The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

First published November 2009 Revised February 2011

Genetic control of leaf development

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Lecture Outline •  Genetic control of leaf identity •  Genetic control of leaf polarity •  Genetic control of differentiation •  Vascular tissues •  Guard cells •  Trichomes

Reprinted by permission from Macmillan Publishers, Ltd: Runions, J. (2003) Cell of the Month. Nat. Rev. Mol. Cell Biol. 4: 603 (Copyright 2003) ; and Hay, A., and Tsiantis, M. (2006) The genetic basis for differences in leaf form between Arabidopsis thaliana and its wild relative Cardamine hirsuta. Nat. Genet. 38: 942-947. Copyright (2006).

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

First published November 2009 Revised February 2011

Leaves are formed at the shoot apical meristem (SAM) SAM

The tiny leaves that surround the meristem are called leaf primordia

SAM

SAM Arabidopsis thaliana Nicotiana tabacum (tobacco) The meristem is usually 0.1 – 1 mm in diameter

Oryza sativa (rice)

Image sources: Poethig , R.S. and Sussex ,I.M. (1985) The developmental morphology and growth dynamics of the tobacco leaf. Planta 165: 158-169. Copyright (1985) Planta. Reprinted with kind permission of Springer Science+Business Media; Itoh, J.-I., Kitano, H., Matsuoka, M., and Nagato, Y. (2000) SHOOT ORGANIZATION genes regulate shoot apical meristem organization and the pattern of leaf rrimordium initiation in rice. Plant Cell 12:2161-2174; Reprinted by permission from Macmillan Publishers, Ltd:. Long, J.A., Moan, E.I., Medford, J.I., and Barton, M.K. (1996) A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 379: 66-69.

Many plants produce leaves in a spiral pattern Leaf primordia are numbered P1, P2 etc from youngest to oldest The next leaf to form is called the incipient primordium, I1

I1

Poethig , R.S. and Sussex ,I.M. (1985) The developmental morphology and growth dynamics of the tobacco leaf. Planta 165: 158-169. Copyright (1985) Planta. Reprinted with kind permission of Springer Science+Business Media.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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An accumulation of the hormone auxin promotes leaf initiation

Indole-3-acetic acid (IAA) a naturally occurring auxin Auxin accumulation precedes leaf initiation Poethig , R.S. and Sussex ,I.M. (1985) The developmental morphology and growth dynamics of the tobacco leaf. Planta 165: 158-169. Copyright (1985) Planta. Reprinted with kind permission of Springer Science+Business Media.

Genetic control of leaf identity How does a leaf primordium become a leaf, rather than part of the meristem?

Leaf primordium Meristem

Leaf primordium

Poethig , R.S. and Sussex ,I.M. (1985) The developmental morphology and growth dynamics of the tobacco leaf. Planta 165: 158-169. Figure 3 Copyright (1985) Planta. Reprinted with kind permission of Springer Science+Business Media.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

First published November 2009 Revised February 2011

Cells in the meristem are indeterminate • Cells in the meristem are functionally distinct from those in the leaf primordia • They are indeterminate and serve as a stem-cell population

Poethig , R.S. and Sussex ,I.M. (1985) The developmental morphology and growth dynamics of the tobacco leaf. Planta 165: 158-169. Figure 3 Copyright (1985) Planta. Reprinted with kind permission of Springer Science+Business Media.

KNOX-1 genes maintain the meristem in an indeterminate state

KNOX-1 Class I KNOX genes (KNOX-1) • (KNOX means Knotted-like homeobox) • Encode transcription factors • Expressed in meristem • Not expressed in incipient primordia • Help maintain indeterminate growth

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

First published November 2009 Revised February 2011

KNOTTED expression

KNOTTED (a KNOX-1 gene) mRNA accumulates in the meristem but not the leaf primordia (arrows) of Zea mays

Jackson, D., Veit, B., and Hake, S. (1994) Expression of maize KNOTTED1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot. Development 120: 405–413. Reproduced with permission.

STM, a KNOX-1 gene, is necessary for meristem formation

Wild-type plant showing leaf formation at the shoot apex.

The shootmeristemless mutant (stm) fails to form a shoot apical meristem during embryogenesis; notice the absence of leaf formation.

