Chilling and Drought Stresses in Crop Plants: Implications, Cross Talk, and Potential Management Opportunities

1Crops grown under open environments often pass through periods of abiotic stress during their life cycle. Such stresses many a time overlap so that the crop growth and productivity of the crops is adversely affected. Plants pass through a series of morphological, physiological, biochemical, and molecular changes in a quest to mitigate such adversities of the abiotic stresses. A lot of work reports the adaptive responses of crops plants to different abiotic stresses wherein emphasis has been laid on individual stress factors (e.g.,;;;;; ). Nonetheless, understanding the plant responses to combined stress factors is inevitable for enhancing the adaptation of plants under field conditions. Crops experience periods of extreme low temperatures in many regions of the world, and are exposed to limited water availability owing to either drought or disturbed water movement and uptake under low ambient temperatures. Such an exposure of plants to chilling and drought simultaneously hampers plant growth, and hence is detrimental to productivity. Studies show that primarily chilling and drought pose a similar impact on stomatal development and leaf growth, nevertheless, the mechanisms of drought-induced changes in some physiological processes are quite different than those induced by chilling. A little is known about the combined effect of chilling and drought stresses on plants, and whether or not plant responses to these are unique or shared is also unclear. Plants may exhibit common molecular and physiological responses on exposure chilling and drought (shared response), others may be specific to a given stress factor. In general, chilling stress thermodynamically declines the kinetics of several physiological as well as metabolic processes occurring in plants. It severely reduces the rate and uniformity of germination, hampers seedling vigor, and delays ontogenic plant development, resulting in severe crop yield losses.

2Drought stress in plants is characterized by reduced leaf water potential and turgor pressure, stomatal closure, and decreased cell growth and enlargement. Drought stress reduces the plant growth by influencing various physiological as well as biochemical functions such as photosynthesis, chlorophyll synthesis, nutrient metabolism, ion uptake and translocation, respiration, and carbohydrates metabolism. Nevertheless, plants experience water deficit not only during drought, but low temperature may also cause turgor stress at cellular level owing to poor root hydraulic conductance and diminished root activity.

3Oxidative stress is crucial in relation to chilling- and drought-induced injuries in plants. Drought and chilling stress exacerbates ROS production in plants’ cell. Excess production and the accumulation of ROS causes oxidative damage at cellular level, disrupts cellular membranes, and leads to enzyme inactivation, protein degradation, and ionic imbalance in plants. The ROS disturb the cellular macromolecules including DNA, and hence may cause deletion of bases due to alkylation and oxidation which are linked with various physiological and biochemical disorders in plants. Plants possess a highly efficient and sophisticated antioxidative defense system to control the overproduction of ROS. The ROS-induced damages and disruption of cellular homeostasis are alleviated by the action of different enzymatic (e.g., catalase, CAT; superoxide dismutase, SOD; peroxidase, POD; glutathione reductase, GR; glutathione peroxidase, GPX) and non-enzymatic (e.g., ascorbic acid, carotenoids, α-tocopherol, and glutathione content) antioxidants. In plants, the levels of ROS are regulated by their production rate as well as the extent of their detoxification by enzymatic and/or non-enzymatic antioxidant systems. Mechanism of ROS production and its scavenging by high antioxidative capacity has been associated with tolerance of plants to abiotic stresses.

4Studies involving chilling and drought as absolute stress factor have been well documented for several crops. Nevertheless, little focus has been laid on their combined effect, and plant response to both of these. Some recent studies ponder that the physiological and molecular response of the crops to combination of two different environmental stresses is unique so that such responses cannot be directly extrapolated when looked in the context of any of these stress applied individually. Based on the available work on the effect of multiple stresses on crop plants, an effort is made to comprehend the current understanding of chilling and drought stress on crop plants. We have tried to give a general overview of shared and unique responses of crop plants to drought and chilling stresses and discuss some possible mitigation strategies to cope with these stresses for minimizing their damage on crop plants.

5Water scarcity and suboptimal temperature at any growth stage of crop plants evoke negative effects on crop growth and development. Such detrimental effects on plants depend on the magnitude of stress and its period and the plant growth stage. Such effects are elicited in terms of morphological, physiological, biochemical, and molecular processes in plants.

6Some field crops are extremely sensitive to chilling and drought particularly during germination and early phases of seedling development. Each seed requires optimum temperature and soil moisture for germination. Chilling stress severely impairs the germination and often lowers the seedling vigor, delays plant development and ultimately causes severe yield losses. Chilling stress is known to thermodynamically limit the kinetics of various physiological as well as metabolic functions in plants. Chilling stress evokes shoot water deficit due to its thermophilic nature and reduced root water uptake.

7Drought stress impairs germination by limiting water imbibition, and reduces seedling vigor. Both chilling stress and limited water supply results in disturbance of osmotic balance, impaired metabolic activity at cellular level and excessive ROS production leading to alterations in DNA, RNA and protein structures, membrane damage, reduced respiration and less ATP production ultimately causing loss in seed germination and vigor.

8Both chilling and drought stress are known to cause significant reductions in plant growth and development. Various studies have reported the negative effects of chilling stress on growth of rice, cotton, tomato, potato, muskmelons, and sugarcane. Likewise, drought stress has also been reported to reduce plant height, leaf area, stem diameter, and plant biomass in different field crops. Our recent investigation on maize (Hussain et al., unpublished) has revealed that chilling as well as drought stress severely damaged the shoot and root growth attributes, compared with control. The interactive damage caused by chilling and drought was found to be additive for all growth attributes.

