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Darrel Fern

Specialised Subjects

Biochemistry, Biology, Biotechnology, Environmental Studies, Forensic Science, Genetics, Health, Plant Science, Sciences, Zoology

With a first degree in biotechnology, a Master’s degree in biochemical engineering from a UK University, and research experience in Stanford University, USA, I have had a very varied and exciting career in the life sciences. I am a scientist with more than five years experience of research, teaching and writing in the areas of biology, genetics and bio-medical sciences. I am passionate about everything that happens in this field of science, and by reading and writing about it, I keep abreast with the latest developments.

Drought, salinity and genes in rice.

1.0 Introduction
The chief crop that occupies the chiefly small holder landscape (less than 2 hectares in area) of Asia, with a cumulative area of ~ 130 million hectares, is rice (Oryza sativa). Smallholder farms are primarily found in dry climatic conditions where the cropping cycles are partially or fully dependent on the availability of rain. Consequently, of the many abiotic barriers that smallholder farmers face in their farming livelihoods, drought takes precedence as one of the most impactful consequences on harvests and consequently their economic sustainability. It is estimated that 18 million metric tonnes of rice amounting to US$3600 is lost annually due to drought-induced harvest losses. Further, the additional effects of drought extend to the coping mechanisms that poor farmers employ for economic sustainability, such as the selling of livestock and personal possessions and to the trickling effects of poverty that increase malnutrition, susceptibility to disease and compromise the education of children.

One clear strategy for the improvement of productivity and livelihoods is to undertake a comprehensive genetic analysis of locally-available rice varieties for the dissection of genes or loci (regions of one or many genes) responsible for the favourable drought-tolerant traits (for example, plants with deeper rooting for continuous water intake), for future incorporation in to plant breeding programmes. Many of the popular locally-adopted rice varieties, which produce strong harvests, are drought susceptible. The production of crosses between a drought-resistant and a susceptible variety will create a progeny population that may contain a portion of the genes that promote drought resistance, which could be identified by a combination of physical growth parameters and subsequently DNA markers (tags) that are tightly linked to the genes/loci of interest. Further back crossing (crossing of drought-resistant progeny with the popular variety) will ensure that the favourable characteristics of the popular variety are retained/co-exist with the incorporated drought-resistant characteristics.

2.0 Drought and the rice plant
Drought can be described using a spectrum of definitions. One of the best descriptions for drought is as follows:  “ Drought is an interval of time, in the order of months or years in duration, during which the actual moisture supply at a given time or place consistently falls short of the climatically appropriate moisture supply” (Palmer, 1965). The collation of data from 107 countries has estimated a staggering 220 million people are affected by drought annually, Sub-Saharan Africa being the worst affected region (Samra, 2004).

It is estimated that 2500 litre s of water is needed to produce 1 kg of rice (Bouman and Aureus, 2009). Therefore, rice as a crop has a high demand for water for strong productivity. The high requirements of rice systems for water can be attributed to four primary functions, namely evapo-transpiration, seepage and percolation, land management practices, and inefficiency of water usage at the hands of farmers which further increases the demand for water in rice cultivation (FAO, 2004). A plant’s resistance/susceptibility to drought is a complex compilation of traits that are determined by several factors including precipitation, evaporation/transpiration, soil nutrient availability, soil properties and other biotic and abiotic stresses (Price et al., 2002).

Previous literature has identified a multitude of physiological mechanisms by which yields are compromised including spikelet sterility, absence of dehiscence, inhibition of pollen tube elongation, reduced starch mobilisation, inferior efficiencies of photosynthesis resulting from stromatal closure and decreases in chlorophyll content and deficiencies in root growth (Bennett et al., 2005; Zhou et al., 2007; Pirdashti et al., 2009).

3.0 Salinity and the rice plant
Salinity is a one of the major obstacles in agriculture with 40% of irrigated lands affected by salinity. Saline soils have a high content of chlorites and sulphates of magnesium, calcium and sodium (Thiruchellam and Pathmaraja, 2003).  Saline-alkali soils usually contain high levels of sodium retention with s odium carbonate the highest salt ingredient that pushes the soil pH up to a maximum of 10.5 (Thiruchellam and Pathmaraja, 2003). Table 2 provides details of the effect of different levels of salinity on a rice plant.

Although rice is considered as a moderately salt-tolerant crop, even an increase of 1 dS/m of EC over 3 dS/m results in a reduction in yield of 12% (Maas and Hoffmann, 1977). The estimated harvest shortfalls are estimated to reach a maximum of 50% when the salt content ranges between 6 – 10 dS/m (Thiruchellam and Pathmaraja, 2003). The many other traits that manifest changes due to high salinisation include reduced tillering, lower number of spikelets per panicle, fertility, and inferior levels of branching in panicles (Walia et al., 2007).

