Osmoprotectants and Metabolite Engineering

Abiotic stress such as dehydration, salt or freezing perturbs the cellular metabolism, as pointed out in numerous physiological experiments using many different plant species (Ingram and Bartels 1996; Bartels and Sunkar 2005). Therefore research strategies have been designed to counteract the metabolic imbalance provoked by abiotic stressors. It is implied in the research strategy that engineering metabolism may counteract negative consequences of stress only within a certain range. Another consideration has to be that engineering of metabolic pathways should not interfere with other pathways, e.g. those which are responsible for biomass or yield.

Most organisms, ranging from microbes to animals and plants, synthesize compatible solutes in response to dehydration. Compatible solutes are non-toxic small molecules which do not interfere with normal cellular metabolism. Depending on the organism a variety of substances have been described as compatible solutes. Examples are sugars or sugar alcohols such as raffinose, galactinol, trehalose or fructan, amino acids such as proline, amines such as glycine betaine or polyamines. Compatible solutes have their main role in turgor maintenance and in osmotic adjustment. Also additional functions have been discussed such as stabilizing cell proteins and structures, scavenging reactive oxygen species, signalling functions or induction of adaptive pathways (Hasegawa et al. 2000; Chen and Murata 2002). However, the exact function is not fully understood. Simple, preferentially one-step transformation strategies were designed to increase the accumulation of these molecules (including in plant species in which osmolytes do not accumulate naturally). This approach was partly successful and stress-tolerant plants were obtained (Table 8.1), although this strategy did not always lead to osmotic adjustment (Serraj and Sinclair 2002). Most approaches relied on transforming plants with a single gene, and this may be the reason that often only marginal stress tolerance was obtained. In nearly all examples osmolyte accumulation in the whole plant was considered but not in specific tissues or specific cells. In a detailed analysis of gene expression in Arabidopsis roots, Dinneny et al (2008) showed that stress genes may be restricted to particular lineages of cells in the Arabidopsis root. Such data may provide better targets for modifying metabolite profiles in the future.

Table 8.1 Selected transgenic crop plants with enhanced tolerance to osmotic stress via metabolite engineering

Gene

Class

Species (origin of gene)

Transgenic crop species

Promoter

Performance of transgenic plant

Additional notes

References

P5CS

SacB

otsA/otsB

mtlD

otsA/otsB

Al-pyrroline-5-carboxylate Vigna aconitifolia Oryza sativa AIPC (ABA synthetase inducible promoter complex)

codA (glycinebetaine) Arthrobacter Tomato CaMV 35S

globiformis

Levan sucrase Bacillus subtilis Beta vulgaris CaMV 35S

Trehalose-6-phosphate synthase/ Trehalose-6-phosphate phosphatase

Mannitol-1 -phosphate dehydrogenase

Trehalose-6-phosphate synthase/ Trehalose-6-phosphate phosphatase

Arginine decarboxylase

Escherichia coli

E. coli

E. coli

O. sativa

Triticum aestivum

O. sativa

Datura stramonium O. sativa

AIPC (ABA inducible promoter complex); rbcS ZmUbil

ZmUbil

ZmUbil

Dehydration and high salinity tolerance

Various abiotic stresses Dehydration tolerance

Dehydration, high salinity and low temperature tolerance

Dehydration and high salinity tolerance

Dehydration, high salinity and low temperature tolerance

Dehydration tolerance

Increased biomass (higher fresh shoot and root weight)

Expression targeted to chloroplasts Increased biomass (leaves and roots) under drought Sustained photosynthetic rate and growth under stress

Sustained growth of calli and whole plants under stress conditions Sustained photosynthetic rate and growth under stress

Unaffected by PEG treatment

Zhu et al.

Park et al.

Capell et al. (2004)

Table 8.2 Transgenic crop plants engineered for enhanced tolerance to osmotic stress using regulatory and signalling genes

Gene

Class

Plant species

Transgenic crop species

Promoter

Performance of the transgenic plant

Additional notes

Reference

OsCDPK7

Calcium-dependent protein kinase

Oryza sativa

O. sativa

CaMV 35S

Dehydration, high salinity and low temperature tolerance

Decreased wilting under stress conditions.