9Chilling stress may cause necrotic lesions on leaves, delay leaf development, prolong cell cycle with decreased cell production, induce wilting, and increase susceptibility to pathogens and diseases. Decreased rate of both cell division and elongation causes smaller leaf area, while slow leaf initiation rate leads to reduced number of leaves under periods of chilling stress. Under drought stress, leaf growth is mainly restricted by decreased leaf water potential. Whilst, interrupted water flow from xylem to other elongating cells and reduced turgor pressure under severe drought stress, leads to reduced cell elongation and smaller leaf area in crop plants.

10Chilling stress restricts root growth by decreasing root length, biomass, and morphology, which ultimately reduces volume of the root system for exploring the nutrients and water. Nevertheless, the reported effects of drought stress on root growth have been diverse. For instance, drought stress increased the root length of Catharanthus roseus and sunflower, but did not affect the root growth of wheat and maize. Increase in root length under drought conditions have been attributed to increased abscisic acid content in roots. stated that root dry biomass of Populus species was reduced under mild and severe drought stress. In general, varieties having characters of deep and prolific root system were better able to sustain growth and yield under water-deficit conditions.

11Water deficit as well as sub-optimal temperatures reduce the growth and metabolic activities leading to reduction in agronomic and yield attributes of the crops. reported that drought and chilling stress severely impaired the barley productivity. The chilling stress in chickpea at reproductive stage led to flower abortion and poor pod setting. In temperate growing areas, cold temperature is responsible for 30–40% reduction in rice yields because chilling stress resulted in degeneration of spikelets, panicle deformation and poor spikelet fertility. In grain crops, low temperature stress during reproductive development may induce flower abscission, ovule abortion, pollen sterility, pollen tube distortion, poor fruit set, and hence reduced final yields. found that chilling stress during stem elongation reduced the internode extension, caused spikelet death, and lowered biomass accumulation and grain yield in wheat. Low temperature at jointing and booting stages of wheat significantly decreased the number of productive tillers per plant, and negatively affected the development of young spikes.

12Drought–induced decrease in yield and yield components of maize, wheat, sugarcane, sunflower, peanut, and cotton has also been reported. In legumes, drought can dramatically reduce the seed yield by limiting flower and pod production, increasing flower and pod abortion, and reducing seed size. It has been estimated that drought stress may decrease the global chickpea production by 33%. In soybean, drought stress significantly reduced the number of branches and total seed yield.

13Conclusively, both drought and chilling temperature adversely affect yield and yield components of crop plants. Reductions in these traits could be the outcome of stress-induced modifications in metabolic and physiological activities and corresponding negative implication for plant reproductive organs with reduced productivity. Overall, onset of either drought or chilling is injurious to plants, nevertheless, plant reproductive stage is perhaps the most sensitive stage to these stress factors than pre- and post-reproductive stages.

14Drought and chilling stresses alter the normal rates of photosynthesis and other gas exchange attributes in crop plants. While working on sugarcane, Sales et al., (2013) found that drought stress alone or in combination with chilling stress severely affected the leaf gas exchange, photochemical activity, CO2 assimilation, generation of energetic pressure at the PSII level, electron transport rate and transpiration. Under chilling stress, low photosynthetic rates in crop plants have been attributed owing to poor stomatal and mesophyll conductance of CO2, impaired chloroplastic development, restricted metabolite transport, decreased quantum efficiency and the quantum yield for CO2 assimilation. argued that chilling stress reduces photosynthetic efficiencies and enhances photo-inhibition process due to over excitation of thylakoid membranes and subsequent impairment of photosynthetic function. Moreover, chilling slows down the rate of CO2 fixation and restricts the NADP+ supplement available to accept electrons from the electron transport chain. On the other hand, drought stress reduces photosynthesis by inhibiting leaf area and decreasing the rate of photosynthesis per unit leaf area. Drought stress slows down carbon fixation rate by directly restricting metabolism or by limiting the CO2 entry into the leaf. Imbalance between capturing and utilization of light, decrease in Rubisco activity, alterations in the photosynthetic pigments, and damage to photosynthetic apparatus are some of the major reasons for drought-induced reductions in photosynthesis. However, the drought-induced limitations of photosynthesis through metabolic distortions are more complex than stomatal limitations, and these mainly occur through reduced synthesis of photosynthetic pigments. During drought stress, limited intercellular CO2 concentration leads to the accumulation of reduced photosynthetic electron transport components that can potentially reduce the molecular oxygen, resulting in the production of ROS, which are deleterious to photosynthetic apparatus. Although, chilling and drought stresses have some unique effects on photosynthetic functions of plants, yet both these stresses are detrimental for plant photosynthetic apparatus and light harvesting mechanism.

15Relative water content, leaf water potential, transpiration rate, stomatal conductance, and leaf and canopy temperature are the important attributes describing plant water relations. reported severe reductions in leaf water potential of sugarcane under the simultaneous occurrence of cold and drought stresses, compared with individual stress factor. Generally, the chilling-sensitive plants exhibit water stress symptoms under low temperatures, which are triggered by reduced root hydraulic conductivity followed by severe decline in leaf water potential and loss of turgor pressure. and also reported that decline in root hydraulic conductivity and loss of stomatal control caused chilling-induced wilting in plants. stated that low temperature-induced reduction in vapor pressure deficit between the leaf surface and the atmosphere decreased transpiration rate and root water uptake drastically. Chilling stress caused shoot water deficits in maize plants by significant reductions in root water uptake instead of leaf transpiration. Likewise, reported that under chilling stress, root water uptake decreased in drought hardened plants similar to non-drought hardened plants, while transpiration rate was lower in drought hardened plants only.

