Vegetative Growth and Organogenesis 555

Vegetative Growth 19 and Organogenesis

lthough embryogenesis and seedling establishment play criti- A cal roles in establishing the basic polarity and growth axes of the plant, many other aspects of plant form reflect developmental processes that occur after seedling establishment. For most plants, shoot architecture depends critically on the regulated production of determinate lateral organs, such as leaves, as well as the regulated formation and outgrowth of indeterminate branch systems. Root systems, though typically hidden from view, have comparable levels of complexity that result from the regulated formation and outgrowth of indeterminate lateral roots (see Chapter 18). In addition, secondary growth is the defining feature of the vegetative growth of woody perennials, providing the structural support that enables trees to attain great heights. In this chapter we will consider the molecular mechanisms that underpin these growth patterns. Like embryogenesis, vegetative organogenesis and secondary growth rely on local differences in the interactions and regulatory feedback among hormones, which trigger complex programs of gene expression that drive specific aspects of organ development.

Leaf Development Morphologically, the leaf is the most variable of all the plant organs. The collective term for any type of leaf on a plant, including structures that evolved from leaves, is phyllome. Phyllomes include the photosynthetic foliage leaves (what we usually mean by “leaves”), protective bud scales, bracts (leaves associated with inflorescences, or flowers), and floral organs. In angiosperms, the main part of the foliage leaf is expanded into a flattened structure, the blade, or lamina. The appearance of a flat lamina in seed plants in the middle to late Devonian was a key event in leaf evolution. A flat lamina maximizes light capture and also creates two distinct leaf domains: adaxial (upper surface) and abaxial (lower surface) (Figure 19.1). Several types of leaves have evolved based on their adaxial–abaxial leaf structure (see WEB TOPIC 19.1).

(A) Shoot structure and leaf polarity (B) Simple leaves Apical

Apical meristem Blade Margin

Midrib Vein Margin Midrib

Petiole Sessile Node Distal (no petiole) Adaxial Stipule Sheath

Petiole Proximal

Axillary bud Abaxial

Basal

Lea et

Rachis

Pinnately trifoliolate Palmate Paripinnate Bipinnate Tripinnate

Figure 19.1 Overview of leaf structure. (A) Shoot struc- leaves. Variations in lower leaf structure include the pres- ture, showing three types of leaf polarity: adaxial–abaxial, ence or absence of stipules and petioles, and leaf sheaths. distal–proximal, and midrib–margin. (B) Examples of simple (C) Examples of compound leaves.

In the majority of plants, the leaf blade is attached to the marginal expansion of leaf tissues, differentiation into the stem by a stalk called the petiole. However, some adaxial and abaxial domains, and morphogenesis along plants have sessile leaves, with the leaf blade attached the proximal-distal axis. Compound leaves are produced directly to the stem (see Figure 19.1B). In most monocots by variations on these developmental pathways. Finally, and certain eudicots the base of the leaf is expanded into we will discuss the gene networks and hormonal signals a sheath around the stem. Many eudicots have stipules, that control the development of the specialized cells of the small outgrowths of the leaf primordia, located on the epidermis and vascular tissue. Plantabaxial Physiology side of 6/E the leafTaiz/Zeiger base. Stipules protect the young Sinauerdeveloping Associates foliage leaves and are sites of auxin synthesis Morales Studio The Establishment of Leaf Polarity TZ6e_19.01during early leaf development. Date 09-10-14 Leaves may be simple or compound (see Figures 19.1B All leaves and modified leaves begin as small protuber- and C). A simple leaf has one blade, whereas a compound ances, called primordia, on the flanks of the shoot apical leaf has two or more blades, the leaflets, attached to a meristem (SAM) (see Chapter 17). All SAMs in higher common axis, or rachis. Some leaves, like the adult leaves plants share a common structure: a central, often dome- of some Acacia species, lack a blade and instead have a shaped domain surrounded by several emerging pri- flattened petiole simulating the blade, the phyllode. In mordia, which may be leaf primordia or, in the case of some plants the stems themselves are flattened like blades an inflorescence meristem, flower primordia. Cells in the and are called cladodes, as in the cactus Opuntia. apical meristem are considered undifferentiated and plu- We will begin our discussion of foliage leaf develop- ripotent. Nevertheless, as we saw in Chapter 17, the cells ment with the production of leaf primordia. Next we will of the SAM are organized into three more or less stable examine blade formation in simple leaves, which involves tissue layers—L1, L2, and L3—although some plants,

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(A) (B) Figure 19.2 Longitudinal section of the Arabidopsis inflorescence meristem and diagrams showing its functional organization. Flower (A) Light micrograph of the Arabidopsis inflo- L1 CZ L2 rescence meristem showing the localization PZ L3 I P of CLAVATA3 (CLV3 ) gene expression (brown Rib stain) (see Chapter 17). (B) Anatomical zona- zone tion of the inflorescence meristem showing the central zone (CZ), peripheral zone (PZ), flower primordium (P), flower primoridium initial (I), L1–L3 layers, flower, and rib zone. (C) Spatial variations in the rate of cell division (indicated by color bar), showing the highest rates in the (C) (D) flower primordia. (D) Proposed distribution of the three main hormones, auxin, cytokinin, and gibberellins, as well as sites of auxin transport, auxin depletion, and gibberellin (GA) degradation. (From Besnard et al. 2011.)

Cell cycle rate will discuss auxin canalization in more detail Low High later in this chapter.) Other hormones, such Auxin Auxin transport as cytokinin, gibberellins, and brassino- Cytokinin Auxin depletion steroids, also play critical roles in maintaining Gibberellin GA degradation SAM structure and activity. The distributions of auxin, cytokinin, and gibberellins in the SAM are shown in Figure 19.2D. such as maize (corn; Zea mays), lack L3. These tissue layers The initiation of leaf primordia was recently shown to can be further demarcated into three histological zones: be light-dependent in a manner that is independent of the central zone (CZ), peripheral zone (PZ), and rib zone photosynthesis; tomato and Arabidopsis apices cease pro- (RZ) (Figure 19.2A and B). ducing new leaf primordia when the plants are grown in To a large extent, positional information dictates the the dark. This cessation is correlated with decreased auxin fate of cells in the SAM. For example, the highest rates synthesis and loss of the polar localization of PIN1 in the of cell division in the inflorescence meristem of Arabi- SAM. However, because organ initiation in dark-grown dopsis are found in the primordium (P) and primordium apices can be restored only after application of both auxin initial (I), followed by the PZ and the CZ (Figure 19.2C). and cytokinin, it appears that cytokinin may be involved Position also determines the patterns of intracellular and in a light-dependent leaf initiation pathway. Phytochrome intercellular signaling. As we discussed in Chapter 17, B has been implicated as the photoreceptor involved in the the different histological zones exhibit distinctive pat- response to light (see Chapter 16), as it regulates auxin terns of gene expression that maintain the growing SAM synthesis and overall auxin levels in the plant. as a stable structure. In addition to hormonal signals, mechanical stress in the SAM has been shown to alter microtubule arrange- Hormonal signals play key roles ments as well as PIN1 distribution, which can affect leaf in regulating leaf primordia emergence Plant Physiology 6/E Taiz/Zeiger primordia initiation (see WEB TOPIC 19.2). SinauerAs we discussed Associates in Chapter 17, polar auxin transport in the INL1 HOUSE layer of the SAM is essential for leaf primordia emer- A signal from the SAM initiates TZ6e_19.02 Date 09-11-14 gence, and is responsible for leaf phyllotaxy (the pattern adaxial–abaxial polarity of leaf emergence from the stem; see Figure 17.32). When Since leaf primordia develop from a group of cells on the shoot apices are cultured in the presence of auxin trans- flank of the SAM, leaves possess inherent positional rela- port inhibitors they fail to form primordia, and application tionships with the SAM: the adaxial side of a leaf primor- of auxin to the SAM results in the induction of primordia dium is derived from cells adjacent to the SAM, while the at the application site. Auxin synthesis via the YUCCA abaxial side is derived from cells farther away. Microsurgi- pathway (see Chapter 15) generates auxin concentration cal studies in the 1950s demonstrated that some type of gradients which, in turn, regulate expression and asym- communication between the SAM and the leaf primor- metric distribution of PIN auxin efflux carriers to enhance dium is required for the establishment of leaf adaxial– or canalize localized polar auxin transport streams. (We abaxial polarity. For example, a transverse incision divid-

(A) (B) mutants phantastica (phan) in snapdragon (Antirrhinum P2 P2 majus) (Figure 19.4A). phan mutants have since been found in other species, including Arabidopsis and tobacco. phan mutants produce leaves with altered adaxial–abaxial symmetry, ranging from abaxialized needlelike leaves that SAM SAM fail to produce lamina, to leaves with blades exhibiting a mosaic of adaxial and abaxial characters (Figure 19.4B). I I The PHAN gene of Antirrhinum, and its orthologs, P1 P1 such as ASYMMETRIC LEAVES1 (AS1) in Arabidop- sis, encode MYB class transcription factors referred to as the ARP (ASYMMETRIC LEAVES1 [AS1], ROUGH Single Two marginal SHEATH2 [RS2], and PHAN) family. ARP genes func- transverse incisions incision tion, at least in part, by helping maintain the repression of KNOX1 (KNOTTED1-LIKE HOMEOBOX) genes in the Figure 19.3 Microsurgical experiment demonstrating the developing leaf (Figure 19.5A). The down-regulation of influence of the SAM on leaf primordium (P) adaxial–abaxial KNOX genes in leaf primordia, which occurs initially in development in potato (Solanum tuberosum). (A) A primor- response to the focused accumulation of auxin at leaf dium initial (I) isolated from the SAM by a transverse inci- initiation sites, is required for adaxial development and sion grows radially and contains only abaxial tissues. (B) A is essential for normal adaxial–abaxial patterning of the primordium initial (I) that has not been completely isolated leaf in many, but not all, species. The importance of this from the SAM shows normal adaxial–abaxial symmetry. (After Sussex 1951.) down-regulation is exemplified by the abnormal leaves of phan and as1 mutants, as well as of plants with KNOX gene mutations that prevent the normal down-regulation of KNOX gene expression in leaves. In Arabidopsis, how- ing the SAM from the primordium initial (I) caused the ever, mutations in AS1 alone do not affect abaxial–adaxial initial to develop radially without forming any adaxial polarity, and thus other factors seem to be involved. Since tissue (Figure 19.3A). The resulting “leaf” was cylindri- ARP genes are expressed uniformly in the leaf primordia, cal and contained only abaxial tissues (it was abaxialized). it is assumed that their role in adaxial fate specification However, two marginal incisions that allowed unimpeded depends on the presence of interacting protein partners. communication between the SAM and the primordium A large part of KNOX1 protein function appears to be initial led to the development of normal adaxial–abaxial mediated by its inhibitory effects on gibberellin levels in symmetryPlant Physiology (Figure 6/E 19.3B Taiz/Zeiger). Later refinements of these sur- the SAM (see Chapter 17; Figure 17.31). While acting to gicalSinauer experiments Associates using laser ablation and microdissec- inhibit gibberellin biosynthesis and to promote gibberellin tionMorales techniques Studio yielded similar results, suggesting that a inactivation, KNOX transcription factors also activate the TZ6e_19.03 Date 09-02-14 signal from the SAM is required for specification or main- cytokinin biosynthetic gene ISOPENTENYL TRANSFER- tenance of adaxial identity. However, the nature of this ASE7 (IPT7), which increases cytokinin levels. signal remains a mystery. Adaxial leaf development requires ARP genes promote adaxial identity HD-ZIP III transcription factors and repress the KNOX1 gene Adaxial development also depends critically on a group Insights into the molecular basis of adaxial and abax- of transcription factors known as HD-ZIP III proteins, so ial identity came from analysis of the loss-of-function named because of the presence of both a DNA-binding

(A) (B)

Figure 19.4 Effects of phan mutations on leaf morphology in Antirrhinum majus. (A) The veg- n-l etative shoot of a wild-type plant with normal leaves. (B) The vegetative shoot of a phan mutant n with narrow (n), needlelike (n-l), and mosaic (m) leaves. (From Waites and Hudson 1995.)

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(A) Leaf polarity

Distal

Abaxial Adaxial Abaxial Adaxial

Laminar HD-ZIPIIIs growth YABBYs Blade KANADIs AS1

AS2 ETT/ARF4

Petiole BOPs

Lower leaf PRS zone

Boundary CUCs SAM meristem

SAM KNOXI

Proximal (B) Leaf margin growth

Adaxial side

HD-ZIPIII AS1/2 Figure 19.5 Gene networks regulating leaf KLU polarity. (A) Regulation of proximal–distal polar- Leaf ity. Various genes involved in proximal–distal margin patterning interact with specific genes in the KAN PRS WOX1 abaxial–adaxial gene network. (B) Gene networks involved in leaf margin growth and adaxial–abax- miR166 ARF3/4 YAB ial polarity. SAM, shoot apical meristem. (See text for discussion.) (A after Townsley and Sinha 2012; Abaxial side B after Fukushima and Hasebe 2013.) homeodomain and a leucine zipper dimerization domain. expressed throughout the leaf, as occurs in some phb and HD-ZIP III transcription factors are also distinguished by phv mutants, abaxial tissues take on adaxial characteris- a putative lipid/sterol-binding domain, suggesting that tics. For example, in mutants in which PHB is ectopically their activity could be regulated by types of signaling expressed in abaxial domains of the leaf, axillary buds, molecules that are currently unknown in plants. These normally limited to the adaxial side of the leaf base, now transcription factors also feature a conserved sequence form on both sides. Conversely, mutations that block the motif that mediates protein–protein interactions, provid- function of PHB and PHV genes in their normal, adaxial ing additional scope for the regulation of their activity. domains of expression lead to loss of adaxial characters, Expression of the HD-ZIP III genes, such as PHAB- but only if the activity of both genes is blocked. Together, ULOSA (PHB) and PHAVOLUTA (PHV), is normally these results suggest that PHB and PHV act redundantly limited to the adaxial domains of the leaf primordia to promote adaxial identities in tissues where they are (see Figure 19.5A). When these genes are abnormally expressed. Plant Physiology 6/E Taiz/Zeiger Sinauer Associates © 2014 Sinauer Associates, Inc. This material cannot be copied, reproduced, Morales Studio/INmanufactured HOUSE or disseminated in any form without express written permission from the publisher. TZ6e_19.05 Date 08-27-14 558 Chapter 19

