MicroRNA expression profiles in response to drought stress in Sorghum bicolor

Accepted Manuscript MicroRNA Expression Profiles in Response to Drought Stress in Sorghum bicolor Nada Babiker Hamza, Neha Sharma, Anita Tripathi, Neeti Sanan-Mishra PII:

S1567-133X(16)30001-1

DOI:

10.1016/j.gep.2016.01.001

Reference:

MODGEP 1003

To appear in:

Gene Expression Patterns

Received Date: 28 June 2015 Revised Date:

18 December 2015

Accepted Date: 4 January 2016

Please cite this article as: Hamza, N.B., Sharma, N., Tripathi, A., Sanan-Mishra, N., MicroRNA Expression Profiles in Response to Drought Stress in Sorghum bicolor, Gene Expression Patterns (2016), doi: 10.1016/j.gep.2016.01.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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MicroRNA Expression Profiles in Response to Drought Stress in Sorghum bicolor

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Nada Babiker Hamza *, Neha Sharma , Anita Tripathi and Neeti Sanan-Mishra * a

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Department of Molecular Biology, Commission for Biotechnology and Genetic Engineering, National Center for Research, P.O. Box: 2404, Khartoum, Sudan b Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, ArunaAsaf Ali Marg, New Delhi, 110067, India *Corresponding author: [email protected]; [email protected]

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ABSTRACT:

The regulatory role of small non-coding RNAs that are 20-24 nucleotides in length has become the foremost area of research for biologists. A major class of small RNAs represented by the microRNAs

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(miRNAs), has been implicated in various aspects of plant development including leaf pattering, meristem function, root patterning etc. Recent findings support that miRNAs are regulated by drought and other abiotic stresses in various plant species. In this study, were report the expression profiling of 8 known abiotic stress deregulated miRNAs in 11 elite sorghum genotypes, under watered and drought conditions. Significant deregulation was observed with miR396, miR393, miR397-5p, miR166, miR167 and miR168. Among these, the expression levels of sbi-miR396 and sbi-miR398 were the highest in all the genotypes.The expression of sbi-miR396 was maximum in the grain Sorghum HSD3226 under well-

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watered conditions and the profile shifted towards HSD3221 under drought stress. Forage accessions, N98 and Atlas, showed an opposite behavior in expression patterns of miR397-5p in drought physiologies.Such dynamic expression patterns could be indicative of prevailing drought tolerant mechanisms present in these sorghum accessions. This data provides insights into Sorghum miRNAs

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which may have potential use in improving drought tolerance in sorghum and other cereal crops.

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Key Words: microRNA, Sorghum bicolor, expression patterns, drought, target

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Introduction Sorghum is an important cereal crop in dryland agriculture because of its use as food and livestock feed. Sorghum grain production ranks fifth globally and it feeds 5 million people, mostly from the poor and under-developed areas of Africa and Asia (Haussmann et al. 2002; Mace et al. 2013; Paterson 2008). Its

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importance has increased with increasing global pressure for food and aberrant climatic changes that has forced researchers to focus on other crops to find an answer to food security challenges, especially for poor people. The crop is known for its hardiness and stability in marginal land farming with extreme tolerance to low input levels of water and fertilizers(Paterson et al. 2009). It is a close relative to sugarcane with the sweet sorghum being tall and producing high biomass in addition to sugar. It has thus

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attracted attention as an emerging bioenergy crop due to high sugar content in its stems (Bowers et al. 2003; Dillon et al. 2007; Vermerris et al. 2007).

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The origin and early domestication of sorghum took place in north-east Africa and the earliest known record of sorghum is near the Egyptian-Sudanese border, which dates back to 8,000 B.C (Kimber et al. 2013). Spread of sorghum resulted in disruptive selection on the basis of agronomic advantages suitable for different locations, but this radial movement across different areas lead to emergence of immense diversity in the sorghum crop. Sorghum landraces and wild relatives of cultivated sorghum from these centers of diversity are therefore rich sources of resistance to diseases, insect pests and other stresses such as high temperature and drought(Rosenow and Dahlberg 2000). Moreover, its relatively small

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diploid genome (735Mbp) makes it an excellent grass model to study C4 plant physiology(Paterson 2008).Preliminary exploration of genomic diversity based on conventional approaches such as RAPD, RFLP, SSR, SNP genotyping have clearly revealed considerable polymorphism between cultivated and wild type Sorghum(Aggarwal et al. 1999; Agrama and Tuinstra 2004; Ng'uni 2011; Smith et al. 2000; Williams et al. 1990; Yoshida 2004). But these studies are limited in number and do not pertain to

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changes in developmental stages or environmental stress (Winter and Kahl 1995). For example, it is important to capture the differential behavior of various accessions to drought conditions, even though

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sorghum is a dry land crop.

