Methods to create more drought resistant Brassica varieties

Methods to create more drought resistant Brassica varieties

Written by Willem Dijkman and Linda van Zomeren, 2010.

Supervised by dhr. T. Elzinga of the Rijksuniversiteit Groningen.
Made in cooperation with Syngenta, Enkhuizen.

Made as part of a students master program at the University of Groningen. 

Abstract

Large parts of the world suffer from a lack of water during some or all periods of the year. Food crop production and food quality is negatively affected by drought, which calls for plant varieties that are more tolerant to this drought. Plant species from the genus Brassica contribute to a large degree to vegetable consumption, but most varieties do not respond well to drought. In this study the following genes and pathways are screened for their use in creating more drought resistant crops: a H+pump, proline accumulation, MAP-kinase manipulation, ABA manipulation and, phosphatidylinositol signal transduction manipulation. Also a new way to asses drought tolerance early in development is reviewed. Altering basal signaling mechanisms is less applicable since that can lead to side effects like developmental abnormalities, making them not applicable in food crops like Brassica. Most promising is the screening of naturally occurring mutation in the genes that regulate the H+pump of the vacuole.

1       Introduction

People all over the world eat vegetables from the genus Brassica, which makes it an important agricultural species. Brassica species are native in western Europe and the Mediterranean but agricultural crops are now grown world wide, mostly in China and India. Two of the most important species are B. napus (rapeseed) and B. oleracea (cabbage, broccoli, cauliflower and other similar vegetables). Brassica napus is grown for its seeds, which contain oil applied in food and in biofuels. Many other Brassica species and varieties are food crops of which roots (Swedish turnips), stems (kohlrabi), leaves (cabbage, Brussels sprouts) or flowers (cauliflower, broccoli) are eaten. The annual production for rapeseed for instance is 3.6 109 kg (2003) of which about one third is produced in China. Cauliflower and cabbages are also grown on a large scale: 1.5 109 kg and 6.5 109 kg annually (2003) respectively (King, 2004). This is hundred times less than the major crops like corn, rice and wheat (WN Atlas Productions, 2001) but still a major contribution to the world food supply.

Biotic and abiotic stresses however decrease the production of Brassica vegetables. One of the major stresses is drought, which can lead to failed harvest, reduced nutritional value and reduced quality of the vegetables. The productivity of major crops is defined by their ability to cope with water stress (Boyer, 1982; Tollenaar and Wu, 1999). Drought can be defined as a meteorological event which entails the absence of rainfall for a period of time, long enough to cause moisture depletion in soil and water deficit with a decrease in water potential in plant tissues (Kramer et al., 1980). In this case, we discuss drought from a more agricultural point of view, therefore defining drought as an environmental condition where scarcity of water is causing decreased production of agricultural crops. Over 45% of the world agricultural fields are subject to frequent or constant drought. Low precipitation, high surface evaporation, poor irrigation availability, weathering of rocks and poor agricultural practices are the major contributors to drought. Drought limits plant growth and development because of a decline in photosynthetic capacity and potentially damage to leaves and roots. Drought leads to osmotic, ionic and consequently to oxidative stress (Chen et al., 2009; Xiong et al., 2002). High salinity, mainly caused by irrigation with saline water, poses the same problems for plants. Because of the relationship in the effects of drought and high salinity, the response of the plant is related in many cases. Genes involved in salt stress response are also involved when plants experience drought stress (Chen et al., 2009).

Plants have developed various mechanisms to escape from or cope with drought stress. These mechanisms can be divided into three groups: drought escape, drought tolerance and drought avoidance (Mitra, 2001). Drought escape is the ability of plants to complete their life cycle before serious water deficit occurs. To be able to do this, a rapid development is required: the plant should mature and flower early, before the lack of water becomes too severe. This is mainly used when water deficit occurs predictably, for example in regions with a wet season and a dry season. Drought tolerance allows plants to withstand low water potential in tissue due to water deficit. Drought tolerance is achieved by the maintenance of turgor through osmotic adjustment, decrease in cell size and an increase in cell elasticity and finally desiccation tolerance by protoplasmic resistance. Drought avoidance is also performed by maintaining of turgor. In contrast to drought tolerance, turgor is not maintained by an osmotic adjustment, but rather through regulating water uptake and water loss. On the one hand more water is taken up by increasing root depth, increasing the efficiency of the root system and increased hydraulic conductance. On the other hand water loss is limited by closure of stomata and lenticula and by reducing evaporation by radiation which can be established by leaf rolling, folding or decreasing leaf size.

