Boron Requirement Cyanobacterial - Plant Physiology · BORONREQUIREMENTIN CYANOBACTERIA Chemicals 0.3 ChemicalswerepurchasedfromMerck. Analytical Methods Culture density was determined

Plant Physiol. (1990) 94, 1554-1 5600032-0889/90/94/1 554/07/$01 .00/0

Received for publication May 7, 1990Accepted July 27, 1990

Boron Requirement in Cyanobacterial

Its Possible Role in the Early Evolution of Photosynthetic Organisms

lidefonso Bonilla*, Mercedes Garcia-Gonzalez, and Pilar MateoDepartamento de Biologia, Facultad de Ciencias, Universidad Aut6noma de Madrid, 28049 Madrid, Spain

ABSTRACT

The effect of boron on heterocystous and nonheterocystousdinitrogen fixing Cyanobacteria was examined. The absence ofboron in culture media inhibited growth and nitrogenase activityin Nodularia sp., Chlorogloeopsis sp., and Nostoc sp. cultures.Examinations of boron-deficient cultures showed changes in het-erocyst morphology. However, cultures of nonheterocystous Cy-anobacteria, Gloeothece sp. and Plectonema sp., grown in theabsence of boron did not show any alteration in growth or nitro-genase activity. These results suggest a requirement of borononly by heterocystous Cyanobacteria. A possible role for thiselement in the early evolution of photosynthetic organisms isproposed.

by cells grown on combined nitrogen (e.g. nitrate or ammo-nium-N) (4). These results suggested an involvement ofboronin nitrogenase activity (15).Our study was designed to determine the boron require-

ment in dinitrogen-fixing Cyanobacteria, which have majordifferences in structures and development among them andto determine the applicability of generalizations regardingboron requirement by the Cyanobacteria. Thus, we chosethree filamentous species with heterocysts that fix molecularnitrogen aerobically (Nostoc sp. UAM 205, Nodularia sp. Ml,and Chlorogloeopsis sp. PCC 6912); a filamentous and non-heterocystous strain that fixes dinitrogen anaerobically (Plec-tonema calothricoides); and a unicellular form that is able tofix dinitrogen aerobically (Gloeothece sp. PCC 6501).

Boron (B) requirement varies markedly among organisms.It is the rare element that, although required in higher plants,has no role in animals or fungi (18). Anderson and Jordan(2) found that B stimulated dinitrogen fixation in Azotobacter,but they did not consider that it could be essential for thisbacterium. Essentiality of boron in algae does not seem to begeneral. Lewin (10, 11) and Smyth and Dugger (23, 24)provided sound evidence that boron was required by marineand freshwater diatoms. However, other investigators (6, 8,16) failed to demonstrate a requirement for boron in Chlorellaand other green algae.

In Cyanobacteria, addition of B was reported to stimulategrowth rates in the absence of combined nitrogen for Nostocmuscorum, Calothrix parietina, and Anabaena cylindrica (5,8). When nitrate was present in the medium, the addition ofboron had only a small effect on the growth rate of theseCyanobacteria. Microcystis aeruginosa, which does not fixmolecular nitrogen, showed no requirement although it ab-

sorbed boron in appreciable amounts (8). Gerloff(8) suggestedthat the smaller response to boron deficiency in cells grownwith nitrate compared with cells grown with dinitrogen was

due to boron contamination from the nitrate salt.We have previously shown that boron was not required for

the growth of the unicellular strain Anacystis nidulans, whichcannot fix molecular nitrogen (14). Even though the micro-nutrient appeared to be required by those Anabaena PCC7119 that depended on dinitrogen fixation, it was not required

' This work was supported by Comisi6n Interministerial de Cienciay Tecnologia, No. PB 86-0323.

