Engineering abiotic stress response in plants for biomass production

One of the major challenges in today's agriculture is to achieve enhanced plant growth and biomass even under adverse environmental conditions. Recent advancements in genetics and molecular biology have enabled the identification of a complex signaling network contributing toward plant growth and development on the one hand and abiotic stress response on the other hand. As an outcome of these studies, three major approaches have been identified as having the potential to improve biomass production in plants under abiotic stress conditions. These approaches deal with having changes in the following: (i) plant–microbe interactions; (ii) cell wall biosynthesis; and (iii) phytohormone levels. At the same time, employing functional genomics and genetics-based approaches, a very large number of genes have been identified that play a key role in abiotic stress tolerance. Our Minireview is an attempt to unveil the cross-talk that has just started to emerge between the transcriptional circuitries for biomass production and abiotic stress response. This knowledge may serve as a valuable resource to eventually custom design the crop plants for higher biomass production, in a more sustainable manner, in marginal lands under variable climatic conditions.

Despite considerable success in crop improvement programs, farmers still lose almost 20 -70% of their potential crop yield because of biotic and abiotic stresses (1). Abiotic stresses are one of the key challenges for plant growth and agricultural productivity in arable lands, with an estimated annual loss of billions of dollars (2,3). This challenge is expected to grow enormously with the proposed expansion of agricultural activities in less fertile and marginal areas, which is now becoming crucial in satisfying the growing food demands (4). Because about 86% of the fresh water is utilized for the production of agricultural biomass, additional pressure is likely to develop on the freshwater resources with the ongoing shifts from the era of "fossil energy" to the era of "energy from biomass" (5). To keep pace with this alarming situation, novel approaches are vital to enhance plant biomass production under adverse environmental conditions (6).
The acclimation of plants to abiotic stress is a complex and coordinated response involving hundreds of genes and their interactions with various environmental factors throughout the developmental period of the plant (7)(8)(9). Accordingly, a thorough understanding of the molecular responses in plants is essential for targeting any improvement in plant biomass or yield. In addition, identifying suitable phenotypes that respond to multiple abiotic stresses appearing either simultaneously or sequentially under field conditions is indeed a tedious task, but it is the need of our time (10). However, to minimize the "yield gap" caused by these abiotic stresses, it is vital to understand the gene regulatory networks operating in plants. We hope that the recent advances in molecular biology, including tissue-specific or developmental stage-specific gene expression, stringently regulated and induced gene expression, site-specific integration of the transgene, and gene pyramiding will assist us in enhancing the photosynthetic efficiency in plants contributing eventually to produce higher biomass when grown under marginal lands (11).
The development of human civilization is inextricably linked to plant biomass, where wood and fiber are used for numerous purposes, including energy production, textiles, paper-making as well as bioenergy resources in the form of biofuels. As of today, there is a tremendous increase in the demand for plant biomass production due to the expanding human population, inadequate food availability, and the need for bioenergy (12). However, with the ongoing debate on "food versus fuel," we need to reassess the options and the resources available to us. Even if we plan to use biofuels to satisfy the 20% of the growing demand for oil products, there will be nothing left to eat. Keeping this fact in mind, it is imperative to design our future food crops that will have the potential to give high yield and biomass even under marginal lands.
Biomass accumulation in plants occurs either by an increase in cell number or by cell expansion (13,14). Dissecting out the regulatory network involved in cell wall biosynthesis provides an attractive strategy to improve plant architecture and biomass (15,16). In recent years, considerable attention has also been given to plant cell wall polymers, which form a major component of plant biomass. The composition and amount of these polymers in the cell wall change with plant development and in response to stress conditions (17). Furthermore, there is a need to delineate the genes responsible for the biosynthesis of different cell wall components, along with the assembly and the regulation of cell wall dynamics and heterogeneity (17). In addition to the above strategy, manipulating endogenous plant hormone content provides still another possibility for improving abiotic stress tolerance as well as biomass of plants. The third crucial area to be explored for plant biomass enhancement and sustainable agriculture is "plant-microbe interaction." Plant roots and microorganisms interact and compete for nutrients, making a complex system within the rhizosphere (18). However, a complete understanding is needed regarding the effect of these microbes in improving abiotic stress tolerance and in inducing higher biomass production in plants (Table 1) (19).