Sudanese region has the largest collection of wild and cultivated sorghum varieties due to early domestication of sorghum in this area. Genetic diversity analysis with the help of ISSR markers has identified 97% polymorphism among 50 sorghum accessions from Sudan (ElAmin and Hamza 2014).There is however very little information about the genetic relationships within the sweet sorghums and grain sorghum genotypes. The molecular characterization of these accessions holds great valuefor exploiting the genetic pool to generate hardy cultivars tolerant to changing climatic conditions. Such studies are important for better crop improvement strategies. Whole genome sequencing of sorghum has re-emphasized the correlation of phenotypic differences among varieties with respect to their genotypic diversity(Mace et al. 2013). This has led to identification of

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several microRNAs (miRNAs) by different groups (Du et al. 2010a; Katiyar et al. 2012; Zhang et al. 2011). The deciphering of miR expression patterns in different cultivars of sorghum will provide a better understanding of their genetic responses. This will help in selecting abiotic stress resistant varieties that can be used for further crop improvement program. In this study, we investigated the effect of drought stress on the expression levels of selected miRNAs across 11 elite accessions of African sorghum. A

differential response of the miRNAs in the varied genotypes. Materials and Methods

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Plant Materials and stress treatment

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functional correlation has been drawn with their predicted targets to obtain meaningful insights into the

11 Sorghum genotypes were used in the study. Seeds of eight grain sorghum genotypes were provided by the Germplasm Bank of the Plant Genetic Resource Unit (The Agriculture Research Corporation, Wad

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Medani), they were collected from three different regions in Sudan, namely, West Darfur (Western Sudan), Red Sea (Eastern Sudan), and Bahr El Jabel (South Sudan).The released variety ArfaGadamak is also a grain sorghum genotype. The other two genotypes include N98 and Atlas, which are breeder lines for sweet sorghum from University of Nebraska.

Sorghum seeds were sown in Shambat Experimental Field, College of Agriculture, University of Khartoum. A randomized block experiment with two replicates was done. Control plants received regular

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irrigation every 10 days whereas drought stress was imposed on the plants by increasing the watering interval to 21 days. Leaves were taken from all controlled and stressed plants and stored in RNase free tubes containing RNA Shield™ (Zymo Research) until RNA extraction was performed. RNA isolation and Stem-loop RT PCR

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Total RNA was extracted from 0.5g of leaf tissues using the Guanidium isothiocynate extraction method of Chomczynski and Sacchi (2006) with slight modifications(Chomczynski and Sacchi 2006). 200ng total RNA was used to synthesize cDNA using miRNA-specific stem-loop primer with Superscript reverse

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transcriptase III (Invitrogen) as per manufacturer’s specifications. Primers used in this study are listed in Table 1. A pulsed RT reaction was performed in a thermalcycler as follows: 30min at 16°C, 60 cycles at 30°C for 30s, 42°C for 30s and50°C for 1s. RT enzym e was inactivated by incubating the reaction at 85°C for5mins. 1µl of direct cDNA was used for PCR using miRNA specific forward primer and universal reverse primer to get a 63bp amplification product.18SrRNA was used as an endogenous control.Relative abundance was calculated as Integrated Density Values (%IDV) by normalizing the obtained values with 18SrRNA.The PCR based expression analysis was performed using three replicates for each set and the calculated standard deviation for each set is shown as error bars on the graphs.

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Target prediction and GO annotation Putative targets for selected miRNAs were predicted by psRNA target webserver (Dai and Zhao 2011) using transcripts from SbGDB (http://plantgdb.org/SbGDB/)(Duvick et al. 2008). Default parameters were employed and mRNA sequences with a score ≤3 were considered as potential targets. All the predicted

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mRNAs were searched in Plant Transcription factor database, Plant TFBD 3.0 to search possible Transcription factors(Jin et al. 2014).Inter-relationships between miRNA and their predicted targets were studied by creating a visual interaction map using Cytoscape 2.8.3(Shannon et al. 2003). For7miRNAs, target prediction was done using hsp size (length of complementary scoring) of 20 butfor miR398, the hsp size was modified to 19.GO annotation and enrichment analysis was carried outby AgriGO with default

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parameters (p-value ≤0.05)(Du et al. 2010b). GO enrichment was done using Singular Enrichment Analysis by employing Plant GO slim. Pathway enrichment analysis for predicted target mRNAs was done using Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology database(Kanehisa and Goto

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2000). Results and Discussions

1. Selection of Sorghum genotypes and the miRNAs

Sorghum from Sudan has been used worldwide as a source for improving germplasm, for drought tolerance, nutritional quality, stalk strength, insect and disease resistance(Rosenow and Dahlberg

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2000).However, no research has been done on these Sudanese genotypes to reveal the role of miRNAs that may be governing these traits. Sorghum is a dry land plant that can avoid stress either by conserving water through an efficient leaf and stomata characteristic (by early closure of stomata, increasing photosynthetic efficiency, reduction of cuticular transpiration, lipid deposition on leaves, leaf area reduction and morphology of leaf surface) or improving water uptake by root or through osmotic

