FUNCTIONS OF AUTOPHAGY IN CROP PLANTS

1. Self-devouring
AUTOPHAGY

2. AUTOPHAGY
IN
CROP PLANT
ABHISHEKA L S
ID No: PGS17AGR7449
Snr. M.Sc., (Agri.)
Genetics and Plant Breeding
UAS, Dharwad

3. INDEX:
1
Introduction
Types of autophagy in plants
History
Autophagy Machinery
Mechanism of autophagy in plants
Functions of autophagy in plants
Future prospects
Conclusion

4. AUTOPHAGY
 Autophagy (Greek word) - “Self-eating”
 Autophagy : Macromolecule degradation pathway that
recycles damaged or unwanted cell materials upon
encountering stress conditions or during specific
developmental processes
 Machinery required for autophagy seems to be conserved from
yeast to plants
2
Tang and Bassham, 2018

5. History:
 Christian de Duve - (1963): Coined the term
„Autophagy‟
“Founding father of autophagy research”
 Yoshinori Ohsumi - (1992): Identified yeast
AUTOPHAGY-RELATED GENE (ATG),
peroxisome turnover and cytoplasm-to-
vacuole targeting pathway (CVT)
He was awarded the Nobel Prize in
„Physiology or Medicine‟ in 2016
 Per Ottar Seglen: Characterize the cup-shaped
structure - phagophore
Frake and Rubinsztein, 2016
3

6. Characterisation of autophagy in yeast:
 Demonstrated that autophagy in yeast
is similar to that in mammalian cells
 The group used electron microscopy to
identify and characterize double-
membraned „autophagosomes‟ as the
precursors of „autophagic bodies‟ in
yeast
4

7. Autophagy pathway in mammalian cells and yeast.
5

8. Continues…,
 Ohsumi and colleagues described Atg16p as the third member of
the Atg5p-Atg12p complex
 Two years later, a second ubiquitin-like conjugation system was
discovered with Atg8p as the ubiquitin-like protein
6

9. Types of autophagy in plants:
1. Micro-autophagy: Cytoplasmic material congregates the vacuole
surface and becomes trapped by invagination of the tonoplast.
Then tonoplast undergoes scission to release autophagic bodies.
2. Macro-autophagy: Cargo is trapped in cytoplasmic vesicles arised
by expansion of a cup-shaped phagophore that encircles
cytoplasm and ultimately seals to generate the double
membrane-bound autophagosome.
Marshal and vierstra, 20187

10. 3. Mega – autophagy: The tonoplast ruptures to release vacuolar
hydrolases directly into the cytoplasm, where they degrade
cytoplasmic material in situ.
It often represents the final stage of programmed cell death
“The most extreme form of autophagy”
Type of autophagy in animals:
Chaperone-madiated autophagy: Independent of vesicles
Marshal and vierstra, 2018
Continue…,
8

11. The Autophagy Machinery:
schematic representation of steps in autophagy
Marshal and vierstra, 2018
9
(Active
complex)

12. Continue…,
Marshal and vierstra, 2018
10

13. Selective autophagy:
“It is an autophagic process that degrades specific
cytoplasmic components such as protein complexes, aggregates,
organelles and pathogens”
Types of selective autophagy:
1. Aggrephagy: Misfolded protien aggregates are degraded by a
specialized autophagic route.
2. Chlorophagy: Quality control mechanism to eliminate
nonfunctional chloroplasts.
3. Mitophagy: Mitophagy is best described in mammals, there is
no clear orthologs in plants.
Marshal and vierstra, 2018
11

14. Continues…,
4. Pexophagy: Under nonstress conditions, pexophagy limits plant
peroxisome abundance.
5. Ribophagy: Biogenesis of ribosomes and subsequent protien
translation are the most energy consuming cellular processes, so
under limited amino acids condition to check translation process
ribophagy will occur.
6. Proteaphagy: Degradation of ubiquitylated protiens via the Ub-
proteasome system.
7. Xenophagy: It helps in enhancement of innate immune response
to protect themselves from pathogen attack.
Marshal and vierstra, 2018
12

15. Regulation of autophagy in plants:
 Gene TOR kinase
 Yeast - two TOR genes
 Plants – only one TOR gene
 Antibiotic Rapamycin inhibits
TOR kinase
 A key regulator of nutrient
stress induced autophagy.
Two different functional complexs
of TOR:
1. TORC2: Controls spatial cell
growth
2. TORC1: Controls temporal
cell growth, negetively
regulating autophagy. Liu and Bassham, 2012
13

16. Methods for monitoring autophagy:
 Immunoblot assays following SDS-PAGE: Here both the
conjugation of ATG12 - ATG5 and the lipidation of ATG8 can
be easily detected based on changes in electrophoretic mobility
Marshal and vierstra, 2018
14