mutants have defective floral and vegetative leaflike organs The expression of HD-ZIP III genes is antagonized in which abaxial characters have been replaced by adaxial by miR166 in abaxial regions of the leaf characters, suggesting that there is functional redundancy Because HD-ZIP III genes promote the acquisition of an among members of the YABBY gene family. The abaxial- adaxial identity in those tissues where they are expressed, promoting activity of YABBY genes is further supported by their expression must somehow be suppressed in abaxial the phenotypes of plants in which YABBY genes are over- regions of the developing leaf. In an effort to explain this expressed. Such plants show ectopic formation of abaxial restricted expression, several analyses have implicated a tissues, and in some circumstances loss of the SAM. class of small regulatory RNAs known as microRNAs (or Despite their redundant action with KANADI genes, the miRs). A microRNA inhibits expression of its target gene function of YABBY genes is more enigmatic and appears by base pairing with a complementary sequence in the to be associated predominantly with growth. In maize, for gene’s transcript, thereby triggering degradation of the example, YABBY genes are expressed in the adaxial leaf mRNA or blocking its translation (see Chapter 2). The domain and hence their role in maize is thought to be to expression of miR166 in abaxial regions of the leaf pri- promote lamina outgrowth rather than abaxialization. mordia has been shown to reduce PHB and PHV transcript levels, thus enabling normal abaxial patterns of develop- Interactions between adaxial and abaxial tissues ment (Figure 19.5B). are required for blade outgrowth The antagonism between HD-ZIP III and miR166 plays As described above, the abaxialized primordia produced multiple roles in different patterning processes, including by surgically isolating primordia from the apical meristem vascular tissue differentiation, endodermis development fail to form leaf blades (see Figure 19.3). Similarly, in phan in the root, and SAM maintenance. mutants, leaf primordia with no adaxial tissues develop into needlelike leaves. Together these observations suggest Antagonism between KANADI and HD-ZIP III is a that lamina outgrowth requires both adaxial and abaxial key determinant of adaxial–abaxial leaf polarity tissues. Indeed, the mosaic leaves sometimes produced Transcription factors in the KANADI family play a central by phan mutants have bladelike outgrowths called lamina role in the specification of abaxial cell identity. KANADI ridges that are formed specifically at the boundaries of genes appear to have overlapping functions with YABBY the adaxial and abaxial domains (Figure 19.6). It has been genes (discussed below), with the most dramatic loss of proposed that the normal lateral growth of the lamina (leaf abaxial identity being observed when loss-of-function blade) is induced by interactions between distinct abaxial mutations of the two types of genes are combined. Con- and adaxial tissue types. According to this model, the pri- versely, abnormal formation of abaxial tissues is observed mary function of PHAN is to enable the development of when KANADI genes are overexpressed. Although it is tissues with an adaxial identity, after which the juxtaposi- not entirely clear how KANADI transcription factors pro- tion of two tissue types triggers lateral growth programs. mote abaxial identity, young embryos that are deficient in KANADI activity exhibit changes in the polar distribution Blade outgrowth is auxin dependent and of PIN auxin efflux carriers that precede any overt changes regulated by the YABBY and WOX genes in development. The suggestion that abaxial development In Arabidopsis, expression of YABBY genes marks the is closely coupled to polar transport of auxin is reinforced by abaxial domain and marginal regions of primordial the observation that members of the AUXIN RESPONSE leaves (see Figure 19.5A). YABBY genes are up-regulated FACTOR gene family, ARF3 and ARF4, are required for the by KANADI, ARF3, and ARF4 transcription factors; con- normal establishment of abaxial fate (see Figure 19.5B and versely, YABBY transcription factors promote the expres- Chapter 15). KANADI genes and HD-ZIP III genes play sion of KAN1 and ARF4 genes, forming positive feedback antagonistic roles in adaxial–abaxial patterning in both loops. In the absence of all YABBY gene activity, leaf pri- leaves and vasculature (see Figure 19.5B). mordia establish adaxial–abaxial polarity but fail to initi- The YABBY gene family of transcription factors, ate lamina outgrowth. These findings indicate that YABBY named after the Australian freshwater crayfish, appears genes mediate the induction of growth activity related to to act redundantly with the KANADI genes. YABBY gene adaxial–abaxial polarity. mutants were among the earliest leaf polarity mutants YABBY transcription factors positively regulate a mem- discovered in Arabidopsis. The first member of this gene ber of the WOX gene family, PRS (PRESSED FLOWER), family identified, CRABS CLAW (CRC), was defined by the which is expressed in the leaf margin and promotes blade phenotype of its Arabidopsis loss-of-function mutant, in outgrowth (see Figure 19.5B). PRS and WOX1 transcrip- which the organization of the carpels (parts of the flower) tion factors function cooperatively, and the prs/wox1 is disturbed. The more general activity of Arabidopsis double mutant exhibits a narrow-leaf phenotype in Ara- YABBY genes is revealed when mutations affecting sev- bidopsis, similar to the leaf phenotype of phan mutants. eral members of this family are combined. These multiple PRS- and WOX1-dependent blade outgrowth is, in part,

Figure 19.6 Leaf development Adabial–abaxial Blade Maturation in relation to the adaxial–abaxial patterning outgrowth boundaries in different types (A) of leaves. The diagrams shows SAM cross-sectional outlines of leaf Adaxial primordia at the establishment of adaxial–abaxial patterning (left), at an early stage in blade outgrowth (middle), and at leaf Abaxial maturity (right). (A) Conventional bifacial leaf, as in wild-type Ara- (B) bidopsis. (B) phan mutant of SAM snapdragon (Antirrhinum majus) and PHAN ortholog mutants of tobacco (Nicotiana sylvestris). (C) Maize milkweed pod1 mutant. Abaxial Note the outgrowths on the surfaces where the adaxial and abaxial tissues come in contact. (From Fukushima and Hasebe (C) Not 2013.) determined Lamina ridges mediated by an as yet unidentified mobile signal(s) pro- tion (see Chapter 17; Figure 17.27). Later in development, cessed by KLU, a cytochrome P450 monooxygenase (see these CUC genes also control the specification of organ Figure 19.5B). KLU promotes cell division activity in aerial boundaries during leaf initiation. As typically happens in organs, including leaves, and a loss-of-function mutant of the case of genes regulating boundary functions, cuc1/cuc2 the KLU gene produces smaller organs. Auxin appears to double mutants display organ fusions and growth arrest. As be another signal acting in blade formation, independent in cotyledon development during embryogenesis, there is of KLU. Multiple loss-of-function mutants of the YUCCA interdependence between CUC gene expression and auxin- (YUC) auxin biosynthetic genes exhibit defective blade dependent leaf primordium initiation. outgrowth, raising the possibility that auxin participates The lower-leaf zone (LLZ) plays an important role in in the regulatory network for directed growth of the leaf. leaves that develop stipules or form leaf sheaths (see Fig- Plant Physiology 6/E Taiz/Zeigerure 19.1). In these cases, the founder cells (which give rise Leaf proximal–distal polarity alsoSinauer depends Associates on Morales Studio to the leaf primordium) recruit additional cells into the specific gene expression TZ6e_19.06 Date 08-29-14primordium through a mechanism that in Arabidopsis In addition to adaxial–abaxial polarity, leaf development is dependent on the expression of orthologs of the WOX also exhibits polarity along its length, called proximal– gene PRS (Pressed Flower). Cells that are recruited distal polarity. Developing leaf primordia can be divided to become stipules or sheath are taken from the flanks of lengthwise into four main zones extending from the meri- the primordium. stem: boundary meristem, lower-leaf zone, petiole, and The region of the leaf primordium destined to become blade (see Figure 19.5A). the petiole is characterized by the expression of BOP (Blade Proximal–distal polarity becomes evident as the pri- on Petiole) genes, which encode transcriptional activators mordium begins to grow out and away from the SAM. The that are required to establish petiole identity in the proxi- boundary meristem, although not considered part of the mal portion of the leaf in Arabidopsis (see Figure 19.5A). leaf, is important for normal leaf initiation. The initiation The double mutant bop1/bop2 lacks the proper distinction of leaves from the peripheral zone requires the creation of between leaf blade and petiole, and both single mutants meristem-to-organ boundaries, buffer zones that separate show laminar development on what would be the peti- these two cell groups with distinct gene expression pro- ole. BOP1 and BOP2 are both expressed in the adaxial grams and morphologies. The boundary meristem itself domain, where they act redundantly to suppress laminar expresses a unique set of transcription factors that partici- outgrowth in the petiole region. pate in the local repression of cell proliferation, a prereq- uisite for the development of physically separate organs. In compound leaves, de-repression of the KNOX1 CUC (CUP-SHAPED COTYLEDON) 1 and 2 genes in gene promotes leaflet formation Arabidopsis encode plant-specific NAC (NAM; ATAF1,2; Compound leaves have evolved independently many CUC2) transcription factors that regulate cotyledon forma- times from simple leaf forms. Despite wide variations in

Figure 19.7 Scanning electron micrograph of tomato shoot tip showing developing compound leaf. Primordia 1 through 4 (P1–P4) are shown. The first primary leaflet pair (PL) and the second leaflet pair (arrow) are visible on P4. (From Kang and Sinha 2010.)

the form and complexity of compound leaves, the developmental mechanisms that lead to their formation have been converged on repeatedly. By delaying the differentiation process, individual leaf primordia can redeploy the gene regulatory networks used by the SAM during leaf initiation to form leaflet primordia, resulting in com- PL pound leaf development (Figure 19.7). Similar to what P2 happens during the initiation of leaf primordia on the SAM, PIN1 proteins focus auxin flow, leading to formation of local auxin maxima on the flanks of the primordia P1 (Figure 19.8). KNOX1 genes are important components of the regulatory network involved in compound leaf development (see Figure 19.8). CUC genes are required for the de-repression P3 P4 of KNOX genes. Cytokinins act downstream of KNOX proteins in promoting leaflet development. For example, overexpression of the cytokinin biosynthetic gene, IPT7, in 100 µm tomato leaf primordia causes an increase in the number of leaflets. Conversely, overexpression of the cytokinin degradation gene, CKX3, results in a decrease in the number of leaflets. A parallel role of KNOX and CUC genes in the formation of leaf serrations is discussed in WEB TOPIC 19.3.

KNOX1 genes, which CUC genes are Lea et outgrowth are repressed in the expressed in the distal suppresses KNOX1 gene Lea et primordia of simple boundary of the expression. Gibberellin leaves, become incipient lea et and levels increase. de-repressed in stimulate PIN1-directed compound leaf auxin ow. Cytokinin primordia. Gibberellin levels increase. Leaf levels decline. primordium Plant Physiology 6/E Taiz/Zeiger Sinauer Associates IN HOUSE GA TZ6e_19.07 Date 08-27-14 Primordium initial Lea et primordia

Meristem 1 2 3 4

KNOX1 expression Figure 19.8 Development of compound leaves. The initial stages of simple CUC expression and compound leaf development are similar. KNOX1 genes are repressed in the Auxin ow primordium initial (1) and are subsequently reactivated (2), thereby maintaining the primordium in an undifferentiated state. Leaflet primordia are then initiated Peak of auxin response in a process resembling the initiation of leaf primordia involving PIN1-mediated GA Gibberellin auxin flow (3 and 4). (From Hasson et al. 2010.)

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Differentiation of cells. Pavement cells are relatively unspecialized epider- Epidermal Cell Types mal cells that can be regarded as the default developmental fate of the protoderm. Trichomes are unicellular or mul- In addition to the palisade parenchyma and spongy meso- ticellular extensions of the shoot epidermis that take on phyll, which are specialized for photosynthesis and gas diverse forms, structures, and functions, including protec- exchange, the leaf epidermis also plays vital roles in leaf tion against insect and pathogen attack, reduction of water function. The epidermis is the outermost layer of cells on loss, and increased tolerance of abiotic stress conditions. the primary plant body, including both vegetative and Guard cells are pairs of cells that surround the stomata, reproductive structures. The epidermis usually consists or pores, which are present in the photosynthetic parts of of a single layer of cells derived from the L1 layer, or pro- the shoot. Guard cells regulate gas exchange between the toderm. In some plants, such as members of the Moraceae leaf and the atmosphere by undergoing tightly regulated and certain species of the Begoniaceae and Piperaceae, turgor changes in response to light and other factors (see the epidermis has two to several cell layers derived from Chapter 10). Other specialized epidermal cells, such as periclinal divisions of the protoderm. lithocysts, bulliform cells, silica cells, and cork cells (Fig- There are three main types of epidermal cells found ure 19.9), are found only in certain groups of plants and in all angiosperms: pavement cells, trichomes, and guard are not as well studied.

(A) Bulliform cells (maize) (B) Monocot leaf (Ammophila sp.)

Bulliform cells

(D) Grass leaf epidermis

Cork cell

Cystolith Silica cell

Figure 19.9 Examples of specialized epidermal cells. (A) Bulliform cells of maize. (B) Rolled leaf of marram Pavement grass (Ammophila sp.). Rolling and unrolling in grass cells leaves is driven by turgor changes in the bulliform cells. (C) Lithocyst cell in a Ficus leaf containing a cystolith, composed of calcium carbonate deposited on a cel- lulosic stalk attached to the upper cell wall. (D) Wheat (Triticum aestivum) leaf epidermis with pairs of silica Guard cells and cork cells interspersed among the pavement cells.

