Mechanisms Regulating SHORT-ROOT Intercellular Movement

Size: px

Start display at page:

Download "Mechanisms Regulating SHORT-ROOT Intercellular Movement"

Erica French
5 years ago
Views:

1 Current Biology, Vol. 14, , October 26, 2004, 2004 Elsevier Ltd. All rights reserved. DOI /j.cub Mechanisms Regulating SHORT-ROOT Intercellular Movement Kimberly L. Gallagher, 1,4 Alice J. Paquette, 2,4 Keiji Nakajima, 3 and Philip N. Benfey 1, * 1 Department of Biology Duke University nontargeted movement involves a protein moving without interacting with the plasmodesmal apparatus. In this case, the process resembles passive diffusion, as for cytoplasmically localized GFP and LFY [4, 9]. Durham, North Carolina Both targeted and nontargeted movement can be 2 Department of Biology regulated, i.e., there is a mechanism for controlling New York University the extent of translocation. For example, transit of both New York, New York targeted and nontargeted proteins appears to be regulated by limiting the availability of the protein in the cytoplasm [9, 13]. Summary Here we use transgenic and genetic approaches to define the movement of SHR (Figure 1) [14, 15]. SHR is Signaling centers within developing organs regulate transcribed in the stele in a subset of cells including morphogenesis in both plants and animals. The putapressed xylem precursors and procambium, but it is not ex- tive transcription factor SHORT-ROOT (SHR) is an orcells in phloem precursors and adjacent pericycle ganizing signal regulating the division of specific stem (Figure 1B) [16]. The SHR protein, however, is presganizing cells in the Arabidopsis root. Comparison of gene tranthe ent throughout the stele, as well as in adjacent tissues: scription with protein localization indicates that SHR endodermis, cortex/endodermis stem cells, and qui- moves in a highly specific manner from the cells of escent center (QC) (Figure 1C) [8, 16]. Translocation is the stele in which it is synthesized outward. Here, we presumed to occur through plasmodesmata, but there provide evidence that SHR intercellular trafficking is is no direct evidence for this. Presence of SHR in these both regulated and targeted. First, we show that subgene, adjacent tissues correlates with expression of a related cellular localization of SHR in the stele is intrinsic to SCARECROW (SCR), which is required for normal the SHR protein. Next, we show that SHR must be patterning of the root. In the shr and scr mutants, the present in the cytoplasm to move, providing evidence ground tissue consists of a single mutant layer [17]. In that SHR movement is regulated. Finally, we describe shr, this layer no longer shows endodermal characterisan informative new shr allele, in which the protein is tics [14]. Thus, SHR movement is associated with pat- present in the cytoplasm yet does not move. Thus, in terning and cell specification. contrast to proteins that move by a process resemently The mechanisms regulating SHR movement are presbling diffusion, a cytoplasmic pool of SHR is not sufficleus unknown. In stele cells, SHR is present in the nu- cient for movement. and cytoplasm. In the endodermis, the protein appears exclusively nuclear. No SHR is detected in the Results and Discussion next cell layer out, the cortex (Figure 2A) [8]. Thus, cytoplasmic SHR may simply diffuse from the stele into the A number of transcription factors involved in plant develthereby endodermis, where it is efficiently nuclear-localized and opment perform non-cell-autonomous actions by trafwhether restricted from further movement. We asked ficking from cell to cell, presumably via plasmodesmata. regulated diffusion is sufficient to describe SHR In the shoot apical meristem, LEAFY (LFY) and KNOT- movement. TED1 (KN1) move within and between cell layers [1 5]. In the developing flower, DEFICIENS (DEF) traffics from one cell layer into another [6]. In the root, CAPRICE Cytoplasmic Accumulation of SHR Is Not Solely (CPC) translocates from atrichoblasts into trichoblasts, a Property of Stele Cells where it regulates root hair formation [7]. Also in the Because subcellular localization might regulate SHR s root, SHORT-ROOT (SHR) moves from the stele outward capacity to move, we first asked whether cytoplasmic into the adjacent ground-tissue layer, where it promotes localization in the stele is a property of the tissue or the endodermis formation [8]. protein. SCR and SHR are GRAS family members with Several different terms have been used to describe 46% similarity to each other outside the divergent N the mechanism by which proteins move intercellularly. terminus. Nuclear localization of GRAS proteins may Targeted movement involves a protein interacting difrom simply be inefficient in the stele. If so, when expressed rectly with the plasmodesma or proteins associated with the SHR promoter, SCR would localize similarly to the plasmodesmal trafficking machinery [9]. Targeted SHR. movement is implicated for viral movement proteins, GFP-SCR driven from the SCR promoter is present KN1, and a family of chaperones [10 12]. In contrast, primarily in the endodermis and appears solely nuclear (Figure 2B). This likely reflects the normal localization of SCR because the GFP fusion can rescue the scr *Correspondence: philip.benfey@duke.edu 3 mutant. GFP-SCR driven from the SHR promoter is Current address: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara found only in the cells in which SHR is transcribed and , Japan. localizes to the nucleus. GFP-SCR was not detected 4 These authors contributed equally to this work. in the cells into which SHR translocates (Figure 2C),