All these mechanisms have drawbacks when applied in food crops. Drought escape will often yield a smaller plant or lower yield compared to a plant that does not use this mechanism. Stomatal closure and a reduced leaf area in drought avoidance not only reduce water loss, but also result in reduced assimilation of carbon dioxide and therefore lower growth and yield. Osmotic adjustment leads to an increased solute concentration but may have detrimental effects, as this adaptation is energetically costly (Mitra, 2001).

Resistance to drought in plants is a complex trait. It is determined by morphological, physiological and biochemical characteristics like described before.

A lot of abiotic stress induced genes have been identified in Arabidopsis thaliana, tobacco (Nicotiana tabacum) and rice (Oryza sativa) (Chen et al., 2009). Much less is however known about other, economical important species like Brassica. Fortunately, knowledge of genes identified and mechanisms unraveled in Arabidopsis can be used for Brassica because of their close relatedness: both are of the Brassicaceae family. Thus far, many of the genes identified in drought response in Brassica have been found to have homologue genes in Arabidopsis (Chen et al., 2009). Their function seems to be conserved along the species. This makes Arabidopsis thaliana a useful model plant for creating more drought resistant Brassica varieties.

Drought resistance is a complex trait, not depending on one single gene or mechanism. In this study several pathways and genes are presented which contributed to drought tolerance in the model plant Arabidopsis thaliana or in Brassica species. The goal of this study is to give insight into mechanisms and genes that could be applicable to breeding more drought resistant Brassica varieties. In chapter 3, the possible mechanisms or genes are discussed and their possible use in Brassica breeding is examined.

2       Rapid assessment of drought tolerance

2.1 Introduction

It can be very valuable to assess drought tolerance of young plants, to estimate their ability to produce a high yield in water limiting conditions in the future. Assessing young plants compared to old plants saves time, because plants do not actually have to reach maturity and produce harvestable organs. Of course it is of major importance to assess plants in a reliable way, to avoid making suboptimal decisions.

When plants are faced with water stress, this can have various consequences for their metabolism. Plant characteristics related to photosynthetic ability have always been viewed as important factors influencing drought tolerance, because photosynthesis is essential for growth of juvenile and adult plants (Kauser et al., 2006). Photosynthesis, which takes place in the chloroplasts, produces reactive oxygen species (ROS) which can destroy pigments, lipids and enzymes. ROS can be inactivated by specialized pigment-protein complexes which protect the photosynthesis apparatus. Carotenoids play a major role in scavenging ROS. Water stress decreases the efficiency of ROS-inactivation and therefore ROS can damage chlorophyll a, decreasing the overall amount of chlorophyll a in the cell. Assessing the amount of caroteniods or chlorophyll a under water stress could indicate if a plant is tolerant to drought (Kauser et al., 2006).

2.2 Assessing drought tolerance using chlorophyll fluorescence

In the study of Kauser et al. (2006) two Brassica napus cultivars that show a distinct difference in drought tolerance (Cyclone and Dunkeld) were compared on various physiological aspects to find a physiological predictor for drought tolerance. Drought tolerance is defined as having the most leaf area and biomass under drought conditions. Three week old plants of both cultivars where kept under water limiting conditions for three weeks. After this treatment, leafs of the plants were measured for osmotic potential, chlorophyll a and carotenoid content, chlorophyll fluorescence and gas exchange parameters. During non-drought treatments, the plants of both cultivars did not differ in these aspects. Under water stress conditions, the more drought resistant cultivar shows a higher level of chlorophyll a and carotenoids, and those plants showed a lower decrease in photochemical efficiency compared to the less drought resistant cultivar. Quantum yield of chlorophyll a seems the best candidate to be used as a selection criterion for drought tolerance in young plants.

