The final split: Regulation of anther dehiscence

2. Flow of Seminar

Pollen development
Gametophytic Genes

Stomium Structure Degeneration of cells M Anther Dehydration Phytoharmones

What are the deviations in male sterile lines ?
GmMS/RGMS | CHA Engineering of male sterile mutants

3. Importance of the Seminar
+ Commercial hybrid seed production requires male sterile lines

+ Better understanding of the processes involved in anther
development provide an effective mechanisms for the efficient
generation of hybrids and general control of fertility

4. Flower development

+ Angiosperms develops complex reproductive structure, flower a {
+ In spite of great diversity of flower, they have a basic construction plan (4 whorls)

+ Most of genetic and molecular studies in Arabidopsis thaliana, Antirrhinum majus

and Petunia hybrida helped in understanding of flower development.

+ Along with these studies, initial cloning of some of the genes involved in flower
development led to proposal of Universal ABC Model (Bowman et al., 1991, Coen

and Meyerowitz, 1991)

5. Se- P - St- C
a
C- St - St- C
» fos
Se- Se -C- C

Se- P - P- Flower
indeterminate flower

ABC model and floral organ identity mutants of Arabidopsis

ae
A >

Carpels

ac”
Leaf like organs in 1 & 4'
Mosaic P/St organ in 28:3'4

Leaf like organ

6. Genes involved in Floral development

Genes
Arabidopsis Antirrhinum
A APETALA1, APETALA2 SQUAMOSA
B APETALA3, PISTILLATA DEFICIENS, GLOBOSA
AGAMOUS PLENA, PERINELLI

+ All genes except AP2 belongs to MADS-box gene family of transcription factors
+ Expression of these genes restricted to their domain of action except AP2

+ AP2 RNA is expressed in 4 whorls but functions only in whorl 1 & 2, because AP2

is translationally repressed by a miRNA which is active in whorls 3 and 4

7. Transcriptional factors and protein interactions

* The name MADS-box family comes from initials of the first four cloned genes of
this kind, MCM1 (from yeast), AGAMOUS (Arabidopsis), DEFICIENS (Antirrhinum)
and SRF (mammals)

+ MADS-box protein are transcription factors, binds to DNA as dimers

How these genes manage to activate specific target genes ? Pattern of expression of ABC genes

8. * Triple mutant of SEP genes (sep1/2/3) led to a flower
composed of endless sepals (like in bc double mutant)

+ B and C functions require the SEP genes during flower
development and the reverse is also true

* Genetic, 2 hybrid assay and the ternary factor trap
experiments strongly support the possibility of the
formation of large complexes viz. and

of floral organ identity proteins in order
to activate their specific target genes

35S::AP1/AP3/PI/SEP

9. ABCE Model
Formation of tetrameric complexes

SEP1/2/3/4

©0606

een 8

Whorl: L 2 3 4
eon. Sepal Petal | Stamen Carpel | Gopal

Petal Stamen

10. Protein
‘stability

Genes Regulation of B & C genes

MIR172

| Translational
| repression

11. Future prospectus in flower development

+ Although the floral quartet model is broadly accepted still lacks many
experimental data for its confirmation

* Quartets of proteins are different in each of the different whorls, indicates
distinct quartets bind to different specific target genes and these genes are
activated in only one whorl

+ NAP gene, direct target gene of AP3 is activated in petals and stamens and
SPL/NZZ (downstream of AG) is activated in stamens and carpels

| What, then, is the functional significance of the different quartets in the different whorls?

