Drought or/and Heat-Stress Effects on Seed Filling in Food Crops: Impacts on Functional Biochemistry, Seed Yields, and Nutritional Quality

1Globally, abiotic stresses drastically affect crop productivity leading to substantial yield losses. According to the report, the decline in food productivity and quality, primarily due to extreme temperatures and water deficit conditions, poses a serious threat to agriculture. Therefore, under the changing climate, minimizing agricultural losses caused by these stresses have become a major challenge and has created a global concern to assure food security. The effects of climate change are exacerbated by the continuous decline in availability and productivity of agricultural land. The world is facing a gradual rise in heat wave frequency leading to warm days and nights, which is projected to exceed 2°C by the end of the 21st century. Abiotic stresses markedly affect the reproductive development of various crops and, ultimately reduce the final economic yields. The effects of drought and high temperature stress on grain yield are complex and include processes such as nutrient assimilation and their mobilization to various reproductive organs, accumulation of stem reserves, gametogenesis, fertilization, embryogenesis, and endosperm as well as seed development. While the presence of these stresses at any growth stage can affect crop yield, the seed filling stage is crucial for determining average seed weight, seed composition and, therefore, the final quantitative and qualitative yield.

2Stresses disrupt germination, vegetative growth, tiller production, dry matter partitioning, reproductive organ development, reproductive processes, grain filling, and grain quality. Reproductive processes and grain filling are more sensitive to both these stresses, and have optimum and ceiling temperatures that are relatively lower than those for vegetative growth and development phases. For instance, exposure of wheat to short episodes (2–5 days) of heat stress (>24°C) at reproductive stage (at start of heading) resulted in substantial damage to florets’ fertility, while mean daily temperature of 35° caused total failures. Rising the duration of high temperature at this stage decreased the grain weight linearly. Similarly, pea, lentil and chickpea performed best at temperatures of 15–25°C at their reproductive stage, and higher temperatures (>32°C) have been found to cause pollen sterility, as in chickpea and lentils. More sensitivity of reproductive stage to heat stress, compared to vegetative stage, is mainly attributed to damage to male components, which are severely impacted as a result of disruption of developmental as well as functional aspects, such as sucrose and starch accumulation in pollen grains. The leaves show more resilience at reproductive ceiling temperatures, probably due to some effective thermotolerance mechanisms, the differential sensitivity of two organ types, which need to be probed further.

3Though both the stresses often exist in combination, yet their interactive effects on crop yield and productivity have received a little attention, apart from a few studies. Not much work has been done exclusively on the impacts of dual stress on seed filling and nutritional composition. When combined, these stresses affect various molecular, biochemical and physiological functions, to more severely impair growth, quality, and yield, compared with their individual effects. Also, the combined heat and drought stress have distinct effects on cellular processes in plants, relative to their individual effects, suggesting stress-specific responses.

4Seed filling is a crucial growth stage for all crops, which involves mobilization and transport processes required for importing various constituents, and many biochemical processes for the synthesis of proteins, carbohydrates and lipids in the developing seeds (Figures 1, 2). Seed filling processes and the accumulation of reserves in the developing and maturing seeds are highly sensitive to environmental changes, which influence the qualitative and quantitative traits of the final yield (Figures 3, 4). Heat and drought stress can hinder the accumulation of various seed constituents, primarily starch and proteins (Figure 3) through inhibiting the enzymatic processes of synthesis of starch and proteins (Triboï et al., 2003) (Figure 2).

5Here, we initially describe the process of seed filling, and later, illustrate how drought and heat stress, either separately and in combination, impact seed yield and quality, and also attempt to explain the advancement in the ‘omics’ technology in addressing these vital challenges. We believe, our efforts would enable the researchers/breeders to evolve strategies to develop stress-resilient, high yielding, and nutritionally superior crops for the future.

6In angiosperms, seed filling is the terminal stage of growth in plants where fertilized ovaries form caryopses/inflorescences, and involves several processes related to the import of constituents, and biochemical processes associated with carbohydrate, protein and lipids synthesis in seeds. The rate and duration of seed filling affect the final seed weight (seed size), a primary component of total seed yield. The seed originates from a double fertilization event, leading to the formation of a triploid endosperm and diploid zygote. The seed develops from the ovule, and has embryo and endosperm/cotyledon, surrounded by the maternally derived seed coat that develops from integuments of the ovule (Figure 1). The primary function of the seed is to protect the embryo, sensing favorable conditions for germination and nourishment of the germinating seedling. The embryo embodies the structures of the future adult plant, and encloses the essential elements and basic processes for the development of a new plant. Seed expansion involves rapid and early cell division of the zygote and triploid nucellus. The division of cells is accompanied by intake of water, which further leads to cell extension (Figure 1). Cell expansion and division starts with more uptake of water, and when cell expansion is completed, cells are destined to maturity (Figure 1). After 2–3 weeks of anthesis, cell division in cotyledons/endosperm halts with net rise in moisture content per seed/grain, which represents maximum seed size. Moisture content is a key factor controlling seed development, seed filling and metabolic activity of developing seed. The processes related to synthesis and accumulation of various seed reserves are largely influenced by the moisture status of the storage cells, depletion of water at this stage disrupts the seed filling. The duration of seed filling is inversely proportional to moisture loss and biomass deposition. Seed development has been excellently described recently, particularly in legumes (see; for more details).

