Discuss the mechanisms responsible for polarity acquisition in lateral organs of plants. What evidence supports your views?

Chris Holland Jesus College

Plant development is based on the formation of modular sections, lateral organs are formed in a reiterative pattern from the periphery of the shoot apical meristem (SAM). Te establishment of polarity in the lateral organs is essential for asymmetric development and research to date indicates it involves both intrinsic and extrinsic factors. Molecular studies have revealed genes are thought to assist in establishing this polarity with many of them being spatially localised transcription factors. In this essay I shall describe a proposed model of organ polarity acquisition and support this with evidence from experiments that have been performed.

Lateral organ development starts with the formation of the SAM. Initially formed during embryogenesis the SAM is the progenitor of the above ground portion of the plant. In order to achieve this the SAM must serve three primary functions:

Lateral organs, such as leaves are produced from the peripheral regions of the SAM
The basal regions of the SAM are responsible for the creation of the stem.
The stem cells of the SAM must replenish those regions from which cells have been recruited and maintain the pool of pluripotent cells necessary for growth.

Through histological analysis the SAM can be divided up into three distinct regions based on cytoplasmic densities and cell division rates: the peripheral zone (PZ), the central (CZ) zone and the rib zone (RZ) (figure 1). Lateral organs are created from tissue in the peripheral zone, stem tissue is derived from the rib zone and the central zone is responsible for replenishing the cells used in the peripheral and rib zones. The SAM is also comprised of three clonally distinct cell layers. The fact that all the above zones are made from three different cell layers implies a lot of communication between the cell layers during the developmental process. The leaves in most eudicot species are comprised of cells from all three layers in the peripheral zone, the second layer (L2) begins periclinal divisions first in the leaf primordia which instructs the other two layers to divide accordingly (L1, L3).

Lateral organs produced by the PZ are usually polar in nature, exhibiting asymmetry in both their proximo-distal and their adaxial-abaxial (dorso-ventral) axis. Such asymmetry is necessary for leaves in order for the correct allocation of the different cell types, such as the chloroplast dense palisade mesophyll cells toward the adaxial side (light harvesting) and the stomatal cells on the abaxial side (gaseous exchange). Although a mature leaf contains more than 20 specifically spatially localised cell type the establishment of tissue polarity in its simplest form only requires generation of two populations of cells with distinct cell types.

Establishing adaxial-abaxial polarity.

Lateral organ primordia initially develop as a small number of cells with, as far as presently known, homologous gene expression. When the primordia becomes more morphologically distinct it is evident that both in terms of gene expression patterns and morphological differentiation that polarity in the leaf has been established. The adaxial side being closest to the SAM and the abaxial side being further away, figure 2.

Initial experiments on young potato leaves revealed that when separated from the SAM they developed into small radial leaves, apparently abaxialised. This suggested that the SAM was in part responsible for lateral organ polarity by the means of some form of diffusible signal. Once such model was that the SAM was responsible for promoting adaxial cell fate and that abaxial cell fate is the default pathway for a leaf. This is also backed by the fact that when older leaf primordia are surgically excised they autonomously develop into phenotypically normal leaves. Note that the model only state that ...

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Although surgical manipulation can tell you a certain amount about the morphological development of plant leaf primordia in order to study the underlying mechanisms determining leaf polarity attention was turned to mutants displaying abnormal leaf development. McConnell et al (2001) noted that the semi dominant mutations in Arabidopsis PHABULSOA and PHAVOLUTA genes phabulosa (phb), and phavoluta (phv) gave rise to plants with abaxial tissues transformed into adaxial ones. Through mutagenesis screening 5 PHB mutants and 4 PHV were found. By mapping the mutations to the ATHB14 and ATHB9 genes respectively they learnt that these genes belonged to a plant specific class of homeodomain-leucine zipper (HD-ZIP)-proteins. The HD-ZIP proteins are characterised by an amino terminal HD-ZIP motif followed by a sterol/lipid binding domain.