12. Sporogenous
cells

13. _14 Stages of Anther Development in Arabidopsis | = | Phase 1: 1-8; cell division and differentiation
= Phase 2: 8-14; filaments elongates, anther

Floral Meristem enlarges, microspores to pollens, degeneration

of anther tissues, anther dehiscence and

L1 pollen release

L3 connective and vascular tissue

E L1; 2-5 stage anticlinal division, Epidermis

Ar (2; periclinal and anticlinal division, 4 clusters of archisporial cells

Sanders et al., (1990), Sexual plant Reproduction. 11: 297-322

14. Outer SPC divide and differentiate to Endothecium

Inner SPC divide and differentiate to Middle layer
and tapetum layer

15. Microspores released from tetrad,
degradation of callose wall.

9-12: microspores develop to pollen grain

Tapetum
degeneration

Pollen mitosis,
Tricellular haploid
pollen grains

13: anther dehiscence, 14: Shrinkage of anther

16. Molecular Genetic analysis of anther cell differentiation

SPL/NZZ (SPOROCYTELESS (SPL)/NOZZLE) for Anther Cell Division and Differentiation:

SPL/NZZ is expressed in stage 3 anthers; subsequently, continues at a high level in
subepidermal cells and at a reduced level in the epidermis.

In stage 5 anthers, expression is strong in the microsporocytes and tapetum
SPL induces sporogenesis
EMS1/EXS and TPD1 Define a Signaling Pathway for Tapetum Differentiation:

Mutations in the EXCESS MALE SPOROCYTES1 (EMS1) gene causes additional male
sporocytes (MMCs) along with a lack of tapetal cells

Total number of male sporocytes = (tapetal cells + male sporocytes) => Normal meisis,
failed inccytokinesis ==> cell degradation => no microspores

Cloning of EMS1 encodes a putative leucine-rich repeat receptor protein kinase, likely
localizes to the cell surface, cell-cell communication —tapetal cell differentiation

TPD1 (TAPETUM DETERMINANT1) gene encodes ligand

18. POLLEN DEVELOPMENT

+ Following meiosis, the microspore becomes vacuolized after release from the
tetrad; migration of the microspore nucleus to one side of the cell

Vegetative cell contains a dispersed nucleus and most
of the cytoplasm from the microspore

M1
e
generative cell has highly condensed chromatin and very
Y ittle cytoplasm

M2 à

In Arabidopsis, maize, and other
plants with trinucleate pollen, the
second mitosis occurs before pollen
maturation

Petunia and tomato, the mature
pollen is binucleate, second mitosis
occurs after pollen germination :*

19. Sporophytic Functions for Pollen Development

+ In Arabidopsis MS1, AMS, and AtMYB103 genes are expressed in the tapetum
required for normal pollen development at the onset of pollen development

+ These genes encode putative transcriptional regulators, may control the
expression of a number of genes critical to normal tapetum structure and/or
function, which are essential for early stages of pollen development

+ Exine is made up of Sporopollenin (MS2) important for cell-to-cell recognition
during pollination and pollen germination.

+ NEF1 may facilitate the transport of molecules required for exine deposition

* DEX1 (defective in exine pattern formation) gene encodes a novel protein that
may bind to calcium and localize to the plasma membrane

* FLP1 (faceless pollen-1); cuticular wax deposition on exine

20. Gametophytic Genes Important for Pollen Development

* Thousands of genes are expressed in the developing pollen
Eg: In rice microarray analysis; 82 genes were highly expressed in pollen
By Affimetric gene chip Rice Genome arrey, Wei et a/ (2010) found 25,062
pollen preferential transcripts
+ SCP gene can regulate the pattern of cell division
* GEM1 gene encodes the MOR protein, which binds to microtubules in first pollen
mitosis

+ In tomato, LeProT1 gene is specifically expressed in mature and germinating
pollen, encodes a proline transporters - uptake of proline and compatible solutes
for pollen development and desiccation

+ Arabidopsis gametophytic rtg (raring-to-go) mutant, pollen grains germinate
precociously within the anther locule beginning at the binucleate stage

21. ANTHER CELL SPECIFICATION

BAM12

3
2
3

y
el (AS ——=>NZZSPL” —>: SERKU2

wos J Bes Microsporogenesis =

JAG > adaxial growth

NAB promotion Ar. Cell No.

