植物自噬作用是一個透過液泡進行細胞質分解的過程並且維持細胞穩定狀態。自噬作用相關蛋白ATG8在自噬小體(autophagosome)的形成過程中扮演中心角色，自噬小體包裹細胞質中的物質並且送入液泡中分解。阿拉伯芥(Arabidopsis thaliana)的ATG8基因家族有九個蛋白異構體(isoform) (AtATG8a–AtATG8i)。對於缺乏磷酸時如何提升植物自噬作用以及AtATG8蛋白如何召集特定物質到液泡分解尚未清楚。我們發現在缺磷時，ATG8f與ATG8h在地上部與根部的表現會被明顯地調升。這個磷酸缺乏時的調升作用在phosphate starvation response 1 (phr1)的突變株中會部分地被抑制，PHR1負責編碼轉錄活化子(transcription activator)以增加缺磷反應基因(Pi starvation responsive genes)的轉錄。我們假設ATG8f與ATG8h是PHR1調控磷酸平衡路徑的下游目標。在此研究中我們發現PHR1並未直接提升ATG8f與ATG8h的基因表現。PHR1活化ATG8f與ATG8h的基因表現可能需要仰賴其他蛋白一同參與，或是藉由PHR1引導第二波的轉錄活化(transcriptional activation)。此外，我們得到atg8f, atg8h, atg8f/atg8h T-DNA插入突變株。在缺磷時，atg8f/atg8h根部組織的自噬通量(autophagic flux)比野生型(wild-type)來得少，意味著ATG8f與ATG8h會調控在缺磷下的自噬作用活性。我們也觀察到無論是否缺磷，atg8f/atg8h的側根數量比野生型少。ATG8f與ATG8h大量表現在主根的維管束組織，尤其是側根即將長出的地方。這些結果提示著ATG8f與ATG8h有潛在調節側根發育的功能。

Plant autophagy is a vacuolar-mediated process of cytoplasmic degradation and maintains cellular homeostasis. Autophagy-related 8 (ATG8) protein plays a central role in the formation of autophagosomes which enclose and deliver the cytoplasmic components into vacuoles for degradation. The Arabidopsis thaliana ATG8 gene family comprises nine isoforms (AtATG8a–AtATG8i). It remains unclear how phosphate (Pi) limitation regulates the induction of plant autophagy and how AtATG8s proteins recruit specific cargos for vacuolar degradation under Pi starvation. We revealed that AtATG8f and AtATG8h are upregulated in the shoot and root by Pi starvation. Such upregulation by Pi starvation can be partially suppressed in the shoot and root of phosphate starvation response 1 (phr1) mutant encoding a transcriptional activator of Pi starvation responsive genes. We hypothesized that AtATG8f and AtATG8h may be the downstream targets of the PHR1-dependent regulatory pathway of Pi homeostasis. In this study, we showed that PHR1 seemed not to directly transactivate the expression of AtATG8f and AtATG8h. PHR1-dependent Pi starvation-induction of AtATG8f and AtATG8h may rely on the cooperation of PHR1 and other components or a second wave of transcriptional activation triggered by PHR1. In addition, we obtained atg8f, atg8h, atg8f/atg8h T-DNA mutants and demonstrated that the autophagic flux was reduced in the Pi-starved root of atg8f/atg8h compared to that of wild-type (WT), indicating that AtATG8f and AtATG8h play a role in regulating autophagic activities under Pi limitation. Moreover, the atg8f/atg8h mutant has fewer lateral root number than WT does regardless of Pi supply. Spatial expression patterns of AtATG8f and AtATG8h are expressed in the vascular tissues of primary roots and strongly at the sites where lateral roots initiate to emerge. These results suggested that AtATG8f and AtATG8h have a potential role in modulating lateral root development.

