Identification of boron transporter genes likely to be responsible for tolerance to boron toxicity in wheat and barley

Tolerance to boron (B) toxicity in cereals is known to be associated with reduced tissue accumulation of B. Genes from roots of B-tolerant cultivars of wheat and barley with high similarities to previously reported B efflux transporters from Arabidopsis and rice were cloned. Expression of these genes was strongly correlated with the ability of tolerant genotypes to lower the concentration of B in roots. The gene from barley located to chromosome 4. Backcross lines containing a B tolerance locus on chromosome 4 showed tolerance in proportion to the level of expression of the transporter gene, whereas those lacking the locus were sensitive to B and had very low levels of gene expression. The results are consistent with a widespread mechanism of tolerance to high B based on efflux of B from root cells.     

Problems associated with the use of foliar analysis for diagnosing boron toxicity in barley

The effects of evapotranspiration conditions on the distribution of B in leaves and critical values for B toxicity measured in shoots were examined in solution culture experiments with barley.Increased water use resulted in increased B accumulation by plants and B was concentrated in the leaf tips. The relationship between shoot DM production and shoot B concentrations was markedly affected by evapotranspiration conditions, but the effect could be removed by not analysing leaf tips. Excluding the leaf tips also decreased the shoot B concentration at which shoot DM production was depressed.Regularly spraying plants with water removed considerable B from leaves without affecting DM production.The present results indicate a number of problems in the establishment of critical values and the use of foliar analysis for diagnosing B toxicity. These problems may account for conflicting reports of critical values and discrepencies between results from glasshouse- and field-cultured plants.     

Mapping of chromosome regions conferring boron toxicity tolerance in barley (Hordeum vulgare L.)

 Boron toxicity has been recognised as an important problem limiting production in the low-rainfall regions of southern Australia, West Asia and North Africa. Genetic variation for boron toxicity tolerance in barley has been characterised but the mode of inheritance and the location of genes controlling tolerance were not previously known. A population of 150 doubled-haploid lines from a cross between a boron toxicity tolerant Algerian landrace, Sahara 3771, and the intolerant Australian cultivar Clipper was screened in four tolerance assays. An RFLP linkage map of the Clipper×Sahara population was used to identify chromosomal regions associated with boron tolerance in barley. Interval regression-mapping allowed the detection of four chromosomal regions involved in the boron tolerance traits measured. A region on chromosome 2H was associated with leaf-symptom expression, a region on chromosome 3H was associated with a reduction of the affect of boron toxicity on root growth suppression, a region on chromosome 6H was associated with reduced boron uptake, and a region on chromosome 4H was also associated with the control of boron uptake as well as being associated with root-length response, dry matter production and symptom expression. The benefits and potential of marker-assisted selection for boron toxicity tolerance are discussed. Received: 18 December 1997 / Accepted: 28 November 1998     

