Alexander B. Zwart

Genetic variation in tolerance to the osmotic stress component of salinity stress in durum wheat

Salinity affects plant growth by the osmotic stress of the salt around the roots as well as by toxicity caused by excessive accumulation of salt in leaves. The aim of this study was to determine whether there is significant genetic variation in tolerance to osmotic stress that can be useful in improving the salinity tolerance of crop plants. Durum wheat is a salt-sensitive crop whose yield is reduced by moderately saline soils. Genetic variation in tolerance to osmotic stress in durum wheat was examined in 50 international durum varieties and landraces by measuring the response of stomatal conductance to salt stress before salts built up in the leaf. Stomatal conductance is a sensitive indicator of the osmotic stress because it is reduced immediately with the onset of salinity, and is the initial and most profound cause of a decline in CO2 assimilation rate. Genetic differences of 2-3-fold were found in the magnitude of the response of stomatal conductance to salt-induced osmotic stress. Higher stomatal conductance in salt related to higher CO2 assimilation rate. There was a positive relationship between stomatal conductance and relative growth rate in salt. This study shows the potential for new genetic gains in salt tolerance in durum wheat.

Advanced engineering of lipid metabolism in Nicotiana benthamiana using a draft genome and the V2 viral silencing-suppressor protein.

The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18∶1 to elongation reactions beginning with 18∶1 as substrate. These V2-based leaf assays produced ∼50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays.

The influence of sampling strategies and spatial variation on the detected soil bacterial communities under three different land‐use types

To determine the influence of pooling strategies on detected soil bacterial communities, we sampled 45 soil cores each from a eucalypt woodland, a sown pasture and a revegetated site in an Australian landscape. We assessed the spatial variation within each land-use plot, including the influence of sampling distance, soil chemical characteristics and, where appropriate, proximity to trees on the soil bacterial community, by generating terminal restriction fragment length polymorphism profiles of the bacterial 16S rRNA genes. The soil bacterial community under the revegetated site was more similar to the original woodland than the pasture, and this result was found regardless of the soil- or the DNA-pooling strategy used. Analyzing as few as eight cores per plot was sufficient to detect significant differences between the bacterial communities under the different plots to be distinguished. Soil pH was found to be most strongly associated with soil bacterial community composition within the plots and there was no association found with proximity to trees. This study has investigated sampling strategies for further research into the transitions of soil microbial communities with land-use change across broader temporal and spatial scales.

Impact of ancestral wheat sodium exclusion genes Nax1 and Nax2 on grain yield of durum wheat on saline soils

Nax1 and Nax2 are two genetic loci that control the removal of Na+ from the xylem and thereby help to exclude Na+ from leaves of plants in saline soil. They originate in the wheat ancestral relative Triticum monococcum L. and are not present in modern durum or bread wheat. The Nax1 and Nax2 loci carry TmHKT1;4-A2 and TmHKT1;5-A, respectively, which are the candidate genes for these functions. This paper describes the development of near-isogenic breeding lines suitable for assessing the impact of the Nax loci and their performance in controlled environment and fields of varying salinity. In young plants grown in 150mM NaCl, Nax1 reduced the leaf Na+ concentration by 3-fold, Nax2 by 2-fold and both Nax1 and Nax2 together by 4-fold. In 250mM NaCl, Nax1 promoted leaf longevity and greater photosynthesis and stomatal conductance. In the uppermost leaf, the Na+-excluding effect of the Nax loci was much stronger. In the field, Na+ in the flag leaf was reduced 100-fold by Nax1 and 4-fold by Nax2; however, Nax1 lines yielded 5-10% less than recurrent parent (cv. Tamaroi) in saline soil. In contrast, Nax2 lines had no yield penalty and at high salinity they yielded close to 25% more than Tamaroi, indicating this material is suitable for breeding commercial durum wheat with improved yield on saline soils.

