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Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tplb20
New branch roots produced by vascular cambium derivatives in woody parental roots of Populus nigra L. a
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D. Chiatante , M. Beltotto , E. Onelli , A. Di Iorio , A. Montagnoli & S. G. Scippa a
Dipartimento di Biologia Funzionale e Strutturale , Università dell’Insubria , Italy
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Dipartimento di Biologia , Università di Milano , Italy
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Dipartimento di Scienze Chimiche ed Ambientali , Università dell’Insubria , Italy
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Dipartimento DISTAT , Università del Molise , Italy Published online: 04 Aug 2010.
To cite this article: D. Chiatante , M. Beltotto , E. Onelli , A. Di Iorio , A. Montagnoli & S. G. Scippa (2010) New branch roots produced by vascular cambium derivatives in woody parental roots of Populus nigra L., Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana, 144:2, 420-433, DOI: 10.1080/11263501003718612 To link to this article: http://dx.doi.org/10.1080/11263501003718612
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Plant Biosystems, Vol. 144, No. 2, June 2010, pp. 420–433
A SELECTION OF PAPERS PRESENTED DURING THE 7TH ISSR SYMPOSIUM, ROOT RESEARCH AND APPLICATION (ROOT-RAP), 2–4 SEPTEMBER 2009
New branch roots produced by vascular cambium derivatives in woody parental roots of Populus nigra L.
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D. CHIATANTE1, M. BELTOTTO1, E. ONELLI2, A. DI IORIO1, A. MONTAGNOLI3, & S. G. SCIPPA4 1
Dipartimento di Biologia Funzionale e Strutturale, Università dell’Insubria, Italy, 2Dipartimento di Biologia, Università di Milano, Italy, 3Dipartimento di Scienze Chimiche ed Ambientali, Università dell’Insubria, Italy, and 4Dipartimento DISTAT, Università del Molise, Italy Taylor and Francis
Abstract In literature, it has been suggested that new branch roots are produced in a woody parental along its axis not only in the proximal zone where a primary anatomical structure is present but even where old branch roots are present, and a thick production of wood internally characterizes the parental root anatomy. This paper confirms this hypothesis by showing, for the first time, the presence of a new branch root primordium forming within the secondary phloem tissues of a woody parental root. The new branch root primordia found in this work are designated secondary branch roots (SBRs) and seem to develop from derivatives of the vascular cambium that abandon the formation of conducting elements to become the mother cells of these new root primordia. We find that traces belonging to SBRs present some anatomical difference compared with those belonging to branch roots deriving from primary tissues designated, according to literature, primary branch roots (PBRs). This difference in traces could help to distinguish between the origin of the SBRs and PBRs present along a woody parental axis, and this could be helpful in understanding how the root system of a woody plant is developed. The possibility of branching, wherever needed, along the root axes, independently of their internal anatomical organization, evokes a new scenario in which woody plants, in analogy with herbaceous plants, continuously modify their root system in an attempt to adapt and to better exploit their rooting environment.
Keywords: Branch root, vascular cambium, poplar, woody plant
Introduction Root architecture plasticity in response to soil characteristics is one of the most important factors conditioning plant colonization of land. In fact, herbaceous species continuously modify their root architecture in response to environmental factors by affecting root growth, root elongation, root turnover, root density and the distribution of branch roots around the axis of each parent root (reviewed by Chiatante & Scippa 2006). Root density and root distribution along the axis of a parent root are associated with regulation of the activities of primary tissues that govern branch root formation, namely pericycle, endodermis and xylar parenchyma (Esau 1965; Peterson & Peterson 1996; Torrey 1996; Vuylstecker et al. 1998; Casson & Lindsey 2003).