Reprinted by permission from Macmillan Publishers, Ltd:. Long, J.A., Moan, E.I., Medford, J.I., and Barton, M.K. (1996) A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 379: 66-69.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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KNOX genes act in part by stimulating cytokinin synthesis Arabidopsis stm1 mutant

stm1 mutant expressing IPT from STM promoter

STM

IPT

CK

The shootmeristemless1 mutant (stm1) fails to initiate a shoot apical meristem. This mutant can be rescued by CK application or by expression of the cytokininbiosynthesis IPT gene at the SAM. STM is a transcription factor that induces expression of an IPT gene. Reprinted from Yanai, O., et al. (2005). Arabidopsis KNOXI Proteins activate cytokinin biosynthesis. Curr Biol. 15: 1566-1571, with permission from Elsevier.

Overexpression of KNOX-1 genes increases leaf complexity and indeterminacy Gain of function in leaf primordia

KNOX-OE

Arabidopsis

WT

KNOX-OE (overexpression)

Tobacco

WT

Maize

KNOX-OE WT KNOX-OE

Chuck, G., Lincoln, C., and Hake, S. (1996) KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis. Plant Cell 8: 1277-1289. ; Reprinted by permission from Macmillan Publishers, Ltd: Hake, S., and Ori, N. (2002) Plant morphogenesis and KNOX genes. Nat. Genet. 31: 121 – 122.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Cells in leaf primordia become determinate • Cells in the primordia are functionally distinct from those in the meristem • They become determinate

Poethig , R.S. and Sussex ,I.M. (1985) The developmental morphology and growth dynamics of the tobacco leaf. Planta 165: 158-169. Figure 3 Copyright (1985) Planta. Reprinted with kind permission of Springer Science+Business Media.

Primordium-specific genes promote differentiation ARP ARP ARP genes • “ARP” is derived from three genes, ASYMMETRIC LEAF1, ROUGH SHEATH2, and PHANTASTICA • ARP genes encode MYB transcription factors • Expressed in cells of leaf primordia •  Promote determinate growth and differentiation

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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The activities of ARP and KNOX-1 genes are mutually antagonistic The two classes of transcription factors are mutually repressive, and help establish a separate identity for the emerging leaf primordium

ARP

KNOX-1

ASYMMETRIC LEAF1 (AS1) mRNA is expressed in cotyledons but not in the meristem ARP

KNOX-1

Reprinted by permission from Macmillan Publishers, Ltd:. Byrne, M.E., Barley, R., Curtis, M., Arroyo, J.M., Dunham, M., Hudson, A., and Martienssen, R.A. (2000) Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis. Nature 408: 967-971.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

Wild type

First published November 2009 Revised February 2011

In the stm mutant, AS1 is expressed in the meristem (arrow)

stm mutant

ARP

Reprinted by permission from Macmillan Publishers, Ltd:. Byrne, M.E., Barley, R., Curtis, M., Arroyo, J.M., Dunham, M., Hudson, A., and Martienssen, R.A. (2000) Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis. Nature 408: 967-971.

Loss of ARP gene function resembles KNOX-1 overexpression rs2

WT

rs2 WT

rs2

Gain of function in leaf primordia

KNOX-OE

Wild-type

as1

Reprinted by permission from Macmillan Publishers, Ltd:. Byrne, M.E., Barley, R., Curtis, M., Arroyo, J.M., Dunham, M., Hudson, A., and Martienssen, R.A. (2000) Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis. Nature 408: 967-971. Reproduced with permission from Schneeberger, R., Tsiantis, M., Freeling, M., Langdale, J. Development (1998) 125: 2857-2865.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Genetic control of leaf complexity

Simple

Pea, tomato and Cardamine hirsuta are genetic models in which to study compound leaves

Compound

Redrawn from Champagne, C., and Sinha, N. (2004). Compound leaves: equal to the sum of their parts? Development 131:4401-4412

Expression of KNOX1 transcription factors correlates with leaf complexity

- KNOX1

+ KNOX1

Wild-type Cardamine hirsuta Reprinted by permission from Macmillan Publishers, Ltd: NATURE GENETICS 38: 942-947. Hay, A., and Tsiantis, M.The genetic basis for differences in leaf form between Arabidopsis thaliana and its wild relative Cardamine hirsuta. Copyright (2006).

