Study of internal molecular structure and gene behaviour of plant development. FLOWERING LOCUS T genes control onion bulb formation and flowering

1. MOLECULAR AND GENETIC
APPROACHES IN PLANT
GROWTH AND DEVELOPMENT
Study of internal molecular structure and gene behavior
Submitted by
S.ADHIYAMAAN (2017603401)
I-M.Sc.,VEGETABLE SCIENCE
DEPT. OF VEGETABLE CROPS
HC & RI, TNAU, CBE.- 641 003

2. CLASSIFICATION
Tissue – group of cells, similar in origin, form and function

3. The plant body
1. RAM
2. SAM
3. LEAF
4. FLOWER
5. FRUIT
6. SEED

4.  Meristems
isodiametric
are populations of small,
(having equal dimensions
on all
sides) cells with embryonic characteristics
•Vegetative meristems are self-perpetuating
•Not only do they produce the tissues that will
form the body of the root or stem, but they
also continuously regenerate themselves
Meristems

5. Some basic cell types
2. Collenchyma
3. Sclerids
4. Bulliform cells
1. Parenchyma

6. 1. Parenchyma
Young parenchyma tissue cut parallel with the epidermis of
Euphorbia pulcherrima (poinsettia). Note the cell contents.
Note the nucleus and
chloroplasts

7. 2. Collenchyma
Apium petiole, collenchyma
Collenchyma is the typical
supporting tissue of the
primary plant body.
It develops from parenchyma.
The cell walls are unevenly
thickened.
It is common in organs like
stems, petioles, laminae or
roots.
Apium is celery – and it is the petiole that you eat!

9. 3. Sclerids
Thickening of the cell wall
Parenchyma Developing sclerid
Sclerenchyma cells are
the principal supporting
cells in plant parts that
have ceased elongation.
Sclerenchyma fibres are
the source material for
many fabrics, e.g., flax,
hemp and jute.

10. 4. Bulliform cells Transverse section of
grass leaf Poa praetense.
During drought water is
lost from the thin walled
bulliform cells and the two
sides of the leaf blade fold
up toward each other so
the leaf is less exposed to
sunlight and is heated less.
Once adequate water is
available, turgor
increases, and the leaves

11. Root Apical Meristem

12. Primary Root Morphology
Lateral roots form primary
meristems in mature regions of
roots,
• form new roots (organs).
• Lateral or branch roots arise
from the pericycle in mature
regions of the root.

13. • Most cell divisions in the root tip are transverse, or
anticlinal, with the plane of cytokinesis oriented at right
angles to the axis of the root (such divisions tend to
increase root length).
• There are relatively few periclinal divisions (mostly at tip),
in which the plane of division is parallel to the root axis
(such divisions tend to increase root diameter).

14. The quiescent center is composed of a group of four cells, also known
as the center cells in the Arabidopsis root meristem.
The quiescent-center cells in the Arabidopsis root usually do not divide
after embryogenesis.

15. Root Apical Meristems (RAM) Contain Several Types
of Stem Cells
 Cortical–endodermal stem cells
 Columella stem cells
 Root cap–epidermal stem cells
 Stele stem cells

16. 1.The cortical–endodermal stem cells form a ring of cells that
surround the quiescent center. These stem cells generate the
cortical and endodermal layers
They undergo one anticlinal division (i.e., perpendicular to
the longitudinal axis); then these daughters cells divide
periclinally (i.e., parallel to the longitudinal axis) to establish
the files that become the cortex and the endodermis

17. 2. The columella stem cells are the cells immediately above
(apical to) the central cells. They divide anticlinally and
periclinally to generate a sector of the root cap known as
the columella.
3. The root cap–epidermal stem cells are in the same tier as
the columella stem cells but form a ring surrounding them.
 Periclinal divisions of the same stem cells, followed by
subsequent anticlinal divisions of the derivatives, produce
the lateral root cap.

18. 4. The stele stem cells are a tier of cells just behind the quiescent-
center cells.
These cells generate the pericycle and vascular tissues.