2.3 Discussion

Measuring chlorophyll a levels in young plants is a promising method to analyze drought tolerance in Brassica varieties. Chlorophyll a levels under water stress are predictive for the future growth of the plant, as chlorophyll a is essential for photosynthesis and thus growth. Maintaining a high level of chlorophyll a thus reflects the plants ability to cope with water stress. The more drought resistant cultivar (Dunkeld) both shows a higher level of chlorophyll a in drought conditions and is known for its higher yield in drought conditions.

Although chlorophyll a is a protein that is very closely involved in photosynthesis, selection on plants with a higher level of this protein does not necessarily have to result in higher yield. Therefore it could be advisable to include more direct tests of yield at some time during the selection process.

3       Genes and Pathways important in drought resistance

3.1 AVP1 H+-pump

3.1.1 Introduction

To maintain turgor during low water potential, plants increase the number of solutes in the cell. The transport of solutes into the cell is dependent on proton gradients between the cell and the extracellular space and between the cytoplasm and the vacuole. In plants, three types of proton pumps are known which generate proton gradients across cell membranes. Two of these pumps, the vacuolar H+-ATPase and the vacuolar H+-pyrophosphatase are of most importance, since the vacuole is essential for turgor of the cell. Using the proton gradients between the vacuole and the cytoplasm, solutes like sugars and (in)organic acids can be actively transported into the vacuole.  This will result in water being drawn into the vacuole by osmosis, resulting in maintaining the cells turgor. Upregulation of one or both the vacuolar proton pumps could therefore be a good way to make plants more drought, and salt, tolerant.

The H+-ATPase pump consist of two domains. V0 is membrane bound and consists of five subunits. V1, which is peripherally associated with the other domain is a complex of eight subunits (Strompen et al., 2005). All these subunits are encoded by individual genes, inducing overexpression of the complex is therefore challenging. All genes have to over expressed at the correct level to achieve higher activity by the multisubunit complex. The other pump, H+-pyrophosphatase, is encoded by a single gene and is therefore much easier to upregulate. In Arabidopsis thaliana, the gene APV1 encodes this pump.

Using transgenic Arabidopsis thaliana and alfalfa plants (figure 1), it has been shown that upregulation of this gene indeed leads to a more salt and drought resistant plant. Plants were transformed with an extra copy of the gene using Agrobacterium to insert the gene into the genome of the plant. This extra copy of the gene caused higher expression of the protein. When the transgenic plants were exposed to 10 days of water deprivation, which was lethal for WT plants, transgenic plants survived and continued normal growth and also produced viable seeds (Gaxiola et al., 2001).

3.1.2 Mechanism

Two mechanisms could be underlying the results obtained from inserting an extra copy of the APV1 gene. The solute concentration in the vacuole increased more rapidly by the treatment, maintaining turgor more efficiently. However, stomatal closure could also play a role in causing these transgenic plants to be more drought resistant. It has been supposed that the higher expression of the H+-pyrophosphatase hinders the generation of cytoplasmic Ca2+ oscillations in guard cells, which leads to the closure of the stomata. This closure results is less transpirational water loss. This mechanism is however not supported by the obtained results, because no differences in abscisic acid (ABA) or Ca2+ concentrations could be measured between the wild type and the transgenic plants. Therefore, the increased drought resistance is due to the increased solute concentration like Na+ and K+ in the vacuole. Sodium uptake is facilitated by increased activity of a vacuolar sodium/proton antiporter because of the higher proton concentration due to the overexpression of the H+-pyrophosphatase. Higher potassium is due to the H+-pyrophosphatase itself since the pyrophosphates can also functions as proton potassium symporter (Maeshima, 2000). The uptake of potassium and sodium by the vacuole leads to an enhanced uptake of water to maintain water potential. Therefore upregulating this antiporter along with the H+-pyrophosphatase might make plants even more drought tolerant.

3.1.3 Conclusion

The desired results are obtained in this experiment: duplication of one single gene yields a more drought tolerant Arabidopsis thaliana strain. To make this method useful for Brassica breeding and food production, some more experiments need to be carried out. The mechanism first has to be applied to Brassica species. A homologue of the APV1 gene is probably present in Brassica but not yet identified. When the genome sequence of Brassica is available this homologue is expected to be identified very easily. Furthermore, the transgenic plants created in this study can not be commercially grown in Europe because of legislation. Without using transgenic technologies, it will be harder to introduce an extra AVP1 gene copy into a genome. Selection on plants already expressing AVP1 on a higher level could be an useful alternative.

