Comparison between qRT-PCR and the deep sequencing in No.116 root exposed to short-term low nitrogen stress.

<p>The y-axis indicate the relative expression levels of twelve selected miRNA in qRT-PCR and in Solexa sequencing analysis. The x-axis indicates twelve selected miRNAs, which are respectively as follows: 1. gma-miR1511-3p; 2. gma-miR390a/c-3p; 3. gma-miR396b/c/d/f/g-5p; 4. gma-miR396b/d/g-3p; 5. gma-miR398a/b-3p; 6. gma-miR4348-5p; 7. gma-miR4413a-5p; 8. gma-miR482a-3p; 9. gma-miR156p-5p; 10. gma-miR171n-3p; 11. gma-miR171o-5p; 12. gma-miR172l-3p. gma-miR1520d-3p and gma-miR156b-5p was chosen as endogenous reference genes.</p

Elucidation of miRNAs-Mediated Responses to Low Nitrogen Stress by Deep Sequencing of Two Soybean Genotypes

<div><p>Nitrogen (N) is a major limiting factor in crop production, and plant adaptive responses to low N are involved in many post-transcriptional regulation. Recent studies indicate that miRNAs play important roles in adaptive responses. However, miRNAs in soybean adaptive responses to N limitation have been not reported. We constructed sixteen libraries to identify low N-responsive miRNAs on a genome-wide scale using samples from 2 different genotypes (low N sensitive and low N tolerant) subjected to various periods of low nitrogen stress. Using high-throughput sequencing technology (Illumina-Solexa), we identified 362 known miRNAs variants belonging to 158 families and 90 new miRNAs belonging to 55 families. Among these known miRNAs variants, almost 50% were not different from annotated miRNAs in miRBase. Analyses of their expression patterns showed 150 known miRNAs variants as well as 2 novel miRNAs with differential expressions. These differentially expressed miRNAs between the two soybean genotypes were compared and classified into three groups based on their expression patterns. Predicted targets of these miRNAs were involved in various metabolic and regulatory pathways such as protein degradation, carbohydrate metabolism, hormone signaling pathway, and cellular transport. These findings suggest that miRNAs play important roles in soybean response to low N and contribute to the understanding of the genetic basis of differences in adaptive responses to N limitation between the two soybean genotypes. Our study provides basis for expounding the complex gene regulatory network of these miRNAs.</p></div

Specific and common response miRNAs in soybean shoots from differential compared libraries.

<p>116SS, 116-shoot short-term treatment; 116SL, 116-shoot long-term treatment; 84SS, 84-70-shoot short-term treatment; 84SL, 84-70-shoot long-term treatment; 116SSC, 116-shoot short-term control; 116SLC, 116-shoot long-term control; 84SSC, 84-70-shoot short-term control; 84SLC, 84-70-shoot long-term control.</p

Specific and common response miRNAs in soybean roots from differential compared libraries.

<p>116RS, 116-root short-term treatment; 116RL, 116-root long-term treatment; 84RS, 84-70-root short-term treatment; 84RL, 84-70-root long-term treatment; 116-root short-term control; 116RLC, 116-root long-term control; 84RSC, 84-70-root short-term control; 84RLC, 84-70-root long-term control.</p

Numbers of up-regulated and downregulated miRNA were summarized in compared libraries.

<p>116RS, 116-root short-term treatment; 116RL, 116-root long-term treatment; 84RS, 84-70-root short-term treatment; 84RL, 84-70-root long-term treatment; 116SS, 116-shoot short-term treatment; 116SL, 116-shoot long-term treatment; 84SS, 84-70-shoot short-term treatment; 84SL, 84-70-shoot long-term treatment; 116RSC, 116-root short-term control; 116RLC, 116-root long-term control; 84RSC, 84-70-root short-term control; 84RLC, 84-70-root long-term control;116SSC, 116-shoot short-term control; 116SLC, 116-shoot long-term control; 84SSC, 84-70-shoot short-term control; 84SLC, g00484-70-shoot long-term control.</p

Statistics of sequenced reads from all libraries.

<p>Clean reads are those remaining after low-quality reads have been removed from total raw reads. Unique reads are different types of clean reads. The number of the total clean reads and unique reads from the sixteen libraries that matched to the genome sequences are also listed.</p><p>Note: 116RS, 116-root short-term treatment; 116RL, 116-root long-term treatment; 84RS, 84-70-root short-term treatment; 84RL, 84-70-root long-term treatment; 116SS, 116-shoot short-term treatment; 116SL, 116-shoot long-term treatment; 84SS, 84-70-shoot short-term treatment; 84SL, 84-70-shoot long-term treatment; 116RSC, 116-root short-term control; 116RLC, 116-root long-term control; 84RSC, 84-70-root short-term control; 84RLC, 84-70-root long-term control;116SSC, 116-shoot short-term control; 116SLC, 116-shoot long-term control; 84SSC, 84-70-shoot short-term control; 84SLC, 84-70-shoot long-term control.</p

Some new miRNAs located in the introns or exons of protein-coding genes.

<p>Some new miRNAs located in the introns or exons of protein-coding genes.</p

Phenotype comparisons of <i>SlGMP2/3</i>-KD and wild-type plants.

<p>(A) Altered cotyledon morphology of <i>SlGMP2/3</i>-KD vs wild-type plants. KD7 and KD17 cotyledons developed lesions (left) at three weeks post germination and accelerated senescence (right) compared with the wild type. (B) Seedlings of wild-type (left) and KD7 (right) plants. The bottom leaves of KD7 plant started to wilt. (C) Plant morphology of the three-month-old wild-type (left) and KD7 (right) plants. Middle and bottom leaves of KD7 plants became dry wilted. (D) Lesion formation on KD7 and KD17 leaves. The leaf lesion areas on the two-month-old plants of KD7 and KD17 (upper panel) are consistent with the areas of dead cells revealed through trypan blue staining (bottom panel). (E) H₂O₂ accumulation in the leaves of KD7 and KD17. H₂O₂ accumulation was revealed via DAB staining in leaves without (upper panel) and with (lower panel) visible lesions from the two-month-old plants of KD7 and KD17. (F) Tomato fruits harvested from four-month-old wild-type and transgenic plants.</p

Relative expression analysis of four members of <i>SlGMP</i> gene family in various tissues of tomato variety Ailsa Craig.

<p>R: root, S: stem, L: leaf, Fl: flower, GF: green fruit, BF: breaker fruit, and RF: red fruit. Data obtained by real-time RT-PCR were normalized against <i>Actin</i>.</p

Expression analysis of four <i>SlGMPs</i> and other AsA biosynthesis-related genes in <i>SlGMP3</i> transgenic plants.

<p>(A) RT-PCR analysis of <i>SlGMP3</i> expression in the young leaves of two <i>SlGMP3</i>-OX lines (upper panel) and 17 <i>SlGMP3</i> RNAi lines (bottom panel). The PCR circle numbers are indicated on the right. (B) Relative expression analysis of four members of <i>SlGMP</i> gene family in the young leaves of lines OX6 and OX19 (left) and lines KD7 and KD17 (right) via real-time RT-PCR. (C) Relative expression analysis of AsA biosynthesis-related genes in the young leaves of lines OX6 and KD17 via real-time RT-PCR. Data were obtained by normalizing against <i>Actin</i> and shown as a percentage of wild-type plants.</p