By using a novel, extremely sensitive and specific gas chromatography-mass spectrometry technique we demonstrate in Pinus sylvestris (L.) trees the existence of a steep radial concentration gradient of the endogenous auxin, indole-3-acetic acid, over the lateral meristem responsible for the bulk of plant secondary growth, the vascular cambium. This is the first evidence that plant morphogens, such as indole-3-acetic acid, occur in concentration gradients over developing tissues. This finding gives evidence for a regulatory system in plants based on positional signaling, similar to animal systems.Formation of patterns is one of the most intriguing phenomena in biology. Pattern development requires that gene activity must be strictly controlled in time and space. Every cell must receive information about its position and express the appropriate genes. The existence of morphogenetic fields has been suggested as a possible source of such information in both animal and plant systems (1-4). This field is thought to consist of one or more diffusable and physiologically active substances (morphogens) which originate from organizing centers. The concentration gradients created would then influence on tissue and organ differentiation. Current examples of morphogens of this sort in animals are retinoic acid in vertebrate limb development and activin in early amphibian development (5, 6). In plants, positional signaling has been discussed mainly on the basis of the orderly induction of leaf and root primordia and the organization of vascular tissues (7), but neither the mechanisms nor the morphogens behind these patterns have been elucidated. Auxins and cytokinins are plant hormones that have been characterized as important signals in plant development, being involved in the induction and maintenance of meristems as well as in plant polarity (8). Not surprisingly, these substances have been proposed to function as positional signals in pattern specification (9, 10). To date, this proposition has not found much support due to the lack of data proving that such endogenous plant hormone gradients are found in plants (11,12).The formation of secondary vascular tissues is a well described phenomenon of patterned growth in plants. This pattern has both radial and longitudinal components (13). Phloem and xylem differentiate radially on each side of the lateral meristem (the vascular cambium). The cambial derivatives which form xylem first pass through a zone of cell expansion and then a zone of secondary wall formation and, finally, a zone of programmed cell death. Phloem derivatives expand and differentiate forming a living tissue (Fig. 1) MATERIALS AND METHODSBlocks (2 x 5 cm) consisting of extraxilary tissues and a few annual rings were chiseled out at breast height during active (late June) and dormant (mid-January) periods from Pinus sylvestris (L.) trees (-120 years old, 19 m tall, 34 cm in diameter at breast height, location 64°14'N, 19°46'E) and immediately frozen in liquid N2. After trimming the blocks, specific ...

In temperate regions the annual pattern of wood development is characterized by the formation of radially narrow and thick walled latewood cells. This takes place at the later part of the growing season when cambial cell division declines. To gain new insight into the regulation of this process, micro-analytical techniques were used to visualize the distribution of indole-3-acetic acid (IAA), soluble carbohydrates, and activities of sucrose (Suc)-metabolizing enzymes across the cambial region tissues in Scots pine (Pinus sylvestris). The total amount of IAA in the cambial region did not change with latewood initiation. But its radial distribution pattern was altered, resulting in an increased concentration in the cambial meristem and its recent derivatives. Thus, initiation of latewood formation and cessation of cambial cell division is not a consequence of decreased IAA concentrations in dividing and expanding cells. Rather, IAA most likely has a role in defining the altered developmental pattern associated with latewood formation. Carbohydrates and enzyme activities showed distinctive radial distribution patterns. Suc peaked in the phloem and decreased sharply to low levels across the cambial zone, whereas fructose and glucose reached their highest levels in the maturing tracheids. Suc synthase was the dominating Suc cleaving enzyme with a peak in the secondary wall-forming tracheids and in the phloem. Soluble acid invertase peaked in dividing and expanding cells. Suc-phosphate synthase had its highest activities in the phloem. Activities of cell wall bound invertase were low. The absence of major seasonal variations indicates that carbohydrate availability is not a trigger for latewood initiation. However, steep concentration gradients of the sugars suggest a role for sugar signaling in vascular development.The annual transition from earlywood to latewood formation is a conspicuous developmental switch in temperate region trees. Latewood is induced during the later part of the growing season, when cell division activity in the cambial meristem declines. It involves a reduction in radial expansion and an increase in wall thickening of the cambial derivatives. Thus, earlywood is characterized by large-diameter and thin-walled tracheids, whereas latewood is composed of narrow diameter tracheids with thick cell walls. The induction of latewood cells and cambial dormancy offers a natural system by which we can gain new insight into the regulation of the basic growth processes of cell division and cell morphology.Early investigators concluded that the initiation of latewood formation was induced by shortening of the photoperiod, and was associated with cessation of apical and needle growth at a time when current year needles had become net exporters of photosynthetic assimilate (Richardson and Dinwoodie, 1960; Larson, 1964; Gordon and Larson, 1968). It was also observed that exogenous IAA could cause cambia that were forming narrow-diameter latewood tracheids to revert to forming large-diameter earlywood tracheids...