Overall this strategy to obtain more drought tolerant plant is genetically possible, results in the desired more drought resistant plants, but not readily applicable in commercially grown Brassica. This pathway has however one major advantage: only one gene has to be targeted.

3.2 ABA-mediated reduction in transpirational water loss

3.2.1 Introduction

As an innate and adaptive response to water deficit, plants synthesize the phytohormone abscisic acid (ABA). This hormone reduces transpirational water loss by causing stomatal closure. Because dicot plants loose 90% of their water through transpirational water loss through their stomata, curbing this water loss could result in better yields of these plants during drought periods (Wang et al., 2009).

3.2.2 ABA manipulation

ABA-mediated stomatal closure is a dosage-dependent process. This means there are three ways to influence this hormone to reduce transpirational water loss. These include: to increase the basal production of ABA, to increase the sensitivity of the stomatal apparatus to ABA and to increase the threshold for ABA production in response to drought. The sensitivity of the stomatal apparatus to ABA is potentially a good candidate to focus on to increase drought tolerance in plants, because this mechanism is moderately well understood (Wang et al., 2009).

Several genes that mediate the sensitivity of the stomatal apparatus in Arabidopsis have been described. These genes could be good candidates to selectively breed into specific Brassica crops. The gene encoding the Arabidopsis b-subunit of farnesyltransferase (ERA1) enhances the plants sensitivity to ABA and increases drought tolerance. Also some loss of function mutation in genes coding a protein phosphatase 2C, ABI1 (Gosti et al., 1999; Saez et al., 2006), a nuclear mRNA cap-binding protein, ABH1 (Hugouvieux et al., 2001), a Ca2+ binding protein, ScaBP5, and its interacting protein kinase, PKS3 (Guo et al., 2002), the a- subunit of protein farnesyltransferase, AtFTA (Running et al., 2004; Wang et al., 2005) and a subunit of holo-elongator, ABO1/ELO2 (Chen et al., 2006). Gain-of-function mutations can also enhance the sensitivity for ABA in genes encoding a stress responsive transcription factor, SNAC1 (Hu et al., 2006), a RING finger E3 ligase, SDIR1 (Zhang et al., 2007), and a R2R3 Myb transcription factor, AtMYB44 (Jung et al., 2008). These mutants in Arabidopsis were found in germination screens with low concentrations of ABA in the growth medium. Plants of the Brassica family could be screened in the same way to find mutations with higher sensitivity to ABA. Scanning the genome of Brassica varieties for the genes found in Arabidopsis could also be an excellent way to find varieties with increased resistance to drought. Selection on plants with mutations in the proposed candidate genes could possibly yield plants with increase resistance to drought.

3.2.3 Discussion

Selecting for increased sensitivity of plants to the phytohormone ABA could also have some adverse effects. In the Arabidopsis ABA-sensitivity mutants seed dormancy is affected (Wang et al., 2009). Many mutants display an abnormal number of floral organs, enlarged meristems and delayed growth. Selection on normal plant function in plants with the desired mutation in ABA sensitivity could resolve these problems, but only if the increased sensitivity to ABA is not caused by the same genes that produce the adverse effects. However, it is likely that the sensitivity to ABA and the abnormal growth are intertwined. Additionally, when drought does not impose a problem, the increased stomatal closure caused by ABA could result in CO2 shortage for photosynthesis and thus reduced growth.

Manipulation of ABA-mediated stomatal closure is a good candidate to reduce transpirational water loss in plants. However, mutants that show no adverse effects along with their ABA sensitivity have not yet been established. Screening for mutants that show increased ABA sensitivity, ABA production or response to water stress seems like a good strategy to obtain plants that have a reduction in transpirational water loss. The adverse site effects experienced thus far are however a drawback of this mechanism.

3.3 Mitogen-activated protein kinases

3.3.1 Introduction

Mitogen-activated protein kinases (MAPK) are highly conserved in eukaryotes. The enzymes transfer information from sensors leading to cellular responses. A large number of genes encoding MAPK pathway components have been revealed by analyzing plant genomes. This indicates the MAPK cascades are abundant players and their action is involved in cell development, proliferation and hormone physiology. It has also been shown MAPK are involved in abiotic and biotic stress signaling (Chen et al., 2009). Manipulating this signaling pathway could therefore yield more drought resistant varieties of plants.

