Plant Vacuole Biogenesis / Bioengineering , their roles, their models and functions

Plant vacuole biogenesis, their roles and models

1. Source: Google.

2. Cui et al. (2020)

3. Cui et al. (2020)

4. UNIVERSITY OF AGRICULTURAL SCIENCES, BANGALORE ADVANCED CENTER FOR PLANT BIOTECHNOLOGY Course: MBB 682 (0+1) Seminar on Vacuole Biogenesis/ engineering in plants their functions, roles and their models Vimanth, S. II Ph.D. (MBB) Seminar 2

5. How it is studied ?

6. How it is studied ? Photoconvertable techniques - BCECF: A pH-Sensitive Fluorescent Dye stands for 2’,7’-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Serial Block Face - Scanning electron microscope (SBF-SEM) 3D Confocal imaging of HPF samples Electron microscopy technology Cryo-electron tomography (Cryo-ET) analysis on cryo-focused ion beam (Cryo- FIB) Shimada et al. (2018)

7. Shimada et al. (2018)

8. Shimada et al. (2018)

9. Formation of Protein Storage Vacuoles (PSV) Shimada et al. (2018)

10. Shimada et al. (2018)

11. Formation of Lytic Vacuoles (LV) Shimada et al. (2018)

12. Shimada et al. (2018)

13. Shimada et al. (2018)

14. Cui et al. (2020)

15. How the soluable proteins , lipids and metabolites are transported into the vacuoles ……?

16. Mechanism of vacuolar Trafficking pathway

17. Mechanism of vacuolar Trafficking pathway Golgi dependent transport pathway Golgi- independent transport pathway

18. Golgi dependent transport pathway

19. The kiss-and-run model considers that moving Golgi apparatus pause and transiently associate with stable ER exit sites dispersed over the ER; then, cargo is transported from the ER to the Golgi apparatus during this transient association.   The secretory unit model considers that moving Golgi apparatus continuously associate with ER exit sites , which facilitates ER export even to moving Golgi stacks. The hybrid model integrates the kiss-and-run and secretory unit models and considers that some ER exit sites are associated and move together with Golgi apparatus , whereas others are independent of the Golgi apparatus Cui et al. (2020)

20. Nucleus Golgi Vacuole

21. ER exit site Ribosomes Sec 12 Gtpase SAR1 GDP COP II TRAPP I

22. ER exit site

23. ER exit site P115 Rab1 gtpase

24. ER exit site P115 Rab1 gtpase Retrograde pathway for recycle of COPII and ER receptor molecules

25. Nucleus Golgi Vacuole

26. Golgi Vacuole TGN

27. Golgi Vacuole TGN

28. Golgi Vacuole TGN

29. Vacuole

30. Vacuole VSR - Type I Integral membrane receptor ·  l arge luminal domain (approximately 550 amino acids) followed. The VSR luminal domain can be divided into three subdomains: protease-associated domain at the N terminus, central domain in the middle, and three EGF repeats at the C terminus. Vacuolar- sorting signals bind the protease-associated and central domains ·    single transmembrane domain ·     short cytoplasmic tail (approximately 40 amino acids).

31. Golgi in-dependent transport pathway Cui et al. (2020)

32. Now the protein is transported inside the vacuole now what ?

33. Vacuolar Processing enzyme V PE are cysteine proteinase, which is having homology mammalian VPE (asparagine enopeptidases), which has critical role to play in endosomal/ lysosomal degradation. VPE is synthesized as an inactive precursor and then activated by self-catalytic removal of an autoinhibitory domain of the C-terminal propeptide , indicating that VPE is a key enzyme for the maturation and activation of vacuolar proteins.

34. VACUOLAR MEMBRANE DYNAMICS INVOLVED IN DEFENSE RESPONSES Two types of vacuolar membrane dynamics that occur during defense responses: ·      Vacuolar membrane collapse to attack viral pathogens ·      Fusion of vacuolar and plasma membranes to attack bacterial pathogens.

35. Disruption of the vacuolar membrane releases the vacuolar lytic enzymes and induces hypersensitive cell death. Lytic vacuoles mediate programmed cell death (PCD) in plants. Because plants lack immune cells, each plant cell must defend itself against invading pathogens. Essentially, all vegetative cells have lytic vacuoles containing hydrolytic enzymes and defense proteins Vacuolar membrane collapse to attack viral pathogens Cui et al. (2020)

36. I t does not prevent bacteria from proliferating outside cells . Plants have evolved a cell- autonomous immune system based on vacuole–plasma membrane fusion to inhibit the proliferation of bacterial pathogens Fusion of the vacuolar and plasma membranes leads to the interconnection of vacuoles and extracellular spaces in leaf cells, which enables the discharge of vacuolar defense proteins into the extracellular space where bacteria proliferate. The extracellular fluid surrounding cells in infected leaves had both antibacterial and cell death–inducing activity. Fusion of vacuolar and plasma membranes to attack bacterial pathogens. Cui et al. (2020)

37. Shimada et al. (2018)

38. Case Study - 1 HVP10 A.K.A as HvNaX - mainly upregulated during salt stress, this HVP10 gene performs sequestration of ions into root vacuoles.