Experiments were carried out to try to recreate the mutant phenotypes observed in the screening. The group used ectopic expression of either the wild type ATHB14 protein or the ATHB14 protein with a 33bp insertion into the sterol/lipid binding domain and a truncated cDNA ending at the first stop in intron 4 (the product expected from transcripts that retain intron 4) under the CaMV 35s promoter. Ectopic expression of ATHB14 resulted in plants with normal leaf phenotype, suggesting that ATHB14 alone was not enough to direct ectopic adaxial development, consistent with ATHB14 requiring a ligand for activation. Half of the 33bp insertion mutants displayed the phb phenotype suggesting that modification to the sterol/lipid binding domain was sufficient to induce the phb phenotype. Repeated experiments with ATHB9 (PHAVOLUTA) gave rise to similar results, indicating ATHB14 and ATHB9 have similar functions.

By looking at expression patterns McConnell found PHB to be expressed in low levels throughout the initial leaf primordium and then higher expression levels subsequently localised to the adaxial side of leaf primordia. Combined this evidence they proposed that low levels are present throughout the young unpolarised leaf primordium. As the leaf develops a ligand for PHB is unequally distributed throughout the primordium, with highest levels present in the cells closest the SAM. This ligand activates PHB protein which in turn promotes adaxial leaf development, by inhibiting the default abaxial development factors. It would also positively feed back on the synthesis and/or stability of its own product. Such a feedback would create a reference point for adaxial positioning thus removing the need for an external ligand allowing it to grow away from the original source of the ligand. This ties in well with the observation that whilst younger leaf primordia are unable to develop adaxial polarity older leaf primordia can autonomously develop into phenotypically normal leaves. However studies have not yet been performed looking at a loss of PHB function which if this hypothesis hold should display an abaxialised phenotype.

Another interesting observation has been made regarding the role that adaxial promoting genes play in SAM maintenance. It was noted by McConnell et al (2001) that the mutants found in the mutagenesis screen not only exhibited adaxialised leaves but also overgrown meristems and even ectopic meristems on the leaf margins. Also studies on the Antirrhinum gene PHANTASTICA revealed loss of function mutations that resulted in abaxialisation and an arrested meristem. These pieces of suggest that adaxial promoting factors signal the SAM to regenerate the cells used when the lateral organ develops.

In contrast to PHANTASTICA and PHABULOSA which appear to promote adaxial cell identity members of the YABBY gene family act in a redundant manner to KANADI to promote abaxial development. Kerstetter et al (2001) screened Arabidopsis for mutants which displayed trichomes (leaf hairs) on the abaxial surface of the first two leaves. This was the KANADI (KAN) mutant phenotype, although it did not alter epidermal cell size or stomatal density it was still believed to play a role in adaxial-abaxial identity. kan mutations were originally identified as enhancers of the mutant floral phenotype crabs claw (crc) (as we shall see later a YABBY gene), a genes that specifies abaxial identity in carpels. It contains a highly conserved domain, the GARP domain, called that because it is present in GOLDEN 2 in maize, Arabidopsis response regulator proteins (ARR) and the Psr 1 protein from Chlamydomonas. The ARR1 and ARR2 GARP domains have been shown act as DNA binding sites thus indicating that KANADI might act as a transcriptional activator. This is also supported by its subcellular localisation achieved through standard GUS staining or GFP tagging. To test the hypothesis that KAN was involved in the regulation of organ polarity it was ectopically expressed using the CaMV 35S promoter. Several of the transformants displayed a range of phenotypes associated with abaxialisation, narrow cylindrical leaves, few trichomes and no vasculature and the internal tissue of transgenic cotyledons resembled abaxial spongy mesophyll tissue cells with large intracellular air spaces. In addition to this transgenic seedlings lacked both a SAM and a hypercotyl, presumably because 35S-KAN peripherises the embryo, eliminating the cell type from which both the SAM and the vascular tissue of the hypercotyl arise. All this evidence supports the conclusion that KAN promotes abaxial identity.

Further work on KANADI mutants revealed two KAN genes, KAN1 and KAN2 (Eshed et al 2001). Single mutants of the closely related genes have little or no effect on plant morphology. However in kan1 kan2 double mutant plants there is a replacement of abaxial cell types with adaxial cell types in most lateral organs. This also correlated with an increase in expression pattern of PHB (and a subsequent reduction in the expression of YABBY genes). Because not all of the abaxial tissues had reverted to adaxial types in the double mutant this implies that another set of genes offer partial redundancy to KAN genes.

