Molecular approaches study the structure and function of genes at a molecular level using methods of molecular biology and genetics. They can identify simple and complex effects of genes on growth regulation and study the interaction of genes and the environment related to crop growth regulation. Key techniques include PCR, recombinant DNA technology, genetic transformation, RNA interference, microarrays, and molecular markers.

1. MOLECULAR APPROCHES IN
CROP GROWTH REGULATION
Sukhjinder Singh
Department of Fruit Science
PAU, Ludhiana

2. “Molecular approaches comes under field
of science that studies the structure and
function of genes at a molecular level and
employs methods of both molecular
biology and genetics.”
Molecular approaches :

3. • To identified simple and complex effects of gene on growth regulation.
• To studying the interaction of gene and environment which are related
to crop growth regulation.
Importance

4. Central dogma

5. • PCR
• Recombinant DNA technology
• Genetic transformation
• RNA interference (RNAi)
• Microarray
• Molecular markers
Techniques of molecular approaches :

6. How plant growth is regulated at the molecular level
 Growth regulation consists of a complex sequence of interconnected events
involving cell division, cell expansion and requiring multiple levels of genetic
regulation.
 Plant hormones regulate plant growth (creates changes in the cell) through effect
on gene expression, transcription and activity of enzymes.
 Plant hormones are signal molecules which determine the formation
of stems, leaves, flowers, shedding of leaves and the development and ripening
of fruit.
Hormones regulate cellular processes in targeted cells at molecular level.
Gonzalez and Dirk (2015)

7. GENERAL MECHANISM OF HORMONE ACTION
The sequence of events initiated by hormones can generally
be resolved into three sequential stages:
(1) the initial signal perception,
(2) a signal transduction pathway, and
(3) the final response.

8. SIGNAL PERCEPTION
• Reaction of the hormone with a receptor site
• Diffusion of Plant hormones through plasmodesmata or through
the apoplastic surface
• Target cell must be capable of detecting the presence of the
hormone
• The formation of this active hormone-receptor complex
completes the signal perception stage

9. SIGNAL TRANSDUCTION
• Activated hormone-receptor complex sets into motion a cascade of biochemical events that leads to the final,
characteristic response.
• Activation of membrane protein called the “G-protein”.
• Alternatively, the G-protein interact with an ion channel that controls the flow of calcium into the cell.
• Once reaches in the cytoplasm, calcium will bind with cytosolic calcium-binding proteins such as
calmodulin.
• The effect of either cAMP or the Ca2+ -calmodulin complex is to activate specific protein kinases.
• Protein kinases phosphorylate other proteins by transferring a phosphate group from ATP.
• Phosphorylation activates the protein, thereby altering the metabolism of the cell
• At this stage it is useful to distinguish between two classes of messengers.
• The hormone is considered a first messenger because it brings the original message to the cell surface.
• Both cAMP and calcium serves as second messengers. Among the most common second messengers are
3′,5′-cyclic AMP (cAMP); 3′,5′-cyclic GMP (cGMP); nitric oxide (NO); cyclic ADP-ribose (cADPR); 1,2-
diacylglycerol (DAG); inositol 1,4,5-trisphosphate (IP3); and Ca2+.The function of second messenger is to
relay information from the plasma membrane to biochemical machinery inside the cell.
• Second messengers also provide for amplification of the original signal.

10. RESPONSE
Second messenger molecules enter the nucleus
Influence gene expression
Directly effects cellular processes such as release
of growth hormones
Development changes

11. HORMONE-BINDING PROTEINS IN PLANTS
Vennis (1985) proposed four generally accepted criteria that must be satisfied in
order to distinguish between nonspecific binding and hormone binding
properties.
• 1. Binding must be hormone specific.
• 2. Receptor should exhibit a high affinity for the hormone.
• 3. Receptors can be saturated by increasing the concentration of hormone
molecules.
• 4. The hormone must bind reversibly with the receptor.