MATERIALS AND METHODS

Organisms

The properties and sources of the Cyanobacteria strainsused in this study are listed in Table I. Chlorogloeopsis PCC6912 and Gloeothece PCC 6501 were obtained from theDepartment of Biochemistry (University of Sevilla, Spain).Plectonema calothricoides, strain No. 1463-4, was from theGottingen University Algal Culture Collection. The Cyano-bacteria Nostoc UAM 205 and Nodularia Ml were recentlyisolated: Nostoc sp. was isolated in soil samples from a ricefield in Valencia, Spain, and Nodularia sp. from a mountainstream in the north of Madrid, Spain.

Culture Conditions

Batch cultures of aerobic dinitrogen-fixing Cyanobacteriawere grown in a medium free of combined nitrogen in 1-Lpolyethylene bottles at 26°C and in the following conditions:Chlorogloeopsis sp. cultures were grown with continuous airbubbling under a constant light intensity of 90 AEm-m2-s-';Nostoc sp. and Nodularia sp. cultures were grown with con-tinuous air bubbling under a constant light intensity of 30E-m-2. s ; Gloeothece sp. cultures were grown without air

bubbling under a constant light intensity of 3 MuE.m2s'.For routine growth of P. calothricoides, stock cultures weremaintained in a nitrate medium (9) and bubbled with airunder a constant light intensity of 90 uE. m-2 s-'. For dini-trogen fixation experiments, cultures were transferred to amedium lacking combined nitrogen as described above. Me-dia without a combined nitrogen source and boron-free cellswere prepared and processed as described previously (1 5).

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BORON REQUIREMENT IN CYANOBACTERIA

Chemicals 0.3

Chemicals were purchased from Merck.

Analytical Methods

Culture density was determined at 600 nm in a Hitachi150-20 spectrophotometer. For dry weight determination,cells were collected on 0.45-,gm filters, washed, and dried at70°C for 24 h.

Nitrogenase Activity

Nitrogenase activity was determined by acetylene reductionand carried out as described previously (15). Ethylene pro-duction was estimated by injection into a Shimadzu GC-8Agas chromatograph.

Anaerobic Induction Experiments

The anaerobic induction of nitrogenase activity in P. cal-othricoides was performed as follows. Cells from the exponen-tial growth phase were harvested, washed with sterile material,and resuspended in a fresh nitrate-free sterile medium. Cul-tures were grown in polyethylene bottles sealed with rubberstoppers and flushed for 10 min with argon before injecting5% CO2 into the flask. The assay was started by injectingacetylene to a 10% concentration. The bottles were main-tained at 26°C under cycles of 16 h light and 8 h dark. Todetermine nitrogenase activity, 0.5-mL gas samples were re-moved with a syringe at the indicated time and their ethylenecontent was measured.

Microscopy

Examination of living filaments by light microscopy was

conducted with preparations mounted in water. Micrographswere taken with Olympus BH-2. For transmission electronmicroscopy, cells were fixed with glutaraldehyde (2%) in 0.1M phosphate buffer, pH 7.2, and postfixed with OS04 (2%).Dehydration was carried out with water-acetone solutions.Samples embedded in Vestopal were sectioned with a dia-mond knife, stained with uranyl acetate, and observed in a

Philips EM 300 transmission electron microscope (7).

I-

'I-

Z LUOE

Jo

Z

0.11

-A +B

i B-B

I I2 4 6

TIME (days)

TIME (doys)

Figure 1. Effect of B deficiency on growth (A) and nitrogenase activity(B) of Nodularia Ml cells. Values are the means ±SD (bars) of fourindependent experiments.

RESULTS

Effect of B on Heterocystous Cyanobacteria

Growth of batch cultures ofNodularia sp. M in a mediumfree of combined nitrogen and without boron was reducedafter 2 d of culture, compared with control cultures in thepresence of B 2 Growth was inhibited by 50% after 6 d ofculture (Fig. IA). Nitrogenase activity in B-deficient cultureswas reduced to about 40% of the activity in B-suppliedcultures within the first 24 h of culture (Fig. 1B). The inhibi-tion in nitrogenase activity was maintained for 6 d, althoughto a lesser extent (Fig. 1B).