Tapetal cell fate

MATURE POLLEN FORMATION ANTHER MATURATION
LAP3 & POLLEN RELEASE
miRNA MS?
= MIA e | ABCWBC27
MYB33/ Ys
N
ams — Pollen wall
pyri——» AMS = MYB26
TDF MSI Tapes PCD. NSTI/NST2
sem ia
NN Case al
breakdown

22. How anther dehisce ?

Microspore release: tapetum
starts to break down,
endothecium expands, and

secondary thickening

pollen expands,
outward pressure,

Enzymatic lysis of the septum

stomium splits,
anther walls retract

anther walls begin dehydrate

23. Stomium structure 4

* The location of which determines the position of
anther opening

+ Prior to dehiscence the stomium undergoes cell death and
splitting; this does not appear to require viable pollen to be
present

24. Stomium and Circular Cell Cluster Development in the Tobacco Anther

Stage | Stage | Stage 1 Stage 4 Stage 6
CD

Stage 10 Stage 12

25. A Novel Cell Ablation Strategy Blocks Tobacco Anther Dehiscence
Thomas et al. 1997, The Plant Cell, Vol. 9, 1527-1545

+ Ablate specific anther cell types involved in the dehiscence process

+ Cytotoxic TA56/barnase (TA56 gene promoter is active within the circular cell cluster,
stomium, and connective regions at different developmental stages)

+ Chimeric barstar genes were fused with
1. TP12 gene promoter, active at high levels in most anther cell types

2. Soybean lectin gene promoter, active earlier in the connective and at lower levels in
the circular cell cluster and stomium, than is the TA56

3. TA20 gene promoter that is active at high levels in most anther cell types but has a
different developmental profile than does the TP12 promoter

Results: TA56/barnase and lectin/barstar genes; Anther developed normally but failed to
dehisce because of extensive ablation of the circular cell cluster, stomium, and contiguous
connective regions

+ TA56/barnase and TA20/barstar genes failed to dehisce as well. Even only the stomium
region was ablated in these anthers.

26. Does Dehiscence process is common to all species ?

The basic process of anther dehiscence appears quite conserved across
species.

+ However, subtle differences exist between the species that influence a
and thus final opening position

+ Dehiscence mechanism (Study on 30 Solanum sps.)
Poricidal; dehiscence through a small apical pore

Poricidal longitudinally dehiscing; apical pore, opening continues do:
longitudinal split

iii. Longitudinally dehiscing; stomium forms on entire anther length

* Distribution of thickening in the endothecium around the stomium
determine the form of stomium opening

+ Variation in structure of the endothecial layer;

> Tomato, endothecium only develops in the distal 1/3" of anther adjacent to the
stomium

> Maize, endothecium completely surrounds the locule

Garcia et al. (2008), Botanical Journal of the Linnean Society. 158: 344-354

27. Degeneration of cells in the anther

+ Enzymatic breakdown of the septum:

* At the cellular level, anther dehiscence is similar to silique dehiscence, involve cell
wall-degrading enzymes

* Several hydrolytic enzymes and proteins linked to cell wall loosening;
polygalacturonases (PGs), b-1,4-glucanases, and expansins

* polygalacturonases

+ Degradation of PMC wall to release microspores from tetrads: QUARTET1
(QRT1), QRT2 and QRT3

+ Silique dehiscence: ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1
(ADPG1) and ADPG2

+ Anther dehiscence: ADPG1, ADPG2, and QRT2

+ All these 3 genes are regulated by JA, ADPG2 by ethylene QRT2 by ethylene
and ABA

Anther dehiscence-related PG activity is likely to be regulated by JA, ethylene, and ABA

28. Programmed cell death (PCD) of the septum and stomium

* The importance of tapetal PCD for successful pollen formation has been Known
However, the anther septum and stomium degeneration and cell death also thought
to be via a PCD-related process

+ Work in Lilium suggests that PCD appears to commence in the tapetal tissues and
then extends to the outer anther tissues, including the middle cell layer, stomium
region, and later, after dehiscence, to the endothecium and connective tissue

+ These cells also exhibited nuclear condensation, shrinkage of the plasma
membrane, and aberrant mitochondrial morphology (as in PCD).