Current Biology 1848 Figure 1. Schematic of SHR Movement within the Arabidopsis Root Apical Meristem Longitudinal (A) and transverse (B and C) views.

Collectively, the pericycle, phloem, xylem, and procambium are referred to as the stele; cortex and endodermis comprise the ground tissue.

2 Current Biology 1848 Figure 1. Schematic of SHR Movement within the Arabidopsis Root Apical Meristem Longitudinal (A) and transverse (B and C) views. From outer to inner, the root is composed of epidermis (dark yellow), cortex (yellow), endododermis (blue), pericycle (purple), phloem (red), xylem (peach), and procambium. Collectively, the pericycle, phloem, xylem, and procambium are referred to as the stele; cortex and endodermis comprise the ground tissue. SHR is transcribed [(B) outlined area] in a subset of stele cells including all but the phloem cells and the pericycle cells adjacent to the phloem poles. SHR protein [(C) outlined area] moves from the cells in which it is synthesized into adjacent cells including the phloem, endodermis, cortex/endodermis stem cells (green), and quiescent center (plum). indicating that SCR-GFP did not move. Thus, subcellular localization of SHR in stele cells is a property of sequence differences between SHR and SCR. The lack of SHR in the cytoplasm of the endodermis (Figure 2A) reveals a difference between these tissues in recognizing these sequences. Cytoplasmic Localization Is Required for SHR Movement Given that the subcellular localization of SHR is intrinsic to the SHR protein and is regulated differently in endodermal and stele cells, we asked whether cytoplasmic localization of SHR is necessary for trafficking or, conversely, whether completely nuclear-localized SHR can move. Because, in the endodermis, SHR is detected exclusively in the nucleus, movement of SHR-GFP from the endodermis into the pericycle would indicate that cytoplasmic localization is not required for movement. Therefore, we expressed SHR-GFP in the endodermis by using the SCR promoter. As previously reported [8], expression of untagged SHR from the SCR promoter results in an increase in the number of ground tissue layers. In these additional layers the SCR promoter is variably expressed. Figure 2D shows a root expressing GFP-tagged SHR from the SCR promoter. In these roots, SHR-GFP was observed only in the nuclei of the ground tissue. We were unable to detect it in the stele, suggesting either that cytoplasmic SHR is required for movement or that SHR trafficking is unidirectional. Ei- ther indicates that SHR movement is regulated. The second approach taken was to reduce the amount of SHR in the cytoplasm. We fused a nuclear-localized version of GFP (nlsgfp) to SHR and drove it from the SHR promoter. This resulted in nearly complete nuclear localization (Figure 2E) and lack of movement. Unlike SHR-GFP, SHR-nlsGFP was not detected in the endodermis, the QC, the phloem precursor cells, or the peri- cycle cells adjacent to them. SHR-nlsGFP expressed from the SCR promoter was detectable, indicating that the NLS did not affect SHR stability in endodermal cells. Also, these roots were phenotypically identical to those expressing SHR-GFP from the SCR promoter (data not shown). Collectively, these data indicate that SHR move- ment requires cytoplasmic localization and suggest that restriction of SHR to endodermal nuclei may be a signifi- cant mechanism regulating SHR movement. Figure 2. SHR Must Be Present in the Cytoplasm to Move (A) SHR-GFP driven by the SHR promoter. SHR-GFP is found both in the nucleus and cytoplasm of stele cells, whereas in the endodermis, SHR-GFP is detected only in the nucleus. (B) GFP-SCR driven by the SCR promoter. GFP-SCR is present in the endodermis and is wholly nuclear localized. The transgene is able to rescue these scr-4 mutant roots. (C) GFP-SCR driven by the SHR promoter is solely nuclear and does not traffic intercellularly. (D) SHR-GFP driven by the SCR promoter is solely nuclear and is not detected in neighboring cells. (E) SHR protein fused to nuclear-localized GFP and driven by the SHR promoter is diminished in the cytoplasm and no longer traffics. Abbreviations are as follows: C, cortex; E, endodermis; and QC, quiescent center. Plasmid construction, generation and growth of transgenic plants, and analysis and imaging methods are described in the Supplemental Data.

