Mariko Shono

Mitochondrial small heat-shock protein enhances thermotolerance in tobacco plants

To clarify the role of mitochondrial small heat‐shock protein (MT‐sHSP) in the heat‐shock response, we introduced the tomato (Lycopersicon esculentum) MT‐sHSP gene under the control of the 35S promoter into tobacco (Nicotiana tabacum), and examined the thermotolerance of the transformed plants. Irrespective of the orientation, sense or antisense, of the gene, the transgenic plants exhibited a normal morphology and growth rate in the vegetative growth stage. When 4‐week‐old seedlings were exposed to sudden heat stress, the sense plants which overexpress the MT‐sHSP gene exhibited thermotolerance, whereas the antisense plants in which the expression of the gene is suppressed exhibited susceptibility.

Molecular and physiological evaluation of transgenic tobacco plants expressing a maize phosphoenolpyruvate carboxylase gene under the control of the cauliflower mosaic virus 35S promoter

The expression of maize (Zea mays) phosphoenolpyruvate carboxylase (PEPC) gene constructs in transgenic tobacco plants (Nicotiana tabacum) was studied. Where transcription was under the control of a CaMV 35S promoter, maize PEPC transcripts of the correct size were detected. Western blot analysis indicated that the transgenic plants contained about twice as much PEPC as non-transformed plants. Furthermore, the enzymatic activity of PEPC in the leaves of these transgenic plants was up to twice as high as that in non-transformed plants. Two forms of PEPC with different kinetic properties were identified in leaf extracts of the transgenic plants: one form (the maize isoform) gave a high apparentKm value for phosphoenolpyruvate (PEP) and a high maximum activity, and the other (the tobacco isoform) exhibited a low apparentKm value for PEP and a low maximum activity. These biochemical differences resulted in several significant physiological changes in the transgenic plants: (1) the growth rate of the transgenic plants was lower than that of non-transgenic plants: (2) chlorophyll content per leaf area was relatively lower in the transgenic plants; and (3) the quantum yield of photosynthesis in the transgenic plants was not affected by changes in leaf temperature.

Molecular cloning of Na(+)-ATPase cDNA from a marine alga, Heterosigma akashiwo.

We cloned novel Na(+)-ATPase (HANA) cDNA from marine alga Heterosigma akashiwo. The full-length HANA cDNA was 4467 bp long and coded for a 1330 amino acid protein with a molecular weight of 146,306. The deduced product exhibited around 40% identity in amino acids with Na(+)/K(+)-ATPase alpha-subunits. A hydrophilic sequence of 285 amino acid residues that showed no homology with any sequence listed in databases existed in the M7--M8 junction of HANA. This is the first report on the primary structure of putative Na(+)-transporting ATPase from plant cells.

Adaptation to High Temperature and Water Deficit in the Common Bean (Phaseolusvulgaris L.) during the Reproductive Period

This paper reviews the adaption to heat and drought stresses in Phaseolus vulgaris, a grain and vegetable crop widely grown in both the Old and New World. Substantial genotypic differences are found in morphophysiological characteristics such as phenology, partitioning, plant-water relations, photosynthetic parameters, and shoot growth, which are related to reproductive responses. The associations between (a) days to podding and leaf water content and (b) the number of pods per plant and seed yield are consistent across different environments and experiments. Leaf water content is maintained by reductions in leaf water potential and shoot extension in response to heat and drought stress. Heat-tolerant cultivars have higher biomass allocation to pods and higher pod set in branches. These traits can be used as a marker to screen germplasm for heat and drought tolerance. In this paper, we briefly review the results of our studies carried out on heat and drought tolerance in the common bean at the Tropical Agriculture Research Front, Ishigaki, Japan.

Partial Purification of a Na+ -ATPase from the Plasma Membrane of the Marine Alga Heterosigma akashiwo

A Na+ -ATPase was partially purified from plasma membranes of the marine alga Heterosigma akashiwo. The plasma membranes of H. akashiwo cells were collected by differential centrifugation with subsequent discontinuous gradient centrifugation. Na+ -ATPase activity was associated with the resultant plasma membrane fraction and was stimulated to the greatest extent in the presence of 100 to 200 mM Na+, 10 mM K+, and 5 mM Mg2+ ions, pH 8.0. The Km value for Na+ ions was 12.2 mM. An apparent Km value for ATP was 880 [mu]M. A 140-kD phosphorylated intermediate was also detected in the same fraction in the presence of both Mg2+ and Na+ ions, and this protein was dephosphorylated upon the addition of K+ ions. We could partially purify the 140-kD protein after solubilization by Suc monolaurate and fractionation by sequential column chromatography on Sephacryl S-300, DEAE-Sepharose CL-6B, and Mono-Q columns. The purified 140-kD polypeptide could also be phosphorylated and be detected after acid sodium dodecyl sulfate-polyacryl-amide gel electrophoresis in the presence of Na+ and Mg2+ ions.