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

Simple leaf

First published November 2009 Revised February 2011

In plants with simple leaves, KNOX1 expression remains off in leaf primordia KNOX1 off in leaves

From Bharathan, G., Goliber, T.E., Moore, C., Kessler, S., Pham T., and Sinha, N.R. (2002) Homologies in leaf form inferred from KNOXI gene expression during development. Science 296: 1858-1860. Reprinted with permission from AAAS.

Simple leaf

Compound leaf In plants with compound leaves, KNOX1 expression turns back on in primordia KNOX1 off in leaves

KNOX1 ON in leaves

From Bharathan, G., Goliber, T.E., Moore, C., Kessler, S., Pham T., and Sinha, N.R. (2002) Homologies in leaf form inferred from KNOXI gene expression during development. Science 296: 1858-1860. Reprinted with permission from AAAS.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Cytokinin contributes to compound leaf development

KNOX1

Wild-type

Increased cytokinin synthesis

Increased cytokinin degradation

Cytokinin biosynthesis

Leaflet proliferation

Shani, E., Ben-Gera, H., Shleizer-Burko, S., Burko, Y., Weiss, D., and Ori, N. (2010). Cytokinin regulates compound leaf development in tomato. Plant Cell 22: 3206–3217.

KNOX1 genes have a recurring role in leaf development

KNOX1

KNOX1 KNOX1 genes are repressed at the site of leaf primordium initiation

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KNOX1

Simple leaves (e.g. Arabidopsis) KNOX1 genes stay off

Compound leaves (e.g. tomato) KNOX1 genes turn on again in developing leaf primordia, conferring prolonged organogenic activity on the leaf edges

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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A PIN1-generated auxin maximum precedes leaflet formation PIN1pro

PIN1:GFP

PIN1 is oriented to direct auxin to the site of leaflet initiation.

DR5pro

YFP

Auxin accumulation precedes leaflet formation.

Reprinted by permission from Macmillan Publishers Ltd: Barkoulas, M., Hay, A., Kougioumoutzi, E., and Tsiantis, M. A developmental framework for dissected leaf formation in the Arabidopsis relative Cardamine hirsuta. Nat. Genet. 40: 1136-1141. copyright (2008)

Polar auxin transport is necessary for compound leaf formation Wild-type

pin1 mutant

pin1 Wild-type mutant

Wild-type NPA treated

In the loss-of-function pin1 mutant of Cardamine hirsuta, or in leaves treated with an inhibitor of polar auxin transport (NPA), leaflet formation is suppressed. Reprinted by permission from Macmillan Publishers Ltd: Barkoulas, M., Hay, A., Kougioumoutzi, E., and Tsiantis, M. A developmental framework for dissected leaf formation in the Arabidopsis relative Cardamine hirsuta. Nat. Genet. 40: 1136-1141. copyright (2008)

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Auxin has a recurring role in leaf development Auxin maxima precede leaf initiation (yellow arrow), leaflet initiation (white) and lobe initiation (red)

Koenig, D., Bayer, E., Kang, J., Kuhlemeier, C., and Sinha, N. (2009) Auxin patterns Solanum lycopersicum leaf morphogenesis. Development 136: 2997-3006. Reproduced with permission.

Genetic control of leaf polarity Most leaves have polarity – they are functionally and anatomically different on their upper and lower surfaces

Adaxial surface – light harvesting

CO2

O2

Abaxial surface transpirational water loss, respiratory gas exchange

Juarez, M. T., Twigg, R.W., and Timmermans, M.C.P. (2004) Specification of adaxial cell fate during maize leaf development. Development 131: 4533-4544. Reproduced with permission.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Establishment of leaf polarity

Leaf

Leaf

Leaf primoridia have inherent polarity because one side is closer to the meristem and one side is farther away from the meristem

Establishment of leaf polarity

Leaf

Adaxial Abaxial

The meristem side is adaxial, and away side is abaxial How does a leaf “know” which side is which?

