Indian Himalayan natural Arabidopsis thaliana accessions with abolished miR158 levels exhibit robust miR173-initiated trans-acting cascade silencing.

Small RNAs (sRNAs) such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) are short 20-24-nucleotide non-coding RNAs. They are key regulators of gene expression in plants and other organisms. Several 22-nucleotide miRNAs trigger biogenesis cascades of trans-acting secondary siRNAs, which are involved in various developmental and stress responses. Here we show that Himalayan Arabidopsis thaliana accessions having natural mutations in the miR158 locus exhibit robust cascade silencing of the pentatricopeptide repeat (PPR)-like locus. Furthermore, we show that these cascade sRNAs trigger tertiary silencing of a gene involved in transpiration and stomatal opening. The natural deletions or insertions in MIR158 led to improper processing of miR158 precursors, thereby blocking synthesis of mature miR158. Reduced miR158 levels led to increased levels of its target, a pseudo-PPR gene that is targeted by tasiRNAs generated by the miR173 cascade in other accessions. Using sRNA datasets derived from Indian Himalayan accessions, as well as overexpression and knockout lines of miR158, we show that absence of miR158 led to buildup of pseudo-PPR-derived tertiary sRNAs. These tertiary sRNAs mediated robust silencing of a gene involved in stomatal closure in Himalayan accessions lacking miR158 expression. We functionally validated the tertiary phasiRNA that targets NHX2, which encodes a Na+ -K+ /H+ antiporter protein, thereby regulating transpiration and stomatal conductance. Overall, we report the role of the miRNA-TAS-siRNA-pseudogene-tertiary phasiRNA-NHX2 pathway in plant adaptation.

Modulation of miRNA expression in natural populations of A. thaliana along a wide altitudinal gradient of Indian Himalayas.

Plant populations growing along an altitudinal gradient are exposed to different environmental conditions. They are excellent resources to study regulatory mechanisms adopted by plants to respond to different environmental stresses. Regulation by miRNA is one of such strategies. Here, we report how different miRNAs are preferentially expressed in the three natural populations of A. thaliana originating from a wide altitudinal range. The expression level of miRNAs was mostly governed by temperature and radiation. Majority of the identified miRNAs expressed commonly in the three populations. However, 30 miRNAs expressed significantly at different level between the low and the high altitude populations. Most of these miRNAs regulate the genes associated with different developmental processes, abiotic stresses including UV, cold, secondary metabolites, etc. Further, the expression of miR397 and miR858 involved in lignin biosynthesis and regulation of secondary metabolites respectively, may be regulated by light intensity. A few miRNAs expressed at increasing level with the increase in the altitude of the site indicating environment driven tight regulation of these miRNAs. Further, several novel miRNAs and isomiR diversity specific to the Himalayas are reported which might have an adaptive advantage. To the best of our knowledge, this is the first report on miRNA expression from natural plant populations.

Global gene expression and pigment analysis of two contrasting flower color cultivars of Canna.

Development of flower color in plants is a complex process. Among others, it is an important trait for ornamental flowering plants. Canna is a flowering ornamental plant of family Cannaceae. To understand the molecular mechanism of flower color development in Canna, RNA sequencing from flower tissues of two contrasting flower color cultivars, Red President (RP) and Tropical Sunrise (TS) was performed. More than 27.0 million and 19.0 million clean reads were obtained from RP and TS, respectively. The combined clean reads were assembled into 147,295 unigenes. The Canna unigenes showed maximum homology with Populus trichocarpa (26.79%). A total of 2702 unigenes expressed differentially between the two cultivars of which 1972 were up-regulated and 730 were down-regulated in RP. Phenylpropanoid and flavonoid biosynthetic processes were the significant processes in RP. Expression of a vast number of transcription factors including MYB, bHLH, ARF, and WRKY were higher in RP than TS. The expression analysis of RNA sequencing data was validated by qRT-PCR analysis. Further, concentration of measured anthocyanidins and flavonols were very low or absent in TS, corroborating largely with our transcriptome data. These findings may help in understanding flower color development in Canna and in future crop breeding program.

High light intensity plays a major role in emergence of population level variation in Arabidopsis thaliana along an altitudinal gradient.

Environmental conditions play an important role in the emergence of genetic variations in natural populations. We identified genome-wide patterns of nucleotide variations in the coding regions of natural Arabidopsis thaliana populations. These populations originated from 700 m to 3400 m a.m.s.l. in the Western Himalaya. Using a pooled RNA-Seq approach, we identified the local and global level population-specific SNPs. The biological functions of the SNP-containing genes were primarily related to the high light intensity prevalent at high-altitude regions. The novel SNPs identified in these genes might have arisen de novo in these populations. In another approach, the FSTs of SNP-containing genes were correlated with the corresponding climatic factors. 'Radiation in the growing season' was the only environmental factor found to be strongly correlated with the gene-level FSTs. In both the approaches, the high light intensity was identified as the primary abiotic stress associated with the variations in these populations. The differential gene expression analysis between field and controlled condition grown plants also showed high light intensity as the primary abiotic stress, particularly for the high altitude populations. Our results provide a genome-wide perspective of nucleotide variations in populations along altitudinal gradient and their putative role in emergence of these variations.

