Presented by:-

POOJA SATPATHY

Introduction

• Hollow tube, develops from a pollen grain when deposited on the stigma of a flower

• Basic function:-

=>It penetrates the style and conveys the male gametes to the ovule.

The pollen tube. DIC image of

a Lilium longiflorum pollen

tube

Kai R Konrad, Michael M Wudick1 and Jose´ A Feijo (2011). Calcium regulation of tip growth: new genes for old mechanisms. Current Opinion in Plant Biology, 14:721–730

Formation of Pollen tube

Diagrammatic representation of a

pollen tube with the typical zonation

A B C D

A => Clear zone B => Subapical domain

C => Nuclear domain D => Vacuolar domain

Kai R Konrad, Michael M Wudick1 and Jose´ A Feijo (2011). Calcium regulation of tip growth: new genes for old mechanisms. Current Opinion in Plant

Biology, 14:721–730

Major factor driving the pollen tube

elongation

1. A steep calcium gradient within the pollen

tube tip

2. The contribution of actin microfilaments to

the elongation process

3. Vesicle trafficking

Calcium in pollen tube elongation

• Necessary

• Impacts on growth direction

• Injection of 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-

tetraacetic acid (BAPTA) buffer (Ca2+ channel blockers),

inhibits elongation and abrogates the Ca2+ gradient

Calcium channels

• Responsible for movement of calcium across membranes

• Calcium-permeable channels are likely to contribute to the

gradient formation

• Transcriptomic data from Arabidopsis pollen revealed the

presence of various putative Ca2+ transport systems, pointing

out to about 20 putative Ca2+ channels

Genes for calcium channels

1) Two-pore channel 1 (TPC1)

• first gene to code for a bonafide Ca2+ channel

in plants

• Expressed on the tonoplast

• Roles in seed germination and stomata

movement

Genes for calcium channels

2) Cyclic nucleotide gated channels (CNGC)

• Forms a second group of putative Ca2+ channels

CNGC18 (best characterized)

Overexpression

Abnormal pollen

tube growth

Gene depletion by T-

DNA insertion

Male sterility

Genes for calcium channels

3) Glutamate receptor-related channels (GLRs)

proper pollen tube growth

fertility

• Third putative group

• Direct electrophysiological, pharmacological and genetic evidence shows Ca2+ transport activity in pollen by AtGLR1.2 and AtGLR3.7

GLR importance in pollen tube growth

• Used model plant Arabidopsis

• Mutant plants (achieved by T-DNA insertion)

Atglr3.7-1 Atglr1.2-1

Pollen tubes grow

slower than wild

type

Reduced number

of seeds per

silique

Abnormal,

deformed tips and

tubes

Reduced number

of seeds per

silique

Result

Erwan Michard, Pedro T. Lima, Filipe Borges, Ana Catarina Silva, Maria Teresa Portes,João E. Carvalho, Matthew Gilliham, Lai-Hua Liu,

Gerhard Obermeyer, José A (2011). Feijó. Glutamate Receptor–Like Genes. Form Ca2+ Channels in Pollen Tubes and Are Regulated by Pistil

D-Serine. SCIENCE, VOL 332; 434-437

D-serine increases [Ca2+]cyt in Arabidopsis

pollen tubes

Erwan Michard, Pedro T. Lima, Filipe Borges, Ana Catarina Silva, Maria Teresa Portes,João E. Carvalho, Matthew Gilliham, Lai-Hua Liu,

Gerhard Obermeyer, José A (2011). Feijó. Glutamate Receptor–Like Genes. Form Ca2+ Channels in Pollen Tubes and Are Regulated by Pistil

D-Serine. SCIENCE, VOL 332; 434-437

(A) Typical YC3.6 cameleon imaging in a

growing Arabidopsis pollen tube.

(B) After D-Ser (5 mM) application, tubes exhibit

an increase in [Ca2+]cyt and an extension of the

gradient toward the subapical zone.

(C and D) Kymographs from the tubes presented

in (A) and (B), respectively.

The slope of the kymograph represents the

growth rate of the tube.

Minimum (*) and maximum (**) [Ca2+]cyt at the tip of the same cell

Ca2+ transport systems

Fig (a): Ca2+ transport systems, sensor proteins and

cytoskeleton elements in pollen tubes. Schematic

drawing of a pollen tube with its polarized structure

• Cytoskeleton elements assemble

as bundles in the shank, allow organelle,

vesicle and cargo transport

• Depolymerization of actin polymers

(profilin)

• Steep tip-focused Ca2+ gradient achieved by

Ca2+-transporting proteins (CNGCs, GLRs)

• Ca2+ and H+ extrusion from the tube occurs

at the shank, through ACA9, a Ca2+ pump

• Intracellular Ca2+ sequestration into the

vacuole occur through the cation-permeable

channel TPC1

• Ca2+ sensor proteins, form a sophisticated

network (Ca2+-dependent protein kinases)

Kai R Konrad, Michael M Wudick1 and Jose´ A Feijo (2011). Calcium regulation of tip growth: new genes for old mechanisms. Current Opinion in Plant Biology, 14:721–730

Ion transport systems and Ca2+ regulatory networks

in plant cells

Fig (b): Ion transport systems and Ca2+ regulatory networks

in plant cells. Schematic drawing of a plant cell pointing out

known Ca2+-binding proteins and their interaction with various

ion transport systems.

