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GABA in plants (article about its mechanism)

GABA in plants (article about its mechanism)


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I am studing to plant physiology exam and i cross over the statment about GABA in plants.

It said that level of GABA increasing my thermal shock. But mechanism of effect on plants is unknown.

Can anybody give me a tipe about proper article about it?


Notes on Photobiology In Plants and Its Mechanism

Chromoprotein (photosensitive photoreceptor, blue pigment protein complex found in almost all flowering plant (angiosperms) was discovered by Borthwick et el (1952) and isolated by Butler et al (1959) from etiolated seedlings of Maize.

It exists in two interconvertible states, blue and yellow green, Pr and Pfr. Pfr is associated with cell membrane while Pr is found in diffused state in cytosol. It causes germination of seeds, inhibits flowering in SDPs and flowering in long day plants. Pr or P600 absorbs light at 660 nm and is changed to Pfr or P730.

The latter absorbs light of 730 nm and is changed back to Pr quickly. Darkness also causes this conversion but it is very slow. Phytochrome is involved in photomorphogenetic responses above 550 nm (except phototropican photonastic movements). It is required in seed germination of some plants, bud dormancy, leaf abscission, synthesis of gibberellins and ethylene, prevention of photo-oxidation, photoperiodism and morphogenesis.

Pr form is stable in dark, absorbs red light of 660 nm and changed quickly into Pfr. Pfr on absorbing far red light of 730 nm or in dark changed into Pr. This photoreversible behaviour is independent of temperature. Pr stimulates flowering in short day plants. Pfr is essential for seed germination.

Mechanism:

At one time, phytochrome was supposed to control photomorphogenic reactions through its control on the metabolism of 2C and 3C compounds (Hendricks, 1964). Later it was thought to operate through genes (Mohar, 1966) or change in membrane permeability (Hendricks and Borthwick, 1967) by functioning as a protein enzyme for membrane structure.

Fondeville et al, (1966) observed that phytochrome controls nyctinastic (light dependent opening and closing) movements of Mimosa leaflets. Similar phytochrome controlled movements were later observed in Albizzia julibrissin and stomata. It was also found that such movements are connected with influx and efflux of K + ions. Therefore, phytochrome causes turgor changes in the cells through – change in active ion transport or membrane properties.

Phytochrome may cause photomorphogenetic response within a few seconds (Newman and Briggs, 1972) or after several hours. The former is called rapid phytochrome response. It seems that the rapid phytochrome response is caused by changes in membrane properties.

For example the excised roots of Barley present in a beaker having a solution of ATP, Mg 2+ and ascorbic acid immediately adhere to the glass surface when exposed to red light. They are released on exposure to far-red light. It shows clearly that phytochrome is located in the cell membrane (Haupt, 1972). It acts by its action on membrane bound enzymes.

Phytochrome operation may also take place through acetylcholin or related compound (Jaffe, 1970). Yamamoto and Tezuka (1972) proposed that phytochrome may act through regulation of NADP + active in the cells. It is just possible that both NADP + and acetycholine-like compounds may be related to some reactions or intermediates of phytochrome action.


Background

In recent years, soil salinization has become an alarmingly severe problem, affecting 10% of the land surface in the world [1] as well as 42.9% of protected soil in China [2]. This issue has become the main obstacle for the sustainable production of protected agriculture. Salt stress harms crops mainly because of the presence of excess ion in soil among them, Na + and Cl − are not essential mineral but are the main ions causing salt stress injury to plants [3]. Recently, the total soil salt content in greenhouse vegetable fields increased by 69.3% (in which Na + and Cl − increased by 140 and 58% respectively) [4]. Tomato (Solanum lycopersicum L.), one of the most widely cultivated vegetable crops, is a moderately salt-sensitive crop [4, 5]. However, soil salinization often severely affects tomato fruit yield and quality by decreasing photosynthetic efficiency and disturbing physiological metabolism due to ion toxicity, osmotic stress, nutrient deficiency, etc. Apparently, compared with the slow progress of breeding [1], the regulation of salt stress tolerance by exogenous substances is a fast and effective method to relieve salt damage in crops, especially by regulating various ion transport pathways and the related metabolism.

Gamma-aminobutyric acid (GABA), a four-carbon non-proteinogenic amino acid, connects the two major metabolic pathways of carbon and nitrogen in plants and its content is significantly higher than that of other non-protein amino acids [3]. It has an important effect on plant growth and abiotic stress resistance as a signal substance or metabolic product by regulating cytoplasmic pH, acting as a temporary nitrogen pool and inducing antioxidant responses [6, 7]. Maintaining cellular ion homeostasis is an important adaptive trait of plant under salt stress [8]. Our previous study showed that exogenous GABA application influenced the absorption and inhibition of mineral elements in cucumber seedlings under NaCl stress and the addition of 5 mmol·L − 1 GABA significantly reduced the accumulation of sodium ions in cucumber roots under salt stress [9]. However, there was no strong evidence that GABA directly reduced Na + to relieve salt stress.

Previous studies have demonstrated that the anabolic metabolism of GABA could be activated by salt stress induction and as a result, GABA accumulation has been observed to increase rapidly in a number of plant species, such as tomato, tea, tobacco, and Arabidopsis [10,11,12,13,14]. Among these plants, the GABA content was enhanced approximately 20-fold in Arabidopsis seedlings under 150 mmol·L − 1 NaCl [13], and GABA levels increased significantly in seedlings of lentils treated with 25–100 mmol·L − 1 NaCl [15]. Furthermore, it has been demonstrated that the synthesis-related accumulation of endogenous GABA is closely related to exogenous GABA supplementation, which increased by 29% in hulless barley and by 1-fold in Caragana treated with 0.5 mmol·L − 1 and 10 mmol·L − 1 GABA, respectively, under salt stress [16, 17]. Therefore, it is believed that endogenous GABA, which is affected by salt stimulation and exogenous GABA induction, plays a vital role in improving plant resistance to salt stress via regulatory metabolic pathways [18,19,20]. However, how exogenous GABA affects endogenous GABA synthesis at transcriptional and metabolic levels remains unknown.

The improved salt tolerance due to GABA in plants is related to many physiological metabolic pathways, including the control of reactive oxygen species (ROS) accumulation in tomato [21], the regulation of redox balance and chlorophyll biosynthesis [22], the enabling of cytosolic K + retention and Na + exclusion in Arabidopsis [1] and the alteration of cell wall composition [13]. We previously reported that GABA synthesis and supplementation are crucial for enhancing salt tolerance by decreasing ROS generation and photosynthesis in tomato [23] and accelerating NO3 − reduction and assimilation in pakchoi [24]. Although there are many physiological metabolic pathways involved in the GABA-related plant salt tolerance, the mechanism by which GABA improves plant salt tolerance has not been elucidated clearly. However, these functions of GABA in plants are performed mainly via a short pathway known as the GABA shunt [7]. During this process, GABA accumulation plays a vital role due to irreversible synthesis catalysed by glutamatedecarboxylase (GAD EC 4.1.l.15) [12, 25] as well as the uptake and transport of exogenous GABA [22].

It has been shown that GAD is the most sensitive gene for GABA metabolism in response to abiotic stress. GAD enzyme activity and gene expression levels are closely related to the GABA-mediated enhancement of plant stress resistance [12]. GAD simultaneously catalyses glutamate (Glu) degradation and GABA synthesis. To date, GAD genes have been cloned and identified in various plant species, including tomato [25], citrus [26], tea [11] and other plants. Moreover, GABA levels are regulated by GAD transcriptional expression and enzyme activity regulation, which contributes approximately 61% to the accumulation of endogenous GABA in NaCl-treated soybean [27] and the GABA content decrease by approximately 50–81% in mature green fruits of SlGAD2-suppressed lines [25]. GAD gene expression significantly increases GABA accumulation in five wheat cultivars under salt and osmotic stress [28]. OsGAD2 was the most important gene for GABA accumulation in rice, exhibiting increased activity in vitro and in vivo, showing that transgenic OsGAD2 had over 40-fold higher activity than wild type (WT) [29]. SlGAD2 and SlGAD3 play key roles in regulating GABA levels in tomato fruit, showing that transgenic over-expression lines contained higher levels of GABA (2.7- to 5.2-fold) than the WT [25]. However, there were significant differences in expression sites in different plants under NaCl. CiGAD1 was expressed in the stem, leaf and seed coat of Caragana intermedia, while CiGAD2 was highly expressed in the bark [17]. The increase in expression of CiGAD1 and CiGAD2 induced GABA accumulation within 24 h of salt treatment [17]. GAD2 expression in all parts of Arabidopsis and tobacco was significantly enhanced, accompanied by increased GAD activity and increased GABA content [12, 13]. The CsGAD gene enhanced the salt and alkali tolerance of melon by increasing leaf GAD activity and GABA content [22]. However, only a limited number of studies have examined the relationship between GADs, GABA and tomato salt tolerance, and the metabolic processes and related metabolism have not been identified.

To experimentally elucidate the relationship between GABA supplementation and tomato plant salt tolerance, we investigated plant growth and changes in Na + flux and accumulation in NaCl-treated with GABA added plants. For the first time, we analysed the transcription level of four GAD genes in tomato leaves and detected the amino acid synthesis (including GABA) and metabolism of ROS in leaves to explore the physiological functions of GABA in salt-damaged tomato seedlings. The objective of the study was to elucidate the regulatory mechanism by which exogenous GABA enhances salt tolerance in tomato plants, which might provide new information regarding the molecular regulation of GAD genes and the subsequent effect of GABA on ion uptake or metabolic processes in tomato plants under high-NaCl conditions.


References

Clark, S. L. Cellular differentiation in the kidneys of newborn mice studies with the electron microscope. J. Biophys. Biochem. Cytol. 3, 349–362 (1957).

Novikoff, A. B. The proximal tubule cell in experimental hydronephrosis. J. Biophys. Biochem. Cytol. 6, 136–138 (1959).

Ashford, T. P. & Porter, K. R. Cytoplasmic components in hepatic cell lysosomes. J. Cell Biol. 12, 198–202 (1962).

Novikoff, A. B. & Essner, E. Cytolysomes and mitochondrial degeneration. J. Cell Biol. 15, 140–146 (1962).

de Duve, C. & Wattiaux, R. Functions of lysosomes. Annu. Rev. Physiol. 28, 435–492 (1966).

Yang, Z. & Klionsky, D. J. Eaten alive: a history of macroautophagy. Nat. Cell Biol. 12, 814–822 (2010).

Klionsky, D. J. et al. A unified nomenclature for yeast autophagy-related genes. Dev. Cell 5, 539–545 (2003).

Nakatogawa, H., Suzuki, K., Kamada, Y. & Ohsumi, Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat. Rev. Mol. Cell Biol. 10, 458–467 (2009).

Ohsumi, Y. Historical landmarks of autophagy research. Cell Res. 24, 9–23 (2014).

Mizushima, N., Yoshimori, T. & Ohsumi, Y. The role of Atg proteins in autophagosome formation. Annu. Rev. Cell Dev. Biol. 27, 107–132 (2011).

Dikic, I. & Elazar, Z. Mechanism and medical implications of mammalian autophagy. Nat. Rev. Mol. Cell Biol. 19, 349–364 (2018).

Levine, B. & Kroemer, G. Biological functions of autophagy genes: a disease perspective. Cell 176, 11–42 (2019).

Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1–222 (2016).

Mizushima, N. Physiological functions of autophagy. Curr. Top. Microbiol. Immunol. 335, 71–84 (2009).

