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Keywords:

  • boron (B);
  • cell cycle;
  • endoreduplication;
  • legume–rhizobia symbiosis;
  • nodule organogenesis;
  • ploidy

Introduction

  1. Top of page
  2. Introduction
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. References

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

  1. Top of page
  2. Introduction
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. References

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

  1. Top of page
  2. Introduction
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. References

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).

image

Figure 1. Effects of boron (B) nutrition on nodule development in Medicago sativa plants inoculated with Sinorhizobium meliloti 1021 (a–d) and in Pisum sativum plants inoculated with Rhizobium leguminosarum bv. viciae 3841 (e–j). (a) M. sativa nodulated root grown in the presence of B (+B), 3 wk postinoculation; (b) M. sativa nodulated root grown in the absence of B (−B), 3 wk post-inoculation; (c) 3-wk-old M. sativa nodules +B; (d) 3-wk-old M. sativa nodules −B; (e) 1 wk postinoculation P. sativum nodulated root +B; (f) 1 wk postinoculation P. sativum nodulated root −B; (g) 2 wk postinoculation P. sativum nodulated root +B; (h) 2 wk postinoculation P. sativum nodulated root −B; (i) 3 wk postinoculation P. sativum nodulated root +B; (j) 3 wk postinoculation P. sativum nodulated root −B. m, Nodule meristem; ir, infected region of red color caused by the presence of leghemoglobine in +B nodules.

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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).

image

Figure 2. Toluidine blue-stained semithin sections of Pisum sativum (a, b) and Medicago sativa (c, d) nodules developed in the presence (a, c) or in the absence (b, d) of boron (B), harvested 3 wk postinoculation, and showing abnormal development and low infection of central tissues in B-deficient nodules. I, nodule meristem composed of small dividing cells; II, infection zone characterized by nodule cell enlargement, bacterial infection and bacteroid division; III, maturation zone where the large nodule cells are fully packed with nitrogen-fixing bacteroids. ct, Central tissues; nc, nodule cortex. Bars, 0.1 mm.

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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).

image

Figure 3. Reverse transcription–polymerase chain reaction (RT-PCR) analysis of expression of cycD3, 10 h and 1 wk postinoculation (a, b), and ccs52a, 1 and 3 wk post-inoculation (c, d) in Medicago sativa-inoculated roots (a, c) and Pisum sativum-inoculated roots (b, d) (for cycD3 analysis 10 h postinoculation) or nodules. +B, nodulated plants grown in the presence of B; −B, nodulated plants grown in the absence of B. Expression of the constitutive msc27 gene was used as a control.

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Table 1.  Ploidy levels (nuclear DNA content) in Pisum sativum and Medicago sativa nodules developed in the presence (+B) or in the absence (–B) of boron
Nuclear DNA contentPisum sativum nodulesMedicago sativa nodules
+B–B+B–B
  1. 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).

2C24.42 ± 8.33a61.12 ± 14.63b38.84 ± 12.09a78.07 ± 21.46b
4C43.96 ± 13.04a30.75 ± 8.21a39.41 ± 9.32a21.49 ± 7.02b
≥ 8C31.62 ± 10.47a8.13 ± 3.69b21.75 ± 6.11a0.44 ± 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.

Acknowledgements

  1. Top of page
  2. Introduction
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. References

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.

References

  1. Top of page
  2. Introduction
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. References
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