16Exposure of drought stress has also been known to substantially decrease the relative water content, turgor pressure, leaf water potential, and transpiration rate in a number of plant species. reported that drought stress significantly declined the water potential of soybean roots, leaves, and pods, compared with control. Likewise, observed that tissue water contents were decreased linearly with increase in drought severity. In short, chilling as well as drought stress cause disturbances in plant water relations; nevertheless such effects are likely to be more severe under the combination of both of these factors.

17In addition to reduced plant growth and productivity, the nutrient uptake behavior of plants is also impaired under chilling and drought stress. Temperature affects the physio-chemical and microbial processes in soils which may modify the plant-nutrient relationships. Poor root system of plants under chilling stress reduces the uptake of several nutrients, including N, P, and K. Under chilling stress, shoot growth of maize seedlings was reduced by the direct effect of low temperature on shoot meristems and by restricted supply of nutrients via roots. concluded that reduced root length, low hydraulic conductance, poor root branching, and thicker root axis under chilling stress lead to reduced mineral nutrient uptake in plants. However, the variations in such effects may arise based on plant species, stress period, physiological plant growth stage, or the frequency of changing the nutrient solutions.

18Drought stress also decreases the availability, uptake, transfer and metabolism of nutrients. Exposure of drought stress in plants generally decreases both the uptake of nutrients by roots and translocation from roots to shoots. Drought-induced reductions in uptake and translocation of macro-nutrients (N, P, and K) have been reported in various plant species. Low soil moisture availability under drought stress reduced the root growth and the rate of nutrient inflow in terms of both per unit of root length and root biomass. Moreover, drought stress causes the difference in active transport and membrane permeability of cations (K+, Ca2+, and Mg2+), thus resulting in decreased absorption of these cations via roots. Drought stress limits the activities of enzymes involved in nutrient assimilation. For instance, the nitrate reductase activities in the leaves and nodules of dhainicha (Sesbania aculeata L.) and common bean (Phaseolus vulgaris L.) were significantly decreased under drought stress. Apart from macro-nutrient, drought can induce the deficiencies of some micro nutrients, i.e., Mn, Fe, and Mo whereas these micronutrients become increasingly available under well-watered conditions due to their conversion to more soluble and reduced forms. In summary; there exists a significant interaction among the nutrient acquisition, soil moisture content and soil temperature. Both chilling and drought stresses decreases the availability, uptake, translocation and metabolism of nutrients in plants. Most of the previous reports have focused on the nutrient relation with individual stress, while rare studies have examined the nutrient acquisition in plants under combine stress factors. Therefore, quantification of the effects of combined chilling and drought stresses on the nutrient uptake, transport, and assimilation need to be investigated further.

19Imbalance between generation and safe detoxification of ROS represents metabolic state that is referred to as oxidative stress. Excess ROS accumulation causes protein oxidation, membrane lipid peroxidation, DNA and RNA damage, and may even lead to cell death. Hydrogen peroxide (H2O2), singlet oxygen (1O2), superoxide radicals (O2-), and hydroxyl radicals (OH-) are the major ROS produced in plants under unfavorable environmental conditions. Generally, the production of ROS is linear with the severity of stress conditions.

20Chilling and drought stresses cause oxidative damage by overproduction of ROS in plant cells. reported that chilling stress negatively affects the membrane properties and enzyme activities resulting in tissue necrosis. Under suboptimal growth temperatures, bundle sheath proteins were more sensitive to oxidative damage than those of mesophyll in maize leaves. Overproduction of ROS under chilling stress cause serious cellular damages by rapidly reacting with essential cellular structures. stated that balance between the formation and detoxification of ROS is crucial to cell survival under chilling stress. Likewise, numerous studies have reported the higher ROS accumulation and oxidative damage under drought stress, owing primarily to limited carbon dioxide fixation and higher photorespiration. documented that considerably higher accumulation of ROS under drought stress resulted from the increased O2 photo reduction rate in chloroplasts.

21To cope with oxidative damage the plants have evolved excellent antioxidative defense systems under unfavorable conditions like drought and chilling stresses. Increased activities/levels of several enzymatic (SOD, APX, POD, CAT, GR, GST, GPX etc.), and non-enzymatic (reduced and oxidized glutathione, ascorbic acid, α-tocopherol, and carotenoids) antioxidants have been reported in plants to maintain the cellular homeostasis and to mitigate the oxidative damage. The specific role of various enzymatic and non-enzymatic antioxidants in drought and chilling stress tolerance of various transgenic plants is summerized in Table 1. Chilling and drought stress tolerance of many crops is regulated by abscisic acid induced higher antioxidant enzyme activities. In contrast, claimed that chilling stress activated the antioxidant defense response by causing damage to membrane components and ROS-induced protein denaturation. Previously, it has been observed that greater activities/levels of antioxidants were well concominant with chilling tolerance in rice, maize, cucumber, and tabbaco. Likewise, several authors documented the role of different enzymatic and non-enzymatic antioxidants in drought tolerance of maize, rice, wheat, coffea, cotton, beans, and alfalfa. This implies that maintaining higher levels of antioxidants may contribute toward stress tolerance in plants.