Midday Drop of Leaf Water Content Related to Drought Tolerance in Snap Bean (Phaseolus vulgaris L.)

Beans are principally grown in areas where plants are regularly exposed to drought stress under high ambient temperature conditions, and the breeding of drought-tolerant plants is drawing concern. However, progress in the development of tolerant lines is slow due to the lack of simple traits associated with drought tolerance. There is a general consensus that water economy is very critical to plant growth and development. It may therefore be possible to improve the drought tolerance of snap bean by identifying the water relations traits associated with higher productivity. In the subtropical islands of Japan, drought stress in the summer adversely affects bean production both for grain and vegetable use. The stress mainly causes low pod setting ratio, early pod abscission and consequently low productivity (Shen and Webster, 1986; Nakano et al., 1998; Suzuki et al., 2001; Tsukaguchi et al., 1999, 2003). Therefore, it is important to identify the characteristic traits associated with pod setting, the number of pods reaching maturity and fi nally seed yield. In this report, we clarify the association of the tissue water content of leaves with pod setting per plant and seed yield, and propose the use of midday drop of leaf water content as a marker to screen snap bean germplasm with drought tolerance. Snap bean has substantial genetic variability in the high water content of leaves at the midday with decreasing water potential (Omae et al., 2004). Slow development of water defi cit may induce osmotic adjustment resulting in the maintenance of appropriate water content of leaf during the period of water defi ciency. As a result, plants can survive longer under a water defi cit. Omae et al. (2004) reported that cultivars showing a smaller midday drop in leaf water content set more pods than the cultivars showing a large midday drop in leaf water content. In this study, we further explored the association of the midday drop of leaf water content with pod production (the number of pods per plant at harvest), seed size (seed weight) and seed yield, because yield of snap bean for vegetable use is a function of the number of pods per plant, and seed yield is a function of both the number of pods per plant and seed size.

Assessing Drought Tolerance of Snap Bean (Phaseolus vulgaris) from Genotypic Differences in Leaf Water Relations, Shoot Growth and Photosynthetic Parameters

Abstract The Leaf Water Relations, Photosynthetic Parameters and Shoot Growth of Five Snap Bean Cultivars Were Assessed During The Drought Period To Determine Their Role In Alleviating Plant Water Deficit Imposed By Withholding Irrigation At Flowering. Soil Water Content of Irrigated Plants Was 18-20% While That of Unirrigated Plants Was 6-10% At 60 Days After Seeding (Das). Leaf Water Potential Was Approximately 0.15Mpa Lower and Relative Water Content Was Approximately 5% Lower In Unirrigated Plants Than In Irrigated Plants At 57 Das. Unirrigated Plants Had A Lower Stomatal Conductance (Gs) and Intercellular Co2 Concentration (Ci). Reduced Leaf Water Potential and Relative Water Content Were Associated With A Decreased Stem Elongation Rate. Plants With A Lower Stem Elongation Rate Had A Higher Specific Leaf Weight and Succulence Index (Suci). Significant Differences Among Five Cultivars of Snap Bean Were Found For All Parameters Measured. Decreased Leaf Water Potential and Stem Elongation Rate Resulting From Drought Participated In Preserving Relative Water Content and Improving Specific Leaf Weight and Suci. Maintenance of Higher Relative Water Content Increased Gs and Ci. Cultivars That Maintained A High Relative Water Content When Leaf Water Potential and Stem Elongation Rate Were Decreased Markedly, Were More Tolerant To Drought Than Those Which A Reduced Relative Water Content and The Leaf Water Potential and Stem Elongation Rate Were Only Slightly Lowered. Reduced Yield (Pods Per Plant and Seed Biomass) Resulting From Drought Was Associated With Reduced Relative Water Content.

Influence of High Temperature on Morphological Characters, Biomass Allocation, and Yield Components in Snap Bean (Phaseolus vulgaris L.)