12. Currently identified different plant hormone receptors
Plant hormone receptors
Hormone Receptor type Receptors
Auxin F-box protein TIR1, AFBs
Abscisic acid G-protein GTG1, GTG2, GCR2,CHLH
Chelatase
Cytokinin Two-component regulators CRE1, AHK2, AHK3
Gibberellins Hormone-sensitive lipase like GID1
Ethylene Two-component regulators ETR1, ERS1, ETR2, EIN4, ERS2
Brassinosteroids Leucine-rich repeat receptor-like kinases BRI1
Jasmonic acid F-box protein COI1
Salicylic acid Unknown
Nitric oxide Unknown
Strigolactones Unknown

13. Growth regulation at molecular level by
hormone signaling

14. AUXIN ACTION
• Exogenously applied auxin during the end of cell division/early cell expansion phase can increase
fruit size of apple due to auxin response gene, ARF106, which maps to a size-related QTL.
(Devoghalaere et al.2012)
• Uptake of auxin
• Binding of auxin to some receptor protein (ABP1)
• Release of some unknown factor/ action of some
receptor complex and IAA complex become
active and release secondary messenger
• Secondary messenger moves in cell wall region
• Action of secondary messenger to induce cell wall
loosening, ultimately decreases the wall pressure
and H2O enter the wall, thus allowing for cell
expansion with prevailing turgor.

15. GENES INVOLVED IN AUXIN REGULATION
1. The AUX/IAA gene family: The expression of most of the AUX/IAA family of genes is
stimulated by auxin within 5 to 60 minutes of hormone addition .
2. The SAUR gene family: Cloned from mung bean, pea, Arabidopsis and has proven for the
lateral transport of auxin during photo-tropism and gravitropism
3. The GH3 gene family : Early-gene family members, identified in both soybean and
Arabidopsis, Mutations in Arabidopsis GH3-like genes result in dwarfism (Nakazawa et al. 2001)
.hour
Further, GH3 expression is a good reflection of the presence of endogenous auxin, a synthetic
GH3-based reporter gene known as DR5 is widely used in auxin bioassays (Ulmasov et al. 1997).

16. GIBBERELLIN IN SEED GERMINATION
GIBBERELLIN SIGNAL TRANSDUCTION:
The biochemical and molecular mechanisms, which are probably
common to all gibberellin responses, have been studied most
extensively in relation to the gibberellin-stimulated synthesis and
secretion of α-amylase in cereal aleurone layers.
1. GA binds to receptor
2. GA-receptor binds to G-protein
3. G-protein activates F-box protein
4. F-box protein binds to DELLA-domain repressor
(GAI and RGA repressor)
5. GAMYB gene expression is activated
6. GAMYB activates alpha-amylase expression
Jacobsen et al. 1995

17. G.A. Enhances the Transcription of α- Amylase mRNA
The two main lines of evidence were as follows:
1. GA3 stimulated α-amylase production was blocked by inhibitors at transcription and translation level.
2. Heavy-isotope and radioactive-isotope-labeling studies demonstrated that the stimulation of α-amylase
activity by gibberellin involved de novo synthesis of the enzyme from amino acids, rather than
activation of pre existing enzyme. (Jacobsen et al. 1995)
In recent studies it was found that calcium and calmodulin act as second messengers. Gibberellin
application is a rise in the cytoplasmic calcium concentration.
Without calcium, α-amylase secretion does not occur, calcium is not on the signaling pathway to α-
amylase gene transcription.
Protein phosphorylation by protein kinases is another component in many signaling pathways. The
injection of a protein kinase substrate into barley aleurone protoplasts inhibited α-amylase secretion,
suggesting the involvement of protein phosphorylation in the α-amylase secretion pathway.
In conclusion, gibberellin signal transduction in aleurone cells seems to involve G-proteins as well as
cyclic GMP, leading to production of the transcription factor GAMYB, which induces α-amylase gene
transcription.

18. GIBBERLLIN ACTION
RESULTS GERMINATION OF SEED
1. GA from the embryo first binds to a cell surface receptor
2. The cell surface GA receptor complex interacts with G-
protein, initiating two separate signal transduction chains
3. A Ca independent pathway involving cGMP, results in the
activation of a signaling intermediate
4. It binds to DELLA represor proteins in nucleus, degraded
when bound to the GA signal
5. Inactivation of DELLA repressors allows the expression of
MYB gene as well as other genes, to proceed through
transcription
6. MYB protein then enters the nucleus and binds to the
promoter genes for alpha amylase and other hydrolytic
enzymes
7. Transcription of alpha amylase and other hydrolytic genes
is activated
8. Alpha amylase and other hydrolases are synthesized on the
rough ER
9. Proteins are secreted via the golgi
10. The secretory pathway requires GA stimulation via a ca-
calmodulin-dependent signal transduction pathway.