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The formation of pavement cells, the default pathway for its rounded morphology. The GMC then undergoes one epidermal cell development, was discussed in Chapter 14 symmetrical division, forming a pair of guard cells sur- (see Figure 14.15). Here we will describe the development rounding a pore—the stomate. Although this lineage is of two types of specialized epidermal cells, guard cells and called the “stomatal lineage,” the ability of meristemoids trichomes, which have been studied intensively as model and SLGCs to undergo repeated divisions means that this systems for pattern formation and cytodifferentiation. lineage is actually responsible for generating the majority of the epidermal cells in the leaves. Guard cell fate is ultimately determined Following amplifying divisions of the meristemoid, by a specialized epidermal lineage the resulting SLGCs can differentiate into pavement cells, Developing plant leaves exhibit a tip-to-base develop- which are the most abundant cell type in the epidermis mental gradient, with cell division prevalent at the base of a mature leaf, or they can divide asymmetrically (spac- of the leaf and differentiation occurring near the tip. In ing divisions) to give rise to a secondary meristemoid. Arabidopsis, guard cell differentiation also follows this The orientation of division in asymmetrically dividing trend, but is ultimately governed by the stomatal cell lin- SLGCs is important for enforcing the “one-cell-spacing eage (Figure 19.10). In the developing protoderm (which rule,” according to which stomata must be situated at least will give rise to the leaf epidermis), a population of meri- one cell length apart to maximize gas exchange between stemoid mother cells (MMCs) is established. Each MMC the leaf and the atmosphere. Incorrect stomatal pattern- divides asymmetrically (the so-called entry division) to ing results when genes controlling critical stages in the give rise to two morphologically distinct daughter cells—a lineage are mutated. larger stomatal lineage ground cell (SLGC) and a smaller meristemoid (see Figure 19.10). An SLGC can either differentiate into a pavement cell or become an MMC and found secondary or satellite lineages. The meristemoid can undergo a variable number of asymmetric amplify- Figure 19.10 Stomatal development in Arabidopsis. ing divisions giving rise to as many as three SLGCs, with Three related transcription factors, SPCH, MUTE, and the meristemoid ultimately differentiating into a guard FAMA, form heterodimers with SCRM and are required for mother cell (GMC), which is recognizable because of the production of meristemoids, GMCs, and guard cells. They are also required for the amplifying and spacing pathways as well (not shown). (After Lau and Bergmann 2012.)

1. A protodermal 2. MMCs undergo 3. Meristemoids 4. Meristemoids may 5. A GMC divides cell commits to the an asymmetric may undergo differentiate into a symmetrically once stomatal lineage division and additional guard mother cell to form a pair of when it becomes a produce a smaller asymmetric (GMC) and the SLGC guard cells (green). meristemoid mother meristemoid (red) divisions. forms a pavement cell (MMC). and a larger cell (white). stomatal lineage ground cell (SLGC). Amplifying

3 Amplifying 1 2 4 5 bHLHs bHLHs bHLHs bHLHs SPCH SPCH MUTE FAMA SCRM SCRM SCRM SCRM

Entry Protodermal Meristemoid Meristemoid Guard cell mother cell SLGC mother cell Guard Pavement cell cell

Other fates: 6 6. An SLGC may pavement cell Spacing or trichome revert to an MMC and undergo an asymmetric division to create a new meristemoid.

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kinase domain and thus are thought to be incapable of Two groups of bHLH transcription factors transducing signals on their own. Like the ERf, the recep- govern stomatal cell fate transitions tor-like protein TMM inhibits stomatal lineage prolifera- The various stages in stomatal development highlight tion and guides spacing divisions in leaves. three specific cell-state transitions: (1) MMC to meriste- The EPIDERMAL PATTERNING FACTOR-LIKE moid, (2) meristemoid to GMC, and (3) GMC to mature (EPFL) protein family is a recently identified group of 11 guard cells. Each of these transitions is associated with, small, secreted cysteine-rich peptides that have been shown and requires the specific expression of, one of three basic to regulate stomatal development. Two founding members helix-loop-helix (bHLH) transcription factors: SPEECH- of the family, EPF1 and EPF2, are stomatal lineage–spe- LESS (SPCH), MUTE, and FAMA (named after the Roman cific factors, and they repress stomatal development at goddess of rumor) (see Figure 19.10). SPCH drives MMC specific stages when ERECTA genes are being expressed. formation and the asymmetric entry division of these According to current models, EPF2 and EPF1 are secreted cells, as well as the subsequent asymmetric amplifying by MMCs/meristemoids and GMCs, respectively, and are and spacing divisions. MUTE terminates stem cell behav- perceived by ERECTA family receptors in surrounding cells. ior by promoting the differentiation of meristemoids into As a result, the ERECTA receptor inhibits stomatal devel- GMCs, and FAMA promotes the terminal cell division opment (see Figure 19.11). In this way the EPF2–ERECTA and differentiation of GMCs into guard cells. In addition, pair regulates the number and density of stomata. Differ- two related bHLH leucine zipper (bHLH-LZ) proteins, ent pairings between EPFL peptides and ERECTA family SCREAM (SCRM) and SCRM2, have been identified as receptors regulate different aspects of stomatal patterning, the partners of SPCH, MUTE, and FAMA. while TMM apparently modulates the signaling pathway. An unexpected wrinkle in the above scenario is the Peptide signals regulate stomatal patterning discovery that the mesophyll also contributes to stoma- by interacting with cell surface receptors tal patterning. One of the EPFL peptides, STOMAGEN, Leucine-rich repeat receptor-like kinases (LRR-RLKs) are a positive regulator of stomatal density, is produced by single-pass transmembrane proteins with an extracellu- the underlying mesophyll and released to the epidermis. lar ligand-binding domain and an intracellular kinase Experiments have shown that the depletion of STOMA- domain for downstream signaling. The ERECTA family GEN results in a decrease in stomatal numbers, indicating (ERf) of receptor-like kinases (RLKs) has three mem- that its function is important for normal stomatal develop- bers—ERECTA, ERL1, and ERL2—all of which control ment. The stomata-inducing phenotype of STOMAGEN the proper patterning and differentiation of stomata. For overexpression or its exogenous application requires example, ERECTA, which is expressed strongly in the TMM, leading to the proposal that TMM may act as a protodermal cells but is undetectable thereafter, restricts receptor for STOMAGEN. However, the mechanism by asymmetric entry division in MMCs (Figure 19.11). which STOMAGEN stimulates stomatal development is A receptor-like protein, TOO MANY MOUTHS (TMM), still unknown. is also required for stomatal patterning. TMM is expressed within the stomatal lineage and appears to provide speci- Genetic screens have led to the identification ficity to the more widely expressed ERECTA gene family of positive and negative regulators (see Figure 19.11). Receptor-like proteins lack a C-terminal of trichome initiation Trichome development has been most thoroughly studied in the rosette leaves of Arabidopsis. Arabidopsis trichomes are unicellular and branched, with a distinctive tricorn EPF2 (three-horned) structure (Figure 19.12). TMM receptor-like Arabidopsis trichomes develop from single protoder- protein domain ERECTA mal cells. The first recognizable change from a proto- Transmembrane Extracellular domain

Figure 19.11 EPF2 peptide signaling negatively regu- Cytoplasm Receptor-like lates stomatal density and patterning. EPF2 is synthesized kinase domain and secreted by meristemoid mother cells and early meristemoids. The presence of extracellular EPF2 is detected by the receptor-like kinase ERECTA of protodermal cells. Together with the receptor-like protein TMM, the EPF2– ERECTA complex activates an intracellular signaling cas- Protodermal Meristemoid cell production cade that represses the production of new meristemoids. (After Lau and Bergmann 2012.)

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Figure 19.12 Arabidopsis trichome showing the typical tricorn branching pattern.

trichome initiation in the intervening protodermal cells. As the leaf expands, new trichomes are initiated at the leaf base, and the previously formed trichomes are further separated by cell divisions of the intervening epidermal cells. Genetic screens for mutants affecting trichome development have led to the discovery of genes regulating trichome patterning—especially trichome density and spacing (Figure 19.13). The mutants generally fall into two classes. One class shows fewer or no trichomes, indica- tive of the absence of proteins that are positive regulators of trichome formation (see Figure 19.13B). These genes include TRANSPARENT TESTA GLABRA1 (TTG1), GLA- dermal cell to an incipient trichome cell is an increase in BRA1 (GL1), and GLABRA3 (GL3). TTG1 encodes a protein nuclear size due to the initiation of endoreduplication, with WD40 repeat domains (a 40 amino acid motif with replication of the nuclear genome in the absence of nuclear conserved tryptophan [W] and aspartate [D] residues) or cell divisions (see Chapter 2). Trichome cell morpho- that generally function as protein–protein interaction genesis is characterized by an initial outgrowth, followed domains. GL1 encodes a MYB-related transcription fac- by two successive branching events resulting in the tricorn tor, and GL3 encodes a bHLH-like transcription factor. morphology. GL1, GL3, and TTG1 function together as a GL1–GL3– Trichomes are initiated at the base of the developing TTG1 protein complex that regulates the expression of leaf, where they are typically separated by three or four other genes. protodermal cells that do not develop into trichomes. The second class of trichome patterning mutants has This regular spacing suggests the existence of develop- either more trichomes or unevenly spaced trichomes (tri- mental fields between neighboring trichomes that inhibit chome clusters), and the corresponding genes therefore

(A) (B)

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(C) (D) Figure 19.13 Trichome patterning mutants of Arabidopsis. (A) Wild-type plant with more or less evenly distributed trichomes on the leaf surfaces. (B) gl1 mutant plant lacking trichomes. (C) try mutant plant exhibiting small trichome clusters (white arrow). (D) try/cpc double mutant with large trichome clusters comprising as many as 40 trichomes. (From Balkunde et al. 2010.)

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encode proteins that act as negative regulators of trichome development (see Figure 19.13C and D). These negative regulators include TRYPTICON (TRY), which encodes a Trichome MYB protein lacking a transcriptional activation domain. Leaf epidermis TRY is expressed in developing trichomes and moves to JA JAZ the surrounding cells, where it inactivates the GL1–GL3– TTG1 complex by displacing GL1 (Figure 19.14). Inactiva- GL3 GL3 tion of the GL1–GL3–TTG1 complex prevents trichome GL1 TTG1 TRY TTG1 formation in the surrounding cells and thus establishes the regular spacing of trichomes on the leaf epidermis. GL1 GLABRA2 acts downstream of the GL1–GL3–TTG1 GL2 TRY complex to promote trichome formation GLABRA2 (GL2) was originally identified as a gene that, when mutated, caused aborted trichomes with aberrant Trichome cell differentiation cell expansion. GL2, which is activated in trichome cells by Figure 19.14 Role of GLABRA2 (GL2) in leaf trichome the GL1–GL3–TTG1 complex, encodes a homeodomain formation. Cells that will form trichomes strongly express leucine zipper transcription factor (see Figure 19.14). GL2 the GL2 and TRY genes (black arrows). GL2 protein acts as expression is thought to represent the rate-limiting step in a positive regulator of trichome cell differentiation. TRY trichome formation. In wild-type plants, high levels of GL2 protein moves to neighboring epidermal cells (blue arrow), promoter activity have been observed in the entire leaf at where it inhibits trichome formation. (After Qing and early leaf development stages; however, later on this activity Aoyama 2012.) is limited to developing trichomes and cells surrounding early-stage trichomes. Extensive analyses of gene expression patterns indicate that a large number of genes are reg- ticipates in trichome initiation by degrading JAZ proteins, ulated downstream of GL2 during trichome differentiation. thereby abolishing the interactions of JAZ proteins with Plant Physiology 6/E Taiz/Zeiger Whereas GL2 promotes trichome formation in the leaf SinauerbHLH Associatesand MYB factors, which activate the transcription epidermis, the gene has the opposite effect in roots. gl2 Moralesof trichome Studio activators (see Figure 19.14). mutants form ectopic root hairs, indicating that the gene TZ6e_19.14 Date 09-12-14 product acts as a suppressor of root hair development. Venation Patterns in Leaves Jasmonic acid regulates Arabidopsis The leaf vascular system is a complex network of intercon- leaf trichome development necting veins consisting of two main conducting tissue Jasmonic acid (JA) and its derivative compounds function types, xylem and phloem, as well as nonconducting cells, as key signaling molecules in trichome formation, and such as parenchyma, sclerenchyma, and fibers. The spa- addition of exogenous jasmonic acid causes an increase tial organization of the leaf vascular system—its venation in the number of leaf trichomes in Arabidopsis. In Ara- pattern—is both species- and organ-specific. Venation bidopsis, jasmonate ZIM-domain (JAZ) proteins repress patterns fall into two broad categories: reticulate venation, trichome formation by binding to GL3 and GL1, key part- found in most eudicots, and parallel venation, typical of ners of the activation complex. Also, jasmonic acid par- many monocots (Figure 19.15).

(A) (B)

Figure 19.15 Two basic patterns of leaf venation in angiosperms. (A) Reticulate venation in Prunus serotina, a eudicot. (B) Parallel venation in Iris sibirica, a monocot.