19. Genes associated with
RaM
HOBBIT (HBT) genes
MONOPTEROS (MP) gene is required for formation of
the embryonic primary root as well as vascular
development
SHORT ROOT (SHR) genes
SCARECROW (SCR) genes

20. Anatomy of dicot root after meristematic growth
(Differentation)
Permanent tissue

21. Shoot Apical Meristem (SAM)
• The vegetative shoot apical meristem generates the stem, as well as
the lateral organs attached to the stem (leaves and lateral buds).
• The shoot apical meristem typically contains a few hundred to a
thousand cells, although the Arabidopsis shoot apical meristem has
only about 60 cells.

22. • The shoot apex consists of the apical meristem plus the most
recently formed leaf primordia
• The shoot apical meristem is the undifferentiated cell
population only and does not include any of the derivative
organs
• The shoot apical meristem is a flat or slightly mounded
region (100 to 300 μm in diameter)

23. Apical meristems
Indeterminate growth
e.g., many tomato
varieties
Determinate growth
e.g., Brinjal

24. Shoot organization - Phytomeres
Phytomer =
modular unit
of the Shoot

25. Shoot
organization
Protoderm
Apical
Meristem
Primary
Meristems
Protoderm
Procambium
Ground Meristem

26. Shoot apical meristem - Importance
• Center of postembryonic growth & development
• Source of all primary meristems
– Protoderm, ground meristem & procambium
• Source of
– Leaves
– Branches
– Tendrils
– Thorns
• Self-renewing mass of cells ≈ stem cells
• Balance cell division and cell differentiation

27. Apical Meristem
Examples
Equisetum
Conifers Angiosperms

28. Shoot apical meristem organization
L1 = tunica
L2 = tunica
Peripheral Zone
Pith or Rib
Meristem
Central Zone
Stem Cells
Organizing Center
L3 = corpus

30. Anatomy of dicot stem after meristematic growth
(Differentation)
Permanent tissue

31. Gene Expression in the Apical Embryo Domain
WUSCHEL (WUS), CLAVATA (CLV) AND SHOOT MERISTEMLESS (STM)

32. WUS, CLV and
STM expression
in the shoot apex
WUS gene
1. Organizing Center of Central Cells (just a few cells)
2. Molecular: Encodes homeodomain protein
3. Molecular Genetic: Induces Expression of CLV3
4. Developmental: WUS specifies stem cells of the SAM,
i.e. maintains stem cells and maintains their identity.
CLV3 gene
1. Stem cells of Central Zone
2. Molecular: Encodes peptide secreted in extracellular space
3. Molecular Genetic: Inhibits WUS expression.
4. Developmental: CLV3 restricts size of Central Cells, i.e. CLV3 restricts size of the
stem cell population.
STM gene
1. Through SAM apical “dome” of cells: central zone and peripheral zone.
2. Molecular:Encodes homeodomain protein
3. Molecular Genetic: Blocks organ formation genes (AS1, AS2)
4. Developmental: Prevents premature differentiation of cells from Peripheral Zone…
thus prevents premature organ initiation.

33. Leaf Development

34. Leaf Development
Stage 1: Organogenesis
Stage 2: Development of suborgan domains
Stage 3: Cell and tissue differentiation

35. LEAF PRIMORDIUM

36. Leaf development

38. These cells differentiate in a genetically
determined pattern that is characteristic of
the species
……..but to some degree modified in response
to the environment

39. The Arrangement of Leaf Primordia is
Genetically Programmed
•There are five main types of phyllotaxy
137.5= Golden angle
Nature by Numbers.mp4
Fibonacci series
-integer sequence

40. A complete flower has four whorls
-Calyx, corolla, androecium, and gynoecium
An incomplete flower lacks one or more of these whorls
Calyx = Consists of flattened sepals Corolla = Consists of fused
petals Androecium = Collective term for
stamens
-A stamen consists of a filament
and an anther
Gynoecium = Collective term for
carpels
-A carpel consists of an ovule,
ovary, style, and stigma
Flower Structure

41. activation
inhibition
Activation of Floral Meristem Identity
Genes
Adult meristem
Floral meristem
Temperature-
dependent
pathway
Autonomous
pathway
Flower-
repressing
genes
Flower-
promoting
genes
Vernalization
Autonomous
gene expression
Repression of Floral
Inhibitors
Cold
Light-
dependent
pathway
Gibberellin-
dependent Gibberellin
pathway
CO
Light
LFY
AP1
ABC
floral organ
identity genes
Floral organ
development

