PIN it on auxin

Abstract

The growth and development of plants is regulated by several external and internal factors including auxin. Its distribution regulates several developmental processes in plants. Auxin molecules function as mobile signals and are involved in the spatial and temporal coordination of plant morphogenesis and in plant responses to their environment. The intercellular transport of auxin is facilitated by transport proteins and the disruption of polar auxin flow results in various developmental abnormalities. In this review, we discuss the developmental and physiological significance of over-accumulation of PIN1 auxin transport facilitator protein in tomato as seen in the enhanced polar auxin transport pct1-2 mutant.

The growth and development of plants is regulated by several external and internal factors. Of the internal cues that regulate developmental processes, auxins were the first substances to be identified.¹ Auxins belong to a group of native plant growth regulatory compounds that play a crucial role in the regulation of basic growth processes such as cell division and cell elongation. At the level of tissues, organs and the whole plant, they exhibit pleiotropic physiological effects.^2–5 In all of these processes, cell-to-cell polar transport of auxin and differential distribution are key aspects of its function.^6–8 Auxin molecules therefore function as mobile signals between cells, tissues and organs and, as such, are involved in the spatial and temporal coordination of plant morphogenesis and in plant responses to their environment. This has led to the idea^9,10 that auxin might function in a similar manner to morphogens, substances that influence morphogenesis and embryogenesis by forming a concentration gradient. Auxin is largely synthesized in apical growing regions of plants, the shoot apex and the young leaves. From these auxin-producing tissues, the major auxin indole-3-acetic acid (IAA) is transported in a polar fashion towards basally located plant tissues and organs.¹¹ The polar transport of auxin has been implicated to be essential for the initiation and maintenance of polarized growth in developing embryos as well as maintaining normal developmental processes in adult plants.^10,12

The polar auxin transport (PAT) is a specialized delivery system in higher plant that is responsible for the transport of auxins (IAA) from its site of synthesis in the shoot (or tip) regions to basal sink tissues, such as the root.¹³ Measurements of auxin biosynthesis have demonstrated that IAA in root tissues is derived predominantly from shoot tissues before 10 d after germination (DAG).^14,15 Transport of auxin in root tissues is more complex than that in other tissues due to the presence of two distinct polarities, i.e., IAA can be transported acropetally (towards the root apex) through the central cylinder and basipetally (from the root apex towards the base) through the outer layers of root cells.^11,16,17 The basipetal flow of shoot-derived auxin to roots is thought to result from the polar distribution of specialized carrier molecules in the plasma membranes. In recent years, molecular-genetic approaches characterizing mutants impaired in auxin transport, have led to the isolation of several genes that strongly support the concept of basally localized auxin efflux carriers.

Since the analysis of the Arabidopsis pin1 mutant in 1991,¹⁸ and the subsequent cloning of AtPIN1 gene,⁶ it is widely accepted that the PIN proteins play an integral role in determining developmental processes in plants by regulating cellular auxin transport.^8,19–24 In Arabidopsis, to date further seven members of the PIN family have been discovered, each of which seems to show high degree of evolutionary and functional conservedness.²⁵ Genes homologous to Arabidopsis PINs have been reported from genomes of several monocotyledonous as well as dicotydelonous species such as Triticum aestivum (wheat), Oryza sativa (rice), Glycine max (soyabean), Medicago trunculata and Solanum tuberosum (potato).²⁵ In Arabidopsis and others, PIN proteins have been shown to be intimately linked with polar auxin transport.^26,27 We have earlier reported that the polycotyledon (pct1-2) mutant of tomato^7,28 shows enhanced polar transport of auxin. Recently we have demonstrated that the pct1-2 mutant also shows enhanced cellular accumulation of PIN1 auxin transport facilitator protein.²⁹ Here we discuss the developmental and physiological significance of PIN1 distribution in tomato.

While auxin occupies an important regulatory role in developmental process, most of the developmental defects associated with its transport have been attributed to a reduced PAT. In Arabidopsis, to date only the max mutant has been reported to show an enhanced rate of auxin flow coupled with increased expression of PIN1.³⁰ The pct1-2 mutant of tomato is the only other mutant reported to show enhanced PAT along with an increased expression of PIN1 protein. The availability of these mutants provides an opportunity for a comparative analysis of the consequences of enhanced PIN1 distribution and PAT in these two biological systems. Stirnberg et al.³¹ and Sorefan et al.³² identified the max mutants on the basis of increased axillary branching, while the pct1-2 was isolated on the formation of multiple cotyledons. Therefore could enhanced PAT provide necessary cellular inputs for determining patterning specificity^33,34 from an early stage in development? The increased PAT in the max mutants results in an increased branching phenotype,³⁰ while the roots of adult pct1-2 plants are bushier than the wild-type. The pct1-2 inflorescence also shows multiple blooming flowers with abnormal phyllotactic arrangement.⁷ During organ formation, it is suggested that efficient auxin transport from the regions of growth is required for active growth,^35,36 and that basal transport of auxin from the meristem epidermis into the growing stem below is essential for shoot meristem function.²⁰ Therefore, if a growing organ cannot transport auxin out into the main stem, then presumably it may be unable to sustain an active meristem. Aloni et al.³⁷ have shown that the developmental pattern of auxin production during floral-bud development suggests that young organs that produce high concentrations of free IAA inhibit or retard organ-primordium initiation and development at the shoot tip. Therefore an increase in the capacity of the main stem to transport auxin would therefore enable it to be more effective as an auxin sink, and would allow for increased active auxin transport out of the meristematic regions into the adjacent vascular bundles in the main stem.³⁰ Such circumstances would thus allow for an increased polar transport of auxin from the apical meristems to the root tissues. The upregulated level of PIN1 in the pct1-2 mutant presumably enables a more efficient transport of auxin from the growing regions into the main stem by freeing up transport capacity in the stem to act as a sink for meristem derived auxin, which could have resulted in the increased organ-primordium initiation leading to an increased number of flowers in the inflorescence of the pct1-2 mutant.