摘要 i
Abstract ii
Acknowledgement iii
Table of contents iv
List of abbreviations viii
Chapter 1. Introduction 1
1.1 An overview of autophagy 1
1.1.1 The role of ATG8 protein in the formation of autophagosome 2
1.1.2 Autophagic flux as determined by ATG8 accumulation 3
1.1.3 Arabidopsis ATG8 gene family 5
1.2 Phosphorus for plant growth 6
1.3 The maintenance of Pi homeostasis in plants 6
1.4 Pi starvation-induced autophagy in the root apical meristem 8
1.5 Aims of the study 9
Chapter 2. Materials and methods 10
2.1 Plant materials and growth conditions 10
2.1.1 Plant material 10
2.1.2 Sterilization of Arabidopsis seeds 10
2.1.3 Plant medium 10
2.1.4 Plant soil matrix 11
2.2 Plasmid construction 11
2.3 Bacterial growth medium 12
2.4 Bacterial host cell strains and plasmid transformation 12
2.4.1 Bacterial host cell strains 12
2.4.2 Plasmid transformation into E.coli 13
2.4.3 Plasmid transformation into agrobacteria 13
2.5 Protoplast isolation and transfection assay 13
2.5.1 Protoplast isolation 13
2.5.2 Protoplast transfection 15
2.6 Transient dual-luciferase assay 15
2.7 Genomic DNA extraction 16
2.8 RNA extraction 16
2.9 Validation of T-DNA insertion mutants 16
2.9.1 PCR genotyping of homozygous T-DNA insertion lines 16
2.9.2 RT-PCR analysis of homozygous T-DNA insertion knockout lines 17
2.10 Real-time quantitative reverse transcription PCR (qRT-PCR) analysis 17
2.11 Pi level measurement 18
2.12 Protein assay 18
2.12.1 Total protein extraction 18
2.12.2 Protein concentration measurement 19
2.12.3 Immunoblotting analysis 20
2.13 Analysis of root phenotypes 20
2.14 Agrobacterium-mediated transformation of Arabidopsis 21
2.15 Transgenic plant selection 21
2.16 Microscopy analysis of GFP-reporter lines 21
2.17 Histochemical staining of β-glucuronidase (GUS) 22
Chapter 3. Results 23
3.1 AtATG8f and AtATG8h promoter activities are not directly activated by PHR1 23
3.2 Characterization of atg8f and atg8h T-DNA insertion knockout mutants 24
3.3 The transcript levels of the other AtATG8 genes are not increased to compensate for the loss of AtATG8f and AtATG8h 25
3.4 The Pi level of atg8f/atg8h is comparable to that of WT 25
3.5 The autophagic flux decreases in the root of Pi-starved atg8f/atg8h 26
3.6 Loss of AtATG8f and AtATG8h suppresses the lateral root formation independent of Pi status 26
3.7 Analysis of AtATG8f and AtATG8h promoter activities under Pi- and N-deficient conditions 27
3.8 Analysis of AtATG8f and AtATG8h promoter activities at the tissue level 29
Chapter 4. Discussion 31
4.1 PHR1 acts upstream of AtATG8f and AtATG8h 31
4.2 Partially functional redundancy of AtATG8f and AtATG8h 32
4.3 Role of AtATG8f and AtATG8h in the maintenance of Pi homeostasis 33
4.4 Role of AtATG8f and AtATG8h in the lateral root development 33
Chapter 5. Figures 35
Figure 1. Transient reporter assay in Arabidopsis protoplast co-expressing PHR1 and AtATG8f/AtATG8h promoter-fused dual-luciferase reporter 35
Figure 2. Characterization of atg8f and atg8h T-DNA insertion mutants 37
Figure 3. Pi levels of atg8f, atg8h, and atg8f/atg8h mutants 38
Figure 4. The ATG8s protein expression of the Pi-starved roots of WT and atg8f/atg8h 39
Figure 5. Phenotypical analysis of root of WT, atg7-3, and atg8f/atg8h 41
Figure 6. Schematic design of AtATG8f and AtATG8h promoters-fused GFP and GUS reporter constructs 42
Figure 7. GFP expression patterns of ProATG8f::GFP transgenic lines 43
Figure 8. GFP expression patterns of ProATG8h::GFP transgenic lines 44
Figure 9. qRT-PCR analysis of GFP and endogenous AtATG8f and AtATG8h expression in ProATG8f::GFP and ProATG8h::GFP transgenic lines 45
Figure 10. Histochemical GUS staining of ProATG8f::GUS transgenic lines 46
Figure 11. Histochemical GUS staining of ProATG8h::GUS transgenic lines 47
Chapter 6. Supplemental data 48
Figure S1. qRT-PCR analysis of AtATGs gene expression in WT seedlings in response to Pi and N starvation 49
Figure S2. PHR1-dependent Pi starvation-induction of ATG8f and ATG8h 50
Figure S3. qRT-PCR analysis of AtATG8s gene expression in the root of WT and atg8f/atg8h 51
Table S1. T-DNA insertion lines for AtATG8f and AtATG8h 52
Table S2. List of primer sequences used for PCR-based gene cloning 53
Table S3. List of constructs used for generating transgenic lines 54
Table S4. Antibiotics used in this study 55
Table S5. List of primer sequences used for PCR genotyping and RT-PCR analyses 56
Table S6. List of primer sequences used for qRT-PCR analysis 57
Table S7. Generation and selection of GUS- and GFP-reporter transgenic lines for ATG8f and ATG8h 58
Table S8. The predicted P1BS element and AtPHR1 binding matrix 59
References 60

1. Barragán-Rosillo, A.C., Peralta-Alvarez, C.A., Ojeda-Rivera, J.O., Arzate-Mejía, R.G., Recillas-Targa, F., and Herrera-Estrella, L. (2021). Genome accessibility dynamics in response to phosphate limitation is controlled by the PHR1 family of transcription factors in Arabidopsis. Proceedings of the National Academy of Sciences 118, e2107558118.