3.3.2 MAPK in drought signaling

Arabidopsis has 20 MAP kinases (MPK), 60 MAP kinase kinase kinases (MAPKKK) and 10 MAP kinase kinases (MKK). In Arabidopsis it has been shown MPK4 and MPK6 (both MPKs) are activated by several stresses including drought. MEKK1, a MAPKKK, is also activated by these stresses although the precise mechanisms and routes are not yet known. For cold and salt stress more is known regarding the MAP kinase kinase MKK2 (figure 2). When mkk2-null mutant plants are grown under ambient conditions no phenotype is shown. These knockout plants are however hypersensitive to cold and salt stress. In addition, when MKK2 is overexpressed plants become more tolerant to cold and high salinity (Nakagami et al., 2005).

Because salt and drought tolerance are often linked, the increased tolerance to high salinity could mean the MKK2-overexpressing plants also have an increased tolerance to drought. However not all kinases in these cascades are responding to both stresses. When MKK9 for instance is inactivated, salt tolerance is increased but plants become not more drought tolerant (Alzwiy and Morris, 2007). So in contradiction to the mkk2-null mutant which is more tolerant when overexpressed, the mkk9 mutant is more salt tolerant when the kinase is inactive. This is due to the fact that mkk9 is a negative regulator. In wild type plant, MKK9 is negatively regulating stress induced expression leading to more tolerance when MKK9 is not present.

Two genes coding for kinases have already been identified in Brassica napus which are upregulated by the plant when it is exposed to drought stress. Artificially overexpressing these genes, for example by selective breeding, might therefore yield a more drought tolerant plant (Chen et al., 2009).map-kinases

 

Figure 2 MAP kinases in Arabidopsis.

Schematic representation of the cascade of 3 kinases. Broken arrows indicate hypothetical connections and question marks unknown components

 


3.3.3 Discussion

Altering the MAPK signaling in Brassica is a promising way to obtain more drought tolerant plants. In Arabidopsis it has already been shown salt tolerance could be obtained after over expression of mkk-genes. No drought resistance was however observed although resistance to drought and to salt is often mediated by the same mechanism. To gain more certainty on the effect of MAPK manipulation, the signaling route induced by drought in relation to MAPK should be unraveled in Arabidopsis and Brassica.

The MAPK signaling system might be a good target for obtaining more drought tolerant plants since the overexpression of a single gene already yielded valuable phenotypes for salt stress. Too many uncertainties on the pathways for drought hinder a clear prospect on the chance of using this method in commercial Brassica production.

3.4 Ca2+ signaling

3.4.1 Introduction

Ca2+ signaling is an important signal for closing stomatal guard cells and thereby restricting transpirational water loss. Brassica napus that have been exposed to high salinity or drought show an upregulation of numerous genes that are part of this signaling pathway. Ca2+ signaling is regulated by a large number of genes and their products (Das et al., 2005). Understanding the exact network of genes and interactions can give candidate genes where selection can be directed at, to obtain more drought resistant Brassica varieties.

3.4.2 Genes of interest

The most important pathway is that of the phosphatidylinositol-specific phospholipase C (PtdIns-PLC)-mediated hydrolysis of PtdIns(4,5)P2 and the production of the second messengers inositol trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG) (Mueller-Roeber and Pical, 2002). The genes Ins(1,4,5)P3 and PtdIns3-P have important functions in Ca2+ signaling and are both regulated by PtdIns 3-kinase (PtdIns 3-K). PtdIns 3-K could therefore be a good candidate for up-regulation by selection (Jung et al., 2002; Meijer and Munnik, 2003).

When subjecting Brassica napus to drought stress (no watering for 6 days until rims of leafs become brown) five transcripts are upregulated: BnPtdIns S1, BnVPS34, PtdIns 4-K, PtdIns4P 5-K and PtdIns-PLC2 (Das et al., 2005). The product of BnVPS34, PtdIns3-P, is required for membrane proliferation and replenishment under certain stress conditions (e.g., salt and drought) that affect membrane integrity (Meijer et al., 2001; Hong and Verma, 1994). Furthermore, PtdIns3-P was also shown to be involved in the abscisic acid-induced generation of reactive oxygen species in guard cells, which is responsible for stomatal closing movement under oxidative stress (Park et al., 2003; Das et al., 2005). Because the products of these genes are very important for Ca2+ signaling and thus for stomatal closure, upregulation of these genes by selection could result in plants that are more drought tolerant. Simple techniques like quantitative PCR and gel electrophoresis on the mRNA of these genes could yield Brassica varieties that are more drought tolerant by a quicker or stronger response to drought stress.

