Gregory R. Cawthray

Chickpea and white lupin rhizosphere carboxylates vary with soil properties and enhance phosphorus uptake

Chickpea and white lupin roots are able to exude large amounts of carboxylates, but the resulting concentrations in the rhizosphere vary widely. We grew chickpea in pots in eleven different Western Australian soils, all with low phosphorus concentrations. While final plant mass varied more than two-fold and phosphorus content almost five-fold, there were only minor changes in root morphological traits that potentially enhance phosphorus uptake (e.g., the proportion of plant mass allocated to roots, or the length of roots per unit root mass). In contrast, the concentration of carboxylates (mainly malonate, citrate and malate, extracted using a 0.2 mM CaCl2 solution) varied ten-fold (averaging 2.3 μmol g−1 dry rhizosphere soil, approximately equivalent to a soil solution concentration of 23 mM). Plant phosphorus uptake was positively correlated with the concentration of carboxylates in the rhizosphere, and it was consistently higher in soils with a smaller capacity to sorb phosphorus. Phosphorus content was not correlated with bicarbonate-extractable phosphorus or any other single soil trait. These results suggest that exuded carboxylates increased the availability of phosphorus to the plant, however, the factors that affected root exudation rates are not known. When grown in the same six soils, three commonly used Western Australian chickpea cultivars had very similar rhizosphere carboxylate concentrations (extracted using a 0.2 mM CaCl2 solution), suggesting that there is little genetic variation for this trait in chickpea. Variation in the concentration of carboxylates in the rhizosphere of white lupin did not parallel that of chickpea across the six soils. However, in both species the proportion of citrate decreased and that of malate increased at lower soil pH. We conclude that patterns of variation in root exudates need to be understood to optimise the use of this trait in enhancing crop phosphorus uptake.

An improved reversed-phase liquid chromatographic method for the analysis of low-molecular mass organic acids in plant root exudates.

Reversed-phase column liquid chromatography (RPLC) was used for the separation and quantification of 10 low-molecular-mass organic acids (malic, malonic, lactic, acetic, maleic, citric, cis-aconitic, succinic, fumaric, and trans-aconitic) in plant root exudates. A mobile phase of 93% 25 mM KH2PO4 at pH 2.5 and 7% methanol at a flow-rate of 1 ml min(-1) resolved all 10 acids in 15 min on a C18 column. Experiments demonstrated a significant (P < 0.05) effect of sample pH on detector response, with peak heights being significantly lower at pH 6.0 compared with pH 2.5, but peak area showed no significant difference. At pH 8.0 and above, both peak height and area differed significantly from injections made at pH 2.5. Limits of detection (LOD) for the 10 acids ranged from 0.05 to 24 microM. Finally, the improved method was applied for the analysis of root exudates from soil cultured field pea, Banksia attenuata, white lupin, and chickpea.

Proteaceae from severely phosphorus‐impoverished soils extensively replace phospholipids with galactolipids and sulfolipids during leaf development to achieve a high photosynthetic phosphorus‐use‐efficiency

Proteaceae species in south-western Australia occur on severely phosphorus (P)-impoverished soils. They have very low leaf P concentrations, but relatively fast rates of photosynthesis, thus exhibiting extremely high photosynthetic phosphorus-use-efficiency (PPUE). Although the mechanisms underpinning their high PPUE remain unknown, one possibility is that these species may be able to replace phospholipids with nonphospholipids during leaf development, without compromising photosynthesis. For six Proteaceae species, we measured soil and leaf P concentrations and rates of photosynthesis of both young expanding and mature leaves. We also assessed the investment in galactolipids, sulfolipids and phospholipids in young and mature leaves, and compared these results with those on Arabidopsis thaliana, grown under both P-sufficient and P-deficient conditions. In all Proteaceae species, phospholipid levels strongly decreased during leaf development, whereas those of galactolipids and sulfolipids strongly increased. Photosynthetic rates increased from young to mature leaves. This shows that these species extensively replace phospholipids with nonphospholipids during leaf development, without compromising photosynthesis. A considerably less pronounced shift was observed in A. thaliana. Our results clearly show that a low investment in phospholipids, relative to nonphospholipids, offers a partial explanation for a high photosynthetic rate per unit leaf P in Proteaceae adapted to P-impoverished soils.

