GENOMICS OF STAY GREEN TRAITS AND THEIR UTILITY IN CROP IMPROVEMENT

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2. Justification
• Climate change / Resilient genotype
• Biotic / Abiotic stresses
• Drought stress
• Morphological/Physiological traits
• Stay green trait
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3. Stay-green traits?
 The ability of a plant to postpone/delayed
senescence and retain their leaves in the active
photosynthetic.
 Therefore be expected to give a higher production
and productivity of grain as well biomass under
biotic and abiotic stress condition.
3
Introduction

4. • The extended foliar greenness during grain filling
and delayed senescence and maintain more photo
synthetically active leaves (Xu et al., 2000).
• Genomics of stay green traits refers to the study of
genes related to photosynthetic activates and
provide the tools to identify the its complexity.
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Introduction

5. 5
 Stay green required specially in a
drought (Rosenow et al. 1983)
and heat stress (Wahid et al. 2007)
environmental condition.
 To keep greenness of leaves alive
for longer period of time,
especially during the grain filling
stage (Spano et al. 2003).
 To maintain or increase higher
grain yield.
Where, when and why stay green traits is required?

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Schematic representation of nitrogen management

7. 7
• The functional stay
green traits is
associated with the
transition from the
carbon capture to the
nitrogen mobilization
phase of foliar
development
Carbon capture/Nitrogen remobilization and
stay green
Source : Thomas H. and Oughum H. (2014)

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 Leaf color turns yellow during senescence due to the degradation
of chlorophylls and photosynthetic proteins.
 Chlorophyll pigment is responsible for greenness of the leaves and
actively involved in photosynthesis reaction.
 Delay chlorophyll degradation during the reproductive stage, the
long time photosynthesis activities, as a result increase the yield.
 The long time photosynthesis increases the grain yield as well
biomass production.
Importance of chlorophyll content plant

9. Chlorophyll degradation process
MCS = Metal-chelating
substance
PPH = Pheophytinase
CLH = Chlorophyllase
PAO = pheide a oxygenase
RCCR = Red-Chlorophyll
Catabolite Reaction
FCC = fluorescent Chlophyll
catabolite
Source : Hortensteiner and Krautler, 2011
MCS
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Chlorophyll B and 7-Hydroxymethyl chlorophyll a reductase
HCAR

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 Alteration of the genetic processes determining the initiation
of senescence and its rate of progress results in a phenotype
which continues to photosynthesize for longer than normal
(i.e. is `functional stay green') and which might therefore be
expected to result in a higher yield.
 By contrast, other types of `stay green' mutant remain green
due to retention of chlorophyll resulting from lesions in its
catabolism, (i.e. is `non functional stay green') but lack
photosynthetic competence (Thomas and Howarth, 2000).
Types of stay green

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(Thomas and Howarth; 2000)
Five way of stay green
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 Key components determining stay green:
 Total plant leaf area : (+ve) correlated with green leaf area at
maturity
 Duration of leaf senescence: (+ve) correlated with green leaf area at
maturity
 Rate of leaf senescence: (-ve) correlated with green leaf area at
maturity
Correlation of stay green traits

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 The wheat stay-green character govern by four recessive genes
that are segregated independently and interacted in an additive
manner (Joshi et al. 2007).
 In rice, this is govern by recessive mutant gene sgr(t) on chrom.
9 (Cha et al. 2002; Jiang et al. 2007) and
 In arabidopsis is also govern by recessive gene fiw on chrom. 4
(Nakamura et al. 2000).
Genetics of the stay green traits

14. Genes related with stay-green traits
14
S0urce: Thomas and Oughum (2014)

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Senescent Stay -greenSenescentStay -green
Stay green traits in corn
Source : www.iita.org
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Senescent Stay -green
Stay green traits in wheat
Source : www. grdc.com.au

17. Senescent Stay -green
Stay green traits in sorghum
Source : Courtesy A. Borrell
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18. Crop improvement
 Delayed senescence is useful only when it contributes
to increase yield (Rosenow and Clark 1981)
 Nitrogen uptake during grain filling is higher in stay-
green genotype than senescent one (Borrell and
Hammer, 2000).
 In some crop it leads to biotic (Rosenow et al. 1983
and Joshi et al. 2007) and abiotic (Harris et al. 2006
and Kumar et al. 2013).
 Stay green exhibit reduced stalk lodging (Rosenow
and Clark 1981; Henzell et al. 1984 and Woodfin et al.
2007).
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Approaches
Conventional
Selections Hybridization
Non
conventional
Gene Silencing
Mutation
Induction
Approaches to develop of stay green trait genotype

