide surveys have predicted that a considerable portion of plant transcription factors are membrane-associated.^10,11 One example is the plant-specific NAC transcription factors. There are more than 100 NAC proteins in Arabidopsis and rice.^12,13 Interestingly, protein structure analysis of the NAC proteins using the ARAMEMNON software have predicted that a strong, a-helical transmembrane motif resides in the C-terminal regions of several NAC proteins,^10,14 suggesting that membrane release is a prerequisite for such NAC MTFs.

Our data indicate that NTM1 is expressed as a dormant, membrane-bound form, and, upon stimulation, it is released from the membranes by proteolytic cleavage.¹⁵ The activated NTM1 form enters the nucleus, where it induces KRPs, causing reduced cell division (Fig. 1). A mutant plant in which a transcriptionally active NTM1 form is constitutively expressed exhibited reduced growth and small plant organs, primarily due to reduced cell division but with enlarged cell size. Consistent with the phenotypic changes, KRPs are up-regulated, and CDK activities and histone H4 expression are down-regulated in the mutant plant. Interestingly, cytokinins, which are known to stimulate cell division, stabilize the NTM1 proteins. Together with previous reports on cytokinin functions in cell division,⁶ our data propose that the promotive effect of the CYCD3-mediated cytokinin signaling on cell division would be countered by the NTM1-mediated induction of KRPs to maintain cell division rate to an appropriate level under a given growth condition. This notion would explain the previous observation in which KRP genes are induced by cytokinins.¹⁶

Figure 1.

A proposed working model for NTM1 function. The membrane-bound form is proteolytically released by RIP, and the transcriptionally active form is translocated into the nucleus. The NTM1 proteins are stabilized by cytokinins. NTM1 processing and/or NTM1 expression would be induced by environmental stresses. Ub, ubiquitin, mNTM1 and nNTM1, membrane-bound and nuclear forms of NTM1, respectively.

Although NTM1 is likely to be activated by calpain or its functional homologues, it is currently unclear how NTM1 processing is regulated. One possibility is that NTM1 may regulate cell division under stress conditions. KRP1 is induced by ABA and cold treatment.¹⁷ Other KRPs might also be induced by environmental stresses, thereby arresting cell division and growth. In this scenario, NTM1 expression or NTM1 processing or both may be affected by environmental stresses. Notably, we recently observed that high salinity and other abiotic stresses greatly induced NTM1 expression (unpublished). Furthermore, many NAC genes are influenced by stress conditions.¹⁸ It will be also interesting to examine whether NTM1 processing is influenced by environmental stresses.

Regulation of NTM1 activity does not seem to be so simple. Cytokinins enhance the protein stability of both the membrane-bound and free NTM1 forms. Furthermore, MG132, an inhibitor of 26S proteasome,¹⁹ mimics cytokinin effects. These observations suggest that cytokinins may regulate the NTM1 stability either by controlling the ubiquitination step or by modulating the 26S proteasome activity. In any case, it is evident that NTM1 activity is regulated at two distinct levels; one at the processing step, and the other at the protein stability level. Identification of the protease responsible for NTM1 processing will further extend our understanding on how NTM1 processing is regulated.

Addendum to: Kim Y-S, Kim S-G, Park J-E, Park H-Y, Lim M-H, Chua N-H, Park C-M. A Membrane-Bound NAC Transcription Factor Regulates Cell Division in Arabidopsis. Plant Cell. 2006;18:3132–3144. doi: 10.1105/tpc.106.043018.