Bovine serum albumin in saliva mediates grazing response in Leymus chinensis revealed by RNA sequencing
- Xin Huang†1, 2, 3,
- Xianjun Peng†1, 2,
- Lexin Zhang1, 4,
- Shuangyan Chen1Email author,
- Liqin Cheng1Email author and
- Gongshe Liu1Email author
© Huang et al.; licensee BioMed Central. 2014
Received: 25 March 2014
Accepted: 3 December 2014
Published: 17 December 2014
Sheepgrass (Leymus chinensis) is an important perennial forage grass across the Eurasian Steppe and is adaptable to various environmental conditions, but little is known about its molecular mechanism responding to grazing and BSA deposition. Because it has a large genome, RNA sequencing is expensive and impractical except for the next-generation sequencing (NGS) technology.
In this study, NGS technology was employed to characterize de novo the transcriptome of sheepgrass after defoliation and grazing treatments and to identify differentially expressed genes (DEGs) responding to grazing and BSA deposition. We assembled more than 47 M high-quality reads into 120,426 contigs from seven sequenced libraries. Based on the assembled transcriptome, we detected 2,002 DEGs responding to BSA deposition during grazing. Enrichment analysis of Gene ontology (GO), EuKaryotic Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed that the effects of grazing and BSA deposition involved more apoptosis and cell oxidative changes compared to defoliation. Analysis of DNA fragments, cell oxidative factors and the lengths of leaf scars after grazing provided physiological and morphological evidence that BSA deposition during grazing alters the oxidative and apoptotic status of cells.
This research greatly enriches sheepgrass transcriptome resources and grazing-stress-related genes, helping us to better understand the molecular mechanism of grazing in sheepgrass. The grazing-stress-related genes and pathways will be a valuable resource for further gene-phenotype studies.
KeywordsLeymus chinensis Grazing BSA deposition RNA-seq Apoptosis Cell oxidative status
Herbivore feeding is a complex process that includes wounding, defoliation, and BSA deposition . Often, the leaves of a plant are completely or partially removed, affecting the photosynthetic activity, secondary metabolism, and carbohydrate relocation of plants [2–5]. Many reports have focused on stress-induced gene expression, photosynthetic capacity, root growth, and nutrient uptake after wounding and defoliation [6–9]. Resistance to herbivores depends largely upon aboveground portion of plants and on leaf-to-leaf wound signaling, which may involve electrical signaling . The propagation of electrical activity leads to the expression of defense genes not only in wounded leaves but also throughout aboveground portion of plants.
Animal saliva deposited on plants (especially on leaves) is another important factor affecting plant recovery after grazing. In 1960, Vittora and Rendina first proposed that interactions between grazers and plants involve the deposition of herbivore saliva during grazing. This hypothesis has since been tested [11–14]. To date, many studies of large-herbivore BSA deposition have focused on macroscopic changes in plants, such as biomass accumulation, tiller, and increased bud initiation after grazing , rather than on changes in gene expression and plant physiology. On the other hand, many growth regulators in insect salivary systems have been fully researched, such as glucose oxidase, β-glucosidase [16, 17], and various growth factors in certain mammalian submaxillary glands (mainly mouse and human), including thiamine , nerve growth factor (NGF), transforming growth factor (TGF), and epidermal growth factor (EGF) , are known to have growth-regulating activity. Growth factors can intervene directly in cellular metabolism by promoting differential gene expression and are expected to be active in a variety of organisms . Injecting growth-promoting substances from grasshopper saliva into Bouteloua gracilis stimulates tiller production . Mouse and human EGF can enhance plant growth rates and promote cell division in the epicotyl . However, to date, no study has reported the effects of BSA deposition by large herbivores such as cows, sheep, and camels.