3 Mechanisms Regulating SHORT-ROOT Movement 1849 Figure 3. A Point Mutation in SHR that Converts T289 to I Reduces SHR Nuclear Localization and Abolishes Movement Longitudinal (A) and transverse (B) optical sections of a root ex- pressing SHRT I-GFP from the SHR promoter. Notice that no protein is detected in cells external to the stele. Arrowheads (B) indicate the phloem poles. (C) The lack of SHRT I-GFP in the cells into which SHR normally traffics is not due to instability of the mutant protein. SHRT I-GFP expressed from the SCR promoter is stable and accumulates in the cytoplasm of endodermal cells. (D) Expres- sion of SHRT I-nlsGFP in the endodermis does not affect radial patterning, indicating that the mutation affects SHR activity in addition to movement. (E H) The SHRT I-GFP protein does not appear to aggregate. High-resolution images of roots expressing SHR-GFP (E and F) or SHRT I-GFP (G and H) from the SHR promoter. In both cases, fluorescence within the stele appears to be distributed evenly throughout the cytoplasm with no indication of aggregation or sequestration of the proteins into vesicles. Images in (E) and (G) were taken with a 100, 1.45 NA objective. Those in (F) and (H) are 2 optical zooms of the images in (E) and (G) respectively. The abbreviations C, E, and QC are as in Figure 2. A Point Mutation that Disrupts SHR Movement The dependence of SHR movement on cytoplasmic localization suggests two models for SHR trafficking. First, SHR movement is nontargeted (passive). Because it is in the cytoplasm, the protein can diffuse through plasmodesmata like GFP and LFY [4, 9], whose movement can be manipulated by modifying the degree of nuclear localization, with an increase correlating with less movement. Second, SHR movement is targeted, and the protein must be in the cytoplasm to interact with proteins that facilitate its movement. We have identified a new allele of shr, shr-5, that points to the second model. This allele has a missense mutation replacing threonine 289 with isoleucine (T289I). The shr-5 phenotype is as severe as that of shr-1, suggesting that shr-5 is a null allele. We introduced this mutation into the SHR-GFP protein (SHRT I-GFP) and drove it from the SHR promoter in both wild-type (Figures 3A and 3B) and shr-2 plants (data not shown). In contrast to SHR-GFP, there was not distinct nuclear localization of SHRT I-GFP in the stele; fluorescence was distributed diffusely throughout the cytoplasm. Moreover, it was not detectable in any of the cells into which SHR normally trafficks (Figure 3B), suggesting that SHR does not move by passive diffusion. Alternatively, the mutant protein may have trafficked into the endodermis and been efficiently degraded there. This might be expected if in wild-type roots a fail-safe mechanism exists that prevents movement out of endodermal cells by efficiently degrading any SHR protein not in the nucleus. To test this idea for the mutant protein, we expressed it directly in the endodermis and found that it accumulated to well above detectible levels (Figure 3C). This finding is inconsistent with a model in which rapid degradation of the mutant protein occurs in the cytoplasm of the endodermis, indicating instead that the lack of SHRT I-GFP reflects a lack of movement into endodermal cells. Thus, although cytoplasmic localization of SHR is required for its movement, it is not sufficient. This is in contrast with the movement of LFY and GFP. Significantly, no mutation of LFY that is able to prevent its translocation has been found. In contrast, KN1, thioredoxin h, and a family of chaperone homologs, all of which target plasmodesmata, have been shown to contain specific sequences that affect movement when mutated [10, 18]. As mentioned earlier, wild-type SHR expressed from the SCR promoter generates multiple ground-tissue lay- ers [8]. When SHRT I-GFP was expressed from this promoter, we observed no effect on root patterning (Figure 3C). Because nuclear localization may be required for its activity as a transcriptional regulator, nlsgfp was fused to the SHRT I protein. The cellular organization of the root was still unperturbed (Figure 3D), suggesting that T289 may regulate activity as well as movement. A possible explanation for both the lack of movement and activity of SHRT I is misfolding. However, there are many indications that SHRT I folds correctly. Misfolding generally leads to degradation or aggregation [19, 20]. The robust fluorescence of SHRT I-GFP (Figures 3G and 3H) as compared to that of wild-type SHR- GFP (Figures 3E and 3F) argues against selective or