3.4.3 Discussion

Although upregulation of genes active in drought stress could potentially increase drought resistance in plants, this has not yet been tested. The genetic variation on activity and regulation of these genes has not been assessed for Brassica, therefore it remains unclear if selection can sort a great effect. If plants with a mutation in one of the mentioned genes are already present, rapid assessment of their drought tolerance could be the next simple step.

3.5 Proline Metabolism

3.5.1 Introduction

Two major organic osmolytes are glycine betaine and proline. These compounds accumulate in plants as a consequence of all kind of environmental stresses including drought. The first mentioned osmolyte is synthesized from choline via betaine aldehyde and is most present in chloroplasts of many crop plants like sugar beet, spinach, barley and wheat. There it protects the thylakoid membrane and maintains thereby photosynthetic efficiency. In Brassica glycine betaine is not used as an osmolyte. Applying exogenous glycine betaine to Brassica also does not improve growth under drought stress (Madan et al., 1995). However, the other major osmolyte, proline, is accumulated in Brassica when exposed to drought (Ashraf and Foolan, 2005). And in contrary to glycine betaine, proline is not present in the chloroplasts but in the cytosol. This amino acid does not only function as an osmolyte for osmotic adjustment, it also serves as a scavenger for free radicals, it is buffering cellular redox potential and it contributes the stabilizing of subcellular structures like membranes and proteins.

3.5.2 Proline accumulation

The accumulation of proline can be due to a higher production, or to a decreased brake down of this amino acid. Two enzymes are involved in the biosynthesis of proline; ornithine aminotransferase (OAT) and pyrroline-5-caboxylate reductase (P5C reductase). These have been studied in salt tolerant Brassica strains. In one of these strains (SR2P1-2), proline levels were not elevated whereas in the other strain (SR3P6-2) proline levels were higher during salt stress. The first strain also showed no increase in the proline synthesizing enzymes. The second strain however had a highly increased OAT level compared to the wild type. In addition, the P5C reductase was more active in this strain (Madan et al., 1995). This increase in P5C reductase level has been observed in drought stress as well (Kohl et al., 1990). Proline can be broken down by proline oxidase, converting proline to glutamate. A decrease in proline oxidase has been reported in salt and drought stresses cells. The increase of OAT and P5C reductase and the decrease of the catabolising enzyme proline oxidase correspondent to the increased proline levels. The enzyme levels however did not match with the proline levels. The enzymes levels were increased by only 3 or 4 fold whereas the proline levels were increased by 34 fold.

In other studies, the proline levels in the Brassica juncea (Indian Mustard), which is drought susceptible, were compared to the levels in Brassica campestris (wild turnip) which is more drought tolerant. It was shown that proline levels in the first species were increased 12 fold as a consequence of water stress, while in the drought tolerant species, the proline level increased much less (only 7 fold). The increase in total protein level was about the same in both species (Gupta and Sheoran, 1979).

In five different Brassica juncea varieties P5C reductase and proline oxidase levels were measured among with proline levels. The most tolerant variety, the P5C reductase and proline level were most increased and the proline oxidase levels were most reduced. The least tolerant species shows a much less increase in proline level and P5C level and a less decrease in proline oxidase (Phutela et al., 2000).

3.5.3 Conclusion

Increased proline levels are related to drought stress. It is however hard to say whether a higher proline production also indicates more drought tolerance. Obtained results are contradictory: the more tolerant Brassica campestris produces less proline then Brassica juncea when stressed. The more tolerant B. juncea variety in contrary produces more proline compared to the less tolerant B. juncea varieties.

In addition to this, not all stressed plant have an increased proline level or increased proline production. Next to this, the synthesis of proline requires energy, which may cause a lower yield.