Gene-expression profiling or transcriptome analysis can provide new insights to understand the molecular mechanism of grazing responses in plants. High-throughput next-generation sequencing (NGS) technologies, such as 454 (ROCHE), Solexa (Illumina), and SOLiD (ABI), have been widely and effectively used to generate large-scale transcriptome data in many plant species [21–28], including sheepgrass (Leymus chinensis) [29, 30]. Sheepgrass is an important forage species in the genus of Leymus, with good quality, high nutrition value, and various stress resistance [31–34]. Its genomic formula was NsNsXmXm and Elymus californicus should be the maternal donor transferred from the genus Elymus to Leymus. One copy of the haploid genome of sheepgrass contains 9.65-Gb, and high-throughput NGS technologies make it possible to generate genome resources at relatively low cost. So far, sheepgrass transcriptome databases have been generated under saline-alkaline treatment  and freezing treatment  using Roche-454 massive pyrosequencing technology. These databases provide numerous DEGs for two stresses. Recently, a comparative transcriptomics analysis of the Illumina sequencing data was conducted, and the results revealed common and distinct mechanisms for sheepgrass responses to defoliation compared to mechanical wounding . Based these transcriptome databases, some grazing responsive genes were cloned and identified, such as LcSUT1 and LcDREB3[37, 38].
Here, we focus on profiling the effects of herbivore saliva on sheepgrass and distinguishing BSA deposition from defoliation in grazing. In our study, we use bovine serum albumin (BSA) instead of bovine saliva to perform the grazing simulation treatment. The components of herbivore saliva are unstable and it usually contains bacterium. BSA is an important protein in bovine saliva. Its homolog has been found in ovine saliva and probably has interactions with plants . In our study, in order to enrich sheepgrass transcriptome resource, accelerate our understanding of the genetic basis of grazing stress, we used Illumina GAIIx technology to sequence sheepgrass transcriptome after defoliation and grazing. We compared defoliation and grazing treatments, and identified the differentially expressed genes (DEGs) responding to BSA deposition and corresponding pathways involved in saliva effects. We performed further biochemical and morphological experiments to verify these results in transcriptome.
Illumina sequencing and Trinity transcriptome assembly of sheepgrass
Statistics summary of Illumina sequencing data generated for sheepgrass transcriptome
Average length (bp)5
We obtained 120,426 contigs (≥200 bp) using the Trinity assembly software. The mean contig size was 634 bp, and the contig size ranged from 201 to 28,343 bp. About one-third of the contigs were longer than 500 bp, and 20,816 contigs were longer than 1,000 bp. Additional file 2 show the quality of the assembly transcripts in more detail (see Additional file 2).
Contig assembly and gene overview
Functional annotation and descriptive profile
The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database resource for the systematic understanding of high-level gene functions in terms of biological networks, such as the cell, organism, and ecosystem, from molecular-level information (http://www.genome.jp/kegg/). The assembled transcripts were searched against the KEGG database using BLASTX with a cut-off E-value of 10−5 to identify the biological pathways related to grazing responses in sheepgrass. We obtained 6,820 matching terms, which were assigned to 275 KEGG pathways in 5 main biological processes. The major pathways were “biosynthesis of amino acids” (ko01230, 191 transcripts), “ribosome” (ko03010, 165 transcripts), “carbon metabolism” (ko01200, 163 transcripts), “purine metabolism” (ko00230, 137 transcripts), “spliceosome” (ko03040, 119 transcripts), “protein processing in endoplasmic reticulum” (ko04141, 118 transcripts), and “RNA transport” (ko03013, 114 transcripts).