42. Model for Flowering
The four flowering pathways lead to an adult
meristem becoming a floral meristem.
They activate or repress the inhibition of floral
meristem identity genes (LFY – LEAFY and AP1 -
APETALA1)
These two genes turn on floral organ identity genes

43. Elliot Meyerowitz
ABC Model – Flower development

45. Model for Flowering
The ABC model proposes that three organ
identity gene classes specify the four
whorls
1.Class A genes alone – Sepals
2.Class A and B genes together – Petals
3.Class B and C genes together – Stamens
4.Class C genes alone – Carpels
When any one class is missing, abnormal
floral organs occur in predictable positions

46. Arabidopsis showing 4 floral
organs
Carpels-C
Stamens - B&C
Petals-A&B
Sepals-A

47. Testing the ABC Model
• Researchers tested the ABC model and found it to be
supported, with the modification that two of the
proteins act to inhibit the production of each other.
• They also found that the DNA sequences of floral organ
identity genes all contain a segment that encodes a DNA-
binding domain called a MADS box.
• They suggested that MADS-box genes are part of the
regulatory cascade that controls the floral organ identity
genes.

48. What are MADS box genes?
• The MADS box is a highly conserved sequence motif
found in a family of transcription factors.
• The conserved domain was recognized after the first four
members of the family, which were MCM1, AGAMOUS,
DEFICIENS and SRF (serum response factor). The name
MADS was constructed form the "initials" of these four
"founders".
• Length of the MADS-box reported by various researchers
varies somewhat, but typical lengths are in the range of
168 to 180 base pairs.

49. THE ABC MODEL FOR FLORAL ORGAN
IDENTITY
APETALA 1

50. No sepals
and petals
No
stamen
No carpels
and stamen

51. The Quartet Model of flower development
Quaternary complexes of MADS
domain proteins

52. Fruit Development
The fruit is a specialized organ, which provides suitable
environment for seed maturation and, often dispersal
mechanisms.
Fruit development can generally be considered to occur in four
phases:
Fruit set phase
Rapid cell division
A cell expansion phase
ripening/maturation phase
GA is a triggering signal

53. Genes
Arabidopsis fruits develop from two fused carpels and are
specialized capsules called siliques
FRUITFULL (FUL) is necessary for proper valve development
and represses SHATTERPROOF 1/2 (SHP 1/2)
(Gu et al., 1998;Ferrándiz et al., 2000a).
SHATTERPROOF 1/2 (SHP1/2) are necessary for valve margin
development (Liljegren et al., 2000).
REPLUMLESS (RPL) is necessary for replum development and
represses SHP1/2 (Roeder et al., 2003)

54. SHP1/2 activate INDEHISCENT (IND) and ALCATRAZ (ALC),
which are both necessary for the differentiation of the
dehiscence zone between the valves and replum (Girin et al.,
2011; Groszmann et al., 2011).
FUL, SHP1/2, RPL, IND, SPT, and ALC all belong to large
transcription factor families.
FUL and SHP1/2 belong to the MADS-box family (Gu et al.,
1998; Liljegren et al., 2000)

55. MADS-box family
MADS-box genes represent a highly conserved gene family of
putative transcription factors in plants
The proteins encoded by these genes are characterized by a
highly conserved domain, which consists of 56 amino acids
K-box involved in protein-protein interactions.
The MADS-box and K-box are separated by a weakly conserved
Intervening (I) region, and a few MADS-box genes have an
amino-terminal extension (N). The Carboxyl-terminal (C)
region may function as a transcriptional activation domain.