One of the goals of our Minireview is to understand the crucial strategies being developed to engineer abiotic stress response and to enhance biomass production in plants. Here, the focus is on the above three major strategies that can be utilized to mitigate abiotic stress response, besides enhancing biomass production. In this Minireview, we present several examples where plant-microbe partnerships have been utilized to cope with the abiotic stress response leading to higher biomass production. Furthermore, we discuss recent advances in molecular studies that reveal key transcriptional switches regulating secondary wall biosynthesis under abiotic stress. We also highlight the key facets of the plant growth regulators having a direct role in the regulation of plant growth under various abiotic stresses. The knowledge of these master switches will simultaneously improve our understanding to develop new strategies to genetically modify the composition and quan-tity of lignocellulosic biomass besides increasing abiotic stress tolerance.

Exploiting plant-microbe interactions
Several studies have demonstrated that plant-microbe interactions influence abiotic stress tolerance along with growth and biomass. Plants form highly complex and diverse associations with co-evolved microbial communities (20). Microbial communities show plant specificity in terms of their morphology and secondary metabolism (21). Diverse mechanisms have been proposed for varied associations of plant growthpromoting bacteria (PGPB) 4 and fungal endophytes, after their colonization in the rhizosphere/endosphere of plants (22). The plant-microbe interactions significantly affect carbon sequestration, nutrient cycling, plant growth, and productivity (23, 24) besides ameliorating plant responses against abiotic stresses (22). Many studies have shown that diverse bacterial species, belonging to different genera, contribute toward tolerance against various abiotic stresses to the host plants resulting in enhanced biomass (25).
There is now good evidence from recent agricultural practices that the use of plant growth-promoting bacteria is a strong, viable, and vital option for overcoming productivity constraints besides mitigating environmental stresses in crops, e.g. soybean, barley, maize, and rice (26). On the one hand, the microbes induce local or systemic inducible response mechanisms in plants to overcome abiotic stress, and on the other hand, they contribute toward sustaining the biomass and growth through uptake, mobilization, and synthesis of nutrients. Hormones such as auxins, cytokinins, and gibberellins along with other organic compounds that stimulate cell growth  (27). A schematic diagram depicting the complex interaction of PGPB in increasing plant growth and biomass under stress is shown in Fig. 1.
Various PGPBs possess not only the ability to induce reactive oxygen species-scavenging enzymes but also to increase the photosynthetic efficiency of plants under abiotic stress, thus leading to enhanced plant growth (28). Seed priming with these PGPB strains significantly improves biomass and nutrient uptake under stress conditions (29). Recently, the multifaceted action of microorganisms has been shown by Khan et al. (30), where enhanced expression of antioxidant enzyme machinery through Bacillus pumilus inoculation led to improved growth under high-salt and boron stress in rice. Furthermore, PGPBs help plants through phytoremediation of excessive salts, leading to improved biomass production under salt stress (31). Bacteria, such as Pseudomonas spp., Burkholderia caryophylli, and Achromobacter piechaudii, were shown to reduce endogenous ethylene levels in plants by producing 1-aminocyclopropane-1carboxylic acid (ACC)-deaminase, thus resulting in increased root growth and improved tolerance of salt and water stress ( Table 1) (31). In addition, synthesis of indole acetic acid (IAA) or enhancing ACC deaminase activity in the rhizosphere by Trichoderma atroviride or Pseudomonas putida in tomato results in improved growth and stress tolerance (32). Similarly, knocking down the expression of the ACCD gene from Trichoderma asperellum showed reduced root elongation in canola seedlings, suggesting its role in root growth promotion (33). Burkholderia phytofirmans strain PsJN improves leaf area, chlorophyll content, photosynthetic rate, and water-use efficiency, ultimately resulting in the enhanced shoot and root biomass under various abiotic stresses in a wide spectrum of host plants, including potato, tomato, and grapevine (34). Considerable progress has been made in unraveling the morphological, physiological, and molecular mechanisms of growth-promoting bacteria-mediated abiotic stress tolerance in plants (35). However, delineating the signaling molecules released by PGPB that enhance plant growth and defense responses still remains a challenge. Similarly, extensive research on enhancing the effectiveness and consistency of microbial inoculants in multiple-stress amelioration under field conditions will help us in the screening of suitable bio-inoculants to improve crop productivity under different environmental vagaries (36). Certainly, novel bacterial strains must be tested in the future to gain better insight into the plant-microbe interaction to achieve this target. However, encompassing different "omics" approaches to study microbe-mediated stress mitigation strategies will further strengthen our knowledge on the mechanisms of plant-microbe interactions with the naturally associated or artificially inoculated microorganisms. Primary targets for optimizing beneficial plant-microbe interactions include quorum sensing, bacterial motility, biofilm formation, and their signaling pathways. Current evidence supports the plant-microbe interaction in mitigating abiotic stress response during varied edaphic and climatic conditions. At present, a range of bacterial formulations are already available commercially for their use as "bioprotection agents" or "biofertilizers" to improve biomass and yield under biotic stresses. Utilizing these microorganisms will further enhance tolerance against various abiotic stresses, thus establishing novel and promising techniques for sustainable agriculture.

