Phosphorus absorption kinetics and exudation strategies of roots developed by three lupin species to tackle P deficiency.

MAIN CONCLUSION: Different lupin species exhibited varied biomass, P allocation, and physiological responses to P-deprivation. White and yellow lupins had higher carboxylate exudation rates, while blue lupin showed the highest phosphatase activity. White lupin (Lupinus albus) can produce specialized root structures, called cluster roots, which are adapted to low-phosphorus (P) soil. Blue lupin (L. angustifolius) and yellow lupin (L. luteus), which are two close relatives of white lupin, do not produce cluster roots. This study characterized plant responses to nutrient limitation by analyzing biomass accumulation and P distribution, absorption kinetics and root exudation in white, blue, and yellow lupins. Plants were grown in hydroponic culture with (64 µM NaH2PO4) or without P for 31 days. Under P limitation, more biomass was allocated to roots to improve P absorption. Furthermore, the relative growth rate of blue lupin showed the strongest inhibition. Under + P conditions, the plant total-P contents of blue lupin and yellow lupin were higher than that of white lupin. To elucidate the responses of lupins via the perspective of absorption kinetics and secretion analysis, blue and yellow lupins were confirmed to have stronger affinity and absorption capacity for orthophosphate after P-deprivation cultivation, whereas white lupin and yellow lupin had greater ability to secrete organic acids. The exudation of blue lupin had higher acid phosphatase activity. This study elucidated that blue lupin was more sensitive to P-scarcity stress and yellow had the greater tolerance of P-deficient condition than either of the other two lupin species. The three lupin species have evolved different adaptation strategies to cope with P deficiency.

Impact of freeze-thaw-induced pit aspiration on stem water transport in the subalpine conifer Abies veitchii.

During winter, subalpine conifers experience frequent freeze-thaw cycles in stem xylem that may cause embolism and pit aspiration due to increased water volume during the sap to ice transition. This study examined the occurrence and ecological impacts of a combination of freeze-thaw-induced pit aspiration and embolism triggered by natural and artificial stem freezing. In subalpine Veitch's fir (Abies veitchii) trees, the fraction of closed pits and embolized tracheids as well as conductivity losses were measured to examine pit aspiration and its effects. When trees incurred mild drought stress in February and early March, 70%-80% of stem conductivity was lost. Cryo-scanning electron microscopy indicated <20% embolized tracheids but ∼90% closed pits. Severe drought stress in late March caused 96% ± 1.2% (mean ± standard error) loss of stem conductivity, while the fraction of embolized tracheids increased to 64% ± 6.6%, and aspirated pit fraction decreased to 23% ± 5.6%. Experimental freeze-thaw cycles also increased pit aspiration from 7.1% ± 0.89% to 49% ± 10%, and the fraction of closed pits was positively correlated to the percent loss of stem hydraulic conductivity. The results indicated that freezing-induced pit aspiration is an important factor for stem xylem dysfunction under mild drought, and upon severe drought in winter; stem water transport is predominantly inhibited by xylem embolism.

Effects of root morphology, respiration and carboxylate exudation on carbon economy in two non-mycorrhizal lupines under phosphorus deficiency.

Under phosphorus (P) deficiency, Lupinus albus develops cluster roots that allow efficient P acquisition, while L. angustifolius without cluster roots also grows well. Both species are non-mycorrhizal. We quantitatively examined the carbon budgets to investigate the different strategies of these species. Biomass allocation, respiratory rates, protein amounts and carboxylate exudation rates were examined in hydroponically-grown plants treated with low (1 µM; P1) or high (100 µM; P100) P. At P1, L. albus formed cluster roots, and L. angustifolius increased biomass allocation to the roots. The respiratory rates of the roots were faster in L. albus than in L. angustifolius. The protein amounts of the non-phosphorylating alternative oxidase and uncoupling protein were greater in the cluster roots of L. albus at P1 than in the roots at P100, but similar between the P treatments in L. angustifolius roots. At P1, L. albus exuded carboxylates at a faster rate than L. angustifolius. The carbon budgets at P1 were surprisingly similar between the two species, which is attributed to the contrasting root growth and development strategies. L. albus developed cluster roots with rapid respiratory and carboxylate exudation rates, while L. angustifolius developed a larger root system with slow respiratory and exudation rates.

Comparison of the response to phosphorus deficiency in two lupin species, Lupinus albus and L. angustifolius, with contrasting root morphology.