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adjustment to lower the osmotic potential(Acevedo and Fereres 1993; Blum 1988). Efficient water uptake is an important determinant of drought tolerance, as it depends on root size (length or mass), activity and

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spatial distribution(Blum and Arkin 1984). An exhaustive study on 40 Sudanese sorghum genotypes under water stress conditions(Assar et al. 2009)identified Arfa Gadamak as a high yielding grain sorghum variety under both normal and drought stress conditions. It was thus, recommended to be used as parent in breeding programs for drier areas. The comparative analysis also revealed higher concentrations of K and Fe in the seeds of tolerant genotypes than the susceptible ones. The concentration of Fe was found to decrease with maturity in the tolerant group butit increased with maturity in the susceptible group. The seeds of Arfa Gadamak variety also contained high level of Zn in its young seedlings(Assar et al. 2002). For this study, 11 elite accessions of sorghum genotypes (Fig. 1) including the Arfa Gadamak, a released variety were selected. The details of these accessions are listed in Table 2. Further, eight conserved

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miRNAs (miRBase release 20) that are known to be deregulated under abiotic stress were chosen for profiling. These include sbi-miR160, sbi-miR166, sbi-miR16,sbi-miR168, sbi-miR393, sbi-miR396, sbimiR397-5p and sbi-miR398.These stress-induced miRNAs are evolutionarily conserved across plant species indicating the conservation of the corresponding miRNA-mediated regulatory mechanisms. It is hypothesized that the behavior of these miRNA-mediated interactions in environmental stresses may be

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governing the physiology of the plants toabiotic stress. This is supported by the opposite expression patterns of specific miRNAsin Arabidopsis and rice under drought stress. Thus, it is important to analyze whether these reported stress responsive miRNAs play tolerance roles in other plant species as well.

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2. Prediction of miRNA targets in Sorghum bicolor and analysis of their interaction Identification of genes regulated by the miRNAs is essential for understanding the biological functions.

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Target prediction was performed with the help of psRNAtarget web server using transcripts from PlantGDB and 80 targets corresponding to 62 unique gene IDs, for all miRNA family members under study were identified. Table 3 enlists the sorted unique transcripts identified for each miRNA family, considering the fact that miRNA family members targeted the same sequence. An inter-relationship between miRNAs and their predicted targets is shown in Fig. 2c. The sbi-miR396 showed maximum number of interactions, whereas sbi-miR167 targeted only a single transcript.Such variability in gene regulatory responses governed by miRNAsindicates that such deregulations can affect growth and

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development in varied ways. Further analysis using PlantTFBD revealed that three miRNAs, sbi-miR160, sbi-miR166 and sbi-miR396 were targeting 28 transcripts encoding for 3 different type of transcription factors (TFs) viz. Auxin Response Factor(ARF), Homeobox-leucine zipper family protein (HD-ZIP) and Growth regulating Factors(GRF), targeted by sbi-miR160, sbi-miR166, and sbi-miR396 respectively. In all, 14 unique gene IDs were transcription factors including 7 GRF, 5 HD-ZIP, and 2 ARFs. This suggest

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that the miRNAs are involved in regulating various developmental processes(Jones-Rhoades et al. 2006). Singular Enrichment Analysis (SEA) of these candidate target genes showed their involvement in a broad

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spectrum of biological processes and regulatory functions, including Metabolic process, developmental processes, Cellular processes, transcription regulatory activity, binding, Catalytic activity, response to stimulus, ant reproductive process etc (Fig. 2a). The identified target transcripts were represented across 66 different GO terms with 55 highly significant terms (p value ≤0.05). A detailed analysis revealed that majority of these (35 categories, 63.63%) belonged to Biological Processes (P) majority coming under cellular and metabolic processes. At least 11 categories (20%) grouped under Molecular Function (F) where a leading part includes binding followed by catalytic activity and 9 categories (16.36%) were included under Cellular Components (C) where the main representation is under cell and cell part (Fig. 2a, b). This analysis was consistent with the KEGG (Kyoto Encyclopedia of Genes and Genomes) orthology analysis, which inferred that the microRNA targets were represented with 12 KO terms (Table

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3). The sbi-miR397-5p, sbi-miR396, sbi-miR393 and sbi-miR166were found to interact with some important pathways such as RNA transport, mRNA surveillance pathway, Aminoacyl-tRNA biosynthesis, Ribosome biogenesis in eukaryotes, Spliceosome, and Plant hormone signal transduction. Target identification and gene ontology analysis revealed important pathways that might be modulated during drought stress. A stable fine tuning of the relative expression of target genes is obtained by a combination

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of transcriptional (mediated by TFs) and post-transcriptional interactions (mediated by miRNAs).Such combinations ensure robustness against stochastic fluctuations and an optimal fine tuning is reached when miRNAs plays the role of master regulator and one of its targets is a TF which regulates other microRNA targets (Riba et al. 2014).