The persistence of these tissues in the roots of herbaceous species throughout the plant’s life implies that, besides the branch roots normally formed during normal development of the root system, new branch roots are formed in loci of the root architecture when and where they are needed. This event has been dissected from an anatomical, cytological and molecular point of view, and therefore, it would be far from the scope of this paper to review such enormous literature. However, it is useful to mention that the majority of these studies have been conducted with roots characterized by a primary structure. In woody species, primary tissues persist only in the proximal region of each root axis (Esau 1965), and these represent a very insignificant portion of the whole root system biomass. In fact, the most
Correspondence: D. Chiatante, Dipartimento di Biologia Funzionale e Strutturale, Università dell’Insubria, Via Dunat 3, 21100 Varese, Italy. Tel: +39 031 2386613. Fax: +39 031 2386630. Email:
[email protected] ISSN 1126-3504 print/ISSN 1724-5575 online © 2010 Società Botanica Italiana DOI: 10.1080/11263501003718612
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Branching in woody parental roots abundant portion of the root system is instead formed by roots having a well-defined secondary structure. This fact raises the question of how it is possible to form new branch roots along a woody parental in places where primary tissues do not persist anymore internally. In literature, it has been recently suggested that the vascular cambium could form new branch roots in a woody parental root (Paolillo & Zobel 2002; Puhe 2003; Paolillo & Bassuk 2005; Paolillo 2006), and this hypothesis is plausible because some vascular cambium initials derive ontogenetically from pericycle cells (Esau 1965), and therefore could have retained the same competence to initiate a new root primordium as their lineage ancestors. Such hypothesis would explain well our previous observations that a woody plant efficiently dissipates mechanical forces into the ground when new branch roots form, where required, in the root system, irrespective of the type of primary or secondary structure within a specific root axis (Chiatante et al. 2002; Chiatante & Scippa 2006). It would also explain several other reports present in the literature that have continuously suggested the occurrence of changes in root architecture in woody plants probably induced by the formation of new branch roots originating from woody parentals (Warning 1934; Thibault 1946; Wilcox 1955; Sinnott 1960; Bogar & Smith 1965; Horsely & Wilson 1971; Kozlowski 1971; Lyford 1980; Alexander & Farley 1983; Fogel 1985, 1990; Beissalah et al. 1988; Jackson & Caldwell 1989; Gruber 1992; Puhe 1994; Bloomfield et al. 1996; Crook & Ennos 1996, 1997; Palma & Barlow 1997; Coutts et al. 1999; Zhang et al. 1999; Forde & Lorenzo 2001; Eissenstat & Yanai 2002; Persson 2002; Hodge 2004). However, in the case of the work of Paolillo and Zobel (2002), Puhe (2003), Paolillo and Bassuk (2005), and Paolillo (2006) also, the formation of a new branch root primordium from the vascular cambium of a woody parental has never been shown and needs to be demonstrated. The aim of this study was to look for clear anatomical evidence that the primordium of a new branch roots forms within the tissues of a parental root in which a secondary structure has been clearly differentiated. For this purpose, we investigated hundreds of serial transverse sections obtained from a number of pieces cut from poplar woody taproots. The anatomical evidence collected in this paper clearly show the occurrence of a root primordium elongating from the taproot vascular cambium derivatives. This root primordium grows internally to the secondary phloem before protruding externally from the bark. Therefore, our work confirms the hypothesis of Paolillo and Zobel (2002), and Paolillo (2006) that some vascular cambium derivatives could become mother cells of a new branch root primordium but do
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not throw light upon the environmental factors responsible for such an event. During our investigations, we have compared various types of branch root traces and have found a number of differences that could be used to distinguish branch roots formed by parental roots characterized by a non-woody structure from those characterized by a woody structure. Materials and methods Plant material Seeds of Populus nigra L. were sown in pots (112 × 20 × 45 cm 3, length × depth × width) in a mixture of soil, peat and agriperlite (3:1:1) during fall 2004 and kept in a greenhouse. In May 2006, five seedlings were excavated and their root system washed to eliminate the growth medium. The root system had a considerable number of first-order branch roots, which differed in diameter, length and stage of development. The oldest first-order branch roots, at the root collar and generally at a more distal position along the taproot axis, were coated with bark tissue. The youngest first-order branch roots were coated with a soft white epidermis at a proximal position. In all samples, between the oldest first-order branch roots, there were several new branch roots coated with an epidermis that emerged directly from the bark tissue. The oldest first-order branch roots coated with bark were designated PBRs and those coated with a soft white epidermis were designated SBRs. For anatomical investigations, we collected portions of five taproots near the root collar in which the presence of bark indicated that a well-developed secondary structure was present internally. The taproot samples used for anatomical studies were cut into 0.5-cm pieces; pieces presenting first-order branch roots with diameters greater than 4 mm were discarded because of the difficulty of obtaining good anatomical preparations and intact serial transverse sections.