The vascular cambium produces secondary xylem and phloem in plants and is responsible for wood formation in forest trees. In this study we used a microscale mass-spectrometry technique coupled with cryosectioning to visualize the radial concentration gradient of endogenous indole-3-acetic acid (IAA) across the cambial meristem and the differentiating derivatives in Scots pine (Pinus sylvestris L.) trees that had different rates of cambial growth. This approach allowed us to investigate the relationship between growth rate and the concentration of endogenous IAA in the dividing cells. We also tested the hypothesis that IAA is a positional signal in xylem development ( Cambial growth involves the production of secondary xylem and phloem elements. The lateral meristem responsible for this growth, the vascular cambium, normally consists of 5 to 15 dividing cells in a radial direction (Larson, 1994), the so-called cambial zone. The rate of cambial growth is the major determinant for the production of wood in forest trees, and it is determined by both the radial number of dividing cells in the cambial zone and the rate of cell division for each of the cambial zone cells (Gregory and Wilson, 1968;Wilson and Howard, 1968; Gregory, 1971). Cambial growth is adjusted to the demands of water transport required by the leaf biomass and to provide the mechanical strength necessary to support the crown and to withstand wind forces (Zimmermann and Brown, 1971). It is also well established that stem-diameter growth is often found to be greatest within the young crown and to decrease gradually down the stem. As a consequence, the amount and pattern of growth along the stem are determined by the size and arrangement of the crown (Larson, 1963; Hall, 1965). Such coordination requires a signaling system that integrates apical with cambial growth.The plant hormone IAA appears to fulfill the requirements for such a signal. Developing buds and young shoots are major sources of IAA, which is transported in a basipetal polar fashion (Kaldeway, 1984; Little and Savidge, 1987). Experiments with exogenous IAA have clearly demonstrated that polarly transported IAA induces formation of primary vascular tissues (Sachs, 1981; Jacobs, 1984; Aloni, 1995) and maintains the structure and cell-division activity of the vascular cambium (Savidge, 1983). IAA also affects the rate of cambial growth, as measured by tracheary element production in both 1-year-old shoots and mature stems in a dose-dependent manner (Little and Savidge, 1987;Little and Sundberg, 1991). These findings strongly suggest a function for IAA as a signal that integrates apical growth with production of vascular tissues. Accordingly, variations in cambial growth patterns along the stem have been explained by the postulated existence of longitudinal concentration gradients of IAA (Larson, 1969; Aloni and Zimmermann, 1983).With the development of accurate techniques for measuring IAA in plant tissues, it has become possible to test these ideas. The physiological relevance of IAA...

Cycling of soluble non-protein N compounds is thought to be indicative of the N-nutritional status of trees. We determined the major N forms in bark, wood and foliage and estimated the dependence of prevalent N forms on N availability in Pinus sylvestris L. trees from northern Sweden. Trees subjected to severe N limitation and trees that had been fertilized with an average 64 kg N ha(-1) year(-1) for 25 years were analyzed. Bark and wood samples were collected by tangentially cryo-sectioning the trunk into 30-microm thick sections, from the bark to the functional xylem. Soluble amino compounds were extracted from the sections for analysis. Sap samples from twigs were obtained by centrifugation, and bark samples from twigs were obtained by tissue extraction. In both needles and bark, arginine dominated the amino-N pool. Because arginine concentrations in needles increased with N fertilization, arginine dominance of the amino-N pool in needles was higher in N-fertilized trees than in control trees. In bark, N fertilization resulted in a large increase in glutamine concentration, so that glutamine accounted for a larger proportion of the amino-N pool in bark in N-fertilized trees than in control trees. Glutamine dominated the amino-N pool in wood of control trees. Nitrogen fertilization resulted in an increased proportion of arginine in the wood amino-N pool. We conclude that the composition of the amino-N pools in bark, wood and foliage is highly sensitive to N supply. The composition of the amino-N pools can contribute to the regulation of tree N-nutritional status, which is mediated by shoot to root signalling by long-distance transport of amino compounds.

Polarly‐transported IAA is regarded as a long distance correlative signal important in many aspects of plant physiology, including, for example, apical dominance and growth and development of vascular tissues. In this study, we investigated the importance of apical sources in supplying stem tissues with IAA. The current‐yearshoots of 4‐year‐old Pinus sylvestris L. saplings were replaced with a source of [13C6]IAA. Subsequent mass spectrometric analysis showed that most of the IAA present at two positions in the subjacent 1‐year‐old internode consisted of [13C6]IAA, while [12C]IAA of endogenous origin formed a minor pool. However, the pool of [13C6]IAA decreased from 90 to 80% of the total free IAA pool [13C6]IAA+[12C]IAA) while being transported down the shoot. This dilution with [12C]IAA indicates that de novo biosynthesis of IAA occurred. An additional defoliation experiment showed that the synthesis took place in stem tissues rather than in the mature leaves. The results confirm the role of apical shoots as the major source of polarly‐transported IAA, but also indicate that synthesis of IAA takes place in stem tissues. This is important when considering IAA balance at the whole plant level.