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3. Expression analysis of miRNAs under drought stress in sorghum genotypes

To identify the level of stress induced deregulation in the miRNA expression patterns endpoint stem-loop

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RT-PCR was performed. The miR166 family is conserved in all land plants and was first reported in sorghum by homology search against the EST database(Katiyar et al. 2012; Zhang et al. 2011). It was observed that the expression patterns of sbi-miR166 varied across different sorghum genotypes (Fig.3a). Its levels were high in the Arfa Gadamak, N98 and Atlas genotypes while its expression level was low in HSD3220 and HSD5373. Under drought stress, the expression levels of sbi-miR166 was up-regulated in the Arfa Gadamak, Atlas, HSD3220 and HSD5373 genotypes, with more than 3 fold change in the grain accessions HSD3220 and HSD5373 (Fig.3a). In accessions HSD3221, HSD3226 and N98 the

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expression levels of sbi-miR166 were down-regulated. The miR166 family was also reported to be drought-responsive among sensitive and tolerant cultivars of soybean(Kulcheski et al. 2011). miR166 along with Ago10 protein affects leaf polarity by modulating shoot apical meristem by changing HD-ZIPIII transcript levels(Lynn et al. 1999; Miyashima et al. 2013; Zhu et al. 2011). It has been clearly shown that HD-ZIPIII transcription factors function in a stress inducible manner under salinity as well as drought

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conditions (Chen et al., 2014). In another report, two sorghum HD-ZIP transcripts were down-regulated under drought stress which indicates that miR166-HD-ZIP module is affected during drought conditions across sorghum genotypes (Johnson et al., 2014). It may also be involved in floral development in

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Arabidopsis(Jung and Park 2007). The high levels of sbi-miR166 in atleast 4 sorghum genotypes including the drought tolerant, high-yielding, grain sorghum variety, Arfa Gadamak, indicates that its induction may be a triggered reaction to cope up with the drought stress. miR166 with its role in defining root, stem and floral architectures, its deregulation, can be a useful marker for identification of drought tolerant sorghum genotypes.

The miR167 family is also deregulated by salinity as well as drought conditions (Liu et al. 2008) hence was selected for the present study. Unlike sbi-miR166, the levels of sbi-miR167 were high in HSD2945, HSD3221, HSD3222, HSD3223 and HSD3226 (Fig.3b). The drought-induced up-regulation was prominent (8-fold) in HSD3220and was also seen in HSD2945, HSD3222, HSD3223 and Atlas. In the other varieties, a significant variation was not observed under drought stress (Fig.3b). However, the levels

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were down-regulated in the drought tolerant Arfa Gadamak. Literature survey also indicates that miR167 is mostly down-regulated in stress. The osa-miR167 is down-regulated in response to ABA, the stress phytohormones (Liu and Chen 2009). Ath-miR167 was reported to be up-regulated by salt stress, whereas zma-miR167 was down-regulated under similar stress conditions (Ding et al. 2009; Liu et al. 2008). In Helianthus annus also miR167 was shown to be down-regulated during drought stress with an

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induction of its target gene under same conditions (Khakesifidi et al., 2015). Recently, miR167, along with miR164 has been linked with water deficit conditions in cassava (Phookaew et al. 2014). In switch grass, a promising biofuel crop like sorghum, miR167 was down-regulated in drought conditions (Sun et al. 2012). MicroRNA167 (miR167) was shown to cleave the transcripts of transcription factor, ARF8 and a negative correlation between mir167 and its target genes was clearly demonstrated. A rice IAA-

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conjugating enzyme, OsGH3-2, was positively regulated by ARF8. This auxin-miR167-ARF8-OsGH3-2 signal transduction pathway, could be, in conjunction with the other microRNA-mediated auxin signals, an important one for responding to exogenous auxin and for determining the cellular free auxin level which

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guides appropriate auxin responses (Yang et al. 2006).

The drought stress also down-regulated the levels of sbi-miR160 in most sorghum genotypes (Fig. 3c)anda 2.5 fold down-regulation was evident in accession HSD52997, from Eastern Sudan. Interestingly, even after the stress-induced deregulation, the expression levels of this miR were more inHSD5373, Arfa Gadamak, N98 and Atlas varieties.miR160 targets the ARF10, ARF16 and ARF17(Liu et al. 2007; SananMishra et al. 2013).Over-expression of miR160 leads to ABA hyposensitivity in germinating seedlings,

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suggesting a cross-regulatory pathway operating during drought stress. In sugarcane, ssp-miR160 and its homologs were either induced or repressed among cultivars (Gentile et al. 2013) while in Populus trichocarpa miR160 family was reported to be down regulated in drought stress (Shuai et al. 2013). This suggests that during dehydration sorghum tends to modulate its metabolic state by changing hormone