39. HVP10_gene Transporter protein

40. HVP10_gene Transporter protein

41. High Low

42. High Low

43. Materials & Methods Golden Promise Var. Rice Barley Nipponbare cv. HVP10 Knockdown Knockout Overexpression 367 bp 2 sg RNA 4369 bp > 2289 bp (8E & 7I)

44. Results RNAi / (Knockdown) Two weeks old barley was subjected to 200mM NaCl solution. Salt stress condition indicates reduced FW, DW, RWC in RNAi Lines (L3,L5, L7) Fu et al. (2022)

45. Crispr-Cas9 / Knockout Sodium flux was greatly reduced as gene was made Knockout, as HVP10 cannot sequestrate sodium ions into root vacuoles estimated by using (MIFE) Fu et al. (2022)

46. Overexpression study in Rice Fu et al. (2022)

47. Conclusion of case study - 1 RT-qPCR and MIFE analysis showed that the gene HVP10 greatly expressed in root region at high salt conditions for sodium ion sequestration. In knockdown mutant (RNAi) 52%, 30% and 11% reduction of SFW, DW and RWC in comparison with wild type under salt stress. In Knockout mutant (Crispr-Cas9) as there was no gene for sodium sequestration, in 2 knockout lines H ions efflux was drastically reduced, In rice over expression lines, they found out that increased seed set rate, and increased grain yield than compared to wild type under salt stress condition.

48. Case Study - 2 Cynovirin -N.

49. Viable alternative Advantages:- scalability; low production cost free of human pathogens, microbial toxins, prions, or oncogenic sequences. plant cellular machinery can biosynthesize, secrete, fold, assemble complex proteins, compartmentalize, and promote eukaryotic post-translational modifications, resulting in high-quality recombinant products, keeping its therapeutic functions. Plant Farming

50. Agarwal et al. (2020)

51. The natural ability of seeds to accumulate proteins endows them with great potential to be an alternative for the biosynthesis and storage of recombinant proteins. Seeds often present an environment with relatively little water, protected from the action of proteolytic enzymes, with a pH close to neutral and a set of organelles capable of stably storing large amounts of proteins , unlike in leaves high moisture and low stability of recombinant products. Why they have selected seeds as protein storage organ ?

52. Why Soybean Plant ?

53. Why Soybean Plant ? Legume Crop Photosensitive crop Soybean used as Bioreactor, increase Stability, Scalability and productivity

54. Examples :-

55. Cynovirin-N Source : Google

56. 80-95% proteins (Glycinin and B-conglycinin) Glycinin and B-conglycinin

57. 80-95% proteins (Glycinin and B-conglycinin) Glycinin and B-conglycinin

58. 80-95% proteins (Glycinin and B-conglycinin) Glycinin and B-conglycinin Heterologous protein coding sequence

59. Vianna et al. (2023)

60. Vianna et al. (2023)

61. Vianna et al. (2023)

62. Conclusion of case study - 2 Harnessed a way for potential production of heterologous protein. Use of PSV as a storage organs for heterologous protein, thus increased scalability, stability without losing its viability. 350ug/g of seed dry weight, that is production of 1 kg of CY-N in an area of 1524 square.meter.

63. Future prospects…..! Vacuole are evolutionarily conserved necessary organelle for all Plants across genus. Since, vacuole doesn’t bear there own genome unlike mitochondria and chloroplast, bioengineering on them will be relatively difficult. The only way scientists can play is to manipulate the transporters proteins that are present on the tonoplast membrane.

64. References :- AGARWAL, R., TRIVEDI, J. AND MITRA, D ., 2020, High yield production of recombinant cyanovirin-N (antiviral lectin) exhibiting significant anti-HIV activity, from a rationally selected Escherichia coli strain.  Process Biochem. ,  93 :1-11. CUI, Y., ZHAO, Q., HU, S. AND JIANG, L. , 2020, Vacuole biogenesis in plants: how many vacuoles, how many models?.  Trends Plant Sci. ,  25 (6):538-548. FU, L., WU, D., ZHANG, X., XU, Y., KUANG, L., CAI, S., ZHANG, G. AND SHEN, Q., 2022, Vacuolar H+-pyrophosphatase HVP10 enhances salt tolerance via promoting Na+ translocation into root vacuoles.  Plant Physiol. ,  188 (2). SHIMADA, T., TAKAGI, J., ICHINO, T., SHIRAKAWA, M. AND HARA-NISHIMURA, I., 2018, Plant vacuoles. Annu.   Rev.   Plant Biol . ,  69 (1):123-145. VIANNA, G.R., CUNHA, N.B. AND RECH, E.L ., 2023, Soybean seed protein storage vacuoles for expression of recombinant molecules.  Process Biochem. ,  71 .

65. Thank You…..