Redistribution of boron in leaves reduces boron toxicity

High soil boron (B) concentrations lead to the accumulation of B in leaves, causing the development of necrotic regions in leaf tips and margins, gradually extending back along the leaf. Plants vary considerably in their tolerance to B toxicity, and it was recently discovered that one of the tolerance mechanisms involved extrusion of B from the root. Expression of a gene encoding a root B efflux transporter was shown to be much higher in tolerant cultivars. In our current research we have shown that the same gene is also upregulated in leaves. However, unlike in the root, the increased activity of the B efflux transporter in the leaves cannot reduce the tissue B concentration. Instead, we have shown that in tolerant cultivars, these transporters redistribute B from the intracellular phase where it is toxic, into the apoplast which is much less sensitive to B. These results provide an explanation of why different cultivars with the same leaf B concentrations can show markedly different toxicity symptoms. We have also shown that rain can remove a large proportion of leaf B, leading to significant improvements of growth of both leaves and roots.Key words: Bor genes, boron tolerance, boron toxicity, efflux pumping, leaf necrosis, membrane transportB-toxic soils are widespread throughout agricultural areas of the world where they cause significant and often substantial reductions in crop quality and yield. The mechanism by which B is toxic to plants is not well understood¹ but toxicity symptoms include reduced root growth which affects uptake of water and nutrients, and the development of necrotic patches on leaves which impairs photosynthesis. Tolerance to B toxicity has been recognized in a number of crops, notably in cereals. In most cases, tolerance is achieved by reduced uptake of B into the root, which then leads to reduced uptake into the shoot. Genetic studies established that in barley, a locus associated with reduced tissue B occurred on chromosome 4 and that this locus could be transferred to other barley cultivars with desirable agronomic traits.²Hayes and Reid³ made a careful study of the characteristics of B uptake in a highly tolerant landrace barley cultivar Sahara, and found that although B was highly permeable, the root B concentration in this cultivar could be maintained at only half that in the external medium, whereas in sensitive cultivars, B was the same in both intracellular and extracellular phases. It was concluded that tolerant cultivars must have a membrane active transporter that exports B from the root. A B exporter, AtBor1 had previously been discovered in Arabidopsis where it was involved in B loading into the xylem⁴ but it was later found to be degraded under high B conditions⁵ and therefore would not be useful in B tolerance.However, other Bor1 homologues were subsequently discovered in Arabidopsis and in rice. Based on homology with rice, Reid⁶ cloned genes from barley and from wheat (HvBor2 and Tabor2 respectively) which were shown to be strongly upregulated in roots of tolerant cultivars, and virtually undetectable in sensitive cultivars. Thus, a simple mechanism to explain tolerance was established; efflux of B from the root reduced the intracellular concentration of B in the root cells, thereby reducing toxicity and improving root growth. At the same time, the lower root content meant that less B was transferred to the shoot, resulting in lower shoot toxicity.Yet there remained several unanswered questions regarding B toxicity. Firstly, it was commonly observed that toxicity symptoms were not reliably correlated with leaf B concentration, and that often after rain, toxicity symptoms became less severe. Nable et al.⁷ had investigated the effect of rain on shoot B concentrations and concluded that although rain did reduce the B concentration in leaves, it did not affect growth and yield. Secondly, field trials with cultivars in which the B tolerance traits were expressed, did not show the improvements in growth and yield that could be observed in glasshouse trials.^8,9Our recent work¹⁰ has provided new insights into these phenomena. Sensitive and tolerant cultivars of both wheat and barley were grown in varying levels of B. Then, ignoring the level of B in the growth solution, leaves of the different cultivars that displayed the same degree of leaf necrosis were selected. This revealed that in the tolerant cultivars, necrosis began to appear at leaf B levels that were two-to five-fold higher than in sensitive cultivars. Since no internal tolerance mechanism had been reported, it was hypothesised that in the tolerant cultivars, internal toxicity was reduced by pumping B from the cytoplasm into the cell wall where B is much less toxic. To prove this hypothesis three types of experiment were conducted. Firstly protoplasts were isolated from leaves of tolerant and sensitive cultivars of barley, and it was shown that when incubated in the same concentration of B, the tolerant cultivar was able to reduce the intracellular B concentration to approximately half that of the sensitive cultivar. Secondly, it was reasoned that if more B was accumulated in the apoplast of the tolerant cultivar, then it should be more quickly released by washing of the leaf; this was confirmed. Thirdly, it was shown that the same efflux transporters that were responsible for B export from the root were also highly expressed in leaves of tolerant cultivars of wheat and barley. The combination of these three experiments provided compelling evidence that redistribution of B in the leaf was a significant factor in B tolerance.The elution experiment also highlighted the fact that because B is highly soluble and has high membrane permeability, it can easily be washed from leaves. Obviously in the field B could be removed from leaves by rain, but no positive effect of this on growth had been quantified. In our experiments, we simulated the average rainfall during the early growing season in a high B region of Southern Australia by spraying plants with calibrated amounts of water for 16 d. At high B concentrations, rain reduced leaf B by around 50% while simultaneously improving growth of shoots by up to 90%. Rather surprisingly, the rain treatment, which had no significant effect on root B concentrations, caused a two-fold increase in root growth, presumably by improving the supply of photosynthate from the shoot.This study has enabled an evaluation of the importance of three main factors in determining the severity of B toxicity; two genetically determined processes, efflux pumping of B in roots and leaves, coupled with the variable leaching of B from leaves by rain (Fig. 1). The results also provide an explanation for the poor correlations observed between toxicity and shoot B concentrations in cereals.^7,11Open in a separate windowFigure 1Summary of processes contributing to reduced B toxicity in wheat and barley. The intensity of shading indicates the level of B in different regions of the plant. Boron (B) enters the leaf via the xylem and continues to accumulate as the leaf grows. When plants are grown in high concentrations of B, the older parts of the leaf become necrotic first while the younger basal tissues continue to expand. In tolerant cultivars, B efflux transporters in leaves pump B from the cytoplasm where it is toxic into the cell walls where it can be tolerated at high concentrations. Sensitive cultivars have a very low capacity for B efflux and therefore retain much higher concentrations inside the cell than in tolerant cultivars. rain can remove large amounts of B from leaves, thereby alleviating toxicity. In roots of tolerant cultivars, the same B efflux transporters that occur in leaves are used to pump B from the cells into the external medium. This reduces the toxicity to roots and limits the amount of B entering the xylem and reaching the leaves.     

