ABSTRACT
The rhodophyte seaweed Porphyra yezoensis, known more commonly world-wide as "nori", is an important commercial crop in Japan. Cultivation of nori in Japan is often affected by outbreaks of "iroochi", a discoloration of the thalli due to a decrease in inorganic nutrients in the culture area that in turn decreases the amount of photosynthetic pigments in the thalli. Treating thalli with inorganic nitrogen can reverse iroochi. In this paper, we report on the characterization of three P. yezoensis genes, a nitrate transporter (PyNRT2) and two urea transporters (PyUT1 and PyUT2), which may be involved in reversing iroochi. The predicted length of the PyNRT2 protein was 479 amino acids (AA), while PyUT1 and PyUT2 were 740 and 680 AA, respectively. PyNRT2 was more similar to NRT2 from a chromophyte than to NRTs from Chlamydomonas and higher plants. The two P. yezoensis UTs had 56% AA identity to each other, and showed the closest relationship to higher plant and yeast DUR3 proteins which formed a subfamily of the sodium-solute symporter protein family. Hydrophobicity plots of the AA sequences showed that the PyNRT2, PyUT1, and PyUT2 included 12, 15, and 16 transmembrane domains, respectively. Southern blot analysis indicated that the P. yezoensis genome has a single NRT2-encoding gene and at least four UT-encoding genes. Expression analysis of PyNRT2 and PyUT genes showed that the messenger RNA level of the PyNRT2 gene reached a maximum after 48 h in the nitrate starvation condition and was then restored to the constitutive level, while expression of the PyUT genes was induced in proportion to treatment times in the nitrate starvation condition. These results suggest that the PyNRT2 and PyUT are responsible for the high-affinity nitrate/urea transport systems that operate under low external nitrate concentrations.
Subject(s)
Nitrogen/metabolism , Rhodophyta/physiology , Seawater , Algal Proteins/chemistry , Algal Proteins/genetics , Algal Proteins/metabolism , Amino Acid Sequence , Cloning, Molecular , DNA, Complementary/isolation & purification , Gene Dosage , Gene Expression Profiling , Gene Expression Regulation/drug effects , Molecular Sequence Data , Nitrates/pharmacology , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rhodophyta/drug effects , Rhodophyta/genetics , Rhodophyta/growth & development , Sequence Analysis, DNAABSTRACT
Genes transformed into plants are usually inherited in a regular Mendelian manner. There are, however, transformants in which the selectable trait fails to segregate as expected. Genetic analysis of the kanamycin-resistance (KanR) trait in >900 independent transformants of Arabidopsis revealed that 9% produced progeny families with an enormous deficiency of KanR individuals. Self-pollination of individual KanR plants from these families revealed lines that continued to segregate for a deficiency of KanR seedlings. In subsequent generations, the segregation ratio in these families stabilized at approximately 1 KanR:3 KanS. Molecular analyses showed that the deficiency of KanR individuals reflected the complete absence of the introduced DNA. Reciprocal backcrosses to untransformed plants showed unequal transmission of the KanR trait through the gametes in these exceptional lines. In five cases, this was primarily a failure of transmission through the microgametophyte (pollen) and in the other two cases, primarily a failure of transmission through the megagametophyte (embryo sac or egg). The number of seeds per silique was reduced by 50% in the latter two lines. We conclude that our exceptional transformants contain T-DNA insertions that delete or disrupt genes essential for gametophytic growth and development.
