Roni Aloni

Gradual shifts in sites of free-auxin production during leaf-primordium development and their role in vascular differentiation and leaf morphogenesis in Arabidopsis.

Abstract. The major regulatory shoot signal is auxin, whose synthesis in young leaves has been a mystery. To test the leaf-venation hypothesis [R. Aloni (2001) J Plant Growth Regul 20: 22–34], the patterns of free-auxin production, movement and accumulation in developing leaf primordia of DR5::GUS-transformed Arabidopsis thaliana (L.) Heynh. were visualized. DR5::GUS expression was regarded to reflect sites of free auxin, while immunolocalization with specific monoclonal antibodies indicated total auxin distribution. The mRNA expression of key enzymes involved in the synthesis, conjugate hydrolysis, accumulation and basipetal transport of auxin, namely indole-3-glycerol-phosphate-synthase, nitrilase, IAA-amino acid hydrolase, chalcone synthase and PIN1 as an essential component of the basipetal IAA carrier, was investigated by reverse transcription–polymerase chain reaction. Near the shoot apex, stipules were the earliest sites of high free-auxin production. During early stages of primordium development, leaf apical dominance was evident from strong β-glucuronidase activity in the elongating tip, possibly suppressing the production of free auxin in the leaf tissues below it. Hydathodes, which develop in the tip and later in the lobes, were apparently primary sites of high free-auxin production, the latter supported by auxin-conjugate hydrolysis, auxin retention by the chalcone synthase-dependent action of flavonoids and also by the PIN1-component of the carrier-mediated basipetal transport. Trichomes and mesophyll cells were secondary sites of free-auxin production. During primordium development there are gradual shifts in sites and concentrations of free-auxin production occurring first in the tip of a leaf primordium, then progressing basipetally along the margins, and finally appearing also in the central regions of the lamina. This developmental pattern of free-auxin production is suggested to control the basipetal maturation sequence of leaf development and vascular differentiation in Arabidopsis leaves. Electronic Supplementary Material is available if you access this article at http://dx.doi.org/10.1007/s00425-002-0937-8. On that page (frame on the left side), a link takes you directly to the supplementary material.

Role of auxin in regulating Arabidopsis flower development.

To elucidate the role of auxin in flower morphogenesis, its distribution patterns were studied during flower development in Arabidopsis thaliana (L.) Heynh. Expression of DR5::GUS was regarded to reflect sites of free auxin, while immunolocalization with auxin polyclonal antibodies visualized conjugated auxin distribution. The youngest flower bud was loaded with conjugated auxin. During development, the apparent concentration of free auxin increased in gradual patterns starting at the floral-organ tip. Anthers are major sites of high concentrations of free auxin that retard the development of neighboring floral organs in both the acropetal and basipetal directions. The IAA-producing anthers synchronize flower development by retarding petal development and nectary gland activity almost up to anthesis. Tapetum cells of young anthers contain free IAA which accumulates in pollen grains, suggesting that auxin promotes pollen-tube growth towards the ovules. High amounts of free auxin in the stigma induce a wide xylem fan immediately beneath it. After fertilization, the developing embryos and seeds show elevated concentrations of auxin, which establish their axial polarity. This developmental pattern of auxin production during floral-bud development suggests that young organs which produce high concentrations of free IAA inhibit or retard organ-primordium initiation and development at the shoot tip.

Foliar and Axial Aspects of Vascular Differentiation: Hypotheses and Evidence

A comparison is made between foliar and axial vascular differentiation. Current thoughts and new evidence are presented on the role of hormones in controlling the differentiation of vascular tissues in organized and tumorous tissues, focusing on the role of auxin and cytokinin in controlling phloem and xylem relationships, vessel size and density, cambium sensitivity, vascular adaptation and xylem evolution in deciduous hardwood trees. The possible role of wounding is also considered. A new hypothesis, namely, the leaf-venation hypothesis, is proposed to explain the hormonal control of vascular differentiation in leaves of dicotyledonous plants. Experimental evidence in support of the hypothesis is presented showing that hydathodes, the watersecreting glands, are the primary sites of auxin synthesis during leaf morphogenesis. Vessel element patterns similar to those found in hydathodes were experimentally induced by exogenous auxin application.