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Italy Spano et al. (2003)
• Chemical mutagen used ethylmethane sulphonate (EMS) for 2 h in 0.3 M
• Durum wheat
• Selected four mutant line (139, 142, 196, 504) from M6 (their similar timing
of flowering but delayed senescence)
• Determined chlorophyll content by chlorophyll meter
(SPAD-502, Minolta, UK)
• Grain weight, yield and nitrogen content

21. The stay green phenotype in durum wheat
21
Italy Spano et al. (2003)
The onset of chlorophyll loss was delayed in the leaves of the mutants, by about 10
d compared with the parental genotype

22. Table 1. Changes in the total chlorophyll content of senescing
leaves of parental (cv. Trinakria) and mutant plants incubated in
continuous darkness for 6 d (0 d-6 d)
22
Italy Spano et al. (2003)
Fully expanded leaves in darkness was faster in the parental line than in the mutants.

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Changes in biochemical parameters during senescence of mutant
Italy Spano et al. (2003)
Two of the mutants (139 and
142) retained about 50% of
their initial chlorophyll content
after 40 d, while the others
remained slightly green.
Decreased later in the mutants
with lines 139 and 142
retaining about 50% of their
initial rate at 40 d.
The CO2 concentration within
the leaf increased after
flowering in all lines

24. Table 2. Grain yields, weights and nitrogen contents of
mutant and parental (cv. Trinakria) control plants
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Increases in 1000 seed weight (by 10-14%) occurred in all the mutants but the embryo weights were lower than in the
control plants
Grain of the four mutants also had lower total nitrogen contents than those of the parent when expressed as mg g±1
dry wt. but differed when expressed as mg grain±1, being slightly higher in 139 and 509 but lower in 142 and 196.
Italy Spano et al. (2003)

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 Four mutants of durum wheat were selected which showed marked
delay in chlorophyll loss from leaves compared to the parental
lines,
 The photosynthetic competence of the mutants was also
determined by measuring parameters related to photosynthesis
from flowering until full senescence.
 The `stay green' mutants of durum wheat had 10-12% increases in
seed weight.
Major findings

26. • Chemical mutagen used N-methyl-N-nitrosourea (MNU).
• Japonica rice cultiver - Hwacheong-wx
• The stay green phenotype was controlled by a single recessive nuclear gene
sgr(t).
• Primer use SSR, STS and RFLP
• Linkage map and map distance analysis by –MAPMARKER /EXP 3.0
• Transgenic trait- stay green
• Marker gene- sgr(t)
26
China Cha et al. (2002)

27. Hwacheong-wx
Mutation
treatment
N-methyl-N-nitrosourea
Development of mutant
 To determine the inheritance mode of the
mutation.
 To determine the chromosomal location of
the stay green mutation
MutantWild type X
F1
I
F2
Milyang23
27

28.  During vegetative growth, no phenotypic difference
was observed between the wild-type and the mutant
plants.
 After heading, the leaf green color of the stay green
mutant was not changed while the wild type turned
yellow completely.
 Stay green mutant was not changed even in dark
induced senescing treatment for 2 weeks, while the
wildtype turned yellow completely.
Wild type mutant
Wild type mutant
28
China Cha et al. (2002)
Phenotypic characterizations of the stay green mutant

29. Table 3 - Comparison of phenotypic characteristics between the parental lines and
the stay green mutant
29
China Cha et al. (2002)

30. Table 4 - Phenotypes of F1 plants populations from the crosses between the stay
green mutant and the wild-type rice varieties.
30
China Cha et al. (2002)
The stay green phenotype was not expressed in F1
plants.

31. Table 5 - Phenotypes of the segregation of F2 populations from the crosses between
the stay green mutant and the wild-type rice varieties
31
China Cha et al. (2002)
F2 populations of all crosses showed a segregation (3:1)
ratio of three wild-type to one stay green phenotype

32. Fig. 1. Time-course changes in the chlorophyll concentration of the upper leaves of
the parental lines and the stay green mutant after heading
 The chlorophyll concentration of
the stay green mutant leaves was
not significantly different from
the parental lines at flowering (0
days after heading).
 With the progress of grain filling,
the phenotypic difference between
the mutant and the parental lines
became clearer in that the leaves of
the parental lines turned yellow,
while those of the mutant
remained green until 50 days after
heading.
32
China Cha et al. (2002)

33.  The photosynthetic
rate of the mutant
green leaves was not
significantly different
from that of the
parental yellowing
leaves after heading.
Fig.2. Time-course changes in the photosynthetic rate of the upper leaves of the
parental lines and the stay green mutant after heading
33
China Cha et al. (2002)