29. Cont...

+ Also observed the accumulation of ricinosomes (precursor of proteases vesicle)

* Cysteine protease has been reported in septum and epidermal cells surrounding
the stomium, endothecium & connective tissue during dehiscence in tomato,
eggplant & tobacco. However, it is not known if this was associated with the PCD
processes

+ In Arabidopsis PROMOTION OF CELL SURVIVAL1 (PCS1) is ectopically expressed
under regulation of the CaMV 35Spromoter, dehiscence fails.

30. 'Endothecium secondary thickening.

+ Endothecium established during stage 5, undergoes expansion stages 6-10 and
develops secondary cell wall thickening during stage 11, at which point bar-like
ligno-cellulosic fibrous bands are deposited

+ Endothecium secondary thickening is essential for providing the mechanical force
for anther dehiscence (Analogous to freezing water within the glass vial)

* The composition of thickening also important

31. + Four types of endothecial secondary thickening: species-specific manner:

1. Annular rib types, single radial rings run in parallel to each other
2. Helical rib types, helix along the periclinal cell axis/U-shaped thickening;
3. Reticulate ribs, irregular thickening forms on every face to form a network

4. Palmate ribs, ribs and a solid plate form in the inner periclinal wall of every cell

32. Regulation of endothecium secondary thickening
+ Arabidopsis male sterile mutants myb26 and the NAC secondary wall thickening
promoting factor1 (nst1) nst2 double mutant. Disruption of endothecial thickening,
failure of anther dehiscence and male sterility

+ There is high conservation in the secondary thickening pathways of floral and
vegetative tissues

* receptor-like protein kinase 2 (rpk2) mutant exhibits endothecial cell collapsing

* Expression analysis in rpk2 mutant identified down-regulation of many genes linked
to pollen development and secondary thickening, including a number of PGs, genes
involved in lignin biosynthesis and a number of stress-related genes (ABA)

+ This implies a direct, or indirect, link between these final events of anther
development and stress responses.

+ AHP4 involved in cytokinin signaling, negatively regulate secondary thickening

33. .

How Dehydration of the anther wall takes place ?

Evaporation via stomata on the adaxial side of the anthers

Active removal of water, depletion of starch from the anther filaments in G.
verrucosa coincided with osmotic retraction of water from the anthers

Differential regions of anther dehydration; conversion of starch to sugar would
serve to increase the osmotic potential of the anther tissues

Localized accumulation of the H*-sucrose transporter, AtSUC1, around the
connective tissues of anthers, which may serve to increase osmotic potential and
induce dehydration of the surrounding regions in the anther

Petunia NECTARY1 (NEC1) and NEC2 genes may function in the upper part ofthe
filament and stomium cells to alter the starch to sugar balance and regulate
water potential in the stomium and nectaries, resulting in anther dehydration

34. Cont...

+ localized accumulation of cations: K* may play a role in attracting water from the
surrounding regions and causing the swelling of endothecium and pollen prior to
anther opening

+ Aquaporin gene family has been shown to mediate the passive movement of
water across membranes. PIP1 and PI2, two tobacco aquaporins, are specifically
expressed in the anther tissues responsible for anther dehydration

35. .

Jasmonic acid

Delayed dehiscence or non-dehiscence have been
observed in mutants defective in JA biosynthetic

enzymes
fatty acid desaturation (fad)
delayed-dehiscence1 (dde1) and dde2
defective in anther dehiscence 1 (dad1)
allene oxide synthase mutants

In general, defects in all stages of the JA pathway cause
similar phenotypes of reduced filament elongation and

a lack of dehiscence

dde1 mutant carries defects in OPDA-reductase 3,
incomplete block in JA pathway- gradual accumulation

of JA via alternate gene family

Stomium opening and fertility can be rescued by
exogenous application of JA during stages 9-11