17. Continues…,
• Chemical inhibitors: Blocks the v-ATPases on tonoplast
responsible for vacuolar acidification
 Inhibitors enhances the pH of the vacuolar enzymes that
suppresses autophagic body breakdown
 Enhances microscopic detection of autophagic bodies and
stabilizes their contents
Marshal and vierstra, 2018
15

18. List of chemical inhibitors Marshal and vierstra, 2018

19. Continues…,
 Fluorescent reporters: Helps in both visual detection of
autophagic structures, and quantitative measures of autophagic
flux
Example: GFP-ATG8 fusions permit detection of autophagosomes
within the cytoplasm and autophagic bodies within the
vacuole by confocal fluorescence microscopy
Marshal and vierstra, 201817

20. Identification and characterization of ATG genes:
ATG genes play key role in autophagy induction
To date, core ATG genes have been identified in 14 crop species
Sequences of ATG genes from Arabidopsis and rice were used as
queries to search against corresponding genomic sequences for
most of 14 crop species
ATG proteins in crops are typically encoded by a single gene
No homologues of ATG3 have been found in grapevine
Autophagy have conserved functions among distinct species but
may also perform species-specific roles Tang and Bassham, 2018
18

21. Autophagy genes in crop species Tang and Bassham, 2018
19

22. Crop species with identified ATG genes and potential processes that require
autophagy Tang and Bassham, 2018
20

23. Leaf
senescence
Seed
development
Reproductive
development
Vascular
development
Nutrient
starvation
Drought
stress
Heat stress
Plant-microbe
interaction
Autophagy
Functions of autophagy in crop plants:
Tang and Bassham, 2018
21

24. Leaf senescence:
 Leaf senescence is considered to be an important developmental
process because of its critical role in remobilizing nutrients from
mature leaves to support developing organs (Ex: seeds)
 The involvement of autophagy in senescence
is supported by upregulation of the ATG
transcripts in older leaves
 Higher accumulation of lipidated ATG8 causes higher level of
autophagy activity
Example: Arabidopsis, 15 ATG genes are upregulated during
senescence. Tang and Bassham, 2018
22

25. Seed development:
 Increased transcript abundance of many ATG genes can be
observed in the endosperm
 Accumulation of ATG8-PE adducts in endosperm will enhance
the seed size
 Autophagy helps in the transport of seed storage proteins
 Breakdown of starch granules during seed germination is
associated with the autophagy pathway
Example: Wheat, electron microscopy showed that prolamins were
transported from the ER to protein storage vacuoles
(PSVs) through an autophagy-like pathway.
Tang and Bassham, 2018
23

26. Reproductive development:
The first evidence connecting autophagy to reproductive
development was found in wheat
The genes ATG7 and ATG9 will cause autophagy-mediated PCD
in tapetum cells leads to nutrient remobilization during pollen
development
 Epidermal cells around the stomium undergoes PCD during
anther dehiscence
Autophagy involved in aborting the florets by causing PCD in
the ovary cells
Example: Tomato, autophagic vesicles were observed in the
epidermal cells surrounding the stomium during their
PCD. Tang and Bassham, 2018
24

27. Vascular development:
 A role for autophagy in xylem development was first
demonstrated in poplar
 The main conductive cells in xylem are tracheary elements
(TEs), which undergo PCD during differentiation
 The small GTP-binding protein RabG3b, co-localized with
ATG8, was shown to be a positive regulator of autophagy and
TE differentiation
 The gene METACASPASE9 (MC9) was recently identified as a
negative regulator of autophagy during TE differentiation and is
thought to restrict autophagic cell death to the target cells
Tang and Bassham, 2018
25

28. Objective: To show the role of autophagy in pollen maturation and
reproductive development in rice.
Material & method: Surface sterilized seeds of rice,
TEM analysis of autophagic bodies,
OsATG- mutants,
in vivo imaging of autophagy.
26

29. The Osatg7-1 mutant exhibits a sterility phenotype
27
Cell culture
FM 4-64
GFP-ATG8
E&F: Delayed
anther
development

30. Autophagy is required for male reproductive development in rice
28
95% 45%
Pollen maturity
60% 0.75%

31. Autophagy occurs in tapetal cells during the male reproductive phase
29
Vacuole
enclosed lipid
bodies
No obviouse
autophagasomal
structures in
tetrad stage
Mature cells
tapetum fully
degraded

32. Autophagy contributes to lipid metabolism during tapetum and pollen maturation
30
LB

33. :
Defects in degradation of tapetum layer and lipid bodies
causes abnormal formation of pollen coat and pollen grain,
resulting in sever male sterility.
31