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Experimental studies of leaf polarity Incipient primordia were surgically isolated from the rest of the meristem by a small incision

P3 I1

I3 P1 P2

I2

Experimental studies of leaf polarity

P3 I1

I3 P1 P2

I2

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The isolated primordium lost polarity (it became entirely abaxialized) and became radially-symmetrical

P3 I1

I3 P1

P2

I2

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Experimental studies of leaf polarity

A more recent experiment showed that laser ablation of only the epidermal cell layer is sufficient for the primordium to lose its adaxial polarity QuickTime™ and a TIFF (Uncompressed) decompressor ar e needed to see this picture.

Reinhardt, D., Frenz, M., Mandel, T., and Kuhlemeier, C. (2005) Microsurgical and laser ablation analysis of leaf positioning and dorsoventral patterning in tomato. Development 132: 15-26. Reproduced with permission.

Establishment of leaf polarity • A signal from the meristem moves through the epidermis into the incipient primordium • The signal conveys the adaxial positional information • The nature of the signal is not known

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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This elusive signal is called the “Sussex signal”

Ian Sussex proposed in the 1950s that a signal from the meristem is required for proper leaf polarity

Reprinted, with permission, from the Annual Review of Plant Physiology and Plant Molecular Biology, Volume 49 (c) 1998 by Annual Reviews www.annualreviews.org

Genetic control of leaf polarity The loss-of-function phantastica mutant of Antirrhinum (snapdragon) gives important clues to the basis of leaf polarity Wild-type

phan

Waites, R., and Hudson, A. (1995) phantastica: a gene required for dorsoventrality of leaves in Antirrhinum majus. Development 121: 2143 – 2154. Reproduced with permission.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Genetic control of leaf polarity

Wild-type leaf

phan mutant leaf

The phantastica mutant has radially symmetrical leaves. PHAN encodes a transcription factor Waites, R., and Hudson, A. (1995) phantastica: a gene required for dorsoventrality of leaves in Antirrhinum majus. Development 121: 2143 – 2154. Reproduced with permission.

Genetic control of leaf polarity

Wild-type leaf

phan mutant leaf

Mutant phan leaves are abaxialized, indicating that PHAN is necessary for adaxial cell fate Waites, R., and Hudson, A. (1995) phantastica: a gene required for dorsoventrality of leaves in Antirrhinum majus. Development 121: 2143 – 2154. Reproduced with permission.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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The phan mutant leaves resemble the surgically isolated leaf primordia P3 I1

I3 P1

P2

I2

Surgical isolation

phan mutant leaf

In Arabidopsis, three redundant genes, PHABULOSA (PHB), REVOLUTA (REV) and PHAVOLUTA (PHV), function similarly to PHAN

Loss of function kanadi mutants have radial, adaxialized leaves • A triple mutant kanadi 1,2 and 3 has radial, adaxialized leaves, showing that KANADI genes promote abaxial cell fate. • This is the opposite function as PHAN • KANADI genes encode transcription factors Eshed Y et al., Izhaki, A., Baum, S.F., Floyd, S.K., and Bowman, J.L. (2004) Asymmetric leaf development and blade expansion in Arabidopsis are mediated by KANADI and YABBY activities. Development 131: 2997-3006. Reproduced with permission.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Domain-specific transcription factor expression controls polarity There is some mutual repression of genes specifying abaxial and adaxial fate

KAN

Abaxial fate

PHAN or PHB/PHV/REV

Adaxial fate

ARP

Mutual repression is an effective way for adjacent cells to adopt and maintain different fates.

KNOX-1

PHB expression is also regulated by a miRNA x AAAAAAA

miR166

PHB mRNA PHB-1D mRNA

O AG AAAAAAA

In wild-type plants, miR166 binds to the PHB mRNA and degrades it on the abaxial side of the leaf primordium

In phb-1d plants, base changes in the PHB mRNA prevent miR166 from binding to it, allowing it to accumulate throughout the leaf primordium

Reprinted by permission from Macmillan Publishers, Ltd: NATURE. Kidner, C.A. and Martienssen, R.A. Nature 428: 81-84, copyright 2004.; McConnell, J.R., Emery, J., Eshed, Y., Bao, N., Bowman, J., and Barton, M.K. Nature 411: 709-713, copyright 2001.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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miRNA control of leaf polarity Additional support for a role of miRNA in leaf polarity comes from the fact that the ago1 mutant has radial leaves; AGO is needed for miR166 function O AG

AAAAAAA

Reprinted by permission from Macmillan Publishers, Ltd: NATURE. Kidner, C.A. and Martienssen, R.A. Nature 428: 81-84, copyright 2004; McConnell, J.R., Emery, J., Eshed, Y., Bao, N., Bowman, J., and Barton, M.K. Nature 411: 709-713, copyright 2001.