Identification and Expression Analyses of miRNAs from Two Contrasting Flower Color Cultivars of Canna by Deep Sequencing.

miRNAs are endogenous small RNA (sRNA) that play critical roles in plant development processes. Canna is an ornamental plant belonging to family Cannaceae. Here, we report for the first time the identification and differential expression of miRNAs in two contrasting flower color cultivars of Canna, Tropical sunrise and Red president. A total of 313 known miRNAs belonging to 78 miRNA families were identified from both the cultivars. Thirty one miRNAs (17 miRNA families) were specific to Tropical sunrise and 43 miRNAs (10 miRNA families) were specific to Red president. Thirty two and 18 putative new miRNAs were identified from Tropical sunrise and Red president, respectively. One hundred and nine miRNAs were differentially expressed in the two cultivars targeting 1343 genes. Among these, 16 miRNAs families targeting 60 genes were involved in flower development related traits and five miRNA families targeting five genes were involved in phenyl propanoid and pigment metabolic processes. We further validated the expression analysis of a few miRNA and their target genes by qRT-PCR. Transcription factors were the major miRNA targets identified. Target validation of a few randomly selected miRNAs by RLM-RACE was performed but was successful with only miR162. These findings will help in understanding flower development processes, particularly the color development in Canna.

Genetic diversity and population structure of Arabidopsis thaliana along an altitudinal gradient.

The natural genetic variation within a plant species is primarily a consequence of its phylogeography and evolutionary history. This variation largely determines its present-day population structure. Arabidopsis thaliana, as a model plant, has been studied in great detail including its probable origin, local as well as global genetic diversity pattern, population structure, adaptation, etc. However, no such studies have so far been reported from the Indian Himalayan region. Here, we describe a comprehensive study on the genetic diversity and population structure of A. thaliana from an altitudinal range of 700-3400 m above mean sea level the highest altitudinal range reported so far. We also compare these populations with previously reported worldwide populations. A total of 48 accessions representing six populations were analysed using 19 microsatellites and 11 chloroplast markers. Genetic diversity analysis indicated populations to be highly diverse and comparable with worldwide populations. STRUCTURE, principal coordinate and isolation by distance (IBD) analyses showed that genetic variation in different populations is structured at geographical and altitudinal level. Further analyses indicate that these populations are genetically distinct from the rest of the world populations. Different parameters of the demographic expansion model support a rapid expansion. Based on mismatch distribution, the initial time of expansion of west Himalayan populations was found to be about 130 000 years. Bayesian analysis of divergence time indicated that these populations have a long evolutionary history in this region. Based on the results of genetic diversity parameters, demographic expansion and divergence time estimation, it appears that west Himalayan populations may be the source of the west-east expansion model.

The internal transcribed spacer (ITS) region and trnH-psbA [corrected] are suitable candidate loci for DNA barcoding of tropical tree species of India.

BACKGROUND: DNA barcoding as a tool for species identification has been successful in animals and other organisms, including certain groups of plants. The exploration of this new tool for species identification, particularly in tree species, is very scanty from biodiversity-rich countries like India. rbcL and matK are standard barcode loci while ITS, and trnH-psbA are considered as supplementary loci for plants. METHODOLOGY AND PRINCIPAL FINDINGS: Plant barcode loci, namely, rbcL, matK, ITS, trnH-psbA, and the recently proposed ITS2, were tested for their efficacy as barcode loci using 300 accessions of tropical tree species. We tested these loci for PCR, sequencing success, and species discrimination ability using three methods. rbcL was the best locus as far as PCR and sequencing success rate were concerned, but not for the species discrimination ability of tropical tree species. ITS and trnH-psbA were the second best loci in PCR and sequencing success, respectively. The species discrimination ability of ITS ranged from 24.4 percent to 74.3 percent and that of trnH-psbA was 25.6 percent to 67.7 percent, depending upon the data set and the method used. matK provided the least PCR success, followed by ITS2 (59. 0%). Species resolution by ITS2 and rbcL ranged from 9.0 percent to 48.7 percent and 13.2 percent to 43.6 percent, respectively. Further, we observed that the NCBI nucleotide database is poorly represented by the sequences of barcode loci studied here for tree species. CONCLUSION: Although a conservative approach of a success rate of 60-70 percent by both ITS and trnH-psbA may not be considered as highly successful but would certainly help in large-scale biodiversity inventorization, particularly for tropical tree species, considering the standard success rate of plant DNA barcode program reported so far. The recommended matK and rbcL primers combination may not work in tropical tree species as barcode markers.