Saijo et al. (2000)

CBF3/DREB1A

ERF/AP2

Arabidopsis

Triticum

rd29A

Dehydration tolerance

Delay in germination, delayed

Pellegrineschi

(subgroup IIIc)

thaliana

aestivum

water stress symptoms, greater number of heads, more branched root architecture.

et al. (2004)

NPK1

MAPKKK

Nicotiana tabacum

Zea mays

CaMV 35S

Dehydration tolerance

Sustained photosynthetic rate under drought. Delayed maturity and increased leaf number. Reduced impact of drought on kernel weight.

Shou et al. (2004)

GF14X

14-3-3

A. thaliana

Gossypium hirsutum

CaMV 35S

Dehydration tolerance

Sustained photosynthetic rate and increased leaf survival under drought. "Stay-green" phenotype (delayed senescence).

Yan et al. (2004)

(subgroup IIIc)

A. thaliana

0. sativa

ZmUbil

Dehydration and high salinity tolerance

Low reduction in Fv/Fm under drought and high salinity. Greater survival under drought. Low level tolerance to low temperature. No growth stunting.

Oh et al. (2005)

ABF3

bZIP

A. thaliana

0. sativa

ZmUbil

Dehydration tolerance

Low reduction in Fv/Fm and greater survival under drought. No enhanced tolerance to low temperature or high salinity. No growth stunting.

Oh et al. (2005)

SNAC1

NAC

0. sativa

0. sativa

CaMV 35S

Dehydration and high salinity tolerance

Increased ABA sensitivity and delayed water stress symptoms. Sustained growth and greater

Hu et al. (2006)

  • continued)
  • continued)

Table 8.2 (continued)

Gene

Class

Plant species Transgenic crop species

Promoter

Performance of the transgenic plant

Additional notes

Reference

HARDY

HvCBF4

SNAC2

OsDREBlF

HDG11

ERF/AP2 A. thaliana

(subgroup Mb)

ERF/AP2 O. sativa

(subgroup IIIc)

Homeodomain-START

A. thaliana

O. sativa

ZmNF-YB2 CCAAT-binding Zea mays Z. mays

CaMV 35S Dehydration tolerance

OsRACT Dehydration tolerance

ERF/AP2 Hordeum O. sativa

(subgroup IIIc) vulgare

O. sativa O. sativa

O. sativa

Nicotiana tabacum

Dehydration, high salinity and low temperature tolerance Osmotic, high salinity and low temperature tolerance Dehydration, high salinity and low temperature tolerance

CaMV 35S Dehydration tolerance

ZmUbil

ZmUbil

CaMV 35S

survival under salinity stress. Enhanced yield under drought stress in the field.

Deeper green, increase in leaf canopy with more tillers. Enhanced water use efficiency. Sustained photosynthetic rate and growth (root and shoot) under drought stress.

Less wilting during drought and greater recovery after stress. Sustained photosynthetic rate and yield under drought stress in the field.

Delayed appearance of stress symptoms, rapid recovery and greater survival rate. No growth stunting.

Improved growth in salinity and PEG media. No yield increase under drought stress in the field.

Sustained growth under stress conditions. Both ABA-dependent and ABA-independent pathways activated.

Extensive root system, reduced leaf stomatal density and enhanced water use efficiency.

Karaba et al. (2007)

Nelson et al. (2007)

8.2.1.1 Amino Acid-Derived Osmoprotectants

Metabolic engineering studies have mainly focused on proline and betaine. Both proline and betaine have been shown to accumulate in several plant species under stress (Sunkar and Bartels 2005). The biosynthetic pathways for both metabolites have been well studied and therefore the genes for manipulating the pathways are available (Bohnert and Shen 1999).