Phospholipid

13-hydroperoxylinolenic acid

‘AOS: CYP74A

12,13-epoxy-octadecatrienoic acid

12-0xo-phytodienoic acid

A3/DDE1
Broxidationx3

Modific

Jasmonic acid

[Signal wansduction

Membrane

Plastid

cis-3-hexenal +
Traumatin

Peroxisome

36. How JA involve in filament elongation, anther dehiscence ?

+ JA synthesized in upper part of filaments, induce water transporter genes (H+-
sucrose transporter, AtSUC1), water uptake from the locules, endothecium, and
connective tissue into this region. This causes anther dehydration and filament
hydrates- subsequently elongates the filament and petal and flower opening

JA Regulation
+ This helps in correct timing of petal opening and anther dehiscence

+ AGAMOUS floral primordial initiation, initiation of microsprogenesis, plays a
regulatory role in late-stage stamen development viz., DAD1 -JA biosynthesis

+ Expression analysis in the opr3 mutant has identified 821 genes (13 are TF) in the
stamen that are regulated by JA application

+ Microarray analysis in anther development in rice showed that 314 genes responded
to either GA or JA treatment, and 24 GA- and 82 JA responsive genes showed
significant changes in expression between meiosis and the mature anther stages.
This suggests significant cross-talk between the JA and GA pathways

37. Gibberlins

Gibberellin contains a tetracyclic
diterpenoid structure, regulates seed
germination, stem elongation, leaf
expansion, pollen and flower
development

GA deficient mutants were have
abnormal anther development led to
male sterility

GA addition to these mutants restored
fertility; opens the door for
manipulation sterility/fertility,
significantly reduces labor and increase
accuracy of producing male sterile lines

Proper knowledge of these processes is
very important

Ks
eop-_PS, entonp + entitaurene KO, entkaurenoic
acid
7 Enzymes
involved Gatto
x
GA — a — an, MO
| ea2oox | gaz
Non-13-hydroxylation GA,, GA; 13-hydroxylation
” | GA200x | sazoox patos
CA M GAx
| GA200x 1 | r20ox
GA3ox
GA20x GA3ox
CA Ar + GA, GA,-GA2OL, Gp,
ÈS | extoe |
GA20x CA,

Ga SA2OX, GA,

38. Contd...

* Tomato: gib1, abnormal flower development.

+ Arabidopsis, ga1, nonviable pollen grain, resulting in poorly developed anthers
* GAs are important for pollen germination and elongation

* GA signaling induces large scale downstream transcriptional changes

Va | | | |
yl lr, ‘
! x Deffect in PCD of
||] ===> |Tapetum,Ubisch body &
HvGAMYB u exine formation

| | J
DE stalin

|
\ "| miRNA159
|

La] =a
a

|
+

Floral organ expansion Anther Development

39. Plays a key role in general floral development, organ formation, and pollen devt.,
coordinating the maturation of pollen and the dehiscence of the anther

Auxin is synthesized within the anther, very high auxin observed in the anther
from stage 10 (tapetal degeneration and the first pollen mitotic division) to stage
12 (bilocular anther)

In cotton glyphosate treatment led to accumulation of auxin, resulted in
problems in anther dehiscence due to reorientation of the cytoskeleton and
alterations in secondary wall thickening in endothecium.

Auxin serving to limit precocious pollen maturation and dehiscence, and to
coordinate the timing of these events

Loss of AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 disrupts JA synthesis by
inactivating DAD1.

