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Integrated Analysis of Small RNA, Transcriptome and Degradome Sequencing Provides New Insights Into Floral Development and Abscission in Yellow Lupine ( Lupinus Luteus L.)

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Integrated Analysis of Small RNA, Transcriptome and Degradome Sequencing Provides New Insights Into Floral Development and Abscission in Yellow Lupine ( Lupinus Luteus L.)

Paulina Glazińska et al. Int J Mol Sci.

Abstract

The floral development in an important legume crop yellow lupine (Lupinus luteus L., Taper cv.) is often affected by the abscission of flowers leading to significant economic losses. Small non-coding RNAs (sncRNAs), which have a proven effect on almost all developmental processes in other plants, might be of key players in a complex net of molecular interactions regulating flower development and abscission. This study represents the first comprehensive sncRNA identification and analysis of small RNA, transcriptome and degradome sequencing data in lupine flowers to elucidate their role in the regulation of lupine generative development. As shedding in lupine primarily concerns flowers formed at the upper part of the inflorescence, we analyzed samples from extreme parts of raceme separately and conducted an additional analysis of pedicels from abscising and non-abscising flowers where abscission zone forms. A total of 394 known and 28 novel miRNAs and 316 phased siRNAs were identified. In flowers at different stages of development 59 miRNAs displayed differential expression (DE) and 46 DE miRNAs were found while comparing the upper and lower flowers. Identified tasiR-ARFs were DE in developing flowers and were strongly expressed in flower pedicels. The DEmiR-targeted genes were preferentially enriched in the functional categories related to carbohydrate metabolism and plant hormone transduction pathways. This study not only contributes to the current understanding of how lupine flowers develop or undergo abscission but also holds potential for research aimed at crop improvement.

Keywords: RNA-seq; abscission; degradome; flower development; miRNA; phased siRNA; yellow lupine.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Development of Lupinus luteus flowers from the upper and lower part of the raceme. An isolated pistil from a given developmental stage is shown under each flower. LF—lower flower, UF—upper flower. Bar 5 mm.
Figure 2
Figure 2
Nucleotide length distribution of small RNAs from all ten libraries: Y-axis represents the percentage frequency of the sRNA sequences identified in this study, the X-axis represents sRNA length.
Figure 3
Figure 3
Identification and evolutionary conservation of known miRNA families in Lupinus luteus. (A) The distribution of known miRNA family sizes in L. luteus. (B) Comparison of known miRNA families in L. luteus and their 52 homologs in Eudicotyledons species present in miRBase (upper panel) and 9 Fabaceae species (lower panel). Known miRNA families of L. luteus identified from small RNA-seq are listed in the top row. The colors represent relative miRNA families classified into different groups with similar conservation. Blue, yellow, magenta, green and orange represent relative miRNA families with homologs across more than 20, 10–19, 5–9, 2–4 species and in 1 species, respectively.
Figure 4
Figure 4
Diversity of miRNA expression (reads per million, RPM) in yellow lupine flowers. Complete data concerning differential miRNA expression in the experiment described herein, divided into six groups, depending on their expression maxima listed in order of appearance from left to right, and top to bottom: over 10,000 RPM, 2000–10,000 RPM, 100–2000 RPM, up to 1 RPM, 1–10 RPM, 10–100 RPM.2.6. Identification of phased siRNA in Yellow Lupine.
Figure 5
Figure 5
Differential miRNA expression in lupine flowers and flower pedicels. (A) Heatmaps of z-scaled miRNA expression (scaled RPM) and log2 fold changes for either position of the flower on the raceme (Log2FC position) or identified between consecutive stages of flower development (Log2FC development). Grey indicates insignificant changes. (B) Heatmaps of miRNA expression, log2 fold changes (Log2FC) and p-values for flower pedicels with an active or inactive abscission zone. The miRNA names are shown on the right vertical axis. Red and green represent the up-regulated and down-regulated miRNAs, respectively.
Figure 6
Figure 6
Diagrams showing distribution of yellow lupine miRNAs in (A) upper flowers, (B) lower flowers, (C) both upper and lower flowers at particular stages of their development, (D) pedicels of abscising flowers or flowers maintained on the plant.
Figure 7
Figure 7
SL RT-qPCR validation of selected sRNAs in L. lupinus. Grey indicates the miRNA expression levels determined by qPCR. Black indicates the miRNA expression levels determined by deep sequencing. Vertical bars indicate standard errors.
Figure 8
Figure 8
Examples of post-transcriptional regulation of miRNA targets in yellow lupine. (A) Ll-miRn1 and SGS3 mRNA, (B) Ll-miR102 and 2-methylacyl-CoA dehydrogenese mRNA, (C) Ll-miR392 and ARF18 mRNA, (D) Ll-miR415 and SCL6 mRNA. The T-plots show the distribution of the degradome tags along the full length of the target gene sequence. The cleavage site of each transcript is indicated by a red dot. Comparison of the expression levels of miRNAs and their targets in flowers from upper and lower whorls of yellow lupine racemes, and flower pedicels, as determined by deep sequencing. In miRNA-mRNA alignments, the red arrows indicate the cleavage site of the target gene transcript.
Figure 8
Figure 8
Examples of post-transcriptional regulation of miRNA targets in yellow lupine. (A) Ll-miRn1 and SGS3 mRNA, (B) Ll-miR102 and 2-methylacyl-CoA dehydrogenese mRNA, (C) Ll-miR392 and ARF18 mRNA, (D) Ll-miR415 and SCL6 mRNA. The T-plots show the distribution of the degradome tags along the full length of the target gene sequence. The cleavage site of each transcript is indicated by a red dot. Comparison of the expression levels of miRNAs and their targets in flowers from upper and lower whorls of yellow lupine racemes, and flower pedicels, as determined by deep sequencing. In miRNA-mRNA alignments, the red arrows indicate the cleavage site of the target gene transcript.
Figure 9
Figure 9
Visualization of GO categories annotated to predicted target genes of known and novel miRNAs in yellow lupine.
Figure 10
Figure 10
KEGG pathways related to plant hormone signal transduction targeted by known and novel miRNAs. Orange indicates DE miRNAs.
Figure 11
Figure 11
MiRNAs and siRNAs participating in yellow lupine flower development and abscission. Scheme based on the results of the current analysis. Arrows pointing upwards and downwards represent sRNAs that are up or downregulated in the transition between two developmental stages, respectively. The plus sign marks significantly expressed sRNAs. Colored circles represent targets found in the degradome, colored squares represent targets found using psRNATarget, as listed below. Multiple markers indicate that the sequence has multiple targets. Abbreviated gene names were acquired from UniProt database, where full target names can be found. Pictures from left to right are as follows: 4-whorl inflorescence of yellow lupine, flower cross-sections and isolated pistils for each stage of development, cross-sections of abscissing and non-abscissing flower pedicels stained with phloroglucinol-HCL solution.

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