Summary - Establishment of polarity in leaves In some cases domainspecific expression is maintained by miRNAs

Different transcription factors specify adaxial and abaxial domains

miR166

KAN

PHAN or PHB/PHV/REV

Abaxial fate

Adaxial fate

Meristem-derived signal

The inner surface of a leaf is specified by a signal derived from the meristem

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Genetic control of leaf size and shape

Leaf form is determined by patterns of cell division and expansion

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Size is determined by growth, shape is determined by differential growth

Uniform growth

Differential growth

Both absolute and relative growth rates are tightly controlled in expanding leaves Image credit: From Lewis Carroll's Alice in Wonderland (1865), illustrated by John Tenniel, from The Victorian Web.

Leaf blade growth in tobacco The pattern of blade expansion can be examined by tracing a grid onto a young leaf

TIME

Poethig , R.S. and Sussex ,I.M. (1985) The developmental morphology and growth dynamics of the tobacco leaf. Planta 165: 158-169. Figure 2 Copyright (1985) Planta. Reprinted with kind permission of Springer Science+Business Media.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Leaf blade growth in dicots Area Length

Leaf expansion stops first at the tip, and later at the base

The shape of the leaf is caused by more growth at the base than the tip Relative growth rate

Poethig , R.S. and Sussex ,I.M. (1985) The developmental morphology and growth dynamics of the tobacco leaf. Planta 165: 158-169. Figure 2 Copyright (1985) Planta. Reprinted with kind permission of Springer Science+Business Media.

Patterns of cell division correlate with blade expansion Cell division arrests first at the tip and later at the base

Cell division is determined by transcription of a cyclin gene, assayed using a reporter construct Cyclin-pro

Day 12

GUS Day 4

Day 8

1 mm Redrawn from Donnelly et al., (1999) Dev Biol 215: 407-419.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Maize leaves grow mostly linearly

Initially the primordium expands isodiametrically (in all directions) At later stages, the leaf grows by uni- or bi-directional cell division and expansion

Image courtesy of J. Derksen, J. Hiddink and E.S. Pierson Copyright Radboud University Nijmegen

Growth in some leaves is more complex In wider leaves, cell divisions contribute to the width of the blade. Complex leaf shapes are formed by persistent growth in isolated regions of the developing blade

Image courtesy of J. Derksen, J. Hiddink and E.S. Pierson Copyright Radboud University Nijmegen

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Leaf shape is under genetic control

Many genes have been identified that contribute to the final form of the leaf through controls of cell division and expansion Image courtesy of J. Derksen, J. Hiddink and E.S. Pierson Copyright Radboud University Nijmegen

Genetic control of cell differentiation

Leaf development involves cell differentiation as well as regulated growth patterns. Remain undifferentiated Differentiate

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Leaves have specialized cells including veins, trichomes and guard cells Trichome

Upper epidermis Palisade mesophyll Veins Spongy mesophyll

Lower epidermis Guard cells

Guard cells

Leaf veins transport water and nutrients

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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The position of the midvein is specified by auxin After a leaf primordium is initiated, auxin moves basipetally and specifies the site of the midvein

Auxin redistributing in a very young primordium – arrows indicate direction of auxin transport

In an older primordium the future midvein is clearly demarcated. (Inset shows “top down” view)

Bayer, E.M, Smith, R.S., Mandel, T., Nakayama, N., Sauer, M., Prusinkiewicz, P., and Kuhlemeier, C. (2009) Integration of transport-based models for phyllotaxis and midvein formation. Genes Dev 23: 373-384.

Second-order veins are formed by further auxin redistributions

Adaxial surface of developing leaf

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Vascular differentiation follows position specification by auxin Day 3 1. 

Auxin redistribution

2. 

Increased [Auxin]

3. 

Pre-procambium differentiation

4. 