Levine, B. & Klionsky, D. J. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev. Cell 6, 463–477 (2004).

Yang, Z., Huang, J., Geng, J., Nair, U. & Klionsky, D. J. Atg22 recycles amino acids to link the degradative and recycling functions of autophagy. Mol. Biol. Cell 17, 5094–5104 (2006).

Kawano-Kawada, M., Kakinuma, Y. & Sekito, T. Transport of amino acids across the vacuolar membrane of yeast: its mechanism and physiological role. Biol. Pharm. Bull. 41, 1496–1501 (2018).

Kirkin, V. History of the selective autophagy research: how did it begin and where does it stand today? J. Mol. Biol. 432, 3–27 (2019).

Johansen, T. & Lamark, T. Selective autophagy: ATG8 family proteins, LIR motifs and cargo receptors. J. Mol. Biol. 432, 80–103 (2019).

Gatica, D., Lahiri, V. & Klionsky, D. J. Cargo recognition and degradation by selective autophagy. Nat. Cell Biol. 20, 233–242 (2018).

Turco, E., Fracchiolla, D. & Martens, S. Recruitment and activation of the ULK1/Atg1 kinase complex in selective autophagy. J. Mol. Biol. 432, 123–134 (2020).

Kirkin, V. & Rogov, V. V. A diversity of selective autophagy receptors determines the specificity of the autophagy pathway. Mol. Cell 76, 268–285 (2019).

Matsuura, A., Tsukada, M., Wada, Y., & Ohsumi, Y. Apg1p, a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae. Gene 192, 245–250 (1997).

Noda, T. & Ohsumi, Y. Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast. J. Biol. Chem. 273, 3963–3966 (1998).

Ragusa, M. J., Stanley, R. E. & Hurley, J. H. Architecture of the Atg17 complex as a scaffold for autophagosome biogenesis. Cell 151, 1501–1512 (2012).

Stjepanovic, G. et al. Assembly and dynamics of the autophagy-initiating Atg1 complex. Proc. Natl Acad. Sci. USA 111, 12793–12798 (2014).

Chew, L. H., Setiaputra, D., Klionsky, D. J. & Yip, C. K. Structural characterization of the Saccharomyces cerevisiae autophagy regulatory complex Atg17-Atg31-Atg29. Autophagy 9, 1467–1474 (2013).

Chew, L. H. et al. Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms. Autophagy 11, 891–905 (2015).

Kamada, Y. et al. Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J. Cell Biol. 150, 1507–1513 (2000).

Kamada, Y. et al. Tor directly controls the Atg1 kinase complex to regulate autophagy. Mol. Cell. Biol. 30, 1049–1058 (2010).

Fujioka, Y. et al. Structural basis of starvation-induced assembly of the autophagy initiation complex. Nat. Struct. Mol. Biol. 21, 513–521 (2014).

Yamamoto, H. et al. The intrinsically disordered protein Atg13 mediates supramolecular assembly of autophagy initiation complexes. Dev. Cell 38, 86–99 (2016).

Yorimitsu, T., He, C., Wang, K. & Klionsky, D. J. Tap42-associated protein phosphatase type 2A negatively regulates induction of autophagy. Autophagy 5, 616–624 (2009).

Yeasmin, A. M. S. T. et al. Orchestrated action of PP2A antagonizes Atg13 phosphorylation and promotes autophagy after the inactivation of TORC1. PLoS One 11, e0166636 (2016).

Memisoglu, G., Eapen, V. V., Yang, Y., Klionsky, D. J. & Haber, J. E. PP2C phosphatases promote autophagy by dephosphorylation of the Atg1 complex. Proc. Natl Acad. Sci. USA 116, 1613–1620 (2019).

Jung, C. H. et al. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol. Biol. Cell 20, 1992–2003 (2009).

Hosokawa, N. et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol. Biol. Cell 20, 1981–1991 (2009).

Ganley, I. G. et al. ULK1·ATG13·FIP200 complex mediates mTOR signaling and is essential for autophagy. J. Biol. Chem. 284, 12297–12305 (2009).

Hara, T. et al. FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. J. Cell Biol. 181, 497–510 (2008).

Hosokawa, N. et al. Atg101, a novel mammalian autophagy protein interacting with Atg13. Autophagy 5, 973–979 (2009).

Mercer, C. A., Kaliappan, A. & Dennis, P. B. A novel, human Atg13 binding protein, Atg101, interacts with ULK1 and is essential for macroautophagy. Autophagy 5, 649–662 (2009).

Bach, M., Larance, M., James, D. E. & Ramm, G. The serine/threonine kinase ULK1 is a target of multiple phosphorylation events. Biochem. J. 440, 283–291 (2011).

Yeh, Y. Y., Wrasman, K. & Herman, P. K. Autophosphorylation within the Atg1 activation loop is required for both kinase activity and the induction of autophagy in Saccharomyces cerevisiae. Genetics 185, 871–882 (2010).

Kijanska, M. et al. Activation of Atg1 kinase in autophagy by regulated phosphorylation. Autophagy 6, 1168–1178 (2010).

Sánchez-Wandelmer, J. et al. Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation. Nat. Commun. 8, 295 (2017).

Papinski, D. et al. Early steps in autophagy depend on direct phosphorylation of Atg9 by the Atg1 kinase. Mol. Cell 53, 471–483 (2014).

Hu, Z. et al. Multilayered control of protein turnover by TORC1 and Atg1. Cell Rep. 28, 3486–3496 (2019).

Pengo, N., Agrotis, A., Prak, K., Jones, J. & Ketteler, R. A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B. Nat. Commun. 8, 294 (2017).

Russell, R. C. et al. ULK1 induces autophagy by phosphorylating beclin-1 and activating VPS34 lipid kinase. Nat. Cell Biol. 15, 741–750 (2013).

Zhou, C. et al. Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy. Cell Res. 27, 184–201 (2017).

Wold, M. S., Lim, J., Lachance, V., Deng, Z. & Yue, Z. ULK1-mediated phosphorylation of ATG14 promotes autophagy and is impaired in Huntington’s disease models. Mol. Neurodegener. 11, 76 (2016).

Park, J. M. et al. The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14. Autophagy 12, 547–564 (2016).

Egan, D. F. et al. Small molecule Inhibition of the autophagy kinase ULK1 and identification of ULK1 substrates. Mol. Cell 59, 285–297 (2015).

Di Bartolomeo, S. et al. The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy. J. Cell Biol. 191, 155–168 (2010).

Jeong, Y. T. et al. The ULK1-FBXW5-SEC23B nexus controls autophagy. eLife 7, 1–25 (2018).

Lin, S. Y. et al. GSK3-TIP60-ULK1 signaling pathway links growth factor deprivation to autophagy. Science 336, 477–481 (2012).

Nazio, F. et al. MTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6. Nat. Cell Biol. 15, 406–416 (2013).

Liu, C. C. et al. Cul3-KLHL20 ubiquitin ligase governs the turnover of ULK1 and VPS34 complexes to control autophagy termination. Mol. Cell 61, 84–97 (2016).

Nazio, F. et al. Fine-tuning of ULK1 mRNA and protein levels is required for autophagy oscillation. J. Cell Biol. 215, 841–856 (2016).

Li, J. et al. Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy. Autophagy 11, 1216–1229 (2015).

Yamamoto, H. et al. Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J. Cell Biol. 198, 219–233 (2012).

Mari, M. et al. An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis. J. Cell Biol. 190, 1005–1022 (2010).

Shirahama-Noda, K., Kira, S., Yoshimori, T. & Noda, T. TRAPPis responsible for vesicular transport from early endosomes to Golgi, facilitating Atg9 cycling in autophagy. J. Cell Sci. 126, 4963–4973 (2013).

Kakuta, S. et al. Atg9 vesicles recruit vesicle-tethering proteins Trs85 and Ypt1 to the autophagosome formation site. J. Biol. Chem. 287, 44261–44269 (2012).

Geng, J., Nair, U., Yasumura-Yorimitsu, K. & Klionsky, D. J. Post-Golgi Sec proteins are required for autophagy in Saccharomyces cerevisiae. Mol. Biol. Cell 21, 2257–2269 (2010).

Ohashi, Y. & Munro, S. Membrane delivery to the yeast autophagosome from the Golgi-endosomal system. Mol. Biol. Cell 21, 3998–4008 (2010).

Backues, S. K. et al. Atg23 and Atg27 act at the early stages of Atg9 trafficking in S. cerevisiae. Traffic 16, 172–190 (2015).

Yen, W. L., Legalds, J. E., Nair, U. & Klionsky, D. J. Atg27 is required for autophagy-dependent cycling of Atg9. Mol. Biol. Cell 18, 581–593 (2007).

Segarra, V. A., Boettner, D. R. & Lemmon, S. K. Atg27 tyrosine sorting motif is important for its trafficking and Atg9 localization. Traffic 16, 365–378 (2015).

Kakuta, S. et al. Small GTPase Rab1B is associated with ATG9A vesicles and regulates autophagosome formation. FASEB J. 31, 3757–3773 (2017).

Orsi, A. et al. Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol. Biol. Cell 23, 1860–1873 (2012).

Duke, E. M. H. et al. Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM). Ultramicroscopy 143, 77–87 (2014).

Lamb, C. A. et al. TBC 1D14 regulates autophagy via the TRAPP complex and ATG 9 traffic. EMBO J. 35, 281–301 (2016).

Longatti, A. et al. TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes. J. Cell Biol. 197, 659–675 (2012).

Popovic, D. & Dikic, I. TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy. EMBO Rep. 15, 392–401 (2014).

Takahashi, Y. et al. Bif-1 regulates Atg9 trafficking by mediating the fission of Golgi membranes during autophagy. Autophagy 7, 61–73 (2011).

Imai, K. et al. Atg9A trafficking through the recycling endosomes is required for autophagosome formation. J. Cell Sci. 129, 3781–3791 (2016).

Søreng, K. et al. SNX 18 regulates ATG 9A trafficking from recycling endosomes by recruiting Dynamin-2. EMBO Rep. 19, e44837 (2018).

Puri, C., Renna, M., Bento, C. F., Moreau, K. & Rubinsztein, D. C. Diverse autophagosome membrane sources coalesce in recycling endosomes. Cell 154, 1285–1299 (2013). This work and Yamamoto et al. (2012) and Mari et al. (2010) together propose that Atg9/ATG9-containing vesicles contribute to the formation of autophagosome precursors.

Kihara, A., Noda, T., Ishihara, N. & Ohsumi, Y. Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. J. Cell Biol. 152, 519–530 (2001).

Araki, Y. et al. Atg38 is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity. J. Cell Biol. 203, 299–313 (2013).

Itakura, E., Kishi, C., Inoue, K. & Mizushima, N. Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. Mol. Biol. Cell 19, 5360–5372 (2008).

Lu, J. et al. NRBF2 regulates autophagy and prevents liver injury by modulating Atg14L-linked phosphatidylinositol-3 kinase III activity. Nat. Commun. 5, 4920 (2014).

Matsunaga, K. et al. Two beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nat. Cell Biol. 11, 385–396 (2009).

Zhong, Y. et al. Distinct regulation of autophagic activity by Atg14L and Rubicon associated with beclin 1-phosphatidylinositol-3-kinase complex. Nat. Cell Biol. 11, 468–476 (2009).

Obara, K., Noda, T., Niimi, K. & Ohsumi, Y. Transport of phosphatidylinositol 3-phosphate into the vacuole via autophagic membranes in Saccharomyces cerevisiae. Genes. Cell 13, 537–547 (2008).