19. Summary of Gibberellin Action in germination
 The proteins GAI and SPY act as repressors of GA responses. Gibberellin acts by deactivating these repressors.
i) The gibberellin (chiefly GA,) combines with a receptor on the outer surface of plasma-membrane of aleurone layer cell.
ii) The GA-receptor complex interacts with a heterotrimeric G protein (also situated on the surface of plasma membrane) and initiates two
separate signal transduction pathways;
(a) Calcium (Ca2+) independent signal transduction pathway-
which involves cGMP as signaling intermediate (secondary messenger) leading to the expression of a-amylase gene .
(b) Calcium (Ca2+) dependent signal transduction pathway –
which involves ca, ca binding protein calmodulin and a protein kinase as signaling intermediates (secondary messengers) leading to the
stimulation of secretion of a-amylase and other hydrolytic enzymes from cells of aleurone layer into the endosperm for starch
degradation.
(The primary messenger is the hormone GA itself).
In conclusion
 α-Amylase secretion is regulated by a calcium-dependent pathway,
 whereas α-amylase gene expression is regulated by a calcium-independent pathway

20. GA signal transduction and stem elongation
The transcriptional factors GAI and RGA act
as repressors of transcription of those genes
that leads to growth.
SPY enhancing the effects of GAI and RGA.
In presence of GA, these repressors are
deactivated or degraded so that transcription of
genes occur that leads to stem elongation
(growth).

21. CYTOKININ SIGNALING
• Recent studies have demonstrated that in plants cytokinin signaling pathway comprised of sensor kinases, histidine
phosphotransfer proteins and response regulators.
• The cytokinin are perceived in plants (at least in Arabidopsis) by three related receptor histidine kinases. The sensor
proteins HISTIDINE KINASE 2 (AHK 2), AHK 3, AHK 4/CYTOKININ RESPONSE 1 (CRE 1)/WOO–
DENGLEG (WOL) a histidine–kinase domain and a receiver domain.
• CRE–family receptors and AHKs are positive, redundant elements in the cytokinin primary signal transduction
pathway.
• (AHP) are encoded by AHP genes in Arabidopsis and their transcription is not affected by cytokinin treatments.
• The AHPs interact with various Histidine sensor kinases and Arabidopsis response regulators (ARRs).
• The AHP play role in mediating phosphotransfer among these (AHKs and ARR) elements.
• Arabidopsis, 23 ARR genes are known to respond to cytokinins.
• Transcription of the type–A of these ARRs is rapidly elevated in response to exogenous cytokinin.
• The transcription of type–B ARRs genes is not altered by cytokinins.
• The type–B ARR proteins have DNA–binding GARP domain
• The type–B ARRs are transcription factors that localize to the nucleus. These are positive elements in cytokinin
signaling.
• The type–AARRs negatively regulate cytokinin signaling.

22. Model for Cytokinin signaling

23. Summary of perception and signal transduction
• Binding of cytokinin to CRE1 or other Related His Kinases
Initiation of phosphorylation
• Phosphorylation and activation of the type-B ARRs (Arabidopsis response
regulators)
• Transcription of Type-A genes which in case over-expression negatively
feedback the signaling pathway
Conclusion:
Type-A and Type-B ARRs interact with various molecules (effectors)
inside the cell and determine the kind of biochemical reactions in response
to cytokinin

24. Mechanism of action of ABA
ABA SIGNALING

25. ABA binding induces the formation of reactive oxygen species, which activate plasma membrane ca
ion channels.
ABA-induced increase in cytosolic calcium concentration and rise in intracellular pH which affect
guard cell plasma membrane channels in two ways:
1. They prevent stomatal opening by inhibiting inward K+ channels and plasma membrane proton
pumps.
2. They promote stomatal closing by activating outward anion channels, thus leading to activation of K+
efflux channels.
(Schroeder et. al .2001)

26. Ripening control in strawberry at molecular level by ABA
Uncolored fruits obtained by:
1. The expression of a gene (FaNCED1), which is key to ABA biosynthesis, was down-regulated
by using a newly established Tobacco rattle virus-induced gene silencing technique, resulting in
a significant decrease in ABA levels and uncolored fruits.
2. In the transgenic RNA interference (RNAi) fruits, in which the expression of a putative ABA
receptor gene encoding the magnesium chelatase H subunit (FaCHLH/ABAR) was down-
regulated by virus-induced gene silencing technique.
 ABA is a signal molecule that promotes strawberry ripening
(Feng Jia et al. 2011)

27. Down-regulation of FaCHLH/ABAR and FaNCED1 gene
expression, inhibits strawberry fruit ripening.
(Two-week-old fruits attached to strawberry plants)
(Feng Jia et al. 2011)
Normal expression of both genes is required for ripening and coloured fruits.