Chlorogloeopsis sp. PCC 6912 cultures grown without Bshowed a progressive decrease in the growth rate when com-pared with B-supplemented cultures (Fig. 2A). When the time

'Abbreviation: B, boron.

Table I. Some Properties of the Cyanobacteria Used for ExperimentsOrganism Nitrogen HeterocystsStructure Fixation Formed

Gloeothece sp. PCC 6501 Unicellular AerobicP. calothricoides Gcttingen Filamen- An-

No. tous aero-1463-4 bic

Nodularia sp. Ml Filamen- Aerobic +tous

Nostoc sp. UAM 205 Filamen- Aerobic +tous

Chlorogloeopsis PCC 6912 Filamen- Aerobic +sp. tous

1 555

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1556 BONILLA ETAL. Plant Physiol. Vol. 94, 1990

A 413 culture (data not shown). When B-deficient cells were washed0.4 - and resuspended in a medium free of B again, their growth

E t -B rate was equal to that of control cultures (Fig. 7A). Further-cn / / more, cultures that were maintained for more than 1 monthE in a B-free medium did not show alterations in their growth

0.2 (data not shown). Nitrogenase activity in Gloeothece sp. cul-tures was similarly unaffected by a lack of B. No differences0 were observed between these cultures after 10 d (Fig. 7B).LO Growth rates and nitrogenase activity were very low compared

with those obtained with other strains. Since Gloeothece was2 4 6 grown in continuous illumination, and since photosynthesis

TIME (days) and nitrogen fixation occur simultaneously under that con-j:sB dition, it is possible that the 02 evolved in photosynthesis

0.¢G3 -partially inhibited nitrogenase activity.P. calothricoides cultures, collected and transferred to a

medium lacking combined nitrogen under anaerobic condi-E/ \ tions, developed nitrogenase activity about 2 d after induction.

z k }/ t\ The specific activity increased rapidly after 7 d and persistedui C4 0.1 ~ / \for 8 d. This pattern of acetylene reduction was similar in B-° 'a i +8 deficient and control cells (Fig. 8).

-E Lt, , , , 4Optical microscopic examination did not show differences2 4 6 between B-deficient and control cultures in any of these

TI ME ( days) strains.

Figure 2. Effect of B deficiency on growth (A) and nitrogenase activity DISCUSSION(B) of Chlorogloeopsis PCC 6912 cells. Values are the means ±SD The essentiality of B for Cyanobacteria is not a general(bars) of three independent experiments, requirement. In this work, we examined the B requirement

course for acetylene reduction was investigated, the lack of Bresulted in decreased acetylene reduction (Fig. 2B). 0.2 AGrowth and nitrogenase activity of Nostoc UAM 205 were +B

not significantly inhibited during the first 3 d of culturewithout B supplementation. However, when these cells were E B+ Bcollected, washed, and resuspended again in a B-free medium, Egrowth was reduced (Fig. 3A), and the lack of B caused a % 0.1drastic decrease in nitrogenase activity (Fig. 3B). In all cases,the growth curves and nitrogenase activities were not corre- 1 2 3lated. The primary effect of B deficiency is an inhibition ofnitrogenase activity that produced nitrogen deficiency. Delayin growth inhibition in B cultures could be caused by utiliza- 2 4 TIME (days)tion of nitrogen resources, especially the photosynthetic pig-ments (phycobiliprotein and Chl) (1, 15). The decrease inpigment content could explain the reduction in photosyn- 0.7 -Bthesis and cessation of growth observed after 3 d of B defi-ciency (data not shown). 0.4Examination of B-deficient cultures ofNostoc sp. and Chlo- _ 3 0.5 - + B

rogloeopsis sp. under the light microscope showed changes incell structure and organization and a yellowing of the cells E 02 /(Figs. 4 and 5). This was previously observed in other heter- tn I 0.3 - -Bocystous filamentous Cyanobacteria, e.g. Anabaena PCC Z7119 (7, 15). Light microscopic examination of Nodularia - -/ -L__ Lcells did not reveal any changes; however, when these cultures ° E 0.1 1 2 3 4were examined by electron microscopy, alterations in the l +Imorphology and ultrastructure of the heterocysts in the B- 1 2 3 T1ME(days)deficient cultures were clearly visible (Fig. 6).