7The quantitative and qualitative characteristics of yield are strongly affected by seed filling process and nutrient reserve accumulation in both developing as well as maturing seeds, and both are responsive to environmental conditions (Figures 2, 3). Seed filling in plants depends upon two sources, transfer of current assimilates directly to seeds and its redistribution from vegetative reserve pools, either pre- or post-anthesis stage (Figures 2, 3). These reserve pools provide substrates essential to maintain transport and supply of assimilates to seeds all through the dark phase of the diurnal cycle, as well as, for duration of the later seed-filling period, when photosynthetic apparatus is becoming senescent, and dry matter accumulation rate of grains exceed dry matter accumulation rate of whole plant. Under normal condition, assimilate reserves during pre-anthesis in stems and sheaths of rice (Oryza sativa) and wheat (Triticum aestivum) contribute around 10–40% to the final seed weight. Remobilization of these reserves to the seed becomes vital in determining seed size on exposure of plants to unfavorable environment or if yield potential is dependent on high biomass accumulation. The contribution of assimilates supply from stem reserves may increase up to 40% during heat and drought stress situations. Drought reduces photosynthesis, hence source strength; moreover the turgor in phloem cells is also reduced by water deficiency, thereby increasing the viscosity of sucrose to inhibit its flow through the conducting cells toward the sinks (seeds). Combined stresses may result in more dehydration to severely slow down the phloem transport. Sucrose transporters (SUTs) have a vital role in the export of sucrose from the leaves to the sinks, the expression of SUT genes is altered by drought (mild–severe) in soybean, barley, wheat, and maize; down-regulation of these transporters has been reported in some cases. On the other hand, the pattern of SUT expression in heat-stressed plants differs from the drought-stressed plants. Arabidopsis AtSUT2 is down-regulated under heat stress (about 15°C increase), while it is up-regulated under drought. In contrast, PtaSUT4 (a symplastic loader poplar) is up-regulated under heat stress (17°C increase), while down-regulated under drought stress, which correlates with reduced sucrose transport from the leaves. Drought resulted in fivefold reduction in cytosolic acid invertase activity in Lupin seeds, suggesting that the amount of sucrose available is reduced in seeds. Heat stress down-regulated OsSUT1 in rice stems, more so in a sensitive cultivar, to reduce the grain quality. In barley too, several genes involved in sucrose and starch biosynthesis were repressed by heat stress, along with down-regulation of SUTs (HvSUT1 and HvSTP3). Thus, photo-assimilation is disrupted because of down-regulation of SUTs in stressed plants. Similar studies on plants subjected to combined drought and heat need to be conducted to find out the types of transporters affected.

8Seed expansion and filling include all processes involved in formation and structural development of mature seed. Seed-making process is highly coordinated and complex, (Figures 1, 2) requiring hormonal regulation (Figure 3), and a constant exchange of signals from-and-to maternal tissues, and between cotyledon/endosperm and embryo. The continual interaction among these three constituents of seed ensures synchronized seed development. Many reports showed substantial changes in hormones content during seed development and filling. Auxin is linked with maize grain filling through controlling cell wall-associated invertase enzyme activity. The increase in auxin during seed filling interval accompanied with increased grain filling rate. The increased auxin is known to enhance sink capacity through enlargement of cell and increased nutrient assimilation. Abscisic acid (ABA), auxin, and cytokinins (CKs) play major role in grain filling through source photosynthate/nutrient re-mobilization and grain development in cereals. Notably, the source activity and sink strength are synchronized and can be altered by hormones and external/environmental stimulus. The grain filling is closely associated with senescence and senescence-related hormone ABA, affecting the time for nutrient mobilization and grain filling in barley, wheat, rice, and sorghum. In barley, ABA can induce the orchestration of gene expression of senescence-related genes. Such activities are also in action during drought stress to accelerate seed filling. In rice and wheat, water stress at grain filling stage caused reduced photosynthetic activity and induced accelerated leaf senescence, resulting in shortened grain filling period due to enhanced ABA level and remobilization of carbon (C)-pool from stem to leaves. Drought-tolerant plants exhibit delayed leaf senescence, thus cultivars with stay-green trait having advantage over terminal drought, which is beneficial to sustain seed filling. Early onset of leaf senescence causes inadequate nutrient supply to grains during grain filling, whereas, late initiation of nutrient mobilization may not support rapid developing seed. This synchronized developmental program is known to involve ABA-inducible NAC-transcription factor (TF), which regulates the expression of genes involved in mineral-nutrient remobilization from leaf to grain, chlorophyll degradation and leaf senescence in rice. A reduced expression of this ABA-regulated NAC – TF caused enhancement in yield in the transgenic rice, plausibly due to prolonged supply of photosynthates/nutrients into the developing grains through fine-tuned ABA biosynthesis pathway. CK also play a key role in grain filling by initiating rapid cell division of endosperm cells. The increased level of CKs probably enhances sink strength by up-regulating cell division related genes via sugar signaling, which involves enhanced phloem unloading and sugar import to endospermic cells through a cell wall-associated enzyme invertase. Overproduction of CK delays early senescence through reduced proteolytic activity and inhibition of N-remobilization, which increases life span and confers drought tolerance in crop plants. Thus, seed filling is determined by diverse plant hormones having specific functions, their interplay might be vital in regulating various processes related to accumulation of seed reserves.