Cheng, J. et al. Yeast and mammalian autophagosomes exhibit distinct phosphatidylinositol 3-phosphate asymmetries. Nat. Commun. 5, 3207 (2014).

Axe, E. L. et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J. Cell Biol. 182, 685–701 (2008).

Ge, L., Zhang, M. & Schekman, R. Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER-Golgi intermediate compartment. eLife 3, 1–13 (2014).

Ge, L. et al. Remodeling of ER-exit sites initiates a membrane supply pathway for autophagosome biogenesis. EMBO Rep. 18, e201744559 (2017).

Ge, L., Melville, D., Zhang, M. & Schekman, R. The ER-Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis. eLife 2, e00947 (2013).

Pattingre, S. et al. Bcl-2 antiapoptotic proteins inhibit beclin 1-dependent autophagy. Cell 122, 927–939 (2005).

Maiuri, M. C. et al. BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between beclin 1 and Bcl-2/Bcl-XL. Autophagy 3, 374–376 (2007).

Takahashi, Y. et al. Bif-1 interacts with beclin 1 through UVRAG and regulates autophagy and tumorigenesis. Nat. Cell Biol. 9, 1142–1151 (2007).

Maria Fimia, G. et al. Ambra1 regulates autophagy and development of the nervous system. Nature 447, 1121–1125 (2007).

Barth, H., Meiling-Wesse, K., Epple, U. D. & Thumm, M. Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p. FEBS Lett. 508, 23–28 (2001).

Guan, J. et al. Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport, pexophagy, and autophagy in Saccharomyces cerevisiae and Pichia pastoris. Mol. Biol. Cell 12, 3821–3838 (2001).

Strømhaug, P. E., Reggiori, F., Guan, J., Wang, C.-W. & Klionsky, D. J. Atg21 is a phosphoinositide binding protein required for efficient lipidation and localization of Atg8 during uptake of aminopeptidase I by selective autophagy. Mol. Biol. Cell 15, 3553–3566 (2004).

Meiling-Wesse, K. et al. Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway. J. Biol. Chem. 279, 37741–37750 (2004).

Proikas-Cezanne, T., Takacs, Z., Dönnes, P. & Kohlbacher, O. WIPI proteins: essential PtdIns3P effectors at the nascent autophagosome. J. Cell Sci. 128, 207–217 (2015).

Obara, K., Sekito, T., Niimi, K. & Ohsumi, Y. The Atg18-Atg2 complex is recruited to autophagic membranes via phosphatidylinositol 3-phosphate and exerts an essential function. J. Biol. Chem. 283, 23972–23980 (2008).

Chowdhury, S. et al. Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex. Proc. Natl Acad. Sci. USA 115, E9792–E9801 (2018).

Zheng, J. X. et al. Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation. Autophagy 8627, 1–14 (2017).

Krick, R., Tolstrup, J., Appelles, A., Henke, S. & Thumm, M. The relevance of the phosphatidylinositolphosphat-binding motif FRRGT of Atg18 and Atg21 for the Cvt pathway and autophagy. FEBS Lett. 580, 4632–4638 (2006).

Nair, U., Cao, Y., Xie, Z. & Klionsky, D. J. Roles of the lipid-binding motifs of Atg18 and Atg21 in the cytoplasm to vacuole targeting pathway and autophagy. J. Biol. Chem. 285, 11476–11488 (2010).

Itakura, E. & Mizushima, N. Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 6, 764–776 (2010).

Velikkakath, A. K. G., Nishimura, T., Oita, E., Ishihara, N. & Mizushima, N. Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets. Mol. Biol. Cell 23, 896–909 (2012).

Mizushima, N. et al. A protein conjugation system essential for autophagy. Nature 395, 395–398 (1998).

Mizushima, N., Sugita, H., Yoshimori, T. & Ohsumi, Y. A new protein conjugation system in human. J. Biol. Chem. 273, 33889–33892 (1998).

Kuma, A., Mizushima, N., Ishihara, N. & Ohsumi, Y. Formation of the ∼ 350-kDa Apg12-Apg5·Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast. J. Biol. Chem. 227, 18619–18625 (2002).

Mizushima, N. et al. Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate. J. Cell Sci. 116, 1679–1688 (2003).

Ishibashi, K. et al. Atg16L2, a novel isoform of mammalian Atg16L that is not essential for canonical autophagy despite forming an Atg12-5-16L2 complex. Autophagy 7, 1500–1513 (2011).

Fujioka, Y., Noda, N. N., Nakatogawa, H., Ohsumi, Y. & Inagaki, F. Dimeric coiled-coil structure of Saccharomyces cerevisiae Atg16 and its functional significance in autophagy. J. Biol. Chem. 285, 1508–1515 (2010).

Fujita, N. et al. Differential involvement of Atg16L1 in Crohn disease and canonical autophagy: analysis of the organization of the Atg16L1 complex in fibroblasts. J. Biol. Chem. 284, 32602–32609 (2009).

Dooley, H. C. et al. WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12-5-16L1. Mol. Cell 55, 238–252 (2014).

Juris, L. et al. PI 3P binding by Atg21 organises Atg8 lipidation. EMBO J. 34, 955–973 (2015).

Hanada, T. et al. The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J. Biol. Chem. 282, 37298–37302 (2007).

Fujita, N. et al. The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. Mol. Biol. Cell 19, 2092–2100 (2008).

Otomo, C., Metlagel, Z., Takaesu, G. & Otomo, T. Structure of the human ATG12 ∼ ATG5 conjugate required for LC3 lipidation in autophagy. Nat. Struct. Mol. Biol. 20, 59–66 (2013).

Fujioka, Y. et al. In vitro reconstitution of plant Atg8 and Atg12 conjugation systems essential for autophagy. J. Biol. Chem. 283, 1921–1928 (2008).

Kabeya, Y. et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720–5728 (2000).

Kabeya, Y. et al. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J. Cell Sci. 117, 2805–2812 (2004).

Kirisako, T. et al. 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 (2000).

Ichimura, Y. et al. A ubiquitin-like system mediates protein lipidation. Nature 408, 488–492 (2000).

Sou, Y. S., Tanida, I., Komatsu, M., Ueno, T. & Kominami, E. Phosphatidylserine in addition to phosphatidylethanolamine is an in vitro target of the mammalian Atg8 modifiers, LC3, GABARAP, and GATE-16. J. Biol. Chem. 281, 3017–3024 (2006).

Nakatogawa, H. Two ubiquitin-like conjugation systems that mediate membrane formation during autophagy. Essays Biochem. 55, 39–50 (2013).

Wild, P., McEwan, D. G. & Dikic, I. The LC3 interactome at a glance. J. Cell Sci. 127, 3–9 (2014).

Slobodkin, M. R. & Elazar, Z. The Atg8 family: multifunctional ubiquitin-like key regulators of autophagy. Essays Biochem. 55, 51–64 (2013).

Nakatogawa, H., Ishii, J., Asai, E. & Ohsumi, Y. Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis. Autophagy 8, 177–186 (2012).

Yu, Z. Q. et al. Dual roles of Atg8-PE deconjugation by Atg4 in autophagy. Autophagy 8, 883–892 (2012).

Nair, U. et al. A role for Atg8-PE deconjugation in autophagosome biogenesis. Autophagy 8, 780–793 (2012).

Hirata, E., Ohya, Y. & Suzuki, K. Atg4 plays an important role in efficient expansion of autophagic isolation membranes by cleaving lipidated Atg8 in Saccharomyces cerevisiae. PLoS One 12, e0181047 (2017).

Kauffman, K. J. et al. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases. Autophagy 14, 992–1010 (2018).

Abreu, S. et al. Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation. EMBO Rep. 18, 765–780 (2017).

He, C. & Klionsky, D. J. Regulation mechanisms and signaling pathways of autophagy. Annu. Rev. Genet. 43, 67–93 (2009).

Yang, Z. & Klionsky, D. J. Mammalian autophagy: core molecular machinery and signaling regulation. Curr. Opin. Cell Biol. 22, 124–131 (2010).

Corona Velazquez, A. F. & Jackson, W. T. So many roads: the multifaceted regulation of autophagy induction. Mol. Cell. Biol. 38, e00303–e00318 (2018).

Gross, A. & Graef, M. Mechanisms of autophagy in metabolic stress response. J. Mol. Biol. 432, 28–52 (2019).

Galluzzi, L., Pietrocola, F., Levine, B. & Kroemer, G. Metabolic control of autophagy. Cell 159, 1263–1276 (2014).

Loewith, R. & Hall, M. N. Target of rapamycin (TOR) in nutrient signaling and growth control. Genetics 189, 1177–1201 (2011).

Budovskaya, Y. V., Stephan, J. S., Deminoff, S. J. & Herman, P. K. An evolutionary proteomics approach identifies substrates of the cAMP-dependent protein kinase. Proc. Natl Acad. Sci. USA 102, 13933–13938 (2005).

Stephan, J. S., Yeh, Y. Y., Ramachandran, V., Deminoff, S. J. & Herman, P. K. The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy. Proc. Natl Acad. Sci. USA 106, 17049–17054 (2009).

Kim, J., Kundu, M., Viollet, B. & Guan, K. L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13, 132–141 (2011).

Egan, D. F. et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 331, 456–461 (2011).

Yi, C. et al. Formation of a Snf1-Mec1-Atg1 module on mitochondria governs energy deprivation-induced autophagy by regulating mitochondrial respiration. Dev. Cell 41, 59–71 (2017).

Kamber, R. A., Shoemaker, C. J. & Denic, V. Receptor-bound targets of selective autophagy use a scaffold protein to activate the Atg1 kinase. Mol. Cell 59, 372–381 (2015).

Vargas, J. N. S. et al. Spatiotemporal control of ULK1 activation by NDP52 and TBK1 during selective autophagy. Mol. Cell 74, 347–362 (2019).

Ravenhill, B. J. et al. The cargo receptor NDP52 initiates selective autophagy by recruiting the ULK complex to cytosol-invading bacteria. Mol. Cell 74, 320–329 (2019).

Turco, E. et al. FIP200 claw domain binding to p62 promotes autophagosome formation at ubiquitin condensates. Mol. Cell 74, 330–346 (2019).

Torggler, R. et al. Two independent pathways within selective autophagy converge to activate Atg1 kinase at the vacuole. Mol. Cell 64, 221–235 (2016).

Zientara-Rytter, K. & Subramani, S. Mechanistic insights into the role of Atg11 in selective autophagy. J. Mol. Biol. 432, 104–222 (2019).

Suzuki, K. et al. The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. EMBO J. 20, 5971–5981 (2001).

Suzuki, K., Akioka, M., Kondo-Kakuta, C., Yamamoto, H. & Ohsumi, Y. Fine mapping of autophagy-related proteins during autophagosome formation in Saccharomyces cerevisiae. J. Cell Sci. 126, 2534–2544 (2013).

Graef, M., Friedman, J. R., Graham, C., Babu, M. & Nunnari, J. ER exit sites are physical and functional core autophagosome biogenesis components. Mol. Biol. Cell 24, 2918–2931 (2013).

Hollenstein, D. M. et al. Vac8 spatially confines autophagosome formation at the vacuole in S. cerevisiae. J. Cell Sci. 132, jcs235002 (2019). This study describes tethering of the Atg1 complex to the vacuolar membrane by Vac8.

Hamasaki, M. et al. Autophagosomes form at ER-mitochondria contact sites. Nature 495, 389–393 (2013).