56. Seed development

57. The seed develops from the ovule and contains the embryo and
endosperm, surrounded by the maternally derived seed coat.
Ovules are borne by both the angiosperms (true flowering plants)
and the gymnosperms (which include the conifers)

58. Maternal tissues appear to have an important influence on seed
development.
An arabidopsis mutant called aberrant testa shape (ats) that lacks
one of the 2 integuments also lacks several cell layers in the testa (3
layers vs. 5 normally).
The seed are abnormally shaped in this mutant and seed shape
shows maternal effect .
Therefore, the seed coat and not the embryo determines the shape
of the seed, and the embryo just grows to fill in the shape
determined by the testa

59. Maternal gene
The maternal gene called FBP7 is specifically expressed in the
ovule and seed coat and is required for normal ovule development.
A mutant screen on a sterile line identified 3 genes that regulate
seed development.
SeedS develop on theSe mutantS in the
abSence of fertilization.
They are called fis for fertilization independent seeds.
The genes appear important for control of seed development by
fertilization.

60. All three genes show parent-of-origin effects (Genome imprinting)
FIE = fertilization independent endosperm, encodes a WD type
POLYCOMB protein
MEDEA encodes a SET domain type POLYCOMB protein
FIS2 = fertilization independent seed2, encodes a zinc finger protein
The maternally inherited gene is expressed and required but the
paternally inherited gene is not expressed or required for seed
development.

61. POLYCOMB proteins are involved in chromatin structure and
regulate (repress) the expression of genes in big portions of the
genome.
ABA is necessary to induce the expression of genes involved in
maturation and desiccation tolerance
Case study
FLOWERING LOCUS T genes control onion b

62. FLOWERING LOCUS T genes
control onion bulb formation and
flowering
Robyn Lee1, Samantha Baldwin2, Fernand Kenel2, John
McCallum2 & Richard Macknight1
Received 20 Jun 2013 | Accepted 6 Nov 2013 | Published 3 Dec 2013
1 Department of Biochemistry, University of Otago, 9016 Dunedin, PO Box 56 Dunedin, New Zealand.
2 Breeding and Genomics, New Zealand Institute for Plant and Food Research, Private Bag 4704, 8140
Christchurch, New Zealand.

63. Introduction
• Onions are one of the oldest vegetables known to mankind and have
been cultivated for more than 4,700 years.
• Vernalization has only been studied at the molecular level in a few
species and, in all cases, results in the inhibition of a repressor, thereby
allowing FLOWERING LOCUS T (FT) to be expressed.
• Flower signal molecule – Florigen

64. Introduction
• FT is produced in the phloem companion cells of the leaf and is transported to the apical
meristem cells.
• where it forms a complex with 14-3-3 proteins and the bZIP transcription factor FD.
• This complex, known as the florigen activation complex, is thought to translocate to the
nucleus where it activates the floral meristem identity genes, thereby inducing
flowering.
• A recent exciting discovery is that the potato FT orthologue StSP6A is involved in the SD
induction of tuberization

65. Methods- Plant materials
• Doubled haploid onion lines CUDH2150 and CUDH2107 were
obtained from the Cornell University.
• Seed lots were sown in spring either directly as seed or as
transplants in Jiffy 7 plug (Jiffy Corp) and grown in Black Magic
seed-raising mix (Yates Orica, New Zealand).
• Onion and Arabidopsis plants were grown under either LD (16 h
light: 8 h dark) or SD (8 h light: 16 h dark) photoperiod in plant
growth rooms maintained at 20 ºC with 30–40% humidity and a light
intensity of - 115 mmolm-2
s-1
.

66. Methods-Identification and cloning of onion FT-like genes.
• Onion FT-like sequences were identified from our transcriptome data deposited at NCBI BioProject using
TBLASTN.
• Full-length cDNA sequences were obtained from six partial FT-like sequences (AcFT1-6) using the Invitrogen
GeneRacer RNA ligase mediated rapid amplification of 50 and 30 cDNA ends (RLM-RACE) kit and these have
been deposited in GenBank Nucleotide core database under accession codes KC485348-53.
• Full-length PCR products of each FT were cloned from ‘CUDH2150’ using pCR8/GW/TOPO TA(primer)
Cloning Kit (Invitrogen).
• Correct orientation was confirmed by sequencing. The TOPO-based clones were then linearized with either
XbaI or XhoI, and recombined into pB2GW7 with LR Clonase (Invitrogen), thereby producing a binary vector
containing CaMV 35S:AcFT.