9Drought stress during the initial stage of seed development reduces ability of kernel/seed sink strength by decreasing the number of endosperm cells and amyloplasts formed, thus reducing grain weight with a decline in endosperm competence to gather starch, in terms of both rate and duration. Likewise, heat stress can significantly influence seed development and thus decreases seed yield in several crops including cereals, legumes. Seed filling is closely related to the process of whole-plant senescence. Usually, drought and heat stress during seed filling causes early senescence and reduces seed-filling duration, and enhances assimilate remobilization from the source to sink, the combined effects are more severe. The stress-induced reduction in assimilate supply strongly influences grain development (Figure 2). Here, we describe how drought or/and heat stresses impact the process of seed filling and ultimately influence seed yield and its quality, citing many definite examples from various crop species, through findings at changes in physiology, biochemistry, proteins, and genes.

10Drought stress limits vegetative growth by reducing leaf water content in various cereals and legumes, which might be markedly influenced by inhibition of stomatal conductance/transpiration. Reduced stomatal conductance resulted in increase in leaf temperatures, both of which induced leaf wilting. Drought stress can cause membrane damage, chlorophyll and photosynthesis, due to stomatal or non-stomatal associated mechanisms. Drought stress impairs mineral uptake and drastically reduces nitrogen fixation in legumes such as in soybean, and pea. Collectively, these adverse effects eventually decrease assimilate production and mobilization to developing seeds in various crops.

11The reproductive stage of growth is more sensitive to drought than the vegetative stage, resulting in fewer flowers, poor pod or fruit set, which decreases seed numbers. Gametogenesis, fertilization, embryogenesis are several impacted, to seriously limit seed development thereby lowering crop yields. Flowering and reproductive developmental stages are among the most disrupted stages during drought stress. Pollen sterility is a common symptom, which decreases pollen germination, hinders pollen tube growth to impair fertilization and reduce seed yield. Stress because of limited water causes carbohydrate deprivation, elevated endogenous ABA levels, and a reduced ability of reproductive sinks to use sucrose and starch. The increase in non-reducing sugars and failure to accumulate starch under drought stress results in ovary abortion, leading to poor grain set and grain yield. A reduction in tissue water potential decreases the activity of acid invertase (a vital enzyme in seed development) in seeds, which inhibits sucrose import. Thus, scarce energy sources and elevated ABA levels result in poor grain set under drought stress. Seed yield was drastically reduced in crops exposed to drought stress at the time of seed filling, for example in several legume crops. Moreover, drought stress at the early phase of seed filling decreased the subsequent germination percentage (approximately 9%) of the progeny in soybean (Glycine max L.), as compared to control plants. Similarly, reported a 5% decline in seed germination, 12% decline in seedling vigor and 19% increase in electrical conductivity of seed leachate in soybean seeds obtained from drought-stressed plants. In peanut (Arachis hypogaea L.), drought stress during seed development moderately reduced seed germination, without any impact on seedling vigor. In chickpea, the medium-sized seeds produced under drought stress have lower germination rates and reduced seed vigor as compared to the control or non-irrigated seedlings. Thus, drought stress at reproductive stage inhibits the production of seeds because of disruption of gamete development and function while at seed filling stage limits the seed size because of inhibitory effects on carbohydrates involving phytohormones ABA.

12Drought stress during seed filling slows down the seed-filling rate and reduces the filling duration to limit seed size. It inhibits cell division in endosperm cells and number of starch granules in grains to reduce their (grains) size. Water stress markedly affects grain composition. For instance, starch content of developing wheat grains declined markedly, when plants were subjected to drought stress, which was attributed to an insufficient supply of photoassimilates, or to direct effects on starch synthesis machinery in grains, influenced by sink dehydration, reduction in endosperm cell numbers, small size of starch grains, and lower amylase contents in starch grains. Starch accumulation involves several complex enzymatic processes, with AGPase, soluble starch synthase (SSS), and starch branching enzyme (SBE) having vital roles. A gene expression study in wheat showed that drought stress reduced the transcript number for the SSS enzyme more than, other enzymes involved in starch biosynthesis. In another study in wheat plants, exposed to drought stress during grain filling, glucose, fructose, and sucrose levels declined significantly in grains of a drought-sensitive genotype, which was accompanied by a sharp reduction in activities of cell wall invertase and soluble invertase. Higher sucrose synthase activity in drought-tolerant variety enhances supply of assimilates to grains to increase the seed size. Various enzymes involved in starch and sucrose metabolism were inhibited to reduce seed starch in chickpea. The enzymatic activities of SSS, granule-bound starch synthase (GBSS), SBE and starch de-branching enzymes (DBE) decreased to different extents in sorghum grains, which inhibited starch accumulation. Sugar metabolism was also inhibited in grains of drought-stressed maize, as indicated by decreased soluble and insoluble invertases activities. In general, because of water limitation, cell number in endosperm or cotyledons decreases, which may occur due to inhibited cell division, however, the finer mechanisms controlling these events need to be probed. Carbohydrates’ accumulation is hampered because of limitations in availability of sucrose from leaves, as well as their synthesis in developing seeds, and/or because of enzymatic inhibitions, to eventually decrease the seed size.