Cheong, H., Nair, U., Geng, J. & Klionsky, D. J. The Atg1 kinase complex is involved in the regulation of protein recruitment to initiate sequestering vesicle formation for nonspecific autophagy in Saccharomyces cerevisiae. Mol. Biol. Cell 19, 433–476 (2008).

Kawamata, T., Kamada, Y., Kabeya, Y., Sekito, T. & Ohsumi, Y. Organization of the pre-autophagosomal structure responsible for autophagosome formation. Mol. Biol. Cell 19, 2039–2050 (2008).

Koyama-Honda, I., Itakura, E., Fujiwara, T. K. & Mizushima, N. Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site. Autophagy 9, 1491–1499 (2013).

Geng, J., Baba, M., Nair, U. & Klionsky, D. J. Quantitative analysis of autophagy-related protein stoichiometry by fluorescence microscopy. J. Cell Biol. 182, 129–140 (2008).

Fujioka, Y. et al. Phase separation organizes the site of autophagosome formation. Nature 578, 301–305 (2020). This study and Fujioka et al. (2014), Yamamoto et al. (2016), Kamber et al. (2015) and Torggler et al. (2016) together show how the Atg1/ULK complex is formed and how multiple copies of the complex are further assembled to form a platform for the initiation of autophagosome formation.

Zhang, G., Wang, Z., Du, Z. & Zhang, H. mTOR regulates phase separation of PGL granules to modulate their autophagic degradation. Cell 174, 1492–1506 (2018).

Yamasaki, A. et al. Liquidity is a critical determinant for selective autophagy of protein condensates. Mol. Cell 77, 1163–1175 (2020).

Sun, D., Wu, R., Zheng, J., Li, P. & Yu, L. Polyubiquitin chain-induced p62 phase separation drives autophagic cargo segregation. Cell Res. 28, 405–415 (2018).

Yang, Y. et al. Cytoplasmic DAXX drives SQSTM1/p62 phase condensation to activate Nrf2-mediated stress response. Nat. Commun. 10, 3759 (2019).

Sánchez-Martín, P. et al. NBR 1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system. EMBO Rep. 21, e48902 (2020).

You, Z. et al. Requirement for p62 acetylation in the aggregation of ubiquitylated proteins under nutrient stress. Nat. Commun. 10, 5792 (2019).

Cloer, E. W. et al. p62-dependent phase separation of patient-derived KEAP1 mutations and NRF2. Mol. Cell. Biol. 38, e00644-17 (2018).

Nishimura, T. et al. Autophagosome formation is initiated at phosphatidylinositol synthase-enriched ER subdomains. EMBO J. 36, 1719–1735 (2017).

Morita, K. et al. Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation. J. Cell Biol. 217, 3817–3828 (2018).

Moretti, F. et al. TMEM 41B is a novel regulator of autophagy and lipid mobilization. EMBO Rep. 19, e45889 (2018).

Shoemaker, C. J. et al. CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor. PLoS Biol. 17, e2007044 (2019).

Zhao, Y. G. et al. The ER contact proteins VAPA/B Interact with multiple autophagy proteins to modulate autophagosome biogenesis. Curr. Biol. 28, 1234–1245 (2018).

Zhao, Y. G. et al. Regulates SERCA activity to control ER-isolation membrane contacts for autophagosome formation article the ER-localized transmembrane protein EPG-3 / VMP1 regulates SERCA activity to control ER-isolation membrane contacts for autophagosome formation. Mol. Cell 67, 974–989.e6 (2017).

Bodemann, B. O. et al. RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly. Cell 144, 253–267 (2011).

Suzuki, S. W. et al. Atg13 HORMA domain recruits Atg9 vesicles during autophagosome formation. Proc. Natl Acad. Sci. USA 112, 3350–3355 (2015).

Sekito, T., Kawamata, T., Ichikawa, R., Suzuki, K. & Ohsumi, Y. Atg17 recruits Atg9 to organize the pre-autophagosomal structure. Genes. Cell 14, 525–538 (2009).

He, C. et al. Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast. J. Cell Biol. 175, 925–935 (2006).

Itakura, E., Kishi-Itakura, C., Koyama-Honda, I. & Mizushima, N. Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy. J. Cell Sci. 125, 1488–1499 (2012).

Kageyama, S. et al. The LC3 recruitment mechanism is separate from Atg9L1-dependent membrane formation in the autophagic response against Salmonella. Mol. Biol. Cell 22, 2290–2300 (2011).

Ravikumar, B., Moreau, K., Jahreiss, L., Puri, C. & Rubinsztein, D. C. Plasma membrane contributes to the formation of pre-autophagosomal structures. Nat. Cell Biol. 12, 747–757 (2010).

Moreau, K., Ravikumar, B., Renna, M., Puri, C. & Rubinsztein, D. C. Autophagosome precursor maturation requires homotypic fusion. Cell 146, 303–317 (2011).

Knævelsrud, H. et al. Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. J. Cell Biol. 202, 331–349 (2013).

Judith, D. et al. ATG9A shapes the forming autophagosome through Arfaptin 2 and phosphatidylinositol 4-kinase IIIβ. J. Cell Biol. 218, 1634–1652 (2019).

Suzuki, K., Kubota, Y., Sekito, T. & Ohsumi, Y. Hierarchy of Atg proteins in pre-autophagosomal structure organization. Genes. Cell 12, 209–218 (2007).

Puri, C. et al. The RAB11A-positive compartment is a primary platform for autophagosome assembly mediated by WIPI2 recognition of PI3P-RAB11A. Dev. Cell 45, 114–131 (2018).

Sakoh-Nakatogawa, M. et al. Atg12-Atg5 conjugate enhances E2 activity of Atg3 by rearranging its catalytic site. Nat. Struct. Mol. Biol. 20, 433–439 (2013).

Fujita, N. et al. Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin. J. Cell Biol. 203, 115–128 (2013).

Nishimura, T. et al. FIP200 regulates targeting of Atg16L1 to the isolation membrane. EMBO Rep. 14, 284–291 (2013).

Harada, K. et al. Two distinct mechanisms target the autophagy-related E3 complex to the pre-autophagosomal structure. eLife 8, e43088 (2019).

Kaminska, J. et al. Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress. Int. J. Biochem. Cell Biol. 79, 494–504 (2016).

Gómez-Sánchez, R. et al. Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores. J. Cell Biol. 217, 2743–2763 (2018).

Tang, Z. et al. TOM40 targets Atg2 to mitochondria-associated ER membranes for phagophore expansion. Cell Rep. 28, 1744–1757 (2019).

Lin, M. G., Schöneberg, J., Davies, C. W., Ren, X. & Hurley, J. H. The dynamic Atg13-free conformation of the Atg1 EAT domain is required for phagophore expansion. Mol. Biol. Cell 29, 1228–1237 (2018).

Stanga, D. et al. TRAPPC11 functions in autophagy by recruiting ATG2B-WIPI4/WDR45 to preautophagosomal membranes. Traffic 20, 325–345 (2019).

Mizushima, N. et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J. Cell Biol. 152, 657–668 (2001).

Nakatogawa, H., Ichimura, Y. & Ohsumi, Y. Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. Cell 130, 165–178 (2007).

Weidberg, H. et al. LC3 and GATE-16 N termini mediate membrane fusion processes required for autophagosome biogenesis. Dev. Cell 20, 444–454 (2011).

Wu, F. et al. Structural basis of the differential function of the two C. elegans Atg8 homologs, LGG-1 and LGG-2, in autophagy. Mol. Cell 60, 914–929 (2015).

Xie, Z., Nair, U. & Klionsky, D. J. Atg8 controls phagophore expansion during autophagosome formation. Mol. Biol. Cell 19, 3290–3298 (2008).

Nair, U. et al. SNARE proteins are required for macroautophagy. Cell 146, 290–302 (2012).

Kraft, C. et al. Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy. EMBO J. 31, 3691–3703 (2012).

Nakatogawa, H. et al. The autophagy-related protein kinase Atg1 interacts with the ubiquitin-like protein Atg8 via the Atg8 family interacting motif to facilitate autophagosome formation. J. Biol. Chem. 287, 28503–28507 (2012).

Alemu, E. A. et al. ATG8 family proteins act as scaffolds for assembly of the ULK complex: sequence requirements for LC3-interacting region (LIR) motifs. J. Biol. Chem. 287, 39275–39290 (2012).

Herhaus, L. et al. TBK1-mediated phosphorylation of LC3C and GABARAP-L2 controls autophagosome shedding by ATG4 protease. EMBO Rep. 21, e48317 (2020).

Scherz-Shouval, R. et al. Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO J. 26, 1749–1760 (2007).

Sakoh-Nakatogawa, M., Kirisako, H., Nakatogawa, H. & Ohsumi, Y. Localization of Atg3 to autophagy-related membranes and its enhancement by the Atg8-family interacting motif to promote expansion of the membranes. FEBS Lett. 589, 744–749 (2015).

Ngu, M., Hirata, E. & Suzuki, K. Visualization of Atg3 during autophagosome formation in Saccharomyces cerevisiae. J. Biol. Chem. 290, 8146–8153 (2015).

Weidberg, H. et al. LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis. EMBO J. 29, 1792–1802 (2010).

Sou, Y. et al. The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice. Mol. Biol. Cell 19, 4762–4775 (2008).

Fujita, N. et al. An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure. Mol. Biol. Cell 19, 4651–4659 (2008).

Tsuboyama, K. et al. The ATG conjugation systems are important for degradation of the inner autophagosomal membrane. Science 354, 1036–1041 (2016).

Nguyen, T. N. et al. Atg8 family LC3/GAB ARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation. J. Cell Biol. 215, 857–874 (2016).

Hayashi-Nishino, M. et al. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat. Cell Biol. 11, 1433–1437 (2009).

Hailey, D. W. et al. Mitochondria supply membranes for autophagosome biogenesis during starvation. Cell 141, 656–667 (2010).

Lamb, C. A., Yoshimori, T. & Tooze, S. A. The autophagosome: origins unknown, biogenesis complex. Nat. Rev. Mol. Cell Biol. 14, 759–774 (2013).

Ylä-Anttila, P., Vihinen, H., Jokitalo, E. & Eskelinen, E. L. 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 5, 1180–1185 (2009).

Uemura, T. et al. A cluster of thin tubular structures mediates transformation of the endoplasmic reticulum to autophagic isolation membrane. Mol. Cell. Biol. 34, 1695–1706 (2014). This work and Axe et al. (2008), Hayashi-Nishino et al. (2009) and Ylä-Anttila et al. (2009) together reveal a direct connection of the isolation membrane to the ER via the omegasome/IMATs.

Baba, M. et al. A nuclear membrane-derived structure associated with Atg8 is involved in the sequestration of selective cargo, the Cvt complex, during autophagosome formation in yeast. Autophagy 15, 423–437 (2019).

Kotani, T., Kirisako, H., Koizumi, M., Ohsumi, Y. & Nakatogawa, H. The Atg2-Atg18 complex tethers pre-autophagosomal membranes to the endoplasmic reticulum for autophagosome formation. Proc. Natl Acad. Sci. USA 115, 10363–10368 (2018).

Valverde, D. P. et al. ATG2 transports lipids to promote autophagosome biogenesis. J. Cell Biol. 218, 1787–1798 (2019).

Osawa, T. et al. Atg2 mediates direct lipid transfer between membranes for autophagosome formation. Nat. Struct. Mol. Biol. 26, 281–288 (2019).

Tamura, N. et al. Differential requirement for ATG2A domains for localization to autophagic membranes and lipid droplets. FEBS Lett. 591, 3819–3830 (2017).