Developmental Physiology of Cluster-Root Carboxylate Synthesis and Exudation in Harsh Hakea. Expression of Phosphoenolpyruvate Carboxylase and the Alternative Oxidase

Harsh hakea (Hakea prostrata R.Br.) is a member of the Proteaceae family, which is highly represented on the extremely nutrient-impoverished soils in southwest Australia. When phosphorus is limiting, harsh hakea develops proteoid or cluster roots that release carboxylates that mobilize sparingly soluble phosphate in the rhizosphere. To investigate the physiology underlying the synthesis and exudation of carboxylates from cluster roots in Proteaceae, we measured O2 consumption, CO2 release, internal carboxylate concentrations and carboxylate exudation, and the abundance of the enzymes phosphoenolpyruvate carboxylase and alternative oxidase (AOX) over a 3-week time course of cluster-root development. Peak rates of citrate and malate exudation were observed from 12- to 13-d-old cluster roots, preceded by a reduction in cluster-root total protein levels and a reduced rate of O2 consumption. In harsh hakea, phosphoenolpyruvate carboxylase expression was relatively constant in cluster roots, regardless of developmental stage. During cluster-root maturation, however, the expression of AOX protein increased prior to the time when citrate and malate exudation peaked. This increase in AOX protein levels is presumably needed to allow a greater flow of electrons through the mitochondrial electron transport chain in the absence of rapid ATP turnover. Citrate and isocitrate synthesis and accumulation contributed in a major way to the subsequent burst of citrate and malate exudation. Phosphorus accumulated by harsh hakea cluster roots was remobilized during senescence as part of their efficient P cycling strategy for growth on nutrient impoverished soils.

Effects of external phosphorus supply on internal phosphorus concentration and the initiation, growth and exudation of cluster roots in Hakea prostrata R.Br.

The response of internal phosphorus concentration, cluster-root initiation, and growth and carboxylate exudation to different external P supplies was investigated in Hakea prostrata R.Br. using a split-root design. After removal of most of the taproot, equal amounts of laterals were allowed to grow in two separate pots fastened together at the top, so that the separate root halves could be exposed to different conditions. Plants were grown for 10 weeks in this system; one root half was supplied with 1 μM P while the other halves were supplied with 0, 1, 25 or 75 μM P. Higher concentrations of P supplied to one root half significantly increased the P concentration of those roots and in the shoots. The P concentrations in root halves supplied with 1 μM P were invariably low, regardless of the P concentration supplied to the other root half. Cluster root initiation was completely suppressed on root halves supplied with 25 or 75 μM P, whereas it continued on the other halves supplied with 1 μM P indicating that cluster-root initiation was regulated by local root P concentration. Cluster-root growth (dry mass increment) on root halves supplied with 1 μM P was significantly reduced when the other half was either deprived of P or supplied with 25 or 75 μM P. Cluster-root growth was favoured by a low shoot P status at a root P supply that was adequate for increased growth of roots and shoots without increased tissue P concentrations. The differences in cluster-root growth on root halves with the same P supply suggest that decreased cluster-root growth was systemically regulated. Carboxylate-exudation rates from cluster roots on root halves supplied with 1 μM P were the same, whether the other root half was supplied with 1, 25 or 75 μM P, but were approximately 30 times faster when the other half was deprived of P. Estimates of root P-uptake rates suggest a rather limited capacity for down-regulating P uptake when phosphate was readily available.

Low levels of ribosomal RNA partly account for the very high photosynthetic phosphorus-use efficiency of Proteaceae species

Abstract Proteaceae species in south-western Australia occur on phosphorus- (P) impoverished soils. Their leaves contain very low P levels, but have relatively high rates of photosynthesis. We measured ribosomal RNA (rRNA) abundance, soluble protein, activities of several enzymes and glucose 6-phosphate (Glc6P) levels in expanding and mature leaves of six Proteaceae species in their natural habitat. The results were compared with those for Arabidopsis thaliana. Compared with A. thaliana, immature leaves of Proteaceae species contained very low levels of rRNA, especially plastidic rRNA. Proteaceae species showed slow development of the photosynthetic apparatus (‘delayed greening’), with young leaves having very low levels of chlorophyll and Calvin–Benson cycle enzymes. In mature leaves, soluble protein and Calvin–Benson cycle enzyme activities were low, but Glc6P levels were similar to those in A. thaliana. We propose that low ribosome abundance contributes to the high P efficiency of these Proteaceae species in three ways: (1) less P is invested in ribosomes; (2) the rate of growth and, hence, demand for P is low; and (3) the especially low plastidic ribosome abundance in young leaves delays formation of the photosynthetic machinery, spreading investment of P in rRNA. Although Calvin–Benson cycle enzyme activities are low, Glc6P levels are maintained, allowing their effective use.