4 Current Biology 1850 Figure 4. A Model for SHR Movement Upon modification of T289, SHR becomes competent to interact with proteins that either translocate it into the nucleus or facilitate its intercellular movement. Lack of modification (or mutation in T289) renders SHR unable to interact either with nuclear importins or with proteins that facilitate SHR intercellular movement. In this model, we postulate the existence of SHR-specific nuclear exportins only in stele cells to maintain a cytoplasmic pool of SHR. threonine mimic. However, T289E had the same effect on SHR-GFP as T289I (data not shown), indicating either that in this context glutamate cannot mimic phosphothreonine or that T289 is not a phospho-acceptor. In conclusion, we showed that movement of SHR requires cytoplasmic localization, but cytoplasmic local- ization is not sufficient, indicating that SHR movement is both regulated and targeted. These results are consis- tent with a model in which modification of T289 is required for both interaction of SHR with nuclear import factors as well as with proteins that facilitate its movement (Figure 4). rapid degradation. In addition, SHRT I-GFP appears evenly distributed throughout the cytoplasm, and (with the exception of the lack of nuclear localization) its appearance is essentially identical to that of SHR-GFP in stele cells, arguing against protein aggregation or sequestration into vesicles (Figures 2A versus 3A and 3E versus 3G). Further support for correct folding is the strong correlation reported between proper folding of proteins fused to the amino terminus of GFP and level of fluorescence [21, 22]. Waldo et al. concluded that GFP fluorescence is a sensitive and robust indicator for proper folding of the attached protein independent of the linker used [22]. Thus, it is highly unlikely that the lack of movement of SHRT I is due to aggregation or misfolding. Instead, the mutation more likely disrupts SHR s ability to interact with proteins that both allow it to move and to function as a transcriptional regulator. Role of T289 To determine whether the effect of T289I on subcellular localization in the stele was specific to SHR, we made the analogous mutation in SCR and expressed the GFPtagged protein using the SHR promoter. We found no difference between mutant and wild-type (data not shown), indicating that the role of this residue in subcellular localization does not extend to SCR. A potential analogy exists between the SHR threonine, which the NetPhos 2.0 server [23] indicates is a phospho-acceptor (score.924), and the conserved tyrosine in STAT proteins, which must be phosphorylated for both proper localization and function. Tyrosine phosphorylation results in dimerization of the STAT protein, which is necessary for nuclear localization and DNA binding [24]. Tyrosine phosphorylation appears to be required only for dimerization because synthetic dimers (which lack the phospho tyrosine) function normally. Thus, mutation of the phospho-tyrosine in a STAT protein does not disrupt protein folding or activity per se [25]. Similarly, phosphorylation of T289 may be required for nuclear localization, intercellular movement, and function of SHR by permitting dimerization or some other protein-protein interaction. We attempted to bypass the potential phosphorylation requirement by replacing T289 with a glutamate, a potential phospho- Supplemental Data Supplemental Data including Experimental Procedures are available at DC1/. Acknowledgments We thank Jeff Long for ppzp222 SCRp::MGFP5ER, Elliot Meyerowitz for pcgn and Agrobacterial strain ASE, and Patricia Zambryski for prtl2 NLS::2xGFP. We also thank Cyndi Bradham, Mitchell Levesque, and Teva Vernoux for helpful comments on the manu- script. This work was funded by a National Institutes of Health (NIH) grant GM43778 to P.N.B. K.L.G. and A.J.P. were partially supported by NIH postdoctoral fellowships GM and GM Received: May 9, 2004 Revised: August 25, 2004 Accepted: August 25, 2004 Published: October 26, 2004 References 1. Kim, I., Hempel, F.D., Sha, K., Pfluger, J., and Zambryski, P.C. (2002). Identification of a developmental transition in plasmo- desmatal function during embryogenesis in Arabidopsis thali- ana. Development 129, Kim, J.Y., Yuan, Z., and Jackson, D. (2003). Developmental regulation and significance of KNOX protein trafficking in Arabidopsis. Development 130, Sessions, A., Yanofsky, M.F., and Weigel, D. (2000). Cell-cell signaling and movement by the floral transcription factors LEAFY and APETALA1. Science 289, Wu, X., Dinneny, J.R., Crawford, K.M., Rhee, Y., Citovsky, V., Zambryski, P.C., and Weigel, D. (2003). Modes of intercellular transcription factor movement in the Arabidopsis apex. Development 130,