Any defects in auxin mediated pathway alone also affects anther dehiscence

40. + Two Petunia genes, PhERS1 and PhERS2, encoding ethylene receptor
homologues are proposed to regulate the timing/synchronization of stomium
degeneration and anther dehiscence

* Tobacco: ethylene-insensitive plants, or treatment with the ethylene perception
inhibitor MCP, which resulted in delayed dehiscence. Which can be accelerated by
treating ethylene as do in JA

41. Morphological effects of stress

+ Increased temperature, have a dramatic effect on pollen fertility and anther
dehiscence

+ This reduction in fertility appears to be at least partly due to tissue-specific auxin
reduction, since auxin treatment can partly compensate for high temperature
stress

+ High temperature stress has also been linked to a reduction in the swelling of
pollen, which provides the force for anther opening

42. high temperatures

A Auxins reverse plant male sterility caused by
Tadashi Sakata’, Takeshi Oshino', Shinya Miura’, Mari Tomabechi', Yuta Tsunaga, Nahoko Higashitani,

e Yutaka Miyazawa, Hideyuki Takahashi, Masao Watanabe, and Atsushi Higashitani?

WE radiate School of Life sciences, Tohoku University, Sendai 980-8577, Japan

yA

Edited by Mark Estelle, University of California, La Jolla, CA, and approved April 1, 2010 (received for review January 23, 2010)

With global warming, plant high temperature injury isbecomingan are quite similar to HT injury to male reprodu
increasinalv serious problem. In wheat. barlev. and various other whereas HT generally increases auxin levels in ce

LP)

+ Under high temp. endogenous auxin levels specifically decreased in developing anther
cells of barly and Arabidopsis, leads to the abortion of pollen development

+ Expression of the YUCCA auxin biosynthesis genes was repressed by HT

+ Application of auxin may help sustain steady yields of crops despite future climate change.

43. Application of auxin (IAA, NAA,
or 2,4-D) reversed the abortion
of pollen development under HT
conditions

A

5-leaf stage
Days aftersowing 0 16 18 19 20 21 22 23 24 Heading 32-35

Panicle length(mm) 1 2 5 10

Control —| High temp. | Control temp.

(at 20C day/ 1ST night) (for 5 days at 30 day/ 25T night)

Control temp. High temp. + mock

44. Seed setting rate (%)

Control temp. High temp. + mock + IAA (10M)

Exogenous auxin rescues
HT injury in Arabidopsis
anther development

100 ET TIAA | Control temp.
Control temp. . [NAA]

80 12.4-D}
60
401 High temp.
2
A 1 .

N L
‘mock 10M 10M 10M

Exogenous auxin reversed male sterility and restored seed setting rate in barley

45. What are the deviations in male sterile lines
CMS

Causes of CMS

e

« Abnormal behavior of the tapetal cell

2

% The structural changes in the stamen filament
+ Callose dissolution
“ Differences of amino acid components in anther

a

% Enzymatic activities

+ In rice to date, 20 independent CGMS have been reported, however, three of
them, wild abortive (WA), Boro Tai (BT) and Honglian (HL) are mostly applied for
commercial hybrid seed production

46. VOLUME 45 | NUMBER 5 | MAY 2013 LETTERS

nature ,

genetics
A detrimental mitochondrial-nuclear interaction causes
cytoplasmic male sterility in rice

n!, Ce Fang?, Qunyu Zhang!,

Hong Xu!-4, Zhenlan Liu, Jingxin Guo!, Heying Li!, L
an Ye!, Xiaoyu Li,

3, Hong Wu!, Hao Wu!, Chonghui Ji", Huigi Zheng!, Yuan!
Li! & Yao-Guang Liu!

* CMS-WA discovered in 1970s in a wild rice (Oryza rufipogon), and this
cytoplasm was backcrossed into indica rice to produce CMS-WA lines

+ In 3-line hybrid seed production in world 99% of CMS lines carry WA cytoplasm
+ Explanations till proposed for CMS system-

1. Mitochondrial energy deficiency

2. CMS protein cytotoxicity and

3. Premature tapetal programmed cell death (PCD)

47. Which gene of mitochondria induce CMS ?

* To identify the factor(s) responsible for CMS-WA, transcripts of the whole CMS-WA mitochondrial
genome examined by RNA blotting, and a probe containing the ribosomal protein gene rpl5.
+ Two Zhenshan 97A (ZS97A) mitochondrial genomic clones that hybridized to rp/5 were sequenced