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levels to cope up with adverse environmental conditions. The miR168 is known to regulate the expression of AGO1 protein, a key component of the RISC (Rhoades et al. 2002; Vaucheret et al. 2006; Vaucheret et al. 2004). Therefore, variations in its

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expression profiles are expected to influence the functionality of other miRNAs. miR168 displays differential expression in both rice and Arabidopsis during drought stress(Liu et al. 2008; Zhou et al. 2010). It was interesting to note that in all the sorghum accession under study, miR168 was deregulated to some extent (Fig.3d) clearly indicating that miRNA action is regulated via a feedback loop driven by miR168. This is supported by an observation in Arabidopsis, where a parallelism exist between the epression patterns of mir168 and its target AGO1 (Sire et al., 2009). So, we do not expect a steep antagonistic behavior between miR168 and AGO1 due to its central role in controlling miR biogenesis. In response to drought stress its miR168 levels were induced by 2-folds in HSD5299 while reduced by 2folds in HSD5373 while smaller changes were observed in the other genotypes.

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The miR393 is a known abiotic stress responsive miR, that is up regulated during salinity, dehydration and cold stress (Liu et al. 2008; Sunkar and Zhu 2004). It is strongly up regulated under drought in other species such as rice and sugarcane (Ferreira et al. 2012; Zhao et al. 2007). The expression patterns of this miRNA showed deregulation among all genotypes under drought stress (Fig.3e). In most varieties the miRNA levels showed a two fold or more increase in response to drought stress, however a contrasting

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behavior was observed in the breeder lines (N98 and Atlas), indicating their poor durability to cope up with drought conditions. miR393 targets transcripts that code for a basic helix-loop-helix (bHLH) transcription factor and for the auxin receptors TIR1, AFB1, AFB2, and AFB3.It is known to directly regulate abiotic stress by determining cellular auxin levels(Xia et al. 2012). AFB3 is part of the ubiquitin protein ligase SCFTIR1/AFB complex that targets and mediates the polyubiquitination and proteasomal

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degradation of the Aux/IAA transcriptional repressors to promote transcription of auxin-responsive genes(Mockaitis and Estelle 2008; Tan et al. 2007). Over-expression of miR393 results in downregulation of TIR1 transcript leading to stunted growth during drought stress in transgenic rice plants (Xia et al.,

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2012). In sugarcane, a close relative of sorghum, TIR1 is validated by qPCR and its expression negatively correlates to the miR393 expression in drought conditions (Ferriera et al., 2012). The suppression of auxin signaling might be a strategy that plants use to enhance their tolerance to abiotic stress. Thus, drought induced-induction of expression of miR393 could contribute towards tolerance of sorghum plants to stress.

In Arabidopsis, miR396 was shown to be responsive to drought as well as salinity(Liu et al. 2008). In an

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earlier report on genome-wide profiling and analysis of miRNAs in rice using a microarray platform it was observed that miR396 was significantly down-regulated in response to drought stress. This was opposite to the observation from drought stressed Arabidopsis (Zhou et al. 2010). In Sorghum, it accumulates to very high levels in the southern and western Sudanese varieties and under drought its transcripts are further up-regulated in the varieties HSD2945, HSD3220 and HSD3221 (Fig.3f). However, in accessions

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HSD3223 and HSD3226 its expression is down-regulated. The remaining varieties showed small changes in the sbi-miR396 expression profile (Fig.3f). The miR396 is known to target Growth Regulating Factor (GRF) gene family which helps in controlling leaf development by regulating cell division and

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differentiation. Over-expression of miR396 in tobacco confers drought tolerance in tobacco (Yang and Du, 2009). miR396 is known to control leaf development by regulating cell expansion through GRFs in Arabidopsis (Liu et al., 2009). Hence, it can be concluded that miR396 is drought responsive miR which may execute its effect by controlling leaf development. This could be an important aspect for further investigation because increase in leaf biomass under water limiting conditions would be an incentive for utilizing sorghum as a biofuel crop. The expression levels of miR397 were relatively low across all the varieties but relatively high levels were observed in HSD3223, HSD3226 andHSD5373. Under drought stress its levels decreased in five varieties including the tolerant Arfa Gadamak variety (Fig.3g). An induction in its expression was seen in

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HSD3222. miR397 is known to target laccases, a gene family involved in lignin biosynthesis and iron/copper sequestration(Abdel-Ghany and Pilon 2008). In sugarcane, a close relative of sorghum, miR397 is known to differentially express in drought conditions. ssp-397 is up-regulated in both tolerant and susceptible cultivars at 2 days of drought conditions, but it is lowered down in tolerant cultivar if stress is increased to 4 days(Ferreira et al. 2012). In both tolerant as well as susceptible sugarcane