13Seeds store carbon for energy, particularly in form of lipids in oil seeds, which are mainly required for germination. Drought stress also affected oil content and quality, linoleic acid and behenic fatty acid contents declined while stearic and oleic fatty acid contents increased in drought-stressed plants of peanuts. There are contrasting reports too, which are possibly due to variations in imposed stress treatments. For instance, in maize, drought stress markedly decreased seed oil content but enhanced linolenic acid and oleic acid contents in oil. There was a reduction in overall seed oil, total tocopherols, flavonoids, and oil phenolics. Drought stress in soybean, during seed filling, decreased oil content up to 12.4%, along with a reduction in oleic acid content. The oil content is affected due to decrease in concentration of digestible carbohydrates such as glucose, fructose, and sucrose under drought conditions, which affects the fatty acids composition due to reduced unloading of sugars from stem to developing seeds. More research is needed to explore the target sites of drought stress in pathways involved in lipids biosynthesis and how lipid quality is regulated under drought stress also requires to be investigated.

14Drought stress impairs symbiotic nitrogen fixation through rising oxygen diffusion resistance to root bacteroides resulting in reduced activity of nitrogenase that decreases nitrogen availability for biosynthesis of proteins, which is a primary reserve in grain legumes, and reduces seed yield. While seed protein quality largely depends on the genotype, it may be influenced by environmental stresses. Alterations in composition of protein fraction due to drought and heat stress are primarily due to changes in quantity of total nitrogen accumulated during seed filling. Some studies in legumes have indicated reduction in accumulation of minerals in developing seeds due to drought stress. For example, in common bean, Fe, Zn, P, and N concentrations decreased under drought stress, which correlated with reduction in total protein content. In another study, in white, red and ‘chitti’ bean cultivars, drought-stress during pod filling resulted in decline in seed nitrogen and protein content, substantially. A marked reduction in starch, protein, and amino acid contents occurs in chickpea seeds, drought-stressed during seed filling, more impact occurs on ‘Kabuli’ than ‘Desi’ types. The free amino acid pool increased but protein-amino acid fraction decreases in cowpea seeds of drought-stressed plants, and inhibition in incorporation of amino acids into protein chain occurs. Proteomic studies in wheat grains, harvested from drought-stressed plants at grain filling stage, indicated a marked change in quality of seed proteins. While globulin and glutenin, remained unaffected, albumin and gliadin concentrations increased significantly under drought conditions. In contrast, some other studies reported increased concentrations of seed protein in response to drought stress in various crop species, for example, in cereals, legumes and oil seeds. These differences may be attributed to intensity and duration of drought stress imposed on plants, and relative to seed dry weight. The increased protein content is linked to altered C-partitioning, especially in cereals, which changes the C/N ratio, to favor more N-assimilation. Thus, these studies indicate crop-specific effects of drought stress on proteins and minerals. Further work is required to investigate the impacts of drought stress on various classes of proteins, amino acid composition, especially in food legumes. Additional work should focus in future on seed quality aspects, especially on mineral transport mechanisms in seeds of drought stressed crops.

15Heat stress affects almost all the stages of the plant beginning from germination until maturity. Exposure to heat stress during pod and seed filling stages results in a substantial decrease in economic yield of crop plants by reduction in seed weight. Decline in seed weight and seed number due to high temperatures has been reported in several crops including legumes, cereals and others. Sustaining grain weight in heat stress conditions during seed filling stage is considered a part of heat stress tolerance mechanism. The sensitivity of seed filling to heat stress may differ according to different crop species. Two traits mainly seed filling rate and potential seed weight may be considered as a selection criteria for heat stress tolerance. High-temperature stress may speed up the rate of seed filling by reducing the duration of this stage and therefore the yield potential. Increase in seed-filling rate resulted in smaller and wrinkled seeds in chickpea and lentil, which mainly occurred due to reductions in the remobilization and translocation of photosynthesis to developing seeds. The time of seed filling reduced in pea, soybean and white lupin, resulting in smaller grains. Specifically, in cowpea, increasing the temperature from 15.5 to 26.6°C decreased seed-filling duration from 21 to 14 days and in chickpea, a 1.7°C increase in temperature reduced seed-filling duration, and accelerated maturity, which decreased seed yield. The reduction in seed size was related to structural and functional reasons. The cotyledon cell number and cell expansion decreased under heat stress, which inhibits the rate of seed-filling and seed size. ABA levels were altered, which correlated with adverse effects on seed filling rate in heat-stressed chickpea. Studies in future need to focus on probing the role of various phytohormones in altering the rate of seed filling as well as structural changes in endosperm and cotyledons in seeds developing under heat stress environment, involving tolerant and sensitive genotypes of various crops.