Nonindigenous Plant Advantage in Native and Exotic Australian Grasses under Experimental Drought, Warming, and Atmospheric CO2 Enrichment

A general prediction of ecological theory is that climate change will favor invasive nonindigenous plant species (NIPS) over native species. However, the relative fitness advantage enjoyed by NIPS is often affected by resource limitation and potentially by extreme climatic events such as drought. Genetic constraints may also limit the ability of NIPS to adapt to changing climatic conditions. In this study, we investigated evidence for potential NIPS advantage under climate change in two sympatric perennial stipoid grasses from southeast Australia, the NIPS Nassella neesiana and the native Austrostipa bigeniculata. We compared the growth and reproduction of both species under current and year 2050 drought, temperature and CO2 regimes in a multifactor outdoor climate simulation experiment, hypothesizing that NIPS advantage would be higher under more favorable growing conditions. We also compared the quantitative variation and heritability of growth traits in populations of both species collected along a 200 km climatic transect. In contrast to our hypothesis we found that the NIPS N. neesiana was less responsive than A. bigeniculata to winter warming but maintained higher reproductive output during spring drought. However, overall tussock expansion was far more rapid in N. neesiana, and so it maintained an overall fitness advantage over A. bigeniculata in all climate regimes. N. neesiana also exhibited similar or lower quantitative variation and growth trait heritability than A. bigeniculata within populations but greater variability among populations, probably reflecting a complex past introduction history. We found some evidence that additional spring warmth increases the impact of drought on reproduction but not that elevated atmospheric CO2 ameliorates drought severity. Overall, we conclude that NIPS advantage under climate change may be limited by a lack of responsiveness to key climatic drivers, reduced genetic variability in range-edge populations, and complex drought-CO2 interactions.

The wheat Lr34 multipathogen resistance gene confers resistance to anthracnose and rust in sorghum

Summary The ability of the wheat Lr34 multipathogen resistance gene (Lr34res) to function across a wide taxonomic boundary was investigated in transgenic Sorghum bicolor. Increased resistance to sorghum rust and anthracnose disease symptoms following infection with the biotrophic pathogen Puccinia purpurea and the hemibiotroph Colletotrichum sublineolum, respectively, occurred in transgenic plants expressing the Lr34res ABC transporter. Transgenic sorghum lines that highly expressed the wheat Lr34res gene exhibited immunity to sorghum rust compared to the low‐expressing single copy Lr34res genotype that conferred partial resistance. Pathogen‐induced pigmentation mediated by flavonoid phytoalexins was evident on transgenic sorghum leaves following P. purpurea infection within 24–72 h, which paralleled Lr34res gene expression. Elevated expression of flavone synthase II, flavanone 4‐reductase and dihydroflavonol reductase genes which control the biosynthesis of flavonoid phytoalexins characterized the highly expressing Lr34res transgenic lines 24‐h post‐inoculation with P. purpurea. Metabolite analysis of mesocotyls infected with C. sublineolum showed increased levels of 3‐deoxyanthocyanidin metabolites were associated with Lr34res expression, concomitant with reduced symptoms of anthracnose.

polypatex: an r package for paternity exclusion in autopolyploids

Microsatellite markers have demonstrated their value for performing paternity exclusion and hence exploring mating patterns in plants and animals. Methodology is well established for diploid species, and several software packages exist for elucidating paternity in diploids; however, these issues are not so readily addressed in polyploids due to the increased complexity of the exclusion problem and a lack of available software. We introduce polypatex, an r package for paternity exclusion analysis using microsatellite data in autopolyploid, monoecious or dioecious/bisexual species with a ploidy of 4n, 6n or 8n. Given marker data for a set of offspring, their mothers and a set of candidate fathers, polypatex uses allele matching to exclude candidates whose marker alleles are incompatible with the alleles in each offspring–mother pair. polypatex can analyse marker data sets in which allele copy numbers are known (genotype data) or unknown (allelic phenotype data) – for data sets in which allele copy numbers are unknown, comparisons are made taking into account all possible genotypes that could arise from the compared allele sets. polypatex is a software tool that provides population geneticists with the ability to investigate the mating patterns of autopolyploids using paternity exclusion analysis on data from codominant markers having multiple alleles per locus.