Histological analysis Taproot pieces were fixed in formaldehyde (4% in 0.1 M phosphate buffer, pH 7) and then rinsed in 0.1 M phosphate buffer, pH 7. Samples were dehydrated in ethanol series – 25%, 50%, 75% and absolute – at 4°C and embedded in Technovit 7100 (Kulzer, Wehrheim, Germany). Eight-micrometre thick cross-sections were obtained using a microtome (RM 2155, Leica, Germany) and doublestained with fast green (2% w/v in ethanol) and safranin red (4% w/v in water) staining cellulose and lignin, respectively. Sections were mounted in Histovitrex and observed with a light microscope (DMRB, Leica, Germany). In order to have a complete image of the branch root and its trace, we
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scanned the transverse sections by means of a Nikon scanner (Coolscan V ED). The scanned jpg image has then been processed with the help of an AdobePhotoshop version 7 software. To better understand the difference existing between PBRs and SBRs, we analysed each branch root in transverse serial sections from end to end. We distinguished two types of PBRs: live-PBRs (LPBRs), which at microscopic examination were found to be healthy at the time of their collection (intact bark layer), and dead-PBRs (D-PBRs), which seemed to have started to decompose (broken bark layer). We also divided SBRs into random-SBRs (RSBRs), which seemed to protrude without a defined pattern, and induced-SBRs (I-SBRs), which were invariably associated with a PBR. We used the word “induced” to indicate that their formation could have been related to the proximity of a PBR, as suggested for other plant species (Paolillo & Zobel 2002).
Results Live primary branch roots The first 150 transverse serial sections of a taproot piece (starting from the proximal end) contained an
L-PBR (Figure 1[a]), with a diameter of 1.3 mm detached from taproot tissues (Figure 1). The diameter of this L-PBR was constituted mainly by the secondary xylem, although an external layer of bark was visible (Figure 1[a]). The distance of this L-PBR from the rest of the taproot tissues decreased in upper transverse serial sections (Figure 1[b] and 1[c]), which suggests that the L-PBR extended obliquely downwards after its protrusion from the taproot axis. All the remaining 150 upper transverse serial sections (Figure 2[a] and 2[c]) obtained from the same taproot piece contained a trace that coincided with this L-PBR. This trace was first seen in the bark (Figure 2[a]), then in the secondary phloem (Figure 2[b]) and then in the secondary xylem (Figure 2[c]). This indicates that the L-PBR continued to grow in an oblique direction even when elongating within the taproot axis. We measured the distances of the L-PBR from the taproot in each transverse section, the length and thickness of the trace left by the L-PBR in the taproot tissues, and the diameters of taproot tissues (secondary xylem and secondary phloem). The diameter of the secondary xylem of the parental root was smaller in correspondence to an L-PBR and larger on the opposite side. Consequently, the centre Figure secondary phloem well tissues separated 2. 1. seem and structure. phloem. Athe tolive from be secondary The primary primary repairing The thetrace parental restxylem branch branch has the of the break also axis of root root parental the started section opened (L-PBR) (L-PBR) parental axis. todoes by disturb growing axis, (c) growing the not As protrusion respectively. the contain ininternally secondary externally (a) and a of trace, (b), the The toxylem. toits but new and its secondary parental the parental the branch (c)distance tissues Asroot phloem root. in root (a) are axis. between Bar: axis. and normally of (a) 200 (b), the (a) The the The parental but µL-PBR organized m.the transverse trace axis and is in to (tr) isits contact section form considerably has parental entered a with secondary of the axis the deranged the L-PBR isbark secondary decreased. structure. layer (lr) by isthe and xylem separated “ph” Bar: L-PBR. the400 indicates secondary and(b) from extends µm.As the the phloem insecondary to section (a), thebut (ph) centre of thephloem of its L-PBR of the parental the parental and parental section axis. “xy” root shows axis The indicates axis. section. L-PBR that “lr” the the indicates has The secondary new developed secondary branch thexylem. transverse root aphloem secondary has (b)formed As section structure instructure, (a), aoftrace but isthestill the L-PBR; that and disturbed L-PBR interrupts a thin “ph” is layer by cut and the the of obliquely, “xy” continuity L-PBR barkindicate is trace, visible although of the although outside secondary it is still the
Figure 1. Live primary branch root (L-PBR) growing externally to its parental root axis. (a) The transverse section of the L-PBR (lr) is separated from the section of its parental axis. The L-PBR has developed a secondary structure, and a thin layer of bark is visible outside the secondary phloem. The parental axis section does not contain a trace, and the tissues are normally organized to form a secondary structure. “ph” indicates the secondary phloem and “xy” indicates the secondary xylem. (b) As in (a), but the L-PBR is cut obliquely, although it is still well separated from the rest of the parental axis. (c) As in (a) and (b), but the distance between the L-PBR and its parental axis is decreased. Bar: 400 µm.
