[Autophagy 3:4, 360-362; July/August 2007]; ©2007 Landes Bioscience
Addendum
Constitutive Autophagy in Plant Root Cells Kanako Yano1 Takao Suzuki2 Yuji Moriyasu1,2,*
Abstract
Key words Arabidopsis, Atg8, autophagy, cell growth, E-64d, green fluorescent protein (GFP), root hair formation, sucrose starvation, vacuole Addendum to:
In previous studies, we reported that autophagy occurs constitutively, irrespective of the nutritional conditions, in the root cells of barley and Arabidopsis,1,2 although it is activated under nutrient‑limiting conditions.2 These studies used the membrane‑permeable cysteine protease inhibitor E‑64d to detect autophagy. On the other hand, the AtAtg8 proteins, the Arabidopsis homologs of the yeast autophagy‑essential protein Atg8, have been reported to transit on the membrane of autophagosomes during autophagy in Arabidopsis cells.3 Thus, utilizing a fusion protein composed of AtAtg8 and green fluorescent protein (GFP), autophagosomes have been visualized by fluorescence microscopy in the root and hypocotyl4,5 and cultured cells6 of Arabidopsis. In the present study, we observed autophagosomes directly in Arabidopsis root cells by expressing the GFP‑AtAtg8 fusion protein and confirmed our previous results. The DNA fragment, expressing the GFP‑AtAtg8 fusion protein under the control of the constitutive cauliflower mosaic virus 35S promoter, was introduced into the binary vector pSMAB701, as will be reported elsewhere (Yano K, Moriyasu Y, manuscript in preparation). The GFP‑AtAtg8 DNA was transferred to Arabidopsis nuclear DNA by Agrobacterium transfection.7 We first observed the root epidermal cells of the transgenic Arabidopsis seedlings that were grown on a nutrient‑sufficient agar medium for 7–9 days. Since the cytoplasm of most root cells showed GFP fluorescence, the fusion protein seemed to be expressed in all the root cells of the transgenic seedlings (Fig. 1A). In some of these cells, we found autophagosomes with GFP fluorescence. Close observation showed that the number of autophagosomes differed among the tissues in the roots. In cells near the root apical meristem, where cell division frequently occurs, autophagosomes were scarcely found (Fig. 1B, Meristem). In the elongation zone, where cell division ceases but cells grow and increase in volume, a small but significant number of autophagosomes were observed (Fig. 1B, Elongation). In the differentiation zone, where cells start to form root hair, more autophagosomes were observed (Fig. 1B, Differentiation). Since we did not observe the differentiation zone in our previous study,2 we tried to confirm a high autophagic activity in the differentiation zone following the method used previously. The Arabidopsis seedlings grown on nutrient‑sufficient agar plates were further cultured in a liquid medium containing E‑64d with their entire roots immersed in the medium. The roots were then excised from the seedlings and stained with neutral red.2
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AtATG Genes, Homologs of Yeast Autophagy Genes, are Involved in Constitutive Autophagy in Arabidopsis Root Tip Cells
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Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=4158
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Original manuscript submitted: 03/09/07 Manuscript accepted: 03/19/07
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*Correspondence to: Yuji Moriyasu; School of Food and Nutritional Sciences; University of Shizuoka; 52-1 Yada, Shizuoka 422-8526 Japan; Tel.: +81.54.264.5226; Fax: +81.54.264.5099; Email:
[email protected]
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Nutritional Sciences; University of Shizuoka; Shizuoka, Japan
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1Graduate School of Nutritional and Environmental Sciences; 2School of Food and
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In previous studies, using a membrane‑permeable protease inhibitor, E‑64d, we showed that autophagy occurs constitutively in the root cells of barley and Arabidopsis. In the present study, a fusion protein composed of the autophagy‑related protein AtAtg8 and green fluorescent protein (GFP) was expressed in Arabidopsis to visualize autophagosomes. We first confirmed the presence of autophagosomes with GFP fluorescence in the root cells of seedlings grown on a nutrient‑sufficient medium. The number of autophagosomes changed as the root cells grew and differentiated. In cells near the apical meristem, autophagosomes were scarcely found. However, a small but significant number of autophagosomes existed in the elongation zone. More autophagosomes were found in the differentiation zone where cell growth ceases but the cells start to form root hair. In addition, we confirmed that autophagy is activated under starvation conditions in Arabidopsis root cells. When the root tips were cultured in a sucrose‑free medium, the number of autophagosomes increased in the elongation and differentiation zones, and a significant number of autophagosomes appeared in cells near the apical meristem. The results suggest that autophagy in plant root cells is involved not only in nutrient recycling under nutrient‑limiting conditions but also in cell growth and root hair formation.