Gene enrichment analysis of DEGs from grazing vs. control
GO second-level functional groups (DEG number 591; background number 9,831)
Amino acid and derivative metabolism
KOG categories (DEG number 337; background number 9,985)
Carbohydrate transport and metabolism
Lipid transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
Energy production and conversion
KEGG pathway ID (DEG number 420; background number 6,820)
02020 Two-component system
02010 ABC transporters
00281 Geraniol degradation
00640 Propanoate metabolism
00330 Arginine and proline metabolism
00500 Starch and sucrose metabolism
00930 Caprolactam degradation
00071 Fatty acid degradation
00380 Tryptophan metabolism
00250 Alanine, aspartate and glutamate metabolism
Differentially expressed genes (DEGs) in response to BSA deposition
Gene statistics of the differentially expressed genes
Type of the differentially expressed genes
Up-regulated genes (p < 0.001)
Down-regulated genes (p < 0.001)
Defoliation (D vs. C)
Grazing (G vs. C)
BSA deposition (G vs. D)
Enriched GO categories of DEGs from BSA deposition (G vs. D)
GO categories (DEG number 377; background number 9,831)
Amino acid and derivative metabolism
External encapsulating structure
Enriched KOG categories of DEGs from BSA deposition (G vs. D)
KOG categories (DEG number 185; background number 9,985)
Energy production and conversion
Lipid transport and metabolism
Amino acid transport and metabolism
Enriched pathways of DEGs from BSA deposition (G vs. D)
Pathway ID (total DEG number 307; background number 6,820)
02020 Two-component system
02010 ABC transporters
00380 Tryptophan metabolism
00281 Geraniol degradation
00780 Biotin metabolism
Cell oxidative and apoptosis related genes in DEGs
Apoptosis-promoting RNA-binding protein TIA-1/TIAR
Serine/threonine protein kinase
Molecular chaperones mortalin /PBP74 /GRP75, HSP70 superfamily
Metacaspase involved in regulation of apoptosis
Aldehyde dehydrogenase family 2 member B4
Exosome complex exonuclease rrp40
Cytosolic Ca2+-dependent cysteine protease (calpain), large subunit (EF-Hand protein superfamily)
Ferric reductase, NADH/NADPH oxidase and related proteins
Glutaredoxin and related proteins
Programmed cell death after clipping and the effect of BSA
Accumulation of oxidative-stress-related factors in grazed sheepgrass
The changes in malondialdehyde (MDA) were similar to those in hydrogen dioxide (H2O2). The MDA levels in the water-treated and OVA-treated group were approximately 3- to 4-fold higher than those in the unclipped and BSA-treated groups, and this difference was significant at p < 0.01. The MDA levels in the BSA-treated cells increased slightly compared to the controls (Figure 7).The superoxide dismutase (SOD) levels in the treated groups did not differ significantly from those of the controls (Figure 7). Thus, the grazing treatment using BSA affected the oxidative-stress-related factors in sheepgrass only slightly.
Analysis of leaf-scar lengths
Grazing is an important and frequent stress for pasture and prairie plants. Plant scientists have long studied the effects of grazing on plants as a single process. However, grazing is a complex process that involves wounding effects caused by herbivore feeding, defoliation effects due to leaf-surface loss during grazing, and the deposition of herbivore saliva onto the surface of plants. Some studies have reported that wounding can stimulate plant growth but clearly differs from grazing [17, 41–43]. Defoliation affects root development in grasses [44, 45] and alters the carbohydrate-metabolism pathway in rice (unpublished). On the other hand, scientists have examined how plants respond to BSA deposition for decades [11, 12]. Saliva has been found to stimulate plant growth, enhance tiller and increase biomass . However little is known about the molecular mechanisms of grazing responses and the genetic and functional differences among three components of grazing. To investigate the genetic profile of the grazing response in sheepgrass and to elucidate the differences in mechanism between the saliva-deposition response and the responses to other grazing components, we analyzed the transcriptomes of control, defoliated, and grazed plants using RNA-seq.
