<i>HbCBF1</i> overexpression activated the expression of <i>COR15a</i> and <i>RD29a</i> genes in <i>Arabidopsis</i>.

<p><b>(A)</b><i>HbCBF1</i>, (B) <i>AtCOR15a</i> and (C) <i>AtRD29a</i> expression in <i>col-0</i>, OE-3, OE-6 and OE-13 plants under control condition (control), 5-day cold-acclimated (cold) or 10-days drought stress (drought). Total RNA was extracted from 2 weeks seedlings and real time PCR was performed with gene specific primers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137634#pone.0137634.s004" target="_blank">S1 Table</a>. The transcripts level was normalized to that of ACTIN 7 gene. Two microgram total RNA was used for sample and the data were presented as mean ± standard error of mean of three biological replicates. Significance was determined by Student’s <i>t</i> test at the probability levels of <i>P</i> < 0.05.</p

Functional Characterization of <i>Hevea brasiliensis CRT/DRE Binding Factor 1</i> Gene Revealed Regulation Potential in the CBF Pathway of Tropical Perennial Tree

<div><p>Rubber trees (<i>Hevea brasiliensis</i>) are susceptible to low temperature and therefore are only planted in the tropical regions. In the past few decades, although rubber trees have been successfully planted in the northern margin of tropical area in China, they suffered from cold injury during the winter. To understand the physiological response under cold stress, we isolated a C-repeat binding factor 1 (<i>CBF1</i>) gene from the rubber tree. This gene (<i>HbCBF1)</i> was found to respond to cold stress but not drought or ABA stress. The corresponding HbCBF1 protein showed CRT/DRE binding activity in gel shift experiment. To further characterize its molecular function, the <i>HbCBF1</i> gene was overexpressed in <i>Arabidopsis</i>. The <i>HbCBF1</i> over expression (OE) line showed enhanced cold resistance and relatively slow dehydration, and the expression of <i>Arabidopsis</i> CBF pathway downstream target genes, e.g. <i>AtCOR15a</i> and <i>AtRD29a</i>, were significantly activated under non-acclimation condition. These data suggest <i>HbCBF1</i> gene is a functional member of the CBF gene family, and may play important regulation function in rubber tree.</p></div

<i>HbCBF1</i> overexpression in <i>Arabidopsis</i>.

<p>(A) Southern blotting of <i>HbCBF1</i> in transgenic plants. (B) <i>HbCBF1</i> expression in OE-3, OE-6 and OE-13 lines. The gene expression was detected by Northern blotting analysis using the <i>HbCBF1</i> probe. (C) Thirty days old seedlings of <i>col-0</i>, OE-3, OE-6 and OE-13 lines showed decreased plant height.</p

Functional Characterization of <i>Hevea brasiliensis CRT/DRE Binding Factor 1 - Fig 1 </i> Gene Revealed Regulation Potential in the CBF Pathway of Tropical Perennial Tree

<p>(A) Full length cDNA sequence of rubber tree <i>C-repeat binding factor 1</i> (<i>HbCBF1</i>) gene and deduced encoding peptides. (B) Conservative domains distribution in HbCBF1 protein. NLS, nuclear localization signal. (C) Phylogenetic tree showing similarity of the deduced <i>Hevea brasiliensis</i> CBF1 protein sequence with those described in other plants. Sequences of each species were downloaded from Genbank by the following accession numbers: <i>Capsicum annuum</i> CBF1, AAZ22480.1; <i>Solanum lycopersicum</i> CBF1, AAS77820.1; <i>Camellia sinensis</i> CBF1, AFN93974.1; <i>Malus domestica</i> CBF1, AAZ20446.1; <i>Manihot esculenta</i> CBF1, AFB83707.1; <i>Raphanus sativus</i> CBF1, ACX48435.1; <i>Arabidopsis thaliana</i> CBF1, AEE85066.1; <i>Populus tomentosa</i> CBF1, ABC79626.1; <i>Vitis vinifera</i> CBF1, AEM37861.1. The phylogenetic tree was constructed using a Mega 6.06 software.</p

Image_1_Genome-Wide Identification and Analysis of Small Nucleolar RNAs and Their Roles in Regulating Latex Regeneration in the Rubber Tree (Hevea brasiliensis).TIF

Small nucleolar RNAs (snoRNAs) are a class of conserved nuclear RNAs that play important roles in the modification of ribosomal RNAs (rRNAs) in plants. In rubber trees, rRNAs are run off with latex flow during tapping and need to be regenerated for maintaining the functions of the laticifer cells. SnoRNAs are expected to play essential roles in the regeneration of rRNAs. However, snoRNAs in the rubber tree have not been sufficiently characterized thus far. In this study, we performed nuclear RNA sequencing (RNA-seq) to identify snoRNAs globally and investigate their roles in latex regeneration. We identified a total of 3,626 snoRNAs by computational prediction with nuclear RNA-seq data. Among these snoRNAs, 50 were highly expressed in latex; furthermore, the results of reverse transcription polymerase chain reaction (RT-PCR) showed the abundant expression of 31 of these snoRNAs in latex. The correlation between snoRNA expression and adjusted total solid content (TSC/C) identified 13 positively yield-correlated snoRNAs. To improve the understanding of latex regeneration in rubber trees, we developed a novel insulated tapping system (ITS), which only measures the latex regenerated in specific laticifers. Using this system, a laticifer-abundant snoRNA, HbsnoR28, was found to be highly correlated with latex regeneration. To the best of our knowledge, this is the first report to globally identify snoRNAs that might be involved in latex regeneration regulation and provide new clues for unraveling the mechanisms underlying the regulation of latex regeneration.</p

Characterization of <i>HbCBF1</i> gene.

