Herbaspirillum seropedicae and sugarcane endophytic interaction investigated by using high pressure freezing electron microscopy

Uso da técnica de congelamento por alta pressão no estudo da interação endofítica de Herbaspirillum seropedicae e cana-de-açúcar

ENVIRONMENTAL AND SOIL MICROBIOLOGY

Herbaspirillum seropedicae and sugarcane endophytic interaction investigated by using high pressure freezing electron microscopy

Uso da técnica de congelamento por alta pressão no estudo da interação endofítica de Herbaspirillum seropedicae e cana-de-açúcar

Lúcia Gracinda da Silva; Flávio Costa Miguens; Fabio Lopes Olivares

Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brasil

^{Correspondence}

ABSTRACT

The interaction between sugar cane plantlets and H. seropedicae was investigated using High Pressure Freezing followed by Freeze Substitution. Microscopical observation showed consistent differences between this approaches when compared with the conventional preparation, specially related to appearance of the bacteria cell and the endophytic attachment to the host cell wall.

Key words:H. seropedicae, high pressure freezing, endophytic interaction.

RESUMO

A interação entre plântulas de cana-de-açúcar e H. seropedicae foi investigada pelo uso da técnica de congelamento por alta pressão seguida de criosubstituição. Observações microscópicas evidenciaram diferenças marcantes entre esta técnica e preparações convencionais, especialmente relacionadas a ultraestrutura da bactéria e às estruturas envolvidas na adesão à superfície da parede celular da planta hospedeira.

Palavras-chave:H. seropedicae, congelamento por alta pressão, interação endofítica.

INTRODUCTION

Herbaspirillum seropedicae are Gram-negative bacteria capable of fixing nitrogen and promoting plant growth in different grasses (1,4,8). Ecological studies have shown that these bacteria can be isolated from the interior of roots, stems and leaves of many grasses, but never from soil samples (7). In addition, studies involving light and transmission electron microscopy have clearly demonstrated the endophytic nature of the plant-bacteria interaction (5,9). At structural level, the interactions between Herbaspirillum seropedicae and sugar cane plants have been receiving attention over the last 13 years. Studies based on chemically fixed samples of roots and shoots have demonstrated that the endophytic bacteria colonize randomly as single cells or micro colonies in the apoplast (intercellular spaces, cell wall and xylem lumen) as cited by James and Olivares (5). In the present study, we used High Pressure Freezing Technique (HPF) followed by Freeze Substitution to investigate the sugar cane / H. seropedicae interaction at microscopic level.

MATERIALS AND METHODS

Bacterial strains and growth conditions

The bacterial strain used in this study was Herbaspirillum seropedicae (BR11175), grown overnight at 30ºC and 120 rpm in Dyg's medium.

Plant Material

Micro-propagated plantlets of sugar cane (3) var. RB72454 were grown in a modified 2% sucrose MS medium (6) and inoculated with 0.1 mL of the suspension containing 10⁸ cell.mL^-1.

Cryo-technique preparation and microscopy evaluation

Plantlets were harvested one week after inoculation, and small pieces of roots and leaves (0.1 - 0.6 mm) were carefully colleted in filter paper and inserted into the support of aluminium covered with hexadecane (1-hexadecene, Fluka, Buchs, Switzerland). The freezing process was performed at -196ºC and at 2,100 Bar (HPM 010 - High Pressure Freezing Machine, Baltec). The frozen samples were transferred to the Freezer Substitution Apparatus (Baltec) and, then fixed with 2% osmium tetroxide in 100% acetone solution for 24h (-85ºC/8h, -60ºC/8h e -20ºC/8h). Samples were washed twice (acetone) and infiltrated with Sppur resin. Polymerised blocks were sectioned, stained and viewed using a transmission electron microscope (TEM) (Zeiss EM 900) operating at 80 KV under standard conditions.

RESULTS AND DISCUSSION

Microscopic observations showed significant differences between the cryo-technique approaches as compared with the conventional preparation, specially in relation to the appearance of the bacteria cell (Figs. 1 and 2) and the type of the endophytic attachment to the host cell wall (Figs. 1c, 1d, 2d, 2e and 2f). H. seropedicae was localized as a single cell or a micro colony inside the apoplast of leaves. Bacteria were also found inside apparently dead vascular parenchyma cells. Root cortex apoplast showed bacteria surrounded by an amorphous matrix of unknown composition (Figs. 2a and 2b), as well as adhered to the plant cell wall by a stalk of electron-dense material (Fig. 2c). Similar results were observed for the first time by Gyaneshwar (2). In both roots and leaves, bacterial cytoplasm has shown numerous poly-b-hydroxybutyrate (PHB) granules. At adhesion sites both bacteria and plant cell walls showed altered aspects (Figs. 1c, 2d, 2e and 2f) and bacterial protrusions could be observed (Fig. 2e). In roots, multivesicular bodies were observed close to the adhesion sites (Figs. 1e and 1f). In conclusion, high-pressure technique was an essential tool to obtain morphological data demonstrating alterations in both bacteria and plant cells during Herbaspirilum seropedicae / sugar cane interactions.

ACKNOWLEDGEMENTS

This work was supported by CNPq, FAPERJ, FENORTE, PRONEX.

This paper corresponds to an "extended abstract" selected for oral presentation in the 22^nd Brazilian Congress of Microbiology, held in Florianópolis, SC, Brazil, in November 17-20, 2003

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Publication Dates

• Publication in this collection
  29 Nov 2004
• Date of issue
  Nov 2003