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Figure 2. Live primary branch root (L-PBR) growing internally to its parental root axis. (a) The L-PBR is in contact with the bark layer and the secondary phloem (ph) of the parental root axis. “lr” indicates the transverse section of the L-PBR; “ph” and “xy” indicate the secondary phloem and the secondary xylem of the parental axis, respectively. The secondary phloem of the parental axis is considerably deranged by the L-PBR. (b) As in (a), but the L-PBR section shows that the new branch root has formed a trace that interrupts the continuity of the secondary phloem structure. The trace has also started to disturb the secondary xylem. (c) As in (a) and (b), but the trace (tr) has entered the secondary xylem and extends to the centre of the parental axis section. The secondary phloem structure is still disturbed by the L-PBR trace, although the tissues seem to be repairing the break opened by the protrusion of the new branch root. Bar: 200 µm.
of the parental root axis near the L-PBR seemed to be diverted towards the periphery. The L-PBR had a maximum diameter of 1.3 mm at its branching point, whereas it was 1.2 mm at its distal apex. The L-PBR was 9 mm long (including its internal trace).
The trace left by this L-PBR within the secondary xylem of the taproot was 1.4 mm high, 0.9 mm wide (near the vascular cambium) and 1.720 mm deep. During its growth within the secondary phloem, the trace was smaller, that is 1.1 mm high, 1.3 mm wide
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and 2 mm deep (radial). On the basis of the measurements of all the sections examined, we reconstructed a three-dimensional model of this LPBR starting from its internal trace in the parent taproot tissues and ending with its orientation after branching (Figure 3). The trace of this L-PBR within the taproot tissues contained vessels and parenchymatic cells that were radially orientated, whereas all other parent taproot tissues were longitudinally orientated (Figure 2[b] and 2[c]). The trace penetrated a wide ray and ended in contact with one of the three arcs forming the primary stele of the parent taproot (arrow in Figure 4). The vascular cambium of the taproot seemed to be confluent with the vascular cambium (dark line in Figure 2[c]) of this L-PBR. Transverse serial sections showed that the trace of this L-PBR extended 0.4 mm upwards in the secondary xylem of the taproot, whereas it was absent from the secondary phloem and bark. However, the vessels in the secondary xylem of the taproot changed their radial orientation from oblique to longitudinal parallel to the taproot axis. The parenchymatic cells in PBRs were full of amyloplasts (Figure 4).
the photographs obtained with three sections chosen among those obtained by cutting the D-PBR axis from end to end: upper end (Figure 5[a]); midsection (Figure 5[b]); and lower end (Figure 5[c]). The tissues of this D-PBR seemed to be in decomposition and its trace extended in the secondary phloem, but not in the secondary xylem. Although the secondary xylem of the taproot was less disturbed by the D-PBR trace, the diameter of vessels in correspondence to this branch root was smaller, and the xylar parenchymatic cells contained more amyloplasts (Figure 5).
Figure 4. The end of an L-PBR trace within the secondary xylem of the parental axis. The arrow indicates one of the three xylary arcs in the centre of the section. The two lines include the trace vessels and the parenchyma cells full of amyloplasts.
phloem Figure 6. arrows indicate of the Transverse parental the vascular axis, serialcambium respectively. sections containing (vc)Bar: of the 200 parental anµSBR. m. root, (a–h)which The arrows extendsindicate externally the and zoneseems of thetomeristematic be in continuity tip of with thethe SBR tissues that in hasthe been vascular cut from cylinder the upper of thisend new (a)SBR to the (pericycle, lower end endoderm (h). (d–f)and were xylar obtained parenchyma). from sections “xy” and passing “ph” through indicate the midsection secondary xylem of theand SBR. theThe secondary double
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xylem Figure of 3. theModel parental of L-PBR axis is indicated formationinincluding light gray, its and tracethe and secondary the external xylem growth. in dark The gray. letters (A, B, C, D, E and F) approximately indicate the position along the root axis of this L-PBR at which transverse sections used for the photographs in Figures 1 and 2 were cut. The secondary
Dead primary branch root We obtained 160 transverse serial sections of a taproot piece containing a D-PBR. Figure 5 shows
Figure to seems 5. beD-PBR. disturbed Anonly abandoned in the midsection branch root. in Transverse which the trace serialofsections this PBR representing is present. the Bar:upper 200 side µm. (a), midsection (b) and lower side (c). The secondary xylem (xy) of the parental axis is not disturbed to the same extent as in the L-PBR trace shown in Figure 2. The secondary phloem
Random secondary branch root We studied several smooth taproot pieces lacking external PBRs and/or SBRs to identify where a new SBR started to form. We obtained clear evidence of the formation of a new SBR, namely, small root primordia growing entirely within the taproot secondary phloem (Figure 6) in several instances. These SBRs were positioned randomly; indeed, we found no evidence of a specific position in the taproot circumference, hence the name “R-SBR”. In the case shown in Figure 6, the taproot piece yielded 130 transverse serial sections, all containing tissues directly attributable to the same R-SBR, which had a diameter of 1 mm. The eight photographs of Figure 6(a)–(h) represent the
Figure 3. Model of L-PBR formation including its trace and the external growth. The letters (A, B, C, D, E and F) approximately indicate the position along the root axis of this L-PBR at which transverse sections used for the photographs in Figures 1 and 2 were cut. The secondary xylem of the parental axis is indicated in light gray, and the secondary xylem in dark gray.
