Maintenance of stress related transcripts in tolerant cultivar at a level higher than sensitive one appears to be a conserved salinity response among plants

Abstract

Response of plants towards salinity is multigenic in nature with its various components playing diverse roles in stress perception, relay or response. For the purpose of dissecting the genetic determinants of salinity response in crops, the family Brassicaceae presents an excellent model since significant inter- and intra-specific variations have been reported for salinity tolerance. Using these intraspecific variations of Brassica, we show that one of the possible mechanism by which a genotype is able to exhibit tolerance better than another is by keeping the basal levels of stress responsive transcripts higher than the sensitive genotype. This is quite reflected when we analyze members of a specific pathway such as SOS pathway or even when we extend the analysis to a range of molecules including those playing important role in stress perception, signal transduction or stress response. However, these investigations need to be extended to genome level transcript analysis to further validate the hypothesis of “well preparedness” in tolerant genotypes and we propose the suitability of Brassica genotypes for this endevours.

Abiotic stresses such as salinity, drought, flood and extremes of temperature pose serious threats to agriculture worldwide. Amongst these stresses, salinity has accounted for 30% loss of useful agricultural land globally and these figures are expected to rise further by 2050.¹ Breeding crops for stress tolerance is one of the core activities of varietal improvement programs across the globe. During the past decade, there has been a clear progression in our understanding of plant science from plant physiology to plant genomics, wherein attempts have been made to connect plant genetics through genomics to plant breeding.^2,3 Initial studies for crop improvement focused only on the transfer of a single gene at a given time, but presently multigene transfer to crop plants is a pleasant reality.^4,5

Plants show great variability in their tolerance towards salinity which is reflected in their differential growth response under salt stress conditions.⁶ Among the cereals, rice is reported to be most sensitive while barley is most tolerant with relative tolerance further varying with growth stage (seedling stage being the most sensitive one). Variation in salinity tolerance is reported to be even greater in dicots than monocots. It is interesting to note that within Brassica species, considerable variation has been reported in salinity tolerance wherein the amphidiploid species have been found to be more tolerant than the diploid species.⁷ With the aim of understanding the molecular basis of salinity tolerance in crops, we have tried to explore the intra-generic variability in our laboratory using Brassica as the test system. We have recently shown that the homologues of SOS pathway are differentially expressed among the various Brassica genotypes, where we could establish a clear correlation between transcript abundance and relative salinity tolerance.⁸ Having established the variation in salinity stress response among the diploids, amphidiploids and the wild relatives of Brassica available with us, the next question we wanted to address was regarding the genetic determinants responsible for the differential salinity response among these Brassica genotypes.

Gene expression profiling holds promise for dissecting the regulatory mechanisms underlying important biological processes. Salinity response in plants is no doubt, multigenic, making it essential to analyze the response at the whole genome level. Such genome level studies have been made possible in model plants such as Arabidopsis and rice with the availability of the complete genome sequence.^9–13 A plethora of data has been generated employing microarray for such genomes (www.uni-tuebingen.de/plantphys/AFGN/atgenex.htm). Plant research is now entering an era where there is a need to combine the tools of molecular, genetic and evolutionary analysis. The need of the hour is to develop model systems in which natural variation in both environment and genome can be used to examine the integration of both genetic and ecological function(s). Such genome wide comparative analyses in recent past have given a meaningful insight into the basis of salinity stress response in case of Arabidopsis and its wild relative Thellungiella halophila.^14,15 However, such efforts can only be extended to other systems such as Brassica, once the high quality whole genome sequence is made available and gene array experiments are conducted at mass scale. The other alternative to overcome this limitation would be to pick up some selected conserved genes which may be used to perform limited transcriptome analysis among the diverse genotypes.

From perception of stress to cellular response, there are multitudes of factors involved, which constitute complex gene regulatory networks. To glean an insight into the stress cascade, representative genes of each class were picked up for northern analysis. The selectively chosen heterologous probes showed a clear specific hybridization signal on northern blot indicating their usefulness in expression analysis in Brassica genotypes (Fig. 1). The heterologous probes for histidine kinase (HK), proline rich extension-like receptor kinase-1 (PERK1) and Lectin like receptor kinase-like protein (LecRK) represent the genes involved in signal perception and their direct or indirect role in the process has been documented several times.^16–18 The basal level of transcripts for these receptors could be detected in all the Brassica genotypes. Most of the receptors showed high constitutive levels but a marginal upregulation under salinity in case of amphidiploids was observed (Fig. 1). This may be an indication of more efficient stress perception mechanisms in amphidiploids as compared to their diploid parents. The Brassica genotypes also exhibited a differential pattern of regulation for stress signaling candidates such as mitogen activated protein kinase (MAPK), serine threonine kinase (STK), calmodulin binding protein (CaM-BP), Zn-finger protein (Zn finger) and basic region/leucine zipper motif (b-ZIP factor) indicating unique pattern of gene regulation specific for each genotype. Previously, many studies have shown that protein kinases are important regulators during osmotic stress in plants.¹⁹ In plants, b-ZIP type of transcription factors regulate various processes such as pathogen defense, light and stress signaling, seed maturation and flower development.²⁰ Finally, a few representative effector genes involved in eliciting the desired stress response including Late embryogenesis abundant (LEA), Glutathione S transferase (GST), Vacuolar ATP synthase (VATP synthase), calmoduline binding protein (CaM-BP) and voltage-dependent anion channel (VDAC) were analysed. These proteins in both plants and animals are associated with tolerance to water stress resulting from desiccation and cold shock.^21,22 A comparative study of the expression profile of these transcripts revealed their differential regulation in diploid and amphidiploids species.

