MicroRNA profiling provides insights into post-transcriptional regulation of gene expression in chickpea root apex under salinity and water deficiency

Activity of root apical meristem (RAM) at the root apex is critical for stress-mediated modulation of root-architecture. Chickpea, like other legumes, possesses a basic open root meristem. Deep sequencing was used to perform microRNA expression profiling in root apex of chickpea (Cicer arietinum L.) in order to investigate post-transcriptional regulation of gene expression in this tissue in response to salinity and water deficit. Five small RNA libraries prepared from chickpea root apices at different stages of stress treatments were sequenced to obtain 284 unique miRNA sequences including 60 novel miRNAs belonging to total 255 families. Two hundred and fiftynine miRNAs were differentially expressed in stress. Six hundred and nine mRNA targets involved in diverse cellular processes were predicted for 244 miRNAs. Stress-responsive expression patterns of selected miRNAs, inverse expression patterns of their target genes and the target-cleavage sites were validated. Three candidate miRNA-target gene relationships were validated in transient expression system in chickpea. The miRNA expression profiling under salinity and water deficiency in a legume root apex and the reported function of their target genes suggested important roles of miRNA-mediated post-transcriptional regulation of gene expression involved in re-patterning of root hair cells, lateral root formation and high-affinity K+-uptake under these stresses.

-Expression level of CYCLINA2, estimated by semi quantitative RT-PCR (32 cycles), in 2 mm region of root apex and in the same size of tissue above 10 mm of root apex. Chickpea elongation factor 1 α (CaEF1α) was used as internal control. A reaction (Con,-RT) was performed without using reverse transcriptase to show absence of genomic DNA contamination. Supplementary Fig. S2-Expression pattern of representative members from each miRNA family in chickpea root and shoot tissues. Northern blot was performed to analyse expression levels of miRNAs in chickpea root and shoot tissues. 15µg of enriched smallRNA from control root and shoot tissues was loaded on denaturing (7M urea) polyacrylamide (15%) gel. Radiolabeled antisense probes were used for hybridization. Ethidium bromide-stained small RNAs were shown for equal loading.

Expression analysis
Target gene expression analysis was done by quantitative RT-PCR (qRT-PCR). First strand cDNAs were synthesized using SuperScript ® III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA) according to manufacturer's protocol. Primers for quantitative real-time PCR(qRT-PCR) analysis were designed from unique regions of transcripts. For miRNA, stem-loop primer was designed by using a 45 nts long universal stem-loop attached with reverse complement of last 5 nts of individual miRNA sequence, which provides binding region to mature miRNA in the pool. cDNA was synthesized by using pulse RT reaction. In brief, 20µl reaction containing 500ng total RNA and 2µm of stem-loop primer was loaded in thermal cycler and incubated for 30min at 16°C, followed by pulsed RT of 60 cycles at 30°C for 30 sec, 42°C for 30 sec and 50°C for 1 Sec. Reaction was incubated at 85°C for 5 min to inactive the reverse transcriptase. This product was used as template for qRT-PCR using an universal reverse primer designed from stem-loop primer. Forward primer was specific to a particular miRNA sequence but the last six nucleotides at 3'-end of miRNA were excluded. At 5'-end, extension of seven nucleotides was added to each forward primer to increase the melting temperature. Primers used in this study are provided in Supplementary 225nM of each forward and reverse primer, and 2X Power SYBRGreen PCR master mix (Applied Biosystems CA, USA). Thermal cycling conditions were 95°C for 2 min followed by 40 cycles of 95°C for 15sec, 60°C for 1 min. One cycle for melt curve included in the last cycle of the program. Cycling condition for melt curve was 95°C for 15sec, 60°C for 1 min and 95°C for 15sec. ELONGATION FACTOR 1-α (EF-1α)gene was used as internal control to normalize the variation in amount of cDNA template. Relative expressions of genes were calculated according to delta-deltaCt method of the system.

Degradome analysis
Validation of miRNA mediated cleavage of target mRNA was done by RNA Ligase mediated-Rapid amplification of cDNA ends (RLM-RACE) method as described by Llave et al. 94 In brief, a RNA oligo was ligated at decapped 5'-end of cleaved mRNA using T4 RNA ligase (NEB, USA). Ligated mRNA was reverse transcribed using SuperScript TM III RT by using oligoDT primers to create RACE ready first strand cDNA with known priming sites at the 5' and 3' ends. This cDNA was amplified with 5' and 3' adapter specific primers to obtain a population of cleaved products strictly ligated to both 5' and 3' adapters. RNA oligo sequence, adapter sequences and used primers are provided in Supplementary table 1. PCR product was directly ligated to pGEMT-Easy vector (Promega., Madison, WI). Positive clones containing fragments of different size, ranging from 250-1000bp were selected and sequenced. Obtained sequences were mapped to chickpea transcripts and annotated by NCBI-BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Validation of candidate miRNA-target relationship
As described by Sparkes et al., 95 a single colony of recombinant agrobacterium strain was cultured in 5 ml of LB media containing antibiotics kanamycin (50 mg/ml) and rifampicin (25 mg/ml) and grown overnight (28°C at 225 rpm). A 15ml LB media suspension was then inoculated with the overnight culture and grown at 28°C to an OD600 of ∼1.0. The cells were harvested by centrifugation at 5000 rpm for 2 min and resuspended in infiltration buffer (10 mM MgCl2 and 100 µM acetosyringone). Cells were washed once with infiltration buffer and then concentration of bacterial suspension was adjusted to a final desired concentration with the same buffer and left at room temperature for at least 2 h. An OD600 of 0.8 was adjusted for transient expression studies. The bacterial suspension was taken in a syringe and infiltrated through the abaxial surface of the leaf. Before infiltration, a small incision was made at the site of infiltration using a sterile needle to enhance the efficiency of infiltration.
Plant was kept overnight in dark and next day it was transferred to light. Three days after infiltration, samples were harvested in triplicate for control (non-infiltrated), empty vector (agrobacterium containing empty vector plasmid) and experimental (infiltration media containing desired construct in agarobacterium) and immediately frozen in liquid nitrogen.
RNA isolation and expression studies was done as described earlier in text.