See the Materials and Methods for experimental conditions. Values followed by a different letter in the +B and –B columns from each plant are significantly different (P ≤ 0.01).
Endoreduplication before cell differentiation fails in boron-deficient legume nodules. Is boron involved in signalling during cell cycle regulation?
Article first published online: 15 MAY 2009
© The Authors (2009). Journal compilation © New Phytologist (2009)
Volume 183, Issue 1, pages 8–12, July 2009
How to Cite
Reguera, M., Espí, A., Bolaños, L., Bonilla, I. and Redondo-Nieto, M. (2009), Endoreduplication before cell differentiation fails in boron-deficient legume nodules. Is boron involved in signalling during cell cycle regulation?. New Phytologist, 183: 8–12. doi: 10.1111/j.1469-8137.2009.02869.x
- Issue published online: 3 JUN 2009
- Article first published online: 15 MAY 2009
- boron (B);
- cell cycle;
- legume–rhizobia symbiosis;
- nodule organogenesis;
The requirement of higher plants for boron (B) was recognized > 80 yr ago (Warington, 1923), but to date only its function related to cell wall stability (caused by borate cross-linking of apiose residues in the pectic polysaccharide rhamnogalacturonan II) has been convincingly demonstrated (O’Neill et al., 2001, 2004). However, reports of abnormal tissue differentiation (Lowatt, 1985) and embryogenesis (as described in Larix by Behrendt & Zoglauer, 1996), together with increasing evidence for the essentiality of B in animal embryo development (Rowe & Eckhert, 1999; Lanoue et al., 2000), support the fact that B is required particularly during the initial phases of cell differentiation and organogenesis. The fact that most plant B is required for developing tissues rather than for mature organs (Bell et al., 2002), and the long-standing report that the first effects of B deprivation appear in meristems (Sommer & Sorokin, 1928), support the hypothesis that signalling mechanisms during cell differentiation and organogenesis are highly sensitive to B deficiency. However, there have been no reports in the literature describing what occurs in meristem function leading to aberrant development under B deprivation. Therefore, investigating the effect of B deficiency on the regulation of organogenetic plant processes could shed new light on the biological role of this micronutrient.
The symbiotic interaction between legume and rhizobia triggers the development of a nodule following a process of organogenesis highly regulated by molecular plant–bacteria interactions (for reviews see Stougaard, 2000; Limpens & Bisseling, 2003) and B deficiency has a strong effect on legume–rhizobia symbioses, affecting not only cell wall structure (Bonilla et al., 1997; Redondo-Nieto et al., 2003) but also rhizobia–legume cell-surface interactions and cell-to-cell signalling during symbiotic events (Bolaños et al., 1996, 2001; Redondo-Nieto et al., 2007, 2008). Nodule organogenesis has to be very accurately regulated to ensure plant and bacteria coupling and a successful symbiosis. It involves the formation of a meristem resulting from cell cycle re-activation in response to Nod factors and the switch of mitotic cycles to several cycles of endoreduplication, cell enlargement and differentiation programs that allow rhizobia to infect host cells, proliferate and acquire the capacity to fix N2 (reviewed by Foucher & Kondorosi, 2000). The high sensitivity of nodule development to B deficiency is in line with a higher B content in nodules than in other legume tissues (Redondo-Nieto et al., 2003), and with evidence that symptoms of B deficiency appear in nodules earlier than in other plant tissues (Bolaños et al., 1994). Therefore, investigating the effects of B deprivation on the activity of key genes involved in the cell cycle during nodule organogenesis can help to increase our understanding of the particular requirement for B during the early stages of development of plants or animals.
Materials and Methods
Growth of pea (Pisum sativum L. cv. Lincoln) and alfalfa (Medicago sativa L. cv. Resis) plants and inoculation with Rhizobium leguminosarum bv. viciae 3841 and Sinorhizobium meliloti 1021, respectively, in B-sufficient or B-deficient conditions, were performed according to previous reports (Redondo-Nieto et al., 2003).
Nodule structure was investigated by light microscopy of semithin (0.5 mm) sections of nodules selected at a comparable stage of development and processed according to Bolaños et al. (1994).
The DNA content of nuclei from different organs was determined using flow cytometry, as described previously (Cebolla et al., 1999), and at least 5000 nuclei were analyzed per measurement in a minimum of three independent experiments.
For gene expression studies, total RNA was extracted from B-sufficient and B-deficient roots 10 h after inoculation, and at 1 wk and 2 wk after inoculation from nodules frozen in liquid nitrogen, and reverse transcription–polymerase chain reaction (RT-PCR) was performed as described by Cebolla et al. (1999) using the following primers: CycD3;1-5′ (5′-GGATGCTTAAAGTCAATGC-3′); CycD3;1-3′ (5′-GGAACACAACCAACAAATC-3′); Ccs52a-5′ (5′-TAGAACGCGGTTGTTTGGAC-3′); Ccs52b-3′ (5′-CTGCTACAAGCATTCCAGAG-3′); Msc27-5′ (5′-GGAGGTTGAGGGAAAGTGG-3′) and Msc27-3′ (5′-CACCAACAAAGAATTGAAGG-3′).