2. Bates, T.R., and Lynch, J.P. (2000). Plant growth and phosphorus accumulation of wild type and two root hair mutants of Arabidopsis thaliana (Brassicaceae). American Journal of Botany 87, 958-963.
3. Beese, C.J., Brynjólfsdóttir, S.H., and Frankel, L.B. (2020). Selective Autophagy of the Protein Homeostasis Machinery: Ribophagy, Proteaphagy and ER-Phagy. Frontiers in Cell and Developmental Biology 7.
4. Briat, J.-F., Rouached, H., Tissot, N., Gaymard, F., and Dubos, C. (2015). Integration of P, S, Fe, and Zn nutrition signals in Arabidopsis thaliana: potential involvement of PHOSPHATE STARVATION RESPONSE 1 (PHR1), Vol 06.
5. Bu, F., Yang, M., Guo, X., Huang, W., and Chen, L. (2020). Multiple Functions of ATG8 Family Proteins in Plant Autophagy. Frontiers in Cell and Developmental Biology 8, 466.
6. Bustos, R., Castrillo, G., Linhares, F., Puga, M.I., Rubio, V., Pérez-Pérez, J., Solano, R., Leyva, A., and Paz-Ares, J. (2010). A Central Regulatory System Largely Controls Transcriptional Activation and Repression Responses to Phosphate Starvation in Arabidopsis. PLOS Genetics 6, e1001102.
7. Castrillo, G., Teixeira, P.J.P.L., Paredes, S.H., Law, T.F., de Lorenzo, L., Feltcher, M.E., Finkel, O.M., Breakfield, N.W., Mieczkowski, P., Jones, C.D., et al. (2017). Root microbiota drive direct integration of phosphate stress and immunity. Nature 543, 513-518.
8. Chen, Q., Soulay, F., Saudemont, B., Elmayan, T., Marmagne, A., and Masclaux-Daubresse, C. (2019). Overexpression of ATG8 in Arabidopsis Stimulates Autophagic Activity and Increases Nitrogen Remobilization Efficiency and Grain Filling. Plant and Cell Physiology 60, 343-352.
9. Chien, P.-S., Chiang, C.-P., Leong, S.J., and Chiou, T.-J. (2018). Sensing and Signaling of Phosphate Starvation: From Local to Long Distance. Plant and Cell Physiology 59, 1714-1722.
10. Chung, T., Phillips, A.R., and Vierstra, R.D. (2010). ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci. The Plant Journal 62, 483-493.
11. Clarkson, D.T. (1996). Marschner H. 1995. Mineral nutrition of higher plants. second edition. 889pp. London: Academic Press, £29.95 (paperback). Annals of Botany 78, 527-528.
12. Cordell, D., Drangert, J., and White, S. (2009). The story of phosphorus: Global food security and food for thought. Global Environmental Change-human and Policy Dimensions 19, 292-305.
13. Denton, D., and Kumar, S. (2019). Autophagy-dependent cell death. Cell Death & Differentiation 26, 605-616.
14. Doelling, J.H., Walker, J.M., Friedman, E.M., Thompson, A.R., and Vierstra, R.D. (2002). The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana. J Biol Chem 277, 33105-33114.