The Induction of Vascular Tissues by Auxin and Cytokinin

The vascular system of the plant connects the leaves and other parts of the shoot, with the roots, and enables efficient long-distance transport between the organs. In higher plants it is composed of two kinds of conducting tissues: thephloemthrough which organic materials are transported and thexylemwhich is the pathway for water and soil nutrients. In angiosperms, the functional conduits of thephloemare thesieve tubes;and those of thexylemare thevessels(4, 37). Vascular development in the plant is an open type of differentiation, it continues as long as the plant grows from apical and lateral meristems. The continuous development of new vascular tissues enables regeneration of the plant and its adaptation to changes in the environment. This differentiation of vascular tissues along the plant is induced and controlled by longitudinal streams of inductive signals (4, 42). In spite of the complexity of structure and development of the vascular tissues (37), there is evidence that the differentiation of both the sieve tubes and the vessels is induced by two hormonal signals, namely: (i) auxin, indole-3-acetic acid (IAA), produced mainly by young leaves (4, 6, 26, 27, 42), and (ii) cytokinin produced by root apices (8, 9, 18). This fact raises the question how these two hormonal signals control the differentiation of complex patterns of phloem and xylem? Nevertheless, it should be emphasized that additional growth regulators, like gibberellin (1) and ethylene (7, 45), may also be involved in vascular differentiation. They are beyond the scope of this article and the reader is directed to reviews on the topic (4, 5, 6, 24, 39, 42, 46).

The Induction of Vascular Tissues by Auxin

The vascular tissues of the plant connect the leaves and other parts of the shoot with the roots. The vascular system is composed of two kinds of conducting tissues: the phloem, through which organic materials are transported and the xylem, which is the conduit for water and soil nutrients. Vascular development in the plant is an open type of differentiation, it continues as long as the plant grows by apical and lateral meristems. Thus, there is a continuous development of new vascular tissues that are in dynamic relationship to one another. In spite of the complexity of structure and development of the vascular tissues, there is evidence that the auxin, indole-3-acetic acid (IAA), is the main limiting and controlling factor for both phloem and xylem differentiation (1, 12, 13). This fact raises a major problem of development, which is, what are the mechanisms by which one stimulus controls the differentiation of complex patterns of phloem and xylem? However, it should be emphasized that other growth regulators may also be involved in vascular differentiation. They are beyond the scope of this article and the reader is directed to recent reviews on this topic (e.g., 19, 21).

Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tissue cultures

The differentiation of sieve and tracheary elements was studied in callus culture of Daucus carota L., Syringa vulgaris L., Glycine max (L.) Merr., Helianthus annuus L., Hibiscus cannabinus L. and Pisum sativum L. By the lacmoid clearing technique it was found that development of the phloem commenced before that of the xylem. In not one of the calluses was differentiation of tracheary elements observed in the absence of sieve elements. The influence of indole-3-acetic acid (IAA) and sucrose was evaluated quantitatively in callus of Syringa, Daucus and Glycine. Low IAA levels resulted in the differentiation of sieve elements with no tracheary cells. High levels resulted in that of both phloem and xylem. IAA thus controlled the number of sieve and tracheary elements, increase in auxin concentration boosting the number of both cell types. Changes in sucrose concentration, while the IAA concentration was kept constant, did not have a specific effect on either sieve element differentiation, or on the ratio between phloem and xylem. Sucrose did, however, affect the quantity of callose deposited on the sieve plates, because increase in the sucrose concentration resulted in an increase in the amount of callose. It is proposed that phloem is formed in response to auxin, while xylem is formed in response to auxin together with some added factor which reaches it from the phloem.

Differentiation of the ray system in woody plants

The regulation of vascular ray differentiation has received limited attention, despite the fact that vascular rays constitute an important part of the secondary body of plants. In this paper we review developmental aspects of the ray system and suggest a general hypothesis for the regulation of ray differentiation and evolution. In studies of ray differentiation, two basic factors should be taken into consideration: 1) the normal gradual increase in ray size in relation to age, distance from the pith, and distance from the young leaves; and 2) the influence of wound effects on the size, structure, and spacing of rays. The relationships between the rate of cambial activity and secondary xylem differentiation are not clearly understood. There are contrasting results on the relationships between ray number and rate of radial growth. The rate of radial growth (= rate of cambial activity) is not the regulating mechanism of ray characteristics. Bünning (1952, 1965) proposed that rays are distributed regularly in the tissue, as the outcome of an inhibitory influence expressed by them. However, Bünning’s hypothesis contradicts a basic feature of the vascular ray system, namely, fusion of rays. Detailed histological studies of the secondary xylem revealed that proximity to and contact with rays plays a major role in the survival of fusiform initials in the cambium (Bannan, 1951, 1953). Such evidence led Ziegler (1964) to suggest that since the cambium is supplied predominantly via the rays, this is an effective feedback regulative system for an equidistant arrangement of the rays. The hypothesis that rays are induced and controlled by a radial signal flow seems to be the best explanation for the structure and spacing of rays. The formation of a polycentric ray—a special case of “ray” initiation inside a vascular ray—supports the idea that radial signal flow occurs within the rays (Lev-Yadun & Aloni, 1991a). This idea is also supported by findings fromQuercus species in which aggregate rays in the xylem disperse naturally in branch junctions and, following partial girdling, leave a longitudinal narrow bridge of cambium and bark as a result of enhanced axial signal flow (of auxin and other growth regulators) (Lev-Yadun & Aloni, 1991b). The longitudinally elongated shape of rays is their response to axial signal flows (mainly the polar auxin flow). Two methods have been used to study the evolution of the ray system: 1) statistical studies of the relationships between vessel and ray characteristics in many species, when vessel characteristics were the evolutionary standard, and 2) comparison of ray characteristics in fossils originating from several geological eras. We suggest that evolution of the ray system reflects changes in the relations between radial and axial signal flows.