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Figure 19.17 Development of the shoot vascular system.  Mid (primary) vein (A) Longitudinal section through the shoot tip of perennial flax (Linum perenne), showing the early stage in the differentiation of the leaf trace procambium at the site of a future leaf primordium. The leaf primordia and leaves are num- Secondary/marginal bered, beginning with the youngest initial. (B) Early vascular veins development in a shoot with decussate phyllotaxy. Dense stippling in the tip indicates SAM, young leaf primordia, and procambial strands. Leaf traces develop basipetally to Tertiary veins the mature vascular system and form a sympodium. The region where the leaf trace diverges from the continuous vascular bundle is called the leaf gap. Numbers correspond Quaternary/freely to the leaf order, starting with the primordia (not all leaves ending veinlets are shown). (After Esau 1953.)

the shoot. Instead, he discovered that the leaf vascular Figure 19.16 Hierarchy of venation in the mature Arabi- bundles, arising from vascular precursor cells called the dopsis leaf based on the diameter of the veins at the site of procambium, were initiated discontinuously in associa- attachment to the parent vein. (After Lucas et al. 2013.) tion with the emerging leaf primordia in the SAM (Figure 19.17A). From there the vascular bundles differentiated downward (basipetally) toward the node directly below Despite the diversity of leaf venation patterns, they all the leaf and formed a connection to the older vascular share a hierarchical organization. Veins are organized into bundle. The portion of the vascular bundle that enters the distinct size classes—primary, secondary, tertiary, and so leaf was later called the leaf trace (Figure 19.17B). on—based on their width at the point of attachment to What Nägeli had discovered was that the continuous the parent vein (Figure 19.16). The smallest veinlets end longitudinal vascular bundles in the stem are actually blindly in the mesophyll. The hierarchical structure of the composed of individual leaf traces. Species may differ in leaf vascular system reflects the hierarchical functions of the exact course of leaf trace development, but the basic different-sized veins, with larger diameter veins function- interpretation of the seed plant shoot primary vascu- ing in the bulk transport of water, minerals, sugars, and lar system as a sympodium of leaf traces appears to be other metabolites, and smaller diameter veins functioning universal. in phloem loading (see Chapter 11). The question of how leaf venation patterns develop Auxin canalization initiates development has long intrigued plant biologists. For the leaf vascular of the leaf trace systemPlant Physiology to carry out 6/E its Taiz/Zeiger long-distance transport functions Several lines of evidence indicate that auxin stimulates Sinauer Associates effectively,Morales Studio its many cell types must be arranged properly formation of vascular tissues. An example is the role of withinTZ6e_19.16 the radial and longitudinal Date 09-08-14 dimensions of the vas- auxin in regeneration of vascular tissue after wounding cular bundle. It is not surprising, then, that the differen- (Figure 19.18A). Vascular regeneration is prevented by tiation of vascular tissues is under strict developmental removal of the leaf and shoot above the wound but can control. In this section we will first describe the develop- be restored by the application of auxin to the cut petiole ment of a leaf’s vascular connection to the rest of the plant. above the wound, suggesting that auxin from the leaf is Then we will discuss how the higher-order venation pat- required for vascular regeneration. As shown in Figure tern of a leaf is established. 19.18B, the files of regenerating xylem elements originate at the source of auxin at the upper cut end of the vascular The primary leaf vein is initiated discontinuously bundle, and progress basipetally until they reconnect with from the preexisting vascular system the cut end of the vascular bundle below, matching the In the mid-nineteenth century, the Swiss plant anatomist Carl Wilhelm von Nägeli made a surprising discovery while tracing the source of vascular bundles in the pri- Figure 19.18 Auxin-induced xylem regeneration around  mary shoot. In the mature part of the stem of seed plants, a wound in cucumber (Cucumis sativus) stem tissue. (A) the longitudinal vascular bundles form a continuous con- Method for carrying out the wound regeneration experi- ducting system that begins at the root–shoot juncture and ment. (B) Fluorescence micrograph showing regenerating ends near the growing tips. Nägeli had assumed that the vascular tissue around the wound. The arrow indicates the vascular system must grow upward (acropetally) from wound site where auxin accumulates and xylem differentia- the preexisting vascular system to the growing tips of tion begins. (B courtesy of R. Aloni.)

(A) (B) 5 5 Procambium 3 3

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(A) The stem was Immediately after the (B) decapitated and the wounding, auxin in lanolin leaves and buds paste was applied to the above the wound stem above the wound. site were removed in order to lower the endogenous auxin. Node Apical bud

Young leaf Plant Physiology 6/E Mature Taiz/Zeiger leaf Auxin in Sinauer Associates lanolin Morales Studio paste TZ6e_19.17 Date 08-28-14 Wound

Vascular strands

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Intact cucumber plant Decapitated and wounded cucumber plant

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presumed direction of the flow of auxin. The upper end of Basipetal auxin transport from the L1 layer the cut vascular bundle thus acts as the auxin source and of the leaf primordium initiates development the lower cut end as the auxin sink. These and similar observations in other systems, such of the leaf trace procambium as bud grafting, have led to the hypothesis that as auxin As we saw in Chapter 18, canalization is often accompa- flows through tissues it stimulates and polarizes its own nied by redistribution of PIN1 auxin efflux carriers. Fur- transport, which gradually becomes channeled–or cana- thermore, the distribution of PIN1 can be used to predict lized–into files of cells leading away from auxin sources; the direction of auxin flow within a tissue. Figure 19.19A these cell files can then differentiate to form vascular tissue. shows the SAM of a tomato plant expressing the Arabi- Consistent with this idea, local auxin application (as dopsis PIN1 protein fused to green fluorescent protein in the wounding experiments described above) induces (GFP). Based on the orientations of the PIN1 proteins, vascular differentiation in narrow strands leading away auxin is directed to a convergence point in the L1 layer from the application site, rather than in broad fields of of the leaf primordium initial (P0). In contrast, auxin is cells. New vasculature usually develops toward, and directed basipetally in the initiating midvein (leaf trace) unites with, preexisting vascular strands, resulting in a of the emerging leaf primordium (P1). connected vascular network. We would therefore predict A model for midvein formation in Arabidopsis is shown that a developing leaf trace acts as an auxin source and in Figure 19.19B. The canalization of auxin toward the the existing stem vasculature as an auxin sink. Recent tip of the leaf primordium (P1) in the L1 layer via PIN1 studies on leaf venation have supported this source– transporters leads to an accumulation of auxin at the tip. sink model, or canalization model, for auxin flow at the Auxin efflux from this region of high auxin concentra- molecular level. tion becomes canalized via PIN1 proteins in the basipetal direction toward the older leaf trace directly below it. This induces the differentiation of the procambium in the basipetal direction. (A) The existing vasculature guides the growth of the leaf trace P0 * Microsurgical experiments have shown that the exist- P1 ing vascular bundle in the stem is required for the direc- tional development of the leaf trace procambium. Figure 19.20A shows PIN1 distribution in the apex of a tomato plant expressing Arabidopsis PIN1 fused to GFP. The leaf trace emerging from the leaf primordium initial (P0) has connected to the existing leaf trace of the leaf primordium below it (P3), as shown diagrammatically in Figure 19.20C. However, if P3 is surgically removed, the leaf trace from P0 connects instead to the vascular bundle of the leaf primordium on the other side of the stem (P2) (Figure (B) P1 19.20B and D). These results suggest that either the exist- SAM Auxin ing vascular bundle is serving as an auxin sink and thus facilitating auxin canalization, or that it is producing a dif- P0 ferent signal that guides the development of the leaf trace.

Figure 19.19 PIN1-mediated auxin flow during midvein formation. (A) Longitudinal section through a tomato vegetative meristem expressing AtPIN1:GFP (green). The red arrows on the left indicate the direction of auxin movement toward the site of the leaf primoridium initial (I1, white star). The red arrows on the right indicate auxin flow toward the emerging leaf primordium (P1). The white arrows show basipetal auxin movement, which initiates the differentia- Procambium tion of the midvein. (B) Schematic diagram of auxin flow Vascular through the L1, L2, and L3 tissue layers and midvein dif- tissue ferentiation during the formation of leaf primordia. Primor- dium initial (P0), primordium (P1). (A from Bayer et al. 2009.)

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Figure 19.20 Preexisting vascular (A) (B) bundle guides basipetal development of the leaf trace. (A and C) In P3 P2 the control Arabidopsis meristem P0 expressing AtPIN1:GFP (green), the P0 newly initiated leaf trace (I1) grows toward, and connects to, the leaf trace associated with P3 directly below. (B and D) When the P3 vasculature is surgically removed (dashed red line), the P0 leaf trace connects instead to the P2 leaf trace on the other side of the stem. (From Bayer et al 2009.)

50 µm 50 µm (C) P3 (D)

P2 P2 P0 P0

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Primary phloem is the first vascular tissue to form The pattern of vein formation follows a stereotypical from procambial cells, and its differentiation begins at the course in Arabidopsis. The first procambium that forms in vascular bundle below and proceeds acropetally into the the leaf primordium—the leaf trace—represents the future leaf primordium. In contrast, primary xylem differentia- primary vein or midvein. Secondary pre-procambium of tion lags behind primary phloem, is discontinuous, and the first pair of looped, secondary veins (orange arrows in proceeds both acropetally into the leaf primordium and Figure 19.21B) develops out from the midvein. The pre- basipetally toward the vascular bundle below. procambium of the second pair of secondary vein loops progresses either basipetally or acropetally. Third and Higher-order leaf veins differentiate higher secondary vein loop pairs progress out from the in a predictable hierarchical order midvein toward the leaf margin and reconnect with other The hierarchical order of leaf vascularization has been extending strands (black arrows in Figure 19.21A). best studied in Arabidopsis. In general, vein development The procambium differentiates from the pre-procam- and patterning progress in the basipetal direction (Figure bium simultaneously along the procambial strand (green 19.21A, black arrow). In other words, venation is generally lines in Figure 19.21A). Xylem differentiation occurs at a more advanced stage of development at the tip of a approximately 4 days later and can develop either con- developing leaf than it is at the base. tinuously, or as discontinuous islands, along the vascular During vein formation, ground meristem cells dif- strand (magenta arrows in Figure 19.21A). ferentiate into pre-procambium cells—a stable interme- Proper differentiation of the vascular tissues within diate state between ground cells and procambium cells the veins depends on normal adaxial–abaxial polarity of that is characterized, in Arabidopsis, by the expression of the leaf. The four circles shown in Figure 19.21C repre- the transcription factor ATHB8. Pre-procambialPlant Physiology cells 6/Eare Taiz/Zeigersent vascular differentiation in the presence and absence isodiametric in shape (approximately cube-shaped)Sinauer Associates and of adaxial–abaxial polarity. The green circle on the left Morales Studio/In House are anatomically indistinguishable fromTZ6e_19.20 ground meristem Daterepresents 09-15-14 the undifferentiated procambial strand. Under cells. Cell divisions of the pre-procambium are parallel to conditions of normal adaxial–abaxial polarity, xylem devel- the direction of growth of the vascular strand, resulting ops on the adaxial side and phloem on the abaxial side. in the elongated cells characteristic of the procambium However, if the leaf has become adaxialized, as is phan (Figure 19.21B). mutants, the xylem cells surround the phloem, whereas

(A) (B)

Secondary pre-procambium

Basipetal overall Procambial Pre-procambium Procambium vascular strands development (C)

Tertiary Xylem and higher secondary Phloem veins Vascular strands Procambium Normal vein Adaxialized Abaxialized pattern vein pattern vein pattern Figure 19.21 (A) Development of vein pattern in young leaves. (B) Formation of procambial cells from a pre- procambium cell. (C) Radial vein pattern in leaves. (Left to right): Procambial strand; normal vein pattern; vein pat- points correspond to locations where serrations (see WEB tern in adaxialized mutants; and vein pattern in abaxialized TOPIC 19.3) and water pores called hydathodes (discussed mutants. (From Lucas et al. 2013.) below) can develop. As the auxin concentration builds up in these regions, auxin efflux induces PIN1-mediated auxin flow away from the convergence points toward the in the abaxialized mutants, such as those of the KANADI primary vein, which in turn causes the differentiation of gene family, phloem cells surround the xylem cells. pre-procambium along the path of auxin flow, eventually forming a secondary leaf vein. In Arabidopsis leaves, ter- Auxin canalization regulates higher-order tiary vein formation can result in loops that connect the vein formation primary and secondary veins. Again, this tertiary vein As it does during leaf trace development, PIN1 is also formation is guided by canalization mediated by PIN1 thought to regulate auxin canalization during the forma- proteins (Figure 19.22B). tion of higher-order leaf veins. PIN1 in the epidermal layer Despite the abundant evidence correlating PIN1 distri- of the developing leaf directs auxin to convergence points bution in the leaf with auxin canalization and vein forma- along the leaf margin (Figure 19.22A). These convergence tion, pin1 mutants have surprisingly mild phenotypes (Fig-

(A) CP (B) CP Plant Physiology 6/E Taiz/Zeiger Sinauer Associates Morales Studio Figure 19.22 Model for higher- TZ6e_19.21 Date 08-29-14 order leaf vein formation in Ara- bidopsis. (A) Auxin accumulates at convergence points (CPs) on the leaf margins, where PIN1 proteins direct auxin transport. CP CP CP CP Canalization of polar auxin transport leads to the differentiation of the procambium of secondary veins. (B) Tertiary veins can form when auxin becomes diverted by PIN1 proteins associated with the midvein. Such tertiary veins may form loops that connect to the secondary veins. The red Auxin concentration arrows indicate the direction of gradient (high–low) PIN1-mediated auxin flow. (After PIN1-mediated auxin ow Petrášek and Friml 2009.)

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(A) (B) (C) Figure 19.23 Mutations that affect auxin transport or auxin biosynthesis alter leaf venation patterns. (A) Wild-type (WT) leaf. (B) pin1/pin6 double mutant. Although the venation pattern of the mutant is defective, it retains the normal hierarchy of veins. (C) yuc1/yuc2/yuc4/yuc6 quadruple mutant. In the absence of significant auxin biosynthesis, the venation pattern is highly reduced. (A and B from Sawchuk et al. 2013; C from Cheng et al. 2006.)

Wild type pin1/pin6 yuc1/yuc2/yuc4/yuc6 ure 19.23). For example, the pin1/pin6 double mutant leaf thode regions along the leaf margin, where YUCCA genes shown in Figure 19.23B has an altered shape and a defec- are known to be expressed (Figure 19.24A). Hydathodes tive venation pattern, but the basic hierarchical structure are specialized pores associated with vein endings at the of the veins is still intact, indicating that other factors also leaf margin, from which xylem sap may exude in the pres- contribute to auxin canalization. For example, other auxin ence of root pressure (see Chapter 4). Figure 19.24B stun- transporters, such as ABCB19, which helps to narrow can- ningly illustrates the canalization of auxin from its site of alized auxin streams by excluding auxin from neighbor- synthesis in the hydathode region to its sink—a developing cells, and AUX1/LAX permeases, which create uptake ing vein. sinks that increase auxin flow (see Chapter 17), may be The importance of auxin synthesis for leaf venation is able to maintain canalization in the absence of PIN1. dramatically demonstrated by the phenotypes of YUCCA gene mutants in Arabidopsis. In contrast to what is seen Localized auxin biosynthesis is critical in the mild phenotype of the pin1/pin6 double mutant for higher-order venation patterns (see Figure 19.23B), the normal venation pattern is almost An additional cause of auxin accumulation on the leaf entirely eliminated in yuc1/yuc2/yuc4/yuc6 quadruple margin, in addition to canalization by PIN1, is based on mutants, in which auxin biosynthesis is substantially localized auxin biosynthesis. As discussed earlier in the reduced (see Figure 19.23C). The few remaining veins chapter, the adaxial–abaxial interface triggers the expres- suggest that either residual auxin is being synthesized sion of the YUCCA (YUC) genes. Auxin production at the by a different biosynthetic pathway or that an auxin- leafPlant margins Physiology is 6/Ethought Taiz/Zeiger to stimulate the expansion of the independent pathway can direct a limited amount of vein lamina.Sinauer Associates Auxin accumulation is concentrated in the hyda- formation. IN HOUSE TZ6e_19.23 Date 09-29-14

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Figure 19.24 Auxin biosynthesis at the hydathodes of Arabidopsis leaves, as indicated by the expression of the GUS reporter gene driven by the auxin-responsive DR5 promoter. (A) An Arabidopsis leaf that has been cleared to reveal the blue stain. (B) Auxin flow and canalization from the hydathode toward a 1 mm 150 µm developing leaf vein. (From Aloni et al. 2003.)

