Genome-wide identification and expression analysis of late embryogenesis abundant protein-encoding genes in rye (Secale cereale L.)

Abstract

Late embryogenesis abundant (LEA) proteins are members of a large and highly diverse family that play critical roles in protecting cells from abiotic stresses and maintaining plant growth and development. However, the identification and biological function of genes of Secale cereale LEA (ScLEA) have been rarely reported. In this study, we identified 112 ScLEA genes, which can be divided into eight groups and are evenly distributed on all rye chromosomes. Structure analysis revealed that members of the same group tend to be highly conserved. We identified 12 pairs of tandem duplication genes and 19 pairs of segmental duplication genes, which may be an expansion way of LEA gene family. Expression profiling analysis revealed obvious temporal and spatial specificity of ScLEA gene expression, with the highest expression levels observed in grains. According to the qRT-PCR analysis, selected ScLEA genes were regulated by various abiotic stresses, especially PEG treatment, decreased temperature, and blue light. Taken together, our results provide a reference for further functional analysis and potential utilization of the ScLEA genes in improving stress tolerance of crops.

Fig 1. Phylogenetic analysis of ScLEA proteins.

Multiple sequence alignment of LEA proteins was performed using MAFFT. The phylogenetic tree was constructed using MEGA X software.

Fig 2. Schematic representation of exon-intron structures of ScLEA genes and conserved domains of ScLEA proteins.

(a) The exon-intron structures were examined using the GSDS online tool. The yellow boxes and black lines represent exons and introns, respectively. (b) The ScLEA protein domains were predicted using the MEME online tool.

Fig 3. Distribution of ScLEA genes on rye chromosomes visualized using Mapchart software.

Fig 4. Schematic representation of the chromosomal distribution and interchromosomal relationships of ScLEA genes derived from segmental duplication.

The gray lines indicate all syntenic gene pairs in the rye genome, and the dark turquoise lines indicate segmental duplicated ScLEA gene pairs. The number of each chromosome is indicated.

Fig 5. RNA-seq expression profiles of 112 ScLEA genes in different tissues of rye (Weining).

The heatmap was constructed using TBtools. The color scale on the right represents relative expression levels: Red represents high level and blue represents low level.

Fig 6. The relative expression levels of six ScLEA genes under different abiotic stresses were analyzed by qRT-PCR.

Ten-day-old seedling leaves were sampled after 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h under 20% PEG6000, 100 μM ABA, 200 mM NaCl, or 100 mM mannitol. The values represent mean ± SEM of three replicates. The significant differences between data were calculated using Student’s t-test, and indicated with asterisks (*P <0.05, ** P<0.01, *** P<0.001).

Fig 7. The relative expression levels of 12 ScLEA genes under cold stress were analyzed by qRT-PCR.

Ten-day-old seedling leaves were sampled after 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h at 0°C or 4°C. The values represent mean ± SEM of three replicates. The significant differences between data were calculated using two-way ANOVA, and indicated with asterisks (** P<0.01, *** P<0.001).

Fig 8. The relative expression levels of nine ScLEA genes under different light conditions were analyzed by qRT-PCR.

Seedlings were grown in the dark for seven days and subsequently transferred to FR light (5 μmol·m^-2·s^-1), R light (17.56 μmol·m^-2·s^-1), B light (13 μmol·m^-2·s^-1), or W light (85 μmol·m^-2·s^-1) for 4 h. The values represent mean ± SEM of three replicates. The significant differences between data were calculated using Student’s t-test, and indicated with asterisks (*P <0.05, ** P<0.01, *** P<0.001).