22Osmotic adjustment is the decrease of osmotic potential owing to the net accumulation of solutes in response to water deficit conditions. Under osmotic stress, plants maintain the water relations by accumulating various osmotically active molecules/ions such as soluble sugars, organic acids, proline, glycinebetaine, potassium, calcium, and chloride ions. Osmoregulation/osmotic adjustment helps plants to sustain growth, photosynthetic rate, and assimilate partitioning under stressful conditions. stated that osmotic adjustment is directly related to the accumulation of osmolytes in different concentrations, which largely depends on stress severity, plant type and the growth stage at which the stress is imposed. As chilling stress has been reported to disturb plant osmotic balance and cause dehydration stress, therefore, under chilling and drought stress, osmotic adjustment assists the plants to maintain higher turgor potential. reported that osmotic adjustment is crucial in postponing the dehydration stress under water limited conditions. Greater accumulation of osmolytes is generally linked with drought tolerance because these facilitate in maintenance of leaf turgor to improve conductance of stomata for efficient CO2 intake, and promote the water uptake ability of roots. reported that protein content in the tissues of chilling-sensitive plants are usually decreased with chilling stress, mainly because of reduced synthesis which, in turn, increases the level of free amino acids particularly proline. Endogenous concentrations of proline were linked with chilling tolerance capability in maize, and drought tolerance in rice, and pea. Proline mainly accumulates in younger leaves and is recognized as the most improtant solute against abiotic stresses. Drought stress alters the endogenous levels of glycinebetaine. Cotton cultivars with higher endogenous glycinebetaine concentrations were better adapted to drought conditions than those which accumulaed less glycinebetaine. Most of the studies have shown that osmotic potential of plants is more affected by drought as compared to chilling stress. However, further studies are needed to understand the alterations in plant osmotic balance under combined drought and chilling stresses.

23Crop growth and productivity is negatively affected by molecular alterations under various environmental stresses. Chilling and drought stress induce the expression of number of genes in plants, however, gene expression in response to both stresses are quite different. In Arabidopsis, expression of DREB1 (also called as CBFs or the c-box binding factors) and DREB2 families was affected in response to chilling and drought stresses, respectively. Likewise, identified the dehydration-responsive element with the core sequence A/GCCGAC as a cis-acting promoter element, which were regulated in response to drought, and chilling stresses. Expression of homeodomain leucine zipper (4D-Zip) protein, which interacts with CaCBFIB was highly regulated by drought and low temperature stresses. stated that cold temperature affects membrane fluidity, disrupts nucleic acid and protein structures, hinders water and nutrient uptake, and causes significant changes in the plant transcriptome. It also drastically disturbs the cellular metabolism either by directly decreasing the rates of biochemical reactions or indirectly by gene expression reprogramming. Chilling stress rapidly increased the levels of cytoplasmic calcium in Arabidopsis and alfalfa due to an influx of calcium from extracellular stores, suggesting that cell signaling and genes expression are altered under chilling stress. observed that expression of majority of pollen-specific genes remained unaffected under chilling stress. However, reported that response of plants to chilling stress was associated with changes in gene transcription of low molecular weight proteins. Differences in genes expression in response to stress also occur among plant parts. found that leaves and roots exhibited differential gene expressions during cold acclimation; and 86% of the cold-induced genes were not shared between leaves and roots. Chilling stress regulates the expression of ETHYLENE RESPONSE FACTOR/APETALA2 family transcription factors (e.g., CBFs), which may bind to cis-elements in the promoters of COR (cold-responsive) genes and induce their expression. Under cold stress, ICE1 (INDUCER OF CBF EXPRESSION1), was constitutively regulated in Arabidopsis, nevertheless, transgenic overexpression of ZAT12 down-regulated the expression of CBFs. Chilling stress also induces the accumulation of proline, which in turn induces the expression of genes having proline-responsive element (PRE and ACTCAT) in their promoters.

24Water deficit conditions also cause the fluctuations in gene expression (up- and down-regulation) in plants. Drought stress altered the expression of dehydrin/late embryogenesis abundant (LEA) genes and molecular chaperones, which protect from protein denaturation in cell. Drought stress regulates the production of ABA in roots that is transported to the shoots leading to stomatal closure and restricted plant growth. Drought-induced increase in ABA level regulates the expression of dehydrin genes (DHN 1/RAB and DHN 2), glycine rich protein gene, and a conserved element in proximal promoter region of gene RAB28. Under drought stress, miR398 was significantly up-regulated in peanut, while, it decreased the expression levels of isoforms Cu/Zn superoxide dismutase viz., AhCSD1-1/AhCSD1-2.1 and AhCSD2. found that drought stress up-regulated the expression of genes involved in osmolyte synthesis, aquaporins, signaling molecules and transcription factors (TFs), and genes coding for LEA proteins. Moreover, TFs of ERF/AP2 family are known to bind with drought-response element present in promoters of many drought-response genes. observed that gene expression of TFs belonging to AP2/ERF, bZIP, HD-ZIP, bHLH, MYB, NF-Y, EAR, NAC, and ZPT2 families was regulated under drought stress. reported that drought stress induces the expression of a variant of histone H1 (called H1-S), which is involved in regulation of stress-responsive gene and plays a crucial role in stomatal closure. reported that drought regulated the DNA demethylation in coding sequence of a glycerophosphodiesterase-like protein gene in tobacco, whereas, DNA hyper-methylation was regulated in pea under drought stress.

25In summary, profound changes in gene expression occur under drought or/and chilling stresses. Many stress-inducible genes are responsive to both drought stress and chilling, while some genes are only regulated specifically by drought or chilling stress. Overall, majority of the transcription factors were up-regulated under these stresses, a few transcription factors having a role in primary growth processes were down-regulated.

26Different abiotic stresses generally induce modify the gene expression in plants which can be stress-specific. Nevertheless, various points of crosstalk may occur amongst stress responses, and interplay of signaling pathways in plants. Cross talk refers to any instance of two pathways from different stress factors that converge and act in synchronization to alleviate the effects of stress. The plant responses to drought and chilling share various common domains including ROS and antioxidant response, calcium signaling, osmotic stress signal perception, transcription factors, hormones, dehydrins, and mitogen-activated protein kinase (MAPK) cascades, to name a few.