Procambium differentiation

Day 4

Day 5 PIN1:GFP

Scarpella et al., (2006) Genes Dev. 2006 20: 1015-1027

DR5pro:GFP

HB8pro:GUS

Et1335pro:GUS

Scarpella, E., Marcos, D., Friml, J., and Berleth, T. (2006). Control of leaf vascular patterning by polar auxin transport. Genes Dev. 20: 1015-1027.

Elongated procambial cells form by parallel cell divisions

Et1335pro::GUS Scarpella, E., Francis, P., and Berleth, T. (2004) Stage-specific markers define early steps of procambium development in Arabidopsis leaves and correlate termination of vein formation with mesophyll differentiation. Development 131: 3445-3455. Reproduced by permission.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Xylem and phloem are derived from the procambium

x

Phloem Procambium Xylem

Turner S, Sieburth LE (2003) Vascular Patterning: March 22, 2003. The Arabidopsis Book. Rockville, MD: American Society of Plant Biologists.

Summary – vein development Auxin transport establishes sites of vein formation A program of gene expression guides cells to differentiate into vascular cells

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Differentiation of cells in the epidermis The leaf epidermis comprises pavement cells, trichomes and guard cells

Nicotiana alata

Image credit Louisa Howard, Dartmouth University

Trichomes are epidermal hairs Some trichomes protect plants by deterring insects and herbivores

Trichomes on a sundew carnivorous plant

Trichomes of stinging nettle Urtica dioica. Photo credit Tom Donald; Jerome Prohaska

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Trichomes are epidermal hairs Some trichomes reflect light to reduce UV and heat absorption. Seed trichomes such as cotton fibers aid seed dispersal

Tidestromia oblongifolia

Gossypium hirsutum

Photo credits 2007 Steve Matson; Mike Doughtery from the National Cotton Council

Trichomes are structurally and developmentally diverse  

   

Nicotiana tabacum

Trichomes can be unicellular or multicellular, branched or unbranched, glandular or nonglandular

Arabidopsis thaliana

Plectranthus ornatus Wagner, G. J., Wang, E., and Shepherd, R. W., New approaches for studying and exploiting an old protuberance, the plant trichome. Ann. Bot. 2004 93: 3-11, by permission of Oxford University Press.; National Research Council Canada; Ascensão, L., Mota, L., and Castro, M.de M. Glandular trichomes on the leaves and flowers of Plectranthus ornatus: morphology, distribution and histochemistry Ann. Bot. 1999 84: 437-447 , by permission of Oxford University Press.

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Glandular trichomes are sources of important chemicals Glandular trichomes produce chemicals that can have antimicrobial, insecticidal or medicinal properties.

Control

Increased production

Control

Modifying gene expression in trichomes can increase their production of chemical compounds. The plant in the center makes more of an insecticidal compound and is resistant to aphids

Reprinted by permission from Macmillan Publishers, Ltd: Nature Biotechnology, copyright (2001). Wang, E., Wang, R., DeParasis, J., Loughrin, J.H., Gan, S., and Wagner, G.J. (2001) Suppression of a P450 hydroxylase gene in plant trichome glands enhances natural-product-based aphid resistance. Nature Biotech. 19: 371– 374.

In Arabidopsis, many trichome mutants have been identified Some mutants underproduce trichomes. WT

gl1

Some mutants overproduce trichomes. Wild-type

cpc try

cpc try etc3

Reprinted from Hülskamp, M., Miséra, S., and Jürgens, G. (1994) Genetic dissection of trichome cell development in Arabidopsis. Cell 76: 555-566. Copyright (1994), with permission from Elsevier Reproduced with permission. Wester, K., Digiuni, S., Geier, F., Timmer, J., Fleck, C., and Hülskamp, M. (2009) Functional diversity of R3 single-repeat genes in trichome development. Development 136:1487-1496.

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In Arabidopsis, trichome spacing is controlled by an inhibitory signal A transcription complex activates expression of the trichome inducer GL2.....

GL2

GL3/ EGL3

Trichome

TTG

GL1

Transcription complex

In Arabidopsis, trichome spacing is controlled by an inhibitory signal A transcription complex activates expression of the trichome inducer GL2..... and a mobile inhibitor moves into adjacent cells to inhibit GL2 expression.