Maeda, S., Otomo, C. & Otomo, T. The autophagic membrane tether ATG2A transfers lipids between membranes. eLife 8, e45777 (2019). This work and Chowdhury et al. (2018), Gómez-Sánchez et al. (2018), Kotani et al. (2018), Valverde et al. (2019) and Osawa et al. (2019) together reveal the membrane-tethering and lipid transfer functions of Atg2/ATG2.

Osawa, T., Ishii, Y. & Noda, N. N. Human ATG2B possesses a lipid transfer activity which is accelerated by negatively charged lipids and WIPI4. Genes to Cells 25, 65–70 (2020).

Baba, M., Ohsumi, Y. & Osumi, M. Analysis of the membrane structures involved in autophagy in yeast by freeze-replica method. Cell Struct. Funct. 20, 465–471 (1995).

Fengsrud, M., Erichsen, E. S., Berg, T. O., Raiborg, C. & Seglen, P. O. Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy. Eur. J. Cell Biol. 79, 871–882 (2000).

Ishihara, N. et al. Autophagosome requires specific early Sec proteins for its formation and NSF/SNARE for vacuolar fusion. Mol. Biol. Cell 12, 3690–3702 (2001).

Shima, T., Kirisako, H. & Nakatogawa, H. COPII vesicles contribute to autophagosomal membranes. J. Cell Biol. 218, 1503–1510 (2019). This work and Ge et al. (2017), Suzuki et al. (2013) and Graef et al. (2013) report the involvement of COPII vesicles in autophagosome biogenesis.

Lynch-Day, M. A. et al. Trs85 directs a Ypt1 GEF, TRAPPIII, to the phagophore to promote autophagy. Proc. Natl Acad. Sci. USA 107, 7811–7816 (2010).

Wang, J. et al. Ypt1 recruits the Atg1 kinase to the preautophagosomal structure. Proc. Natl Acad. Sci. USA 110, 9800–9805 (2013).

Lipatova, Z. et al. Regulation of selective autophagy onset by a Ypt/Rab GTPase module. Proc. Natl Acad. Sci. USA 109, 6981–6986 (2012).

Davis, S. et al. Sec24 phosphorylation regulates autophagosome abundance during nutrient deprivation. eLife 5, 1–22 (2016).

Wang, J. et al. Ypt1/Rab1 regulates Hrr25/CK1δ kinase activity in ER-Golgi traffic and macroautophagy. J. Cell Biol. 210, 273–285 (2015).

Tan, D. et al. The EM structure of the TRAPPIII complex leads to the identification of a requirement for COPII vesicles on the macroautophagy pathway. Proc. Natl Acad. Sci. USA 110, 19432–19437 (2013).

Abada, A., Levin-Zaidman, S., Porat, Z., Dadosh, T. & Elazar, Z. SNARE priming is essential for maturation of autophagosomes but not for their formation. Proc. Natl Acad. Sci. USA 114, 12749–12754 (2017).

Ogasawara, Y. et al. Stearoyl-CoA desaturase 1 activity is required for autophagosome formation. J. Biol. Chem. 289, 23938–23950 (2014).

Ogasawara, Y., Kira, S., Mukai, Y., Noda, T. & Yamamoto, A. Ole1, fatty acid desaturase, is required for Atg9 delivery and isolation membrane expansion during autophagy in Saccharomyces cerevisiae. Biol. Open 6, 35–40 (2017).

Andrejeva, G. et al. De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy. Autophagy https://doi.org/10.1080/15548627.2019.1659608 (2019).

Schütter, M., Giavalisco, P., Brodesser, S. & Graef, M. Local fatty acid channeling into phospholipid synthesis drives phagophore expansion during autophagy. Cell 180, 135–149 (2020).

Biazik, J., Ylä-Anttila, P., Vihinen, H., Jokitalo, E. & Eskelinen, E. L. Ultrastructural relationship of the phagophore with surrounding organelles. Autophagy 11, 439–451 (2015).

Shpilka, T. et al. Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis. EMBO J. 34, 2117–2131 (2015).

Li, D. et al. Storage lipid synthesis is necessary for autophagy induced by nitrogen starvation. FEBS Lett. 589, 269–276 (2015).

Velázquez, A. P., Tatsuta, T., Ghillebert, R., Drescher, I. & Graef, M. Lipid droplet-mediated ER homeostasis regulates autophagy and cell survival during starvation. J. Cell Biol. 212, 621–631 (2016).

Nguyen, N., Shteyn, V. & Melia, T. J. Sensing membrane curvature in macroautophagy. J. Mol. Biol. 429, 457–472 (2017).

Romanov, J. et al. Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation. EMBO J. 31, 4304–4317 (2012).

Kaufmann, A., Beier, V., Franquelim, H. G. & Wollert, T. Molecular mechanism of autophagic membrane-scaffold assembly and disassembly. Cell 156, 469–481 (2014).

Knorr, R. L. et al. Membrane morphology is actively transformed by covalent binding of the protein Atg8 to PE-lipids. PLoS One 9, e115357 (2014).

Nath, S. et al. Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3. Nat. Cell Biol. 16, 415–424 (2014).

Monastyrska, I., Rieter, E., Klionsky, D. J. & Reggiori, F. Multiple roles of the cytoskeleton in autophagy. Biol. Rev. 84, 431–448 (2009).

Mi, N. et al. CapZ regulates autophagosomal membrane shaping by promoting actin assembly inside the isolation membrane. Nat. Cell Biol. 17, 1112–1123 (2015).

Kast, D. J., Zajac, A. L., Holzbaur, E. L. F., Ostap, E. M. & Dominguez, R. WHAMM directs the Arp2/3 complex to the ER for autophagosome biogenesis through an actin comet tail mechanism. Curr. Biol. 25, 1791–1797 (2015).

Kaksonen, M., Toret, C. P. & Drubin, D. G. Harnessing actin dynamics for clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 7, 404–414 (2006).

Knorr, R. L., Dimova, R. & Lipowsky, R. Curvature of double-membrane organelles generated by changes in membrane size and composition. PLoS One 7, e32753 (2012). This study proposes that the expanding isolation membrane bends into a spherical shape on the basis of the physical properties of the lipid bilayer.

Nice, D. C., Sato, T. K., Stromhaug, P. E., Emr, S. D. & Klionsky, D. J. Cooperative binding of the cytoplasm to vacuole targeting pathway proteins, Cvt13 and Cvt20, to phosphatidylinositol 3-phosphate at the pre-autophagosomal structure is required for selective autophagy. J. Biol. Chem. 277, 30198–30207 (2002).

Zhao, D. et al. Atg20- and Atg24-family proteins promote organelle autophagy in fission yeast. J. Cell Sci. 129, 4289–4304 (2016).

Kanki, T. & Klionsky, D. J. Mitophagy in yeast occurs through a selective mechanism. J. Biol. Chem. 283, 32386–32393 (2008).

Knorr, R. L., Lipowsky, R. & Dimova, R. Autophagosome closure requires membrane scission. Autophagy 11, 2134–2137 (2015).

Vietri, M., Radulovic, M. & Stenmark, H. The many functions of ESCRTs. Nat. Rev. Mol. Cell Biol. 21, 25–42 (2019).

Takahashi, Y. et al. An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure. Nat. Commun. 9, 2855 (2018).

Zhen, Y. et al. ESCRT-mediated phagophore sealing during mitophagy. Autophagy 16, 826–841 (2019).

Takahashi, Y. et al. VPS37A directs ESCRT recruitment for phagophore closure. J. Cell Biol. 218, 3336–3354 (2019).

Zhou, F. et al. Rab5-dependent autophagosome closure by ESCRT. J. Cell Biol. 218, 1908–1927 (2019). This work and Takahashi et al. (2018), Zhen et al. (2019) and Takahashi et al. (2019) propose that the ESCRT machinery is involved in isolation membrane pore closure.

Backues, S. K., Chen, D., Ruan, J., Xie, Z. & Klionsky, D. J. Estimating the size and number of autophagic bodies by electron microscopy. Autophagy 10, 155–164 (2014).

Lamb, C. A., Longatti, A. & Tooze, S. A. Rabs and GAPs in starvation-induced autophagy. Small GTPases 7, 265–269 (2016).

Itoh, T. & Fukuda, M. Roles of Rab-GAPs in regulating autophagy. in Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging (ed. Hayat, M. A.) Ch. 6, 143–157 (Elsevier, 2017).

Martens, S., Nakamura, S. & Yoshimori, T. Phospholipids in autophagosome formation and fusion. J. Mol. Biol. 428, 4819–4827 (2016).

Dall’Armi, C., Devereaux, K. A. & Di Paolo, G. The role of lipids in the control of autophagy. Curr. Biol. 23, R33–R45 (2013).

Itakura, E., Kishi-Itakura, C. & Mizushima, N. The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell 151, 1256–1269 (2012).

Matsui, T. et al. Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17. J. Cell Biol. 217, 2633–2645 (2018).

Gao, J., Reggiori, F. & Ungermann, C. A novel in vitro assay reveals SNARE topology and the role of Ykt6 in autophagosome fusion with vacuoles. J. Cell Biol. 217, 3670–3682 (2018).

Licheva, M. et al. Reconstitution reveals Ykt6 as the autophagosomal SNARE in autophagosome–vacuole fusion. J. Cell Biol. 217, 3656–3669 (2018).

Kimura, S., Noda, T. & Yoshimori, T. Dynein-dependent movement of autophagosomes mediates efficient encounters with lysosomes. Cell Struct. Funct. 33, 109–122 (2008).

Johansson, M. et al. Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-p150Glued, ORP1L, and the receptor βIII spectrin. J. Cell Biol. 176, 459–471 (2007).

Jordens, I. et al. The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors. Curr. Biol. 11, 1680–1685 (2001).

Wijdeven, R. H. et al. Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway. Nat. Commun. 7, 11808 (2016).

Takáts, S. et al. Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila. Mol. Biol. Cell 25, 1338–1354 (2014).

Takáts, S. et al. Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion. PLoS Genet. 14, e1007359 (2018).

Bas, L. et al. Reconstitution reveals Ykt6 as the autophagosomal SNARE in autophagosome-vacuole fusion. J. Cell Biol. 217, 3656–3669 (2018).

Jiang, P. et al. The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin 17. Mol. Biol. Cell 25, 1327–1337 (2014).

Wang, C. W., Stromhaug, P. E., Kauffman, E. J., Weisman, L. S. & Klionsky, D. J. Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during the tethering/docking stage. J. Cell Biol. 163, 973–985 (2003).

Gao, J., Langemeyer, L., Kümmel, D., Reggiori, F. & Ungermann, C. Molecular mechanism to target the endosomal Mon1-Ccz1 GEF complex to the pre-autophagosomal structure. eLife 7, e31145 (2018).

McEwan, D. G. et al. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins. Mol. Cell 57, 39–54 (2015).

Tabata, K. et al. Rubicon and PLEKHM1 negatively regulate the endocytic/autophagic pathway via a novel Rab7-binding domain. Mol. Biol. Cell 21, 4162–4172 (2010).

Wang, Z. et al. The vici syndrome protein EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. Mol. Cell 63, 781–795 (2016).

Diao, J. et al. ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes. Nature 520, 563–566 (2015).

Chen, D. et al. A mammalian autophagosome maturation mechanism mediated by TECPR1 and the Atg12-Atg5 conjugate. Mol. Cell 45, 629–641 (2012).

Liu, X. et al. The Atg17-Atg31-Atg29 complex coordinates with Atg11 to recruit the Vam7 SNARE and mediate autophagosome-Vacuole fusion. Curr. Biol. 26, 150–160 (2016).