15. Dong, J., Ma, G., Sui, L., Wei, M., Satheesh, V., Zhang, R., Ge, S., Li, J., Zhang, T.-E., Wittwer, C., et al. (2019). Inositol Pyrophosphate InsP8 Acts as an Intracellular Phosphate Signal in Arabidopsis. Molecular Plant 12, 1463-1473.
16. Dong, Y., Silbermann, M., Speiser, A., Forieri, I., Linster, E., Poschet, G., Allboje Samami, A., Wanatabe, M., Sticht, C., Teleman, A.A., et al. (2017). Sulfur availability regulates plant growth via glucose-TOR signaling. Nature communications 8, 1174-1174.
17. Escamez, S., André, D., Sztojka, B., Bollhöner, B., Hall, H., Berthet, B., Voß, U., Lers, A., Maizel, A., Andersson, M., et al. (2020). Cell Death in Cells Overlying Lateral Root Primordia Facilitates Organ Growth in Arabidopsis. Current Biology 30, 455-464.e457.
18. Freed, C., Adepoju, O., and Gillaspy, G. (2020). Can Inositol Pyrophosphates Inform Strategies for Developing Low Phytate Crops? Plants 9.
19. Garske, B., Stubenrauch, J., and Ekardt, F. (2020). Sustainable phosphorus management in European agricultural and environmental law. Review of European, Comparative & International Environmental Law 29, 107-117.
20. Geng, J., and Klionsky, D.J. (2008). The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. 'Protein modifications: beyond the usual suspects' review series. EMBO reports 9, 859-864.
21. Gruber, B.D., Giehl, R.F.H., Friedel, S., and von Wirén, N. (2013). Plasticity of the Arabidopsis root system under nutrient deficiencies. Plant physiology 163, 161-179.
22. Hellens, R., Allan, A., Friel, E., Bolitho, K., Grafton, K., Templeton, M., Karunairetnam, S., Gleave, A., and Laing, W. (2005a). Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1:13. Plant methods 1, 13.
23. Hellens, R.P., Allan, A.C., Friel, E.N., Bolitho, K., Grafton, K., Templeton, M.D., Karunairetnam, S., Gleave, A.P., and Laing, W.A. (2005b). Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1, 13.
24. Huang, L., Yu, L.-J., Zhang, X., Fan, B., Wang, F.-Z., Dai, Y.-S., Qi, H., Zhou, Y., Xie, L.-J., and Xiao, S. (2019). Autophagy regulates glucose-mediated root meristem activity by modulating ROS production in Arabidopsis. Autophagy 15, 407-422.
25. Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., et al. (2000). A ubiquitin-like system mediates protein lipidation. Nature 408, 488-492.
26. Inoue, Y., Suzuki, T., Hattori, M., Yoshimoto, K., Ohsumi, Y., and Moriyasu, Y. (2006). AtATG Genes, Homologs of Yeast Autophagy Genes, are Involved in Constitutive Autophagy in Arabidopsis Root Tip Cells. Plant and Cell Physiology 47, 1641-1652.
27. Jung, H., Pusan National University, Busan, Republic of Korea, Kim, J.H., Pusan National University, Busan, Republic of Korea, Shin, K.D., Pusan National University, Busan, Republic of Korea, Kim, J., Pusan National University, Busan, Republic of Korea, Lee, H.N., Pusan National University, Busan, Republic of Korea, and Chung, T., Pusan National University, Busan, Republic of Korea (2017). Autophagic Flux Analysis of Arabidopsis Seedlings Exposed to Salt Stress. v. 60.
28. Kaufmann, A., Beier, V., Franquelim, Henri G., and Wollert, T. (2014). Molecular Mechanism of Autophagic Membrane-Scaffold Assembly and Disassembly. Cell 156, 469-481.
29. Kijanska, M., Dohnal, I., Reiter, W., Kaspar, S., Stoffel, I., Ammerer, G., Kraft, C., and Peter, M. (2010). Activation of Atg1 kinase in autophagy by regulated phosphorylation. Autophagy 6, 1168-1178.
30. Kim, S.-H., Kwon, C., Lee, J.-H., and Chung, T. (2012). Genes for plant autophagy: functions and interactions. Mol Cells 34, 413-423.