Plant critical levels for the evaluation of boron toxicity in spring barley (Hordeum vulgare L.)

Summary Pot experiments on B toxicity in spring barley (Hordeum vulgare L., cv. Trumpf) using sandy soils indicated that there are significant relationships between B content of the leaves and of the shoots respectively in the toxic range and degree of damage of the leaves at stage 7–8 of the Feekes scale, which may be used to derive plant critical levels of B toxicity. Symptoms due to B excess begin to develop on the leaves (leaf tip necroses) relatively independently of the ontogenetical stage of development as soon as the B content of the leaf tissue reaches 60–80 mg/kg DM.The corresponding symptom-related toxic plant critical level of shoots (i.e. the B content of the whole shoot at which the first damage in leaves begins to occur) ranges from 30 mg/kg shoot DM (related to older leaves) to 80 mg/kg shoot DM (related to younger leaves). Grain yield is significantly reduced only when the B content of shoots at Feekes stages 7–8 exceeds the yield-related toxic plant critical level (yield reduction to 90% of the optimum yield) of 120–130 mg/kg shoot DM.B contents of the shoots at Feekes stages 7–8 from 80–120 mg/kg shoot DM define the range at which plants have marked toxicity symptoms, but at which there are no yield reductions.     

An investigation into inter-plot interactions, in experiments with mildew on barley, using balanced designs

Serial designs, balanced for effects of neighbours, were used in 1975–1977 to investigate interactions between plots of spring barley given different treatments for the control of powdery mildew (Erysiphe graminis DC. f. sp. hordei Mérat). Differences in amounts of disease, between similarly treated plots, could be related largely to the treatments applied to the neighbouring plots and to wind directions. Amounts of disease in plots were usually increased if the upwind neighbour was untreated but untreated plots themselves were also affected by neighbours, having least disease where the upwind neighbour was sprayed early. Differences in mildew due to treatment were reflected in grain yield. Yields also provided evidence of interactions between plots. Interference, as well as altering the average response to treatments, can also contribute to variability in experiment results and lead to substantial losses in efficiency. The yields in 1975 and 1976 provided strong evidence of profiles of fertility. In the analyses, adjustment by covariates, employed to allow for these profiles, gave substantial net gains in accuracy.     

Enhanced tolerance to boron toxicity in two-rowed barley by marker-assisted introgression of favourable alleles derived from Sahara 3771

Boron (B) is an essential micronutrient for higher plant, but toxic levels can seriously diminish grain yield in cereal crops by affecting root growth, and thus restricting water extraction from the subsoil. Amelioration of high concentrations in soils is expensive and not always feasible, so breeding for B tolerance is the most viable alternative. This article reports the marker-assisted (MAS) transfer of favourable alleles from an unadapted six-rowed barley (Hordeum vulgare L.) variety, Sahara 3771, into two-rowed lines adapted to southern Australia. During the backcrossing process, the SSR marker, EBmac679, located on chromosome 4H was used to control the target region in foreground selection, but no background selection was applied. Gene introgression was confirmed with 40 BC₆F₁-derived doubled haploid lines segregating for the SSR marker EBmac679. We used a combination of molecular and conventional assays to unequivocally classify the 40 BC₆F₁-derived DH lines as B tolerant or sensitive, and then compared their means for grain yield measured over 2 years and four locations. Results showed modest improvements in grain yield of lines carrying B tolerance genes at some B toxic environments, and negative impact at others. Our results also showed that malting quality profile was not adversely affected through the introgression of the B tolerance allele from Sahara 3771, allowing the newly developed material to be used by breeding programs without risk of a penalty on malt quality.     