16High temperatures during seed filling may stimulate leaf senescence to reduce photosynthetic capacity, which impacts seed development and reduces growth and yield traits in grain legumes. Senescence occurs due to disruption of cellular structure and organization, with a substantial reduction in chlorophyll, which also terminates the photosynthetic process. Besides, causing senescence, increased temperature accelerates scorching and abscission of leaves. Heat stress decreases photosynthetic activity and induces premature senescence, which decreases synthesis and distribution of assimilates to seeds. A rise in temperature (30–35°C) for a few days repressed photosynthesis and electron flow, interrupted metabolic pathways, damaged seed set and seed development, and ultimately reduced seed yield in chickpea. Rise in temperature inhibited sucrose metabolism in leaves and impaired sucrose supply to developing seeds, as in chickpea, mung bean, which might be a primary reason of shriveled seeds under heat stress. Moreover, the rate of chlorophyll decline from the leaf was strongly coordinated with contents of non-structural carbohydrates and nitrogen as well as their remobilization efficiencies. Damage to membranes of leaves is a common effect of heat stress, along with increase in ethylene production, which may accelerate the senescence. Heat stress increases leaf senescence by disrupting chloroplasts and damaging chlorophyll due to direct or indirect mechanisms such as photo-oxidation, which severely inhibits photosynthetic ability to reduce biomass and seed yield. Moreover, carbon fixation is inhibited severely by heat stress due to reduction in activities of PEP carboxylase and RuBP carboxylase in photosynthetic organs during seed filling, as observed in wheat. These studies collectively indicate that heat-stresses induces leaf senescence, inhibits net photosynthetic rate, chlorophylls, hastens seed filling, disrupts sucrose-starch conversion and causes loss of sink activity to decrease the seed weight and quality.

17The seed filling rate in plants is mainly dependent on two carbon resources (1) currently synthesized assimilates from photosynthesis, and (2) carbohydrate (assimilates) reserves translocated to the seed from vegetative tissue in leaves and stem. Impairment of photosynthesis during heat stress, reduces the currently available assimilates to the seed. Thus, stem reserves’ mobilization play a crucial role; hence, the stored carbohydrates become the chief source of transported materials, contributing around 75–100% to grain yield during stress environment. The mobilization of stored reserves from leaves and stem strongly correlates with carbohydrate metabolism, involving synthesis of sucrose and its utilization. More studies are needed to explore the genotypes of various crops having ‘Stay-green’ ability in leaves under heat stress to sustain seed filling.

18Over 65% of seed dry weight is accounted for by starch, therefore, decrease in seed yield is mainly caused by decline in starch accumulation. Heat stress during grain filling markedly decreased starch accumulation in wheat and rice by altering the expression of starch-related genes, which contributed toward reduction in seed size. Total non-structural carbohydrates also decreased, which changed the proportion of soluble sugars to starch. Sugars such as fructose, sugar nucleotides and hexose phosphate levels also declined due to heat stress, as in wheat. The decrease in sugars in heat-stressed plants may be related to enhanced assimilate utilization rather than production. In some cases, increase in sugar levels was reported, which was related to up-regulation of starch hydrolyzing enzymes such as α-amylase during seed filling. Moreover, due to enhanced activity of the α-amylase enzyme, rice produce chaffy grains under heat stress environment. The transcripts of the β-glucosidase gene decrease due to heat stress to impact the seed composition in soybean. Thus, heat stress adversely affects the accumulation of carbohydrates by influencing their metabolic pathways, the changes are crop-specific, and depend upon exposure to heat stress. Comprehensive studies are needed in the future involving SUTs and various genes related to starch-sugar inter-conversions in seeds of heat-stressed plants of various crops to know the accumulation patterns of various carbohydrates.

19The amount of starch and proteins, accumulating in each seed, depends on total number of endosperm cells, determined early in stage of seed filling, and the final size of cells, which is regulated by the rate and duration of seed filling. Heat stress during early seed development reduced the endosperm cell number, however, at the later stage, heat stress impaired starch synthesis either due to limited supply of assimilates to seeds or as a result of the direct effects on biosynthesis processes in the seed. Endosperm structures were altered and storage products were degraded due to heat stress (35°C for 5 days) during the seed filling in barley and maize seeds while in rice seeds, heat stress accelerated the growth rate of endosperm resulting in chalky grains. In dicots like soybean, heat stress negatively affected cotyledon cell number, cell expansion, and hence seed filling rate, which reduced seed weight. Thus, heat stress limited the production of storage cells in monocots and dicots, which further restricted the accumulation of various seed reserves. The mechanisms affecting the cell number in endosperm and cotyledons in seeds of heat-stressed plants are not well understood, an elaborative study is in need.

