Role of Helicases inImparting Tolerance to Abiotic Stress
Abiotic stress condition often affects the cellular gene expression machinery. Therefore, the molecules that are involved in the processing of nucleic acids including helicases are also likely to be affected. Multiple DNA helicases are present in the cell and are involved in gene regulation at various developmental stages as well as in stress conditions. These DNA unwinding enzymes may have different substrates as well as structural requirements (Matson et al, 1994; Tuteja and Tuteja, 1996). Though a number of different helicases have been reported from E. coli, bacteriophages, viruses, yeast, calf thymus and humans the biological role of only a few DNA helicases have been explored (Lolunan and Bjomson, 1996; Tuteja and Tuteja, 2004). Helicase genes are now being reported as powerful gene for developing stress tolerant crops. Most helicases are members of DEAD- box protein super-family and play essential roles in basic cellular processes such as replication, repair, recombination, transcription, ribosome biogenesis and translation initiation. Transient opening of the stable duplex DNA is an essential prerequisite step in many biological processes such as DNA replication, repair, recombination and transcription. DNA helicases catalyses the unwinding of energetically stable duplex DNA (DNA helicase) or inter and intra molecular base -paired duplex RNA (RNA helicase) structures by disrupting the hydrogen bonds between the two strands and thereby plays an important role in all aspects of nucleic acid metabolisms. Gene pdli45, the first plant DNA helicase has been cloned, overexpressed and characterized in detail (Hoi et al, 2008). The potential role of PDH45 (pea DNA helicase 45) in overcoming salinity stress was explored (Sanan- Mishra et al, 2005). They have proved that PDH45 over expressing transgenic lines showed high salinity tolerance and the Tj transgenic plants were able to grow to maturity and set normal viable seeds under continuous salinity stress without any reduction in plant yield in terms of seed weight. The authors have proposed a dual mode of action for PDH45 (Table 3).
There are various reports published on the isolation of a pea DNA helicase 45 (PDH45) and its novel role in abiotic stress tolerance in model plant tobacco (Plant J, 24, 1-13; PNAS, USA 102, 509-514). The exact mechanism of helicase- mediated salt tolerance is not yet understood. However, based on the properties of PDH45 that were studied earlier (Rocak and Linder, 2004) and those known for DEAD-box proteins (Hasegawa, 2000; Tuteja and Tuteja, 2004). It is revealed that two sites of action of this dual helicase: 1) it may act at the translation level to enhance or stabilize protein synthesis; or 2) it may associate with DNA multisubunit protein complexes to alter gene expression. After observing the proof of concept in model plants, the PDH45 has been used to transform the bacteria and to different varieties of rice and groundnut crops. The results show that PDH45 also provide the salt tolerance in bacteria, rice and groundnut. Interestingly, there were no yield losses.
Signal Transduction Genes
Signaling pathway is complex as it involves the coordinated action of various genes in a single pathway or diverse pathways. Calcium is a prime candidate, which functions as a central node in mediating the coordination and synchronization of diverse stimuli into specific cellular responses. Thus, the proteins, which sense cytoplasmic Ca2+ perturbations and relay this information to downstream molecules, serve as an important component of signaling. In plant cell many calcium sensors have been recognized which include calmodulin (CaM) and calmodulin-related proteins (Luan et al, 2002), Ca2+-dependent protein kinases (CDPKs) (Sanders et al, 2002) and the relatively recently discovered sensor CBL (calcineurin Blike) protein (Liu and Zhu, 1998). CBLs are characterized by 4 helix-loop-helix calcium binding domains termed as EF hands. Currently, 10 isoforms of CBL have been dis-covered in Arabidopsis and named as CBL due to then' sig-niWcant sequence similarity to animal calcineurin B. Despite this sequence similarity, Arabidopsis lacks calcineurin in its data bank (Gong et al., 2004). Various isoforms of CBL are up-regulated in stress condition. CBLs specially interact with a class of kinases known as CBL-interacting protein kinase (CIPKs) to transduce the signal via phosphorylation of downstream signaling components.
One of the merits for the manipulation of signaling factors is that they can control a broad range of downstream events that can result in superior tolerance for multiple aspects (Umezawa et al, 2006). Alteration of these signal transduction components is an approach to reduce the sensitivity of cells to stress conditions, or such that a low level of constitutive expression of stress genes is induced (Grover et al., 1999). Over expression of functionally conserved At-DBF2 (homolog of yeast DBf2 kinase) showed striking multiple stress tolerance in Arabidopsis plants (Lee et al., 1999). Pardo et al. (1998) also achieved salt stress- tolerant transgenic plants by over expressing calcineurin (a Ca2+ Calmodulin dependent protein phosphatase), a protein phosphatase known to be involved in salt-stress signal transduction in yeast. Transgenic tobacco plants produced by altering stress signaling through functional reconstitution of activated yeast calcineurin not only opened-up new routes for study of stress signaling, but also for engineering transgenic crops with enhanced stress tolerance (Grover et al, 1999). Over expression of an osmotic-stress-activated protein kinase, SRK2C resulted in a higher drought tolerance in A. thaliaiia, which coincided with the upregulation of stress-responsive genes (Umezawa et al., 2004). Similarly, a truncated tobacco mitogen-activated protein kinase kinase kinase (MAPKKK), NPK1, activated an oxidative signal cascade resulting in cold, heat, salinity and drought tolerance in transgenic plants (Table 3; Kovtun et al, 2000; Shou et al, 2004). However, suppression of signaling factors could also effectively enhance tolerance to abiotic stress (Wang et al, 2005). This hypothesis was based on previous reports indicating that a and b subunits of famesyltransferase ERA1 functions as a negative regulator of ABA signaling (Cutler et al., 1996; Pei et al, 1998). Conditional antisense downregulation of a or b subunits of protein famesyl transferase, resulted in enhanced drought tolerance of Arabidopsis and canola plants.