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Based on the abundance of evidence from other stud- Shoot Branching and Architecture ies, we can reconstruct the process of vein formation as follows: The shoot and inflorescence architecture of flowering 1. Auxin is synthesized by YUCCA proteins and accu- plants is determined to a large extent by the branching mulates in the hydathode regions. patterns established during postembryonic development. The earliest vascular plants branched dichotomously at 2. Auxin efflux from the margin induces PIN1 forma- the SAM, producing two equal shoots. This condition tion and polar orientation in nearby cells, promot- exists today in some lower vascular plants (Figure 19.25) ing auxin flux away from the site of auxin synthesis. and a few angiosperms, such as certain cacti. 3. ABCB exporters enhance canalization by exclud- In contrast, shoot architecture in seed plants is charac- ing auxin from all but a narrow zone that leads terized by multiple repetitions of a basic module called the directly to the developing leaf vein, while AUX1/ phytomer, which consists of an internode, a node, a leaf, LAX uptake transporters create sinks that enhance and an axillary meristem (Figure 19.26). Modification of auxin flows. the position, size, and shape of the individual phytomer, 4. Auxin is taken up by the developing cells of the and variations in the regulation of axillary bud outgrowth, vein, which maintains auxin flux until the vein is provided the morphological basis for the remarkable fully differentiated. diversity of shoot architecture among seed plants. Veg- etative and inflorescence branches, as well as the floral primordia produced by inflorescences, are derived from (A) axillary meristems initiated in the axils of leaves. During vegetative development, axillary meristems, like the apical meristem, initiate the formation of leaf primordia, resulting in axillary buds. These buds either become dormant or develop into lateral shoots depending on their position

SAM

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Figure 19.25 Dichotomous branching in the primitive Root cap vascular plant Psilotum nudum (image shows sporangia). (A) Shoot showing dichotomous branching. (B) Shoot tip showing the formation of two SAMs during branch formation. A, Figure 19.26 Schematic drawing of a phytomer, the basic shoot apical meristem. (B from Takiguchi et al. 1996.) module of shoot organization in seed plants.

© 2014 Sinauer Associates, Inc. This material cannot be copied, reproduced, manufactured or disseminated in any form without express written permission from the publisher. Plant Physiology 6/E Taiz/Zeiger Sinauer Associates IN HOUSE TZ6e_19.25 Date 09-08-14 Plant Physiology 6/E Taiz/Zeiger Sinauer Associates Morales Studio TZ6e_19.26 Date 09-11-14 Vegetative Growth and Organogenesis 573

along the shoot axis, the developmental stage (A) Wild type (B) ls mutant of the plant, and environmental factors. During reproductive development, axillary meristems initiate formation of inflorescence branches and flowers. Hence, the growth habit of a plant depends not only on the patterns of axillary meristem formation, but also on meristem identity and its subsequent growth characteristics. Axillary meristem initiation involves many of the same genes as leaf initiation and lamina outgrowth Auxin biosynthesis, transport, and signaling are all required for the initiation of axillary meristems, as demonstrated by the fact that mutants defective in these pathways fail to form new axillary meristems. Axillary meristem initiation involves three main steps: correct positioning of initial cells, delineation of the meristem boundaries, and establishment of the meristem proper. Figure 19.27 The tomato mutant lateral suppressor (ls) shows As discussed earlier in the chapter, PIN1- defects in axillary bud formation. (A) A wild-type plant. (B) The ls mediated auxin transport helps determine the mutant. Axillary buds fail to form in most of the leaf axils. (Courtesy of sites of leaf primordia, and it is also important Klaus Theres.) for axillary meristem formation. Not surprisingly, genetic evidence indicates considerable overlap in the gene networks involved in shoot tip, which ultimately determine shoot architecture. the initiation of leaf primordia, leaf margin serrations, The main hormones involved are auxin, cytokinins, and and axillary meristems. For example, mutations in the strigolactones (see Chapter 15). All three hormones are LATERAL SUPPRESSOR (LAS) genes from tomato (Sola- produced in varying quantities in the root and shoot, but num lycopersicum) (Figure 19.27) and Arabidopsis cause translocation of the hormones allows them to exert effects a complete block in axillary bud formation during the far from their site of synthesis (Figure 19.29). vegetative phase of development, and similar results have been observed in rice (Oryza sativa). Consistent with this finding, LAS mRNA has been shown to accumulate in the axils of leaf primordia, where new axillary meristems develop (Figure 19.28). LAS expression patterns P4 are similar to those for CUC genes, which, as discussed earlier, regulate embryonic shoot meristem formation and specify lateral organ boundaries. Two other genes that are P3 known to be required for normal axillary bud formation in Arabidopsis are the bHLH protein gene REGULATOR SAM P2 OF AXILLARY MERISTEM FORMATION (ROX) andPlant the Physiology 6/E Taiz/Zeiger MYB transcription factor gene REGULATOR OF AXILSinauer- Associates P1 IN HOUSE LARY MERISTEMS (RAX). TZ6e_19.27 Date 09-08-14 Auxin, cytokinins, and strigolactones regulate axillary bud outgrowth Once the axillary meristems are formed, they may enter a phase of highly restricted growth (dormancy), or they Auxiliary may be released to grow into axillary branches. The “go bud initials or no go” decision is determined by developmental pro- gramming and environmental responses mediated by plant hormones that act as local and long-distance signals. Figure 19.28 Accumulation of LATERAL SUPPRESSOR Interactions of hormonal signaling pathways coordinate mRNA in the axillary bud regions of an Arabidopsis shoot the relative growth rates of different branches and the tip. P1–P4 = leaf primordia. (From Greb et al. 2003.)

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Auxin Figure 19.29 Long-distance transport of three hormones that regulate shoot branching: auxins, cytokinins, Cytokinins and strigolactones. Auxin is produced primarily in young Strigolactones expanding leaves and is transported basipetally by PIN1- Auxin synthesis PIN Xylem mediated polar auxin transport. Strigolactones and cyto- proteins kinins are synthesized mainly in the root and can be trans- Basipetal located acropetally to the shoot in the xylem. These two transport hormones can also be synthesized in shoot tissues adjacent to axillary buds. (After Domagalska and Leyser 2011.) Acropetal transport

More than a century of experimental evidence suggests that in plants with strong apical dominance, auxin produced in the shoot tip inhibits axillary bud outgrowth. In plants with strong apical dominance, mutants with decreased rootward auxin transport exhibit increased branching, and treatment of the shoot apex with auxin transport inhibitors results in increased branching. Addi- tion of auxin to the shoot at the point of apical excision inhibits outgrowth, while application of auxin transport inhibitors to the stem releases the axillary buds below Cytokinin and from apical dominance (Figure 19.30A). Gardeners take strigolactone advantage of this phenomenon when they “pinch back” synthesis chrysanthemums with strong apical dominance to create dense, dome-shaped bushes of blossoms. Strigolactones act locally to repress axillary bud growth Strigolactones are thought to act in conjuction with auxin Auxin is synthesized predominantly in young leaves during apical dominance. Arabidopsis mutants defec- and the shoot apex and is transported rootward in a spe- tive either in strigolactone biosynthesis (max1 [more axil- cialized polar auxin transport stream via ABCB and PIN lary growth1], max3, or max4) or signaling (max2) show proteins in the vascular cylinder and starch sheath or increased branching without decapitation (Figure 19.30B). endodermis (see Chapter 18). Auxin can also be transported in the phloem, where it moves by mass flow from source to sink (see Chapter 11). Strigolactones are transported out of sites of synthesis Figure 19.30 Axillary bud outgrowth is inhibited by auxin  Plant Physiology 6/E Taiz/Zeiger and strigolactones. (A) Classic physiological experiments via plasma membrane ABC transporters Notand surehave beenwhat the dashed arrows at the top of the root area Sinauer Associates demonstrating the role of auxin in apical dominance. In de- shownMorales to Studio move in the xylem from root toare shoot. supposed Cytoki- to betipped shoots the axillary bud is released from apical domi- TZ6e_19.29 Date 08-28-14 nins, too, are transported from sites of synthesis to the nance. Replacing the missing shoot tip with auxin prevents xylem by an ABC transporter, and they can also move in bud outgrowth. Applying an inhibitor of polar auxin trans- the phloem. Consequently, there is considerable scope for port to the stem causes bud outgrowth below the applica- long-distance communication via these hormones. tion site. (B) Grafting experiments carried out with mutants defective in strigolactone biosynthesis or signaling that Auxin from the shoot tip maintains have increased branching. Grafting the shoots of the strigo- apical dominance lactone biosynthesis mutants (max1, max3, or max4) to wild- The role of auxin in regulating axillary bud growth is most type roots restored shoot branching of the mutant to wild- easily demonstrated in experiments on apical dominance. type levels. Grafting the roots of the strigolactone signaling mutant max2 onto the shoots of the wild type and synthesis Apical dominance is the control exerted by the shoot tip mutants max1, max3, or max4 also prevented bud out- over axillary buds and branches below. Plants with strong growth, demonstrating that max2 can produce the signal in apical dominance are typically weakly branched and show the roots, even though it cannot respond to it. The branch- a strong branching response to decapitation (removal of inhibiting hormone can also be produced in the shoot, as the growing or expanding leaves and shoot tip). Plants grafting of the wild-type shoot to the strigolactone- with weak apical dominance are typically highly branched deficient roots (max1, max3, or max4) did not increase the and show little, if any, response to decapitation. number of branches. (After Domagalska and Leyser 2011.)

(A) Stump Auxin Auxin transport inhibitor

Intact plant De-tipped De-tipped PAT inhibitor plus IAA

(B)

Wild type max2 mutant max1;3;4 mutant max1;3;4 shoot, wild type root

max2 shoot, WT shoot, max1;3;4 shoot, WT shoot, wild type root max2 root max2 root max1;3;4 root © 2014 Sinauer Associates, Inc. This material cannot be copied, reproduced, Plant Physiology manufactured6/E Taiz/Zeiger or disseminated in any form without express written permission from the publisher. Sinauer Associates Morales Studio TZ6e_19.30 Date 09-02-14 576 Chapter 19

O O Figure 19.31 Model for strigolactone ubiquitin ligase signaling. (After Janssen and Snowden 2012.) MAX2A, DAD2, PSK (Skp), and O O Cullin are all components of the SCFMAX2 ubiq- O uitin ligase complex. Strigolactone 1. The α-/β-fold hydrolase D14 binds and reacts with Target strigolactone, changing its conformation to the active form, D14*. D14* (α-/β-fold 2. D14* interacts with D14 hydrolase) the F-box protein MAX2 (α-/β-fold and the other partners hydrolase) Target of the SCFMAX2 MAX2A ubiquitin ligase (F-box) PSK (Skp) Cullin complex.

4. D14* hydrolyses strigolactone MAX2A and releases the products of (F-box) PSK hydrolysis. D14 disengages from (Skp) Cullin the SCFMAX2 complex and returns Ubiquitination to its original conformation, O O allowing it to respond to fresh 3. Target protein(s) are strigolactone signal. recognized by the D14*–SCFMAX2 complex OH Target and are ubiquitinated.

Ubiquitin Grafting the biosynthesis mutant shoot onto a wild-type root restores apical dominance, indicating that strigolactone can move from the root to the shoot. However, breaking apical dominance. Consistent with this hypoth- root-derived strigolactone is not required for bud repres- esis, the expression of two cytokinin biosynthetic genes sion, since wild-type shoots grafted onto strigolactone- (IPT1 and IPT2) increases in the second nodal stem of deficient roots have normal apical dominance. This result peas following decapitation, suggesting that auxin from suggests that the strigolactones that repress bud growth the shoot apex normally represses these genes. This was normally come from within the shoot. confirmed by incubating excised stem segments with and Genes regulating strigolactone biosynthesis and recep- without auxin; IPT1 and IPT2 expression persisted only in tion are conserved among higher plants. Strigolactones are segments incubated without auxin. In addition, applica- produced in the plastids from b-carotene by three sequen- tion of the auxin transport inhibitor 2,3,5-triiodobenzoic tially acting plastid enzymes that must be co-located in acid (TIBA) around the internode led to increased IPT1 the same cell: D27, a carotenoid isomerase, and CCD7 and and IPT2 expression below the site of application, demon- CCD8, which are carotenoid cleavage dioxygenases (see strating that these genes are normally repressed by auxin Chapter 15). The product, an apocarotenoid named car- transported down from the shoot apex. Thus, it appears lactone, can move between cells but must undergo two that the cytokinins involved in breaking apical dominance oxygenation steps to produce a bioactive strigolactone. are synthesized locally at the node, not transported from The oxygenation steps are catalyzed by a cytosolic cyto- the root. chrome P450. A simplified model for the antagonistic interactions Strigolactones are perceived by a protein complex con- between cytokinin and strigolactone is shown in Figure taining an a-/b-fold hydrolase protein and an F-box protein 19.32. Auxin maintains apical dominance by stimulating (D14 and MAX2, respectively) (Figure 19.31). The signal- strigolactone synthesis via the MAX4 gene. In eudicots, ing mechanism appears to be similar to that for gibberellin strigolactone then activates the gene for BRANCHED1 signaling (see Figures 15.33 and 15.34) and involves target- (BRC1), a transcription factor which suppresses axillary ing proteins for degradation by ubiquitination. bud growth. Besides activating BRC1, strigolactone also Plant Physiology 6/E Taiz/Zeiger inhibits cytokinin biosynthesis by negatively regulat- CytokininsSinauer Associates antagonize the effects of strigolactones ing the IPT genes. In contrast, cytokinin inhibits BRC1 DirectMorales application Studio of cytokinin to axillary buds stimulates action, and prevents auxin-induced strigolactone biosyn- TZ6e_19.31 Date 09-02-14 their growth, suggesting that cytokinins are involved in thesis. In rice, the BRC1 homolog FINE CULM1 (FC1) is

