Yusuke Arakawa

The responses of red clover (Trifolium pratense L.) to iron deficiency: a root Fe(III) chelate reductase

Abstract Red clover ( Trifolium pratens e L. cv. Kenland) was found to be an Iron (Fe)-efficient plant while it was sensitive to deficiencies of Cu, Zn and Mn or toxicities of Cu and Mn. Under Fe deficiency, the activity of Fe(III) chelate reductase in roots displayed two distinct peaks during 24 days of treatment. The root Fe(III) chelate reductase was induced within 1 day, during which no proton extrusion was observed. Both roots and shoots are involved in the reductase induction. The roots alone were able to rapidly induce reductase activity, while the shoots were necessary for the later enhancement of reductase activity. It is proposed that the rapidly-induced root reductase plays an important role in overcoming Fe deficiency in calcareous soils. The relationships between reductase induction, proton extrusion, and the role of shoots and roots are also discussed.

A copper-deficiency-induced root reductase is different from the iron-deficiency-induced one in red clover (Trifolium pratense L.)

There is increasing evidence that Cu deficiency can induce root reductase activity, but the ecological and physiological significance of this is unknown. This study compared the characteristics of root reductase activity induced by Cu deficiency with those induced by Fe deficiency in red clover (Trifolium pratenseL. cv. Kenland), a Fe-efficient plant. Effects of other nutritional stresses were also investigated for comparison. Compared with the effect of Fe deficiency, Cu deficiency induced only a moderate level of root reductase activity, while other nutrient stresses had no effect, or even inhibited the root reductases activity, especially in the case of Zn deficiency. Compared with Fe deficiency-induced Fe(III)-chelate reductase, Cu deficiency-induced reductase displayed a different pattern of induction. The activity of the Cu deficiency-induced reductase in intact plants increased with time; in decapitated plants it showed a distinct peak at a later stage of the treatment. The Fe concentration in the roots was significantly increased under Cu deficiency. Furthermore, the reductase activity was presented in the entire root system, contrary to what was observed for the Fe-deficiency-induced reductase activity, which was confined to the root apex. Cu deficiency did not increase proton extrusion from the roots, even when growth was significantly affected. The present results suggest that in red clover Cu deficiency induces a root reductase that is different from the reductase induced by Fe deficiency.

An Alfalfa Gene Similar to Glutathione S-Transferase Is Induced in Root by Iron Deficiency

Abstract In order to isolate genes influenced by the iron (Fe) status of plants, we constructed a cDNA library from the roots of Fe-starved alfalfa (Medicago sativa L.) plants. The library was then screened with a subtracted probe. Based on the results of hybridization, we selected a full-length clone cDNA, designated as Isr1 (iron starvation-responsive gene 1). The Isr1 consisted of 815 nucleotides containing an open reading frame of 642 bp and encoded a protein with 214 amino acids, which showed a homology to glutathione S-transferase (GST, EC 2.5.1.18). A significant increase in the amount of Isr1 mRNA was observed in roots after 8 h of Fe starvation. The amount of Isr1 transcript, however, decreased rapidly on the second day, then increased again after the third day. The amount of Isr1 transcript was not influenced by the resumption of the Fe for 1 d. Possible functions of the gene product are discussed.

26Al MICROANALYSIS IN THE LEAF CELL ORGANELLES OF RUZIGRASS AND BARLEY BY ACCELERATOR MASS SPECTROMETRY (AMS)

The response to aluminum (Al) application was compared between an Al-tolerant plant, ruzigrass (Brachiaria ruziziensis), and two cultivars of barley (Hordeum vulgare L.), Al-tolerant ‘Dayton’ and Al-sensitive ‘Kearney’, of Al at 1.0 mM level. The micro content of Al transportation and accumulation in leaf cell organelles (nuclei, mitochondria, and chloroplast) was determined by the 26Al tracer technique with accelerator mass spectrometry (AMS), which could detect atoms of the order of 107 of 26Al. In a short-time-exposure experiment, plants were immersed into 0.1 mM 27Al (containing 101 dpm/300 mL 26Al) for 5 hours. Then the leaves were separated and 27Al and 26Al were determined. Growth of shoots and roots of ruzigrass was not influenced at 1 mM AlCl3, but the growth of barley cultivars (Dayton and Kearney) was seriously affected. There was only a small difference in Al content in shoot (about 5 g g−1) among plant species or cultivars when total leaf 27Al was determined by the inductively coupled plasma atomic emission spectrometry (ICP/AES) method at 0.1 mM level. While large differences in the Al content between plant cultivars in the leaf subcellular organelles were found by using 26Al with the AMS method, the amount was in the order of 1–10 pg per 1 mg of organelle protein. Ruzigrass accumulated about 4 or 5 times higher content (2.8 ng mg−1 of protein) of 27Al in the nuclei and 2 or 3 times higher content (0.2 ng mg−1 of protein) in the mitochondria than the two barley cultivars did. There was no difference in Al accumulation in the chloroplast among these three plant cultivars.