Presently the direction of research is more towards isolation of master switches, which can control these stress genes. As cytosolic calcium up-regulation is more or less a universal phenomenon associated with stress signaling, thus the calcium sensors, which decode these Ca2+ signatures and relay the information down stream, may act as master switches in controlling various stress genes. Moreover, mutations in these calcium sensors like AtCBLl and their interacting protein kinases have been shown to cause aberrations in the expression of some of the major stress responsive genes like RD29A, KINL KIN2 and RD22 indicating their immense significance in stress signaling (Pandey et al, 2004).
Targeting Pathways: Tandem Expression of Genes
Under natural field conditions plants have to cope with different stress combinations at different developmental stages and for varying duration. Tolerance to abiotic stress is a consequence of genetic and environmental interactions through a complex network that implies physiological, molecular and biochemical responses. Modifying the expression of different components simultaneously has the potential to generate responses apt to the complexity of a combination of stresses. There are only few examples where the simultaneous co-expression of different components of the same pathway has been tried. Increase in biosynthesis of proline was achieved by co-expression of E. coli P5C biosynthetic enzymes gamma-glutamyl kinase 74 (GK74) and gamma-glutamylphosphate reductase (GPR) and the antisense transcription of pro line dehydrogenase (ProDH) in Arabidopsis and tobacco (Stein et al, 2011). The transgenic plants displayed improved tolerance to heat stress associated with the accumulation of cell wall proline-rich proteins (Stem et al, 2011). Simultaneous co-expression of dehydroascorbate reductase (DHAR), glutathione reductase (GR) or glutathione-S-transferase (GST) and glutathione reductase (GR) in tobacco plants also resulted in the increased tolerance of the transgenic plants to a variety of abiotic stresses (Martret et al, 2011). In tobacco seeds, higher antioxidant enzymes activity driven by the simultaneous over expression of the CuZnSOD and APX genes in plastids, allowed the increase of germination rates and longevity of long-term stored seeds under combined stress conditions (Lee et al, 2010), demonstrating the enormous potential of simultaneous gene expression in plant engineering (Table 3).
Modifying Function: Engineering C4Photosynthetic Pathway into C3 Crops
Abiotic stress is the major factor limiting photosynthetic activity, resulting hi growth and yield reduction. The photosynthesis machinery also affects metabolic processes such as carbon and nitrogen partitioning (Ainsworth and Bush, 2011) and oxidative stress regulation (Foyer and Shigeoka, 2011). The projected effects of climate change in rising ambient temperatures and CCl concentrations will have influence plant C02 assimilation (and yield), and photorespiration. Research efforts are focused on obtaining Kranz anatomy (Hibberd et al, 2008), especially in lice which have an intermediate anatomical characteristics between C3 and C4 plants (Sage and Sage, 2009). While most genes controlling bundle density in C4 plants are still unknown, it has been postulated that about 20 genes are involved (reviewed by Peterhansel, 2011).The ability of the C4 photosynthetic pathway to suppress ribulose 1,5-bisphosphate (RuBP) oxygenation and photorespiration represents the most efficient form of photosyn-thesis on Earth (Sage 2004). In recent years, efforts have been given to engineer C4 photosynthesis into C3 crops (Sage and Zhu, 2011). The expression of genes encoding enzymes such as phosphoenol pyruvate carboxylase (PEPC), the chloroplastic pyruvate orthophosphate dikinase (PPDK), and NADP-malic enzyme (NADP-ME) into rice (Ku et al, 2007), tobacco (Hausler et al., 2002) and potato (Rademacher et al., 2002) unproved photosynthetic rate and yield. Although considerable efforts have been made, the over expression of either single or multiple C4-enzyme related genes in C3 plants have resulted in contradictory results (Shao et al, 2011). Thus, in order to obtain C4 crops, new transformation methods together with additional efforts to better understand the function of C4 enzymes in a proper leaf anatomy (Furbank et al., 2009) are needed. Another important aspect that has to be addressed is source/ sink relationships. From an evolutionary perspective C3 plants have modified their sink size proportionally to the source size (i.e. photosynthesis organs). Thus, more efficient carbon fixation via C4 pathway in the transformed plants would require adapting the sinks to attain efficient harvest index (Murchie et al., 2009).