Figure 3. Effect of B deficiency on growth (A) and nitrogenase activityEffect of B on Nonheterocystous Cyanobacteria (B) of Nostoc UAM 205 cells. After 4 (A) or 3 (B) d, cells of culture

grown without B were transferred to a medium in the presence orThe absence of B in the culture medium did not alter the absence of B. Values are the means +SD (bars) of four (A) and three

growth of Gloeothece sp. PCC 6501 during the first 5 d of (B) independent experiments.

---




A

I.4

;,...4

E ;.

Figure 4. Effect of B deficiency on Nostoc UAM205 cultures. A, control cells; B, B-deficient cells.Bar markers represent 5 um.

5i.,

0'A.. f

L

I.,

for some heterocystous and nonheterocystous dinitrogen-fix-ing Cyanobacteria. Among the differences found were: nitro-genase activity and growth of Gloeothece sp. were not affectedby the lack of B in the growth medium. As was the case forGloeothece sp., the absence of B did not change the nitrogen-ase activity of Plectonema sp. cells. However, B was requiredfor both nitrogenase activity and growth in Nodularia sp.,Chlorogloeopsis sp., and Nostoc sp. These results are in agree-ment with previous ones that showed such a requirement inAnabaena sp. PCC 7119 cells (7, 15). Different responses werefound with regard to a B requirement in Chlorogloeopsis sp.,Nodularia sp., and Nostoc sp. The differences could be due todifferences in B sensitivity of the strains used, as occurs withhigher plants (19), or due to some biochemical, structural,and/or developmental differences in these organisms. Alter-natively, the 'history' of the species may influence the re-

sponse: Chlorogloeopsis sp. PCC 6912 is a strain from an algalculture collection; however, Nodularia sp. Ml and Nostoc sp.UAM 205 are wild type strains that were recently isolated andthat may have developed mechanisms enabling them to ac-climate to environmental changes. Furthermore, Nostoc sp.forms filamentous aggregates (3), which, in our experiments,might have retained the B from the washes during the prepa-ration of B-free cells ( 15).The fact that some Cyanobacteria require B as an essential

nutrient while others do not is not unusual. An evolutionarystudy of the acquisition of an essential role for B in themetabolism of vascular plants showed that, as is true in theCyanobacteria, the B requirement differs from species tospecies ( 13). McClendon ( 17) proposed that the relative abun-dance of an element is a decisive factor in the origin of itsnutritional essentiality and considered the B requirement to

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Plant Physiol. Vol. 94, 1990

Figure 5. Effect of B deficiency on Chloro-gloeopsis PCC 6912 cultures. A, control cells;B, boron-deficient cells. Bar markers represent5Mm.

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.xLe

have been acquired over time, given that B was of limitedavailability in primitive oceans (13). Nevertheless, sodium,one of the most abundant elements in the lithosphere, is notessential for higher plants with the exception ofthe C-4 plants,the most modem species. But sodium is an essential nutrientfor the heterocystous Cyanobacteria, which are among themost ancient living genera. In these organisms, sodium isrequired for photosynthesis and the bicarbonate transportingsystem (22).The fact that B is also essential for the heterocystous Cy-

anobacteria (principally Nostocacean), which were predomi-nant organisms during the Middle Pre-Cambrian Period some2 billion years ago, would indicate that B was an essentialelement during the early evolution of life, even if its availa-bility was relatively limited in the primitive ocean.The different taxa probably developed a B requirement