• Interaction of Calcineurin B-like

interacting protein kinases (CIPKs)

with Calcineurin B-like proteins

(CBLs), activation of plasma

membrane- (AKT1, SOS1)

• CPK-triggered activation of a plasma

membrane NADPH-oxidase results in

the production of reactive oxygen

species (ROS) that subsequently

activates Ca2+-permeable channels in

the plasma membrane

• These channels, in concert with Ca2+

pumps, regulate the intracellular Ca2+

concentration

Kai R Konrad, Michael M Wudick1 and Jose´ A Feijo (2011). Calcium regulation of tip growth: new genes for old mechanisms. Current Opinion in Plant Biology, 14:721–730

Actin in pollen tube elongation

• Actin, essential for the polarized tip growth

• Along with myosin motors, support vesicular transport and

other crucial processes

• Latrunculin B and cytochalasin B (inhibit actin

polymerisation), blocks pollen tube elongation

Actin Binding Proteins (ABPs)

• Involved in :-

=> Actin’s polymerization

=> Depolymerization

=> Stability

=> Organisation in bundles or networks

=> Fragmentation and destruction

Actin Binding Proteins (ABPs)

1. Actin-related protein 2 and 3 (Arp2/3) complex

• Action=> stimulates actin assembly from pre-existing actin filaments to

produce branched actin networks

2. Profilin

• Action => -Binds monomeric actin

-modulates actin nucleation

-enhances actin polymerization

• Increase in amount => disrupts actin organization => inhibits tube growth

• Ca2+ affect profilin activity spatially and temporally

Actin Binding Proteins (ABPs)

3. Actin depolymerizaing factor (ADF)

• Action => -binds to actin filament cooperatively

-severs and stimulates the depolymerization of actin filaments

• Mild overexpression=> disrupts actin organization=> inhibits tube growth

• Inhibited by phosphorylation at a conserved serine residue in the N-terminal

region; under the control of a Rho GTPase signaling cascade

4. Formins

• Action => -stimulate actin assembly de novo, produce linear actin cables

-activity enhanced by interacting with profilin

• Slight increases in AFH1 (Arabidopsis formin homology 1) levels, growth is

moderately stimulated

• Overexpression of AFH1=> induces actin cables and pollen tube depolarization

Actin Binding Proteins (ABPs)

5. Villins/gelsolins

• Action => -actin severing proteins

-some also show bundling, capping, and actin-nucleating activity

• Overexpression of lily ABP29 => obliterate the actin cytoskeleton

• reduction in ABP41 (by antibody injection in lily pollen tubes) results in

growth inhibition

6. Capping proteins

• Action => bind and modulate polymerization on the rapidly growing end of

actin filaments

• phosphatidic acid (PA) inhibits AtCP1, a heterodimeric CP from Arabidopsis,

increase actin polymerization

GTPases: in vesicle trafficking

Rab GTPases

• Organize intracellular membrane trafficking by:-

=> Membrane budding and vesicle formation

=> Tethering vesicles to target areas

=> promoting fusion of vesicles with target membrane

• NtRab2 (pollen predominant Rab2 from Tobacco)

- vesicle trafficking between ER and Golgi bodies, transporting cell membrane and secretory proteins

- Mutation=> Inhibit proteins transport=> suppress pollen tube elongation

• NtRab11b (member of Rab11 subfamily of Tobacco)

- in apical accumulation of transport vesicles

- DN or CA expression of NtRab11b=> inhibits pollen tube growth, disrupts growth directionality and cause male fertility

Termination of extension of pollen

tube

• AtCSLA7 (Cellulose synthases, assembly of cellulose

microfibrils) from A. thaliana

=> Ubiquitously expressed β-glycosyltransferase that affects

late stages of growth and/or termination of extension of the

pollen tube

References

• Kai R Konrad, Michael M Wudick1 and Jose´ A Feijo (2011). Calcium regulation of tip growth: new genes for old mechanisms. Current Opinion in Plant Biology, 14:721–730

• Alice Y. Cheung and Hen-mingWu (2008). Structural and Signaling Networks for the Polar Cell Growth Machinery in Pollen Tubes. Annu. Rev. Plant Biol. 59:547–72

• Erwan Michard, Pedro T. Lima, Filipe Borges, Ana Catarina Silva, Maria Teresa Portes,João E. Carvalho, Matthew Gilliham, Lai-Hua Liu, Gerhard Obermeyer, José A (2011). Feijó. Glutamate Receptor–Like Genes. Form Ca2+ Channels in Pollen Tubes and Are Regulated by Pistil D-Serine. SCIENCE, VOL 332; 434-437

• Alexander Krichevskya, Stanislav V. Kozlovskya, Guo-Wei Tianb, Min-Huei Chena, Adi Zaltsmana, Vitaly Citovskya(15 March 2007). How pollen tubes grow. Volume 303, Issue 2, Pages 405–420.

• Leonie Steinhorst, Jörg Kudla (July 2013). Calcium - a central regulator of pollen germination and tube growth. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research,volume 1833, Issue 7, Pages 1573–1581