An investigation of the toxicity of insecticides to birds' eggs using the egg-injection technique

Twenty-five insecticides have been tested for their toxicity to hen embryos at various concentrations, using an egg-injection technique. Of the two major groups, the organophosphorus compounds are much more toxic than the organochlorine. Most organophosphorus compounds lower the hatch rate and cause teratogenic effects at 100 ppm. Most organochlorines do not harm the embryo at high dosages (up to 500 ppm), with notable exceptions to this among the cyclodienes. However, starvation of the hatched chicks suggests that the majority of organochlorine compounds can kill at this stage, though fed chicks survive. The solvent used affected toxicity; in general, insecticides are more toxic to the embryo when dissolved in corn oil than when dissolved in acetone. If these findings can be applied to wild birds, it can be assumed that the vast majority of insecticides are harmless to birds' eggs. They are either not toxic in the concentrations so far found in this country, or else unlikely to pass through the mother bird to the egg.     

Genotypic variation in response of barley to boron deficiency

Responses of a range of barley (Hordeum vulgare L.) genotypes to boron (B) deficiency were studied in two experiments carried out in sand culture and in the field at Chiang Mai, Thailand. In experiment 1, two barley genotypes, Stirling (two-row) and BRB 2 (six-row) and one wheat (Triticum aestivum L.) genotype, SW 41, were evaluated in sand culture with three levels of applied B (0, 0.1 and 1.0 μM B) to the nutrient solution. It was found that B deficiency depressed flag leaf B concentration at booting, grain number and grain yield of all genotypes. In barley Stirling, B deficiency also depressed number of spikes plant^-1, spikelets spike^-1 and straw yield. However, no significant difference between genotypes in flag leaf B concentration was found under low B treatments. Flag leaf B concentration below 4 mg kg^-1 was associated with grain set reduction and could, therefore, be used as a general indicator for B status in barley. In experiment 2, nine barley and two wheat genotypes were evaluated in the field on a low B soil with three levels of B. Boron levels were varied by applying either 2 t of lime ha^-1 (BL), no B (B0) or 10 kg Borax ha^-1 (B+) to the soil prior to sowing. Genotypes differed in their B response for grain spike^-1, grain spikelet^-1 and grain set index (GSI). The GSI of the B efficient wheat, Fang 60, exceeded 90% in all B treatments. The B inefficient wheat SW 41 and most of the barley genotypes set grain normally (GSI >80%) only at the B+. In B0 GSI of the barley genotypes ranged from 23% to 84%, and in BL from 19% to 65%. Three of the barley with severely depressed GSI in B0 and BL also had a decreased number of spikelets spike^-1. In experiment 3, 21 advanced barley lines from the Barley Thailand Yield Nursery 1997/98 (BTYN 1997/98) were screened for B response in sand culture with no added B. Grain Set Index of the Fang 60 and SW 41 checks were 98 and 65%, respectively, and GSI of barley lines ranged between 5 and 90%. One advanced line was identified as B efficient and two as moderately B efficient. The remaining lines ranked between moderately inefficient to inefficient. These experiments have established that there is a range of responses to B in barley genotypes. This variation in the B response was observed in vegetative as well as reproductive growth. Boron efficiency should be considered in breeding and selection of barley in low B soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative accumulation of manganese and boron in barley tissues

Summary The uptake of Mn and B by barley plants was studied in a 5-week period in growth chambers. Fluorescent light was provided with an intensity of 3200 foot-candles in a 12-hour day length and the entire plants were grown at temperatures of 10°, 15°, or 20°C. The root medium consisted of a base nutrient solution in which Mn or B was added in the following concentrations: 0, 0.1, 0.5, 2.0, and 5.0 ppm. Five plants were grown in volumes of 20 liters of solution. At the end of the growth period the shoots and roots were analyzed for Mn and B. The Mn content of the roots increased with temperature and with the Mn concentration of the external solution while the B content remained virtually static regardless of temperature or solution concentration. The shoots were divided into young, mature, and old leaves. The Mn and B content of the old leaves showed increases which varied both with temperature and concentration. Similar results were obtained with young and mature leaves. The failure of B to accumulate in the roots was discussed. It was suggested that boric acid, with a very low degree of dissociation, is present largely in a molecular form and does not participate in the customary metabolic activity connected with ion uptake and accumulation in roots.     

