Calcium Antagonist TMB-8 Inhibits Cell Wall Formation and Growth in Pea

The effects on auxin-stimulated growth and cell wall formationof 8-(N, N-diethylamino)-octyl-3, 4, 5-trimethoxybenzoate.HCI(TMB-8), an intracellular Ca²⁺ antagonist, were investigatedin abraded stem segments from aetiolated seedlings of Pisumsativum L. cv. Alaska. Incubation of segments at pH 6.0 with200 mmol m^–3 TMB-8 resulted in a 50% inhibition of auxin-stimulatedgrowth. Added Ca²⁺ did not restore normal auxin-stimulated growth,presumably because of its well-known stiffening effect on thecell wall. In segments incubated at a pH (7–2) which preventedelongation, auxin promoted the incorporation of [³H]glucoseinto the cell wall relative to total uptake of label. TMB-8abolished about 60% of the total incorporation of label intocell walls in the presence of auxin, but was not effective inthe absence of auxin. Exogenous CaCl₂ reversed the inhibitoryeffect of TMB-8 on relative cell wall incorporation in a parabolicmanner, with a 50% reversal at about 100 mmol m^–3 andcomplete reversal at 1.0 mol m^–3 Ca²⁺. Other ions tested(Mg²⁺, Mn²⁺, Cu²⁺, Zn²⁺) were without substantial effect atconcentrations of 0.5 mol m^–3. Both apparent uptake ofCa²⁺ and consequent reversal of TMB-8 inhibition of cell wallincorporation were blocked by the Ca²⁺ channel blockers verapamiland La³⁺. The data provide further evidence that auxin-stimulatedgrowth is dependent upon continued cell wall incorporation,and suggest that a Ca²⁺ messenger system may be involved inthe promotory actions of auxin on cell wall synthesis and long-termgrowth. Key words: Auxin, calcium, cell wall synthesis     

Post‐translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold‐induced sweetening of potato tubers

Cold‐induced sweetening (CIS) is a serious post‐harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark‐coloured and bitter‐tasting product and generating the probable carcinogen acrylamide as a by‐product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS‐susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS‐resistant line increased susceptibility to CIS. The results show that post‐translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS.     

A High Arctic soil ecosystem resists long‐term environmental manipulations

We evaluated above‐ and belowground ecosystem changes in a 16 year, combined fertilization and warming experiment in a High Arctic tundra deciduous shrub heath (Alexandra Fiord, Ellesmere Island, NU, Canada). Soil emissions of the three key greenhouse gases (GHGs) (carbon dioxide, methane, and nitrous oxide) were measured in mid‐July 2009 using soil respiration chambers attached to a FTIR system. Soil chemical and biochemical properties including Q₁₀ values for CO₂, CH₄, and N₂O, Bacteria and Archaea assemblage composition, and the diversity and prevalence of key nitrogen cycling genes including bacterial amoA, crenarchaeal amoA, and nosZ were measured. Warming and fertilization caused strong increases in plant community cover and height but had limited effects on GHG fluxes and no substantial effect on soil chemistry or biochemistry. Similarly, there was a surprising lack of directional shifts in the soil microbial community as a whole or any change at all in microbial functional groups associated with CH₄ consumption or N₂O cycling in any treatment. Thus, it appears that while warming and increased nutrient availability have strongly affected the plant community over the last 16 years, the belowground ecosystem has not yet responded. This resistance of the soil ecosystem has resulted in limited changes in GHG fluxes in response to the experimental treatments.     

Cell Wall Acidification and its Role in Auxin-Stimulated Growth

The role of cell wall acidification in auxin-stimulated growthwas examined in abraded hypocotyl segments of etiolated Cucumis.sativus seedlings. Acidification of the medium by these segmentswas strongly inhibited by a pretreatment and the continued presenceof 1?0 mol m^–3 vanadate, widely used as an inhibitor ofplasma membrane ATPase activity. Elongation of segments in pH6?5 buffer was almost completely inhibited by such a treatmentwith vanadate, and the promotion of growth by indole-3-aceticacid (IAA) seen in the absence of vanadate was completely abolished.However, both inhibited and uninhibited segments showed a pronouncedelongation in response to pH 4?0) buffer. In pH 4?0 buffer,in contrast to the results obtained at pH 6?5, IAA significantlypromoted growth in both the presence and absence of vanadate.The results indicate that IAA can promote growth in the absenceof endogenous acidification, but that an acid wall is necessaryfor wall loosening to occur. Key words: Acidification, auxin-stimulated growth, Cucumis sativus, vanadate     