Proline

Many plants accumulate proline in response to osmotic stress (Delauney and Verma 1993). Two biosynthetic proline pathways exist in plants: the ornithine-dependent pathway and the glutamate-dependent pathway. The glutamate pathway seems to be the predominant pathway for proline synthesis, especially under stress conditions (Delauney and Verma 1993).The other important reaction that controls proline levels is the oxidation of proline by proline dehydrogenase to pyrroline-5-carboxylate (Nanjo et al. 1999). The transgenic plants overexpressing proline biosynthetic enzymes demonstrated the involvement of proline in response to water deficit (Kishor et al.1995; Roosens et al. 2002). Increased degradation of proline via pyrroline-5-carboxylate reductase resulted in increased sensitivity to water stress (de Ronde et al. 2000). Transgenic rice (see Chap. 22) and wheat plants (Chap. 16) that accumulate proline showed better stress tolerance to dehydration and salinity, respectively (Zhu et al. 1998; Sawahel and Hassan 2002).

Glycine Betaine

Glycine betaine is a quarternary ammonium compound that occurs in a variety of plants, animals and microorganisms (Chen and Murata 2008). Glycine-betaine is synthesized in plants via a two-step oxidation of choline. In spinach and some other plants the oxidation is carried out by two chloroplastic enzymes: choline mono-oxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). The first reaction is the oxidation of choline to betaine aldehyde and the second reaction oxidizes betaine aldehyde to betaine (Fitzgerald et al. 2009). The overexpression of betaine biosynthesis genes either derived from bacteria or from plants in generating drought tolerance in plants, which do not naturally synthesize glycine betaine (Chen and Murata 2002). Examples are transgenic Arabidopsis, Brassica napus, tobacco, rice or tomato (Sakamoto et al. 1998; Mohanty et al. 2002; Shirawasa et al. 2006; Park et al. 2007). In some plants low accumulation of betaine was observed, which was explained by a limited supply of choline (Nuccio et al. 1998). The effectiveness of this approach seems also be dependent on the correct compartment in which betaine accumulates, as demonstrated for tomatoes, in which chloroplastic accumulation of betaine is more effective than cytosolic (Park et al. 2007). The different results show that understanding metabolic fluxes in plants is an important prerequisite for successful genetic engineering. Several agronomically important crops such as wheat, potato or tomato do not accumulate glycine betaine naturally and would therefore be good targets for engineering betaine biosynthesis.

8.2.1.2 Sugar-Related Osmoprotectants

Fructans

Fructans are oligo- or polyfructose molecules that accumulate in vacuoles of many plants growing in temperate climates. Sugar beet and tobacco plants that were transformed with the bacterial fructan biosynthesis gene showed improved tolerance to drought stress (Pilon-Smits et al. 1999). However, this approach has not been transferred to other crop plants.

Polyols

Polyols are hydroxylated sugar alcohols with osmoprotective properties which accumulate in response to abiotic stress in various plants. The synthesis of these compounds involves simple pathways and therefore it was possible to transfer the biosynthesis genes to transgenic plants in order to test their potential for stress tolerance (Bohnert and Chen 1999; Bartels and Sunkar 2005). The overproduction of polyols such as mannitol, D-ononitol, inositol or sorbitol in transgenic plants enhanced stress tolerance (Bartels and Hussain 2008). It is assumed that the polyols confer stress tolerance through osmotic adjustment. However, the level of polyols did not always correlate with stress tolerance, therefore other mechanisms have also been suggested like reactive oxygen scavenging or signalling. Targeting polyol biosynthesis was one of the earliest concepts for engineering plants with improved stress tolerance (Bohnert and Chen 1999). Although Abebe et al. (2003) demonstrated that expressing the gene encoding mannitol dehydrogenase in wheat improved the performance under drought and salinity stress, there are no further reports of using this concept for crops. The reason for this is that some of the polyol compounds have undesired effects like growth defects and necrosis (Sheveleva et al. 1998). These authors showed that there is a competitive effect between transgene and host metabolism. This underlines the necessity to understand metabolic fluxes before successful applications to agricultural plants.