Auxin The initial signal for axillary bud growth may be an increase in sucrose availability to the bud Recent evidence indicates that sucrose itself may serve MAX4 as the initial signal in controlling bud outgrowth (Fig- IPT ure 19.34). In pea plants, axillary bud growth is initiated approximately 2.5 h after decapitation. This is 24 h prior to Strigolactone any detectable decline in the auxin level in the stem adja- Cytokinin cent to the axillary bud, which suggests that a decrease in auxin from the tip occurs much too slowly to initiate bud BRC1 outgrowth. In contrast, studies using [14C]sucrose demonstrated Axillary bud growth that the concentration of leaf-derived sucrose in the stem adjacent to the bud begins to decline in as little as 2 h after Figure 19.32 Hormonal network regulating apical domi- decapitation. This decline is due to the uptake of sugars nance. Auxin from the shoot apex promotes strigolactone by the axillary bud. Thus, following decapitation, bud out- synthesis in the nodal area via the MAX4 gene. In eudicots, growth on the lower stem is initiated prior to auxin deple- strigolactone up-regulates the BRANCHED1 (BRC1) gene tion but after sucrose depletion in the stem adjacent to the and down-regulates IPT genes. BRC1 inhibits axillary bud bud. As a result of decapitation, the endogenous carbon growth. Strigolactone also inhibits cytokinin biosynthesis, supply to the axillary buds increases within the timeframe which otherwise would prevent BRC1 production. (From El- sufficient to induce bud release. Apical dominance is thus Showk et al. 2013.) regulated by the strong sink activity of the growing tip, which limits sugar availability to the axillary buds. How- ever, sustained bud outgrowth requires the depletion of the target of strigolactone signaling, while in maize the auxin in the stem adjacent to the bud as well. TEOSINTE BRANCHED1 (TB1) is the primary gene regulating branching. This gene is responsible for a major trait Integration of environmental and hormonal involved in the domestication of maize, transforming the branching signals is required for plant fitness highly branched maize progenitor teosinte into the more In some cases, a plant can adjust its default shoot branch- desirable reduced branching phenotype of modern maize ing pattern in response to environmental conditions. Two (Figure 19.33). classic examples are the shade avoidance response and

(A) (B) Plant Physiology 6/E Taiz/Zeiger Sinauer Associates Morales Studio TZ6e_19.32 Date 08-28-14

Figure 19.33 Comparison of teosinte (Zea mays ssp. parviglumis) and modern maize (Zea mays ssp. mays). Teosinte (Zea mays ssp. parviglumis) Maize (Zea mays ssp. mays) (Left photo courtesy of Paul Gepts.)

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Auxin Sugars nutrient conditions. The increase in strigolactone sup- Auxin Sugars presses axillary bud outgrowth. Reduced branching in response to nutrient deficiency is adaptive because Bud release the plant is able to focus its resources on development and growth of the main shoot and existing branches, rather than on promoting the growth of additional branches that cannot be supported by the nutrient supply. Axillary bud dormancy in woody plants is affected by season, position, and age factors Auxin Sugars Auxin Sugars Woody perennial plants produce dormant buds pro- tected by specialized bud scales in response to a variety Bud release Bud release of environmental and age factors (Figure 19.35). Major and growth and growth X environmental factors that influence bud dormancy include temperature, light, photoperiod, water, and nutrients. Bud position and plant age are also important factors. The circadian clock and flowering genes such as FT, CO, and TFL1, together with phytochrome A, are involved in controlling dormancy in deciduous trees in relation to photoperiod and chilling requirements. In aspen trees, for example, an established target of Intact Decapitated this regulatory system is the cell cycle in buds. Even in Figure 19.34 Apical dominance is regulated by sugar avail- herbaceous plants, pathways that regulate flowering in ability. After decapitation, sugars, which normally flow toward response to photoperiod interact with pathways regu- the shoot tip via the phloem, rapidly accumulate in axillary lating axillary bud outgrowth. For example, strigolac- buds, stimulating bud outgrowth. At the same time, the loss tone branching mutants of garden pea (Pisum sativum) of the apical supply of auxin results in a depletion of auxin show dramatic shifts in the position and number of in the stem. However, auxin depletion is relatively slow and axillary branches when grown under different photo- therefore the growing buds in the upper shoot are affected before those lower on the stem. In this model, auxin is involved predominately in the later stages of branch growth. (After Mason et al. 2014.)

the nutrient deficiency response. Both of these responses involve the regulatory pathways described above. Plants avoid shade through enhanced shoot elongation and suppressed branching. Shade avoidance involves phy- Plant Physiology 6/E Taiz/Zeiger Sinauertochrome Associates B signaling in response to the decreased R:FR Bud scales Moraleslight ratio Studio that results when sunlight is filtered through TZ6e_19.34green leaves containing Date 09-02-14 chlorophyll (see Chapter 18). Genetic studies in Arabidopsis have shown that phytochrome B requires both auxin and strigolactone signaling pathways, as well as the budspecific BRC1 and BRC2 genes to inhibit axillary bud outgrowth under shading conditions. The response to nutrient deficiency is mediated by strigolactones. Well-nourished plants are bushy, whereas plants growing under poor nutrient conditions tend to be weakly branched. The involvement of strigolactones in this branching response presumably relates, evolutionarily, to the role of these hormones in enhancing nutrient acquisition. Mycorrhizal plant species secrete strigolactones into the rhizosphere to promote the mycorrhizal symbiosis and enhance nutrient uptake. The details vary among different species, but even in non-mycorrhizal plant species, Figure 19.35 Dormant axillary buds of horse chestnut strigolactone levels in the shoot are elevated under low (Aesculus hippocastanum) surrounded by bud scales.

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periods (even prior to flower opening), and flowering genes affect branching at cauline nodes in Arabidopsis. Plants can modify their root system architecture to optimize water and nutrient uptake Root System Architecture Root system architecture is the spatial configuration of the entire root system in the soil. More specifically, root Plant root systems are the critical link between the system architecture refers to the geometric arrangement growing shoot and the rhizosphere, providing both vital of individual roots within the plant’s root system in the nutrients and water to sustain growth. In addition, roots three-dimensional soil space. Root systems are composed anchor and stabilize the plant, enabling the growth of of different root types, and plants are able to modify and aboveground vegetative and reproductive organs. Because control the types of roots they produce, the root angles, roots function in heterogeneous and often changing soil the rates of root growth, and the degree of branching. conditions, roots must be capable of adapting to ensure Variations in root system architecture within and across a steady flow of water and nutrients to the shoot under a species have been linked to resource acquisition and variety of conditions. Recent research on the structure of growth. As illustrated in Figure 19.36, root system archi- root systems has been driven by advances in root system tecture varies widely among species, even those living in phenotyping (see WEB TOPIC 19.4). These and other stud- the same habitat. ies have shown that plants have evolved complex control mechanisms that regulate root system architecture. Figure 19.36 Diversity of root systems in prairie plants.

8 (D) (E) (I) 7 (H) (N) (Q) 6 (J) 5 (F) (M) (O) 4 (C) (K) (P) (S) 3 (B) (G) (L) (R) 2 (T) (U) 1 (A) Feet 1 2 3 4 5 6 8 7 9 10 11 12 13 14 15 (A) Kentucky blue grass (G) Heath aster (L) Side oats gramma (Q) Rosin weed (Poa pratensis) (Aster ericoides) (Bouteloua curtipendula) (Silphium perfoliatum) (B) Lead plant (H) Prairie cord grass (M) False boneset (R) Purple prairie clover (Amorpha canescens) (Spartina pectinata) (Kuhnia eupatorioides) (Petalostemum purpureum) (C) Missouri goldenrod (I) Big blue stem (N) Switch grass (S) June grass (Solidago missourienis) (Andropogon gerardii) (Panicum virgatum) (Koeleria cristata ) (D) Indian grass (J) Pale purple coneflower (O) White wild indigo (T) Cylindric blazing star (Sorghastrum nutans) (Echinacea pallida) (Baptisia leucantha) (Liatris cylindracea) (E) Compass plant (K) Prairie dropseed (P) Little blue stem (U) Buffalo grass (Silphium laciniatum) (Sporobolus heterolepis) (Andropogon scoparius) (Buchloe dactyloides) (F) Porcupine grass (Stipa spartea)

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(A) (B) Figure 19.37 Root system of a 14-day-old maize seedling composed of primary root derived from the embryonic radicle, the seminal roots Crown roots derived from the scutellar node, postembryonically formed crown roots that arise at nodes above the mesocotyl, and lateral roots. (B) Mature maize root system. (A from Hochholdinger and Tuberosa 2009.)

Lateral roots Seminal roots

Monocots and eudicots differ in their root system architecture tative eudicot is depicted in Figure 19.38 where the tap, Before examining the complexities of root system archi- branch, basal, and adventitious roots can be seen. tecture, it is important to understand how monocot and eudicot root systems are organized and how they differ. Root system architecture changes in response Both monocot and eudicot root systems are roughly simi- to phosphorous deficiencies lar in structure, consisting of an embryonically derived Phosphorus is, along with nitrogen, the most limiting primary root (the radicle), lateral roots, and adventitious mineral nutrient for crop production (see Chapters 5 and roots. However, there are significant differences in their 13). Phosphorus limitation is a particular problem in tropi- root systems. Monocot root systems are generally more cal soils, where the highly weathered acidic soils tend to fibrous and complex than the root systems of eudicots, bind phosphorus tightly, rendering much of it unavail- especially in the cereals. For example, the maize seedling able for acquisition by roots. Plant root systems undergo root system consists of a primary root that develops from well-documented morphological alterations in response to the radicle, seminal roots (adventitious roots that branch phosphorus deficiency. These responses can vary some- from the scutellar node), and postembryonically derived what from species to species, but in general they include a crown roots (Figure 19.37). The primary and seminal reduction in primary root elongation, an increase in lateral roots are highly branched and fibrous. The crown roots, root proliferation and elongation, and an increase in the Plantalso Physiology called “prop 6/E roots,” Taiz/Zeiger are adventitious roots derived number of root hairs. Sinauer Associates INfrom HOUSE the lowermost nodes of the stem. Although crown Phosphorus, always in the form of the phosphate TZ6e_19.37roots are relatively unimportantDate 09-08-14 in seedlings, in contrast anion, is immobile in soil because it binds tightly to iron to the primary and seminal roots, crown roots continue to and aluminum oxides on the surfaces of clay particles or form, develop, and branch throughout vegetative growth. is fixed as biological phosphorus within soil microorgan- Thus the crown root system makes up the vast majority of isms. Hence, the majority of the phosphorus, especially the root system in adult maize plants. in low-phosphorus soils, is trapped in the surface hori- The root system of a young eudicot consists of the pri- zons (layers) of the soil. Phosphorus deficiency can trigger mary (or tap) root and its branch roots. As the root system “topsoil foraging” by plants. Some bean genotypes, for matures, basal roots arise from the base of the tap root. In example, respond to phosphorus deficiency by producing addition, adventitious roots can arise from subterranean more adventitious lateral roots, decreasing the growth stems or from the hypocotyl, and can be considered to be angle of these roots (relative to the shoot) so that they loosely analogous to the adventitious crown roots in the are more shallow, increasing the number of lateral roots cereals. The root system of a soybean plant as a represen- emerging from the tap root, and increasing root hair den-

Figure 19.38 Soybean root system showing primary (tap) root, branch roots, basal roots, and adventitious roots. (Courtesy of Leon Kochian.) Adventitious root sity and length (Figure 19.39). These changes in root system architecture combine to locate more of the roots in the topsoil where the majority of Basal the phosphorus resides. These and similar dis- root coveries of “phosphorus-efficient” genotypes have enabled researchers to breed for root system architecture traits in bean and soybean that better adapt these crops to low-phosphorus soils. Branch Once roots are placed in soil with adequate sup- root plies of phosphorus, they still must solubilize and absorb the phosphate. These tasks are facilitated by a range of biochemical processes, such as the release of organic acids into the soil to solubilize phosphate from aluminum and iron phosphates, the release of phosphatases to solubilize organic Tap root phosphorus, acidification of the rhizosphere, and

Figure 19.39 Topsoil foraging of phosphorus by phosphorus- efficient bean genotypes. (After Lynch 2007.)

Plant Physiology 6/E Taiz/Zeiger Sinauer Associates Non-adapted genotypes Adapted genotypes IN HOUSE TZ6e_19.38 Date 09-02-14Aerenchyma Topsoil More adventitious roots

Smaller root diameter

Shallower basal roots Subsoil More dispersed laterals

Greater root biomass

Mycorrhizas More exudates: Organic acids, protons, Longer, denser, phosphatases root hairs

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an increased abundance of phosphate transporters on the not yet been identified. The arrival of these stress signals plasma membrane (see Chapters 5 and 13). Phosphorus- at target cells in source leaves triggers additional signal- efficient plants are enhanced in these biochemical adapta- ing events. Subsequently, long-distance signals from tions, maximizing their ability to extract phosphorus from the shoot—including siRNAs and miRNAs, mRNAs, low-phosphorus soils. proteins, sucrose, and other unidentified signals—are transported via the phloem to various sinks, where they Root system architecture responses to regulate plant growth and phosphorus homeostasis. phosphorus deficiency involve both local These sinks include both root and shoot apical meri- and systemic regulatory networks stems. For example, the miRNA miR399 has been shown Both local and systemic regulatory networks are involved to be induced and transported via the phloem to the root in the adaptation of root system architecture to phospho- under phosphorus-stress conditions, where it suppresses rous deficiency. Individual roots are able to respond locally expression of a putative ubiquitin E2 conjugase, which is to phosphorus-deficient patches in the rhizosphere; hor- involved in the degradation of root phosphate transport- mones play important roles in reprogramming root devel- ers. The miR399-mediated suppression of ubiquitination opment locally to facilitate more efficient phosphorus cap- in response to phosphorus deficiency results in the pro- ture by that part of the root system. However, if a plant motion of root phosphate transport. In addition, genes experiences prolonged phosphorus deficiency, systemic acting downstream of the miR399 signaling pathway signaling comes into play. regulate phosphate loading into the xylem and encode a The systemic regulation of phosphorus-deficiency plasma membrane phosphate transporter. responses is summarized in Figure 19.40. This initially Thus far it appears that all of the phosphorus-deficiency involves long-distance transport of signals from the root miRNAs that are transported to the root via the phloem to the shoot via the xylem. The root-to-shoot signals may are involved in the regulation of root phosphate trans- include the phosphate ion itself, as well as sugars, cytoki- port processes rather than root development. However, nins, strigolactones, and possibly other signals that have it is likely that other, as yet unidentified, phloem-mobile signals play a role in altering root system architecture. As the model in Figure 19.40 indicates, phosphorus deficiency Figure 19.40 Phosphate sensing involves communica- increases the abundance of IAA18 and IAA28 mRNAs, tion between the root and shoot. Phosphate deficiency in and it has been shown that these mRNAs are transported the soil results in the movement of various stress signals to the root in tomato plants, altering root auxin sensitivity in the xylem to the shoot (black arrows), where they alter and lateral root formation. development and trigger phosphorus homeostatic mechanisms. Additional signals originating in source leaves then move via the phloem to sink leaves and roots, where they can affect development and phosphorus-stress responses

(purple arrows). Pi, phosphate. (After Zhang et al. 2014.) 2. Shoot-derived long-distance signals (e.g., siRNAs, mRNAs, 3. Hormonal signals proteins, and sucrose) are can affect branching transported via the phloem patterns and from source leaves to sink phosphorus leaves and roots, where they homeostasis. regulate growth, development, and phosphorus homeostasis.