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cultivars, expression of miR397 and its target gene Laccase 23-like protein exhibited statistically significant opposite behavior, indicating it to be an actual cleavage product generated by miR397 (Ferreira et al., 2012). miR397 also displayed a contrasting expression pattern among sensitive (upregulated) and tolerant (down-regulated) genotypes of soyabean(Kulcheski et al. 2011). An up regulation of miR397-5p under dehydration would lead to increased lignification of cell wall, limiting the uptake of

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nutrients and release of solutes from cell, for its survival in water limiting conditions.

miR398 holds special place among plant miRNAs as it was the first miR reported to be linked with stress

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tolerance in plants(Sunkar et al. 2006). It is directly associated with stress regulatory networks with varied responses to drought, salinity, ABA, oxidative stress, metal ion deficiency etc (Zhu et al. 2011). Considerably high expression of miR398 was observed in all sorghum accession. In rice, miR398 shows leaf-preferential expression in young seedings(Mittal et al. 2013) and this could explain its high levels in the sorghum seedlings. Its levels were induced in response to drought stress except for genotypes HSD2945, HSD3221, HSD3223, HSD5299 and HSD5373 (Fig. 3h). A notable down-regulation of 2-fold was observed in accession HSD5299, which indicates that it can be helpful in quenching ROS more

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effectively during stress response so as to decrease cellular damage. In Arabidopsis, miR398 is known to target Cu/Zn superoxide dismutase (CSD1/CSD2), which help in ROS detoxification, an important process for stress resistance and plant survival (Beauclair et al. 2010); a reduced level of miR398 improves tolerance to oxidative stresses (Sunkar et al. 2006). ROS is produced under all kinds of abiotic stresses to fight adverse conditions, including water deficit. In Medicago trunculata, miR398 is up-

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regulated under water deficit (Trindade et al. 2010). miR398 is a drought inducible miRNA in Triticum dicoccoides with respect to tissue and drought duration.

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To summarize, the expression levels of sbi-miR396 and sbi-miR398 were the highest in all the genotypes. Under drought stress, there was not much change in the expression patterns of sbi-miR398and highest expression was seen in the breeder line, Atlas(Fig. 4a). Whereas the expression of sbi-miR396 was maximum in the grain Sorghum HSD3226 under well-watered conditions and the profile shifted towards HSD3221 under drought stress (Fig. 4a). Similarly there was not much change in the overall profiles of sbi-miR160 but the levels of sbi-miR166were dramatically high in Atlas under drought stress. On comparing the expression profiles across species under control conditions suggested that miR168 is behaving most dynamically in all species followed by miR393 and miR160 (Fig. 4b). Interestingly, miR168 profiles were high in HSD5373 and Atlas but under drought stress it shifted sharply towards HSD5299

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(Fig. 4b, c). This observation re-establishes the fact that miRNAs can act specifically both in space and time.

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Conclusion Plant miRNAs play an important role in regulating responses to diverse abiotic stress, including dehydration, freezing, salinity, alkalinity, and high temperature. In general the expression levels of miRNAsare deregulated by the environmental stresses. Differences in the expression patterns are the effect of the nature, duration and severity of the stress and the physiology of the plant under stress.

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Sorghum is an important cereal in dry and semi-arid areas of world and is an emerging biofuel crop too. As compared to other important cereal crops, focused studies on specific miRNAs under environmental stress have not been described in sorghum. Hence, a deeper understanding of the role of miRNAs in

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sorghum under stressful environment will help in developing better crop varieties of sorghum which are sturdier and more tolerant to changing climatic conditions. In this study, we obtained the expression changes of 8 conserved miRNAs that have been demonstrated to be involved in salt or drought stress in previous studies. The comparisons were made across 11 different Sudanese’s sorghum exposed to drought stress.

It is interesting that, in general, we did not find a uniform pattern for the miRNA variations though dramatic

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changes in expression of specific miRNAs were observed in the Sorghum genotypes. Overall, this points to the existence ofa fine-tuning mechanism rather than a dramatic control of expression exerted by miRNAs under drought stress. This fine-tuning mechanism may be influencing the growth and development process, giving rise to the variations between the Sorghum genotypes. This can be well exemplified by the fact that many of the miRNA studied are known to target the ARF genes, for example,

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ARF6 and ARF8 are targeted by miR167, whereas ARF10, ARF16 and ARF17 are targeted by miR160(Mallory et al. 2005; Rhoades et al. 2002; Wang et al. 2005). ARF proteins bind to auxin response promoter elements and mediate gene expression responses to the plant hormone auxin (Hagen and