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Figure 4. End of an L-PBR trace within the secondary xylem of the parental axis. The arrow indicates one of the three xylary arcs in the centre of the section. The two lines include the trace vessels and the parenchyma cells full of amyloplasts.
complete outline of this new R-SBR from end to end. The central cylinder of this new R-SBR elongated in the secondary phloem, and its basal tissues were in contact with the vascular cambium of the parent taproot. Figure 6(a) and 6(h) show transverse sections in which the upper and lower rhyzodermis of the R-SBR are visible as a darker group of cells within the secondary phloem. Figure 6(b)–(g) show the apical meristem of this R-SBR, whereas the central vascular cylinder is visible only in Figure 6(b)–(d). Interestingly, the vascular cambium of the parent taproot seems to be connected with the central cylinder of this R-SBR (double arrows in Figure 6[d] and 6[f]). In other taproots pieces, R-SBRs were at a later stage of development, and their meristematic tips were in contact with the external layer of bark (arrow in Figure 7). In this case, R-SBRs were completely surrounded by the parenchymatic cells of the taproot that displayed swelling of undifferentiated tissue (white circle in Figure 7). This swelling sometimes occurred externally (black arrow in Figure 8 Panel A and Panel B) and could probably have caused the break in the bark heralding protrusion of this new R-SBR (white arrow in Figure 8 Panel B). with the8. of anof SBR that protrudes through the meristematic cells and elongates downwards (white arrow). The barphloem in at panel is 1 mm andof3has mm in panel Figure production 7.addition of Protrusion Parenchyma parenchymatic tissue an R-SBR cells. forming The through aswelling swelling the bark caused around layer a break an of SBR. theinparental the The bark. arrow root indicates axis. Panel the A:position The arrow within indicates the secondary the position which at A which a swelling an SBR parenchymatic grown and B. reached cells hasthe caused bark layer. a breakThe in the white bark circle layer. includes Panel B: anThe areablack of thearrow periphery indicates of the theparental same condition axis that described is swollenindue panel to the A,
Induced secondary branch root In some taproot pieces, an SBR protruded from the parent taproot axis above the position of an L-PBR branching along the same rank. These SBRs were designated I-SBRs (see Materials and methods section). A feature common to I-SBRs and R-SBRs was that their traces occurred only within the secondary phloem of the parent taproot and never in the secondary xylem. In the representative example of an I-SBR shown in Figure 9, we collected 270 transverse serial sections from end to end. The root had a diameter of 1.3 mm, which remained constant from its branching point to the conical tip, and internally, there was a typical actinostele with a thick cortex and a small vascular cylinder (Figure 9 Panel B and Panel C). The trace of this I-SBR in the secondary phloem of the parent taproot was 0.9 mm high, 1.85 mm wide and 2.260 mm deep (radial). The vascular cambium of the taproot formed a continuous line with the periphery of the central cylinder of the I-SBR, which contained primary tissues, namely, pericycle, endoderm and xylar parenchyma (white arrow in Figure 9 Panel A). The measurements of all the parameters referring to this I-SBR enabled us to reconstruct the model
phloem Figure 9.of the Formation parentalofaxis an I-SBR. (ph) includes Panel A: theThe trace centre of thecylinder I-SBR.of Panels the new B and branch C: Aroot transverse is in contact section with ofthe thevascular I-SBR, which cambium hasderivatives a diameter (vc) of 1.38 of the mm. parental Bar: 500 axis.µThe m. secondary xylem of the parental axis (xy) penetrates the vascular cylinder of the I-SBR (white arrow). The secondary
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Figure 5. D-PBR. An abandoned branch root. Transverse serial sections representing the upper side (a), midsection (b) and lower side (c). The secondary xylem (xy) of the parental axis is not disturbed to the same extent as in the L-PBR trace shown in Figure 2. The secondary phloem seems to be disturbed only in the midsection in which the trace of this PBR is present. Bar: 200 µm.