Shoot 5. Transcripts of IAA18 and IAA28 move through the Root phloem and can inhibit lateral root growth.

1. Root-derived stress signals (Pi, cytokinins, strigolactones) triggered 4. Sucrose and other signals from the by phosphorus de ciency are shoot can regulate lateral root transported in the xylem to the initiation, aerenchyma formation, root shoot, affecting shoot growth and hair development, and phosphate architecture, such as branching. transport.

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Mycorrhizal networks augment root system Bark architecture in all major terrestrial ecosystems Periderm As we discussed earlier in Chapter 5, fungal mycorrhizas are nearly ubiquitous in nature and play an important role in the mineral nutrition of individual plants. In addi- Fusiform tion, recent studies have shown that entire communities cell of plants are typically linked by mycorrhizal associations, Ray cell which form nutritional networks. A mycorrhizal network is defined as a common mycorrhizal mycelium linking the roots of two or more plants. For decades, scientists have been fascinated by evidence that mycorrhizal networks can transfer organic and inor- ganic nutrients, especially phosphate, between the root systems of otherwise separate individuals. Long-distance nutrient transfer through direct hyphal pathways appears to occur by mass flow driven by source–sink gradients generated by nutrient differences between plants. Through their effects on plant nutrition, mycorrhizal networks have been shown to facilitate seedling establishment, promote vegetative growth, and enhance plant responses to biotic and abiotic stress in a wide range of ecosystems. At the ecosystem level, mycorrhizal networks play an important role in carbon, nutrient, and water cycling. Growth In recent years the use of molecular techniques has shed light on the nature and extent of mycorrhizal networks among forest trees and between overstory and understory plants. For example, microsatellite DNA sequences (short Cork Cambial zone tandem repeats) have been employed as molecular mark- Phellogen Xylem ray ers to study the spatial topology of genets (clonal colonies) Phloem ray Secondary xylem of ectomycorrhizal fungi in the genus Rhizopogon. In a for- Primary phloem Primary xylem est of Douglas-fir (Pseudotsuga menziesii), the majority of Secondary phloem Pith trees in a 30 × 30 m plot were found to be interconnected by a complex mycorrhizal network of Rhizopogon vesiculo- Figure 19.41 Internal anatomy of a woody stem. The sus and R. vinicolor. The most highly connected tree was zone of the vascular cambium (red region) consists of a sin- linked to 47 other trees through eight R. vesiculosus genets gle layer of cambial stem cells and its immediate derivatives and three R. vinicolor genets. The interconnectivity of the on either side, and is surrounded by an outer layer of sec- trees in this forest illustrates the surprising complexity of ondary phloem cells (black) and an inner layer of secondary nutrient flow in forest ecosystems, which will need to be xylem cells (light green). The primary phloem (dark blue), taken into account in future studies of the effects of cli- primary xylem (dark green), and pith (light blue) are also mate change on forest productivity. shown. The periderm includes the phellogen (tan cell layer) and phellem (cork cells, in brown). The bark layer includes all tissues external to the vascular cambium. Most angio- Secondary Growth sperm and gymnosperm tree species also contain radial files of ray cells that play a role in nutrient transport and All gymnosperms and most eudicots—including woody storage. (From Risopatron et al. 2010.) shrubs and trees, as well as large herbaceous species— develop lateral meristems that cause radial growth (growth in width) in stems and roots. Growth that results arisen repeatedly during the evolution of vascular plants, from lateral meristems is called secondary growth (Figure and many extinct groups exhibit conspicuous secondary 19.41; see also Figure 1.5). Two types of lateral meristems vascular tissues. Monocots as a group lack a vascular cam- Plant Physiology 6/E Taiz/Zeiger are involved in secondary growth: the vascular cambium, biumSinauer and Associates therefore do not exhibit secondary growth. Even which produces secondary vascular tissues, and the cork tree-formMorales Studio members of the Palmae lack a vascular cam- cambium, or phellogen, which produces the outer pro- biumTZ6e_19.41 and increase their Date width 08-29-14 solely by means of a pri- tective layers of the secondary plant body called the peri- mary thickening meristem, located in the “meristem cap” derm. Secondary growth via the vascular cambium has just below the leaf primordia, which produces additional

Terminal bud Figure 19.42 The development (A) of secondary vascular tissue. (A) Pri- Epidermis mary growth in woody stems occurs Cortex in the spring, followed by secondary growth. (B) Orientations of the Primary cell division planes in the cambial phloem zone maintain the proper balance This Bud scale Vascular between growth in diameter versus year’s cambium growth circumference. Cambial cells initially Pith Primary xylem divide anticlinally to produce new initials and increase the circumference of the cambium. The same Primary growth initials also divide periclinally to produce xylem and phloem mother cells, Cork always leaving behind another initial. Primary Periderm Secondary xylem Cork cambium xylem Cortex Primary phloem primary tissues, including vascular bundles. The primary thickening Secondary Last year’s phloem meristem persists in some species, growth producing additional parenchyma Pith Vascular cambium tissue and vascular bundles. The transition from primary to Secondary growth secondary growth in gymnosperms Scars left by bud and eudicots is readily visible along the shoot axis (Figure scales from 19.42A). In poplar, for example, primary growth occurs previous year in the top eight internodes, approximately 15 cm from Growth Axillary bud from two the SAM. Primary growth then gives way to secondary years ago Leaf scar (woody) growth that produces secondary xylem and secondary phloem. The primary and secondary growth zones are separated both spatially and temporally, are easily dis- cernible, and develop rapidly (within 1–2 months) in fast- (B) growing species such as poplar. Anticlinal divisions Periclinal divisions produce an add new initials to initial and a xylem or phloem The vascular cambium and cork cambium the cambium mother cell are the secondary meristems where secondary Phloem growth originates Secondary growth originates in the vascular cambium—a lateral meristem that displays perennial growth patterns Xylem in woody species. Many herbaceous species also have a Vascular vascular cambium, but its formation is usually conditional cambium (e.g., in response to stress) or is very short-lived. The vascular cambium consists of meristem cells (cam- Phloem cells bial initials) organized in radial files that form a continuous cylinder around the stem. Cambial initials divide to Phloem mother/ produce xylem, phloem, and ray mother cells, which in daughter cells turn undergo several rounds of divisions to form a zone of relatively undifferentiated cells that usually comprise Cambial initials six to eight cell files and are known as the cambium zone. Xylem mother/ These cells then differentiate into several cell types. The daughter cells vascular cambium of all modern extant seed plants is bifacial—that is, it produces xylem inward and phloem Xylem cells outward (see Figure 1.5). In addition to secondary growth involved in secondary phloem and xylem, most woody eudicots and gym-

© 2014 Sinauer Associates, Inc. This material cannot be copied, reproduced, Plant Physiology manufactured6/E Taiz/Zeiger or disseminated in any form without express written permission from the publisher. Sinauer Associates Morales Studio TZ6e_19.42 Date 09-08-14 Vegetative Growth and Organogenesis 585

nosperms develop a secondary cambium known as cork thought to indicate the position of the cambium initials, cambium or phellogen that gives rise to the periderm (see which are otherwise morphologically indistinguishable Figure 19.41). Collectively the periderm consists of phel- from the other cells in the cambium zone. The peak of logen, phellem, and phelloderm. Phellem, or cork, is the anticlinal division is typically within the first to second multilayered protective tissue of dead cells with suberized cell file proximal to the phloem and is usually used to pin- walls, which is formed outward by the phellogen. The phel- point the approximate position of the vascular cambium. loderm is living parenchyma tissue that is formed inward. In a typical bifacial cambium, the periclinal divi- The term bark, often applied incorrectly to the periderm sions produce phloem outward and xylem inward in the alone, actually consists of all the tissues outside the vas- woody stem. The proliferation of xylem is dispropor- cular cambium, including functional secondary phloem, tionately geater and in many ways more complex since crushed nonfunctional secondary phloem, crushed pri- it encompasses the full life cycle of the tracheary cells in mary phloem, and the periderm (phellogen, phellem, and a matter of days. In addition to phloem and xylem cells, phelloderm). Bark tends to peel easily from a tree because the vascular cambium produces ray cells, parenchyma the vascular cambium, with its dividing cell layers, is much cells that serve as conduits for lateral transport in the more fragile than the secondary tissues on either side. stem and for storage during unfavorable conditions such The degree of phellogen activity giving rise to phellem as winter dormancy. Ray cells can be arranged in one varies among tree species, with cork oak (Quercus suber) (uniseriate) or multiple (multiseriate) files to form a tis- representing an extreme example that contains a perma- sue known as rays that traverses the phloem, cambium, nent phellogen layer producing cork or phellem indefi- and xylem (see Figure 19.41). nitely. The thick, corky layer probably protects the main trunk from dehydration in the hot and dry Mediterranean Phytohormones have important roles in climate. regulating vascular cambium activity and differentiation of secondary xylem and phloem Secondary growth evolved early As with many other processes in plants, hormones play in the evolution of land plants important roles in the regulation of secondary growth. Fossil vestiges of primitive secondary growth activity can Several hormones provide positional cues and signals be found very early in plant evolution, possibly predating for growth and differentiation of different cell types and modern seed plants. Secondary growth likely predates tissues (Figure 19.43). Here we focus on four hormones the evolution of gymnosperm plants and is speculated to because a significant amount of experimental evidence be the ancestral life form of all modern angiosperms. For supports their role in regulation of secondary growth. example, the woody perennial Amborella genus is the most However, this does not imply that they have more signifi- basal lineage in the angiosperm clade, thought to be the cant roles than other hormones. ancestor of all modern angiosperms. The woody perennial Although auxin movements in trees has not been habit has been lost and reacquired during the evolution extensively studied, it is assumed that auxin is produced of seed plants, with some lineages showing intermediary in leaves and apical meristems and transported via polar forms, known as insular woodiness. Secondary woody auxin transport to the stem and vascular cambium. Mea- growth has evolved into various forms and shapes that surements of auxin concentrations from vascular cam- are likely adaptive in nature. For example, many lianas bium into differentiating xylem and phloem in both display flat stems resulting from differential proliferation angiosperm and gymnosperm trees have shown that the of xylem tissues in particular parts of the stem circum- peak of the gradient is located in the cambium initials, and ference. Alternatively, stems of some lianas (most nota- tapers toward the differentiating xylem and phloem. The bly from the Bignonieae family) maintain a cylindrical decrease is steeper toward the phloem and much more shape but produce internally wedge-shaped sectors of gradual toward the xylem. This concentration gradient parenchyma tissues. These changes, seen predominantly across the cambial zone has lead to the speculation that in lianas, are thought to facilitate stem flexibility, wound the role of auxin in xylem and phloem differentiation is healing associated with twisting, and recovery from loss based on a radial morphogen gradient. of xylem conductivity associated with severe twisting. The critical role of auxin is also supported by exogenous treatments showing that auxin application in Secondary growth from the vascular cambium decapitated trees, in which the vascular cambium has gives rise to secondary xylem and phloem become inactive, leads to reactivation of the cambium. Vascular cambium displays two main division pat- More recently, direct manipulation of the auxin response terns—anticlinal (perpendicular to the stem surface) and in transgenic poplar trees has shown that auxin sensitiv- periclinal (parallel to the stem surface) (Figure 19.42B). ity is critical for both periclinal and anticlinal divisions in Anticlinal divisions add more cells to the cambium to the cambium and affects the growth and differentiation accommodate the increasing girth of the stem and are of xylem cells.

Figure 19.43 Hormones are involved in regu- Vascular stem cells initial lating key stages of secondary vascular tissue development. ARABIDOPSIS HISTIDINE PHOS- PHOTRANSFER PROTEIN (AHP6) acts as a cytoki- Stem cell initiation/speci cation nin signaling inhibitor that restricts the domain of cytokinin activity, thus allowing protoxylem differentiation in a spatially specific manner.