4 Conclusion and discussion

            The mechanisms plants have to withstand drought are extensive and diverse. Some of these mechanisms have been described well whereas others pathways are still under investigation. Next to this, many mechanisms might not be discovered yet. Even though there are still a lot a mechanisms to elucidate, the knowledge that is found already can give us a wealth of information to start creating more drought tolerant Brassica varieties.

In this study we reported several mechanisms are candidate mechanisms of drought resistance which can be applied in Brassica. Some mechanisms seem more promising than others, because of their easy use or the lack of severe side effects that can reduce the plants quality. The most promising mechanism in our opinion is overexpression of the vacuolar proton pump H+-pyrophophatase encoded by the gene APV1. This gene is responsible for making Arabidopsis thaliana more drought tolerant by increasing the amount of solutes in the vacuole. Because of the close relatedness of Brassica with Arabidopsis, chances are that this gene or a variant on it can have the same effect in Brassica. The elegance of this method lies in the fact that this proton pump is regulated by just one gene. Directed selection or screening for mutants that have overexpression of this gene is therefore much easier than when a trait is regulated by a gene complex.

Another promising technique is to screen for mutants that show an increased production of proline. Proline is a common compatible solute that can retain water in the cells. This substance also protects the cells against Reactive Oxygen Species, preventing damage to proteins and membranes. Although contradictory data have been obtained related to precise effect and potential side-effects of proline accumulation, overproduction of proline is a good way to increase drought tolerance in plants. The benefit of this method is again that overexpression can be obtained by a mutation in a single gene.

The methods we presented on the manipulation of signaling pathways are ABA-mediated reduction of water loss, MAP kinase manipulation and Ca2+ signaling. The signaling pathway manipulations are promising because of their large effects on drought tolerance. However, as to now, these methods do not seem to be applicable because of the severe side effects that have been observed. The large effects that are observed when changing signaling pathways on drought tolerance are also observed in the growth and structure of the plants. Until the pathways can be changed in such a way that only the effect on drought tolerance can be obtained, but not the other effects, these methods are not readily applicable. ABA-mediated reduction of water loss is caused by making ABA receptors more sensitive to the plant hormone ABA, which is released under drought stress. Stomatal guard cells respond to ABA by closing the stomata, reducing water loss. However, interfering in this pathway often leads to adverse effects, like development abnormalities and delayed growth. Ca2+ signaling is a part of this same ABA-mediated reduction of water loss pathway, but because it is further downstream of the cascade, it may have less adverse side effects. Manipulation of this pathway has proven to be more difficult than ABA sensitivity manipulation, while side effects are still found. Cell signaling mediated by MAP kinases is involved in drought response of many plant species. Changes in this signaling cascade may therefore lead to more drought resistant varieties. The precise signaling pathway has not yet been elucidated, but manipulations do show some promising results. However, this has not been tested in Brassica and side effects have not been studied.

Overall it can be concluded overexpression of the AVP1 gene to produce more of the proton pump H+-pyrophophatase seems most promising. The apparent lack of side effects and the fact that only one gene needs to be altered, makes this a method to be looked into. It has however to be taken into account that drought tolerance is a complex trait. Making Brassica varieties more drought tolerant simply by altering one single gene, which is genetically easier, might cause side effect like less yield or an altered phenotype. No obvious side effects have been reported, but still subtle side effects affecting yield could still occur.

Naturally occurring mutations in the genes reviewed in this report could be searched for in the normal stock of seeds or plants, making sure no valuable mutations are accidentally lost from the stock. By focusing on certain genes, plants do not need to be grown to adulthood or subjected to drought to select them for further tests or further breeding. This is expected to minimize the time until finding valuable mutations and reduce costs of breeding. Although looking at mutated genes is a promising and effective way to screen for plants with valuable mutations, focusing solely on these genes could mean passing other genes or mechanisms that could also be valuable. Furthermore, when finding a mutation in a candidate gene, extensive tests for drought tolerance, yield and other plant performances need to be carried out to make sure the mutation has an overall positive effect.

Taken as a whole, using genes as possible candidates for selecting drought resistant strains is promising for reducing costs and reducing the time needed to find new varieties, but is not the holy grail of plant breeding and selection. There is still need for extensive performance tests on the plants that are selected for, although smart screening can increase general success for creating drought resistant Brassica varieties.

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