2,4-D resistance in wild radish: reduced herbicide translocation via inhibition of cellular transport.

Highlight Reduced translocation of 2,4-D confers resistance in wild radish, and is due to inhibition of phloem loading, rather than enhanced metabolism or sequestration of the herbicide.

Analysis of Dimethylsulphoniopropionate (DMSP), Betaines and other organic solutes in plant tissue extracts using HPLC

An HPLC method using a cation-exchange column and UV detection for the simultaneous determination of dimethylsulphoniopropionate (DMSP), glycinebetaine, prolinebetaine, proline and arginine in plant tissue extracts is described. Recoveries of DMSP and glycinebetaine in either 5% perchloric acid, or methanol:chloroform:water (12:5:1) extracts of sugarcane leaf tissue were 94.0–95.4%. Proline was resolved in the 5% perchloric acid extract (recovery was 93.8%), whereas in the methanol:chloroform: water extract quantification of proline was not possible due to interference by unidentified compounds. Retention times of DMSP and arginine were increased when the pH of the mobile phase was decreased from 4.6 to 3.5, and the adjustment of the pH of the mobile phase was shown to be necessary in order to resolve DMSP in extracts from sugarcane leaves. The quantification of DMSP in sugarcane leaves using the HPLC method was compared with an indirect GC method, and the values obtained using GC were 1.14–1.65-fold higher. This discrepancy remains to be resolved. The detection limit for DMSP using the HPLC technique was about 2 µmol/g dry weight, while for the GC method it was 0.04 µmol/g dry weight. Copyright

Crassulacean acid metabolism enhances underwater photosynthesis and diminishes photorespiration in the aquatic plant Isoetes australis

• Underwater photosynthesis by aquatic plants is often limited by low availability of CO(2), and photorespiration can be high. Some aquatic plants utilize crassulacean acid metabolism (CAM) photosynthesis. The benefits of CAM for increased underwater photosynthesis and suppression of photorespiration were evaluated for Isoetes australis, a submerged plant that inhabits shallow temporary rock pools. • Leaves high or low in malate were evaluated for underwater net photosynthesis and apparent photorespiration at a range of CO(2) and O(2) concentrations. • CAM activity was indicated by 9.7-fold higher leaf malate at dawn, compared with at dusk, and also by changes in the titratable acidity (μmol H(+) equivalents) of leaves. Leaves high in malate showed not only higher underwater net photosynthesis at low external CO(2) concentrations but also lower apparent photorespiration. Suppression by CAM of apparent photorespiration was evident at a range of O(2) concentrations, including values below air equilibrium. At a high O(2) concentration of 2.2-fold the atmospheric equilibrium concentration, net photosynthesis was reduced substantially and, although it remained positive in leaves containing high malate concentrations, it became negative in those low in malate. • CAM in aquatic plants enables higher rates of underwater net photosynthesis over large O(2) and CO(2) concentration ranges in floodwaters, via increased CO(2) fixation and suppression of photorespiration.

Herbicide Resistance Endowed by Enhanced Rates of Herbicide Metabolism in Wild Oat (Avena spp.)

Abstract The biochemical basis of resistance to the acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicide diclofop-methyl was investigated in a resistant wild oat population (R1), which does not exhibit a resistant ACCase. Rates of foliar uptake and translocation of [14C]-diclofop were the same in the R1 vs. susceptible (S) populations. However, the level of phytotoxic diclofop acid was always found to be lower in the R1 vs. S plants, with a concomitant higher level (up to 1.7-fold) of nontoxic polar diclofop metabolites in R1 relative to the S plants. These results indicate that a non–target-site-based mechanism of enhanced rate of diclofop acid metabolism confers resistance in population R1. Moreover, the high-performance liquid chromotography elution profile of the major diclofop metabolites in R1 is similar to that of wheat, suggesting resistance in individuals of population R1 involves a wheat-like detoxification system mediated by cytochrome P450 monooxygenases. In addition, lower level of tissue diclofop acid was also observed using nonradioactive ultra-performance liquid chromatography–mass spectrometry analysis in resistant individuals of three other resistant wild oat populations (R2, R3, and R4) known to posses ACCase gene resistance mutations. These results establish that either one or at least two independent resistance mechanisms (target-site ACCase resistance mutations and non–target-site enhanced rates of herbicide metabolism) can be present in individual wild oat plants. Nomenclature: Diclofop-methyl; wild oat, Avena fatua L. AVEFA.