Vascular stem cells Stem cell Auxin Procambium/cambium maintenance

Cytokinin has also been implicated in regu- Cell proliferation Cytokinin lation of secondary growth (see Figure 19.43). A specific decrease in the cytokinin concentration in the cambium zone of transgenic poplar Cell fate determination Gibberellin trees led to significant impairment of radial growth and cell division in the cambium. This Brassino- result was correlated with the expression in the steroid cambium zone of a gene encoding the cytoki- Phloem precursor Xylem precursor nin receptor and primary response regulator involved in cytokinin signaling. This suggests Auxin that cytokinin is an important regulator of cell Cell differentiation Cell differentiation proliferation in the cambium. Ethylene is yet another hormone that has AHP6 been strongly implicated as playing a regulatory role in secondary growth. The concentration of the ethylene precursor, 1-aminopropane-1-car- boxylic acid (ACC), was found to be high at the Companion Sieve Cytokinin Proto- Meta- cambium zone, but unlike with auxin and gib- cell element xylem xylem berellin, no gradient was detected. Ethylene treatment and ACC feeding experiments demonstrated that ethylene is a positive regulator of Gibberellins also play major and distinct role in sec- cambial activity, radial growth, and secondary ondary growth. Like auxins, bioactive gibberellins exhibit xylem formation. These results are also consistent with a concentration gradient across the wood-forming zone, the results of transgenic manipulation of ethylene biosyn- but unlike in auxin, the peak is shifted toward the devel- thesis and response in poplar. Ethylene plays an important oping xylem. Exogenous treatment of decapitated seed- role in the formation of tension wood, a specialized type lings lacking auxin with gibberellins resulted in the acti- of reaction wood in angiosperms formed in response to vation of cambium cell divisions. However the dividing stem bending or tilting. Expression of both ethylene bio- cells lost their typical shape and failed to differentiate into synthesis and signaling genes is elevated in the tension xylem. Simultaneous application of auxin and gibberellins wood–forming zone, and ethylene insensitive transgenic prevented the abnormalities observed in the gibberellin poplars fail to produce tension wood. treatment alone, and stimulated cambium division to an extent not seen in the gibberellin and auxin treatment Genes involved in stem cell maintenance, alone, suggesting that the two hormones act synergisti- proliferation, and differentiation regulate Plantcally Physiology(see Figure 6/E 19.43). Taiz/Zeiger secondary growth SinauerMetabolic Associates profiling and expression of several genes Like other developmental processes in plants, secondary Morales Studio TZ6e_19.43from the gibberellin Date biosynthetic 08-04-14 pathway indicate that growth involves several key steps. It can be divided into gibberellin metabolism in the wood-forming tissues also three developmental stages: involves transport of gibberellin precursors from the •• Maintenance of the initial cell microenvironment, or phloem laterally through the rays into the differentiating stem cell niche xylem where they are then converted to bioactive forms •• Proliferation and growth of cells derived from the stem (see Figure 19.43). Both exogenous treatments and trans- cells genic manipulations indicate that gibberellins have a positive effect on elongation of fiber cells, suggesting a role •• Differentiation of the dividing cell into different cell for gibberellins in xylem cell differentiation and growth. types, tissues, and organs

It is therefore not surprising that the developmental and during prolonged (seasonal) unfavorable or lethal condi- growth patterns during secondary growth are governed tions, such as these encountered during winter months by processes and genes that are similar to those that reg- in the temperate and boreal regions. To endure the dehy- ulate the development of the SAM. This similarity has dration and freezing stress during the winter months, aided the molecular dissection of the mechanisms of sec- trees alternate between periods of active growth and ondary growth. dormancy. The annual transition from active growth to Perhaps the best studied of these processes is the main- dormancy in the cambium results in the formation of tree tenance of the stem cell niche. In the SAM of Arabidop- rings that record the amount of lateral growth of the tree sis, KNOX1 transcription factors, such as SHOOT MERI- each year. The molecular mechanisms that control cam- STEMLESS (STM) and BREVIPEDICELLUS (BP), are bium growth during growth–dormancy cycles are poorly involved in maintaining stem cell identity. Orthologs of understood. Phytohormones such as auxin and gibberel- the STM and BP genes in poplar, known as ARBORKNOX lins are thought to play major roles in the reactivation and 1 and 2, play similar roles in the cambium. Overexpression cessation of growth in the cambium. of the two genes in transgenic plants leads to delayed dif- Growth seasonality also imposes a significant chal- ferentiation and enlargement of the cambium zone. lenge with respect to nutrient use, storage, and recycling. Proliferation of the dividing cells in the SAM is typi- Nitrogen is the most abundant macronutrient in plants. cally regulated by genes such as AINTEGUMENTA. Although all plant species have mechanisms to recycle, AINTEGUMENTA is an AP2-type transcription fac- store, and remobilize nitrogen during the growing sea- tor involved in regulation of organ size in Arabidopsis son, the seasonal cycling of nitrogen is a hallmark of through the activation of cell proliferation. In aspen, an the perennial life habit. For example, nitrogen from the ortholog of the AINTEGUMENTA gene was found to be senescing leaves is stored in the form of bark storage pro- highly expressed in the cells that display high cell prolif- teins (BSPs) in small vacuoles of the phloem parenchyma eration in the cambium zone. (inner bark). These proteins are synthesized early in the The best example of similarity between the regulation of autumn but are rapidly mobilized during the spring as the SAM and the cambium is arguably at the differentiation growth is reinitiated. The signaling mechanisms involved step. The HD-ZIP III and KANADI transcription factors are still unclear but may involve the transport of hormonal play important roles in defining the adaxial and abaxial signals from the SAM. polarity in the emerging leaf. This polarity results in the Physiologically, wood serves transport, storage, and differentiation of xylem on the adaxial side of the leaf vein, mechanical functions, and thus the response to various and phloem on the abaxial side (see Figure 19.5). The HD- environmental factors reflects changes that best accom- ZIP III and KANADI genes also regulate the patterning of modate these functions. These three functions are also the primary vascular bundles, as well as the differentiation reflected in the main cell types that are encountered in of secondary vascular tissues later in development, probably the xylem. For example, in a typical angiosperm the trans- through their effects on polar auxin transport. port, mechanical, and storage functions are carried out The three developmental stages considered above also by vessels, fibers, and parenchyma cells, respectively. The require spatial separation. This is achieved through tran- proportion of these cell types changes dramatically in scription factors that define the developmental boundar- response to different stress factors and reflects compen- ies. One such class of transcription factors involved in the satory shifts that reinforce one function or another. regulation of the SAM is LATERAL ORGAN BOUND- The mechanical function of wood is strongly reinforced ARIES (LBD). LBD genes help establish the boundary during reaction wood formation (see Figure 15.1D and meristem (discussed earlier in the chapter), which sepa- E). Reaction wood forms when stems are displaced from rates the undifferentiated cells in the SAM from the differ- their vertical position. In angiosperm trees, reaction wood entiating tissues of the leaf primordium. Members of the develops on the top part of the stem and is known as ten- LBD family were found to play a similar role in secondary sion wood. Tension wood is distinct from wood developed growth by separating the cambium zone from the differ- under vertical orientation in that it contains more fibers entiating secondary phloem and xylem. (e.g., the cells that serve the mechanical function), and its cell walls are enriched with highly crystalline cellulose Environmental factors influence vascular cambium that reinforces the mechanical function. In contrast, water activity and wood properties deficit or significant osmotic stress results in changes Plants are sessile and require robust responses to unfa- that support the transport function and are in a way the vorable conditions for survival (see Chapter 24). This is opposite of those in tension wood. Wood developed under particularly important for woody perennial plants such as drought conditions typically shows increased vessel den- trees that can occupy a site for hundreds and even thou- sity and cell walls that produce more lignin than cellulose. sands of years. One distinct challenge that trees face is These changes improve the water transport and retention climate seasonality, which poses risks to trees’ survival function of the xylem.

SUMMARY

After embryogenesis and germination, vegetative meristemoids, meristemoids to GMCs, and GMCs to growth is controlled by developmental processes mature guard cells (Figure 19.10). involving molecular interactions and regulatory • Stomatal-lineage cells and mesophyll cells excrete feedback. These mechanisms create root and shoot peptide signals that interact with transmembrane polarity, allowing plants to produce lateral organs receptors to regulate stomatal patterning (Figure (e.g., leaves and branching systems), which form an 19.11). overall vegetative architecture. • Genes in protoderm cells regulate trichome differen- Leaf Development tiation and distribution (Figures 19.12–19.14). • The development of flat laminas in spermatophytes • The GL2 transcription factor is the rate-limiting ele- was a key evolutionary event; since then, phyllome ment in trichome formation. morphology has diversified dramatically (Figure 19.1). • Jasmonic acid regulates development of leaf trichomes in Arabidopsis. The Establishment of Leaf Polarity • In addition to positional information, hormone distri- Venation Patterns in Leaves bution also affects leaf primordia emergence (Figure • Leaf venation patterns indicate the spatial organiza- 19.2). tion of the vasculature (Figures 19.15, 19.16). • Adaxial–abaxial polarity in a leaf primordium is es- • Triggered by auxin that is transported downward, tablished by a signal from the SAM (Figure 19.3). leaf veins are initiated separately from established • ARP transcription factors interact with protein vasculature and grow down to rejoin it, directed by partners to promote adaxial identity and repress the the vascular bundle in the stem (Figures 19.17–19.20). KNOX1 gene (Figures 19.4, 19.5). • Similarly to initial development of veins, develop- • Adaxial identity is also supported by HD-ZIP III tran- ment of higher-order veins proceeds from tip to base scription factors, which are abaxially suppressed by and is regulated by auxin canalization. However, the microRNA miR166. auxin transport is less dependent on PIN1 (Figures 19.21–19.23). • Specification of abaxial identity is promoted by KANADI and YABBY gene families and is antago- • Localized auxin biosynthesis allows for development nized by HD-ZIP III. of higher-order veins (Figure 19.24). • Normal blade growth depends on juxaposition of adaxial and abaxial tissues and is regulated by auxin Shoot Branching and Architecture and by YABBY and WOX genes (Figure 19.5). • Shoot architecture can be based on continuously • Leaf primordia also differentiate proximally–distally branching, equal shoots, or on repeating units of into a boundary meristem, lower-leaf zone, petiole, hierarchical shoots, leading to axillary branches (Fig- and blade (Figure 19.5). ures 19.25, 19.26). • • Similar genes and transcription factors govern com- Branch initiation involves some of the same genes pound leaf formation (Figures 19.7, 19.8). and hormones as leaf initiation and outgrowth (Fig- ures 19.27–19.29). Differentiation of Epidermal Cell Types • There is strong experimental and empirical evidence that auxin and strigolactones from the shoot tip • The epidermis is derived from the protoderm (L1) and maintain apical dominance (Figures 19.30, 19.31). has three main cell types: pavement cells, trichomes, and stomatal guard cells, as well as other cell types • Cytokinins break apical dominance and promote axil- (Figure 19.9). lary dominance (Figures 19.32, 19.33). • Not only guard cells, but the majority of leaf epider- • Sucrose also serves as an initial signal for axillary bud mal cells, arise from specialized meristemoid mother growth (Figure 19.34). cells (MMCs), stomatal lineage ground cells (SLGCs), • Environmental signals can override default hor- meristemoids, and guard mother cells (GMCs) (Fig- monal signals to shape vegetative architecture. For ure 19.10). example, woody perennial plants produce dormant • Basic helix-loop-helix (bHLH) transcription factors buds in response to temperature and to availability govern the cell-state transitions from MMCs to of water, nutrients, and light (Figure 19.35).

Root System Architecture Secondary Growth • Species-specific root system architecture optimizes • Growth in width is accomplished by the vascular water and nutrient uptake (Figure 19.36). cambium and cork cambium, which are secondary meristems that give rise to secondary vascular tissue • Monocot roots systems are composed largely of (Figures 19.41, 19.42). seminal and crown roots, while eudicot root systems are derived largely from the primary (tap) root (Fig- • Secondary growth predates the appearance of ures 19.37, 19.38). gynosperms. • Phosphorus availability can alter root system archi- • Auxin, gibberellins, cytokinins, and ethylene regu- tecture both locally and systemically (Figures 19.39, late vascular cambium activity and differentiation of 19.40). secondary vascular tissues (Figure 19.43). • Mycorrhizal relationships with root systems are ter- • Genes regulate the cellular microenvironment restrially ubiquitous. for stem cell maintenance, proliferation, and differentiation. • Vascular cambium activity is sensitive to environmental factors that ultimately influence wood properties.

WEB MATERIAL

• WEB TOPIC 19.1 Bifacial, Unifacial, and Equifa- • WEB TOPIC 19.3 Leaf Serrations Are Coordinated cial Leaves Bifacial, unifacial, and equifacial leaves by the Action of a CUC2–Auxin Feedback Loop can be distinguished based on their anatomical and While marginal serrations are modified by many morphological differences. genes, the key components are auxin and CUC2. • WEB TOPIC 19.2 Mechanical Stress Alters Micro- • WEB TOPIC 19.4 Advances in Root System Pheno- tubule Orientation and PIN1 Distribution in the typing Modern root system image capture methods SAM The apical meristem can be thought of as a include two-dimensional (2D) and three-dimensional giant cell whose shape generates stress patterns that (3D) imaging techniques. can influence PIN1 localization. available at plantphys.net

Suggested Reading Balkunde, R., Pesch, M., and Hülskamp, M. (2010) Tri- Fukushima, K., and M. Hasebe1 (2014) Adaxial–abaxial chome patterning in Arabidopsis thaliana: From genetic polarity: The developmental basis of leaf shape diver- to molecular models. Curr. Top. Dev. Biol. 91: 299–321. sity. Genesis 52: 1–18. Bayer, I., Smith, R. S., Mandel, T., Nakayama, N., Sauer, M., Greb, T., Clarenz, O., Schafer, E., Muller, D., Herrero, R., Prusinkiewicz, P., and Kuhlemeier, C. (2009) Integra- Schmitz, G., and Theres, K. (2003) Molecular analysis tion of transport-based models for phyllotaxis and mid- of the LATERAL SUPPRESSOR gene in Arabidopsis re- vein formation. Genes Dev. 23: 373–384. veals a conserved control mechanism for axillary meri- Besnard, F., Vernoux, T., and Hamant, O. (2011) Organo- stem formation. Genes Dev. 17: 1175–1187. genesis from stem cells in planta: Multiple feedback Hay, A., and Tsiantis, M. (2010) KNOX genes: Versatile loops integrating molecular and mechanical signals. regulators of plant development and diversity. Develop- Cell. Mol. Life Sci. 68: 2885–2906. ment 137: 3153–3165. Byrne, M. E. (2012) Making leaves. Curr. Opin. Plant Biol. Heisler, M. G., Hamant, O., Krupinski, P., Uyttewaal, M., 15: 24–30. Ohno, C., Jönsson, H., Traas, J., and Meyerowitz, E. M. Caño-Delgado, A., Lee, J. Y., and Demura, T. (2010) Regula- (2010) Alignment between PIN1 polarity and micro- tory mechanisms for specification and patterning of plant tubule orientation in the shoot apical meristem reveals vascular tissues. Annu. Rev. Cell Dev. Biol. 26: 605–637. a tight coupling between morphogenesis and auxin Domagalska, M.A., and Leyser, O. (2011) Signal integra- transport. PLOS Biol. 8(10): e1000516. DOI:10.1371/jour- tion in the control of shoot branching. Nat. Rev. Mol. nal.pbio.100051 Cell Biol. 12: 211–221.

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