Mutant analysis had already shown that another group of genes from the YABBY family might also play a role in specifying abaxial cell fate. The Arabidopsis YABBY family is composed of six members which are likely to encode transcriptional regulators. There are several lines of evidence to support the hypothesis that members of the YABBY gene family are involved in promoting abaxial cell fate in lateral organs. They exhibit a polar expression pattern in each of the above ground lateral organs. FILAMENTOUS FLOWER (FIL), YABBY2 and YABBY3 are expressed in a polar manner in all lateral organs produced by the SAM and flower meristems. CRABS CLAW (CRC) and INNER NO OUTER (INO) appear to be specialised members of the gene family in that their expression is restricted to the carpels and nectaries or outer integuments respectively. Transcripts of the YABBY gene family members are detectable only in the abaxial domains of lateral organs when their primordia emerge and begin to differentiate from the apical meristem. Secondly when the fate of either adaxial or abaxial organs are altered through genetic modification YABBY gene expression correlates with abaxial cell fate. For example in phb mutants (dominant ectopic expression) FIL expression is greatly reduced (possibly due to the inhibitory actions of PHB). Thirdly ectopic expression of family members is enough to cause adaxial epidermal tissues to differentiate with abaxial ell fate. Finally loss of expression of both FIL and YABBY3 is enough to disrupt polar development in lateral organs.

Therefore the relationship between these two abaxial promoting pathways is not exactly clear. While YABBY genes are activated in the leaf primordia in kan1 kan2 mutants, KANADI activity is still required for correct lateral organ development, suggesting that KANADI is slightly upstream of YABBY function. However with loss of expression of both FIL and YABBY3 being enough to disrupt polar development in lateral organs it implies there is a more complex relationship between the two that is not currently understood (Eshed et al 2001). (He talks here about possible molecular mechanisms but I don’t quite understand them)

Other genes might be important in the finer acquisition of lateral organ polarity although not much work has been done on them and so there’s little evidence to concrete their roles. However some interesting observations have been made. McHale (Planta 1992) noted that leaves that lack proper lamina expansion are produced in lam1 mutants of tobacco. These leaves display abaxial-adaxial polarity at their midrib, but blade tissue lacks adaxial cell types. Using periclinal chimeras (I am not sure what these are), it was shown that signals in the L3 can restore adaxial tissues in the L2 but that these signals can only act over a short range. This data suggests that LAM1 acts downstream of the establishment of polarity in lateral organs and may encode a component in the signalling pathway that mediates communication between the abaxial and adaxial domains. There is also some evidence that KNOX genes may be involved in setting up initial domains for polarity and that some difference occurs between the way orthologues (specifically ROUGH SHEALTH 2 and PHANTASTICA) of monocots and eudicots play a part in control of leaf axis.

Summary

Given the current evidence Eshed et al (2001) produced a model of polarity establishment in lateral organs with the spatial and temporal aspects mapped onto a potato apical meristem.

Factors, possibly sterol or lipid are produced by the SAM which in turn activate PHABULOSA thus promoting adaxial cell fate, regeneration of the meristem and by indirect factors inhibits KANADI. KANADI is believed to be involved in promoting abaxial cell fate and possible direct inhibitory action on the stability of the PHABULOSA transcripts. YABBY is thought to act in a parallel pathway with KANADI to promote abaxial cell fate but the exact interaction between the two pathways has not been elucidated. Finally the collaboration between the adaxial and the abaxial factors establish polarity within the leaf and facilitate a normal phenotypic development.

By assessing all this evidence I believe there is a long way to go before the entire pathway is elucidated. Although evidence exists for some components of the pathway in only a few cases, such as KANADI and PHABULOSA am I convinced of their roles in the pathway although the mechanisms behind them are still evading the scientific community.

Bibliography

Bowman and Eshed (2000) Formation and maintenance of the Shoot Apical Meristem. Trends in Plant Sciences. Vol 5, pp 110-114

Bowman JL (2000) Axial patterning in leaves and other lateral organs Current opinion in genetics and development Vol 10 pp 399-404

Eshed Y, Baum SF, Perea JV, Bowman JL. (2001) Establishment of polarity in lateral organs of plants. Curr Biol. Aug 21;11(16):1251-60.

McConnell JR, Emery J, Eshed Y, Bao N, Bowman J, Barton MK. (2001) Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots. Nature. Jun 7;411(6838):709-13.

Kerstetter RA, Bollman K, Taylor RA, Bomblies K, Poethig RS. (2001) KANADI regulates organ polarity in Arabidopsis. Nature. Jun 7;411(6838):706-9.