Sequence quality and annotation
Illumina RNA-seq technology had been widely used in genome-wide analyses of cotton (Gossypium hirsutum), radish (Raphanus sativus), Brassica juncea, and Brassica pekinensis[46–49]. Here, we obtained more than five million raw reads from most samples. Using the Trinity transcriptome-assembly software, a total of 120,426 assembled transcripts were obtained from seven sample libraries (untreated control, defoliation, and simulated grazing). Of these transcripts, 14,240 were annotated by BLASTX and functional-bioinformatics analyses, including the GO, KOG, and KEGG databases. No genome of sheepgrass or close relative species was available, so most of our transcripts cannot hit known proteins. In addition, a relative stringent blast parameter (E-value < 1e-5) might discard a part of known hits. We obtained 74,087 GO terms, 9,985 KOG terms and 7,240 KEGG pathway terms for all transcripts combined. The gene-transcription profiles of sheepgrass after grazing were stored in an annotated-gene catalog to provide a molecular understanding of grazing responses. The remaining un-annotated transcripts may represent a sheepgrass-specific gene pool. These results provide a solid foundation for further studies of the molecular mechanisms of grazing responses and for identifying grazing-related genes in this species.
Gene enrichment analysis and the effects of grazing
Grasslands and grass re-growth after grazing are very important for both the ecosystem and human dairy food supply on this planet. Grazing is a processing that have multiple components including at least wound, defoliation, and BSA deposition. Grazing often removes completely or partially the leaf part of plants. After grazing, plants have to transport the carbohydrate such as sucrose and other energy substance due to root sink demand [50–52]. In our enrichment analysis (Table 2), “transporter activity” and “amino acid and derivative metabolism” in GO categories, “Amino acid transport and metabolism” and “Lipid transport and metabolism” in KOG categories, and KEGG amino acid metabolism pathways were significant enrichment in grazing treatment. The results indicated that amino acid metabolism involved in plant grazing response. The amino acid metabolism pathways may contribute to protein biosynthesis and plant recovery after grazing, and the related genes are worth further study.
Differential genetic profiles in response to BSA deposition
Sheepgrass is a dominant grassland species in northeastern China and Inner Mongolia and is known for its adaptability to grazing and excellent forage quality among perennial grasses . In this study, 2,759 genes were expressed differently between the control and defoliation libraries, indicating that these genes responded to defoliation. Similarly, 3,156 genes were expressed differently between the control and grazing libraries, indicating that these genes responded to the combined effects of defoliation and BSA deposition. Furthermore, 2,002 genes were expressed differently between the defoliation and grazing libraries, indicating that these genes responded to BSA deposition. The only difference between these two treatments was the liquid deposited on leaves.
As shown in Table 3, most of the DEGs were down-regulated. In the KOG enrichment analysis (Table 5), the down-regulated DEGs were enriched in lipid transport and metabolism; energy production and conversion; and amino-acid transport and metabolism. Thus, several functionally linked metabolic pathways were down-regulated in response to grazing. This result is consistent with a proteomic analysis of rice after ovine BSA deposition , in which the authors found that most photosynthesis-related, energy-related, and carbohydrate-metabolism related proteins were down-regulated. BSA deposition on plants is accompanied by a multitude of stresses including oxidative stress, pathogenesis, and wounding [55–57].
We examined transcript expression in sheepgrass at three time points following grazing. The gene-expression analysis helped to clarify how the expression of the DEGs adjusts to grazing stress. When G2 (2 h after treatment), G6 (6 h after treatment), and G24 (24 h after treatment) were compared to C (no treatment), 2,367 genes were differentially expressed after 2 h, 2,285 genes were differentially expressed after 6 h, and 1,692 genes were differentially expressed after 24 h. Among these, 1,074 genes were differentially expressed at all three time points, indicating that about half of the DEGs were stable during the first 24 h after grazing. The key pathways involved in grazing-response mechanisms may contain these genes.
In plants, apoptosis is induced by multiple stresses, including salt, nitric oxide, oxidative stress, and wounding [58–61]. This study also shows apoptosis-related DEGs in response to grazing. Based on functional-enrichment analysis, the apoptosis pathway is significantly involved in the saliva-deposition response. In the DNA-fragmentation experiment, we found fewer DNA fragments and delayed DNA fragmentation following BSA deposition compared to defoliation.