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Guilfoyle, 2002; Liscum and Reed, 2002;Tiwari et al., 2003). Similarly, miR393 also influences the auxin pathway by negatively regulating TIR1 and AFB2 mRNAs. This leads to the stabilization of Aux/IAA repressors bringing about concomitant repression of auxin signaling. Depending on the type, intensity and duration of environmental stress the variations in auxin signaling cast their influence on embryogenesis, root architecture and floral development (Hardtke et al., 2004; Mallory et al., 2005; Sessions et al., 1997; Wang et al., 2005). It is known that under various adverse environmental conditions, ROS homeostasis can lead to oxidative damage and cell death(Van Breusegem and Dat 2006). The plant antioxidant system consists of a number of enzymes that maintain the home ostasis by amultifaceted network of ROS producing and ROS-scavenging enzymes(Mittler et al.).miR398 targets two closely related Cu/Zn SODs (CSD1 and

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CSD2) which are known to involve in oxidative stress detoxification(Sunkar et al. 2006) and cytochrome C oxidase subunit V which is involved in electron transport system of the mitochondrial respiratory pathway(Sunkar and Zhu 2004). An interaction between auxin and ROS signaling has been suggested during salinity by using tir1 afb2 mutant(Iglesias et al. 2010).In conclusion, our results suggest that miRNAs play important role in governing the plant’s growth under abiotic stresses in vegetative tissues.

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Further studies mapping the miR and their target expression domains in coordination with the plants behavior under stress will be required to give deeper evidence on the regulatory pathways operative under stress.

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Acknowledgements

NBH is thankful to CV Raman International Fellowship programme for African Researchers, DST, Govt of

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India for Senior Fellowship. The authors thank ICGEB, India for providing the necessary facilities in carrying out this research work. The Senior Research Fellowship offered by the CSIR to NS is greatly

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Legends to Figures Fig. 1 The diversity of some seed accessions from Western Darfur and Bahr EL Jabel used in the study:a-HSD2945; b-HSD3220; c-HSD3221; d-HSD3222; e-HSD3223; f-HSD3226

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Fig. 2Analysis of the predicted target transcripts for sorghum miRNAs a Details of distribution across different categories bDistribution of GO termsP: biological process; F: molecular function; C: cellular component c miR-target regulatory model based on their interactions

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Fig.3 Expression analysis of 8 miRNAs in 11 sorghum genotypes under control and drought stress a miR166 b miR167 c miR160 d miR168 e miR393 f miR396 g miR397 h miR398. Standard deviations calculated for atleast three experimental replicates are shown as error bars for each dataset. Trendline (shown in pink) shows the basal level of miR expression in each case

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Fig. 4 Comparative analysis of miR expression across different miRNAs and/or stress condition a levels of miR396 and miR398 under control and drought condition b levels of miR160, miR166, miR167, miR168, miR393 and miR397 under control condition c levels of miR160, miR166, miR167, miR168, miR393 and miR397 under drought condition

Table 1 List of Pimers used in the study

SLP166 Fwd166 SLP167 Fwd167

Fw168

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATAC AGCACG

AC C

SLP168

Sequence 5'-3'

SLP160

Fwd160 SLP393

Fwd393 SLP396c-5p Fwd396c-5p SLP397

CGTCGGACCAGGCTTCA GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACT CAGAT CGCAATGAAGCTGCCAGCATG GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACG TCCCG TCGCTTGGTGCAGATCG GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACT GGCAT ACTGCCTGGCTCCCTGT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATAC GATCAA GAGGATCCTCCAAAGGG GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACA AGTTC GTGCCTTCCACAGCTTTCTT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACC ATCAA

EP

Primer Name

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Fwd397

Fwd398

GCTCATTGAGTGCAGCG GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACA AGGGG GTTGCATGTGTTCTCAGGTCA

Universal Reverse Primer

GTGCAGGGTCCGAGGT

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SLP398

AC C

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Table 2. Details of sorghum genotypes used in the study No. Genotype Name Category

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Sample Abbreviation Sources

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1

HSD 2945

Bahr EL Jabel

1

West Darfur

2

West Darfur

3

Cultivated

HSD 3220 2

Cultivated HSD 3221

Cultivated

4

HSD 3222

Cultivated

5

HSD 3223

Cultivated

6

HSD 3226

Cultivated

7

HSD5299

Cultivated

8

HSD5373

Cultivated

9

ArfaGadamak

Released Variety

Sudan

10

N98

Breeder line

University of Nebraska (Katiyar et al.) of University

11

Atlas

Breeder line

RI PT

3

4

West Darfur

5

West Darfur

6

SC

West Darfur

7

Eastern Sudan

8

9 10

11

Nebraska (Katiyar et al.)