27Identification of cross talk between different signaling cascades is important in strengthening our knowledge on plants responses to a specific stress condition. In recent era, researches dealing with the cross talk between different abiotic stress signaling pathways have paid attention on genes/gene products involved in two different stress conditions, and thus are known to be part of a shared response. The transgenic up- or down-regulation of these genes indicate that they play key role in enhancing tolerance to multiple abiotic stresses. Therefore, these genes can be crucial in conferring plant tolerance against combined stress factors often prevailing under field conditions.

28Among four transcriptional regulatory systems in plants, two are ABA-dependent while two are ABA-independent, and the crosstalk between these regulatory systems has been shown by molecular and genetic evidence in various reports. Genetic analyses suggested that there is no clear line of delineation between ABA-dependent and ABA-independent pathways, and the contributing components may often cross talk or even congregate in the signaling pathway. Generally, the signaling pathways associated with chilling stress are reported to be rather less dependent on ABA than those involved in drought stress response. However, it is evident that some cross-talk, at least, does occur between different abiotic stress signaling pathways as the transcription of members of similar gene families is regulated and identical products are rather accumulated. For instance, the hydrophilic proteins (COR78 and COR15a) were accumulated in Arabidopsis thaliana, in response to both chilling and drought stress.

29Genomic analyses of stress-induced gene expressions using microarrays also have recently revealed crosstalk in stress-responsive genes. Many drought-responsive genes were regulated by high-salinity stress or ABA; however, only 10% of drought-responsive genes were indeed cold-inducible as well. In fact, ABA is involved in regulation of osmotic stress-responsive genes but it does not specifically play a key role in low-temperature stress response. Presence of ABRE (cis acting element) is generally important for ABA induced gene expressions.

30As both drought and chilling result in cellular dehydration, it is not uncommon to expect some overlap in various signaling pathways focused on delivering protection against cellular dehydration. This commonality presumably occurs due to the involvement of the CBF regulon in the abiotic stress response. reported that CBF4/DREB1D expression was induced by the occurrence of osmotic-stress. The CBF/DREB1 family genes are mainly regulated by chilling stress, while CBF4 has been reported to be activated by drought stress which provides crosstalk between CBF/DREB1 and DREB2 regulatory systems. The drought-induced expression of CBF4 is controlled by ABA-dependent pathways, indicating that CBF4 may function in drought stress response that relies on ABA accumulation.

31Calcium (a secondary messenger) and ROS network (which balances scavenging with production) are the ubiquitous components of both chilling and drought stress signaling pathways. Genes involved in Ca and ROS signaling constitute a crucial part of common molecular response of concurrent stresses in plants. Generally, rapid increase in Ca levels in plant cells has been reported with ABA, drought, and chilling. Plants are also evolved in high degree of control over ROS toxicity, to extent level that ROS are exploited as signaling molecules. The plant cell senses ROS through redox-sensitive transcription factors (e.g., heat-shock factors), which trigger functional proteins involved in the reinstatement of cellular homeostasis.

32investigated the drought- and low temperature-responsive genes in barley. About 44% genes specifically responded to drought, while only 3.8% were chilling specific, and 9.5% genes were shared between both stresses. Of 2,622 up-regulated genes, 13.0% were chilling specific, and 32.7% were drought specific suggesting that drought induced nearly three times as many stress specific changes as chilling. Genes involved in osmotic stress signal perception (bZIP, MYB29, and MYB-CC), calcium signaling proteins, signal transduction (e.g., MAPK), protection of membranes and proteins (e.g., LEA proteins), and antioxidative defense mechanisms (e.g., glutathione related genes) were commonly regulated in chilling and drought stresses (Table 2). Chilling stress specifically activated the genes involved in cell cycle and DNA processing, signaling cascades and transcriptional control, protein modification and destination, oxidative stress, cellular transport, and cold shock response (Table 2). However, genes related to transcriptional regulation, protection of membranes and proteins, cell growth and cell cycle, water and ion uptake and transport, and ABA synthesis were specifically activated by drought stress (Table 2). concluded that accumulation of dehydrins is regulated under chilling, salinity, and ABA exposure along with drought stress. They further reported that extremely hydrophilic proteins as dehydrins not only protect biomembranes in ripening seeds (LEA proteins) but also accumulate in the tissue of drought-tolerant plants during cold adaptation.

33Each stress is a multigenic trait and, thus, the manipulation of such genes may lead to alteration of a large number of genes as well as gene products. A deep insight into transcription factors regulating these genes, the products of the major stress-evoked genes, and cross talk between different pathways and responses should remain an area of future research activities. The fact that these cross talk genes regulate both chilling and drought response of plants highlights their importance in enhancing plant tolerance to combined stress factors. However, this should be validated by conducting studies on concurrent stresses wherein expression of cross talk genes needs to be examined.

34Both drought and chilling stresses have adverse effects on the growth and productivity of crop plants, and plant tolerance to these stresses might have certain protective mechanisms in shared. Plants often develop a cascade of strategies through physiological, biochemical, and molecular modifications at cell level to cope with these stresses. Studies have shown that mechanisms of plant tolerance to chilling and drought stresses can be regulated through developing tolerant plant genotypes, genetic modifications, seed treatments, application of plant growth regulators and compatible solutes and use of plant mineral nutrients.

35Integration of conventional, molecular and omic-based techniques can successfully be employed to develop tolerant plant genotypes. Conventional breeding has been instrumental in developing chilling tolerant cultivars in various crops. Various rice varieties, e.g., ‘Koshihikari’ in Japan, ‘Silewah’ in Indonesia and ‘Padi Labou Alumbis’ in Malaysia were released for chilling stress tolerance by conventional breeding method. Based on both open air and controlled conditions, reported that ‘EP80 × Puenteareas’ population in maize was an important source of low temperature tolerance. Implementation of the modern omics approaches and identification of QTLs/genes for chilling tolerance can significantly support crop improvement strategies aimed to develop high yielding cultivars under low-temperature condition. In rice, selection using different parameters led to the development of low temperature tolerant genotypes such as ‘HSC55,’ ‘M103,’ and ‘Jyoudeki’ based on low spikelet sterility. reported that phenotyping selection and SSR makers’ identification methods are helpful in screening of plant genotypes against chilling stress. They found three QTLs responsible for seed setting percentage under chilling stress by using a recombinant inbred line (RIL) population developed by tropical japonica × temperate japonica.