GL2

GL3/ EGL3

TTG

Trichome

TRY CPC ETC1

GL1

Transcription complex

Inhibitor

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No transcription, no differentiation

Inactive transcription complex

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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The Arabidopsis trichome developmental pathway is derived from an older biosynthetic pathway Pigment Biosynthesis

No Pigment Biosynthesis

Redrawn from: Serna, L., and Martin, C. (2006) Trichomes: different regulatory networks lead to convergent structures. Trends Plant Sci. 11: 274–280.

Many pathways for trichome formation Molecular studies suggest that Arabidopsis trichomes arose independently from those of most other plants Furthermore, within a single species, multicellular and unicellular trichomes are formed differently, indicating that there are many ways to make a trichome........

Reprinted by permission from Macmillan Publishers, Ltd: Runions, J. (2003) Cell of the Month. Nat. Rev. Mol. Cell Biol. 4: 603 (Copyright 2003)

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Guard cell differentiation and patterning OPEN

Guard cells regulate leaf pores through which water vapor and gasses move

CLOSED Bar = 30 µm Nadeau JA, Sack FD (2002) Stomatal Development in Arabidopsis: September 30, 2002. The Arabidopsis Book. Rockville, MD: American Society of Plant Biologists.

Stomatal patterning in grasses

Grass leaves have parallel veins (indicated by black arrows) separated by mesophyll cells. Guard cells form in certain cell files (indicated by red arrows) between the veins. Image courtesy of J. Derksen, J. Hiddink and E.S. Pierson Copyright Radboud University Nijmegen

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Guard cell development in grasses

Guard cells (light blue) are supported by subsidiary cells (orange) which form from adjacent cell files Image courtesy of J. Derksen, J. Hiddink and E.S. Pierson Copyright Radboud University Nijmegen

Guard cell development in grasses

Asymmetric division in GC file produces Guard Mother Cell (GMC)

GMC induces division in adjoining cells

GMC divides to produce guard cells

Redrawn from Sack, F.D., and Chen, J.-D. (2009) Pores in Place..Science 323: 592-593.

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In the pan1 mutant, subsidiary cell formation is abnormal Wild-type

pan1 PAN1 encodes a receptorlike protein expressed in subsidiary-cell precursors. These studies may reveal the mechanism by which the differentiating guard cell communicates with the adjoining cells

From Cartwright, H.N., Humphries, J.A., Smith, L.G. (2009) PAN1: A receptor-like protein that promotes polarization of an asymmetric cell division in maize. Science 323: 649–651. Reprinted with permission of AAAS.

Guard cells differentiation in Arabidopsis Meristemoid Mother Cell Asymmetric division

Differentiation

Guard Mother Cell

Guard Cell Pair

Symmetric division

Protodermal cell

Guard cells are formed through a tightly controlled series of cell divisions Barton, M.K. (2007) Making holes in leaves: Promoting cell state transitions in stomatal development. Plant Cell 19: 1140- 1143.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Guard cells differentiate through a tightly controlled series of cell divisions SPEECHLESS Asymmetric division

Meristemoid Mother Cell Differentiation

Guard Mother Cell

Guard Cell Pair

Symmetric division

Protodermal cell SPEECHLESS is necessary for the initiation of the guard cell developmental pathway

Wild-type

speechless

SPEECHLESS-OX

Barton, M.K. (2007) Making holes in leaves: Promoting cell state transitions in stomatal development. Plant Cell 19: 1140- 1143.

Guard cells differentiate through a tightly controlled series of cell divisions SPEECHLESS Asymmetric division

Meristemoid Mother Cell Differentiation

Guard Mother Cell

Guard Cell Pair

Symmetric division

MUTE Protodermal cell

Loss of Function

Gain of Function

MUTE promotes differentiation of guard mother cells. In plants that over-express MUTE, all epidermal cells differentiate as guard cells Barton, M.K. (2007) Making holes in leaves: Promoting cell state transitions in stomatal development. Plant Cell 19: 1140- 1143.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

First published November 2009 Revised February 2011

Guard cells differentiate through a tightly controlled series of cell divisions SPEECHLESS Asymmetric division

Meristemoid Mother Cell

Guard Mother Cell

Differentiation

MUTE Protodermal cell

In fama loss-of-function mutants, continued GMC division generates rows of undifferentiated guard cell precursors

SPEECHLESS, MUTE and FAMA all encode basic helixloop-helix transcription factors

Guard Cell Pair

Symmetric division

FAMA

Loss of Function

Gain of Function

Barton, M.K. (2007) Making holes in leaves: Promoting cell state transitions in stomatal development. Plant Cell 19: 1140- 1143.