reported in Figure 10, which summarizes the findings we observed in the 270 sections examined. The I-SBR model illustrates how this I-SBR bent downwards after its protrusion and then elongated parallel to its parent taproot axis (Figure 10[B]) before moving away (Figure 10[C]). Figure used for10. panels Model A, Bofand I-SBR C in formation, Figure 9. including its trace within the secondary phloem (in dark gray) of the parental axis and its external growth. The letters A, B, and C approximately indicate the position along the root axis of this I-SBR at which transverse sections were cut and
Anatomical comparison between the traces formed by PBRs and SBRs We investigated the differences between traces formed in the parent taproot tissues by PBRs and SBRs at a higher magnification. These observations confirmed that the traces produced by SBRs did not affect the organization of the secondary xylem because they did not penetrate into the xylary components of the taproot (Figure 11 Panel A and
Panel B). On the contrary, the secondary xylem seemed to extend externally by forming a V-shaped trace pointing to the periphery (arrow in Figure 11 Panel A and Panel B). The vessels and parenchymatic cells in the secondary xylem of the taproot continued to have a normal axial orientation (double arrow in Figure 11 Panel C). This contrasted with traces formed by L-PBRs, which extended internally in a V-shape fashion (Figure 11 Panel D) and penetrated deep in the secondary xylem within a wide medullar ray (Figure 11 Panel E). The xylary components of the L-PBR trace were radially orientated (arrow in Figure 11 Panel E). The vascular cambium of the taproot was in contact with the vascular cambium of the branch root (PBR) or with the external tissues (pericycle, endoderm and xylar parenchyma) of the central cylinder (SBR). A
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Figure 6. Transverse serial sections containing an SBR. (a–h) The arrows indicate the zone of the meristematic tip of the SBR that has been cut from the upper end (a) to the lower end (h). (d–f) were obtained from sections passing through the midsection of the SBR. The double arrows indicate the vascular cambium (vc) of the parental root, which extends externally and seems to be in continuity with the tissues in the vascular cylinder of this new SBR (pericycle, endoderm and xylar parenchyma). “xy” and “ph” indicate the secondary xylem and the secondary phloem of the parental axis, respectively. Bar: 200 µm.
common feature of PBR and SBR traces was that the parenchyma cells of the secondary xylem were rich in amyloplasts (single arrow in Figure 11 Panels C–E). Discussion
Figure 7. Parenchyma tissue forming a swelling around an SBR. The arrow indicates the position within the secondary phloem at which an SBR has grown and reached the bark layer. The white circle includes an area of the periphery of the parental axis that is swollen due to the production of parenchymatic cells. The swelling caused a break in the bark.
Our data clearly show the formation of a new branch root primordium taking place within the secondary phloem tissues of a parental root that has differentiated its secondary structure. In this way, our work confirms what had been suggested by other authors (Paolillo & Zobel 2002; Paolillo & Bassuk 2005; Paolillo 2006) as a potential feature of woody roots in several coniferous and broadleaved tree species. This finding impinges on our understanding of the sequence of events responsible for the full development of root architecture in a woody plant. In fact, it is now clear that woody root architecture results from two different mechanisms of root branching. The first is active at an early stage of root axis development and depends essentially upon primary
tissues (pericycle, endoderm and/or xylar parenchyma) in the parental axis; the second starts later and depends probably upon the induction of an unknown number of vascular cambium derivatives in the secondary structure of the parental axis. Despite the ontogenetic lineage difference between the tissues responsible for the formation of branch roots, both mechanisms contribute to the final root architectural outline of a woody plant. However, we do not know whether these two mechanisms are related and whether they respond to the same intrinsic and/or extrinsic factors (Malamy & Benfey 1997a, 1997b; Malamy 2005; Chiatante & Scippa 2006).
SBR Figure withintrace. double the 11. arrow secondary The Magnification indicates panel phloem Econducting show of (ph). the PBRtrace Panel components and of SBR B: a PBR The traces. sectioned with midsection Panel its own A transversally, and shows conducting panel theBcentral and represent components thecylinder single the magnification radially arrow (vc) inindicates oriented. contact of with xylary two Barsections the inparenchymatic panels vascular passing A, cambium B and through cells D: 200 containing ofanthe SBR µparental m; upper bar amyloplasts. inaxis end panels (arrow). (A)Cand Panel andmidsection Panel E: D 10 shows Cµshows m.(B). the trace aThe magnification secondary (tr) of a PBR, xylem of the with (xy) zone theofof arrow the theparental secondary pointing axis toxylem penetrates the vascular of the(arrows parental cambium in axis Aof and the near B)parental the where SBR an axis trace SBR that (tr) issurrounds formed. that extends The the
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Figure 8. Protrusion of an R-SBR through the bark layer of the parental root axis. Panel A: The arrow indicates the position at which a swelling of parenchymatic cells has caused a break in the bark layer. Panel B: The black arrow indicates the same condition described in Panel A, with the addition of an SBR that protrudes through the meristematic cells and elongates downwards (white arrow). The bar in Panel A is 1 mm and 3 mm in Panel B.