36Under drought stress conditions, estimating final grain yield is a simple and easy method for screening tolerant crops. Promising tolerant genotypes can be identified by direct selection of grain yield under water deficit conditions. Hence, conventional breeding approach includes the selection of crop plants for its yield and related attributes under drought conditions. In general, breeding strategies have been employed to manipulate the genetic makeup of crops for enhancing their tolerance against drought, however, marker assisted selection (MAS) proved better than classical breeding for screening traits associated with drought tolerance and enhancing drought tolerance in plants. The molecular basis of drought tolerance is generally based on QTL analysis and segregation mapping. The identification of QTLs associated with drought tolerance is an important tool for MAS of plants with desired characters. Marker assisted recurrent selection involves the accumulation of more superior alleles responsible for drought tolerance in plants. A large number of QTLs have already been identified for several drought tolerance traits, nevertheless, epiQTLs and Epistatic QTLs discovered in future can be used for molecular breeding. Sequential or simultaneous transfer of QTLs at different developmental stages of rice may lead to cultivars with increased drought tolerance capacities. Overexpression of an LEA gene has been reported to increase drought tolerance in rice. Furthermore, while discussing the constraints in breeding approaches, reported that low heritability of grain yield and lack of effective selection criteria are the main hindrances to develop drought tolerant cultivars. Similarly, reported that many previous trials on drought stress tolerance failed to impose severe stress, therefore true and precise drought-tolerant lines were not selected. Although, conventional breeding methods for drought tolerance have been used for a long time, nevertheless, advancements in genomics and molecular breeding approaches would have the significant role in the development of drought tolerant cultivars. Moreover, selection of crop cultivars for their unique responses to drought and chilling stress might be helpful for breeding programs to develop stress tolerant plant types.

37Plants respond to drought or chilling stress through a series of mechanistic changes in morpho-physiological features. Many genes that respond to drought or low temperature at the transcriptional level have been previously identified. However, the products of some specific stress-responsive genes could have significant roles in mitigating stress-induced damage through still elusive mechanisms. stated that ABA is generally produced under both drought and chilling stresses and has significant contributions in stress tolerance in plants. They further stated that overexpression of various regulatory elements induces drought and low temperature tolerance in plants. reported that overexpression of DREB1A enhanced drought and chilling tolerance in tobacco plants. Furthermore, genes encoding transcription factors were recognized better for improving stress tolerance in plants than various other genes responsible for cold tolerance in Arabidopsis and rice. stated that identification of QTLs involved in chilling stress tolerance may directly relate to acquire valuable genetic diversity in the physiological features in plants. Low-temperature restrictions have been overwhelmed by the support of cold-tolerant genes in genetically modified crops. Chilling stress induces three C-repeat binding factor genes or dehydration-responsive elements such as DREB1B (AtCBF1), DREB1C (AtCBF2), and DREB1A (AtCBF3) in Arabidopsis. Cold tolerance in tobacco can also be enhanced by the transgenic overexpression of the chloroplast omega-3 fatty acid desaturase gene. also reported the involvement of omega-3 desaturases to establish chilling tolerance in plants. described that the overexpression of Lefad7 in transgenic tomato could enhance the chilling stress tolerance. reported that plant response to low temperature is linked with the extent of change of gene transcription of low molecular weight proteins. described that CBF binds with COR genes carrying CRT/DRE elements, and the over-expression of these COR genes have an imperative role for chilling tolerance and cold acclimation in plants. These findings signify the roles of cold-inducible genes to protect plant cells against cold stress.

38Mechanism of drought tolerance is related to the drought induced regulations of multiple genes expression. Drought tolerant plants developed by recent genomics approaches require clear understandings of the genetic basis of drought tolerance, and identification of transcriptions factors associated with drought tolerance is important in this regard. Recently, an active form of DREB2 was found to transactivate target stress-evoked genes that led to better drought stress tolerance in Arabidopsis. also reported the involvement of water stress induced transcription factors (DREB2A and DREB2B) in the expression of various genes responsible for drought stress tolerance in plants. reported that responses of ABA-responsive elements-binding proteins (AREB) at both transcriptional and post-transcriptional levels of drought also determine the drought tolerance abilities of the plants. Moreover, described the importance of aquaporins in regulating the plant-water relations and could be promising targets for developing drought tolerant plant genotypes. Differential responses of two contrastive barley genotypes were found in low-molecular dehydrins under water deficit conditions which were involved in drought stress tolerance. enlighten the necessities to advent the whole genomics to identify the key genes involved in the regulations of drought stress tolerance in plants. Since, drought tolerance is a genetically controlled phenomenon, therefore, identification of QTLs for membrane stability and other functionally related phenomenal genes needs to be explored in future by using bio-informatics whilst many of them has already been characterized.

39Some studies have been shown that some genes expressed by both drought and cold stress concurrently whilst some are only responsive to individual stress. There have been several efforts in improving tolerance toward combine stress factors such as concurrent chilling and drought stresses. The improved tolerance to chilling and water deficit stress in Arabidopsis was attained by overexpression of CBF4. In addition, the overexpression of DREB1A has been found to have linked with drought and chilling stress in wheat, groundnut and tobacco plants. In order to improve tolerance against chilling and drought stress, applications of various molecular and engineering strategies are needed. It is well evident that the integration of traditional and molecular breeding approaches with genetic engineering and MAS may help the scientists to improve individual and concurrent environmental stresses in crop plants.