Results and Discussion
Nodules induced in roots of alfalfa (M. sativa) (Fig. 1a–d) and pea (P. sativum) (Fig. 1e–j) plants developed aberrantly in the absence of B, suggesting that cell cycle regulation was lost during nodule organogenesis. Following primordium formation in the presence of B (+B; Fig. 1e), nodules progressively differentiated a central infected region (ir) that was observed as a pink colour owing to the presence of the oxygen carrier leghemoglobin, while a persistent meristem (m) remained in the apical zone of alfalfa (Fig. 1c) and pea (Fig. 1g,i) +B nodules. However, typical nodule organogenesis failed under B deficiency, and –B nodules were smaller in size than the control +B nodules. Moreover, they were spherical, pale in colour and different zones of development were indistinguishable (Fig. 1e shows –B alfalfa nodules and Fig. 1f,h,j shows –B pea nodules).
The small size exhibited by −B nodules could be the result of low mitotic activity; however, examination of nodules 3 wk post-inoculation using light microscopy (Fig. 2), apparently suggest a problem of cell size, rather than of cell number, in –B nodules. B-sufficient nodules clearly showed the typical zones of development described by Newcomb et al. (1979) (Fig. 2a,c). Zone I corresponds to the apical meristem composed of small dividing cells; zone II is the infection zone, where enlarged cells are being invaded by rhizobia; and zone III includes the largest cells full of bacteroids. By contrast, B deficiency led to nodules with no well-defined zones of development. Pea –B nodules were almost devoid of bacteria and only a few infected cells had an enlarged size, in which most of the inner tissue was composed of small cells (Fig. 2b). This effect of B deficiency was even more evident in alfalfa nodules, which showed an uninfected central tissue that was composed of small meristem-like undifferentiated cells (Fig. 2d).
Therefore, RT-PCR experiments were carried out to analyze the expression of key genes involved in regulation of the cell cycle and cell differentiation during nodule organogenesis. Expression of the cycD3 gene (Fig. 3a,b), which is involved in re-activation of the cell cycle in response to Nod factors, was detected 10 h after inoculation of alfalfa or pea plants and was maintained in nodules harvested 1 wk postinoculation. The expression of this cyclin gene under B deficiency was not significantly different, and even slightly higher, than in +B plants, confirming that the mitotic activity was not diminished by B deprivation. Therefore, the reason for the reduced size of –B nodules might be found in cell enlargement rather than in cell division processes. The ccs52a gene, involved in cell cycle arrest and transition of mitotic to endoreduplication cycles (Cebolla et al., 1999), was expressed in +B alfalfa nodules 1 wk postinoculation and the expression increased 3 wk postinoculation (Fig. 3c). However, the expression of ccs52a was almost undetectable in –B nodules throughout the time course of the experiment. Similar results of the effect of B deficiency on the expression of ccs52a gene expression were found in pea nodules (Fig. 3d). This gene encodes an anaphase-promoting complex activator. Anaphase-promoting complex is involved in polyubiquitination of mitotic cyclins B before its degradation by the proteasome, resulting in inactivation of mitotic CDKs (cyclin dependent kinases) before the M-phase of the cell cycle and in endocycles (Kondorosi et al., 2005). Therefore, the anaphase-promoting complex should not be activated in B-deficient nodules, cyclins should not be ubiquitined and degraded, and the mitotic cycle should still be active, as indicated by the huge number of small cells in −B nodules (Fig. 2b,d). Confirming that endoreduplucation cycles fail under B deficiency, measurement of the nuclear DNA content by flow cytometry (Table 1) indicated that the ploidy level is reduced in B-deficient pea or alfalfa nodules, which showed an increase in the number of 2C nuclei, a tendency (statistically significant in alfalfa nodules) for a diminished population of 4C nuclei and a pronounced decrease in the number of nuclei with a DNA content of ≥ 8C that were almost absent in alfalfa –B nodules. Moreover, activation of ccs52a and polyploidy are required for cell differentiation (Vinardell et al., 2003), and therefore most cells of the central tissues from –B nodules must remain in an undifferentiated ‘meristem-like’ state, as shown in Fig. 2(c,d).
