Akio Watanabe

OsHMA3, a P1B‐type of ATPase affects root‐to‐shoot cadmium translocation in rice by mediating efflux into vacuoles

• The cadmium (Cd) over-accumulating rice (Oryza sativa) cv Cho-Ko-Koku was previously shown to have an enhanced rate of root-to-shoot Cd translocation. This trait is controlled by a single recessive allele located at qCdT7. • In this study, using positional cloning and transgenic strategies, heavy metal ATPase 3 (OsHMA3) was identified as the gene that controls root-to-shoot Cd translocation rates. The subcellular localization and Cd-transporting activity of the gene products were also investigated. • The allele of OsHMA3 that confers high root-to-shoot Cd translocation rates (OsHMA3mc) encodes a defective P(1B) -ATPase transporter. OsHMA3 fused to green fluorescent protein was localized to vacuolar membranes in plants and yeast. An OsHMA3 transgene complemented Cd sensitivity in a yeast mutant that lacks the ability to transport Cd into vacuoles. By contrast, OsHMA3mc did not complement the Cd sensitivity of this yeast mutant, indicating that the OsHMA3mc transport function was lost. • We propose that the root cell cytoplasm of Cd-overaccumulating rice plants has more Cd available for loading into the xylem as a result of the lack of OsHMA3-mediated transportation of Cd to the vacuoles. This defect results in Cd translocation to the shoots in higher concentrations. These data demonstrate the importance of vacuolar sequestration for Cd accumulation in rice.

Mutations in Rice (Oryza sativa) Heavy Metal ATPase 2 (OsHMA2) Restrict the Translocation of Zinc and Cadmium

Widespread soil contamination with heavy metals has fostered the need for plant breeders to develop new crops that do not accumulate heavy metals. Metal-transporting transmembrane proteins that transport heavy metals across the plant plasma membrane are key targets for developing these new crops. Oryza sativa heavy metal ATPase 3 (OsHMA3) is known to be a useful gene for limiting cadmium (Cd) accumulation in rice. OsHMA2 is a close homolog of OsHMA3, but the function of OsHMA2 is unknown. To gain insight into the function of OsHMA2, we analyzed three Tos17 insertion mutants. The translocation ratios of zinc (Zn) and Cd were clearly lower in all mutants than in the wild type, suggesting that OsHMA2 is a major transporter of Zn and Cd from roots to shoots. By comparing each allele in the OsHMA2 protein structure and measuring the Cd translocation ratio, we identified the C-terminal region as essential for Cd translocation into shoots. Two alleles were identified as good material for breeding rice that does not contain Cd in the grain but does contain some Zn, and that grows normally.

A single recessive gene controls cadmium translocation in the cadmium hyperaccumulating rice cultivar Cho-Ko-Koku

The heavy metal cadmium (Cd) is highly toxic to humans and can enter food chains from contaminated crop fields. Understanding the molecular mechanisms of Cd accumulation in crop species will aid production of safe Cd-free food. Here, we identified a single recessive gene that allowed higher Cd translocation in rice, and also determined the chromosomal location of the gene. The Cd hyperaccumulator rice variety Cho-Ko-Koku showed 3.5-fold greater Cd translocation than the no-accumulating variety Akita 63 under hydroponics. Analysis of an F2 population derived from these cultivars gave a 1:3 segregation ratio for high:low Cd translocation. This indicates that a single recessive gene controls the high Cd translocation phenotype. A QTL analysis identified a single QTL, qCdT7, located on chromosome 7. On a Cd-contaminated field, Cd accumulation in the F2 population showed continuous variation with considerable transgression. Three QTLs for Cd accumulation were identified and the peak of the most effective QTL mapped to the same region as qCdT7. Our data indicate that Cd translocation mediated by the gene on qCdT7 plays an important role in Cd accumulation on contaminated soil.

Isolation of novel types of Arabidopsis mutants with altered reactions to cadmium: cadmium-gradient agar plates are an effective screen for the heavy metal-related mutants

We are interested in elucidating the molecular mechanisms underlying plant reactions to the toxic heavy metal cadmium (Cd). To this end, we devised a new screening strategy using agar plates with a gradient of Cd concentrations, termed Cd-gradient agar plates (CGAPs), to isolate Arabidopsis mutants that displayed altered reactions to the metal. Arabidopsis M2 seeds, derived from ethyl methanesulfonate (EMS) treated seeds, were germinated on the CGAPs such that the primary root of each seedling elongated against increasing concentrations of Cd on the surface of the plate. Under these conditions, the lengths of the primary roots reliably demonstrated the degree of Cd tolerance of individual seedlings. The use of CGAPs also allowed close observation of the root reaction of each seedling to Cd without causing lethal damage. The screen identified three mutant lines, MRC-32, MRC-22 and MRC-26, which showed distinctly different characteristics. MRC-32 plants exhibited enhanced tolerance to Cd and contained Cd at higher concentrations than wild-type (WT) plants treated with the heavy metal. The whole root system of MRC-22 plants showed a Cd-phobic response. MRC-26 plants accumulated less Cd in their aboveground tissues than WT plants, suggesting that they were defective in transporting the heavy metal from roots to aboveground tissues. We also determined the likely chromosomal location of each mutation.