Regulation of cell wall biosynthesis
Around 430 million years ago, vascular plants appeared on land and co-evolved with the ability to develop a secondary cell wall. Plant cell wall functions as an "exoskeleton" by providing mechanical and structural support to the entire cell as well as acting as a physical barrier against abiotic and biotic stresses (17,37). The principal components of a secondary cell wall are cellulose, hemicelluloses, and lignin; interestingly, they are quite unique to each cell type and vary in their amount and composition along with development and in response to various biotic and abiotic stresses (17,37). Thus, we can conclude that plants have inherently evolved intricate mechanisms to THEMATIC MINIREVIEW: Plant biomass under abiotic stress regulate biosynthetic pathways for cell wall components and have assembled them for the proper functioning of the cell. For example, plants show higher production of lignin biosynthesis enzymes during abiotic stresses (37). Similarly, severe dwarfing and altered wood anatomy were observed by silencing 4coumarate-coenzyme A ligase, which reduces the lignin content in tracheal elements (38). At present, there is fair interest in unraveling the molecular processes regulating secondary wall development in plants as it is the most abundant plant biomass in the form of fiber and timber (39).
Immense progress has been made for the identification and functional validation of cell wall polysaccharide biosynthetic genes (including those for cellulose, glucomannan, xyloglucan, xylan, and pectin), as well as sugar nucleotide donors of these pathways (Table 1) (17). In plants, cellulose microfibrils, composed of ␤-1,4-glucan chains, mainly contribute toward the above-ground biomass, and their biosynthesis and accumulation play a vital role in providing defense against climatic vagaries (40). CesA (cellulose synthase) gene superfamily regulates the synthesis of cellulose in plants (40). Brassinosteroid signaling activates the BES1 (BRI1-EMS-Suppressor-1) transcription factor, which binds to the E-box (CANNTG) in the promoter of CesA causing its up-regulation and thus resulting in enhanced cellulose accumulation in Arabidopsis (41). Similarly, CESA1 kinase activity was shown to be enhanced by the degradation of its inhibitor BRASSINOSTEROID INSENSITIVE2 (BIN2) (42). In addition, introgression of the BR receptor containing chromosome segment (7DL) from Agropyron elongatum results in enhanced drought tolerance in wheat (43). However, an increase in UDP-Glc (a substrate for synthesis of various sugars, necessary for different wall polymers) levels by overexpressing sucrose synthase (SuSy) and UDP-glucose pyrophosphorylase (UGPase)-encoding genes leads to enhanced drought tolerance and cellulose accumulation (44). Similarly, heavy metal stress in rice and wheat causes enhanced lignin accumulation in the cell wall, which improves defense response against biotic and abiotic stresses (45).
Transgenic Arabidopsis plants overexpressing CaXTH3, encoding a xyloglucan biosynthesis gene (xyloglucan endotransglucosylase/hydrolases), have abnormal leaf morphology and severely wrinkled leaf shape and enhanced tolerance against water stress (46). ␤-Expansin encoding gene EXPB2 showed higher expression under water stress and seemed to be involved in improvement of root system architecture under water deficits in soybeans (47). Similarly, silencing of NAC2 or its downstream gene EXPA4 has been shown to result in reduced drought tolerance during the petal development in rose (48). In contrast, overexpression of RhEXPA4 conferred an improved phenotype with shorter stems, curly leaves, compact inflorescences, and enhanced drought tolerance in Arabidopsis (48). However, higher transcript abundance of cinnamoyl-CoA reductase in maize inhibited cell wall extensibility and root growth under water deficiency (49). Similarly, expansin acts as a key component in heat stress tolerance as its higher expression in Agrostis leads to increased cell wall elasticity, which maintains cellular functions (50). Arabidopsis MYB41 and rice R2R3-type MYB transcription factor MPS (MULTIPASS) are induced under salinity, which then enhances expression of cell wall biosynthesis genes, during vegetative and reproductive stages, while suppressing the transcript of expansin and endoglucanase genes (51). Similarly, overexpression of TOR, a Ser/ Thr kinase of the phosphatidylinositol 3-kinase-related kinase family leads to enhanced cellular biomass and stress tolerance (52). However, AtTOR RNAi lines show high sensitivity toward osmotic stress (52).