4,552 nt

2,395 nt

yrps14

Probe 1

orf284 (100%) À orf224(81%) 0'288 |

Structu
evolved ri

Unknown origin

48. Nuclear transformation of candidate gene(s) of CMS fused with a mitochondrial transit signal (MTS), driven
by the CaMV35S promoter into the male-fertile japonica variety Zhonghua11 (ZH11) and Arabidopsis thaliana.

sterility requires mitochondrial

localization of WA352

b mrs-wass2 MTS- OsCOX11 RNAi
12) Nontransgenic GFP-WA352 (T,) 1)

É
i ®

49. + CMS-WA is genetically sporophytic, WA352 acts in the diploid anther cells to
cause CMS, can be restored by Rf genes, Rf3 or Rf4

how the Rf genes affect WA352 expression ?
using 3 NILs with WA cytoplasm and the nuclear background of ZS97A but that

carry different genotypes of Rf genes

Rf3 functions post-translationally

oe

e .

a
+ A ge eS
Pp zsns 4

PP ze

ate

2,395
2,300
1,527
1,500

eS

In CMS-WA plants, WA352 mRNAs
were ubiquitously expressed but
WA352 accumulated in anthers
only at the microspore mother cell
(MMC) stage and not in leaves

50. ZS97A stage

WA352 interact with COX11 lead

“In Saccharomyces cerevisiae COX11 SL has a rc

+ Tapetum d midlle microspore stage until the bicellular

pollen stage. In ZS97A, tapetum degeneration
started at the early microspore stage and
finished before the

pollen deve
ZS97A (MMO)

| eiodsommu SIE] erodsosmmu eIppIN uodsorıu Ayeg

uslod Jelena

51. e
!

\
Ne

¿“Genomic
rearrangements

Mitochondrion

VAL

Sn
Fertility >
> >> > > restoration "\

y * WA352 arosed ~

y (Tapetum at

eee MMC stage)
ci
ot

.

ED Gra) Gz) of “Cyto. BOS Cytosol

== —— + — 2 oo
| al «+ Ma

“Ris Rf4 OsCOX1
Nucleus

1 Premature tapetal PCD and degeneration

Male sterility

+
e

a

4
+

U

0.7-2 mill. Years ago
in O. rufipogon and
O. nivara further to
indica cultivars

Maintained by
widespread Rf
gene(s)

N

52. Restorer of fertility gene in CGMS

* Rf gene only expressed in the presence of the sterile cytoplasm

+ Rf genes mostly encodes pentatricopeptide repeat (PPR) proteins

+ The first isolated Rf gene was on maize Rf2 which codes for aldehyde
dehydrogenase

+ In rice, Rf-1 gene codes for a 791-aa protein containing mitochondrial targeting
sequence in the N-terminal region and 35-aa PPR motif

+ Till now the role of PPR is not known

+ But evidences support its function in the gene expression of organelles by binding
to specific transcripts

+ Eg: positional cloning, Rf1 gene in rice contain duplicate ORF (Rf-1A and Rf-1B)
with PPR motif. Rf-1A has a mitochondrial targeting signal and possess transcript
from an atp6/orf79 region of the mitochondrial genome

53. TGMS and PGMS

What is the relationship between Male sterility and Environmental factors ?

* Genes: pms1, pms2 and pms3 (PGMS); tms1, tms2, tms3(t), tms4-1, tms4-2,
tms5, tms6-1, tms6-2, rtms1 & ms-h

+ Enzymes: Adenine phosphoribosyl transferase (APRT), convert adenine into
adenosine-3’-phosphate (AMP) associated with the purine salvage pathway.

< In TGMS rice line Annong S-1, transcription of APRT gene is down-regulated
by HT stress (282C), results in pollen abnormal development

* TGMS is associated with premature PCD of the tapetum (Ku et al., 2003)

54. v Male sterility under
short-day conditions

csa(SD)|

Y” carbon starved
anther (csa) mutant;
mutation in an R2R3
MYB transcription (point
mutation) regulates
sugar partitioning from
photosynthetic tissues
to anthers to promote
pollen maturation.