Analysis of phloem exudate collected from fruit-bearing stems of coconut palm: Palm trees as a source of molecules circulating in sieve tubes

Abstract Sieve tubes have been attracting widespread research interest because of their possible role in mediating physiological signals within the whole plant. However, progress in research into the function of sieve tubes has been limited by the low volume of sap available. To overcome this problem, we attempted to collect phloem exudate from tropical coconut palm trees (Cocos nucifera L. cv. Namhom). As much as 3 to 15 mL of exudate per hour was collected from the cut surface of the plants fruit-bearing stem. Our analyses revealed that the characterized profiles of sugars (sucrose: 339 mM), amino acids (total concentration: 17.1 mM), cations (potassium: 48.3 mM), and proteins (total concentration: 0.1 /-lg /-lL-1) in the exudate were mostly consistent with those of phloem sap or phloem exudate collected from rice plants, castor bean plants, etc. This exudate was assumed to reflect the composition of the phloem sap from the source organs of coconut palm trees. The large volume of exudate collected contributed significantly to the analyses of the various compounds in the stream of sieve tubes.

Identification of QTLs controlling low-temperature germination of the East European rice (Oryza sativa L.) variety Maratteli

Germinability under low temperature is one of the most important traits in seedling establishment in direct-sowing culture of rice. The objective of this study was the identification of genes responsible for higher and faster germination under low temperature, with the aim of breeding new rice varieties for direct-sowing culture. We identified four quantitative trait loci (QTLs) responsible for germination properties under low temperature in an East European rice variety, Maratteli. Maratteli-derived alleles at qLTG3-1 and qLTG3-2 on chromosome 3 and qLTG11-1 on chromosome 11 increased germination rate under low temperature. In contrast, the Maratteli allele at qLTG1-1 on chromosome 1 reduced the rate. Near-isogenic lines (NILs) with the Akitakomachi genetic background harboring qLTG3-1 and qLTG11-1 alleles from Maratteli accelerated germination and a NIL with qLTG1-1 delayed germination under low temperature, resulting in enhanced or suppressed germination rates, respectively. These QTLs altered the germination profile of Akitakomachi. Hence, Maratteli is a better source of genes for the improvement of germination under low temperature. We conclude that the low temperature germinability of various Japanese varieties inferior in this character can be improved by marker-assisted selection without modification of other superior traits. These NILs may also contribute to uncovering the molecular mechanisms of cold tolerance at the germination stage.

Functional analysis of the C-terminal region of the vacuolar cadmium-transporting rice OsHMA3

Rice OsHMA3 is a vacuolar cadmium (Cd) transporter belonging to the P1B‐ATPase family and has a long (273aa) C‐terminal region. We analyzed the function of the region related to Cd using the transgenic Arabidopsis Col‐0 ecotype, which is sensitive to Cd. The OsHMA3 variant containing a truncated (58aa) C‐terminal region did not confer Cd tolerance, whereas an OsHMA3 variant containing a longer truncated (105aa) C‐terminal region conferred Cd tolerance to transgenic Arabidopsis. We conclude that the C‐terminal region, particularly the region containing the first 105aa, has an important role in OsHMA3 activity.

Nutrient concentrations of phloem sap of aromatic coconut grown under various nutrient conditions

To clarify the phloem transport of nutrients, the nutrient concentration of phloem sap of aromatic coconut palm was studied at Petchaburi Horticultural Research Center in Thailand. An experiment in randomised complete block design with three replications for aromatic coconut (Cocos nucifera) cultivation was carried out in Gray Podzolic soil (Ustoxic Dystropept) with the application of N-P-K: 0-0-0, 0-0.26-0.83, 0.4-0.26-0.83, 0.6-0.26-0.83, 0.8-0.26-0.83, 0.6-0-0.83, 0.6-0.18-0.83, 0.6-0.35-0.83, 0.6-0.26-0, 0.6-0.26-0.66, 0.6-0.26-1, 0.8-0.35-1 kg plant−1 year−1. In the second year of production, phloem sap was collected from the cut tips of fruit-bearing stems of coconut palms. The nutrient concentration of phloem sap was analysed by means of atomic absorption spectrometry.

Detection of antioxidant enzyme activities in the phloem sap of sugar palm.

Sieve tubes have been suggested to play an important role in the defensive system of higher plants. In this study, we investigated activities of several antioxidant enzymes possibly implicated in the system, using the phloem sap from sugar palm (Arenga saccharifera Lan.). The enzymes whose activities were detected in our native-PAGE (polyacrylamide gel electrophoresis) studies were superoxide dismutase (SOD; EC 1.15.1.1), ascorbate peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2). The results suggest that an ascorbate-glutathione cycle functions in the sieve tubes to alleviate oxidative damages.

Sugar palm, a novel model plant for phloem sap analysis

Because of their possible role in mediating physiological signals within a whole plant, sieve tubes have been attracting interest from a wide range of researchers. However, the very small volume of phloem sap available for analysis has kept research in this field away from full development. To avoid this problem, we collected phloem sap from sugar palm (Cocous nucifera Linn. cv. Namhom) in the present study. The sap collected from this tropical plant amounted to as much as 3 to 15 ml per hour. This large volume of the sap greatly helped us to analyse various compounds in it. The characterised profiles of proteins, sugars, amino acids, etc. in the sap will be presented.