31. Kirisako, T., Baba, M., Ishihara, N., Miyazawa, K., Ohsumi, M., Yoshimori, T., Noda, T., and Ohsumi, Y. (1999). Formation process of autophagosome is traced with Apg8/Aut7p in yeast. J Cell Biol 147, 435-446.
32. Kirisako, T., Ichimura, Y., Okada, H., Kabeya, Y., Mizushima, N., Yoshimori, T., Ohsumi, M., Takao, T., Noda, T., and Ohsumi, Y. (2000). The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J Cell Biol 151, 263-276.
33. Klionsky, D.J., Abdalla, F.C., Abeliovich, H., Abraham, R.T., Acevedo-Arozena, A., Adeli, K., Agholme, L., Agnello, M., Agostinis, P., Aguirre-Ghiso, J.A., et al. (2012). Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445-544.
34. Klionsky, D.J., Abeliovich, H., Agostinis, P., Agrawal, D.K., Aliev, G., Askew, D.S., Baba, M., Baehrecke, E.H., Bahr, B.A., Ballabio, A., et al. (2008). Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4, 151-175.
35. Krouk, G., Lacombe, B., Bielach, A., Perrine-Walker, F., Malinska, K., Mounier, E., Hoyerova, K., Tillard, P., Leon, S., Ljung, K., et al. (2010). Nitrate-Regulated Auxin Transport by NRT1.1 Defines a Mechanism for Nutrient Sensing in Plants. Developmental Cell 18, 927-937.
36. Kwon, S.I., Cho, H.J., Jung, J.H., Yoshimoto, K., Shirasu, K., and Park, O.K. (2010). The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis. The Plant Journal 64, 151-164.
37. Li, F., and Vierstra, R.D. (2012). Autophagy: a multifaceted intracellular system for bulk and selective recycling. Trends Plant Sci 17, 526-537.
38. Liu, Y., Burgos, J.S., Deng, Y., Srivastava, R., Howell, S.H., and Bassham, D.C. (2012). Degradation of the endoplasmic reticulum by autophagy during endoplasmic reticulum stress in Arabidopsis. The Plant cell 24, 4635-4651.
39. Liu, Y., Villalba, G., Ayres, R.U., and Schroder, H. (2008). Global Phosphorus Flows and Environmental Impacts from a Consumption Perspective. Journal of Industrial Ecology 12, 229-247.
40. Müller, R., Morant, M., Jarmer, H., Nilsson, L., and Nielsen, T.H. (2007). Genome-wide analysis of the Arabidopsis leaf transcriptome reveals interaction of phosphate and sugar metabolism. Plant physiology 143, 156-171.
41. Müller, J., Toev, T., Heisters, M., Teller, J., Moore, Katie L., Hause, G., Dinesh, Dhurvas C., Bürstenbinder, K., and Abel, S. (2015). Iron-Dependent Callose Deposition Adjusts Root Meristem Maintenance to Phosphate Availability. Developmental Cell 33, 216-230.
42. Maillard, A., Diquélou, S., Billard, V., Laîné, P., Garnica, M., Prudent, M., Garcia-Mina, J.-M., Yvin, J.-C., and Ourry, A. (2015). Leaf mineral nutrient remobilization during leaf senescence and modulation by nutrient deficiency. Frontiers in Plant Science 6.
43. Malamy, J.E., and Benfey, P.N. (1997). Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124, 33-44.
44. Marat, A.L., and Haucke, V. (2016). Phosphatidylinositol 3-phosphates-at the interface between cell signalling and membrane traffic. The EMBO journal 35, 561-579.
45. Marion, J., Le Bars, R., Besse, L., Batoko, H., and Satiat-Jeunemaitre, B. (2018). Multiscale and Multimodal Approaches to Study Autophagy in Model Plants. Cells 7.
46. Marshall, R.S., Hua, Z., Mali, S., McLoughlin, F., and Vierstra, R.D. (2019). ATG8-Binding UIM Proteins Define a New Class of Autophagy Adaptors and Receptors. Cell 177, 766-781.e724.
47. Martens, S., and Fracchiolla, D. (2020). Activation and targeting of ATG8 protein lipidation. Cell Discovery 6, 23.
48. Maruyama, T., and Noda, N.N. (2017). Autophagy-regulating protease Atg4: structure, function, regulation and inhibition. J Antibiot (Tokyo).
49. Maruyama, T., and Noda, N.N. (2018). Autophagy-regulating protease Atg4: structure, function, regulation and inhibition. The Journal of Antibiotics 71, 72-78.