34. Fig. 3. Molecular genetic mapping of the stay green sgr(t) gene on chromosome 9 in
rice
 Molecular markers on chromosome 9 and
chose the candidate markers presumably
around the sgr(t) locus.
 Between the stay green mutant and
Milyang23 as the parents, two SSR markers,
RM160 and RM189, showed polymorphic
bands.
 The sgr(t) gene was mapped between RFLP
markers RG662 and C985, with distances of
1.8- and 2.1- cM, respectively,
 Using MAPMAKER/EXP
34
China Cha et al. (2002)

35.  The stay green phenotype in japonica rice is controlled by a single
recessive nuclear gene.
 Photosynthetic activity and senescence is proceeding normally in
the mutant so this mutation is similar, but not identical, to the non
functional type C stay green
 Dispite the mapped sgr(t) locus to the long arm of chromosome 9
between RFLP markers RG662 and C985 at 1.8- and 2.1-cM
intervals, respectively.
Major findings
35

36. China Zhou et al. (2011)
Model plant Medicago truncatula
Transgenic trait Stay green
Marker gene MtSGR
Suppression gene MsSGR
Plasmid construct pANDA35HK-SGR /MsSGR-RNAi (Binary vector)
Transgenic by RNAi technology
Transposable element Tnt1
SG conformation PCR, Southern bolt analysis
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37. 37
Phenotypic characterization
China Zhou et al. (2011)

38. Characterization of Chlorophyll degradation in wild type with mutant (NF2089) during
dark-induced senescence.
38
Chlorophyll characterization
China Zhou et al. (2011)

39. Chloroplast structures observed using transmission electron microscopy at 0 d (A, B, E, and F) and
10 d (C, D, G, and H) after dark treatment are shown.
▫ G, Grana stack;
▫ P, plastoglobule;
▫ S, starch granule.
39
China Zhou at el. (2011)
Ultrastructure of chloroplasts in the wild type (A–D) and the NF2089 mutant (E–H).
Wild type
NF2089
mutant

40. Table 6. Genetic segregation analysis of the NF2089 mutant
NF2089 × Wild-type
Mutant Wild-type-like Mutant : Wild-type-like
(green color) (yellow color)
F1 seeds 148 457 1 : 3.09
F2 plants 46 143 1 : 3.11
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 The F1 plants did not show the stay-green phenotype.
 Segregation ratio was observed closed to 1:3 in F1 seeds and F2 plants.
 Stay green a gene recessive mutant and that the mutation was caused by the loss of function of a single gene.
China Zhou at el. (2011)

41. A. Diagram of the MtSGR gene (1,734 bp) structure showing the four exons (blocks),
three introns (lines), and positions of Tnt1 insertions.
B. PCR amplification of MtSGR from genomic DNA of the wild type (WT) and
NF2089 showing the presence of a 5.3-kb Tnt1 insertion in the mutant.
C. The expression of MtSGR was abolished in the mutant.
D. The transcription levels of MtSGR in wild-type plants showed that the expression of
MtSGR was up-regulated during dark-induced senescence. (0, 5, and 10) indicate
days after dark treatment (DAD).
Molecular cloning of the MtSGR gene
41
China Zhou et al. (2011)

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Isolation of an SGR Gene (MsSGR)
Suppression of MsSGR Expression
Quantitative RT-PCR analysis
Five transgenic lines
(SGRi-10, SGRi-17, SGRi-21, SGRi-29, and SGRi-39)
RNAi Technology
China Zhou et al. (2011)

43. Twenty transgenic lines were identified through
PCR analysis
43
China Zhou et al. (2011)

44. • Quantitative RT-PCR analysis of MsSGR gene expression in transgenic lines.
• Five transgenic lines, had SGR transcript levels reduced by more than 60% when
compared with the empty vector control line.
44
China Zhou et al. (2011)
Twenty transgenic lines were identified through
PCR analysis

45. • Southern-blot analysis of KpnI-digested genomic DNA from leaves of the wild type and
MsSGR-RNAi transgenic lines. The DNA was probed with 699 bp of gus linker
fragment from the pANDA35HK vector.
• Regenerated positive lines were truly independent transformants. Both single-
copy and multiple-copy integrations of the transgene were observed in the
transgenic lines.
45
China Zhou et al. (2011)

46. • Dark incubation of alfalfa
MsSGR-RNAi transgenic
lines.
• Detached leaves of the
wild type, empty vector
control (CTRL), and
• MsSGR-RNAi transgenic
lines in the dark. Numbers
(0, 5, 10, 15 and 20)
indicate days after dark
treatment (DAD).
46
China Zhou et al. (2011)

47. Chlorophyll Content and Physiological Changes in Transgenic
Alfalfa during Dark Incubation
50.6% 52.8% 55.6%30.0% 60.2%
 No difference was observed in chlorophyll loss after 5 d of dark treatment. Significant changes were observed, however, after 10 d of
dark treatment. By the 20th d of dark treatment. only 5% of chlorophylls remained in the control leaves, while more than 50% of
chlorophylls remained in most of the transgenic line.
 No difference in Chl a/b ratio between transgenic and control plants was observed after 0 and 5 d of dark treatment. However, after 10
and 20 d of darkness, the RNAi lines showed large decreases in Chl a/b ratios .
47
China Zhou et al. (2011)