The ultimate demonstration that woody parentals can form routinely new branch roots changes the scenario where this event was thought to depend only exceptionally on the occurrence of specialized meristematic tissues at the periphery of the parental axis (Warning 1934; Esau 1940; Thibault 1946) or on specific conditions such as the loss of roots after flooding or pruning (Wilcox 1955; Bogar & Smith 1965; Kozlowski 1984). On the contrary, in future, this event must be considered to be a normal feature of the root system of all woody species. In the light of these new concepts, the models used to describe root architectures in woody species must be adapted to include the event of a new root branching in the woody portion of all root axes. Even the terminology used for specific roots may have to be revised. For example, “adventitious” (Paolillo & Zobel 2002) no longer seems appropriate to indicate a new root branching from a woody root parental because the
term was coined to indicate the formation of new roots on a stem (Hayward 1938; Esau 1965; Fahn 1990). Similarly, the term “accessory”, suggested previously by us to indicate the emission of new roots to increase anchorage (Chiatante & Scippa 2006), no longer seems appropriate because it suggests that this event only occurs when a certain type of response is needed. We now propose the term “secondary branch root” (SBR) to indicate that new roots can be formed from a woody parental at any time during its life. Thus, “SBR” becomes the opposite of “primary branch root” (PBR), proposed by Paolillo and Zobel (2002), to indicate a branch root formed by primary tissues when a primary structure is present in the parental root. Using these two definitions, one may summarize that the root architecture in a herbaceous plant is due to PBRs, whereas in woody plants, it is due to the sum of PBRs and SBRs.
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Figure 9. Formation of an I-SBR. Panel A: The centre cylinder of the new branch root is in contact with the vascular cambium derivatives (vc) of the parental axis. The secondary xylem of the parental axis (xy) penetrates the vascular cylinder of the I-SBR (white arrow). The secondary phloem of the parental axis (ph) includes the trace of the I-SBR. Panels B and C: A transverse section of the I-SBR, which has a diameter of 1.38 mm. Bar: 500 µm.
Our data confirm the importance of investigating root traces to distinguish between a PBR and an SBR (Paolillo & Zobel 2002; Paolillo & Bassuk 2005; Paolillo 2006). In PBRs, the trace is Vshaped, with vessels fully integrated within the conducting systems of the parent root. Furthermore, the connection between the xylary components of the trace and the secondary xylem of the parental axis continues even after the trace disappears from the secondary phloem. Similar traces have been reported for the persistent roots of other plant species (Paolillo & Zobel 2002; Paolillo & Bassuk 2005; Paolillo 2006). Our work suggests that when a PBR stops thickening, the connection
with the secondary xylem of the parent taproot is lost. In this case, the PBR could remain attached to its parental root axis for a certain time before entering its final decomposition stage that precedes its shedding. In this case, the thickening of the axis in the parental root completely encases the “abandoned” PBR with the growing secondary tissues, and this would explain the finding of branch roots whose axes seem to run within a tunnel excavated in the secondary tissues of their parent root (arrow in Figure 12). An SBR trace is completely different. In fact, an SBR trace is oriented in a direction opposite to that of a PBR, that is the angle of the V-shape trace
taproot FigureThe PBR. 12. axislack is Morphology 1.3 of acm. connection of a PBR between that has the been two conducting abandoned but systems has not is demonstrated been shed from byits theparental fact thatroot. the The PBRarrow is covered indicates by its theown PBRbark whose tissue. diameter The secondary no longer phloem increases, andwhereas the barkthe ofdiameter the parental of the root secondary have been xylem removed of itsby parental hand scalpel root continues to showtothe increase PBR better. and wraps The diameter completely of the
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Figure 10. Model of I-SBR formation, including its trace within the secondary phloem (in dark gray) of the parental axis and its external growth. The letters A, B, and C approximately indicate the position along the root axis of this I-SBR at which transverse sections were cut and used for Panel A, B and C in Figure 9.