50. Matthus, E., Wilkins, K.A., Swarbreck, S.M., Doddrell, N.H., Doccula, F.G., Costa, A., and Davies, J.M. (2019). Phosphate Starvation Alters Abiotic-Stress-Induced Cytosolic Free Calcium Increases in Roots. Plant physiology 179, 1754-1767.
51. Misson, J., Thibaud, M.-C., Bechtold, N., Raghothama, K., and Nussaume, L. (2004). Transcriptional regulation and functional properties of Arabidopsis Pht1;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants. Plant Molecular Biology 55, 727-741.
52. Nakamura, Y. (2012). Phosphate starvation and membrane lipid remodeling in seed plants. Progress in lipid research 52, 43-50.
53. Naumann, C., Muller, J., Sakhonwasee, S., Wieghaus, A., Hause, G., Heisters, M., Burstenbinder, K., and Abel, S. (2018). The Local Phosphate Deficiency Response Activates ER Stress-dependent Autophagy. Plant Physiol.
54. Nilsson, L., MÜLler, R., and Nielsen, T.H. (2007). Increased expression of the MYB-related transcription factor, PHR1, leads to enhanced phosphate uptake in Arabidopsis thaliana. Plant, Cell & Environment 30, 1499-1512.
55. Nishigaki, T., Sugihara, S., Kobayashi, K., Hashimoto, Y., Kilasara, M., Tanaka, H., Watanabe, T., and Funakawa, S. (2018). Fractionation of phosphorus in soils with different geological and soil physicochemical properties in southern Tanzania. Soil Science and Plant Nutrition 64, 291-299.
56. Noda, N.N., Ohsumi, Y., and Inagaki, F. (2010). Atg8-family interacting motif crucial for selective autophagy. FEBS Letters 584, 1379-1385.
57. O'Malley, R.C., Barragan, C.C., and Ecker, J.R. (2015). A user's guide to the Arabidopsis T-DNA insertion mutant collections. Methods in molecular biology (Clifton, NJ) 1284, 323-342.
58. Ohsumi, Y. (2001). Molecular dissection of autophagy: two ubiquitin-like systems. Nature Reviews Molecular Cell Biology 2, 211-216.
59. Okazaki, Y., Otsuki, H., Narisawa, T., Kobayashi, M., Sawai, S., Kamide, Y., Kusano, M., Aoki, T., Hirai, M.Y., and Saito, K. (2013). A new class of plant lipid is essential for protection against phosphorus depletion. Nature Communications 4, 1510.
60. Osabe, K., Harukawa, Y., Miura, S., and Saze, H. (2017). Epigenetic Regulation of Intronic Transgenes in Arabidopsis. Scientific Reports 7, 45166.
61. Péret, B., Clément, M., Nussaume, L., and Desnos, T. (2011). Root developmental adaptation to phosphate starvation: better safe than sorry. Trends in Plant Science 16, 442-450.
62. Parzych, K.R., and Klionsky, D.J. (2014). An overview of autophagy: morphology, mechanism, and regulation. Antioxidants & redox signaling 20, 460-473.
63. Puga, M.I., Mateos, I., Charukesi, R., Wang, Z., Franco-Zorrilla, J.M., de Lorenzo, L., Irigoyen, M.L., Masiero, S., Bustos, R., Rodríguez, J., et al. (2014). SPX1 is a phosphate-dependent inhibitor of Phosphate Starvation Response 1 in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 111, 14947-14952.
64. Ren, C., Liu, J., and Gong, Q. (2014). Functions of autophagy in plant carbon and nitrogen metabolism. Frontiers in plant science 5, 301-301.
65. Schlücking, K., Edel, K.H., Köster, P., Drerup, M.M., Eckert, C., Steinhorst, L., Waadt, R., Batistič, O., and Kudla, J. (2013). A New β-Estradiol-Inducible Vector Set that Facilitates Easy Construction and Efficient Expression of Transgenes Reveals CBL3-Dependent Cytoplasm to Tonoplast Translocation of CIPK5. Molecular Plant 6, 1814-1829.
66. Schneider, C.A., Rasband, W.S., and Eliceiri, K.W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9, 671-675.
67. Seo, E., Woo, J., Park, E., Bertolani, S.J., Siegel, J.B., Choi, D., and Dinesh-Kumar, S.P. (2016). Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4. Autophagy 12, 2054-2068.