Auxin, or more specifically IAA, has been associated with phototropism for over half a century. Scientists have provided compelling evidence that the formation of a lateral auxin gradient across an organ might allow for polar changes in growth and development.³⁸ The Cholodny-Went theory holds that increase of auxin concentration along the shaded side of a phototropically stimulated plant would result in a shoot that bends toward the light because of auxin-induced growth.³⁹ Although numerous studies have been carried out to test this hypothesis, there is not yet much compelling evidence indicating a direct cause-and-effect relationship between changes in auxin concentration and phototropism. However, mutant analyses have provided some insight into the relationship between auxin transport and tropic growth responses.^22,40–42 Several possibilities have been suggested that link hypocotyl phototropism to changes in auxin transport involving the phototropins.⁴³ The first suggests that since proteins regulating phototropic responses viz. phototropin1 (phot1) are located in the plasma membrane,⁴⁴ it is ideally situated for a possible direct interaction with proteins responsible for changes in auxin transport. It is also possible that phototropin-dependent signaling results in relocation of auxin efflux complexes from the basal end of the cell to the sides, redirecting the usual polar shoot-to-root flow of auxin to a lateral flow. This theory is particularly inviting as characterization of PIN3, revealed that it is required for stem and root tropic responses.²² Moreover, PIN3 exhibits a higher lateral than basal wall localization is stems. The disruption of the basal localization of PIN1, upon exposure of to unidirectional blue light²⁴ lends further support to this theory. Furthermore, this delocalization of PIN1 is not seen in the non-phototropic phot1 mutant of Arabidopsis under similar conditions. The multidrug resistant (mdr) mutant of Arabidopsis having reduced polar auxin transport, due to mislocalization of PIN1 also show enhanced phototropic and gravitropic responses.⁴⁵ While changes in auxin transport have been linked to phototropic responses in higher plants,^43,46 one of the changes to occur in phototropically stimulated stems appears to be changes in expression of certain genes. The requirement of an auxin-responsive transcriptional factor (ARF) for proper phototropic response and auxin-mediated responses suggests that changes in gene expression and abundance of the encoded proteins play important roles in the phototropic system.^46,47 Microarray studies of Brassica oleracea have also revealed differential expression of several genes when subjected to phototropic stimulus.⁴⁸ The accumulation of specific mRNAs was seen in the region farthest from the incident phototropic stimulation along with the formation of a lateral gradient of auxin in the hypocotyls in response to tropic stimulation. The delayed and reduced responsiveness of the tomato pct1-2 mutant to phototropic blue light stimulus (Fig. 1) suggest that the enhanced distribution of PIN1 proteins favor the polar distribution of auxin as against the lateral flow thereby increasing the time threshold needed for an auxin gradient to be established in order to facilitate phototropic or geotropic curvature.⁴⁹ The findings of Al-Hammadi et al.⁷ and Noh et al.⁴⁵ suggest that PAT and PIN1 distribution possibly play similar role(s) in regulating the geotropic and phototropic responses in both tomato and Arabidopsis hypocotyls.

Figure 1.

Phototropic curvature of hypocotyl of etiolated wild type and pct1-2 seedlings exposed to blue light at a low fluence rate of 0.5 µmolm⁻²s⁻¹. The pct1-2 seedlings show an increased time threshold for showing phototropic response as compared to the wild type seedlings.