20An enhanced rate of seed filling under heat stress may be due to increased enzyme and metabolic processes, which accelerates and compresses the overall process of seed development at elevated temperatures. Increase in rate of seed dry matter accumulation may be a compensation for the decline in its duration. Heat stress decreased the duration and amount of protein accumulation, but the rate of accumulation was unaffected, as in some studies. The storage protein composition was also altered under heat stress due to changes in the amount of total nitrogen accumulated during seed filling in wheat. Protein quality deteriorated in heat-stressed plants, for example, dough quality declined in wheat under heat stress, because of reduction in aggregation properties, caused by a decline in high molecular weight glutenins, and rise in gliadin accumulation. Moreover, the ratio of gliadins and glutenins increased while the ratio of large polymers decreases in wheat grain flour. Similarly, in pea, high temperature denatured and aggregated the seed storage proteins (globulins, legumin, and vicilin) in pea; which might be due to loss of covalent and non-covalent interactions. In the same way, heat stress denatured β-conglycinin in soybean and damaged globulin and phaseolin. Seed protein fractions, especially albumins and globulins were adversely impacted by heat stress in lentil seeds, thus reducing the overall seed quality. The damage to the structure and metabolism of proteins due to heat stress also inhibited the activities of enzymes involved in protein synthesis, as in Andean lupin (Lupinus mutabilis).

21Fatty acid composition and content are variably affected by heat stress in a crop-specific response. For instance, different temperatures (10, 16, 21, 26.5°C), imposed on plants of rapeseed (Brassica napus), safflower (Carthamus tinctorius), flax (Linum usitatissimum), sunflower (Helianthus annuus) and castor bean (Ricinus communis) plants, during seed development, did not affect the composition of fatty acid in the oil of safflower and castor bean, but reduced the amounts of unsaturated fatty acids. The oil composition changed because of the impact of heat stress on fatty acid biosynthesis in sunflower and in soybean, heat stress (35°C) during seed-filling decreased oil content by 2.6%, compared with seeds from plants exposed to 29°C. Considering that, heat stress effects are likely to be more prominent in future, it would be vital to understand the target sites of heat stress in the biochemical pathways, related to accumulation of proteins and lipids in developing seeds of various food crops.

22Both drought and heat stress, when applied jointly, reduce leaf water content more severely, resulting in early leaf wilting, acute chlorosis and membrane damage, drastic inhibition of photosynthesis and production of assimilates in leaves was recorded by various studies, for instance, in chickpea; lentil and barley. Though the impact of each of this stress, when applied singly on plants may differ, depending upon its intensity and duration, nevertheless, some common symptoms appear as overall reduction in vegetative biomass, reproductive growth and yield-traits. The effects of heat and drought stresses may differ, when applied singly; for example, heat stress during seed filling may accelerate or even suppress the seed filling process, and reduce the duration of filling to inhibit the accumulation of various reserves. Drought stress slowed down the seed filling process due to water limitations, but eventually resulted in inhibitory effects on seed size and numbers. While these two stresses affect the seed filling differently, each one leads to reduced seed size due to reduction in the cell number in endosperm/cotyledons, inhibitions or acceleration of seed filling rate and various biochemical processes, as in wheat and maize. Variations may also exist in the relative composition of seed reserves in response to either of these stresses, nevertheless, in general, seed quality was adversely affected by both the stresses.

23The combined effects of heat and drought stress are an excellent example of two different abiotic stresses occurring in the field simultaneously. Little is known about on the combined effects of heat and drought stress on crops, with most studies reporting very severe effects on crop growth and productivity. The impact of the simultaneous occurrence of two stresses is more pronounced during early part of reproductive processes, especially micro-and mega-sporogenesis, pollen and stigmatic function, anthesis, pollination, pollen tube growth, fertilization, and early embryo development, compared to individual drought or heat stress. Failure in any of these processes will drastically decrease the fertilization rate or leads to early embryo abortion, thus resulting in lower seed or grain numbers and finally limiting the crop yield. Substantial losses observed in crop yields under combined stress environment have been attributed to several reasons, such as metabolic changes, reductions in the period of crop developmental stages. Subsequent decline in light perception, decreased phenology, and perturbation of processes involved in carbon assimilation such as transpiration, photosynthesis and respiration may contribute to fewer, malformed and smaller seeds, which are economically futile.