|Nuclear DNA content||Pisum sativum nodules||Medicago sativa nodules|
|2C||24.42 ± 8.33a||61.12 ± 14.63b||38.84 ± 12.09a||78.07 ± 21.46b|
|4C||43.96 ± 13.04a||30.75 ± 8.21a||39.41 ± 9.32a||21.49 ± 7.02b|
|≥ 8C||31.62 ± 10.47a||8.13 ± 3.69b||21.75 ± 6.11a||0.44 ± 0.38b|
The overall results suggest that B is required particularly at the initial phases of organogenesis, not for maintaining mitotic activity in meristems but for eliciting mechanisms leading to cell differentiation. But, why is cell differentiation highly sensitive to B deficiency? There is a known relationship among B, phytohormones and apical dominance (Wang et al., 2006), but it seems to be insufficient to explain the high B requirement for nodule organogenesis and it cannot explain abnormal animal organogenesis. Several studies on B in plant and animal development and metabolism point to a role of B in extracellular matrices and/or in membrane functions (Brown et al., 2002). Nodule development is strongly driven by membrane-related functions and, in a recent paper (Redondo-Nieto et al., 2007), B was found to be related to the presence of membrane glycoproteins of cells in the zone II of pea nodules, where cells exit from the meristem and start to differentiate (Foucher & Kondorosi, 2000). It has been proposed that membrane glycoproteins stabilized by borate are involved in cell-to-cell signalling during symbiosome development and in nodule, plant and even animal organogenesis (Redondo-Nieto et al., 2008); therefore, besides hormones being affected by B deficiency, it is possible that membrane B-dependent glycoproteins in nodule cells are the target that elicits ccs52a induction and endocycles before cell differentiation. Therefore, further progress should involve strategies, including plant mutagenesis, to identify target molecules for B to affect cell differentiation.
This work was supported by Ministerio de Educación y Ciencia BIO2005-08691-CO2-01, BIO2008-05736-CO2-01 and by MICROAMBIENTECM Program from Comunidad de Madrid. María Reguera is the recipient of a Contract from Comunidad de Madrid.
- 1996. Boron controls suspensor development in embryogenic cultures of Larix decidua. Physiologia Plantarum 97: 321–326. ,
- 2002. Boron requirements of plants. In: GoldbachH, RerkasemB, WimmerMA, BrownPH, ThellierM, BellRW, eds. Boron in plant and animal nutrition. New York, NY, USA: KluwerAcademic/Plenum Publishers, 63–86. , ,
- 1996. Effects of boron on Rhizobium–legume cell-surface interactions and nodule development. Plant Physiology 110: 1249–1256. , ,
- 2001. Lectin-like glycoprotein PsNLEC-1 is not correctly glycosylated and targeted in boron deficient pea nodules. Molecular Plant–Microbe Interactions 14: 663–670. , , , ,
- 1994. Essentiality of boron for symbiotic dinitrogen fixation in pea (Pisum sativum)-Rhizobium nodules. Plant Physiology 104: 85–90. , , , , , ,
- 1997. The aberrant cell walls of boron deficient bean root nodules have no covalently bound hydroxyprolin-/proline-rich proteins. Plant Physiology 115: 1329–1340. , , , , , , , , .
- 2002. Boron in plant biology. Plant Biology 4: 205–223. , , , , , , , ,
- 1999. The mitotic inhibitor ccs52 is required for endoreplication and ploidy-dependent cell enlargement in plants. EMBO Journal 18: 4476–4484. , , , , , ,
- 2000. Cell cycle regulation in the course of nodule organogenesis in Medicago. Plant Molecular Biology 43: 773–786. ,
- 2005. Ubiquitin-mediated proteolysis. To be in the right place at the right moment during nodule development. Plant Physiology 137: 1197–1204. , ,
- 2000. Functional impairments in preimplantation mouse embryos following boron deficiency. FASEB Journal 14A: 539. , , , .
- 2003. Signaling in symbiosis. Current Opinion in Plant Biology 6: 343–350. ,
- 1985. Evolution of xylem resulted in a requirement for boron in the apical meristems of vascular plants. New Phytologist 99: 509–522. .
- 1979. The early morphogenesis of Glycine max and Pisum sativum root nodules. Canadian Journal of Botany 57: 2603–2616. , , .
- 2001. Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth. Science 249: 846–849. , , , .
- 2004. Rhamnogalacturonan II: structure and function of a borate crosslinked cell wall pectic polysaccharide. Annual Reviews of Plant Biology 55: 109–139. , , , .
- 2007. Developmentally regulated membrane glycoproteins sharing antigenicity with rhamnogalacturonan II are not detected in nodulated boron deficient Pisum sativum. Plant, Cell & Environment 30: 1436–1443. , , , ,
- 2008. Boron dependent membrana glycoproteins in symbiosome development and nodule organogenesis. A model for a common role of boron in organogenesis. Plant Signaling & Behavior 3: 298–300. , , ,
- 2003. Relationship between boron and calcium in the N2-fixing legume–rhizobia symbiosis. Plant, Cell & Environment 26: 1905–1915. , , , ,
- 1999. Boron is required for zebrafish embryogenesis. Journal of Experimental Biology 202: 1649–1654. , .
- 1928. Effects of the absence of boron and of some other essential elements on the cell and tissue structure of the root tips of Pisum sativum. Plant Physiology 3: 237–260. ,
- 2000. Regulators and regulation of legume root nodule development. Plant Physiology 124: 531–540.
- 2003. Endoreduplication mediated by the anaphase-promoting complex activator CCS52A is required for symbiotic cell differentiation in Medicago truncatula nodules. Plant Cell 15: 2093–2105. , , , , , , , , , et al .
- 2006. Involvement of auxin and CKs in boron deficiency induced changes in apical dominance of pea plants (Pisum sativum L.). Journal of Plant Physiology 163: 591–600 , , ,
- 1923. The effect of boric acid and borax on the broad been and certain other plants. Annals of Botany 37: 629–672.