The retaliatory mechanisms in molecular plant physiology at the incidence of salt stress are diverse and modulated according to the plant genotype. The effect of salinity on a plant cell can be described by its response to ionic and osmotic stresses imposed by the higher accumulation of salts (Moradi and Ismail, 2007). The increase in Na+ and Cl- ions in guard cells, which induces a reduction in turgor pressure, is projected to be one of the primary causes of diminished stromatal conductance (increased resistance) (Moradi and Ismail, 2007). Consequently, the net photosynthetic rate of the plant decreases due to the reduced conductance of CO2 to photosynthetic tissue. A separate physiological change that is instigated due to salt stress is the increased efflux of reactive oxygen species (ROS). ROS- scavenging enzymes such as the superoxide dismutases (SODs) manifest reduced activity in rice seedlings during salt stress. Due to the susceptibility of plant physiology to ROS with the oxidation of enzymatic systems including photosynthetic proteins and premature programmed cell death (PCD), the maintenance of pools of anti-oxidants such as ascorbate and glutathione is upregulated during salt stress, with the enhanced transcription of genes mediating the regeneration processes of key anti-oxidants (Chen and Gallie, 2004; Moradi and Ismail, 2007).

4.0 Drought and salinity resistance
Previous literature has highlighted several key characteristics that appear to be upregulated during drought periods including rapid leaf rolling, stromatal closure, higher water use efficiencies, secretion of a thicker epicuticular wax, osmotic adjustments, dehydration tolerance, enhanced membrane stability, thicker and deeper rooting and photoinhibition resistance (Babu et al., 2009; Price et al., 2002). Deep rooting is of particular importance to upland varieties in drought- prone farm land, but is of less significance in low land ecosystems which contain hardpans that restrict root growth (Price et al., 2002). In general, many of the observed phenotypes appear to adhere to Le Chatalier’s Principle where the system (plant) initiates several adjustments to minim ise the damage caused by the perturbation (drought) such as the rolling of leaves and stromatal closure (Price et al., 2002).

All drought- resistant traits appear to originate from three separate mechanisms, tolerance to, escape from (early flowering) and avoidance of water stress (maintenance of water potential), or two or more of the pathways in tandem (Levitt et al., 1980; Price et al., 2002). In the study by Levitt et al. (1980) drought tolerance is further divided into dehydration tolerance and dehydration avoidance. However, there is no consensus on drought resistance at the holistic (whole plant) or reductionist (gene) levels, although the duration of drought appears to play a leading role in the choice of coping mechanism: It is suggested that short drought periods invoke a ‘tolerance’ or ‘escape’ strategy, whereas longer durations of water stress appear to incite drought avoidance mechanisms such as increased water uptake and conductance and decreases in water potential that arises from the accumulation of osmolytes (Degnkolbe et al., 2009; Price et al., 2002).

It is postulated that there is strong genotype variability of both drought stress tolerance and avoidance. In addition, there is an adaptable component that supplements the innate drought-resistive mechanisms in a growth stage (vegetative/reproductive) specific manner and the phenology of the cultivar, i.e. the changes in physiology both at the whole plant and molecular levels due to climatic variability play a crucial role in the plant’s capacity to withstand water stress. Significant variations in the onset of drought and the severity and duration of water stress (high environmental variability) provide a further obstacle to the adaptability of cultivars and, consequently, the extent of independence of, as well as interplay between, pathways that mediate stress-resistive responses at the molecular level could be of vital importance to the extent of drought resistance (Degenkolbe et al., 2009).

5.0 The molecular basis for drought and salinity resistance
Several high throughput studies have unearthed a wealth of microarray data on genes that are actively transcribed during drought. Studies on Arabidopsis thaliana and rice have identified several drought-induced gene families that can be broadly classified as drought-tolerant/resistant genes and regulatory gene families that encode for transcription factors and upstream regulatory enzymes including protein kinases and phosphatases. The families of drought-resistant proteins that have upregulated expression during drought conditions include chaperones, RNA- binding proteins, osmolyte biosynthesis proteins and detoxification enzymes (Shinozaki et al. 2007). In a separate study, a multitude of gene families were demonstrated to upregulate their expression as a response to drought, namely those encoding for transcription factors, proteins that confer protection against oxidative stress (superoxide dismutases, glutathione S-transferases), metal detoxification proteins (metalothioneins), transporters (ATP- binding cassette proteins, aquaporins), late embryonic stage proteins, heat shock proteins, signal transducers and regulatory enzymes (kinases, phosphatases) (Gorantla et al., 2006).

Drought- inducible pathways appear to have a high degree of crosstalk with cold-stress and salinity-induced pathways. It is inferred that a majority of abiotic stress- induced pathways in plants are absisic acid (ABA) mediated and are upregulated via several upstream transcription factors including the AREB, MYC and MYC proteins (Gorantla et al., 2006; Shinozaki et al., 2007). Parallel to ABA- dependent drought tolerance, the ABA-independent pathways also appear to play a role in drought-induced gene expression through the actions of DREB (AP2/ERF) and NAC transcription factors (Shinozaki et al., 2007). Many of the drought- inducible transcription factors and downstream proteins that mediate drought tolerance have been incorporated into drought-susceptible varieties through conventional breeding programmes and via the production of transgenic lines containing the foreign genes of interest, with varying degrees of success (Shinozaki et al., 2007; Karaba et al., 2007).