Resistance to boron toxicity amongst several barley and wheat cultivars: A preliminary examination of the resistance mechanism

The mechanism of resistance toB toxicity in barley and wheat was studied in a solution culture experiment using several cultivars displaying a large range of sensitivity to excessB supply. Plants were cultured for 35 d atB concentrations ranging from normal to excessive (15 to 5000 M, respectively) then examined for dry matter production and theB distribution between roots and shoots.In both species, increasedB supply was accompanied by increased tissueB concentrations, development ofB toxicity symptoms and depressed growth. At each level ofB supply, however, resistant cultivars accumulated considerably lessB than did sensitive cultivars, in both roots and shoots. Even at the lowestB supply, at which noB toxicity symptoms developed and growth was not affected, resistant cultivars maintained relatively low tissueB concentrations. No cultivar displayed an ability to tolerate high tissueB concentrations.These results indicate that sensitivity toB toxicity in barley and wheat is governed by the ability of cultivars to excludeB. If theB concentrations of tissues is used to indicate resistance toB toxicity, then cultivars have the same ranking whether cultured at a normal or excessB supply.     

An investigation of the mineral content of barley grains and seedlings

The Ca, Mg, K, and P content of dry barley (Hordeum vulgare) grains and seedlings was investigated using energy dispersive x-ray analysis and neutron activation analysis. Energy dispersive x-ray analysis of protein bodies in aleurone cells showed that these bodies contained very little Ca in relation to P, Mg, and K. Neutron activation analysis also showed that the endosperm contained very little Ca in relation to the other three elements. Surface sterilization and soaking treatments brought about slight loss of Ca but substantial loss of K from embryos. Over 6 days of growth the seedling plant gained minerals from the endosperm.     

Hormonal regulation of barley nuclease: investigation using a monoclonal antibody

Abstract. A monoclonal antibody prepared against barley ( Hordeum vulgare L., cv. Himalaya) nuclease (EC 3.1.30.2) was characterized with solid-state enzyme-linked immunosorbent assays and immuno-blotting. The antibody was specific for intracellular and secreted nuclease. Hormonal regulation of the synthesis and secretion of nuclease in isolated aleurone layers was investigated by immunoprecipitation of biosynthetically-labelled nuclease using polyclonal antibodies and by immunoblot analyses using the monoclonal antibody, respectively. Gibberellic acid (GA₃) induced the de novo synthesis and secretion of nuclease in a time-and concentration-dependent manner. Nuclease was detected in aleurone layers incubated in 1 mmol m⁻³ GA₃, after 24 h. The maximum rates of nuclease synthesis and secretion occurred 36–48 h after hormone treatment. A minimum concentration of 10⁻⁶ mol m⁻³ GA₃ was required for nuclease synthesis and secretion, whereas the maximum rate of nuclease secretion occurred at concentrations of 10⁻⁵ mol m⁻³ and higher. In the presence of abscisic acid, the synthesis and secretion of nuclease from GA₃-treated aleurone layers was almost completely inhibited. Based on these findings, the authors conclude that all nuclease within and secreted from aleurone layers treated with GA₃ is the result of its de novo synthesis.     

Contrasting responses to boron deficiency in barley and wheat

Plant and Soil - To determine if boron (B) deficiency, commonly reported to depress grain set in wheat, has the same effect in barley, a set of experiments compared five wheat and seven barley...     

The involvement of aquaglyceroporins in transport of boron in barley roots

Boron (B) enters cells as the uncharged boric acid, a small neutral molecule with sufficient lipid solubility to cross cell membranes without the aid of transport proteins. The extent to which the observed uptake rates for B in plants can be explained by this simple physical process was examined by applying treatments expected to inhibit the membrane transporters most likely to be involved in B transport. These experiments established that at least 50% of B uptake could be facilitated by transporters. The B transport characteristics of two barley aquaglyceroporins, HvPIP1;3 and HvPIP1;4, were investigated using yeast complementation assays. Expression of both genes in yeast resulted in increased B sensitivity. Transport assays in yeast confirmed that HvPIP1;3 and HvPIP1;4 are both capable of transporting B. The physiological role of these HvPIP1 genes in B transport is uncertain since their expression was not responsive to B nutritional status, and they continued to be expressed under toxicity conditions.     

