Rootstock genetics and improvement: tolerance to abiotic stress
Abstract
Australia's major grape-growing regions will face increased salinity problems in the future resulting from a warmer drier climate. Chloride exclusion is a major trait for salt tolerance and this study has led to a world-first breakthrough in the quest to understand the molecular mechanism that regulates this trait in grapevine. We utilised a combination of segregating mapping populations and transgenic vines with altered gene expression to identify not only the first locus for chloride exclusion but also the underlying casual gene. This knowledge will enable the development of a genetic marker to predict chloride exclusion in the rootstock breeding program.
Summary
Climate change predictions suggest that the major wine regions of Southern Australia are likely to face reduced rainfall combined with increased temperatures and more frequent heat wave events, thus increased evaporation. Coupled with the likelihood of reduced availability and affordability of quality irrigation water, there is a high chance that the issue of irrigation related salinity in vineyards will increase.
Salinity stress can lead to vines accumulating chloride and sodium ions at levels which become toxic and disrupt cellular and metabolic processes. In the leaves, this can manifest as visual marginal leaf burn symptoms resulting in impaired photosynthetic activity and reduced yields. Accumulation of high concentrations of chloride and sodium ions in the berries can also have detrimental impacts on wine quality and marketability.
Ion excluding rootstocks have the ability to limit the uptake of chloride and/or sodium into the shoot and are therefore a key tool for mitigating the negative effects of salinity on the vine. Guided by studies in other plant species, we previously identified the major gene, HKT1:1, that controls sodium exclusion in grapevine rootstocks and we developed a robust genetic marker for predicting this trait in the rootstock breeding program.
The primary aim of this project was to identify the major gene controlling chloride exclusion in grapevine rootstocks in order to develop a reliable genetic marker to this key trait. Unlike the case for sodium, comparably little research focus has been paid to chloride exclusion in other crop species and hence major genes controlling chloride exclusion in plants are yet to be identified. This study has led to a world-first breakthrough in the quest to understand the molecular mechanism that regulates chloride exclusion in plants.
We first identified a locus, named CLEX for Cl- exclusion, that accounts for 85-89% of the variation in Cl- exclusion in a self-pollinated population derived from a (V. berlandieri x V. vinifera cv. Sultana) x V. vinifera cv. Biancone genotype. We showed that this segregating trait, first observed in juvenile seedlings, is carried through to pot grown vines propagated from cuttings, as well as mature vines in the field. We also showed the variation attributed to the CLEX locus is dependent on the root system and is fully conferred to a grafted scion, making it an excellent target for rootstock improvement. In this population the dominant (stronger) CLEX allele for exclusion is derived from V. berlandieri and a recessive (weaker) allele is derived from V. vinifera.
In a K51-40 (V. champinii ‘Dogridge’ x V. riparia ‘Gloire de Montpellier’) x 140 Ruggeri (V. berlandieri ‘Boutin B’ x V. rupestris ‘du Lot’) F2 population, the CLEX locus was found to explain 72% of the variation in Cl- exclusion. This result not only confirmed that this locus is likely to be the major locus for Cl- exclusion across different grapevine species, but also led to the identification of single nucleotide polymorphisms to act as the basis of genetic markers to predict this trait in the breeding program.
Of the 72 predicted genes located in the CLEX locus, two stood out as potential candidate genes underlying this trait. CHX20.1 and CHX20.2 belong to a large family of genes known as Cation/H+ exchangers, of which their roles in plants are poorly characterised. Recent evidence in soybean, however, suggests that a homologue of CHX20.1, known as GmSALT3, encodes a shoot-based Cl- exclusion mechanism in that species. To determine if CHX20.1 or CHX20.2 contribute to Cl- exclusion in grapevine we genetically transformed Shiraz with constructs designed to reduce their expression. Analysis of these transformed lines revealed CHX20.1 plays a major role in Cl- exclusion, occurring primarily within the root stele, being active at both low and high salt treatments (Fig. 2.1) and potentially functioning in restricting the entry of Cl- into, or its retrieval from, the xylem. CHX20.2 on the other hand, is expressed in the petiole and appears to have a minor influence on the regulation of Cl- concentration in the laminae, but only at low salt treatments. These results suggest that CHX20.1 is the major gene underlying the variation in Cl- exclusion in the V. berlandieri x V. vinifera and K51-40 x 140 Ruggeri populations. CHX20.1 is also highly expressed in the bunch rachis, and therefore may play a role in regulating the Cl- concentration of the berry. This hypothesis was not able to be tested in this study but will be important in the future to determine the importance of this gene to scion breeding and improvement.
The CHX20.1 alleles derived from V. berlandieri and V. vinifera differ by 19 amino acids, one or more of which are likely to cause functional differences in the transport properties of the encoded proteins. In order to identify the key amino acid differences underlying this trait, our collaborators at the University of Adelaide, Dr Yue Qu and Professor Matthew Gilliham, sought to develop an assay to characterise the function of CHX20.1 proteins. While the E.coli and Arabidopsis based systems that were tested both showed promise for characterising CHX20.1 function, further work is required to perfect one or both of these assay systems. This will be required in order to identify the causal amino acids underlying functional differences if a perfect genetic marker for Cl- exclusion is to be designed and targeted to the exact genetic mutations underlying variation in Cl- exclusion in different rootstock genotypes.
Strategic crosses using 140 Ruggeri were performed to begin converting the scientific knowledge gained here into increased capacity for salt tolerance in the rootstock breeding program for durable and broad-spectrum resistance to the major soil pests, phylloxera and nematodes. We also investigated other germplasm as potential novel sources of Cl- exclusion which led to the identification of V. longii as a potentially superior source of Cl- exclusion over 140 Ruggeri and V. berlandieri. Future work is needed to; 1) determine the suitability of V. longii in rootstock breeding, 2) to confirm the presence of, and determine the role of, CHX20.1 in V. longii, and 3) to begin the introgression of Cl- exclusion from V. longii into the rootstock breeding program.