Rapid cellular responses to auxin and the regulation of growth

Abstract The cellular responses rapidly evoked by auxin are reviewed, and related to a consideration of how growth rate is regulated in excised segments and in whole dicotyledonous plants. Two processes, synthesis of proteins and of cell wall components, are both promoted by auxin and essential for auxin-stimulated growth, whereas other processes show little promotion by auxin or do not appear essential for growth. Current models for the cellular regulation of growth by auxin are briefly discussed, and a new model presented. Auxin is suggested to act by bringing about a transient increase in cytosolic Ca²⁺ levels, which through the stimulation of protein kinases converts a cytoplasmic protein factor to an active state capable of binding auxin. The protein-auxin complex induces mRNA synthesis, which effects the increased synthesis of cell wall components and their incorporation into the wall, resulting in wall loosening and growth. It is proposed that the factor limiting growth in floating excised segments may initially be cell wall pH, but that this is not the case in whole plants and growth is instead mediated by increased protein and matrix cell wall synthesis. Differences are noted between monocotyledonous coleoptiles and dicotyledonous stems in some metabolic processes possibly involved in auxin growth responses, and it is cautioned that observations made on one tissue may not necessarily be applicable to the other. Care should also be taken in applying conclusions drawn from studies on excised tissue to the interpretation of growth regulation in the whole plant.     

Effect of Vanadate on Microsomal ATPase Activity, Acidification of the Medium and Auxin-stimulated Growth in Pea and Cucumber

The relationship between ATPase activity, medium acidificationand auxin-stimulated growth in segments of pea stem (Pisum sativumL., cv. Alaska) and cucumber hypocotyl (Cucumis sativus L.,cv. Long Green Ridge) was investigated using sodium orthovanadate,widely used as a selective inhibitor of plasma membrane-associatedATPase activity. ATPase activity of cucumber microsomal preparationswas about seven times lower than similar preparations from pea(on a mg microsomal protein basis) and was much more effectivelyinhibited by vanadate. Similarly, acidification of the mediumby abraded cucumber segments occurred to a lesser extent thanwith pea and showed a greater inhibition by vanadate. Both growthin controls and auxin-stimulated growth of cucumber segmentswere strongly inhibited by vanadate, whereas in pea auxin-stimulatedgrowth was reduced by only half and controls showed little inhibition.Acidification of the medium by segments of both species wasfound to occur readily even in controls and showed little promotionin the presence of IAA, although growth in both species wasrapidly and significantly promoted by IAA. These results indicatethat acidification is brought about by a plasma membrane-associatedATPase, and suggest that while acidification is an essentialfactor for auxin-stimulated growth it may not be the mechanismby which the growth rate is controlled. ATPase, Cucumis sativus, indole-3-acetic acid, Pisum sativum, vanadate     

Medium Acidification by Auxin- and Fusicoccin-Treated Peeled Stem Segments from Etiolated Seedlings of Pisum sativum

Several reports have suggested that peeling off the epidermisfrom dicotyledonous stems also removes the capacity of thesesegments to show an auxin-induced elongation response. Thiscould be due either to the removal of the auxin-responsive tissue,or to the loss of H⁺ into the medium when the impermeable cuticleis removed. Medium acidification by peeled etiolated segmentsof Pisum sativum L. cv. Alaska, and the effects of indoleaceticacid (IAA) and fusicoccin (FC) on this process, were thus investigatedin an attempt to resolve this problem. It was found that mediumacidification occurred even with control segments, and thatonly in the presence of the divalent cations Ca²⁺ or Mg²⁺ didIAA give any enhancement of acidification. Under these conditions,the main effect of IAA was a reduction in the lag time betweenIAA addition and an observed acidification response. Additionof these ions is not, however, required to obtain normal auxin-inducedgrowth. Similarly, peeled segments of mature non-elongatingtissue also acidified the medium. In contrast, FC gave someenhancement of medium acidification even in the absence of Ca²⁺and Mg²⁺. With both IAA and FC, lag times before observableacidification occurred were reduced (from about 40–50min to 20 min and 2/emdash 4 min respectively) in the presenceof Ca²⁺ or Mg²⁺. These ions appeared to exert similar effects.IAA, when added once a stabilized pH had been attained, hadlittle effect on pH, whereas FC caused a further drop of about0.5 unit. It thus appears that auxin has little appreciableeffect on the acidification of the medium caused by peeled segments,either of non-elongating or of growing stems, and we concludethat peeling must therefore remove the auxin-responsive tissuein pea stem.