Trehalose

Trehalose is a non-reducing disaccharide found in many different organisms. The sugar functions as a reserve carbohydrate and as a stress protectant, particularly in yeast or microorganisms. Trehalose does not accumulate to high levels in most plants probably due to degradation by trehalase (Goodijn and van Dun 1999). The synthesis of trehalose is a two-step reaction, starting with glucose-6-phosphate and uridine diphosphoglucose using trehalose phosphate synthase and trehalose-6-phosphate phosphatase as catalysing enzymes. By overexpressing both genes in transgenic plants, stress-tolerant plants have been obtained (Table 8.1; Bartels and Hussain 2008). Often the trehalose level did not correlate with stress tolerance and plants were observed with altered growth and morphogenic phenotypes. Investigations showed that the metabolic intermediate trehalose-6-phosphate is responsible for the aberrant phenotypes, as it functions as a signalling molecule regulating sugar and starch metabolism (Paul 2007). Recent experiments indicated that negative consequences may be overcome by choosing a suitable promoter and by transforming chloroplasts. In this way transgenic plants have been obtained with improved drought stress tolerance but no negative side-effects (Garg et al. 2002; Jang et al. 2003). Like the engineering of polyol levels, the trehalose metabolic network needs to be better understood before such an approach can be accepted as a way to improve stress tolerance in agricultural plants.

8.2.2 Regulatory and Signalling Genes: Tools to Engineer Drought Stress Tolerance

Studies of abiotic stress-activated signalling cascades have resulted in the identification of potential regulatory genes, such as transcription factors and protein kinases. The transformation of plants using regulatory genes is an attractive approach for producing abiotic stress-tolerant plants. Since the products of these genes regulate gene expression and signal transduction under stress conditions, the expression of these genes can activate the expression of many stress-tolerance genes simultaneously. For example, transcription factors are able to recognize and bind to regions of DNA that have a specific sequence in the promoters of the genes they regulate. Thus, by altering the expression levels of a transcription factor, entire biological pathways can be modified. Similarly, altered expression of protein kinases may enable phosphorylation dependent changes of multiple protein substrates by changing enzyme activity, cellular location, or association with other proteins. One potential drawback of this approach is the increased likelihood of unintended or pleiotropic effects when regulatory/signalling genes are genetically engineered. Such effects tend not to be desirable in crops and strategies to ameliorate these effects may need to be considered. Here we discuss examples where transcription factors and protein kinases have been used to engineer enhanced tolerance to drought stress conditions in crop plant species.

8.2.2.1 DREB/CBF: a Landmark Discovery in the Manipulation of Abiotic Stress Tolerance

The ability to manipulate co-regulated stress tolerance-associated genes at the transcriptional level was first realised when common cis elements were discovered in abiotic stress-responsive promoter regions and the associated transcription factors that specifically bound to the cis elements were identified. An early example was the discovery of the dehydration-responsive element/C-repeat (DRE/CRT) as a cis-acting element regulating gene expression in response to dehydration (salt, drought, cold stresses) in Arabidopsis (Yamaguchi-Shinozaki and Shinozaki 1994). Subsequently, transcription factors DREB1/CBF1-3 and DREB2, belonging to the ERF/AP2 family (subgroup IIIc; Nakano et al. 2006), were reported to bind to DRE/ CRT elements (Stockinger et al. 1997; Liu et al. 1998). A major breakthrough was made when Kasuga et al. (1999) transformed Arabidopsis with a cDNA encoding DREB1A/CBF3 driven by either the constitutive CaMV 35S promoter or an abiotic stress-inducible promoter. The overexpression of this gene activated the expression of many stress-tolerance genes such as late embryogenesis abundant (LEA) genes and A1-pyrroline-5-carboxylate synthetase (P5CS). In all cases, the transgenic plants were more tolerant to drought, salt, and freezing stresses.