Abstract High temperature in summer is a major limiting factor for the growth of snap bean in the subtropical islands of Okinawa, Japan. The effect of temperature on biomass production, yield components, and morphological characters were studied in five snap-bean cultivars and strains in the phytotron. Plants were initially grown at 24/20ºC (day/night temperature, 12/12hr) and transferred to 24/20ºC, 27/23ºC (control) or 30/26ºC at the onset of flowering (34 days after sowing). High temperature (30/26ºC) increased single pod weight and the number of flowers and branches, but reduced the number of pods/plant, pod set ratio, and plant weight. ‘Haibushi’, a heat-tolerant cultivar, had higher pod weight/plant, number of pods/plant, pod weight/pod, pod set ratio, number of branches, and rate of biomass allocation to pods, but lower rates of biomass allocation to leaves, stems, and roots than ‘Kentucky Wonder’, a heat-sensitive cultivar, in all temperature regimes. The number of flowers, biomass, and accumulative temperature affected both the yield components (number of pods/plant, single pod weight) antithetically. The yield components were estimated by a stepwise multiple regression analysis. The number of pods/plant was estimated from the number of flowers, leaf weight, pod set ratio per branch, and plant weight with a reasonable precision (R2=0.78). Single pod weight was determined (R2=0.69) from pod set ratio, number of branches, root weight, and accumulative temperature. The results indicated that higher biomass allocation to pods and higher pod set in branches, which were observed in heat-tolerant cultivars at all temperature regimes, were most effective for the estimation of heat tolerance in snap bean.

Influence Of Temperature Shift After Flowering on Dry Matter Partitioning In Two Cultivars of Snap Bean (Phaseolus Vulgaris)That Differ In Heat Tolerance

Abstract The Partitioning of Dry Matter (Ratio of Dry Weight of Individual Parts To That of Total Dry Matter) Was Analyzed In Snap Bean Cultivars, Haibushi, A Heat-Tolerant Cultivar, and Kentucky Wonder, A Heat-Sensitive Cultivar, At Four Temperatures After Flowering on The Subtropical Island of Ishigaki, Japan. The Temperature Regimes Included 27/23ºC (Day/Night) As Normal, 24/20ºC As Low, 30/26 As High, and 33/29ºC As Extremely High. Most Growth Traits Increased After Flowering Time (35 Das), Displaying A Plateau At 68-75 Das. The Total Dry Matter Was Similar Under All Temperature Conditions, But Differed With The Cultivar. Haibushi Had A Higher Value of Total Dry Matter Than Kentucky Wonder, Which Was Mainly Due To Higher Pod Dry Weight Although Stem and Root Dry Weights Were Lower In Haibushi. A Sharp Decline of Dry Matter Partitioning To Pods Was Observed At 33/29 ºC. In The Temperature Range of 24/20 To 30/26ºC, Haibushi Showed Higher Partitioning To Pods Than Kentucky Wonder, Independent of Temperature. on The Other Contrary, Kentucky Wonder Showed Higher Partitioning To Pods At 27/23ºC Than At 24/20ºC. These Results Showed That The Partitioning of Dry Matter, Which Varied With The Cultivar and Temperature, Played An Important Role In Achieving Higher Harvest Index In The Heat-Tolerant Than In The Heat-Sensitive Cultivars.

MOLECULAR CLONING OF P-TYPE ATPASES ON INTRACELLULAR MEMBRANES OF THE MARINE ALGA HETEROSIGMA AKASHIWO

Two cDNA clones (HAA13 and HAA1) which include conserved regions of genes of P-type ATPases were isolated from the marine alga Heterosigma akashiwo by a method that included the polymerase chain reaction. The longer cDNA (3286 bp), HAA13, consisted of an open reading frame that encoded a 106 kDa polypeptide of 977 amino acids with several possible transmembrane domains and conserved regions of eukaryotic P-type ATPases. One transmembrane domain had a leucine zipper structure. HAA1 was not a full-length gene (2054 bp) and lacked the 5′ region, but it also included the conserved regions and putative transmembrane domains. Antibodies against the polypeptides encoded by HAA13 and HAA1 that have been expressed in Escherichia coli reacted with 100 kDa and 95 kDa polypeptides, respectively, on intracellular membranes of H. akashiwo cells. Immunostaining of H. akashiwo cells revealed that the HAA13 antigen was distributed on membranes around chloroplasts and the HAA1 antigen was located on small vesicles.