Yu, L. et al. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 465, 942–946 (2010).

Rong, Y. et al. Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvation. Proc. Natl Acad. Sci. USA 108, 7826–7831 (2011).

Rong, Y. et al. Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation. Nat. Cell Biol. 14, 924–934 (2012).

Du, W. et al. Kinesin 1 drives autolysosome tubulation. Dev. Cell 37, 326–336 (2016).

Fernández, Á. F. et al. Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice. Nature 558, 136–140 (2018).

Nakamura, S. et al. Suppression of autophagic activity by Rubicon is a signature of aging. Nat. Commun. 10, 847 (2019).


How to beat the heat: Memory mechanism allows plants to adapt to heat stress

"If you can't stand the heat, get out of the kitchen," as the old saying goes. But for organisms that can't leave the proverbial kitchen when things get too hot, there's another way: researchers from Japan have discovered that plants can gain heat tolerance to better adapt to future heat stress, thanks to a particular mechanism for heat stress 'memory'.

In a study published in Nature Communications, researchers from Nara Institute of Science and Technology have revealed that a family of proteins that control small heat shock genes enables plants to 'remember' how to deal with heat stress.

Climate change, especially global warming, is a growing threat to agriculture worldwide. Because plants can't move to avoid adverse conditions, such as potentially lethal high temperatures, they need to be able to deal with factors such as heat stress effectively to survive. Therefore, improving the heat tolerance of crop plants is an important goal in agriculture.

"Heat stress is often repeating and changing," says lead author of the study Nobutoshi Yamaguchi. "Once plants have undergone mild heat stress, they become tolerant and can adapt to further heat stress. This is referred to as heat stress 'memory' and has been reported to be correlated to epigenetic modifications." Epigenetic modifications are inheritable changes in the way genes are expressed, and do not involve changes in the underlying DNA sequences.

"We wanted to discover how plants retain a memory of environmental changes," explains Toshiro Ito, senior author. "We examined the role of JUMONJI (JMJ) proteins in acquired temperature tolerance in response to recurring heat within a few days."

JUMONJI proteins are histone demethylases. Demethylases are enzymes that remove methyl groups from molecules such as proteins, particularly histones, which provide structural support to chromosomes. The team revealed that plants are able to maintain heat memory because of lowered H3K27me3 (histone H3 lysine 27 trimethylation) on small heat shock genes.

"We found that these proteins are necessary for heat acclimation in Arabidopsis thaliana. These results, along with future studies, will further clarify the mechanisms of plant memory and adaptation," says Yamaguchi.

This research will be relevant to genetic research in a number of fields, including biology, biochemistry, ecology, and environmental and agricultural sciences, and is applicable to the study of animals as well as plants. Understanding the epigenetic memory mechanism revealed in this study will help in working with heat tolerance to maintain the food supply in natural conditions.


Discussion

Physiological and proteomic mechanisms with different temperatures and exogenous GABA through GABA shunt

Plants have evolved complex mechanisms to deal with low temperature. When plants sense low temperature, a series of protective mechanisms are triggered – including decreasing malondialdehyde (MDA) content, which is considered to be an indicator of plant oxidative stress synthesizing cryoprotectant molecules and low-molecular-weight nitrogenous compounds and improving the scavenging activity of reactive oxygen species (ROS) [40,41,42]. In addition, chlorophyll fluorescence imaging can be employed to evaluate cold tolerance in numerous plants. [43] These alterations help plants keep a metabolic balance of substance and energy in cold environments. Under low temperature, tea plants adopted a ‘survival mode’ as shown by higher free proline content, decreased MDA content, regulating the antioxidant activity and reduced photosynthesis, resulting in growth arrest (Additional file 9: Figure S1 and Additional file 10: Figure S2). GABA can be quickly accumulated and is involved in adapting to low-temperature stress [44, 45]. However, in our study, the endogenous GABA content decreased when plants were exposed to low temperature. We speculate that endogenous GABA degraded to promote synthesis of other stress response substances and feedback to the TCA cycle at low temperature, which showed that other amino acid contents and the DAPs in the TCA cycle were up-regulated at low compared to optimum temperature.

The GABA metabolism in tea plants could regulate the GABA precursor glutamate and PAs contents to relieve anoxia stress in our previous study [14]. No significant changes in GABA-T, GAD and DAO activities, Put and Spm levels for the two different temperatures compared to controls, while application of exogenous GABA. The PAO activity clearly decreased compared to control for optimum temperature at day 4, and the SPD concentration was higher at low temperature than the control (Additional file 11: Figure S3 and Additional file 12: Figure S4). The results imply that application of exogenous GABA can feed back to the TCA cycle to promote the carbon and nitrogen cycle. The amino acid contents except for alanine and lysine all declined (Additional file 7: Table S7). A reversible transformation of an amino acid from glutamic acid to pyruvic acid was catalyzed by alanine aminotransferase (AlaAT) form 2-oxoglutarate and alanine. The AlaAT-related protein (Q9S7E9) showed no significant change in treatment T2 compared to T1, which matched the report showing that AlaAT1 broke down excess alanine [46]. Lysine is synthesized by a special branch of the family pathway of aspartic acid. Carbon atoms from lysine to acetyl coenzyme A, which then enters the TCA cycle, generatingα-ketoglutaric acid [47]. These results imply that, at optimum temperature, application of exogenous GABA influenced the GABA level resulting in response of metabolism pathways to the alert in carbon and nitrogen transport.

The iTRAQ-based protein analysis was a great information-rich approach for hunting the stress-induced dynamic proteins for pinning down the key metabolic pathways, it may play important roles in the response of improving resistance to low temperature in tea plants with application of exogenous GABA. The results indicated that application of exogenous GABA at low temperature compared to control profoundly changed metabolic pathways, including amino acids biosynthesis, flavonoid biosynthesis, glyoxylate and dicarboxylate metabolism, carbon fixation in photosynthetic organisms and the pentose phosphate pathway. These pathways included most of the DAPs in treatments T4/T3 however, pathways such as TCA cycle, glutathione metabolism, ascorbate and aldarate metabolism, and purine metabolism were also important. To our knowledge, this is the first report on dynamic proteomic responses to exogenous GABA application at low temperature in tea plants.

Flavonoid metabolism regulated with exogenous GABA at low temperature

Flavonoids are important secondary metabolites in plants and play important roles in many functions, which included pigment and antioxidant activities. Five DAPs related to flavonoid biosynthesis were observed in treatment T4/T3 (Fig. 7a). Anthocyanins are a major class of flavonoids, whose functions are very diverse which include antioxidant activity, UV rejection, defense of plant pathogens, leguminous nodulation, man fertility, optical signal and auxin transport govern [48]. Under salt treatments, the proline content increased while the wheat genotypes have higher anthocyanin content [49]. Low temperature induces anthocyanin synthesis in various species [50], it was also reported that overexpression of flavonol glycosides and anthocyanins in plants under abiotic stress can effectively remove ROS and improve drought tolerance of plants. [51, 52] Of the three anthocyanin-related genes selected for RT-qPCR, CsBAN was up-regulated and CsCHS and CsF3H were down-regulated (Fig. 7b), which indicating that the expression of CsCHS, CsF3H and CsBAN involved in anthocyanin biosynthesis, could be induced by cold treatment [53]. Our results also indicate that the effects of exogenous GABA for the three genes involved in anthocyanin synthesis in tea plants at low temperature differed, as did the genes in the process of mRNA expression and protein expression in the flavonoid metabolism, and should be further studied.

Effect of application of exogenous GABA at low temperature on (a) DAPs and (b) RT-qPCR analysis of genes related to flavonoid biosynthesis. Transcript abundance was calculated according to the difference in cycle threshold values between the target gene and β-actin transcripts normalized by the 2 −ΔΔCT method. The mRNA levels of the genes in tea leaves at 0 h were set as 1.0. Data represent the mean value ± standard deviation. Means with different letters significantly differ from each other (p ≤ 0.05). BAN: NAD(P)-binding Rossmann-fold superfamily protein CHI1: chalcone flavanone isomerase 1 CHS: chalcone synthase F3H: flavanone 3-hydroxylase FLS1: flavonol synthase 1

Amino acid metabolism and ascorbate (AsA)/glutathione cycle and exogenous GABA at low temperature

The levels of GABA, proline, tryptophan, histidine, asparagine and alanine showed the greatest changes in abundance in response to GABA application at low temperature compared to control (Additional file 7: Table S7). Combining with the metabolome and proteome, we found no significant relation between them. There are few published studies of amino acid export in the xylem under saline conditions [54]. After being treated with exogenous GABA at low temperature for 7 days, except for glycine, contents of all amino acids in leaves rose greatly.

Glutamine synthetase (GS) is an enzyme in the process of plant primary nitrogen assimilation, since glutamine and other related nitrogenous compounds are converted by it catalyzing ammonium, which is also a substrate for protein synthesis. The reabsorption of ammonia in photorespiration is the main role of chloroplast GS, and chloroplastic GS aided nitrogen assimilation in chloroplasts of YL [55]. GS was down-regulated here, and most amino acid contents increased, indicating that GS played an important role in the process that GABA works as a signal molecule when plants exposed to cold stress. Cysteine synthase catalyzes the synthesis of cysteine from O-acetylserine and disulfides, which serves as the only amino acid containing disulfide bonds (S–S) that protecting cellular environments from oxidative stress [55]. Cysteine synthase and spermidine synthase might be associated to the rise molecular chaperone activity and molecular chaperone and the decline in oxidative stress [56]. In the present study, cysteine and spermidine contents increased significantly but no differences between treatment T4 and T3, suggesting regulatory mechanisms occurring in response to cysteine and spermidine and involving both transcriptional and post-translational levels (Fig. 8a).

Effect of application of exogenous GABA at low temperature on (a) DAPs and (b) RT-qPCR analysis of genes related to amino acid metabolism and AsA/glutathione cycle. Transcript abundance was calculated according to the difference in cycle threshold values between the target gene and β-actin transcripts normalized by the 2 −ΔΔCT method. The mRNA levels of the genes in tea leaves at 0 h were set as 1.0. Data represent the mean value ± standard deviation. Means with different letters significantly differ from each other (p ≤ 0.05). Abbreviations: ASA1: anthranilate synthase alpha subunit 1 CM2: chorismate mutase 2 GGAT2: glutamate--glyoxylate aminotransferase 2-like GLN1–4: glutamine synthetase 1–4 GPX7: glutathione peroxidase 7 GSTF10: glutathione S-transferase PHI 10 GSTF9: glutathione S-transferase PHI 9 LAP2: Cytosol aminopeptidase family protein LGALDH: L-galactose dehydrogenase MDAR5: monodehydroascorbate reductase 5, chloroplastic-like METK4: S-adenosylmethionine synthetase 4 PGK3: phosphoglycerate kinase precursor RPE: ribulose-5-phosphate-3-epimerase SK1: shikimate kinase 1 TKL-2: transketolase 2

Cell process and in vitro stress produce AsA, a step in the biosynthetic pathway of which is the catalysis of GDP-mannose-3′, 5′-diisomerase (GME) between GDP-galactose and GDP-mannose [57]. Spinach L-galactose dehydrogenase (L-GalDH) show invertible inhibition by AsA, the final product of biosynthetic pathway [58]. AsA peroxidase (APX) plays a main role in cellular H2O2 metabolism. GME, L-GalDH and leucyl amino peptidase had lower expression in tea plants, but APX, glutathione S-transferase and glutathione exhibited an opposite trend (Fig. 8a), which implied that exogenous GABA application might regulate AsA and glutathione metabolism by different mechanisms. Sensitive genes in amino acid metabolism and the AsA/glutathione cycle had their relative expressions determined using RT-qPCR (Fig. 8b) and these were consistent with the related DAPs.