independently and long after the groups diverged. Most algaeand fungi do not require B, with the exception of severaldiatom species, in which B is an important structural com-ponent of the cell wall (10, 11). Lewis (12) developed ahypothesis to explain the fact that some taxonomic groupshave an absolute requirement for B, whereas others do not.He pointed out that fungi and algae contain large concentra-tions of compounds that complex with borate. This wouldhave prevented B from exerting a regulatory role in themetabolism of these organisms (21). However, sucrose formsonly a very weak complex with borate, and, after sucrose hadbeen adopted as a major carbohydrate in the Chlorophyta,phloem developed in land plants, which evolved from thisgroup, as a tissue in which the complexing of B with solublecarbohydrates was minimal. Since B was not sequestered bycomplexing with carbohydrates, and, with the evolution of

BONILLA ET AL.1 558

ti




'-, --I, , ,-B

w E 0.1

Zc~

Figure 8. Time course of anaerobic induction of nitrogenase activityin P. ca/othricoides in the presence or absence of B. Values are themeans +SD (bars) of 3 independent experiments.

Figure 6. Electron micrographs of Nodularia Ml grown in the pres-ence (A and B) or in the absence (C and D) of B. Note the alterationin heterocyst envelope of B-deficient cultures (arrows). Bar markersrepresent 1 fim.

0.2

0.1

1 2 3 6 7 9TIME ( days )

1 2 3 4 6 7 8 9 10TIME (days)

Figure 7. Growth (A) and nitrogenase activity (B) of Gloeothece PCC6501 cells grown in the presence or absence of B. Previously thecultures were grown in a medium without boron for 5 d. Values arethe means ±SD (bars) of 3 independent experiments.

xylem through which B was passively carried to the end ofthe transpiration stream, it acquired a regulatory role in plantmetabolism (12, 13).There is some evidence that B is not essential for nondini-

trogen fixing forms of Cyanobacteria (8, 14) or for the non-heterocystous, dinitrogen-fixing forms, Gloeothece sp. andPlectonema sp. However, a lack of B does have an inhibitoryeffect on heterocystous Cyanobacteria.

Because boric acid forms esters with cis-diols (21), wesuggested a possible role for B in the stabilization of theglycolipid inner layer of heterocysts by interacting with their-OH groups (7, 15) as has been proposed for higher plant cellmembranes (18, 20). Boron deficiency could lead to an alter-ation in the heterocyst envelope, which would facilitate 02diffusion and result in an inhibition of nitrogenase activity.This hypothesis is consistent with the inhibitory effect that Bdeficiency has only on heterocystous Cyanobacteria. Further-more, a drastic alteration in the protecting 02-diffusion en-velope of the heterocysts in Nodularia sp. cells has also beenshown in this study.Taken together, these results suggest that the essentiality of

B for Cyanobacteria is restricted to heterocystous species andthat B is involved in heterocyst function. A role for thiselement in the stabilization of heterocyst structure couldexplain this requirement only in heterocystous Cyanobacteria,and, given their biological antiquity, this might well suggestthat B was necessary in the early history of life.

Finally, the findings show that the Cyanobacteria are ade-quate models for the study of mineral nutrient requirementsin relation to the origin of life.