Figure 1.

Analysis of transcript abundance of selected genes involved in stress response as receptors, signaling components, transcription factors and effectors in shoot tissues of various Brassica genotypes in response to 200 mM NaCl. Northern blots containing 20 µg total RNA from shoots were hybridized with various probes viz; HK (DQ248962); LecRK (EF206780); PERK1 (EF206788); MAPK (EF206781); STK (EF206784); CaM-BP (EF206782); b-ZIP transcription factor (EF206783); Zn-finger (EF206785); LEA protein (EF206789); GST (EF206786); VDAC (EF206787) and VATP synthase (EF206790). The representative ethidium bromide (EtBr) stained gel has been shown as the loading control in the bottom panel. ‘−’ and ‘+’ on top of the lanes indicate the absence and presence of 200 mM NaCl stress for a period of 5 h, respectively.

Heat map analysis of transcript accumulation of these representative genes (mentioned above) picked up from all the levels of stress cascade has given some insight into the much complex network underlying stress response in the various Brassica genotypes. From this study, we can hypothesize that the possible reason for the ability of amphidiploids to withstand stress more effectively as compared to diploids can partly be due to their better evolution at the primary level of stress cascade i.e., they have a more efficient stress perception mechanism. Similar results have also been reported previously during the comparative genome analysis between Arabidopsis and its salt tolerant wild relative Thellungiella (salt cress) where it was suggested that stress inducible signaling pathway is constitutive and active in salt cress even under normal growth conditions without stress.^14,15 Similar to salt cress, our heat map analysis of constitutive vs. salinity induced transcript abundance of various stress related genes also reflected that transcript levels for selected stress responsive genes are relatively higher under normal growth conditions in amphidiploid species (B. juncea var. CS52; Fig. 2). These observed differences between diploids and amphidiploids gene expression patterns could be either the result of higher dose of a given gene (assuming that both genomes in the amphidiploids are expressed) or alternatively, from a change in regulatory machinery of the gene (if one genome is assumed to be silenced). However, the latter possibility seems to operate in our case since we have observed that some selected SOS genes also show reverse pattern of expression between diploid and amphidiploids under stresses other than salinity (Pareek, et al. unpublished data).

Figure 2.

Heat map showing relative transcript abundance under control and salinity stress conditions for genes involved in stress perception, relay and response as revealed from northern probing was analyzed using Mayday software. Note that most of the transcripts (mainly GST, b-ZIP transcription factor, PERK1, HK and VATP synthase) are more abundant amongst amphidiploids even under control conditions and show further induction in response to salinity. Heatmap on left (unstressed) has been sorted in increasing order from top to bottom based on transcript abundance value for B. juncea. The sequence of genes in the heatmap on right (salinity stress) has been kept same for comparison sake. Expression values of SOS1, SOS2 and SOS3 for the above heatmap have been taken from our recent publication.⁸

Based on these results and those published by others groups in various plant species, we may infer that maintenance of stress related transcripts in tolerant cultivar at a level higher than sensitive one appears to be a conserved salinity response among plants (Fig. 3). Our recent work related to transcriptome analysis in contrasting cultivars of rice²³ and Brassica⁸ has also supported this interesting hypothesis. However, it is imperative to extend the analysis towards proteomics and metabolomics before we can establish this fundamental aspect of salinity response.

Figure 3.

Cartoon depicting the “well preparedness” of the tolerant cultivar in terms of gene regulation (by keeping high transcript levels under non-stress i.e., uninduced conditions). This hypothesis has been validated in case of rice,²³ Arabidopsis¹⁴ and Brassica,⁸ and thus appears to be a conserved salinity response among plants.

Addendum to: Kumar G, Purty RS, Sharma MP, Singla-Pareek SL, Pareek A. Physiological responses among Brassica species under salinity stress show strong correlation with transcript abundance for SOS pathway-related genes. J Plant Physiol. 2009;166:507–520.