Benzoxazinoids (BXs) are specialized metabolites with protective properties that are synthesized predominantly by Poaceae species, including rye (Secale cereale). Among factors known to influence BXs production, prolonged low temperature has not been studied previously. In this study, the influence of cultivation at 4 °C, which is essential for vernalization, on the concentration of BXs (HBOA, DIBOA, GDIBOA, DIMBOA, GDIMBOA, and MBOA) and the expression level of genes involved in the BX biosynthesis pathway (ScBx1–ScBx5 and ScIgl) in three rye inbred lines was investigated. After cultivation for seven weeks at 4 °C, the expression level of all analyzed genes and BX concentrations had decreased compared with those at the initiation of treatment (21 days after germination) in control and cold-treated plants. At this time point, the decrease in BX concentrations and gene expression was lower in cold-treated plants than in untreated plants. In contrast, at 77 days after germination, the gene expression levels and BX concentrations in untreated plants had generally increased. Investigation of the vernalization impact on rye BXs accumulation, as well as on Bx gene expression, may aid with determination of the most suitable winter lines and cultivars of rye for cultivation and breeding purposes.

Among the natural mechanisms used for wheat hybrid breeding, the most desirable is the system combining the cytoplasmic male sterility (cms) of the female parent with the fertility-restoring genes (Rf) of the male parent. The objective of this study was to identify Rf candidate genes in the wheat genome on the basis of transcriptome sequencing (RNA-seq) and paralog analysis data. Total RNA was isolated from the anthers of two fertility-restorer (Primépi and Patras) and two non-restorer (Astoria and Grana) varieties at the tetrad and late uninucleate microspore stages. Of 36,912 differentially expressed genes (DEGs), 21 encoding domains in known fertility-restoring proteins were selected. To enrich the pool of Rf candidates, 52 paralogs (PAGs) of the 21 selected DEGs were included in the analyses. The expression profiles of most of the DEGs and PAGs determined bioinformatically were as expected (i.e., they were overexpressed in at least one fertility-restorer variety). However, these results were only partially consistent with the quantitative real-time PCR data. The DEG and PAG promoters included cis-regulatory elements common among PPR-encoding genes. On the basis of the obtained results, we designated seven genes as Rf candidate genes, six of which were identified for the first time in this study.

Cyst nematodes are important herbivorous pests in agriculture that obtain nutrients through specialized root structures termed syncytia. Syncytium initiation, development, and functioning are a research focus because syncytia are the primary interface for molecular interactions between the host plant and parasite. The small size and complex development (over approximately two weeks) of syncytia hinder precise analyses, therefore most studies have analyzed the transcriptome of infested whole-root systems or syncytia-containing root segments. Here, we describe an effective procedure to microdissect syncytia induced by Globodera rostochiensis from tomato roots and to analyze the syncytial proteome using mass spectrometry. As little as 15 mm² of 10-µm-thick sections dissected from 30 syncytia enabled the identification of 100–200 proteins in each sample, indicating that mass-spectrometric methods currently in use achieved acceptable sensitivity for proteome profiling of microscopic samples of plant tissues (approximately 100 µg). Among the identified proteins, 48 were specifically detected in syncytia and 7 in uninfected roots. The occurrence of approximately 50% of these proteins in syncytia was not correlated with transcript abundance estimated by quantitative reverse-transcription PCR analysis. The functional categories of these proteins confirmed that protein turnover, stress responses, and intracellular trafficking are important components of the proteome dynamics of developing syncytia.

Leaf rust (LR) caused by Puccinia recondita f. sp. secalis (Prs) is a highly destructive disease in rye. However, the genetic mechanisms underlying the rye immune response to this disease remain relatively uncharacterised. In this study, we analysed the expression of four genes in 12 rye inbred lines inoculated with Prs at 20 and 36 h post-treatment (hpt): DXS (1-deoxy-D-xylulose 5-phosphate synthase), Glu (β-1,3-glucanase), GT (UDP-glycosyltransferase) and PR-1 (pathogenesis-related protein 1). The RT-qPCR analysis revealed the upregulated expression of the four genes in response to Prs in all inbred lines and at both time-points. The gene expression data were supported by microscopic and macroscopic examinations, which revealed that eight lines were susceptible to LR and four lines were highly resistant to LR. A relationship between the infection profiles and the expression of the analysed genes was observed: in the resistant lines, the expression level fold changes were usually higher at 20 hpt than at 36 hpt, while the opposite trend was observed in the susceptible lines. The study results indicate that DXS, Glu, GT and PR-1 may encode proteins crucial for the rye defence response to the LR pathogen.

Two genes, Bx1 and Igl, both encoding indole-3-glycerol phosphate lyase (IGL), are believed to control the conversion of indole-3-glycerol phosphate (IGP) to indole. The first of these has generally been supposed to be regulated developmentally, being expressed at early stages of plant development with the indole being used in the benzoxazinoid (BX) biosynthesis pathway. In contrast, it has been proposed that the second one is regulated by stresses and that the associated free indole is secreted as a volatile. However, our previous results contradicted this. In the present study, we show that the ScIgl gene takes over the role of ScBx1 at later developmental stages, between the 42nd and 70th days after germination. In the majority of plants with silenced ScBx1 expression, ScIgl was either expressed at a significantly higher level than ScBx1 or it was the only gene with detectable expression. Therefore, we postulate that the synthesis of indole used in BX biosynthesis in rye is controlled by both ScBx1 and ScIgl, which are both regulated developmentally and by stresses. In silico and in vivo analyses of the promoter sequences further confirmed our hypothesis that the roles and modes of regulation of the ScBx1 and ScIgl genes are similar.

According to current opinion, the first step of benzoxazinoids (BXs) synthesis, that is, the conversion of indole-3-glycerol phosphate to indole, occurs exclusively in the photosynthesising parts of plants. However, the results of our previous work and some other studies suggest that this process may also occur in the roots. In this study, we provide evidence that the first step of BXs synthesis does indeed occur in the roots of rye seedlings. We detected ScBx1 transcripts, BX1 enzyme, and six BXs (2-hydroxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-1,4-benzoxazin-3-one, (2R)-2-O-β-d-glucopyranosyl-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one glucoside, 2,4-dihydroxy- 7-methoxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside, and 6-methoxy-2-benzoxazolinone) in the roots developed from seeds deprived of the coleoptile at 2 days after sowing (i.e., roots without contact with aerial parts). In roots regenerated in vitro, both ScBx1 transcripts and BX1 enzyme were detected at a low but still measurable levels. Thus, BXs are able to be synthesised in both the roots and above-ground parts of rye plants.