No abstract

MicroRNAs (miRNAs) are non-protein coding RNAs of ~20–24 nucleotides in length that play an important role in many biological and metabolic processes, including the regulation of gene expression, plant growth and developmental processes, as well as responses to stress and pathogens. The aim of this study was to identify and characterize novel and conserved microRNAs expressed in methyl jasmonate-treated Scots pine needles. In addition, potential precursor sequences and target genes of the identified miRNAs were determined by alignment to the Pinus unigene set. Potential precursor sequences were identified using the miRAtool, conserved miRNA precursors were also tested for the ability to form the required stem-loop structure, and the minimal folding free energy indexes were calculated. By comparison with miRBase, 4975 annotated sequences were identified and assigned to 173 miRNA groups, belonging to a total of 60 conserved miRNA families. A total of 1029 potential novel miRNAs, grouped into 34 families were found, and 46 predicted precursor sequences were identified. A total of 136 potential target genes targeted by 28 families were identified. The majority of previously reported highly conserved plant miRNAs were identified in this study, as well as some conserved miRNAs previously reported to be monocot specific. No conserved dicot-specific miRNAs were identified. A number of potential gymnosperm or conifer specific miRNAs were found, shared among a range of conifer species.

Silver birch (Betula pendula Roth.) is an ecologically and economically important deciduous tree species in Northern Europe. In vitro culture initiation and shoot rejuvenation from birches in their reproductive phase is difficult and often unsuccessful. Currently, markers to evaluate the degree of juvenility of birch in vitro shoots, which could be used to track the rejuvenation process and to determine factors affecting it, have not been developed. The aims of this study were to assess expression of juvenility related microRNAs and target genes during micropropagation of silver birch, and to investigate factors affecting juvenility of birch genotypes with different in vitro morphogenic ability. Potential precursor sequences and target genes of the microRNAs miR156 and miR172 were determined by alignment to the birch unigene set. Ten potential miR156 and miR172 precursor sequences were identified, and were tested for the ability to form the required stem-loop structure. Based on precursor sequences, primers were designed for real time PCR analysis of precursor miRNA expression. Expression patterns of two miR156 family precursors (miR156_511 and miR156_789) and one miR172 precursor (miR172_1931) and two target genes (BpSPL1 and BpAP2) had the best correlation with juvenility/maturity in the analysed in vitro propagated silver birch samples. Expression patterns of these miRNA precursors and target genes were also investigated in samples cultured under different in vitro conditions. This study provides an initial survey of molecular markers for assessment of phase change in birch in vitro micropropagation.

The induction of defense responses in Pinus sylvestris L. seedlings by methyl jasmonate (MeJA) was investigated in three experiments. Two different MeJA application methods were tested, and induction of defense responses was assayed by seedling inoculation with Heterobasidion annosum (Fr.) Bref. and Lophodermium seditiosum Minter, Staley and Millar. In the first experiment, five-year-old P. sylvestris ramets of one clone were directly treated with MeJA, followed by inoculation with H. annosum. In the second experiment, open-pollinated Scots pine seedlings were treated with MeJA by direct spraying and vaporization, and inoculation with H. annosum was done using a slightly modified protocol. In the third experiment, open-pollinated Scots pine seedlings were treated with MeJA by vaporization and inoculated with L. seditiosum. Direct application of MeJA induced seedling mortality, and in some cases, decreased resistance to inoculation with H. annosum. Application of MeJA by vaporization was less stressful for seedlings, and resulted in increased resistance to both H. annosum and L. seditiosum. In addition, an unforeseen Neodiprion sertifer (Geoffroy) and Hylobius abietis L. infestation provided anecdotal evidence of the efficacy of MeJA in inducing resistance to insect pests as well. Further studies are required on the induction of resistance to additional diseases and pests. Induced resistance could be used as a possible protective mechanism for Scots pine seedlings prior to planting during reforestation of stands to increase vitality and survival.