6.0 Breeding for drought and salinity tolerance
Drought and salinity tolerance can be designated as complex traits due to the interplay of multiple pathways that mediate a plant’s capacity to withstand these abiotic stresses (Witcombe et al., 2008). Consequently, the identification and characterisation of alleles/QTL that confer tolerance of either drought or salinity, suffer from the complexities of secondary traits and molecular mechanisms mediating stress tolerance, low heritability of traits under stress, variations in field studies and time constraints of crop cycles (Ribaut et al., 1997). However, several traits have been utilised as marker phenotypes of drought resistance including cell membrane stability, ABA content, stromatal regulation, leaf water potential and root morphology (Yue et al., 2006). Research to discover drought-resistant rice cultivars has identified several varieties with superior capacities to withstand abiotic stresses and has produced several loci of interest for introgression to popular high-yielding varieties.

Many QTL associated with drought tolerance have been identified in several other studies. An individual QTL on chromosome 9 was demonstrated to be associated with spikelet fertility and expanded growth of both roots and shoots during drought (Jena and Mckill, 2008). In another study, 32 QTL contributing to grain yields and associated traits (spikelet number per panicle, spikelet fertility, and panicle number) were identified with up to 14% phenotypic variance (Zou et al, 2005). In a mapping population of a Lemont (indica) and Teqing (japonica) cross, alleles that were transferred from the Lemont line to the recurrent parent (Teqing) conferred superior levels of drought tolerance (Xu et al., 2005). An individual QTL (qtl 2.1) localised to a ~ 10 cM region on chromosome 12, accounting for 51% of phenotypic variance, was demonstrated to contribute broad levels of drought tolerance in a Vandana (50% japonica, 50% aus) and Way Rarem (indica) cross (Bernier et al., 200 7). In the study by Yue et al. (2005), a large- effect QTL on chromosome 9 which contributed to yield and biomass was identified, but there was low association of the QTL to phenotypic variance (14– 25%).  A separate QTL centred on chromosome 1 was responsible for superior yields with 32% phenotypic variance (Kumar et al, 2007).

Salt tolerance is predominantly found in traditional indica varieties. Most rice, in particular the japonica varieties are susceptible to salt stress, which can range from moderate to high depending on the inorganic parameters of the soil (Lee et al., 2007). Salt tolerance is a polygenic trait in traditional varieties such as Pokkali, Nona Bokra and Kararata and it has further been demonstrated that there is strong heritability of the salt- tolerant phenotype (Gregorio and Sensdhira, 1993; Lee et al., 2007). In spite of the favourable circumstances for breeding, the transfer of stress- tolerant alleles and QTL from the traditional varieties to japonica counterparts has been laborious due to the co-inheritability of other undesirable agronomic traits in to the progeny population (Lee et al., 2007).

Several QTL have been mapped that provide higher vigour in saline soil conditions. One of the strongest QTL (saltol) localised to chromosome 1 has been demonstrated to be accountable for 64– 80% of the variance in phenotype (Bonilla et al., 2002). The saltol locus has been identified in several varieties including Pokkali and Nona Bokra (Bonilla et al., 2002). An independent QTL, containing the skc1 gene encoding for a HKT- type transporter has been demonstrated to mediate K+ homeostasis under salt stress (Jena and Mckill, 2008). A recombinant in-bred line derived from a cross between Milyang 23 (indica) and Gihobyeo (japonica) was demonstrated to contain high levels of salt tolerance at the seedling stage due to the presence of two QTL (qST1 and qST3) which mediated 35.5– 36.9% of the phenotypic variation (Lee et al., 2007). The qST1 locus was obtained from the Gihobyeo parent, whereas the qST3 allele was derived from the Milyang 23 parent (Lee et al., 2007). A further 10 QTL that governed five heritable traits that contribute to salt tolerance, namely Na+ and K+ uptake, Na+ and K+ concentrating mechanisms and maintenance of the Na+:K+ ratio were identified by Koyoma et al. (2001). Three further QTL mapped to chromosomes 1,6 and 7, associated with Na+ and K+ uptake, were characterised from the F2 and F3 generations of a cross between a salt-tolerant indica variety, Nona Bokra and the salt-susceptible japonica variety, Koshihikari, that conferred higher levels of survival of seedlings (Lin et al., 2004). In the same study, an additional eight QTL were demonstrated to contribute to traits mediating favourable root and shoot architecture. Large- effect QTL for shoot Na+ and K+ concentration were mapped to the qSNC-7 and qSKC1 QTL, respectively, with 48.5% and 40.1% of phenotypic variation explained by the respective loci (Lin et al., 2007).  A further three QTL on chromosomes 1,5 and 7 that contribute to salt tolerance were identified by three separate studies (Lin et al., 1998; Zhang et al., 1995; Prasad et al., 2000). All of the above salt- tolerant QTL appear to counter salt stress exclusively during the vegetative stage and no QTL responsible for salt tolerance during reproductive stages have been identified so far.

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