48. Forage nutritive quality analysis
Compared with the control, Most of the transgenic lines (except SGRi-17) had increased crude protein
content the level of increase in crude protein content
48
China Zhou et al. (2011)

49. Major findings
• Silencing of MsSGR led to the production of stay-green transgenic alfalfa.
• The number of young nodules on the mutant roots have higher than in the wild
type. The expression levels of several nodule senescence markers were reduced in
the sgr mutant.
• This beneficial trait offers the opportunity to produce premium alfalfa hay with a
more greenish appearance and retained more than 50% of chlorophylls during
senescence and had increased crude protein content.
• This study illustrates the effective use of knowledge gained from a model system
for the genetic improvement of an important commercial crop.
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50. China Jiang at el. (2007)
Model plant Japonica rice
Gene sgr on chrom. 9
Mutagen used γ - ray (60Co) irradiation
Markers RM3803 and RM6862
Cloned in pCAMBIA1301 (BAC)
Promoter CaMV35S
Via Agrobacterium tumefaciens -mediated
SGR conformation PCR, marker gene of hygromycin phospho-transferase (Hyg) and
primer
5’-CGATCTTAGCCAGACGAGCGGGTTC-3’
3’-GCTGGGGCGTCGGTTTCCACTTCGG-5’
50

51. • No significant differences were observed between wild-type and sgr mutant plants during the vegetative
growth period but After grain-filling, the leaves of wild-type rice plants turned yellow while the sgr mutant
leaves remained green
• when transferred to permanent darkness for 5 days the wild-type leaves turned yellow, whereas the sgr
mutant leaves were still green.
The ‘stay green’ phenotype
51
China Jiang at el. (2007)

52. Mutant isolation, cloning and construction
52
China Jiang at el. (2007)
 The stay green phenotype was not expressed in
F1 plants, but F2 populations exhibited a 3:1
segregation ratio of the wild type to the stay
green phenotype when crossed with indica rice
MutantWild type X
F1
I
F2

53. Cloning of the SGR gene
 The sgr locus found on long arm of chromosome 9 between the two
markers RM3803 and RM6862 and 3 STS marker developed between
them.
 Gene was fine-mapped and linked on STS3 in a bacterial artificial
chromosome (BAC).
53
China Jiang at el. (2007)

54.  The leaves of the two independent T1 families turned yellow after 5 days of dark treatment (Figure d)..
e. The transgenic event was identified by PCR analysis of the dominant selectable marker gene Hyg.
54
China Jiang at el. (2007)
Conformation of transgenic plant

55. Over-expression of SGR
 Phenotypically the pale green color of flag
leaves and grains of two transgenic lines
(35S: SGR-1 and 35S: SGR-5)
 The expression was confirmed by
biochemical staining of the product of the
marker gene β-glucu-ronidase.
 The increased SGR transcripts were
analyzed by RT-PCR.
55
China Jiang at el. (2007)

56. Major findings
• Stay green rice mutant, sgr, compared with the wild type, exhibited a marked delay
in Chlorophyll loss from leaves during grain-filling and after dark induced
senescence.
• The mutant leaves did not maintain photosynthetic competence for any longer than
the wild type leaves, and this is therefore referred to as a non-functional stay green
mutant (type C) .
• Loss of function of SGR affected Chlorophyll breakdown and feedback, inhibiting
degradation of thylakoid membranes in sgr mutant leaves during senescence.
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Achievements

58. 58
 To increase the productivity, one of the possible ways is to increase or
retain the photosynthetic activity during grain filling.
 Stay green traits is beneficial for increasing production, productivity and
quality of grains and biomass under stress conditions.
 Physical and chemical treatment of mutagen should be beneficial for
developing stay green trait in major crops to give better yield in stressed
environment.
 Silencing of gene approach will be beneficial for development of stay
green trait in crops for better yield under stressed environment.
Conclusion

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 Effective treatments to induce mutagen may be benificial to create
variability for stay green trait and can be use by reverse genetic in major
crops to give better yield in stressed environment.
 There need to explore stay green trait extensively in breeding programs
via conventional and non-conventional method in the climate change
scenario for fulfilment of future food requirement.
 Although much progress has been made on the field of RNAi over the past
few years, the full potential of RNAi should be utilized for development
of stay green lines under crop improvement programmes.
 Development of new generation stable lines/crops for future by using
conventional and non-conventional methods.
Future thrusts

60. The future's bright, The future's ... green
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