28. ABA Regulates Strawberry Fruit Ripening
(Feng Jia et al. 2011)

29. Perception and mode of action of ethylene
• Perception of ethylene occurs through a chain of events involving proteins
that were first identified in Arabidopsis. Many key components of ethylene
signal transduction pathway have been identified using effect of ethylene on
dark–grown seedlings known as the ‘triple response’.
• In Arabidopsis thaliana, the triple response is characterized by
(i) inhibition of hypocotyls and root elongation,
(ii) a thickened hypocotyls and
(iii) an exaggerated apical hook.
• Populations of mutagenized Arabidopsis were screened for seedlings that
displayed altered triple–response phenotype.
• This approach resulted in the identification of several ethylene–insensitive
mutants.

30. • These mutants include etr 1 (ethylene response), etr 2, ein 2 (ethylene–insensitive),
ein 3, ein 4, ein 5, ein 6, hls 1 (hookless) and eir 1 (ethylene insensitive root).
Mutants were also identified that exhibited a triple response in the absence of ethylene.
These include ctr 1 (constitutive triple response) and ran 1 (responsive to antagonist).
• Ethylene is perceived by a family of five membrane–bound receptors (ETR 1, ETR 2,
ERS 1, ERS 2, EIN 4) that have similarity to two–component regulators. ETR 1 was
the first plant hormone receptor to be identified.
• Binding of ethylene to ethylene receptors results in an (inactive) configuration.
• This prevents interaction of ethylene with the negative response regulator CTR1. As a
result, ethylene responses are initiated.
• Conversely (if ethylene is absent), ETR 1 binds to CTR 1, which prevents ethylene
signaling. Thus an important feature of the ethylene signaling pathway is that it
contains both positive and negative regulators, some proteins thereby serving to induce
the responses while other suppress them
Guo and Ecker (2004)

31. • The proper timing of the onset and release of dormancy impacts the survival,
productivity and spatial distribution of temperate perennials.
• Molecular mechanisms that govern the dormancy and growth changes in perennial
plants in response to seasonal climatic variation remains largely unanswered.
A molecular framework for seasonal growth dormancy
regulation in temperate perennial plants
Case study 1
Shim et al.2014

32. Regulatory programs
PHYs and PHY-
interacting
transcription
factors (PIFs).
Auxin receptor
F-box
proteinTIR1.
Dormancy
associated MAD
box (DAM)
transcription
factors, inducing
endodormancy.
Case study 1

33. (Hare Krishna,2012)
5.Growth cease
6. Dormancy
induced
Phytochrome
1. Onset of low temperature & shorter day
length and high temperature
2. Signal perception &
Transduction
3.Metabolic
activity decrease
4. High level of
Endogenous PG
inhibitors accumulation
7. Dormancy
maintained
8. Dormancy released

34. • Understanding of the molecular mechanisms controlling the annual growth and dormancy cycle
has the potential to help mitigate the impact of climate change on plant productivity and survival
by providing vital information about how temperate perennials utilize the environmental cues to
trigger adaptive mechanisms.
• Identification of expression based molecular markers for dormancy regulation may facilitate
cultivar selection and breeding for development of regionally suited crops in accord with changes
in the global climate.
Results :

35. Selected biological process of differentially expressed genes during dormancy release
of horticultural woody crops identified by genome-wide transcriptomic analysis Yamane ,2014

36. Genes dormant buds:
• DORMANCY-ASSOCIATE MADS-box genes (DAM)
DAM4 & DAM6
- Raspberry, Japanese Apricot, and Peach.
• Up regulation - during induction of dormancy
• Down regulation – during release of dormancy
Epigenetic regulation of bud dormancy events in perennial
plants
Rios et al., 2014
Case study 2

37. Chestnut:
DNA methylation (DNAme)
Acetylation of histone (H4ac)
Peach
General and specific modifications of chromatin in dormant and
dormancy-released buds.
Peach:
Acetylation of H3 (H3ac)
Trimethylation of H3 at K4 (H3K4me3)
Trimethylation of H3at K27 (H3K27me3)
Case study 2