White lupin (Lupinus albus) produces cluster roots, an adaptation to low soil phosphorus (P). Cluster roots exude large levels of P-solubilizing compounds such as citrate and malate. In contrast, narrow leaf lupin (L. angustifolius) is closely related to L. albus, but does not produce cluster roots. To examine the different strategies for P acquisition, we compared the growth, biomass allocation, respiratory properties and construction cost between L. albus and L. angustifolius under P-deficient conditions. Both Lupinus species were grown in hydroponic culture with 1 or 100 µM P. Under the P-deficient regime, L. albus produced cluster roots with little change in biomass allocation, while L. angustifolius significantly increased biomass allocation to roots. The rate of cyanide-resistant SHAM (salicylhydroxamic acid)-sensitive respiration was high in cluster roots and very low in roots of L. angustifolius. These results suggest a low alternative oxidase (AOX) activity in L. angustifolius roots, and thus, ATP would be produced efficiently in L. angustifolius roots. The construction cost was highest in cluster roots and lowest in L. angustifolius roots. This study shows that under P deficiency, L. albus produces high-cost cluster roots to increase the P availability, while L. angustifolius produces large quantities of low-cost roots to enhance P uptake.

Two CLE genes are induced by phosphate in roots of Lotus japonicus.

Genes of CLE (CLAVATA3/ESR-related) family encode peptide ligands that regulate plant development in response to external stimuli such as rhizobial infection and the nitrate application as well as various internal stimuli. To investigate whether LjCLE gene(s) may involve in plant response to inorganic phosphate (Pi), we analyzed Pi responses of 39 LjCLE genes in hydroponically grown Lotus japonicus plants (ecotype Miyakojima 'MG-20'). Two LjCLE genes, LjCLE19 and 20, were up-regulated specifically and greatly in roots of L. japonicus by Pi addition to the hydroponic solution. When the external Pi level increased, expressions of LjCLE19 and 20 increased before the increase in the Pi content in plants. On the other hand, when the external Pi level decreased, the Pi content in plants decreased first, then expression levels of LjCLE19 and 20 decreased. Based on our results, we discuss the relationship between LjCLE19 and 20 and the tissue Pi levels in plants. This is the first report showing induction of specific CLE genes by phosphate.

plenty, a novel hypernodulation mutant in Lotus japonicus.

Nitrogen fixation in nodules that contain symbiotic rhizobial bacteria enables legumes to thrive in nitrogen-poor soils. However, this symbiosis is energy consuming. Therefore, legumes strictly control nodulation at both local and systemic levels. Mutants deficient in such controls exhibit a range of phenotypes from non-nodulation to hypernodulation. Here, we isolated a novel hypernodulation mutant from the M(2) progeny derived from Lotus japonicus MG-20 seeds mutagenized by irradiation with a carbon ion beam. We named the mutant 'plenty' because it formed more nodules than the wild-type MG-20. The nodulation zone in the plenty mutant was wider than that in the wild type, but not as enhanced as those in other previously reported hypernodulation mutants such as har1, klv or tml of L. japonicus. Unlike these hypernodulation mutants, the plenty mutant developed nodules of the same size as MG-20. Overall, the plenty mutant exhibited a unique phenotype of moderate hypernodulation. However, a biomass assay indicated that this unique pattern of hypernodulation was a hindrance to host plant growth. The plenty mutant displayed some tolerance to external nitrates and a normal triple response to ethylene. Grafting experiments demonstrated that the root of plenty was responsible for its hypernodulation phenotype. Genetic mapping indicated that the PLENTY gene was located on chromosome 2.

Induction of Terminal Oxidases of Electron Transport Chain in Broccoli Heads under Controlled Atmosphere Storage.

Controlled atmosphere (CA) storage, that is, at low O2 and high CO2 concentrations, effectively extends the shelf life of horticultural products. The influence of CA storage (O2/CO2: 2.5%/6.0% or 2.5%/0.0%) and in normal air (both at 1 °C for 21 d) on the physicochemical (O2 uptake, mass loss and L-ascorbate) and biological properties of broccoli (Brassica oleracea var. italica, Plenck, 1794) via amounts and activities of terminal oxidases of the electron transport chain was investigated. Mass loss, a sensitive index of freshness for broccoli heads under CA, was significantly lower under CA than under normoxia (p < 0.05). Mass loss was depressed 7 d earlier under CA, including 6.0% CO2 than under CA without CO2. High CO2 effectively depressed the degradation of L-ascorbate. During storage, the activity of the alternative oxidase (AOX) was lower under CA than in normal air (p < 0.05), while the amount of cytochrome c oxidase (COX), and the AOX/COX activity ratio (based on oxygen isotope discrimination), were not affected during storage. Our results indicate that CA storage effectively retained the freshness of broccoli heads by depressing the induction of AOX. However, depression of AOX amount was not associated with CO2 around broccoli heads.