The apoptosis-promoting RNA-binding proteins TIA-1 and TIAR (RRM superfamily) was detected among the DEGs. These proteins promote DNA fragmentation in digitonin-permeabilized thymocytes and are pro-apoptotic factors that influence some aspect of RNA metabolism . In our expression-mode analysis, TIA-1/TIAR was significantly down–regulated in the grazing treatment compared to the control and defoliation treatments. Correspondingly, less DNA ladder was seen after BSA deposition.
The expression of the serine/threonine kinase PAK4 increases the phosphorylation of the pro-apoptotic protein BAD and inhibits the activation of caspase, which protects cells against apoptosis . Hsp70 and many other heat-shock proteins can overcome both caspase-dependent and caspase-independent apoptotic stimuli and confer immortality in various cell types . Metacaspases are evolutionarily distant caspase homologs that are found outside the Metazoa and are known to play key roles in programmed cell death (PCD) . However, whether metacaspases in plants function as caspases is controversial . These apoptosis-inhibiting genes were all up-regulated only in the grazing treatment.
ATPases, including Na+, K+, and H+ ATPases, play critical roles in apoptosis [67, 68]. Apoptotic stimuli impair Na+- and K+-ATPase activity as a mechanism of neuronal death mediated by concurrent ATP deficiency and oxidative stress . Several genes were annotated as “apoptotic ATPases” in the KOG functional analysis and their expression modes are shown in Table 7.
Additional apoptosis- or PCD-related genes were detected among the DEGs. Cullin controls non-lysosomal-mediated protein degradation and thus cell death . Aldehyde dehydrogenase-2 (ALDH2) converts acetaldehyde into acetate, and over-expression of an ALDH2 transgene prevents acetaldehyde-induced cell injury and apoptosis . The exosome-complex exonuclease rrp40 forms part of the exosome, which is important to the RNA-processing machinery of eukaryotes and functions in RNA degradation in both the nucleus and the cytoplasm . A Ca2+-dependent cysteine protease (CDP) is associated with anoxia-induced root-tip death in maize .
Programmed cell death or apoptosis is an integral part of plant ontogenesis and plays a fundamental role in plant development. According to the above described genes, we suggest that there are some important pathways of apoptosis, in response to BSA deposition during grazing in plants.
Cellular oxidative stress is a common challenge for plants that usually accompanies wounding  or senescence  and is closely associated with apoptosis . Many apoptosis-inducing agents are either oxidants or stimulators of cellular oxidative metabolism. In our measurements of cellular oxidative status, H2O2, and MDA but not SOD increased substantially after grazing. However, cells from sheepgrass leaves subjected to BSA deposition showed significantly low H2O2 and MDA levels. In addition, we detected cellular oxidative-control genes among the DEGs.
Major ROS-scavenging enzymes in plants include superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX), and peroxiredoxin (PrxR) . We found no SOD genes among the DEGs, indicating that SOD expression was stable following the grazing process. This finding is consistent with the cellular oxidative-status experiment. Furthermore, we found no PrxR DEGs. However, the DEGs included APX, CAT, and GPX genes (Table 7). These three ROS-scavenging enzymes were up-regulated in the grazing treatment compared to the control. Their expression was also relatively higher in the grazing treatment than in the defoliation treatment, possibly explaining the low H2O2 and MDA levels after BSA deposition.
To investigate the molecular mechanism of grazing responses, we performed transcriptome sequencing and analysis to identify DEGs in sheepgrass subjected to simulated BSA deposition. Our results show that BSA deposition triggers differential gene expression compared to defoliation and other grazing components. Based on a functional analysis of the saliva-deposition DEGs, the cellular-antioxidant and apoptotic pathways apparently respond to grazing stress. Macroscopic changes confirm the effects of these two pathways in sheepgrass. Although the connection between the two pathways requires further evidence, we believe that the saliva-deposition-induced pathways work together to contribute to plant recovery after grazing.