5 Mechanisms Regulating SHORT-ROOT Movement Kim, J.Y., Yuan, Z., Cilia, M., Khalfan-Jagani, Z., and Jackson, D. (2002). Intercellular trafficking of a KNOTTED1 green fluorescent protein fusion in the leaf and shoot meristem of Arabidopsis. Proc. Natl. Acad. Sci. USA 99, Perbal, M.C., Haughn, G., Saedler, H., and Schwarz-Sommer, Z. (1996). Non-cell-autonomous function of the Antirrhinum floral homeotic proteins DEFICIENS and GLOBOSA is exerted by their polar cell-to-cell trafficking. Development 122, Wada, T., Kurata, T., Tominaga, R., Koshino-Kimura, Y., Tachibana, T., Goto, K., Marks, M.D., Shimura, Y., and Okada, K. (2002). Role of a positive regulator of root hair development, CAPRICE, in Arabidopsis root epidermal cell differentiation. Development 129, Nakajima, K., Sena, G., Nawy, T., and Benfey, P.N. (2001). Intercellular movement of the putative transcription factor SHR in root patterning. Nature 413, Crawford, K.M., and Zambryski, P.C. (2000). Subcellular localization determines the availability of non-targeted proteins to plasmodesmatal transport. Curr. Biol. 10, Aoki, K., Kragler, F., Xoconostle-Cazares, B., and Lucas, W.J. (2002). A subclass of plant heat shock cognate 70 chaperones carries a motif that facilitates trafficking through plasmodesmata. Proc. Natl. Acad. Sci. USA 99, Creager, A.N., Scholthof, K.B., Citovsky, V., and Scholthof, H.B. (1999). Tobacco mosaic virus. Pioneering research for a century. Plant Cell 11, Kragler, F., Monzer, J., Xoconostle-Cazares, B., and Lucas, W.J. (2000). Peptide antagonists of the plasmodesmal macromolecular trafficking pathway. EMBO J. 19, Kragler, F., Curin, M., Trutnyeva, K., Gansch, A., and Waigmann, E. (2003). MPB2C, a microtubule-associated plant protein binds to and interferes with cell-to-cell transport of tobacco mosaic virus movement protein. Plant Physiol. 132, Helariutta, Y., Fukaki, H., Wysocka-Diller, J., Nakajima, K., Jung, J., Sena, G., Hauser, M.T., and Benfey, P.N. (2000). The SHORT- ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell 101, Pysh, L.D., Wysocka-Diller, J.W., Camilleri, C., Bouchez, D., and Benfey, P.N. (1999). The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW-LIKE genes. Plant J. 18, Sena, G., Jung, J., and Benfey, P.N. (2004). A broad competence to respond to SHORT-ROOT necessitates tight regulation over its cell-cell movement. Development 131, Benfey, P.N., Linstead, P., Roberts, K., Schiefelbein, J.W., Hauser, M.T., and Aeschbacher, R.A. (1993). Root development in Arabidopsis: Four mutants with dramatically altered root morphogenesis. Development 119, Ishiwatari, Y., Fujiwara, T., McFarland, K.C., Nemoto, K., Hayashi, H., Chino, M., and Lucas, W.J. (1998). Rice phloem thioredoxin h has the capacity to mediate its own cell-to-cell transport through plasmodesmata. Planta 205, Kopito, R.R., and Sitia, R. (2000). Aggresomes and Russell bodies. Symptoms of cellular indigestion? EMBO Rep. 1, Wickner, S., Maurizi, M.R., and Gottesman, S. (1999). Posttranslational quality control: folding, refolding, and degrading proteins. Science 286, Wang, H., and Chong, S. (2003). Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex. Proc. Natl. Acad. Sci. USA 100, Waldo, G.S., Standish, B.M., Berendzen, J., and Terwilliger, T.C. (1999). Rapid protein-folding assay using green fluorescent protein. Nat. Biotechnol. 17, Blom, N., Gammeltoft, S., and Brunak, S. (1999). Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J. Mol. Biol. 294, Hoey, T., and Schindler, U. (1998). STAT structure and function in signaling. Curr. Opin. Genet. Dev. 8, Milocco, L.H., Haslam, J.A., Rosen, J., and Seidel, H.M. (1999). Design of conditionally active STATs: insights into STAT activation and gene regulatory function. Mol. Cell. Biol. 19,

Lecture 4: Radial Patterning and Intercellular Communication.

Lecture 4: Radial Patterning and Intercellular Communication. Summary: Description of the structure of plasmodesmata, and the demonstration of selective movement of solutes and large molecules between