LITERATURE CITED

1. Allen MM, Smith AJ (1969) Nitrogen chlorosis in blue-greenalgae. Arch Microbiol 69: 114-120

2. Anderson GR, Jordan JV (1961) Boron. A non essential growthfactor for Azotobacter chroococcum. Soil Sci 92: 113-1 16

3. Bold HC, Wynne MJ (1985) Introduction to the Algae. PrenticeHall, Englewood Cliffs, NJ, pp 34-69

4. Bonilla I, Mateo P, Fernandez-Valiente E, Sanchez-Maeso E,Martinez F (1984) Essentiality of boron for Anabaena PCC

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0

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Plant Physiol. Vol. 94, 1990

7119 grown in nitrogen fixation conditions. In Proceedings ofthe Fourth Congress of the Federal European Society of PlantPhysiology, Strasbourg, pp 373-374

5. Eyster C (1952) Necessity ofboron for Nostoc muscorum. Nature170: 755

6. Fernandez E, Sanchez E, Bonilla I, Mateo P, Ortega P (1984)Effect ofboron on the growth and cell composition of Chlorellapyrenoidosa. Phyton 44: 125-131

7. Garcia-Gonzalez M, Mateo P, Bonilla 1 (1988) Boron protectionfor 02 diffusion in heterocysts of Anabaena sp. PCC 7119.Plant Physiol 87: 785-789

8. Gerloff GC (1968) The comparative boron nutrition of severalgreen and blue-green algae. Physiol Plant 21: 369-377

9. Kratz WA, J Myers (1955) Nutrition and growth of several blue-green algae. Am J Bot 42: 282-287

10. Lewin JC (1966) Physiological studies of the boron requirementof the diatom Cylindrotheca fusiformis. J Exp Bot 17:473-479

11. Lewin JC (1966) Boron as a growth requirement for diatoms. JPhycol 2:160-163

12. Lewis DH (1980) Boron lignification and the origin of vascularplants-a unified hypothesis. New Phytol 84: 209-229

13. Lovatt CJ (1985) Evolution of xylem resulted in a requirementfor boron in the apical meristems of vascular plants. NewPhytol 99: 509-522

14. Martinez F, Mateo P, Bonilla I, Fernandez E, Garate A (1986)Growth of Anacystis nidulans in relation to boron supply. IsrJ Bot 35: 17-21

15. Mateo P, Bonilla I, Fernandez-Valiente E, Sanchez-Maeso E(1986) Essentiality of boron for dinitrogen fixation in Ana-baena sp. PCC 7119. Plant Physiol 81: 430-433

16. McBride L, Chorney W, Skok J (1971) Growth of Chlorella inrelation to boron supply. Bot Gaz 132: 10-23

17. McClendon JH (1976) Elemental abundance as a factor in theorigins of mineral nutrient requirements. J Mol Evol 8:175-195

18. Parr AJ, Loughman BC (1983) Boron and membrane functionin plants. In DA Robb, WS Pierpoints, eds, Metals and Micro-nutrients Uptake and Utilization by Plants. Academic Press,London, pp 87-107

19. Pilbeam DJ, Kirkby EA (1983) The physiological role of boronin plants. J Plant Nutr 6: 563-582

20. Pollard AS, Parr AD, Loghman BC (1977) Boron in relation tomembrane function in higher plants. J Exp Bot 28: 831-841

21. Raven JA (1980) Short- and long-distance transport ofboric acidin plants. New Phytol 84: 231-249

22. Sanchez-Maeso E, Fernandez-Pifias F, Garcia-Gonzalez M, Fer-nandez-Valiente E (1988) Sodium requirement for photosyn-thesis and its relationship with dinitrogen fixation and theexternal CO2 concentration in Cyanobacteria. Plant Physiol85: 585-587

23. Smyth DA, Dugger WM (1980) Effect of boron deficiency on86Rb uptake and photosynthesis in the diatom Cylindrothecafusiformis. Plant Physiol 66: 692-695

24. Smyth DA, Dugger WM (1981) Cellular changes during borondeficient culture of the diatom Cylindrothecafusiformis. Phys-iol Plant 51: 111-117

1 560 BONILLA ET AL.



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Boron Requirement Cyanobacterial - Plant Physiology · BORONREQUIREMENTIN CYANOBACTERIA Chemicals 0.3 ChemicalswerepurchasedfromMerck. Analytical Methods Culture density was determined