67. • To construct the SUC2 promoter-driven constructs, first the 35S promoter in
pB2GW7 was replaced with the SUC2 promoter from pAF12.
• pB2GW7 was digested with SacI/EcoRI to release the 35S promoter.
• The attRI site was lost in this reaction, so it was replaced with a PCR fragment
that contained SacI–XbaI sites at the 50-end and an EcoRI site at the 30-end.
Ligated together, SacI/ EcoRI, this reforms pB2GW7 without the 35S promoter.
• The SUC2 promoter was PCR amplified from the pAF12 vector, with SacI and
XbaI sites on the 50- and 30- ends respectively, and cloned into the promoterless
pB2GW7 vector at these sites.
Methods-Identification and cloning of onion FT-like genes.

68. Plant transformation
• SUC2:AcFT and 35S:AcFT binary vectors were transformed into Agrobacterium tumefaciens GV3101 by
electroporation. Arabidopsis were transformed using the method of Martinez-Trujillo et al. as follows:
• Agrobacterium harbouring the binary vector were streaked onto LB media plates with selective
antibiotics and incubated at 28 ºC for 2 days. The bacteria were then scraped off the plate and
resuspended in 10 ml of infiltration media (0.5 MS (Duchefa), 0.05% Silwett L-77 (Lehle Seeds), 5%
sucrose) to an OD600 an droplets of this solution were pipetted onto unopened flower buds of
Arabidopsis thaliana ft-1 mutants.
• This was repeated three times in 2–3-day intervals. The resulting seed was sown on seed-raising mix and
transformants selected by spraying the young seedlings with Basta herbicide (glufosinate
ammonium; Bayer) at 120 mg/l

69. • Onions were transformed with a binary vector containing the AcFT1 or AcFT4 cDNA under the
control of the 35S promoter, along with the eGFP-ER reporter gene and the Bar selectable marker-,
using the method of Eady et al. as follows:
• Immature embryos from onion seeds (‘Pukekohe LongKeeper’) incubated overnight at 4 ºC were isolated
and cut into -1mm lengths before transfer into 50 ml liquid P5 medium.
• Agrobacterium, resuspended in 0.4 ml liquid P5 to an OD550 of 0.4–0.6, was added and embryos were
vortexed for 30 s, and then placed under vacuum (B20 in Hg) for 30 min before blot drying on filter
paper and transferred to P5 medium solidified with 0.4% Phytagel (Gellan Gum).
• After 6 days cocultivation, embryo pieces were transferred to P5 plus 2.5 mg /1
phosphinothricin and 200 mg /1 timentin. These embryo pieces were cultured in the dark under the
same conditions as described for the production of secondary embryos. Cultures were transferred to fresh
medium every 2 weeks.
Onion transformation

70. • After three to four transfers, growing material was transferred to P5 plus 5mg /1
phosphinothricin and grown for a further 8 weeks.
• During this time, pieces of putative transgenic tissue that reached -2mm2
were
transferred to regeneration medium.
• Shoot cultures were maintained for 12 weeks and developing shoots were
transferred to ½ MS medium plus 20 mg /1 geneticin to induce rooting.
• Rooted plants were either transferred to ½ MS plus 120 g /1 sucrose to induce
bulb formation or to soil in the glasshouse (12 h 12–23 C day, 12 h 4–16 C night).
• Expression of the transgenes were confirmed by reverse transcriptase–PCR using
primers.

71. Analysis of gene expression
• Plant tissue was harvested into liquid nitrogen. Total RNA was extracted using the Invitrogen Plant RNA Purification
Reagent, according to the manufacturer’s instructions, but with an addition 30 min incubation in the reagent for onion
tissue.
• RNA concentrations were determined using a NanoDrop 8000 (ThermoScientific).
• Reverse transcription was carried out with 1 mg of total RNA in 20 ml volumes using Invitrogen SuperScriptIII
according to the manufacturer’s instructions.
• First-strand cDNA was diluted 30-fold, and 3 ml of this was used in each 10 ml reaction.
• Real-time PCR was performed using the Roche LC480 machine and Light-Cycler 480 SYBR Green I Master mix
(Roche). Relative gene expression levels were calculated using the 2 (2delta delta C(T)) method using Roche LC480
software.
• b-tubulin was used as the reference gene.