Growth-promoting effect of root exudates of Fe-deficient alfalfa on Rhizobium meliloti

Root exudates were collected from the Fe-deficient hydroponic culture of alfalfa by using an amberlite XAD-4 resin column. The in vitro growth of Rhizobium meliloti (RM) in yeast extract mannitol liquid medium supplemented with root exudates at different concentrations (0 to 200 mg L-1) was monitored by counting the number of viable cells by the dilution plate technique. Root exudates at 1, 10 and 100 mg L-1 promoted the in vitro growth of RM, but that at 200 mg L-1 inhibited growth. Some compounds purified from the root exudates promoted growth at 0.1mg/L in the liquid medium. One of the purified compounds was identified as 4’,7-dihydroxyflavanone by mass spectrometry and 1H-nuclear magnetic resonance. Thus, alfalfa may control the RM growth under Fe-deficient condition.

Effect of Iron Deficiency on Translatable mRNA Populations in Root of Medicago Sativa (L.)

Messenger RNA was isolated from alfalfa (Medicago sativa L.) root tissue at various times after the start of iron deficiency treatment and translated in vitro in a massenger-dependent wheat germ extract. [35S]-methionine-labelled translation products were then analyzed by 1- and 2-D gel electrophoresis. The changes in mRNA population were detectable in the plant subjected to iron deficiency prior to the occurrence of iron-deficient symptoms. A new translation product with pI=5.4 and Mrs=29kDa was found in day 10 and day 12 samples taken from iron-deficient root. On the other hand, a translation product with pI=5.3 and Mrs=42kDa were suppressed in all iron-deficient samples. Translation products with pI=5.6–6.3 and Mrs=43kDa gradually decreased in day 10 and day 12 samples taken from iron-deficient root. Whether these changes observed are specific to iron deficiency remains to be seen.

Improvement of soil fertility by fallow flooding for groundwater conservation on the middle reaches of the Shirakawa River in Kumamoto, Japan

Fallow flooding at converted paddy fields on the middle stream of the Shirakawa river basin, Kumamoto Japan is encouraged by the local government since the practice is effective for artificial groundwater recharge. In this study, we investigated the effects of fallow flooding on the water quality of percolating water, soil fertility, and succeeding carrot (Daucus carota L.) production. The flooding was conducted at a converted paddy field in 2007. Nitrate concentration of percolating water surged immediately after the start of flooding, due to the leaching of residual nitrate in the soil layer; then dropped to less than the concentration of irrigation water, supposedly due to denitrification thereafter. Denitrification rate was calculated as 0.22 g-NO3 −-N m−2d−1; such a rate may alleviate the nitrate contamination caused at the very beginning of flooding. Regardless of severe leaching conditions, soil nutrient contents were maintained or rather increased in the drained soil. Carrot yield 158 days after sowing in the test field was 67 Mg ha−1, far exceeding the yield of 41 Mg ha−1 in the untreated adjacent field. Flood fallowing, known as an efficient practice for suppressing pathogenic nematodes and/or continuous cropping injury, appears to be a favorable practice for improvement of soil fertility.

Characteristics of root Fe 3+ -reduction and root exudate secretion under Fe-deficient conditions in alfalfa

The induction of Fe3+-reduction in intact root tissue and ferric phosphate dissolution by root exudates were examined periodically during culture of alfalfa in Fe-free medium. The Fe3+-reduction rate was highest (0.72 μmol g-1 hr-1) on day 10 of culture, and rapidly decreased thereafter. Fe3+-reductase activity might be negatively modulated by high Cu or Mn accumulation in the alfalfa plant. In contrast, ferric phosphate dissolution by root exudates increased abruptly on day 10 and was maintained at 13–33 nmol g-1 d-1 thereafter. The root exudate accounted for only 0.2% of Fe3+-reduction. In conclusion, ferric phosphate dissolution is controlled by different biochemical processes, but Fe3+-reductase activity is influenced by high intracellular Cu or Mn accumulation.