34. Abiotic stress:
1. Nutrient starvation
2. Drought stress
3. Heat stress
 Over expression of genes ATG18a and ATG8i conferred to
increased tolerance to nutrient starvation
 Increased production of anthocyanin helps in preventing ROS
burst, contributing to higher stress tolerance
 Upregulation of ATG5, ATG7 and ATG18a genes induces
autophagy during drought condition
 Any mutation in ATG5 or ATG7 led to reduced induction of
autophagy, leading to compromised heat tolerance
Tang and Bassham, 201832

35. Identified regulators of autophagy during drought and heat stress in tomato
Tang and Bassham, 201833
ERF: ethylene response
factor.
AOX: alternative
oxidases.
HsfA1a: heat shock
transcription factor.
DRE: drought
responsive
element
HSE: heat
shock
element

36. Objective: To study the role of autophagy in pepper tolerance to
heat and other abiotic stresses.
Material & method:
Pepper thermotolerent line R9 &
thermosensitive line B6, Heat &
other abiotic stresses treatments.
34
R9 & B6: heat stress – 40 degree
celsius
R9: 4 degree celsius (3 h)
dark for 2d- carbohydrate starvation
200 mM NaCl (3 h) – Salt stress

37. Accumulation of autophagosomes under abiotic stresses
35
LysoTracker Red staining DND-99
E-64 (5-6 leaf stage)
N
S
D
H
C
CS
Normal condition

38. Expression profiles of 29 CaATG genes in response to abiotic stresses.
36
Did not shown significant
response to any condition.
Stress dependent changes
Genes unaffected in some
cases and upregulated in
onother cases

39. Heat-induced autophagosome punctates accumulation in pepper
37

40. Expression profiles of CaATG genes during heat stress in pepper leaf
38
26 genes
upregulated
17 genes
upregulated

41. :
Under abiotic stresses of salt, drought, heat, cold, and
starvation, the accumulation of autophagosome punctates increased
markedly showing the possibility of autophagy participation in the
pepper response to abiotic stresses.
39

42. Plant-pathogen interaction:
Autophagy can serve both a “prosurvival” and a “prodeath” role
upon pathogen infections
Autophagy functions in a prosurvival role during necrotrophic
infection, so it restrict the HR-PCD and prevent runaway cell
death
Autophagy serves a prodeath role upon biotrophic pathogen
infection
Tang and Bassham, 2018
40

43. The dual role of autophagy during plant–pathogen interactions in crops
Tang and Bassham, 201841
Anti-microbial Pro-microbial

44. Objective: To study the role of autophagy genes in wheat immune
responses to fungal pathogens.
Material & method: Plant materials and fungal strain, Virus-
induced gene silencing (VIGS), Evaluation of
powdery mildew resistance.
42

45. Autophagy-related ATG6 genes function in wheat autophagy
43
Yeast expression vector

46. 44
Samples: mutants
Continues…,
TaATG6s – regulating autophagy
process enhances Pm21 –
triggered immune response to
powdery mildew.
Pm21
Low level of AB Enhanced level of AB

47. Expression patterns of wheat autophagy-related ATG6 genes
45
TaATG6s are closely
related to the wheat‟s
responses to abiotic stress
factors.
Two leaf stage seedlings

48. 46
Continues…,
TaATG6s genes are involved in the wheat immune
response to powdery mildew

49. Knocking down wheat autophagy-related ATG6 genes weakly compromises the
broad-spectrum resistance gene Pm21-triggered resistance response to Blumeria
graminis f. sp. tritici (Bgt) 47
TaATG6s play a positive
role in Pm21- triggered
wheat resistance response
to Bgt
Trypan blue

50. :
Wheat ATG6s are implicated in immunity to powdery
mildew, playing positive role in the Pm21-triggered resistance
response.
48

51. Symbiotic interaction:
 Research with the common bean (Phaseolus vulgaris) indicates
that autophagy may be involved in symbiotic interactions
 Trehalose is one of the greatly induced metabolites during the
rhizobium–legume symbiotic interaction
 Autophagy related genes ATG3 and PI3K is upregulated in the
root hair
 Genes ATG3 and PI3K will silence the trehalase enzyme and
will increase the trehalose sugar content in root hairs
 Higher trehalose content leads to increased bacterial viability,
nodule biomass and N assimilation
Tang and Bassham, 2018
49

52. Future perspectives:
 New findings such as the involvement of autophagy in
reproductive development are increasing our understanding of
autophagy but much work is still needed
 The identification and characterization of new regulatory
mechanisms is a critical area for future research
 Some important regulators characterized in Arabidopsis have not
yet been well-studied in crops, for example, TOR and Snf1-
related protein kinase 1 (SnRK1)
 The transcriptional control of autophagy should be another
fruitful area for further research.
50

53. Conclusion:
 Considering its importance in development and stress responses,
autophagy is a promising target to manipulate for agricultural
benefits like higher yield
 Increased expression of ATG genes may be valuable in
agricultural applications, as this can confer a number of benefits
to plants, including enhanced growth, higher yield and increased
stress tolerance
51