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Eastern Sudan

Table 3. Target gene prediction and KEGG orthology annotations for the 8 miRNAs under study

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sbi-miR167

Target Description

Sb01g019130

Cleavage

ARF 16

NA

NA

Sb10g027790

Cleavage

NA

NA

Sb02g020860

Translation

ARF 16 lipid phosphate phosphatase 2

NA

NA

Sb04g001100

Translation

expressed protein

K11883

Sb01g013710

Cleavage

HD-ZIP

Sb01g019120

Cleavage

HD-ZIP

Sb01g050000 Sb03g002660

Cleavage Cleavage

HD-ZIP HD-ZIP

Sb08g021350

Cleavage

Sb04g026450

Cleavage

Sb10g031030

Cleavage

Sb01g028080

Translation

Sb06g014420

Cleavage

HD-ZIP Protein of unknown function (DUF640) Stabilizer of iron transporter SufD/ Polynucleotidyltransferase Tetratricopeptide repeat (TPR)-like superfamily protein auxin signaling F-box 2

Sb03g001240

Cleavage

sbi-miR168

Class II aminoacyl-tRNA and biotin synthetases superfamily protein

TE D

sbi-miR393

KO ID

Sb09g003870

Cleavage

Sb03g040355

Translation

Sb04g033860 Sb01g009330

K09338

Ribosome biogenesis NA

K09338

NA

K09338 K09338

NA NA

K09338

NA

F-box/RNI-like superfamily protein

NA

NA

NA

NA

NA

NA

NA

NA AminoacyltRNA biosynthesis

K01876

K14485

Plant hormone signal transduction NA

K06063 NA

Spliceosome NA

Sb01g012170

Cleavage

GRF 2

NA

NA

Sb04g030770 Sb04g034800

Cleavage Cleavage

GRF 5 GRF 5

NA NA

NA NA

Sb10g001350

Cleavage

GRF 5

NA

NA

Sb10g006690 Sb01g019970

Cleavage Translation

GRF 5 hypothetical protein

NA NA

NA NA

Sb10g010620

Cleavage

MuDR family transposase

NA

Sb01g012650

Cleavage

poly(A) polymerase 1

K14376

NA mRNA surveillance

EP

NA

Cleavage Cleavage

carboxyl-terminal domain (ctd) phosphatase-like 2 chromatin protein family GRF 1

AC C

sbi-miR396

Pathways

RI PT

sbi-miR166

Inhibition

SC

sbi-miR160

Target

M AN U

miRNA

Sb04g023010

Cleavage

Sb09g011890

Cleavage

Proline-rich spliceosomeassociated (PSP) family protein / zinc knuckle (CCHC-type) family protein Rhodanese/Cell cycle control phosphatase

19

K13128

NA

NA

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Translation

Sb01g048750

Cleavage

Sb03g004310

Translation

Sb01g039690

Cleavage

laccase 17

Sb03g039520 Sb03g039530

Cleavage Cleavage

laccase 17 laccase 17

Sb09g022510

Cleavage

laccase 17

Sb01g010910

Translation

protein-protein interaction regulator family protein

NA

NA

NA

Cleavage

AC C

EP

TE D

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Sb01g035350

NA

20

NA

RI PT

Copper/zinc superoxide dismutase * Transcription Factors are highlighted in blue color.

sbi-miR398

NA

NA

NA

NA NA

NA NA

NA

NA RNA transport, mRNA surveillance

K13114

SC

sbi-miR397-5p

Sb03g024530

superfamily protein Tetratricopeptide repeat (TPR)-like superfamily protein CBL-interacting protein kinase 9 cellulose synthase 1

K04565

Peroxisome

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Fig.1

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transcription factor activity RNA binding hydrolase activity, acting on acid… hydrolase activity, acting on acid… transcription regulator activity nucleotide binding nucleic acid binding hydrolase activity protein binding catalytic activity binding nucleus organelle intracellular organelle intracellular membrane-bounded… membrane-bounded organelle intracellular intracellular part cell cell part embryonic development secondary metabolic process reproductive structure development reproductive process reproductive developmental… signal transduction growth cellular amino acid and derivative… anatomical structure… response to endogenous stimulus post-embryonic development multicellular organismal process multicellular organismal… anatomical structure development developmental process regulation of metabolic process regulation of macromolecule… regulation of gene expression transcription regulation of cellular process biological regulation biosynthetic process cellular biosynthetic process regulation of biological process nitrogen compound metabolic… nucleobase, nucleoside,… macromolecule biosynthetic… cellular macromolecule… gene expression cellular macromolecule metabolic… macromolecule metabolic process primary metabolic process cellular process metabolic process cellular metabolic process

M AN U

SC

b

EP

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c

AC C

Biological Processes

Cellular Component

Molecular Function

a

0

RI PT

Fig. 2

20

40

60

80

22

100

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Fig. 3

a

b

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Fig. 4

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HIGHLIGHTS (3 to 5; each point with max 85 characters)

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1. Expression analysis of eight conserved miRs in sorghum under control and drought conditions showed differential behavior across 11 gentoypes 2. The expression levels of sbi-miR396 and sbi-miR398 were the highest in all the genotypes 3. No regular pattern could be observed for all the genotypes under study suggesting differential intrinsic capabilities of various sorghum genotypes 4. The study emphasizes the need for miR-target based characterization of sorghum genotypes to develop varieties with improved drought tolerance.