Arabidopsis CBF/DREB proteins are also heterologously effective in crops such as Brassica napus (Jaglo et al. 2001), tomato (Hsieh et al. 2002), wheat (Pellegrineschi et al. 2004), and rice (Oh et al. 2005), up-regulating the corresponding target genes and enhancing stress tolerance in transgenic plants. These results established the DREB/ CBF pathway as a useful target for the biotechnological improvement of abiotic stress tolerance in both monocotyledons and dicotyledons. Despite the fact that DREB/CBF related responses appear conserved, plant species vary greatly in their abilities to survive adverse effects from exposure to environmental constraints. In the initial experiments by Kasuga et al. (1999), it was observed that constitutive overexpression of DREB 1A/CBF3 in Arabidopsis resulted in severe growth retardation under normal growth conditions. Constitutive expression of Arabidopsis DREB1A/CBF3 and OsDREB1F in rice, however, resulted in neither growth inhibition nor visible pheno-typic alterations (Oh et al. 2005; Wang et al. 2008). A similar lack of pleiotropic effects was also observed for the basic leucine zipper (bZIP) transcription factor ABF3 when ectopically expressed in rice (Oh et al. 2005). This phenomenon may have occurred because lower levels and/or fewer numbers of target genes are activated by DREB1A/CBF3 or ABF3 in rice than in Arabidopsis, and hence, the effects on plant growth might be minimized in rice. Oh et al. (2005) also postulate that rice is evolutionarily more tolerant to the expression of stress-regulated genes than dicots, including Arabidopsis. Where pleiotropic effects are observed, however, it appears more appropriate, at least in the case of DREB/CBF genes, to use an inducible promoter. For example, the stress-inducible regulation of DREB1A/CBF3 via the rd29A promoter repeatedly had minimal effects on plant growth under well watered conditions (Kasuga et al. 1999; Pellegrineschi et al. 2004).

Arabidopsis CBF3/DREB1A expression in transgenic rice increases tolerance to drought and high salinity, but with relatively low levels of tolerance to low-temperature exposure (Oh et al. 2005). These data are in direct contrast to CBF3/ DREB1A expression in transgenic Arabidopsis, which functions primarily to enhance freezing tolerance. This is presumably because Arabidopsis plants that are capable of cold acclimatization have evolved differently from rice plants that are unable to undergo cold acclimatization (Jaglo et al. 2001). Furthermore, the

HvCBF4 protein from barley appears to be more efficacious than CBF3/DREB1A from Arabidopsis in conferring stress tolerance to transgenic rice (Oh et al. 2007). When compared with CBF3/DREB1A, HvCBF4 overexpression in rice showed similar levels of tolerance to drought and high salinity, but a higher level of tolerance to low temperature. These data suggest functional differences between members of the DREB/CBF family and highlights the variation in stress tolerance between transgenic plant species. This is probably related to the complexity and nature of the target genes or "regulon" that is present in the plant genome and the capacity of the transcription factor to activate or repress each target gene.

8.2.2.2 SNAC1/2: Stress-Responsive Plant-Specific Transcription Factors with Distinct Mechanisms of Action

The NAC gene family encodes plant-specific transcription factors that were initially linked with regulation of plant development; however a role in abiotic stress tolerance has since been established (Olsen et al. 2005). This discovery was based on the identification of a salt- and drought-induced gene, ERD1, that was found to be regulated in an ABA-independent manner via a novel regulatory pathway for drought and high salinity adaptation (Nakashima et al. 1997). A MYC-like cis element was found necessary for induction of ERD1 that was recognized by three transcription factors of the NAC family (Tran et al. 2004). In addition to ERD1, many other salt and/or drought stress-induced genes were also regulated by NAC proteins which correlated with enhanced drought tolerance in Arabidopsis overexpression lines (Tran et al. 2004).

More recently, Hu et al. (2006) reported a NAC transcription factor significantly enhancing drought and high salinity tolerance in rice: STRESS-RESPONSIVE NAC 1 (SNAC1). SNAC1 -overexpressing rice plants exhibited significantly enhanced yield (22-34% higher seed setting than control) in field conditions under drought stress conditions at the reproductive stage, while displaying no yield penalty. The transgenic rice displayed noticeably improved drought and salt tolerance at the vegetative stage. The transgenic rice plants are more sensitive to abscisic acid (ABA) and lose water more slowly through stomatal movement, yet display no significant difference in the rate of photosynthesis. The SNAC1-overexpressing rice plants also showed improved salt tolerance, further emphasizing the usefulness of SNAC1 in a broad abiotic stress tolerance improvement (Hu et al. 2006).