Plant materials, growth conditions, and treatments
All sheepgrass plants (Zhongke No. 3) were obtained from the field 4 weeks before the initiation of the experiment. Seedlings in good condition were collected and transplanted into trays in the greenhouse. The trays were filled with a mixture of vermiculite and commercial potting soil at a ratio of 1:2. The plates were placed in a greenhouse at 25°C and 70% relative humidity. The aboveground portion of each plant was cut off, and plants were allowed to re-grow to the 5- or 6-leaf stage. To initiate the experimental treatments, two-thirds of the aboveground portion of each plant was cut off. For the defoliation treatment, water was then daubed on the cut ends of the leaves. For the grazing treatment, a BSA solution (1 mM) was daubed on the cut ends of the leaves. The remaining aboveground portions of the plants were collected 2, 6, and 24 h after the clipping and daubing treatments. The corresponding parts of the control (unclipped) seedlings were collected at the same time. All harvested seedlings of each treatment were immediately frozen in liquid nitrogen and stored at −80°C. The clipping and daubing treatments were conducted at 10:00 AM and all the materials collection was conducted in daylight hours to reduce the effort of circadian rhythmicity. In total, seven samples were obtained: C (control); D2, D6, and D24 (2, 6, and 24 h after defoliation, respectively); and G2, G6, and G24 (2, 6, and 24 h after grazing, respectively).
RNA-seq library preparation and Illumina sequencing
Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and NucleoSpin® RNA Clean-up kit (CapitalBio Company, China) according to the manufacturer’s instructions. The RNA quality was assessed by agarose-gel electrophoresis, and the RNA absorbance was measured by spectrophotometry. The ratio of the absorbance at 260/280 nm was then used to determine the RNA quality. 6 μg RNA of each sample was used for transcriptome sequencing. The RNA was processed for use on an RNA-seq platform (Illumina, Inc, San Diego, CA, USA) by the Chinese National Human Genome Center at Shanghai (CHGC).
mRNA [poly(A) RNA] was then purified from total RNA using Micropoly(A)Purist™ mRNA purification kit (Ambion, Cat.No.1919, Foster, CA, USA). The mRNA was fragmented and converted into a RNA-seq library using the mRNAseq library construction kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s instructions. 2×100 bp paired-end sequencing was performed using the Illumina Genome Analyzer II x (Illumina GAIIx, San Diego, CA, USA).
Sequence filtering and assembly
Sequence reads from all samples were cleaned using the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). First all the reads containing ‘N’ were discarded using a perl script, then adapter sequences were removed using the fastx_clipper program, followed by removal of quality < 5 bases from the 3′ end with fastq_quality_trimmer, requiring a minimum sequence length of 50 bp. Finally the reads with at least 90% bases > quality 20 were chosen using fastq quality filter for further assembly. De novo transcriptome assembly was performed using Trinity RNA-seq assembly v2013-02-25 with default parameters [PMID: 21572440].
Meanwhile, all sequence reads generated by Illumina sequencing were aligned to the reference transcriptome dataset using SOAP2 software .
Based on the Trinity assembly results, the number of reads for each contig from each sample (control, defoliation, and grazing) was converted to reads per kilobase per million (RPKM) . The MARS (MA-plot-based method with random-sampling model) module in the DEGseq package was used to calculate the differential expression of each contig between the analyzed samples . The package DEGseq is a free R package for identifying DEGs from RNA-seq data. We used an FDR (false discovery rate) to determine the threshold p-value. An FDR < 0.001 was considered to indicate a significant difference in expression between the control and treated samples.
Functional annotation, classification, and pathway analysis of DEGs
The sheepgrass transcriptome sequencing data had previously undergone Gene ontology (GO) annotation using a BLASTP search against the Swiss-Prot and TrEMBL databases with an E-value ≤ 1e-5 . A GO functional classification was performed using WEGO software (http://wego.genomics.org.cn) to understand the distribution of gene functions in grazed sheepgrass .