TCA cycle, glyoxylate cycle and carbon fixation in photosynthetic organs and GABA shunt and exogenous GABA at low temperature

The TCA cycle includes important approaches to plant synthetic amino acid, energy supply and different biological movement course [59]. Malate dehydrogenase (MDH) catalyzes the effect of NAD+ on NADH oxidation of malic acid was reproduced in mitochondrial matrix during TCA cycle [60]. Environmental stresses including drought, heat, salinity and aluminum can decrease the MDH level in various plant species [61,62,63]. Exogenous GABA application at low temperature resulted in lower MDH level compared with the control, suggesting that exogenous GABA improved the low-temperature-induced reduction in MDH to catalyze the inhibited malate to oxaloacetate during malate metabolism. All of the DAPs in the TCA cycle showed greater down-regulation in treatment T4 compared to T3 (Fig. 9a).

Effect of application of exogenous GABA at low temperature on (a) DAPs and (b) RT-qPCR analysis of genes related to TCA cycle, glyoxylate cycle and carbon fixation in photosynthetic organisms. Transcript abundance was calculated according to the difference in cycle threshold values between the target gene and β-actin transcripts normalized by the 2 −ΔΔCT method. The mRNA levels of the genes in tea leaves at 0 h were set as 1.0. Data represent the mean value ± standard deviation. Means with different letters significantly differ from each other (p ≤ 0.05). Abbreviations: AGT1: alanine-glyoxylate transaminase FBA2: fructose-bisphosphate aldolase 2 GAPB: glyceraldehyde-3-phosphate dehydrogenase B subunit GDH3: glutamate dehydrogenase 3 GGAT2: glutamate--glyoxylate aminotransferase 2-like GLDP2: glycine decarboxylase P-protein 2 GLO1: glyoxalase NADP-ME4: NADP-malic enzyme 4 PDH-E1: pyruvate dehydrogenase E1 PMDH1: peroxisomal NAD-malate dehydrogenase 1 RBCL: ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit RBCS-2B: Ribulose bisphosphate carboxylase small chain 2B SDH1–1: succinate dehydrogenase 1–1

In addition, MDH participates in amino acid synthesis because of the reactions of malic acid, acetoacetic acid and aspartic acid [64]. With the significant decrease in MDH and succinate dehydrogenase and increases in aspartic acid, the contents of threonine, isoleucine, lysine, alanine, valine and serine were elevated with exogenous GABA application at low temperature. It was reported that the levels of alanine, valine and serine were affected by the stimulation of elevated endogenous GABA under abiotic stresses: Bermuda grass under heat stress [65] and tobacco under salt [66]. The GABA shunt was considered to be a part of the TCA cycle during respiration [67,68,69], which is also important in primary carbon and nitrogen metabolism [70]. Increased GABA promoted the alanine substance accumulation, which entered the proline metabolism and TCA cycle. All amino acid contents except for glycine were increased by elevated GABA at low temperature, which suggested that GABA increased the carbon and nitrogen metabolism. However, the proteins related to the GABA shunt showed no significant changes, but the GABA precursors and the enzyme activity of GABA metabolism clearly changed.

Glyoxylate cycle was firstly discovered in microorganisms, which provided substrates for biosynthetic processes and respiration [71]. In addition to lipids, glyoxylate cycle is another carbon source necessary for growth after germination [72]. Most of the proteins involved in the glyoxylate cycle were down-regulated in response to low temperature with exogenous GABA compared to the control. Furthermore, the exogenous GABA could relieve the cold damage to photosystem II, which was crucial in carbon metabolic [73]. There are many signaling molecules, such as nitrate, ammonium, sugar, amino acids and organic acids, also contain interactions between carbon and nitrogen metabolism [74]. Nitrogen uptake and metabolism is contributed by carbon metabolism since it requires carbon skeletons, reducing power, ATP, reductants and co-transporters. [73, 75,76,77]. Their control may be closely linked and coordinated at the level of gene expression [78]. However, only aminomethyl transferase and fructose-bisphosphate aldolases (FBAs) were up-regulated in the TCA cycle, glyoxylate and dicarboxylate metabolism, and carbon fixation in photosynthetic organisms in treatment T4 compared to T3. Fructose 1,6-bisphosphate aldolases, which are important in the Calvin-Benson cycle (CBC), were dramatically altered when tomato seedlings suffer from heat/cold stresses [79]. This might imply that exogenous GABA application could improve photosynthesis to regulate low-temperature response through FBA expression and enzyme activity.

The above evidence suggests that, as a signal molecule, GABA regulates the glyoxylate cycle, the TCA cycle and carbon fixation in photosynthetic organ metabolism in a complex manner, especially in the carbon and nitrogen cycle. It will be interesting to establish how tea plants respond to low temperature via different mechanisms and how the signal molecule GABA influences the operating mechanisms. Several genes in the TCA cycle, glyoxylate cycle and carbon fixation in photosynthetic organisms had their relative expression determined by RT-qPCR (Fig. 9b) and the expression mode of genes was similar to the response of related DAPs.

Oxidative pentose phosphate pathway and purine metabolism and exogenous GABA at low temperature

The energy and metabolic intermediates in the biosynthesis are mainly derived from the pentose oxidation pathway. However, further details are needed on how the pathway and its effects on other processes in plants. Nitrate is the major source of nitrogen that plants need, and the location of nitrate assimilation significantly affect the plant energy budget [73]. This implies that exogenous GABA application could affect the energy budget in tea plants. Only FBA in the oxidative pentose phosphate pathway was up-regulated in treatment T4 compared to T3 here. Lu et al. [80] reported that all FBA genes in Arabidopsis thaliana, except AtFBA6, were up-regulated in response to cold stress. The carbon skeleton of the amino acid synthesis pathway arises from different sectors of the respiratory pathway. Some root in the oxidative pentose phosphate pathway (or, in the light, the CBC) and glycolysis (Ery4P and PEP for the synthesis of aromatic amino acids), some stem from the final product of glycolysis (pyruvate for alanine) and other organic acids from the TCA cycle [73]. For treatment T4 compared to T3, FBA was not only in glycolysis cycle but also in CBC, FBA2 and FBA3 genes were up-regulated, but the DAPs in the TCA cycle were down-regulated. Testing how the application of exogenous GABA in tea plants affects the respiration pathway of amino acid biosynthesis through reverse genetic pathway under low temperature, is further needed.

Purine nucleotides are produced by two distinct routes in plants: de novo and salvage pathways. The de novo synthesis employs 5-phosphoribosyl-1-pyrophosphate, aspartate, glycine, glutamine, HCO 3− and 10-formyltetrahydrofolateas for building blocks, which could be found in all plants, such as tea-leaves however, the salvage pathways are more diverse and less well understood [81, 82]. Tea plants engender special nitrogen compounds including theanine and caffeine and their effects on human health have been studied in detail [83]. In order to against herbivores and pathogens, young shoots will accumulate caffeine and leaves may be treated as a chemical defense of young soft tissue [84]. All the DAPs in purine metabolism, except ureidoglycolate amidohydrolase, were up-regulated (Fig. 10a).

Effect of application of exogenous GABA at low temperature on (a) DAPs and (b) RT-qPCR analysis of genes related to oxidative pentose phosphate pathway and purine metabolism. Transcript abundance was calculated according to the difference in cycle threshold values between the target gene and β-actin transcripts normalized by the 2 −ΔΔCT method. The mRNA levels of the genes in tea leaves at 0 h were set as 1.0. Data represent the mean value ± standard deviation. Means with different letters significantly differ from each other (p ≤ 0.05). Abbreviations: ALDH1: Aldehyde dehydrogenase CYFBP: Fructose-1,6-bisphosphatase FBA3: fructose-1, 6-bisphosphate aldolase

Thus, the evidence suggests that exogenous GABA application either directly or indirectly stimulated flux into amino acid and caffeine biosynthesis and regulated the plant energy budget. This affected the resistance to cold in tea plants and the quality of tea flavor through the oxidative pentose phosphate pathway and purine metabolism. Key genes in the oxidative pentose phosphate pathway and purine metabolism were selected for RT-qPCR, which showed that expression mode of genes were similar to that of related DAPs (Fig. 10b). Above all, the effects of exogenous GABA at low temperature on physiological index and DAPs in metabolism pathways, were summarized on Fig. 11.

Effect of exogenous GABA at low temperature on physiological index and DAPs in metabolism pathways. Models of physiological index and possible metabolism pathways by exogenous GABA compared to that without GABA under low temperature in the tea plants. Antioxidant activities had no significant difference (green), Polyamine contents increased slightly (blue), while other index and the metabolism pathways above were affected by exogenous GABA (red)


Discussion

The identification of phytochemicals in herbal materials is a critical step during the process of system biology analysis. Herbal materials are often subjected to extraction, concentration, and/or purification, resulting in the phytochemical compositions alteration. The phytochemical data from current databases (e.g., TCMSP, http://tcmspw.com/tcmsp.php TCMID, http://www.megabionet.org/tcmid/) may not be used directly for system biology investigation. Additional methods for phytochemical identification, such as UPLC-Q-TOF/MS, should be a complementary tool to obtain more accurate results of phytochemical compositions (Shen et al., 2013). In current work, a series of flavonoid glycosides and saponins were identified from SZJ extract, in which the spinosin derivatives including 6’’’-vanilloylspinosin, 6’’’-para-hydroxylbenzoylspinosin, 6’’’-sinapoylspinosin, 6’’’-para-coumaroylspinosin, 6’’’-feruloyspinosin, 6’’’-(-)-phaseoylspinosin, and 6’’-O-feruloylspinosin are rare in other plant species. The studies on their bioactivities and effective targets have been so poorly reported that there is not enough data for system biology analysis. In addition, their chemical structures are complicated and contain multi-chiral centers that bring a great challenge to obtain the potential targets through reverse virtual fishing technique. Traditional system biology analysis would take ADME (absorption, distribution, metabolism, and excretion) screening strategy that may exclude these glycosides with low oral bioavailability and low drug-likeness (Yue et al., 2017a Yue et al.,�). For instance, ginsenosides are the dominant phytochemicals in ginseng and are thought to be contributed to its multiple bioactivities (Ru et al., 2015 Kim et al., 2017). However, the above analysis strategy, i.e., ADME screening, would exclude the ginsenosides along with their contributions on efficacy when performing systematic research of ginseng. Therefore, such an analytical strategy is incomplete and not systematic. In fact, it has been well demonstrated that the metabolites of the ginsenosides are responsible for the specific bioactivities (Chen et al., 2018 Kim, 2018). Similarly, the chemoinformatics and pharmacoinformatics approach indicated that jujubogenin was the effective GABAA agonist, neither jujuboside A nor jujuboside B (Chen, 2009). Gut microbes play an important role in favoring phytochemicals transformation into metabolites endowed with biological activity (Dey, 2019). As a result, the strategy that involves the metabolites of glycosides in gastrointestinal environment (e.g., gut microbes) will be a more reasonable approach to understand the actual efficacy and mechanism of herbal materials in which the glycosides are considered as the main active components.