A closely related stress-responsive NAC transcription factor gene termed SNAC2 was subsequently isolated from upland rice (Hu et al. 2008). Transgenic rice overexpressing SNAC2 showed significantly improved tolerance to cold, as well as to salinity and dehydration stresses. SNAC2 differs from SNAC1, however, in a several aspects, which illustrates how transcription factor gene family members can have broadly different functional characteristics. Unlike SNAC1, overexpression of SNAC2 showed no significant effect on drought resistance in the field conditions even though the transgenic plant showed improved tolerance to osmotic stress by PEG treatment. In addition, SNAC2 overexpression can enhance cold tolerance while overexpression of SNAC1 had no significant effect on improving cold tolerance, even though SNAC1 is induced by cold (Hu et al. 2008). Furthermore, the transcriptional target genes of SNAC1 and SNAC2 are different, such that transcript profiles of differentially regulated genes in the SNAC1- and SNAC2-overexpressing lines revealed broadly no overlap (Hu et al. 2008). This appears to be the basis for the difference of the two overexpressing transgenic plants in stress tolerance, which is further supported by the observation that flanking sequences of the core DNA binding sites in the putative SNAC1 and SNAC2 target genes are different (Hu et al. 2008). One caveat to the comparative studies involving SNAC1- and SNAC2-overexpressing lines is that the transcription factors were under different regulatory control (see Table 8.2). While both the CaMV 35S and ZmUbi1 promoter sequences are considered to be constitutive (Odell et al. 1985; Christensen et al. 1992), subtle differences in the promoter activity may influence the functional properties of each transcription factor.

8.2.2.3 HARDY: Engineering Water Use Efficient Rice

Water use efficiency (WUE), measured as the biomass produced per unit transpiration, describes the relationship between water use and crop production. In water-limiting conditions, it is agronomically desirable to produce increased biomass, which contributes to crop yield, using less water. Although genetic variation for WUE may vary in crop plants, so far, the engineering of major field crops for improved WUE has been challenging. One notable success involved the expression of the Arabidopsis subgroup IIIb AP2/ERF-like transcription factor, HARDY (HRD; Karaba et al. 2007). The HRD gene is normally active in inflorescence-stage tissue, however ectopic HRD expression leads to an enhancement in root and leaf structure, which is recognized as an adaptive mechanism for drought tolerance and WUE in crops.

The overexpression of HRD in rice generates plants with significantly higher biomass, independent of drought stress (Karaba et al. 2007). With the increase in shoot biomass in the HRD-overexpressing lines, there is a reduction in the specific leaf area and the leaf area per unit dry weight, suggesting an increase in leaf thickness or tissue density. The increased number of cells in the bundle sheath is likely to contribute to increased photosynthetic assimilation. HRD overexpression increases root biomass under drought stress, indicating an ability to adapt by inducing roots to harvest the scarce water. The increase in photosynthesizing area and carbon assimilation contributes significantly to canopy photosynthesis, resulting in high biomass. This result appears to be related to an increase in leaf mesophyll, bundle sheath, and root cortical cells, enhancing the capacity of both source and sink tissue. In rice, ectopic HRD expression causes significant increases of whole-plant WUE in well watered and drought conditions, however it remains to be determined whether such a strategy will increase WUE in different crops.

8.2.2.4 HD-START: a Developmental Regulator Conferring Drought Tolerance

Transpirational water loss through the stomata is a key determinant of drought tolerance. Stomatal movement is a response to environmental changes and is controlled by guard cell turgor which is influenced by many endogenous and exogenous factors (Assmann and Wang, 2001; Schroeder et al. 2001). Phenotypic screening of gain-of-function Arabidopsis mutants led to the discovery of enhanced drought tolerance 1 (edt1) that was found to have elevated levels of HDG11, a gene that plays an important role in water homeostasis and encodes a homeodomain (HD)-START transcription factor (Yu et al. 2008). Overexpression of HDG11 in tobacco resulted in improved drought tolerance, improved root architecture, and reduced stomatal density, all of which contributed to improved water homeostasis (Yu et al. 2008). In the original edt1 mutant, HDG11 expression resulted in higher proline levels and superoxide dismutase activity, which contributed to enhanced osmotic adjustment and reactive oxygen species detoxification. In addition, higher abscisic acid content was observed that led to a reduced rate of water loss (Yu et al. 2008). Taken together, the water homeostasis-related phenotypes conferred by ectopic expression of HDG11, as well as the reported absence of unwanted pleio-tropic effects, make this gene an excellent candidate for genetic engineering of drought tolerance in crop plants.