Brachypodium distachyon genotypes vary in resistance to Rhizoctonia solani AG8

Brachypodium distachyon (L.)P.Beauv. (Bd) has previously been developed as a pathosystem model for the wheat root rot pathogen Rhizoctonia solani Kühn anastomosis group 8 (AG8). Here we explore variation in resistance to R. solani AG8 in Bd, to determine whether genomic tools could be used to find Bd genes involved in the grass defence response, with the aim of using this information for the improvement of Rhizoctonia root rot resistance in wheat. We looked for variation in resistance to R. solani AG8 in a diverse Bd natural accession collection and in Bd T-DNA insertion lines selected based on putative mechanisms reported for tagged genes. All lines were susceptible to the pathogen. Repeatable and significant variation in resistance was measured in both groups, with greater variation in resistance found across the natural accessions than in the T-DNA lines. The widest and most repeatable variation in resistance was between lines Koz-3 and BdTR 13a. The ratio of R. solani AG8-inoculated to uninoculated root length for line Koz-3 was 33% greater than the same ratio for line BdTR 13a. The increased resistance of Koz-3 was associated with nodal root initiation in response to the pathogen. A negative correlation between seedling vigour and resistance was observed, but found not to be the sole source of variation in resistance to R. solani AG8. The only T-DNA line with significantly greater resistance to R. solani AG8 than the reference line had an insertion in a putative galactosyltransferase gene; however, this result needs further confirmation. Genetic resistance to Rhizoctonia root rot is not available in wheat cultivars and only a few instances of quantitative resistance to the pathogen have been described within close relatives of wheat. Brachypodium distachyon offers potential for further investigation to find genes associated with quantitative resistance and mechanisms of tolerance to R. solani AG8.

Environmental water allocations are insufficient to control an invasive wetland plant: evidence from a highly regulated floodplain wetland

Summary 1. Regulation of the world’s rivers and alteration of natural flow regimes have led to the degradation of floodplains and wetlands. Here, we investigate plant–flood interactions in a floodplain wetland to determine whether a return to more natural flooding regimes using environmental water allocations can contribute to restoration from a degraded state. 2. We use detailed field surveys to quantify the effects of flooding on a native rush, Juncus ingens, which has become invasive in floodplain grasslands in south-eastern Australia. We then apply these results to determine the likelihood of generating the flood regime required to control J. ingens in its new range, considering the regulated nature of the river system. 3. Complete submergence for several months substantially reduced J. ingens stem densities, survival and recovery. However, rhizomes were able to survive, with new stems produced during subsequent partial submergence. Under current regulated conditions, the volume of environmental water needed to cause substantial stem decline, and potentially rhizome death, is extremely large – particularly for tall stems – even when used in conjunction with a natural flood peak. 4. Synthesis and applications. Although repeated submergence events may cause stem decline of the invasive plant Juncus ingens, the present day operational constraints of the regulated river make it difficult to deliver the required volume and depth of water using environmental water allocations. Furthermore, shallow floods may actually provide favourable conditions for growth. This research is particularly relevant for other restoration projects where historical disturbance regimes can only be partially mimicked. Whilst a managed flood regime can be successful in achieving a range of conservation and restoration goals, there may be situations in which river regulation can limit the ability to effectively manage particular species using environmental water allocations alone.

Does Late Maturity Alpha-Amylase Impact Wheat Baking Quality?

Late maturity α-amylase (LMA) and pre-harvest sprouting (PHS) are both recognized as environmentally induced grain quality defects resulting from abnormally high levels of α-amylase. LMA is a more recently identified quality issue that is now receiving increasing attention worldwide and whose prevalence is now seen as impeding the development of superior quality wheat varieties. LMA is a genetic defect present in specific wheat genotypes and is characterized by elevated levels of the high pI TaAMY1 α-amylase, triggered by environmental stress during wheat grain development. TaAMY1 remains present in the aleurone through the harvest, lowering Falling Number (FN) at receival, causing a down-grading of the grain, often to feed grade, thus reducing the farmers’ income. This downgrading is based on the assumption within the grain industry that, as for PHS, a low FN represents poor quality grain. Consequently any wheat line possessing low FN or high α-amylase levels is automatically considered a poor bread wheat despite there being no published evidence to date, to show that LMA is detrimental to end product quality. To evaluate the validity of this assumption a comprehensive evaluation of baking properties was performed from LMA prone lines using a subset of tall non-Rht lines from a multi-parent advanced generation inter-cross (MAGIC) wheat population grown at three different sites. LMA levels were determined along with quality parameters including end product functionality such as oven spring, bread loaf volume and weight, slice area and brightness, gas cell number and crumb firmness. No consistent or significant phenotypic correlation was found between LMA related FN and any of the quality traits. This manuscript provides for the first time, compelling evidence that LMA has limited impact on bread baking end product functionality.