9522(SD) csa(LD) Zhang et al (2013), Proc Natl Athd Sci USA.

55. CHA

+ Inducing male sterility is by the application of chemicals
* CHAs used in rice:

Ethylene

Arsenates

Oxanilates

SRE

Plant growth hormones

+ CHAs may be unsafe for human health

* This prompted many scientists to explore the relationship of male sterility/fertility
and metabolic biosynthesis pathway

56. Engineering of male sterile mutants

+ MS induced by interfering the development and metabolic synthesis of the
tapetum or pollen in transgenic plants

+ Sense or antisense suppression to inhibit essential genes
+ Expressing aberrant mitochondrial gene products

+ Use of gene that encodes a protein which is able to disrupt cell function
(Barnase/Barstar system)

* Change the levels of metabolites needed for the anther and pollen development
“+ Eg: early stages of pollen development of tobacco are blocked by the

expression of extracellular invertase Nin88 gene (promoter Nin88), results in
MS in tobacco

+ Expression of 6-ketothiolase (phaA from Acinetobacter) in chloroplast genome
(chl. promoter psbA) of tobacco accelerates anther devt. and pollen grains
collapse

57. Bio-engineered MS 4

Maintainer line Male-sterile Restorer line Bic-engineered
Male-fertile parent parents Male-fertile parent Male-sterile parents
Ms/ms ms/ms is/Ms ms/ms
MS plants were
genereated by RNAi
i Phytohormones

approach by fusing two Selfing treatment on flowers
anther-specific promoter, Ms/ms : ms/ms to restore male-sterility
Osc4 and Osg6b with male fertile 1 : 1 male sterile
allene oxide synthase

(OsAOS) that catalyzes the

i i in ri Ms/Ms
biosynthesis of JA in rice 50% Male tere
jale-fertile progeny progeny

y

To be removed before
anther dehiscence

y

Time/labour-consuming jobs
Higher cost of seed production

e
= HZ

Bae et al. (2010), Transgenic rice plants carrying RNA interference constructs of AOS (allene oxide synthase) genes show |

severe male sterility, Plant Breed., 129: 647-651.

58. Barnase/Barstar System of Male sterility

Barnase gene...produees RNASE up =

is fic: Herbicide

(tapetum layer) Resistance
promoter (Phosphinothricin)

(Gene construct)

Endothecium

% Middle layer

tt

Tapetum

CE Chimaerie | rbonuch sooner
imaeric ribonuclease gene

a expression within the anther selectively degene =

Bacillus

A . destroys the tapetal cell layer that
imyloliquefaciens surrounds the pollen sac, prevents
pollen formation, and leads to male PMCs/pollen
sterility.

become
sterile

59. TRANSGENIC GENETIC MALE STERILITY

„ ““Endothecium

i Ora

Tapetum
persistant
Cm
amyloliquefaciens | |
PMC: Bauen
>
fort le

Barstar gene

60. | TRANSGENIC GENETIC MALE STERILITY 'GARNASE-BARSTAR SYSTEM |
STEP 1 7
Maintenance x
of male sterility

Sterile

ertile
Herbicide Herbicide
Resistant Sensitive

a
a 50% Barnase-Bar/- (male sterile, rest. o
50% I

Killed , Killed , Killed , Killed

STEP 2
Production of X Barstar/Barstar Fertile
Hybrid | storer

Barnase-Bar/Barstar (male fertile)

F1 Hybrid 50% -/Barstar (male fertile)

100% male fertile { 50%

61. |_ Filament Extension / Anther & Flower Opening

Tapetal Breakdown / Stomium Split
A E E O HEERES ER

Microspore Release

YUC2 YUC6 expression- auxin accumulation in anther

Endothecium expansion

Secondary thickening formation in anther