Stomatal patterning is determined by inhibition of differentiation Wild-type Patterning mutants sdd

tmm

er; erl1; erl2

yoda

A proposed genetic pathway for stomatal patterning and development showing positive and negative regulators

Bergmann, C.D., and Sack, F.D. (2007) Stomatal development. Annu. Rev. Plant Biol. 58: 163-181; Hunt,L., and Gray, J.E. (2009) The signaling peptide EPF2 controls asymmetric cell divisions during stomatal development. Current Biology 19: 864–869.

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The Plant Cell, February 2011 © 2011 The American Society of Plant Biologists

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Stomatal precursors produce extracellular inhibitory peptides Stomatal precursor

Stomatal precursors produce one or more secreted peptides that are cleaved by a peptidase and then binds extracellular domain of receptors of adjoining cells. This inhibitory signal prevents the adjoining cells from differentiating as guard cells

Peptidase

Receptor Undifferentiated cell

Inhibition of guard cell pathway

Redrawn from Gray, J.E., Casson, S., and Hunt, L. (2008) Intercellular peptide signals regulate plant meristematic cell fate decisions. Sci. Signal. 1 (49) pe53.

Stomatal development requires light

Kang, C.-Y., Lian, H.-L., Wang, F.-F., Huang, J.-R., and Yang, H.-Q. (2009). Cryptochromes, phytochromes, and COP1 regulate light-controlled stomatal development in Arabidopsis. Plant Cell 21: 2624-2641.tim

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Stomatal density is also determined by environmental CO2 levels Transferring a plant to a higher-CO2 environment causes the newly initiating leaves to produce fewer stomata, but does not affect stomatal density of the mature leaves Mature leaves

Stomatal density Low CO2

Young leaves

High CO2

Mature leaves perceive CO2 levels and transmit information to developing leaves High CO2 Low CO2

High CO2 – mature leaves Low CO2 – developing leaves

The nature of this signal is not yet known

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Summary – Cell differentiation Remain undifferentiated Differentiate

Positional information (auxin, signals from adjoining cells, environmental cues)

Communicate with adjoining cells

Transcriptional activation of cellspecific gene expression patterns

Summary – leaf development

Efroni, I., Eshed, Y., and Lifschitz, E. Morphogenesis of simple and compound Leaves: A critical review. Plant Cell 22: 1019-1032.

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KNOX-1 genes contribute to indeterminacy Simple leaves (e.g. Arabidopsis) KNOX-1 genes stay off

KNOX-1

Compound leaves (e.g. tomato) KNOX-1 genes turn on again in developing leaf primordia, conferring prolonged organogenic activity on the leaf edges

KNOX-1 KNOX-1 genes are repressed at the site of leaf primordium initiation

KNOX-1

Establishment of leaf polarity involves coordinated expression of transcription factors

KAN

PHAN or PHB/PHV/REV

Abaxial fate

Adaxial fate

Meristem-derived signal

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Auxin distribution specifies the position of leaf, leaflet and lobe initiation as well as vein development

Koenig, D., Bayer, E., Kang, J., Kuhlemeier, C., and Sinha, N. (2009) Auxin patterns Solanum lycopersicum leaf morphogenesis. Development 136: 2997-3006. Reproduced with permission.

Trichome and guard cell spacing is controlled by an inhibitory signal Spacing patterns of trichomes and guard cells involves extracellular inhibitory signaling

Inhibitory signal

Trichome

Inhibitory signal

Bergmann, C.D., and Sack, F.D. (2007) Stomatal development. Annu. Rev. Plant Biol. 58: 163-181; Hunt,L., and Gray, J.E. (2009) The signaling peptide EPF2 controls asymmetric cell divisions during stomatal development. Current Biology 19: 864–869.

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Small changes in gene expression and growth patterns are sufficient to produce a wide diversity of leaf forms.

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