In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation was performed using the KEGG Automatic Annotation Server (KAAS) with the bi-directional best-hit method. For KEGG, KAAS annotates every submitted sequence with a KEGG orthology (KO) identifier representing an orthologous group of genes directly linked to an object in the KEGG pathways and BRITE functional hierarchy [86, 87]. KEGG pathway enrichment analysis was conducted using KOBAS 2.0 (http://kobas.cbi.pku.edu.cn/, ).
Finally, EuKaryotic Orthologous Groups (KOG) annotation was carried out against the NCBI KOG database with a typical cut-off E-value ≤ 1e-5. The KOG annotations of the DEGs were classified into 25 protein functions and compared between the defoliation and grazing treatments. KOG enrichment analysis was conducted through hypergeometric distribution testing using the Phyper function in the R software package (http://www.rproject.org/). The Bonferroni correction was used to adjust the p-values. The significantly enriched functional clusters were selected based on a corrected q-value (< 0.05).
DNA extraction and DNA-fragmentation assay
Using the same plant material, the cut ends of the leaves were collected every day for 9 days after clipping and daubing with water or BSA. Unclipped leaves were used as controls. Each sample for DNA extraction consisted of ten 1-cm-long leaf pieces. The leaves were flash-frozen in liquid nitrogen and stored at −80°C. The leaf tissues were ground to a fine powder in liquid nitrogen using a mortar and pestle, and the DNA was extracted using a plant genomic-DNA extraction kit (TIANGEN, Beijing, China) according to the manufacturer’s protocol. The total DNA was treated with RNase A (TaKaRa Bio Inc., Dalian, China) to remove any contaminating RNA. The isolated DNA, which was mainly derived from apoptotic cell bodies, was electrophoresed on a 2.0% agarose gel at 50 V for 3 h. The DNA fragments, which consisted of 160–200 bp multimers, were visualized under ultraviolet light after staining with ethidium bromide.
Measurement of cell oxidative factors concentrations in the leaves
Using the same plant material, the leaves were clipped and daubed with distilled water, BSA solution or OVA solution. Three independent replicates were performed. Three days later, 1 cm of the cut end of each completely expanded leaf was collected, and approximately 30 cut ends were pooled. We chose the first complete leaf ends for further experiments. The corresponding parts of unclipped seedlings were collected as controls. The samples were frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. Approximately 0.2 g of each crude extract was added to 5 ml of pre-chilled PBS (50 mM at pH 7.8), thoroughly mixed, and centrifuged for 20 min at 4,000 g at 4°C. The supernatants were simultaneously assayed using the TBA (to measure MDA), KI (to measure H2O2), and NBT (to measure SOD) methods [89–91]. The absorbance values were measured using a 2600 UV spectrophotometer (UNICO, Shanghai, China).
Leaf-scar measurements and phenotype comparisons
Using the same plant material, two-thirds of the first and last completely expanded leaves on each seedling were cut off at 10:00 AM. After clipping, the cut surfaces of the leaves were immediately daubed with water, 1 mM BSA or 1 mM OVA. Approximately 120 completely expanded leaves were removed per treatment. Three days later, the leaf scars were measured, and statistical analyses were performed to evaluate the effects of BSA on the first and last completely expanded leaves. Three independent replicates were performed. The statistical analyses were conducted using SAS 9.0 (SAS Institute, Cary, NC, USA) to compare the differences among the three treatments.
We are grateful to Huajun Zheng for his assistance in RNA sequencing and data support. This work was supported by the National Basic Research Program of China (“973”, 2014CB138704), the National High Technology Research and Development Program of China (“863”, 2011AA100209), and Project of Ningxia Agricultural Comprehensive Development Office (NTKJ −2013-04; NTKJ −2014-04).
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