40Plants respond to drought and chilling stress via series of agronomic and physiological processes. Exogenous application of compatible solutes, plant growth hormones, plant mineral nutrient and seed priming can be effective in alleviating the adverse effects of drought and chilling stresses.

41Compatible solutes protect the plants from osmotic stress by not posing any detrimental effects on enzymes, membranes, and other macromolecules even at higher concentrations. Compatible solutes include glycinebetaine, soluble sugars, proline, sugar alcohols, trehalose and some organic acids. Many of the osmolytes are organic solutes in nature whilst some of the essential ions such as K+.

42Amongst various osmolytes, proline has been found to have multiple roles in plant stress regulation and tolerance potential. Proline and polyamines have been reported to impart chilling tolerance in maize. Proline retains the potential to relieve low temperature injury in cold-sensitive plants. Although, most of the chilling sensitive plants also accumulate proline but the levels of accumualation are not enough to induce cold tolerance, therefore, exogenous application of proline can be a potential option to enhance its level in plants. The beneficial role of proline in augmenting drought tolerance in rice and pea cultivars has been well reported.

43Soluble sugars and sugar alcohols serve as osomoregulators, signelling molecules, and cryoprotectants in plants and helps in cleansing stress-generated ROS. Chilling tolerance has been observed by increase of total soluble sugars in many plant species. Soluble sugars reduce osmotic potential and enhance water absorption under water limited conditions, and thus improve stress tolerance in plants.

44Exogenous application of glycinebetaine play critical roles in abiotic stress tolerance in plants by modifying antioxidant activities, membrance integrity, osmotic adjustment and ROS detoxification. reported that glycinebetaine accumulation was positively related to the cold stress tolerance in plants. Glycinebetaine improved the growth of Solanum tuberosum lants under low temperature conditions and freezing tolerance in A. thaliana. Its foliar applicationwas helpful in drought tolerance of several plant species. In short, application of campatible solutes such as glycinebetaine, soluble sugars, proline, and polyamines can be helpful in imparting drought and chilling tolerance in crop plants.

45Plant growth hormones have a dynamic role for increasing the ability of plants to acclimatize against abiotic stress conditions. Auxins (IAA), gibberellins (GA3), cytokinins (Cks), ethylene, abscisic acid (ABA), salicylic acid (SA), jasmonates (JA), and brassinosteroids (BRs) have been widely studied in plants under different abiotic stresses. These plant hormones are being used efficiently under stress conditions for enhancing crop production. Plants normaly synthesize various types of phytohormones under individual and concurrent abiotic stresses depending upon the strength of their defense mechanism. ABA is synthesized in response to both drought and chilling and contribute in stress tolerance of crop plants. ABA, JA, and SA often activate phosphoprotein cascade pathways that led to expression of genes associated with cold stress tolerance in plants. Greater chilling tolerance has been recorded in ABA-treated maize, and levels of ABA are increased in chilling tolerant genotypes. Under drought stress, ABA plays a key role in plant signaling betweeen shoot and root, that results in water-saving antitranspirant response, notably stomatal closure and reduced leaf expansion. ABA is also involved in robust root growth and other morphological modifications under drought stress. During drought stress, increased accumulation of ABA decreases the Cks contents while triggers the ABA/Cks ratio in plants. Under water stress conditions, plant growth regulator including BRs and ABA significantly improved chlorophyll content and increased water potential in soybean. reported that application of BRs led to considerable improvements in plant tolerance against chilling stress in terms of plant growth, photosynthesis and antioxidant system in tomato plants. stated that drought tolerance in rice was concomitant with the accumulation of Cks. Drought tolerance induced by Cks has also been observed in tobacco and wheat. reported that drought tolerance can also be enhanced by the application of GA3 in maize. Exogenous GA3 application breaks the rosette by rapid enlargement of differentiated tissues under cold stress. Similarly, JA has been reported to trigger the plant defense responses to abiotic stresses including drought and chilling. Exogenous application of methyl jasmonate improved chilling tolerance in wheat by modulating antioxidant defense system and production of soluble protein content. The relationship between enhanced ethylene levels and chilling tolerance in crop plants has also been reported by and.

46Salicylic acid is also considered as poterntial chemical compound for drought and chilling tolerance in several plant species. SA application was found to be effective in alleviating the chilling and freezing injury. In maize, the harmful effects of low-temperature stress were decreased by the application of SA. Low concentrations of applied SA have also been reported to improve drought tolerance in crop plants. reported that significant improvement in performance of winter wheat was related with SA-induced higher activities of CAT under drought stress. Exogenous auxin application improved wheat yield significantly under drought stress conditions, and overepxression of auxin efflux carrier gene (OsPIN3t) in rice was linked with enhanced drought tolerance.

47Non-hormonal growth regulators, e.g., triazoles, paklobutrazol, chlorocholinchloride, meflidid, and unikonazol are also used to enhance drought and chilling tolerance in crop plants. observed that foliar applications of paclobutrazol remained effective in improving drought tolerance in tomato seedlings. These evidences suggest the exogeneous application of various plant growth regulators is a potent method to improve the drought and chilling tolerance in plants. However, future research should focus on developing genetically engineered or transgenic plants that have the ability to produce specific hormones by using biotechnological and molecular techniques. These transgenic plants would be capable to grow sucessfully under stressful environments.