Integrating the knowledge of key regulatory genes with their cell-specific promoters and transcriptional regulation of secondary cell wall biosynthesis through the cascade of activators, repressors, and feedback regulators is expected to enable the development of designer plants with enhanced biomass under stress conditions (16,17). A schematic diagram depicting the transcriptional regulatory network modulating secondary cell wall formation in plants is shown as Fig. 2. These studies also provide deep insight into the complex transcriptional machinery to genetically modify cell wall composition for tolerance against abiotic stresses.

Manipulation of phytohormone levels
Of the various factors regulating plant biomass production under abiotic stresses, manipulation of plant hormone content is the most efficient approach (53). Of these hormones, auxins, cytokinins (CKs), gibberellins (GAs), BRs, and abscisic acid are markedly involved in the regulation of plant growth and biomass production during stress conditions (53) . Fig. 3 depicts the cross-talk between various phytohormones leading to enhanced stress tolerance resulting in higher biomass production in plants. Dissecting the molecular mechanism of these hormone biosyntheses and regulations provides an insight into the complexity of various processes such as time, rate, and extent of cell division and cell expansion to manipulate the regulation of meristematic division and plant growth under stress conditions (Table 1) (54). Although CKs have an important role in delaying the natural senescence in many plants (55), the concentration of the bioactive CKs decreases during exposure to water and salt stresses (56).
Plants exhibiting negative regulation of cytokinin activity, such as plants overexpressing CKX (cytokinin oxidase) or mutation in the cytokinin receptors, show smaller shoot apical meristem and decreased leaf area (57). Similarly, it is known that knocking down CKX2 expression in rice results in the maintenance of photosynthetic rate, panicle branching, and reduction in yield gap under salinity stress conditions (58). Most of the studies on transgenic plants with pSARK promoter-regulated isopentenyltransferase gene expression have reported delayed leaf senescence, higher photosynthetic activity, and enhanced biomass and/or yield-related parameters under drought stress in tobacco (59), rice (60), broad bean (61), and creeping bent grass (62), peanut (63), and under salt stress in cotton (64).
Under heavy metal and salt stress, IAA increases shoot as well as root growth in plants (27). Transgenic poplars overexpressing an abiotic stress-responsive YUCCA6 gene, which is involved in tryptophan-dependent IAA biosynthesis pathway using stress-inducible SWPA2 promoter, exhibit rapid shoot elongation and reduced main root development with enhanced root hair formation (65). Earlier studies demonstrated that down-regulation of SPL8 (squamosa promoter-THEMATIC MINIREVIEW: Plant biomass under abiotic stress binding-like protein-8) transcription factor significantly increased branching by promoting axillary bud development and enhanced forage biomass yield, besides enhancing salt and drought tolerance in alfalfa (66); these transgenic plants showed reduced GA accumulation, whereas spl8 mutants showed significantly higher GA transcript abundance. Furthermore, GA2-ox6, which is the prime GA deactivation enzyme, is significantly up-regulated by SPL8 (66). These results suggest that SPL8 regulates GA signaling, and GA2-ox is a possible key node of this signaling network (67). Under saline conditions, GA application improved stomatal conductance, water use efficiency, and growth and yield in tomato plants, thus supporting the above hypothesis (68).