Influence of a Modified Atmosphere on the Induction and Activity of Respiratory Enzymes in Broccoli Florets during the Early Stage of Postharvest Storage.

Modified atmosphere packaging and controlled atmosphere storage (hypoxia conditions) extend shelf lives of horticultural products by depressing the O2 uptake rate. We investigated the relationship between atmospheres and alternative oxidase (AOX) to cytochrome c oxidase (COX) activities (on the basis of oxygen isotope discrimination) and the relative amounts of two respiratory enzymes, AOX and COX, during the early stage of storage. Broccoli florets, with high O2 uptake rates, were stored under hypoxia and normoxia at 25 °C. O2 uptake rates, weight loss, and yellowing of broccoli florets were significantly lower when stored under hypoxia than when stored under normoxia. Significantly more AOX proteins were produced during storage under normoxia, but COX proteins were more consistent than those of AOX proteins. Hypoxia may depress the expression of AOX and prolong the shelf life. Oxygen isotope discrimination was elevated under hypoxia after 50.5 h. AOX production in broccoli was controlled more by changing atmospheres than by COX.

Effects of polyploidy on photosynthetic properties and anatomy in leaves of Phlox drummondii.

Polyploidy affects photosynthesis by causing changes in morphology, anatomy and biochemistry. However, in newly developed polyploids, the genome may be unstable. In this study, diploid (2×) and synthetic autotetraploids in initial (4×-C0) and 11th generations (4×-C11) of Phlox drummondii Hook were used to study the effects of chromosome doubling and genome stabilisation on leaf photosynthesis and anatomical properties. The light-saturated photosynthetic rate on a leaf area basis at 360 µmol CO2 mol-1 air (A360) was highest in 4×-C11 leaves, intermediate in 4×-C0 leaves, and lowest in 2× leaves. Rubisco amounts, CO2-saturated photosynthetic rate at 1200 µmol CO2 mol-1 air at PPFD of 1000 µmol m-2 s-1 (A1200, representing the capacity for RuBP regeneration), cumulative surface areas of chloroplasts facing intercellular spaces (Sc), all expressed on a leaf area basis, were all higher in 4× leaves than in 2× leaves, and stomatal conductance (gs) at 360 µmol CO2 mol-1 air was only higher in the 4×-C11 leaves. A360 for the 4×-C11 leaves was greater than that in the 4×-C0 leaves despite having similar amounts of Rubisco. This was presumably associated with a greater RuBP regeneration capacity, as well as an increase in Sc and gs, which would increase the CO2 concentration of Rubisco. These results indicate that the higher rate of photosynthesis in 4×-C11 leaves was not an immediate outcome of chromosome doubling; rather, it was due to adjustment and adaptation during the process of genome stabilisation.

Effects of Eupatorium yellow vein virus infection on photosynthetic rate, chlorophyll content and chloroplast structure in leaves of Eupatorium makinoi during leaf development.

Infection of Eupatorium yellow vein geminivirus (EpYVV, formerly called tobacco leaf curl virus, TLCV) causes variegation in Eupatorium makinoi Kawahara et Yahara leaves. We examined changes in photosynthesis during leaf development to clarify what is the primary event when photosynthesis is suppressed in virus-infected E. makinoi leaves. The gas-exchange rate, leaf absorptance, chlorophyll (Chl) and nitrogen contents, leaf anatomy and chloroplast ultrastructure were compared between virus-infected and uninfected E. makinoi leaves at various developmental stages. These photosynthetic properties did not differ between infected and uninfected leaves when they were young. However, when expanded, infected leaves showed lower maximum quantum yield of photosynthetic CO2 uptake in the incident photosynthetically active photon fluence rate (PPFR), which was attributed to their lower Chl contents. The Chla / b ratio was higher and the grana had fewer thylakoids in the infected leaves, which are features common to Chl b-deficient mutants that have defects in Chl synthesis. Our results suggested that, in E. makinoi leaves, EpYVV infection primarily impairs Chl biosynthesis. Possible mechanisms of the suppression of photosynthesis in E. makinoi leaves by virus infection are discussed.

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.