The three-dimensional structure of vascular tissues in Agrobacterium tumefaciens-induced crown galls and in the host stems of Ricinus communis L.

The three-dimensional pattern of phloem and xylem in 10-d-to two-month-old tumors induced by Agrobacterium tumefaciens (C58) and in adjacent Ricinus communis L. stem tissues was studied in thick sections by clearing with lactic acid and by staining with lacmoid. The crown galls contained two types of vascular strands: treelike branched bundles, which developed towards the tumor surface in fast-growing regions, and globular bundles in the slowly developing parts. Both types of vascular bundles contained xylem and phloem and were continuous with the vascular system of the host plant. The tumor bundles were interconnected by a dense net of phloem anastomoses, consisting of sieve tubes but no vessels. These vascular patterns reflect the apparent synthesis sites, concentration gradients and flow pathways of the plant hormones additionally produced in the tumors upon expression of the T-DNA-encoded genes. The A. tumefaciens-induced crown gall affected vascular differentiation in the host stem. In the basipetal direction, the tumor induced more xylem differentiation directly below it, where the crown-gall bundles joined the vascular system of the host. In the centripetal direction, the crown gall caused the development of pathologic xylem characterized by narrow vessels, giant rays and absence of fibers. On the other hand, most probably as a consequence of its gibberellic acid content, the host plant stimulated a local differentiation of regenerative phloem and xylem fibers with unique ramifications, only at the base of the tumor. However, fibers were absent from the main body of the crown gall. The study shows that A. tumefaciens-induced crown galls are characterized by a sophisticated network of vascular tissues in the tumor and are accompanied by a perturbated vessel system in the host. The hormonal mechanisms controlling vascular differentiation in the tumor and neighboring host tissues are discussed. In addition, the “gall constriction hypothesis” is proposed for explaining the mechanism which gives priority in water supply to the growing gall over the host shoot.

Leaf-Induced Gibberellin Signaling Is Essential for Internode Elongation, Cambial Activity, and Fiber Differentiation in Tobacco Stems

In plants, the directional flow and sites of hormone accumulation facilitate organ development. Identifying leaves as the source of a mobile signal that induces gibberellin accumulation and signaling revealed the hormone’s roles in plant primary and secondary growth. It is demonstrated that leaf-induced gibberellin is required for stem elongation, cambial activity, and fiber formation. The gibberellins (GAs) are a group of endogenous compounds that promote the growth of most plant organs, including stem internodes. We show that in tobacco (Nicotiana tabacum) the presence of leaves is essential for the accumulation of bioactive GAs and their immediate precursors in the stem and consequently for normal stem elongation, cambial proliferation, and xylem fiber differentiation. These processes do not occur in the absence of maturing leaves but can be restored by application of C19-GAs, identifying the presence of leaves as a requirement for GA signaling in stems and revealing the fundamental role of GAs in secondary growth regulation. The use of reporter genes for GA activity and GA-directed DELLA protein degradation in Arabidopsis thaliana confirms the presence of a mobile signal from leaves to the stem that induces GA signaling.

Calmodulin-binding transcription activator 1 mediates auxin signaling and responds to stresses in Arabidopsis

Auxin is a key plant hormone that regulates various aspects of plant development. However, the mechanisms integrating auxin growth effects with stress responses are not fully understood. In this study, we investigated the possible role of calmodulin-binding transcription activator 1 (CAMTA1), an Arabidopsis thaliana calcium/calmodulin-binding transcription activator, in auxin signaling and its responses to different stresses. Plants harboring the AtCAMTA1 promoter fused to the GUS reporter gene revealed cell-specific expression patterns reminiscent of auxin responses. The responsiveness of CAMTA1 to auxin was further assessed by chemical disturbances in polar auxin transport, and by RT-PCR analysis of gene expression of dissected leaf sections from plants exposed to the auxin transport inhibitor NPA. Furthermore, the intensity and cell-specific expression patterns of CAMTA1 changed significantly and differentially on exposure to increasing salt concentrations and heat. Transcriptome analysis of a camta1 T-DNA insertion mutant revealed 63 up-regulated genes, of which 17 are associated with auxin signaling. Finally, analysis of hypocotyl elongation in the presence and absence of auxin revealed that camta1 T-DNA insertion mutants and CAMTA1-repressor lines are hyper-responsive to auxin compared to wild-type seedlings. Thus, CAMTA1 participates in auxin signaling and responds to abiotic stresses.