The Arabidopsis homolog of RXT3 (REGULATOR of TRANSCRIPTION 3), named HISTONE DEACETYLASE COMPLEX1 (HDC1) shows direct interaction with histone deacetylases HDA6 and HDA19 (69). The hda6, hda19, and hdc1-1 mutant showed hypersensitivity toward NaCl and ABA during seedling stage (69). However, HDC1 overexpression reduces NaCl and ABA sensitivity and increases biomass (70). Transgenic tomato plants overexpressing ABA-responsive complex (ABRC1) from the barley HVA22 showed improved growth and yield in addition to enhanced tolerance against cold, drought, and salt stress (71). Furthermore, AtERF11 was shown to negatively modulate the ABA-mediated regulation of ethylene biosynthesis; furthermore, its overexpression conferred ABA hypersensitivity during post-germination growth under stress conditions (72). ABA3/LOS5 encodes a Mo-cofactor sulfurase that catalyzes abscisic aldehyde to ABA conversion, and its constitutive expression in rice leads to significantly higher yield during drought stress under field conditions (73). Similarly, overexpression of MoCo sulfurase gene in soybean resulted in enhanced biomass and yield along with improved drought tolerance attributed to increased ABA accumulation, reduced water loss, and induced antioxidant enzymatic machinery (74). Furthermore, 9-cis-epoxycarotenoid dioxygenase (NCED) has been shown to catalyze the conversion of neoxanthin to xanthoxin. In addition, T-DNA insertional nced3 mutants displayed impaired drought tolerance and limited ABA accumulation under water stress, whereas tobacco plants constitutively expressing NCED1 enhanced ABA accumulation in leaves, which leads to improved drought and salt   (75). Similarly, in tomato the constitutive overexpression of NCED1 displayed enhanced ABA accumulation, reduction in assimilation rates, leaf chlorosis, and higher biomass because of counteracting positive effects of ABA on leaf expansion and increased water status (76).
Similarly, BR pre-treated seeds demonstrated significantly higher accumulation of dry mass and antioxidant enzyme activity in alfalfa under salt stress (77) and ameliorated growth and survival of Robinia pseudoacacia during water stress (78). In addition, BR application causes enhanced seedling growth in sorghum under osmotic stress (79). Knockout T-DNA insertion mutant of Osgsk1 (a rice GSK3/SHAGGY-like protein kinase gene, ortholog of AtBIN2/AtSK21, and negative regulator of BR signaling) depicted enhanced tolerance toward abiotic stress, whereas OsGSK1 overexpression resulted in stunted growth in Arabidopsis (80). In addition, hormones target various members of protein families playing a significant role in growth either individually or in a co-regulated manner, thereby indicating the synergistic action of metabolic pathways (81). Biotechnological manipulation of these key proteins could allow not only the adaptation under adverse environmental conditions but also determine the flux in phytohormone biosynthesis for securing, in the long run, improved food production.

Future outlook
With the apparent changes in the global environment, agricultural production systems are liable to change. Thus, there may be a need to produce cost-effective biomass to replace the existing fossil fuel in the near future. At the same time, to ensure availability of enough food for 9 billion people is indeed a daunting task. One possible solution to this challenge could be to make use of marginal lands with low input crops capable of providing high-biomass yield. Attempts are already being made to modify the plant architecture in such a way that will led to "transgressive overyielding" of plant biomass. Furthermore, there are numerous options to explore plant-microbe interactions for enhanced biomass production in marginal and arable lands. It is noteworthy that most of these interactions are currently unexplored, making it important to closely observe the soil, the rhizosphere, and the endophyte populations. Engineered plants and their beneficial symbionts will pave the way for future strategies to modify them for higher biomass production to meet the demands of a growing population in a changing climate scenario. Similarly, understanding how cellular differentiation occurs during cell wall development will provide deep insight into designing cell wall architecture to enhance biomass under environmental stresses. More importantly, identifying the key transcription factors directly regulating secondary cell wall biosynthesis genes will not only provide valuable clues to understand the evolution of secondary wall biosynthesis in vascular plants but also to uncover the complexity of the dynamic changes during cell wall development and abiotic stress response. Similarly, the phytohormones have been found to be involved directly in plant responses to different stresses. However, the identification and maintenance of optimal dose/response ratio of hormones still remain a tedious task, because the hormonal balance should be moderate to maintain homeostasis to provide abiotic stress tolerance and retain growth and biomass. Much work has been done in the past decades on the molecular pathways modulating hormone biosynthesis and signaling and dissecting out their role in response to varied climatic conditions. Our Minireview will thus be fruitful in suggesting ways to genetically manipulate hormone biosynthesis pathways for abiotic stress tolerance and for enhanced crop productivity. In the future, it will be highly desirable to study the response of plants toward a combination of stresses mimicking the field conditions, as no stress ever comes alone!