8.2.2.5 Plant Nuclear Factor Y B Subunits: Field-Validated Drought Tolerance in Maize

Systematic analysis of Arabidopsis transcription factor families led to the identification of candidate genes that have the potential to improve tolerance to environmental stress in crop species (Riechmann et al. 2000; www.mendelbio.com). This high-throughput (HTP) screening approach resulted in the discovery of AtNF-YB1, a subunit of the nuclear factor Y (NF-Y complex), which mediates transcriptional control through CCAAT DNA elements and confers abiotic stress tolerance when constitutively expressed in Arabidopsis (Nelson et al. 2007). NF-Y is a conserved heterotrimeric complex consisting of NF-YA, NF-YB, and NF-YC subunits (Mantovani, 1999). Following the discovery of AtNF-YB1, an orthologous NF-YB gene from Zea mays (ZmNF-YB2) was identified that similarly coordinates plant responses to drought tolerance (Nelson et al. 2007). This discovery illustrates functional conservation of the underlying drought tolerance pathway across the dicot and monocot lineages as well as validating the HTP biotechnology discovery process. Drought tolerance was also obtained in field trials with maize lines constitutively expressing the ZmNF-YB2 protein, demonstrating the potential of this strategy for improving drought tolerance in commercial crop plants (Nelson et al. 2007).

8.2.2.6 Rice Calcium-Dependent Protein Kinase 7: Multiple Abiotic Stress Tolerance with Minimal Pleiotropic Events

Cytoplasmic Ca2+ levels in plant cells increase rapidly in response to abiotic stress, including drought (Sanders et al. 1999). Following Ca2+ influx, signals are mediated by combinations of protein phosphorylation/dephosphorylation cascades, involving members of the Ca2+-dependent protein kinase (CDPK) family. Overexpression of one member of the CDPK family, OsCDPK7, results in cold, salt, and drought tolerance in rice plants (Saijo et al. 2000). Analysis of the transgenic rice revealed enhanced salt/drought induction of the genes of late embryogenesis abundant (LEA) proteins, which appears to contribute, at least in part, to the improved abiotic stress tolerance in rice plants (Saijo et al. 2000). This observation is consistent with results from a previous study where the ectopic expression of the barley group 3 LEA protein, HVA1, was shown to confer both salt and drought stress tolerance to transgenic rice plants (Xu et al. 1996). OsCDPK7 is thought to be subject of post-translational control and/or requires the expression of other proteins in order to function, since the presence of OsCDPK7 is not sufficient to induce expression of stress-associated target genes. Consistent with this theory, no significant pleiotropic effects were reported with regard to development, growth and yield penalty of the OsCDPK7 overexpression lines in untreated conditions (Saijo et al. 2000).

8.2.2.7 Tobacco Protein Kinase: Sustained Yield in Maize under Water-Limited Conditions

Tobacco protein kinase (NPK1) is a tobacco mitogen-activated protein kinase kinase kinase (MAPKKK). The catalytic domain of NPK1 specifically activates a bypass of C kinase (BCK1)-mediated signal transduction pathway in yeast, indicating that the catalytic function of NPK1 is conserved among different organisms (Banno et al. 1993). NPK1 is located upstream of the oxidative pathway and can induce expression of heat shock proteins and glutathione-S-transferase in Arabidopsis and maize (Kovtun et al. 2000). Activation of oxidative stress tolerance genes is a strategy to protect the photosynthesis machinery from damage caused by drought, thus stabilizing source-sink relationships and improving the yield potential in water-limited conditions. Transgenic maize plants showed an increase in drought tolerance including higher photosynthetic rates, higher leaf numbers, and higher kernel weights compared with the control (Shou et al. 2004). Tolerance to drought stress in maize was improved through the constitutive expression of NPK1 that activates the oxidative signalling pathway, however the effect on yield components such as kernel number was less apparent in the study. This was most likely due to the application of pollen from non-stressed maize plants. Although this ensured seed set, the potential effects of NPK1 on reducing the anthesis-silking interval under drought stress was not determined. Further studies are needed to explore the full potential of NPK1 on source-sink relationships in different maize germplasm.

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