48Mineral nutrients play important role in improving plant tolerance against stress conditions. To alleviate the adverse effects of abiotic stresses, nitrogen (N) fertilization has been reported to have significant role in stress alleviation. Nitrogen as nitric oxide (NO) is a highly reactive that protects plant against stress conditions by acting as scavenger to ROS. Under drought stress, phosphorus (P) helps plants to maitain leaf water potential which, in turn, enhances stomatal conductivity and photosynthetic rates. Supplementation of K doses and its uptake in plant parts could be beneficial for obtaining reasonable yields under drought conditions. K-induced increases in proline and free sugar contents in rice were reported by under water deficit conditions. reported that low temperature injuries can be avoided in carnation plants by supplimenting K in irrigation water.

49Calcium has been reported enhance the concentrations of amino acids, polyamines (putrescine and spermidine) and chlorophyll content in red spruce under low temperature stress. reported that Ca is an essential nutrient for stomatal closure in chilling tolerant genotypes. Application of Ca in Vicia faba enhanced plant biomass, plant water relations, and chlorophyll content while reduced membrane leakage. Magnesium (Mg) also improve root morphological charcaters which helps to increase uptake of water and nutrients via roots, whlist boron nutrition enhances sugar transport in the plant body and improves seed germination and grain formation process. Application of Se was found to improve plant tolerance against chilling and drought stress. Silicon application improved the plant water status and biomass accumulation in sorghum and wheat under drought stress. These studies suggest that use of plant mineral nutrient is a potiential option to achieve better crop growth and productivity and to alleviate the dterimental effects of drought and/or chilling stresses in a sustainable way.

50Seed priming is considered to improve the abiotic stress tolerance (including chilling and drought) in different plant species. Seed priming promotes the germination related metabolic functions without radicle protrusion from seeds. It is a useful technique to acclimatize against stress conditions by modulating physio-biochemical processes in developing seedlings. Seed- priming with plant hormones and other chemical compounds can play effective role in alleviating the adverse effects of drought and chilling stresses at earlier growth stages. Seed priming activated antioxidant defense system thus improved seed germination and early growth in tobacco seedlings. Seed priming with ascorbic acid, SA and H2O2 improved seedling establishment under suboptimal temperature. found that the seed priming with selenium and SA were effective in improving chilling tolerance in rice cultivars. Likewise, seed priming either with 2.5% K2HPO4 or 2.5% K2HPO4 + KNO3 were observed to induce chilling tolerance at early growth stages than non-primed seeds. Seed priming with SA improved relative water and chlorophyll contents, antioxidant activities, plant biomass and grain yield of wheat, whereas selenium priming regulated activities/levels of enzymatic and non-enzymatic antioxidants in rapeseed under drought stress. The priming of tomato and bean seeds with 0.1–0.5 mM SA improved plant tolerance to drought and low temperature stresses. Significant improvements in physio-biochemical features and the expression of rice drought-responsive RD1 and RD2 genes of AP2/ERF family in drought tolerant and sensititve rice genotypes were observed when seeds were primed with methyl jasmonate, SA, and paclobutrazol. Above evidence suggest that seed priming offers a realistic solution to increase the stress tolerance of plants against the drought and suboptimal temperatures.

51Emergence of intricate stress combinations and their impacts on crop growth and productivity in modern day agriculture are the outcomes of global climate change. Climate change is a multi-facet field that could have long-term impacts in the form of different abiotic stresses. Both drought and chilling stresses are amongst those abiotic stresses that have deleterious effects on crop plants. Plants depict a wide range of responses to these stresses (Figure 1) that could lead to significant reductions in crop yields or some time complete crop failure. Actually co-occurrence of drought and chilling stresses is a major challenge for crop production under field conditions instead of individual stress. Both stresses are also characterized by unique changes in plant growth and development starting from seed germination to yield. The ability of plants to withstand these stresses also differs from species to species. Above evidences affirms that plant responses to combined stresses are unique than those to individual stress factor. Being unique, chilling stress can change the root morphology more adversely than drought stress. In contrast, reduced crop yields as a result of poor dry matter accumulation, reduced flower and pod formation, increased flower and pod abortion, and small seed size are the outcomes of both stresses. Other noticeable effects are diminished nutrient uptake behavior, CO2 diffusion into chloroplast and photosynthetic system in plants. In general, interaction between drought and chilling conditions have found to be additive for almost all plant growth and physiological attributes leading to enhanced damage under combine stresses. In order to overcome the effects of chilling and drought, plants induce biochemical, physiological and molecular modifications that improve tolerance against these stresses. Concurrent occurrence of chilling and drought cause the osmotic as well as oxidative stress on shared basis. Plants counter-balance these adversities by accumulation of compatible solutes and ROS detoxifying proteins, and regulating the activities of antioxidant enzymes. Gene expression is also changed in response to both drought and chilling stresses; some of these genes are regulated only by drought and some genes are chilling-inducible only. To date, various novel approaches have been tested in minimizing the negative effects of combine stresses. Despite of the main improvements there is still a big room for improvement in combination of drought and chilling tolerance. For proper understanding of the plants responses to drought and chilling stress, the experiments should be designed under field conditions. Furthermore, future studies should focus on developing genetically engineered plants by using molecular and biotechnological approaches that have the capability to create a particular response against drought and chilling stress. Transcriptomics and proteomics analysis, genomic sequencing and bioinformatics can help to determine the common and unique genes modulated under stress condition. Thus, these techniques can improve the sophisticated and efficient network in plant response to drought and chilling stresses and subsequently help in the improvement of plant tolerance and productivity.

52HH, SH, and LW conceived the idea of the review and prepared the initial outline. HH, SH, UA, SA, and SM gathered the literature and contributed to writing the different sections. AK and LW provided the technical guidance and editing support.

53The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.