72. •Result

73. Onion has at least six FT-like genes
• Phylogram of FT-like protein sequences
from different monocot plants. Four
subgroups are indicated.
The sequences are from
• onion (Allium cepa, Ac),
• rice (Oryza sativa, Os),
• maize (Zea mays, Zm),
• barley (Hordeum vulgare, Hv) and
• spring orchid (Cymbidium goeringii, Cg).
 FTs with asterisks have been shown to
promote flowering.

74. Onion has at least six FT-like genes
• Complementation of the Arabidopsis ft-1 mutant with
onion FT-like genes expressed under the constitutive
35S promoter (blue) or phloem-specific SUC2
promoter (yellow).
• Flowering time as determined by number of rosette
leaves at flowering of representative lines grown in
LD.
• (c) Photo of 35S:AcFT lines grown in LD, with
AcFT1 flowering and floral buds visible on WT and
35S:AcFT2 plants (plants are in the same order as the
panel b graph).

75. AcFT1 and AcFT4 expression suggests they
control bulbing.
• Expression of AcFT2 in leaf tissue from seedlings with 3–4
leaves (young), plants with 10 leaves before bulb formation
(mature), and bulbing plants (bulbing) and central bud tissue
from bulbs (bulb tissue) non-vernalized (-V) or vernalized in
the dark for 3 months (+V), leaves from flowering plants
(flowering bulb).
• Samples were taken from plants grown at 20 ºC under SD (8 h
light: 16 h dark) and LD (16 h light: 8 h dark) photoperiods
(except bulbing plants, as bulbs do not form in SD).
• Data represent an average ±s.e.m. of three biological
replicates, with transcripts normalized to b-tubulin.
AcFT2 expression correlates with flowering.

76. AcFT1 and AcFT4 control bulb formation
in transgenic onions.
(a) Expression of AcFT1 and
AcFT4 in leaf tissue from young
plants with 3–4 leaves (young),
plants with -10 leaves before bulb
formation (mature) and bulbs from
previous season grown to have 4–5
fully expanded leaves (bulbs).
AcFT1 is induced and AcFT4 is repressed by conditions that induce bulb formation
Long day condition only promote bulb formation.
AcFT 4 Inhibit bulb formation

77. Expression of AcFT1 and AcFT4 after transfer
from SD to LD
• Mature onion seedlings were grown under SD for
14 weeks and then transferred to LD.
• Leaf samples were taken. All plants were grown at
20 ºC under SD (8 h light: 16 h dark) or LD (16 h
light: 8 h dark) photoperiods.
• The data represent an average ±s.e.m. of three
individual plants, with transcripts normalized to b-
tubulin.

78. Overexpression of AcFT4 prevents bulbing and inhibits
expression of AcFT1
• a) 35S:AcFT1 plants form bulb-like structures in tissue culture
(independent lines are shown in separate photographs) and in
comparison
• (b) 35S:AcFT4 plants that are the same age, show normal
vegetative growth in tissue culture. (NO BULB)
• (c) Relative expression of AcFT1, AcFT2 and the bulb marker
1-SST in representative 35S:AcFT41 and 35S:AcFT4 lines
grown in tissue culture and bulb tissue from a mature wild-
type plant.
• (d) 35S:AcFT4 plants (NO BULB)
• (e) control plants (derived from calli generated in same
experiment as 35S:AcFT4 plants but not expressing GFP or
AcFT4) grown in glasshouse conditions and photographed in
late summer (28 Feb 2013).
• (f) Expression ofAcFT4 and AcFT1 in representative
35S:AcFT4 and control plants.

79. Discussion
• Our evidence indicates that in juvenile plants and those
grown under non-inductive photoperiod, AcFT4 functions
to prevent the upregulation of AcFT1.
• Once the plants are mature and daylength reaches a critical
length, AcFT4 is downregulated and AcFT1 is upregulated
leading to the initiation of bulb formation

80. • Interestingly, AcFT4 also has a number of amino-acid changes that
are predicted to prevent its interaction with the 14-3-3 protein that
mediates the interaction between FT and FD

81. • FT genes control both floral induction and bulb formation in onion.,
also play a wider role in controlling developmental decisions.