68. Shin, H., Shin, H.-S., Dewbre, G.R., and Harrison, M.J. (2004). Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments. The Plant Journal 39, 629-642.
69. Shin, K.D., Lee, H.N., and Chung, T. (2014). A revised assay for monitoring autophagic flux in Arabidopsis thaliana reveals involvement of AUTOPHAGY-RELATED9 in autophagy. Mol Cells 37, 399-405.
70. Tanida, I., Minematsu-Ikeguchi, N., Ueno, T., and Kominami, E. (2005). Lysosomal Turnover, but Not a Cellular Level, of Endogenous LC3 is a Marker for Autophagy. Autophagy 1, 84-91.
71. Ticconi, C.A., Delatorre, C.A., and Abel, S. (2001). Attenuation of Phosphate Starvation Responses by Phosphite in Arabidopsis. Plant Physiology 127, 963-972.
72. Tiessen, H. (2008). Phosphorus in the Global Environment. In, pp. 1-7.
73. Torres-Martínez, H.H., Rodríguez-Alonso, G., Shishkova, S., and Dubrovsky, J.G. (2019). Lateral Root Primordium Morphogenesis in Angiosperms. Frontiers in Plant Science 10, 206.
74. Vance, C.P., Uhde-Stone, C., and Allan, D.L. (2003). Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytologist 157, 423-447.
75. Vergne, I., and Deretic, V. (2010). The role of PI3P phosphatases in the regulation of autophagy. FEBS letters 584, 1313-1318.
76. Wang, P., Mugume, Y., and Bassham, D.C. (2018). New advances in autophagy in plants: Regulation, selectivity and function. Semin Cell Dev Biol 80, 113-122.
77. Wang, P., Nolan, T.M., Yin, Y., and Bassham, D.C. (2020). Identification of transcription factors that regulate ATG8 expression and autophagy in Arabidopsis. Autophagy 16, 123-139.
78. Wang, W., Xu, M., Wang, G., and Galili, G. (2017). Autophagy: An Important Biological Process That Protects Plants from Stressful Environments. Frontiers in plant science 7, 2030-2030.
79. Williamson, L.C., Ribrioux, S.P.C.P., Fitter, A.H., and Leyser, H.M.O. (2001). Phosphate Availability Regulates Root System Architecture in Arabidopsis. Plant Physiology 126, 875.
80. Wu, F.-H., Shen, S.-C., Lee, L.-Y., Lee, S.-H., Chan, M.-T., and Lin, C.-S. (2009). Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method. Plant Methods 5, 16.
81. Wun, C.-L., Quan, Y., and Zhuang, X. (2020). Recent Advances in Membrane Shaping for Plant Autophagosome Biogenesis. Frontiers in Plant Science 11, 565.
82. Xie, Z., Nair, U., and Klionsky, D.J. (2008). Atg8 controls phagophore expansion during autophagosome formation. Mol Biol Cell 19, 3290-3298.
83. Xiong, Y., Contento, A.L., and Bassham, D.C. (2005). AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana. The Plant Journal 42, 535-546.
84. Yang, Z., and Klionsky, D.J. (2009). An overview of the molecular mechanism of autophagy. Current topics in microbiology and immunology 335, 1-32.
85. Yin, Z., Pascual, C., and Klionsky, D.J. (2016). Autophagy: machinery and regulation. Microb Cell 3, 588-596.
86. Yoo, S.-D., Cho, Y.-H., and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols 2, 1565-1572.
87. Yoshii, S.R., and Mizushima, N. (2017). Monitoring and Measuring Autophagy. International Journal of Molecular Sciences 18, 1865.
88. Yoshimoto, K. (2012). Beginning to Understand Autophagy, an Intracellular Self-Degradation System in Plants. Plant and Cell Physiology 53, 1355-1365.
89. Yoshimoto, K., Hanaoka, H., Sato, S., Kato, T., Tabata, S., Noda, T., and Ohsumi, Y. (2004). Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy. Plant Cell 16, 2967-2983.
90. Zavodszky, E., Vicinanza, M., and Rubinsztein, D.C. (2013). Biology and trafficking of ATG9 and ATG16L1, two proteins that regulate autophagosome formation. FEBS Letters 587, 1988-1996.