24Seed-filling duration declines more in combined stresses than individual treatments, as evidenced in wheat, chickpea and lentil. Both the stresses can occur simultaneously after anthesis to limit seed-filling duration, and result in poor-quality grains in wheat, due to the substantial reduction in seed dry weight, seed numbers, and starch content. The rate of transport of non-structural carbohydrates in endosperm tissue decreases, as in wheat, in response to dual heat and drought stress. There is severe reduction in starch accumulation due to combined stresses imposed during seed filling, which has been attributed to more drastic inhibition of starch synthesizing enzymes, compared to individual stress treatments, as in chickpea and lentil, consequently seeds become shriveled. Combined heat and drought stress can reduce nitrogen pool due to a decline in free amino acids containing various transfer substances related to metabolism of nitrogen and other osmotic compounds. The response to dual stress situation is crop specific, for example, in soybean plants, subjected to dual stress, seeds showed higher protein contents, but lower oil contents than controls/individual stresses,. In cereals such as barley and wheat, the combined stress treatment reduced starch accumulation but increased protein content, as compared to single stress treatment. In Brassica species, exposed to combined heat and drought stress, seed protein content increased while seed weight decreased at higher temperature (35/18°C), in comparison to moderate temperature (28/18°C). Considering that heat and drought stress are frequently experienced together and would be a major risk for the grain crops in future, more studies are needed to evaluate the impacts of these two stresses on mechanisms at various organizational levels affecting seed yield and quality in various crops.

25Though, seed filling stage is one of the most important phases that determine yield, based on the available information regarding the stress sensitivity of enzymatic processes involved in accumulation of starch, the complete picture is missing. The individual and combined effects of heat and drought stress on crop production and yield is a complex phenomenon that includes processes as diverse as assimilation and supply of nutrients to various reproductive organs, accumulation of stem reserves, gamete formation, fertilization and embryo development, and endosperm and seed development. Developmental studies, if integrated with “omic” studies would determine the complex gene expression pattern during seed filling and unravel the molecular basis of the impacts of heat and drought stress on seed quality and composition (Figures 2, 3). To deduce the extremely susceptible molecular process underlying the seed filling phenomenon and the effect of drought and heat stress episode(s) on seed filling, different approaches involving genomics, transcriptomics, proteomics, micromics and epigenomics are imperative to identify not only the key responsible genes but also other regulatory molecules/proteins influencing make them responsive to genetic improvement.

26Using microarray technology in rice, ∼21,000 genes, which are involved in the successive stages of grain filling, have been identified, and a majority of them relate to the metabolic pathways of carbohydrate and fatty acids. Cluster analysis and correlation studies reveal the association of 269 genes with grain filling. The information about the rice genome sequence has helped in the recognition of promoter regions that control these genes, and has led to the discovery of common cis-elements shared in the promoter region in the grain filling genes. Using grain gene cluster-analysis, it has been revealed that AACA element appears to be dominant among 103 available promoters. These eventually led to deciphering nine transcription factors that help in regulating gene expression. Study in the Barley caryopses using Affymetrix 22K Barley1 Gene Chip, at 21 days post-anthesis stage reveal that 2020 genes were differentially expressed under heat stress. The genes with a role in biosynthesis of storage compounds and cell growth are down-regulated indicating disruption of seed development. Further, genes for production of sugars increased, which provide evidence for high production of compatible solutes as well as feed-back induced substrate accumulation for biosynthesis of storage compounds. Metadata analysis in the same study reveals that embryo and endosperm are the primary targets of heat stress response. The impact of high temperature on grain filling during the milky stage of rice has been elucidated by. The genes involved in starch synthesis, GBSS and SBE show decreased expression while the starch consuming α-amylase show increased expression. In rice caryopses, exposed to high temperature, the transcription level of genes encoding for an enzyme ADP-Glc pyrophosphorylase (AGPase) declines, but not in the SSS isoforms levels. The variations noticed in transcript levels were correlated to the observed biochemical differences between the starch grains formed both under normal and high temperatures, mainly, with reduced amylose content, side chain elongation of amylopectin and smaller grain size.

27Similar observations have been reported when drought stress is applied during the first few days after pollination, where inhibition of endosperm division occurs which is related to reduced kernel size at maturity, such as in maize and wheat. The endosperm and placental/pedicel tissues of maize, drought-stressed for 5–9 days after pollination, have been examined using cDNA microarray. A marked difference occurs in the response of both tissue types: in the pedicel, 89% of the 79 transcripts affected show up-regulation whereas, in the endosperm, 82% of the 56 transcriptionally altered genes show down-regulation. In case of pedicel, transcription levels of stress-related genes [e.g., heat shock proteins (HSPs) and chaperone genes] are enhanced whereas, in the endosperm, the genes involved in cell division, cell wall degradation and growth are down-regulated. In another study, the expression of starch and sucrose metabolism pathway genes like a-Amy3 gene encoding α-amylase, and an alpha-glucosidase (ONG2), catalyzing the hydrolysis of the raw starch granules, in wheat is altered during drought stress at grain filling stage.