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Plant Cell Physiol 2006; 47:1641-52
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Y. Inoue, T. Suzuki, M. Hattori, K. Yoshimoto, Y. Ohsumi and Y. Moriyasu
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2007; Vol. 3 Issue 4
Constitutive Autophagy in Plant Root Cells
We found the accumulation of cytoplasmic materials in the vacuoles and/or lysosomes of the cells in the differentiation zone as well as in the elongation zone (image not shown), suggesting that autophagy occurs in the differentiation zone as efficiently as in the elongation zone. Thus, the results confirm the previous finding that autophagy constitutively occurs in plant root cells even under nutrient‑sufficient conditions,1,2,8 and further suggest that autophagy is involved in cell growth and cell differentiation, such as root hair formation. We suppose that constitutive autophagy in root cells is involved in the enlargement of the central vacuole, which occurs together with cell growth. Autophagy in the elongation zone, where the expansion of the central vacuoles occurs, is compatible with this notion. In contrast, the size of the cells that start to form root hair and those that contain mature root hair was nearly the same, which is consistent with a detailed analysis of plant root growth.9 That is, cells engaging in root hair formation appeared to cease cell growth. Thus, the results that autophagy occurs in the differentiation zone as efficiently as in the elongation zone superficially suggest that autophagy is not necessarily involved in cell growth and vacuole expansion. Root hair formation, however, can be regarded as a type of vacuole expansion that accompanies cell growth.10 Indeed, a part of the plasmalemma of epidermal cells evaginates in root hair formation. Such evagination accompanies the evagination of vacuolar membrane, and as a result, a forming root hair is occupied by a tubular vacuole connecting to the central vacuole. Therefore, it is likely that autophagy in the differentiation zone is eventually involved in such cell growth and vacuole expansion. We next tried to confirm another previous result that autophagy is activated in root cells under nutrient‑limiting conditions. The root tips (10 mm long) excised from Arabidopsis seedlings were cultured in a sucrose‑free medium for 6 h and observed by fluorescence microscopy. The number of autophagosomes increased in all three regions of the root tips (Fig. 1C). The cells near the apical meristem, where autophagosomes are seldom found under nutrient‑sufficient conditions, also showed a significant number of autophagosomes under the starvation conditions (Fig. 1C, Meristem). In the differentiation zone, the number of autophagosomes per cell increased approximately two‑fold (Fig. 1C, Differentiation). These findings suggest that, as in cultured plant cells, autophagy is activated in plant root cells under nutrient‑limiting conditions, and support the notion that one of the functions of autophagy is the degradation of cytoplasmic materials for recycling molecules for biosynthesis, and for supplying substrates for respiration. References
Figure 1. Observation of autophagosomes in Arabidopsis root cells expressing the GFP‑AtAtg8 protein. (A) A wide view of the part of a root observed by light microscopy with Nomarski optics (Nomarski) and fluorescence microscopy (Fluorescence). Bar, 100 mm. (B) The three parts enclosed by rectangles in (A) are magnified: the differentiation zone, where root hair is formed (Differentiation); the elongation zone, where cells grow and increase in size (Elongation); and a region near the root apical meristem, where cell division occurs (Meristem). Arrows indicate putative autophagosomes. Bar, 50 mm. (C) Root tips (10 mm long) were excised from Arabidopsis seedlings and cultured in sucrose‑free (‑sucrose) and nutrient‑sufficient (+sucrose) media for 8 h. The differentiation zone (Differentiation) and a region near the meristem (Meristem) were observed by confocal‑laser microscopy. Arrows indicate putative autophagosomes. Bar, 50 mm.
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1. Moriyasu Y, Hattori M, Jauh GY, Rogers JC. Alpha tonoplast intrinsic protein is specifically associated with vacuole membrane involved in an autophagic process. Plant Cell Physiol 2003; 44:795‑802. 2. Inoue Y, Suzuki T, Hattori M, Yoshimoto K, Ohsumi Y, Moriyasu Y. AtATG genes, homologs of yeast autophagy genes, are involved in constitutive autophagy in Arabidopsis root tip cells. Plant Cell Physiol 2006; 47:1641‑52. 3. Bassham DC, Laporte M, Marty F, Moriyasu Y, Ohsumi Y, Olsen LJ, Yoshimoto K. Autophagy in development and stress responses of plants. Autophagy 2006; 2:2‑11. 4. Thompson AR, Doelling JH, Suttangkakul A, Vierstra RD. Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol 2005; 138:2097‑110. 5. Yoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi Y. Processing of ATG8s, ubiquitin‑like proteins, and their deconjugation by ATG4s are essential for plant autophagy. Plant Cell 2004; 16:2967‑83. 6. Contento AL, Xiong Y, Bassham DC. Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP‑AtATG8e fusion protein. Plant J 2005; 42:598‑608. 7. Clough SJ, Bent AF. Floral dip: A simplified method for Agrobacterium‑mediated transformation of Arabidopsis thaliana. Plant J 1998; 16:735‑43.
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Constitutive Autophagy in Plant Root Cells 8. Sláviková S, Shy G, Yao Y, Glozman R, Levanony H, Pietrokovski S, Elazar Z, Galili G. The autophagy‑associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants. J Exp Bot 2005; 56:2839‑49. 9. van der Weele CM, Jiang HS, Palaniappan KK, Ivanov VB, Palaniappan K, Baskin TI. A new algorithm for computational image analysis of deformable motion at high spatial and temporal resolution applied to root growth: Roughly uniform elongation in the meristem and also, after an abrupt acceleration, in the elongation zone. Plant Physiol 2003; 132:1138‑48. 10. Dolan L, Davies J. Cell expansion in roots. Curr Opin Plant Biol 2004; 7:33‑9.
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