Effect of boron application on barley in a sierozem sandy soil

Summary In a pot culture experiment on a sierozem sandy soil (pH 8.2) rates of added B at 3 ppm although decreased root yield significantly but shoot and grain yield was unaffected even at 6 ppm added B, even though shoot B concentration was as high as 360 ppm and Ca/B ratio as low as 11. At 6 ppm applied B, shoot yield was increased by 18.5 per cent, whereas grain yield was at par with control. The results suggested that Ca/B ratio in barley straw was not a reliable index for determing the magnitude of B problem in the soil.     

Hydrogen sulfide alleviates aluminum toxicity in barley seedlings

Aims

Aluminum (Al) toxicity is one of the major factors that limit plant growth. Low concentration of hydrogen sulfide (H₂S) has been proven to function in physiological responses to various stresses. The objective of this study is to investigate the possible role of H₂S in Al toxicity in barley (Hordeum vulgare L) seedlings.

Methods

Barley seedlings pre-treated with sodium hydrosulfide (NaHS), a H₂S donor, and subsequently exposed to Al treatment were studied for their effects on root elongation, Al accumulation in seedlings, Al-induced citrate secretion and oxidative stress, and plasma membrane (PM) H⁺-ATPase expression.

Results

Our results showed that H₂S had significant rescue effects on Al-induced inhibition of root elongation which was correlated well with the decrease of Al accumulation in seedlings. Meanwhile, Al-induced citrate secretion was also significantly enhanced by NaHS pretreatment. Al-induced oxidative stress as indicated by lipid peroxidation and reactive oxygen species burst was alleviated by H₂S through the activation of the antioxidant system. Moreover, Al-induced reduction in PM H⁺-ATPase expression was reversed by exogenous NaHS.

Conclusions

Altogether, our results suggest H₂S plays an ameliorative role in protecting plants against Al toxicity by inducing the activities of antioxidant enzymes, increasing citrate secretion and citrate transporter gene expression, and enhancing the expression of PM H⁺-ATPase.     

An investigation into the relations between some growth parameters and yield of barley

Twenty-five field experiments on barley were done at four sites from 1970 to 1975, with an additional site in 1975, comparing five rates of nitrogen application. The crops were sampled at c. 14-day intervals from about the four-leaf stage to maturity to follow changes with time in the dry weights of the constituent plant parts and to measure fertile tiller number, grain number per ear and grain weight. Some data have been selected from this body of information and regression analysis has been used to assess the possible importance of pre- and post-anthesis growth in determining the yield of barley. Ear emergence was taken as an approximate indicator of anthesis. Yield was closely related (r=+0–96) and almost equal to the amount of dry matter accumulated before ear emergence for twenty-one of the experiments, with a regression coefficient of 0–95. In two of the remaining experiments the regression coefficient was greater, 1–14; and smaller in the other two, o-8i. Plant population is also shown to affect the regression coefficient. Despite these anomalies grain yield was more closely related to plant dry weight at ear emergence than to the increase in weight after ear emergence (r =+0–79). The increase in plant dry weight after ear emergence was frequently less than the yield of grain, suggesting that the plant can compensate for inadequate photosynthesis during grain filling. The result of this compensation is a reduction in straw dry weight. This implies that previously assimilated dry matter is transported to the grain, or that respiratory losses from the straw are not replaced by current photosynthesis. These observations suggest that yield may be limited by sink capacity rather than by photosynthesis after ear emergence. The main component of sink capacity, grain number per m² was closely related to yield (r =+0–95) and is known to be determined at or before anthesis. Grain size was shown to be related to grain number per ear (r= 4- 0–99), suggesting that grain size is also, at least partially, determined at ear emergence. Therefore, all the yield components are determined, wholly or partly before ear emergence.     

Jasmonic acid alleviates boron toxicity in Puccinellia tenuiflora,a promising species for boron phytoremediation

Plant and Soil - Although elevated [CO2] causes an increase of photosynthesis in the short-term, this increase is often attenuated over time due to decreased photosynthetic capacity of the leaf in...