Phenolic compounds in ectomycorrhizal interaction of lignin modified silver birch.

BACKGROUND: The monolignol biosynthetic pathway interconnects with the biosynthesis of other secondary phenolic metabolites, such as cinnamic acid derivatives, flavonoids and condensed tannins. The objective of this study is to evaluate whether genetic modification of the monolignol pathway in silver birch (Betula pendula Roth.) would alter the metabolism of these phenolic compounds and how such alterations, if exist, would affect the ectomycorrhizal symbiosis. RESULTS: Silver birch lines expressing quaking aspen (Populus tremuloides L.) caffeate/5-hydroxyferulate O-methyltransferase (PtCOMT) under the 35S cauliflower mosaic virus (CaMV) promoter showed a reduction in the relative expression of a putative silver birch COMT (BpCOMT) gene and, consequently, a decrease in the lignin syringyl/guaiacyl composition ratio. Alterations were also detected in concentrations of certain phenolic compounds. All PtCOMT silver birch lines produced normal ectomycorrhizas with the ectomycorrhizal fungus Paxillus involutus (Batsch: Fr.), and the formation of symbiosis enhanced the growth of the transgenic plants. CONCLUSION: The down-regulation of BpCOMT in the 35S-PtCOMT lines caused a reduction in the syringyl/guaiacyl ratio of lignin, but no significant effect was seen in the composition or quantity of phenolic compounds that would have been caused by the expression of PtCOMT under the 35S or UbB1 promoter. Moreover, the detected alterations in the composition of lignin and secondary phenolic compounds had no effect on the interaction between silver birch and P. involutus.

One tissue, two fates: different roles of megagametophyte cells during Scots pine embryogenesis.

In the Scots pine (Pinus sylvestris L.) seed, embryos grow and develop within the corrosion cavity of the megagametophyte, a maternally derived haploid tissue, which houses the majority of the storage reserves of the seed. In the present study, histochemical methods and quantification of the expression levels of the programmed cell death (PCD) and DNA repair processes related genes (MCA, TAT-D, RAD51, KU80, and LIG) were used to investigate the physiological events occurring in the megagametophyte tissue during embryo development. It was found that the megagametophyte was viable from the early phases of embryo development until the early germination of mature seeds. However, the megagametophyte cells in the narrow embryo surrounding region (ESR) were destroyed by cell death with morphologically necrotic features. Their cell wall, plasma membrane, and nuclear envelope broke down with the release of cell debris and nucleic acids into the corrosion cavity. The occurrence of necrotic-like cell death in gymnosperm embryogenesis provides a favourable model for the study of developmental cell death with necrotic-like morphology and suggests that the mechanism underlying necrotic cell death is evolutionary conserved.

Interaction with ectomycorrhizal fungi and endophytic Methylobacterium affects nutrient uptake and growth of pine seedlings in vitro.

Tissues of Scots pine (Pinus sylvestris L.) contain several endophytic microorganisms of which Methylobacterium extorquens DSM13060 is a dominant species throughout the year. Similar to other endophytic bacteria, M. extorquens is able to colonize host plant tissues without causing any symptoms of disease. In addition to endophytic bacteria, plants associate simultaneously with a diverse set of microorganisms. Furthermore, plant-colonizing microorganisms interact with each other in a species- or strain-specific manner. Several studies on beneficial microorganisms interacting with plants have been carried out, but few deal with interactions between different symbiotic organisms and specifically, how these interactions affect the growth and development of the host plant. Our aim was to study how the pine endophyte M. extorquens DSM13060 affects pine seedlings and how the co-inoculation with ectomycorrhizal (ECM) fungi [Suillus variegatus (SV) or Pisolithus tinctorius (PT)] alters the response of Scots pine. We determined the growth, polyamine and nutrient contents of inoculated and non-inoculated Scots pine seedlings in vitro. Our results show that M. extorquens is able to improve the growth of seedlings at the same level as the ECM fungi SV and PT do. The effect of co-inoculation using different symbiotic organisms was seen in terms of changes in growth and nutrient uptake. Inoculation using M. extorquens together with ECM fungi improved the growth of the host plant even more than single ECM inoculation. Symbiotic organisms also had a strong effect on the potassium content of the seedling. The results indicate that interaction between endophyte and ECM fungus is species dependent, leading to increased or decreased nutrient content and growth of pine seedlings.

Polyamine metabolism during exponential growth transition in Scots pine embryogenic cell culture.

Polyamine (PA) metabolism was studied in liquid cultures of Scots pine (Pinus sylvestris L.) embryogenic cells. The focus of the study was on the metabolic changes at the interphase between the initial lag phase and the exponential growth phase. PA concentrations fluctuated in the liquid cultures as follows. Putrescine (Put) concentrations increased, whereas spermidine (Spd) concentrations decreased in both free and soluble conjugated PA fractions. The concentrations of free and soluble conjugated spermine (Spm) remained low, and small amounts of excreted PAs were also found in the culture medium. The minor production of secondary metabolites reflected the undifferentiated stage of the embryogenic cell culture. Put was produced via the arginine decarboxylase (ADC) pathway. Futhermore, the gene expression data suggested that the accumulation of Put was caused neither by an increase in Put biosynthesis nor by a decrease in Put catabolism, but resulted mainly from the decrease in the biosynthesis of Spd and Spm. Put seemed to play an important role in cell proliferation in Scots pine embryogenic cells, but the low pH of the culture medium could also, at least partially, be the reason for the accumulation of endogenous Put. High Spd concentrations at the initiation of the culture, when cells were exposed to stress and cell death, suggested that Spd may act not only as a protector against stress but also as a growth suppressor, when proliferative growth is not promoted. All in all, Scots pine embryogenic cell culture was proved to be a favourable experimental platform to study PA metabolism and, furthermore, the developed system may also be beneficial in experiments where, e.g., the effect of specific stressors on PA metabolism is addressed.

Mycobacteria are hidden endophytes in the shoots of rock plant [Pogonatherum paniceum (Lam.) Hack.] (Poaceae).

A mycobacterium was isolated from micropropagated Pogonatherum paniceum and identified as a close relative of Mycobacterium cookii. The endophyte diversity in the shoots of potted and micropropagated P. paniceum plants was studied by culture-independent techniques. Group- and strain-specific PCR demonstrated that the P. paniceum plants harboured the isolated Mycobacterium strain as a minority. Altogether 101 clones of the PCR products were sequenced. The shoots of potted P. paniceum plants harboured unculturable endophytes in the families Phyllobacteriaceae, Hyphomicrobiaceae, Sphingobacteriaceae, Enterobacteriaceae, Alcaligenaceae and Mycobacteriaceae. Among the unculturable Mycobacteriaceae strains related to Mycobacterium chubuense, M. poriferae, M. obuense, M. fortuitum, M. neoaurum, M. diernhoferi, M. intracellulare and M. cookii were identified. Three unique sequences that clustered with M. llatzarense and M. mucogenicum were identified in micropropagated plants. According to the results, the shoots and micropropagated tissues of rock plant are inhabited by mycobacteria, which should stimulate further studies on the diversity of unculturable mycobacteria in edible crop plants.