extends towards the periphery of the parental root. These traces never follow the path of a wide medullar ray and are similar to the “peripheral traces”, as described by Paolillo and Zobel (2002). A common element of all SBRs observed in this work is the continuity of their primary tissues (pericycle, endoderm and xylar parenchyma) with the taproot vascular cambium. This factor enables full integration between the two xylary systems and an SBR to become a persistent root with its own secondary structure. Our experiments were not designed to evaluate the turnover length of SBRs; however, it seems reasonable to speculate that the turnover length depends on the role played by these roots, that is the roots needed for mechanical support become persistent roots with a longer turnover (Chiatante et al. 2002), whereas roots involved in plant nutrition become ephemeral roots with a variable turnover length. Sporadically, we found that some SBRs form above a PBR. This event also occurs in other woody species and is often associated with the formation of cluster roots (Bogar & Smith 1965; Wilcox 1968; Paolillo 2006). The PBR trace and/or large medullar rays have been implicated in the formation of cluster roots (Bogar & Smith 1965). We did not find cluster roots along the taproot axes; nevertheless, we cannot exclude that the vascular cambium or the large
medullar rays could have played a role in the formation of our SBRs. The occurrence of a stable pattern in root branching, which could be used to detect a growth unit along the parental axis similar to the phytomer found along the stem axis, is still a matter of debate (Draye 2002; Fitter 2002; Hou et al. 2004; Chiatante & Scippa 2006). However, it has been established that the morphology of the apical meristem of each branch root (Coutts 1989) and/or gravitropism (Coutts 1989) affect the “liminal angles” (Sachs 1874) and positions at which the branch root protrudes from the parental root axis. Simple morphological analysis did not reveal a specific pattern of SBR branching in our samples, and we were not able to investigate the cellular and molecular events related to SBR formation, which could have provided some indication of factors involved in deciding where a new SBR must be formed. In this context, the break in the bark of the parent roots occurs just before protrusion of the SBR and, therefore, it is too late to predict the site where the vascular cambium derivatives have been induced to form a root primordium. This is why we have not investigated the earlier stage of SBR formation and have no information about the intrinsic and extrinsic factors responsible for this event. Similarly, we do not know why the diameter of the vascular cambium of the parental axis decreases in correspondence to the site of PBR formation but not of SBR formation. The only known cellular event taking place in the parental root coincident with the production of a new SBR seems to be the accumulation of starch in the parenchyma tissue of the secondary xylem. This fact suggests that the production of a new SBR in a particular position of the parental root axis is preceded by the accumulation in that zone of a considerable amount of carbohydrates. Conclusion and perspective The picture confirmed by our work is that, in analogy with herbaceous plants, woody plants can continuously change root architecture to better adapt to a variable rooting environment. For a nutritional purpose, the formation of new branch roots from woody root axes allows trees to limit the extension of the soil explored by its roots by returning cyclically in the same volume of soil (Paolillo & Zobel 2002). This type of reiterative branching of woody roots also has obvious advantages for the mechanical anchorage of a tree in the soil. In fact, the mechanical loading forces affecting the root system of a tree are very variable (i.e. variations due to changes in wind direction and/or canopy structure) and require continuous mechanical adjustments. By forming new branch roots along a
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Figure 11. Magnification of PBR and SBR traces. Panel A and Panel B represent the magnification of two sections passing through an SBR upper end (A) and midsection (B). The secondary xylem (xy) of the parental axis penetrates (arrows in A and B) the SBR trace (tr) that extends within the secondary phloem (ph). Panel B: The midsection shows the central cylinder (vc) in contact with the vascular cambium of the parental axis (arrow). Panel C shows a magnification of the zone of the secondary xylem of the parental axis near where an SBR is formed. The double arrow indicates conducting components sectioned transversally, and the single arrow indicates xylary parenchymatic cells containing amyloplasts. Panel D shows the trace (tr) of a PBR, with the arrow pointing to the vascular cambium of the parental axis that surrounds the SBR trace. The Panel E shows the trace of a PBR with its own conducting components radially oriented. Bar in Panels A, B and D: 200 µm; bar in Panels C and E: 10 µm.
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Figure 12. Morphology of a PBR that has been abandoned but has not been shed from its parental root. The arrow indicates the PBR whose diameter no longer increases, whereas the diameter of the secondary xylem of its parental root continues to increase and wraps completely the PBR. The lack of a connection between the two conducting systems is demonstrated by the fact that the PBR is covered by its own bark tissue. The secondary phloem and the bark of the parental root have been removed by hand scalpel to show the PBR better. The diameter of the taproot axis is 1.3 cm.
woody parental, a tree can redeploy new root buttresses in a specific position of the root system in which roots were not present or have been shed because they were no longer needed (Chiatante et al. 2002; Chiatante & Scippa 2006). Experiments are now underway in our laboratory to find an experimental model enabling us to address the following questions: (1) Are all vascular cambium derivatives inducible to become mother cells of a new lateral root? and (2) What cellular, molecular and physiological events occur during the formation of a new root primordium from vascular cambium derivatives? Acknowledgements The authors thank J. A. Gilder for text editing. Financial support was received from MIUR (PRIN 2005), Università dell’Insubria (FAR) and COST Action E38.
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