The GABAA receptors are chloride channels and are composed of several subunit classes (α, β, γ, δ, and ϵ) (Olsen and Sieghart, 2008). GABAergic neurotransmission plays an important role in anxiety status. Previous studies have shown that deficit of GABAA receptors and reduction of GABA transmission were observed in people with anxiety-like symptoms (Horowski and Dorow, 2002 Nutt and Malizia, 2004 Hasler et al., 2008). In contrast, positive modulation of GABAA receptors and enhancement of GABA transmission have shown anxiolytic effects. Classic benzodiazepines reduce anxiety by interacting with the GABAA receptors via the benzodiazepine binding site, which is present at the interface of 㬑, 㬒, 㬓, or 㬕 subunits and γ subunit of GABAA receptors (Möhler, 2012). Other classes of compounds, GABA, barbiturates, and alcohol also could act at different benzodiazepine binding sites to increase the opening of the chloride channel resulting in enhancement of inhibitory synaptic transmission (Harris, 1990 Schousboe and Redburn, 1995). The results of our system biology study suggested the GABAA receptors signaling is a significant pathway involving in anxiolytic effect of SZJ. In fact, pharmacologic study has found that spinosin, a major C-glycoside flavonoid in SZJ, exerted anxiolytic-like effects via modulation of GABAA and 5-HT receptors (Liu et al., 2015). Similarly, 6′′′-feruloylspinosin and spinosin have been reported to significantly enhance the expression of GABRA1 and GABRA5 mRNA in rat hippocampal neurons (Qiao et al., 2016). In addition, it has been found that stimulation of jujuboside A at 50 µg/mL could increase the mRNA transcription levels of GABRA1, GABRA5, GABRB1, and GABRB2 in hippocampal neurons (You et al., 2010 Wang et al., 2015) however, long time stimulation of jujuboside A at a high dose of 100 µg/mL result in the decrease of GABRA1 and GABRB2 mRNAs expression (You et al., 2010). These results suggested a two-way modulatory effect of SZJ on GABRA1 and other GABAA receptors. Such benefits were similar to what we found in this work, that is, SZJ extract enhanced mRNA level of GABRA1 in non-H2O2 treated SH-SY5Y cells, but inhibited the H2O2-induced overexpression of GABRA1. Therefore, combining with the results from literatures and our results, it was suggested that SZJ exhibited anxiolytic effects through modulating GABAA receptors, in which a two-way modulation of GABRA1 may play an important role.

It was well established that the alteration of various behaviors in anxiety disorders including appetite, mood, sleep, and cognitive function have been linked to the serotonergic system (Liu Y. et al., 2018 Liu et al., 2019). Serotonin receptors are prevalent throughout the nervous system and the periphery, and they potentially control the serotonergic neurotransmission throughout the brain and neuronal activity to alleviate neuropsychiatric disorders (Okazawa et al., 1999). Generally, the activation of HTR1A, HTR2A receptors can produce anxiolytic effects, whereas inactivation of them increases anxiety-like behaviors (Clinard et al., 2015 Spiacci et al., 2016). Involvement of other 5-HT receptors including HTR1B, HTR1B, and HTR2C in the mechanisms of anxiety have also been recognized (Graeff et al., 1996 Griebel et al., 1997 McCorvy and Roth, 2015). Similarly, our system biology analysis found that the serotonergic synapse pathway was dominant in anxiolytic effects mechanism of SZJ extract, in which different subtypes of 5-HT receptors were involved. Notably, as the same effect on GABRA1, SZJ extract also showed a two-way modulation on HTR1A and HTR2A in our RT-qPCR test. Genetic studies in animal models have suggested that anxiety-like behavior can increase when the HTR1A function is eliminated or overexpressed (Overstreet et al., 2003). Hence, these results suggested the involvement of modulating serotonergic synapse pathway, specifically two-way modulation of HTR1A and HTR2A in anxiolytic effects of SZJ.

In addition, the cannabinoid receptors (CNR) are extensively expressed in areas of the nervous system and have been found closely associated with anxiety behavior (Akirav, 2011). It has been well illustrated that endocannabinoid (eCB) reduces the serotonin release in the central nervous system and increases the expression and function of HTR1A in the hippocampus via the activation of CNR1 (Haj-Dahmane and Shen, 2011 Patel et al., 2017). Beyond CNR1, eCB system could exert actions on other targets including CNR2, transient receptor potential vanilloid receptor type 1 (TRPV1), or cyclooxygenase-2 (COX2) to participate in improvement of anxiety (Patel et al., 2017). In addition, cyclic AMP-responsive element-binding protein (CREB) has been suggested to be crucial for the role of HTR1A in modulating anxiety-related behaviors via mediating hippoacampus structural plasticity (Zhang J. et al., 2016). Intriguingly, this systematic analysis work showed phytochemicals in SZJ extracts potentially act on the above mentioned targets including CNR1, CNR2, TRPV1, COX2, and CREB. These results, to some extent, suggested that the mechanism of action of SZJ in anti-anxiety may also involve those pathways/targets that indirectly modulate eCB and serotoninergic systems. More attention needs to be paid to those targets/pathways in further experimental studies on anxiolytic effects of SZJ.

The phytochemicals in herbal medicines are the substantial basis for their pharmacologic actions. Those phytochemicals with good bioactivity and high content are considered to be the chemical markers in quality control of the herbal medicines. Jujuboside A and spinosin are used to quality markers of SZJ crude drug in Chinese Pharmacopoeia (Edition 2020). Combining the results from literatures reports (Han et al., 2009 Abdoul-Azize, 2016) and our results, the modulations of GABAergic and serotoninergic systems seem the major mechanisms of SZJ exerting anxiolytic effects, as well as the traditional efficacy of nourishing heart and calming mind. Based on that, we abstracted the phytochemicals-targets-pathway sub-networks of GABAergic and serotoninergic synapse pathways. As shown in Figure 7, it demonstrated that metabolites of C-glycosides (spinosin, etc.) and jujubosides (jujuboside A, etc.) including apigenin, kaempferol, naringenin, genkwanin, and jujubogenin were involved in modulation of GABAergic and serotoninergic synapse pathways. The result provides the extra evidence to support that C-glycosides and jujubosides are responsible for the anxiolytic effects of SZJ, and they support jujuboside A and spinosin as chemical markers for quality control of SZJ and its preparations. Beyond the C-glycosides and jujubosides, the involvements of triterpenic acid (e.g., betulinic acid) and alkaloid (zizyphusine) were also observed in modulation of GABAergic and serotoninergic synapse pathways. Specifically, betulinic acid is a function of the modulating GABAergic system via multiple subtypes of GABAA receptors, whereas zizyphusine is a function of the modulating serotoninergic system via multiple subtypes of 5-HT receptors. Notably, it has been reported that zizyphusine was identified as one of the principal components in SZJ by principal component analysis (Sun et al., 2014). And according to records in TCMIP, zizyphusine is exclusively derived from Ziziphus jujube (fruit or seeds), and its bioavailability and druglikeness is much better than C-glycosides and jujubosides. These findings recommend that the involvement of zizyphusine in quality control of SZJ extract and pharmacologic actions in anxiolytic effect is worth investigating in the future. Because the pharmacologic study of zizyphusine is poor at present, more attention could be paid to research and development of zizyphusine as a potential natural anti-anxiety drug.

Figure 7 Abstracted phytochemicals-targets-pathway sub-networks of serotoninergic synapse (A) and GABAergic pathways (B). Cytoscape (Version 3.6.1) was applied to construct the sub-networks.

There are some limitations of our current research. First, we did not conduct the behavioral test to confirm the anxiolytic-like effect of SZJ. Such benefit of SZJ was concluded on the base of previous pharmacodynamic studies, as well as clinical practice experience of traditional medicine. Our in vitro mRNA expression evaluation of GABAA and 5-HT receptors only used a single concentration of SZJ extract, which corresponds to approximately 90% cell viability in CellTiter-Glo assay. Leveraging the integrated approach of system biology, UPLC-Q-TOF/MS and RT-qPCR, present work contributed to the illustration of potential mechanism of action involved in the anxiolytic-like effect of SZJ. However, further in-depth preclinical studies are warranted to verify the results obtained from the current analysis.


GABA Supplements

Currently gamma aminobutyric acid is commercially available as a dietary supplement, both natural and synthetic. Natural GABA is created by a fermentation process that uses a bacteria called Lactobacillus hilgardii.

Many people consume it to sleep better and decrease anxiety. It is also famous in athletes, as it seems to contribute to fat loss and to the development of muscle mass.

This is because it produces an intense increase in growth hormone, which is critical for muscle. In addition, it allows better sleep, something that those who do bodybuilding need.

However, the use of this supplement is subject to controversy. Many believe there is a lack of scientific evidence about its benefits.

In addition, it appears that it is difficult for GABA in blood to cross the blood-brain barrier to reach the brain. Therefore, it could not act on the neurons of our nervous system.


GABA-mimetic effect on neurodegeneration and neuroregenerative potential

Behavioral experiments have lent support to the GABA-mimetic activity of Ashwagandha root extract. GABAergic neurodegeneration due to neuroleptic-induced excitotoxicity and oxidative stress is one of the etiopathological mechanisms in the pathophysiology of tardive dyskinesia (Gunne et al., 1993) and GABA agonists are shown to be effective in ameliorating the symptoms of tardive dyskinesia. The beneficial effect of Ashwagandha root extract might be due to its GABA mimetic activity. Ashwagandha, its constituents and the metabolites of its constituents promote the growth of nerves after taking it for 7 days.

An intriguing study demonstrated that chronic oral administration of withanoside IV attenuated the axonal, dendritic and synaptic losses and memory deficits induced by amyloid peptide Aβ(25�) in mice (Kuboyama et al, 2006). After oral administration in mice, withanoside IV was metabolized into sominone, which induced marked recovery in neurites and synapses and also enhanced axonal and dendritic outgrowth and synaptogenesis. These effects were maintained for at least 7 days after discontinuing withanoside IV administration. These data suggest that withanoside IV, and its metabolite, sominone, may have clinical usefulness as antidementia drugs.

Another team found that the methanol extract of Ashwagandha (5 mg/ml) significantly increased the percentage of cells with neurites in human neuroblastoma SK-N-SH cells. The effect of the extract was dose-and time-dependent. mRNA levels of the dendritic markers MAP2 and PSD-95 by RT-PCR were found to be markedly increased by treatment with the extract. Immunocytochemistry demonstrated the specific expression of MAP2 in neurites extended by the extract. These results suggest that the methanol extract of Ashwagandha promotes the formation of dendrites (Kulkarni et al., 1993).


Aluminium Toxicity and Its Tolerance in Plant: A Review

Aluminium (Al) toxicity is one of the major abiotic stress problems around the globe where acidic soil is present. Al shows a toxic effect between the soil pH 4.5 and 5.5. Root growth inhibition is the most prodigious symptom of Al toxicity in plants. Aluminium toxicity adversely affects the plant growth and development which ultimately reduces the yield. However, the extent of toxicity depends on the genotype of the plant, that is the plant is either the Al-sensitive or Al-tolerant type. Plants have several mechanisms to cope with the toxic effects of aluminium which include exclusion mechanism and internal tolerance mechanism. This review discusses the harmful impacts of aluminium on morphological, anatomical, physio-biochemical, and molecular aspects of the plant. This review also discusses the strategies to reduce the toxic effects of aluminium in plant and various aluminium-responsive genes which can be used in genetic manipulation for better crop development.

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Watch the video: Δημήτρης Κούβελας Γιατί με διώκει ο εισαγγελέας Ο Βασιλακόπουλος ούτε 10 για διαδικασίες φαρμάκων 48 (May 2022).


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