The defining property of auxins has been their capacity to stimulate cell expansion upon external application.⁵⁰ Blilou et al.²³ have provided evidence of the effect of PIN activities on final cell size in Arabidopsis. They infer that interactions between changes in auxin distribution and abundance of carrier components could presumably affect meristem size, elongation and cell size in roots. The induction of the AUXIN BINDING PROMOTER1 (ABP1) of Arabidopsis in tobacco leaf strips resulted in an increased capacity for auxin-mediated cell expansion, whereas induction of ABP1 in intact plants resulted in leaves with a normal morphology, but larger cells. Similarly, constitutive expression of maize ABP1 in maize cell lines conferred on them the capacity to respond to auxin by increasing cell size.¹⁶ The enhanced distribution of PIN1 in the pct1-2 seedlings has consequently resulted in a net increase in the auxin transport capacity. As a result, similar to the observations of Jones et al,¹⁶ the roots of pct1-2 also show increased cell size (Fig. 2). In comparison, we have also observed that the roots of the lanceolate (lan) mutant of tomato⁵¹ which has reduced PAT,⁵² show narrower cells. Interestingly in contrast to the cells in the root, the cotyledons and hypocotyls of the pct1-2 mutant show a reduction in cell size.⁷ These observations indicate that auxin distribution and its transport play an important role(s) in orchestrating developmental processes in tomato seedlings. Deciphering the roles of auxin in such developmental processes would lend newer insight into the myriad of cellular events determined by this important phytohormone.

Figure 2.

Immunolocalization of PIN1 proteins in root tips of three-day-old wild-type (A and D), pct1-2 (B and E) and lan (C and F) seedlings. Laser scanning confocal microscopy was used to monitor signals in fixed tissue sections. The cell size in the pct1-2 mutant is larger as compared to both the wild-type and lan seedlings. PIN1 signal (green fluorescence) was indirectly visualized with fluorophore labeled anti-PIN1 antibodies. Bars = 100 µm.

Auxin level and its distribution is believed to be a key signal in regulating major patterning events during embryogenesis such as establishment of apical-basal axis, central-peripheral axis and the establishment of bilateral symmetry. Amongst these three events, the pct1-2 mutant is characteristically defective in the establishment of bilateral symmetry accompanied by formation of multiple cotyledons, during the early stages of embryogenesis.⁷ In Arabidposis several studies have indicated that regulation of auxin flux and maxima in embryogenesis is regulated by at least three PINS, PIN1, PIN4 and PIN7, which are differentially expressed in the zygote to globular stage embryos. The abundance and distribution of these PINs determine an initial accumulation of auxin in the embryo. In the latter stages of embryogenesis, these PINs also regulate the basal efflux of auxin from the embryo determining root meristem. Most importantly during development of late globular embryo, the reversal of polar localization of PIN1 protein in the epidermis marks the site(s) of cotyledon initiation.^53–55 We presume that overexpression of PIN1 in the pct1-2 mutant leads to formation of more than two foci of auxin accumulation sites causing formation of multiple cotyledons. Such a correlation between auxin accumulation and PIN1 localization with reference to cotyledon initiation has been observed in somatic embryos of Picea abies leading to multiple cotyledon initiation.^56,57

Though an association of increase in cotyledon number with increase in PAT is seen for the pct1-2 mutant of tomato, it is at variance with the pinoid mutant of Arabidopsis, which shows polycot seedlings but reduced PAT in inflorescence stems.⁵⁸ Benjamins et al.⁵⁹ have suggested that PINOID acts as a positive regulator of PAT whereas our previous reports indicate PCT1-2 to be a negative regulator of auxin flow by regulating the abundance of PIN1 proteins. In such a case, these two might affect different physiological processes and still show similar phenotypic effects. It could be equally plausible that enhanced PAT in Arabidopsis is not related to development of polycotyly as seen in the tt4 mutant of Arabidopsis.⁶⁰ Further investigations into the cellular and molecular events that influence patterning in the early stages of development in the pct1-2 mutant would shed more light on the role of PAT and PINs in regulating the developmental processes in tomato.

Since the last decade several experimental evidences have confirmed the central importance of polar auxin transport in regulating plant growth and developmental responses. The specificity of the delivery of auxin from its sites of synthesis to the growing organs is critical in controlling such differential growth responses of plants. By combining genetic, molecular and physiological technologies plant biologists are arriving at the identification of genes and networks acting to control differential growth processes. In tomato, information regarding the role of auxin and its transport in regulating developmental processes is still scanty. Few studies on the role of auxin transport in tomato^7,61,62 have elucidated that the correct distribution of auxin in plant tissues is critical for the proper growth and development. There are fewer studies that have highlighted the PIN localization and cellular distribution of PINs in tomato.^29,63 From studies on Arabidopsis and other plants, researchers on auxin transport agree that the PIN network influences the directional auxin flux (polar auxin transport) providing cells, in any part of the plant, with specific positional and temporal information. Thus, the PIN network, works in concert with the auxin signaling system(s) to coordinate plant development. However the distribution of PIN genes throughout the plant kingdom and the complicated cellular processes involved in their localization indicate that the function of PINs is more than simply mediating auxin efflux. Uncovering these role(s) of PIN proteins in tomato and how they relate to auxin transport in determining plant developmental processes is the likely challenge to drive research on tomato auxin transport in the coming years.

Abbreviations

PAT

polar auxin transport

pct

polycotyledon

PIN

pin-formed

SAM

shoot apical meristem