38. The study done was aimed to characterize Satsuma mandarin (Citrus unshiu
Marc.) GA 2-oxidase genes encoding enzymes with GA inactivation activity
because the accumulation of active GAs is regulated by the balance between their
synthesis and inactivation.
CuGA2ox4, CuGA2ox2/3, and CuGA2ox8 were differentially expressed in
various tissues in Satsuma mandarin and that these genes functioned like GA 2-
oxidase genes in transgenic Arabidopsis.
Gibberellin 2-Oxidase Genes from Satsuma Mandarin (Citrus unshiu Marc.)
Caused Late Flowering and Dwarfism in Transgenic Arabidopsis.
Kotoda et al.,2016
Case study 3

39. Expression patterns of CuGA2ox4 (A), CuGA2ox2/3 (B), and
CuGA2ox8(C)
•New leaves (NL)
•Old leaves (OL)
•Shoot apices (SA)
• Flower buds(FB)
•Young fruit (YF)
• Juice sacs (JS)
•Peel (PL)

40. • CuGA2ox4, CuGA2ox2/3, and CuGA2ox8 were differentially expressed in various
tissues in Satsuma mandarin and that these genes functioned like GA 2-oxidase genes
in transgenic Arabidopsis.
• Further study of GA biosynthetic genes including GA 2-oxidase genes will provide
insight into the mechanism of flowering, fruit development, seedlessness, biennial
bearing, and the peel puffins citrus such as Satsuma mandarin.
Result:

41. • Little is known of the precise physiological or genetic basis of the phenomenon of rootstock-induced
dwarfing in apple (Malus · domestica Borkh).
• Gene Dw1 was mapped.Dw1 is the first reported mapped locus controlling the dwarfing ability of the
apple rootstock.
• Dw1 is a major component of dwarfing, as most of the dwarfing and semidwarfing rootstocks carried
the dwarfing allele of this locus.
Genetic Markers Linked to the Dwarfing Trait of Apple Rootstock ‘Malling 9’
Pilcher et al.,2008
Case study 4

42. • Massive young fruit abscission usually causes low and unstable yield in litchi (Litchi chinensis
Sonn.)
• This study aimed at identification of molecular components involved in fruitlet abscission in litchi,
for which reference genome is not available at present.
• Profiling was performed to screen and identify candidate genes involved in fruit abscission
induced by girdling plus defoliation (GPD).
• qRTPCR was used to explore the expression pattern of 15 selected candidate genes in the
abscission zone, pericarp, and seed.
Li et al.,2015
Case study 5
An improved fruit transcriptome and the candidate genes involved in
fruit abscission induced by CHO stress in litchi

43. Preliminary framework of the gene network involved in litchi fruit abscission induced by carbohydrate
stress.

44. • Fruit ripening is a highly coordinated developmental process that
coincides with seed maturation.
• The ripening process is regulated by thousands of genes that control
progressive softening and/or lignification of pericarp layers,
accumulation of sugars, acids, pigments, and release of volatiles.
Molecular approaches for regulation of fruit
ripening- A review
Porika et al.,2015
Case study 6

45. Non climacteric fruit
Osorio et al., 2013

46. Genes and their predicted function related to papaya
ripening
Case study 6

47. Genotype Activity Function Crop References
ACO Amino carboxylicacid cyclopropane
oxidase
Ethylene biosynthesis Strawberry Kess et al, 2007
ACS ACC synthase Ethylene biosynthesis Strawberry Kess et al, 2007
CTR1 Constitutive triple response gene controls
kinase activity
Ethylene signalling - Gapper et al, 2013
EIN EIN binding protein Reduces the ethylene sensitvity Mandarin Gapper et al, 2013
RAP2-4 Ethylene-responsive transcription factor-
fruit ripening via light and ethylene
signaling pathway
Ethylene biosynthesis Papaya cv. Eksotika Shin et al, 2011
Adomet S-adenosylmethonine synthetase Ethylene biosynthesis Papaya cv. Eksotika Shin et al, 2011
FFT ß-fructofuranosidase-invertase enzyme
breakdown the sucrose to fructose and
glucose
Starch mobilization Mango Betfield et al, 2005
R1 ɑ-glucan-water-dikanse-phosphorylation of
starch polymers both at developing and
ripening stage
Starch mobilization Mango Betfield et al, 2005
List of genes involved in fruit ripening and their regulatory activities

48. Limitation
• Cost : A major obstacle
Cost associated with identifying and utilizing
multiple genes is high.
• Well trained manpower
• Health hazards – molecular involve use of radioactive isotopes for
labeling.