<p>(A) Southern blotting analysis of <i>HbCBF1</i> gene in <i>Hevea brasiliensis</i> genome. (B) <i>HbCBF1</i> expression in response to cold, drought stress or ABA treatment. Twenty microgram total RNA from the leaves of the seedlings treated with cold, drought or ABA for the indicated time were used for Northern blotting analysis. <i>HbCBF1</i> full length cDNA was used as probe. EtBr-stained rRNA was used as an internal standard to monitor equal loading of total RNA.</p

Table_1_Genome-Wide Identification and Analysis of Small Nucleolar RNAs and Their Roles in Regulating Latex Regeneration in the Rubber Tree (Hevea brasiliensis).XLSX

Small nucleolar RNAs (snoRNAs) are a class of conserved nuclear RNAs that play important roles in the modification of ribosomal RNAs (rRNAs) in plants. In rubber trees, rRNAs are run off with latex flow during tapping and need to be regenerated for maintaining the functions of the laticifer cells. SnoRNAs are expected to play essential roles in the regeneration of rRNAs. However, snoRNAs in the rubber tree have not been sufficiently characterized thus far. In this study, we performed nuclear RNA sequencing (RNA-seq) to identify snoRNAs globally and investigate their roles in latex regeneration. We identified a total of 3,626 snoRNAs by computational prediction with nuclear RNA-seq data. Among these snoRNAs, 50 were highly expressed in latex; furthermore, the results of reverse transcription polymerase chain reaction (RT-PCR) showed the abundant expression of 31 of these snoRNAs in latex. The correlation between snoRNA expression and adjusted total solid content (TSC/C) identified 13 positively yield-correlated snoRNAs. To improve the understanding of latex regeneration in rubber trees, we developed a novel insulated tapping system (ITS), which only measures the latex regenerated in specific laticifers. Using this system, a laticifer-abundant snoRNA, HbsnoR28, was found to be highly correlated with latex regeneration. To the best of our knowledge, this is the first report to globally identify snoRNAs that might be involved in latex regeneration regulation and provide new clues for unraveling the mechanisms underlying the regulation of latex regeneration.</p

Gel mobility shift assay of recombinant HbCBF1 protein indicated CRT/DRE binding activity.

<p><b>(A)</b> Probes sequences used in this analysis. Bolded nucleotides are CRT/DRE motif, while underlined nucleotides are the mutation sites. <b>(B)</b> HbCBF1 bound with CRT/DRE elements specifically <i>in vitro</i>. Four microgram total proteins of GST (lane 1 from the left) or GST::HbCBF1 (lane 2–9 from the left) were used for the binding with labeled hot COR15a probe. One hundred and twenty-five folds molar unlabelled cold competitor probes were added as indicated (labe 3–9 from the left) in each reactions. The retarded binding bands and free non-binding probe were indicated as "B" and "F" respectively.</p

<i>HbCBF1</i> overexpression conferred cold resistance and relatively slow dehydration in <i>Arabidopsis</i>.

<p>(A) The electrolyte leakage rates of <i>col-0</i>, OE-3, OE-6 and OE-13 lines under nonacclimated (warm) or 5-days cold—acclimated conditions (cold). Electrolyte leakage was measured using leaves from two-week-old seedlings frozen to -7°C at a refrigerating rate of 1°C per h from -1°C. The star symbol (*) indicates significant difference between each individual OE line and wild type plants (WT). (B) Representative seedlings of <i>col-0</i>, OE-3, OE-6 and OE-13 lines recovered from 18 hours frozen at -6°C. The survival rates of each line were listed. (C) The dehydration rates of the detached leaves from <i>col-0</i>, OE-3, OE-6 and OE-13 seedlings. The star (*) symbol indicates significant difference between WT and each individual OE line, while plus symbols (+) shows significant difference between WT and individual OE-6 and OE-13 line. Data in A and C were presented as mean ± standard error of mean of five biological replicates. Significance was determined by Student’s <i>t</i> test at the probability levels of <i>P</i> < 0.05. Comparisons were made between the wild type plant and each individual overexpressing line. For B, totally thirty to fifty plants were tested for each line and about ten seedlings were grown in each pot.</p

Image_2_Genome-Wide Identification and Analysis of Small Nucleolar RNAs and Their Roles in Regulating Latex Regeneration in the Rubber Tree (Hevea brasiliensis).TIF

Small nucleolar RNAs (snoRNAs) are a class of conserved nuclear RNAs that play important roles in the modification of ribosomal RNAs (rRNAs) in plants. In rubber trees, rRNAs are run off with latex flow during tapping and need to be regenerated for maintaining the functions of the laticifer cells. SnoRNAs are expected to play essential roles in the regeneration of rRNAs. However, snoRNAs in the rubber tree have not been sufficiently characterized thus far. In this study, we performed nuclear RNA sequencing (RNA-seq) to identify snoRNAs globally and investigate their roles in latex regeneration. We identified a total of 3,626 snoRNAs by computational prediction with nuclear RNA-seq data. Among these snoRNAs, 50 were highly expressed in latex; furthermore, the results of reverse transcription polymerase chain reaction (RT-PCR) showed the abundant expression of 31 of these snoRNAs in latex. The correlation between snoRNA expression and adjusted total solid content (TSC/C) identified 13 positively yield-correlated snoRNAs. To improve the understanding of latex regeneration in rubber trees, we developed a novel insulated tapping system (ITS), which only measures the latex regenerated in specific laticifers. Using this system, a laticifer-abundant snoRNA, HbsnoR28, was found to be highly correlated with latex regeneration. To the best of our knowledge, this is the first report to globally identify snoRNAs that might be involved in latex regeneration regulation and provide new clues for unraveling the mechanisms underlying the regulation of latex regeneration.</p