28Beside the transcriptomics, micromics approach also provides information about the post-transcriptional gene regulation of the seed filling through identification of different microRNAs (miRNAs). The role of miRNAs as master regulators in controlling the gene expression has been studied in various crop species. Different studies have identified the crucial role of miRNAs and their regulation in seed filling process through their target identification in different crop species like rice, wheat and maize, etc.. Members of miR156 family show exclusive expression during grain filling process in rice by targeting the squamosal promoter binding protein like (SPL) family genes. SPL16 regulates the cell proliferation at the time of grain filling in rice and the increased expression of SPL16 is directly proportional to the grain yield. The role of SPLs in different yield related traits like grain size, grain quality, yield, panicle branching, tillering and plant height has been described in rice. Overexpression of miR397 shows its role in grain size by down-regulating its target, lac that encodes laccase protein. Likewise, increased expression of miR156, miR164, miR166, miR167, and miR1861 during grain development and grain filling stages signifies their regulatory roles. Studies about the influence of miRNAs during grain filling under heat and drought stress periods are very limited. In one such study in wheat, the expression of miRNAs- miR159a, 159b, miR160, and miR171a during heat stress at grain filling stage decreases. More studies are needed to understand the finer role of micro RNAs involved in regulation of seed filling under drought as well as heat stress.

29Proteomic reference maps are available for various crops during grain filling and maturation stages, e.g., wheat and maize endosperm and barley grain. Exploring the individual as well as combined impacts of drought and heat stress on protein composition can be useful for improving protein quality in crops. Heat stress effects have been studied thoroughly on hexaploid wheat grains at the protein levels where various proteins involved in starch metabolism decrease whereas HSPs increase. Subsequently, differentially 121 proteins are reported to reveal considerable alterations in response to drought stress in the proteome of wheat grain,, of which 57 have been identified. More than half of the proteins identified are thioredoxin targets, unveiling the link between drought and oxidative stress. Another study on wheat, exposed to heat stress during grain filling stage show increase in expression of proteins involved in signal transduction, photosynthesis, antioxidant enzyme, ATP synthase, HSPs, and other nitrogen metabolism related proteins in tolerant genotype, as compared to sensitive genotypes, indicating their crucial role in tolerance. Proteomics studies in the rice grain development show the association and acquisition of different metabolic pathway proteins including glycolysis, citric acid cycle, lipids and proteins in the mature grains. Assessment of seeds developing under drought or/and heat in contrasting genotypes would reveal vital information about the type of transcripts and proteins influenced by stresses, which would be beneficial for their genetic manipulation to improve stress tolerance.

30‘Stay green’ trait can be a convenient trait to use as an indicator of sustainable supply of assimilates and utilization of stem reserves, which can also be regarded as a mutually exclusive strategy to promote seed filling under stressful conditions. Many QTLs or candidate genes related to leaf senescence have been identified in cereals and might prove crucial for genetic modifications in breeding. Five QTLs in two bread wheat genotypes are identified on chromosomes 1B, 2B, 3B, 5A, and 6B under high-temperature stress during grain filling. A QTL for the heat susceptibility index for the grain filling period is present in close association with QTL region for productive tillers under late-sown conditions and grain filling duration on chromosomes 1B and 5A, respectively. Similarly, a major QTL, qDTY1.1 is present on chromosome 1 for grain yield for terminal drought stress in rice. This shows the positive effect on grain yield during drought stress conditions. Another major QTL on linkage group 2 (LG 2) for terminal drought tolerance, identified in pearl millet, is also considered as a marker for grain yield improvement across different terminal stress conditions. Thus, these QTLs can be used for molecular wheat breeding programs for conferring heat as well as drought tolerance.

31Seed-filling processes are adversely affected by heat and drought stress in all crop species, resulting in poor-quality seeds and reduced seed yields. The frequency of these two stresses occurring at the same time is increasing, for both for summer- and cool-season crops, which is highly detrimental to the qualitative and quantitative aspects of yield. Hence, future research should focus on investigating the dual effects of drought and heat, involving various physiological, biochemical and molecular approaches. Future endeavors should also pay attention on screening the existing germplasm of various crops under combined stress environment to identify tolerant genotypes and their subsequent incorporation into breeding programs. It is vital to understand and dissect various components influencing seed-filling processes under separate and combined stress environments to unveil varying responses of different crops to these two stresses. Identification of sensitive sites (embryonic stages, hormonal changes and biochemical pathways for seed reserves, signaling molecules, proteins and genes) related to seed-filling processes in stressed plants, especially under the combined stress, would provide useful cues in developing strategies to improve seed quality. As photosynthetic activity mainly determines crop productivity, the breeding for ‘stay-green’ trait is essential to combat drought as well as heat stress. ‘Omics’ studies are in progress that will be useful for identifying the genes, proteins, and metabolites in developing seeds that are, impacted by heat or drought stress. Modeling the stages of development, growth, grain productivity, grain quality and sink-source relations will enable better insights on the physiological and genetic nature of stress tolerance, ultimately resulting in enhanced grain yields and quality in crops. Improved models can enhance the likelihood of predicting crop performance in future challenging climates, which will largely help to identify traits that can be exploited through breeding to produce sustainable climate-resilient genotypes with acceptable yield under stressed environments.

32All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

33The 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.