Keisuke Matsui

The HALTED ROOT gene encoding the 26S proteasome subunit RPT2a is essential for the maintenance of Arabidopsis meristems

In higher plants, post-embryonic development is dependent on the activity of the root and shoot apical meristem (RAM and SAM). The quiescent center (QC) in the RAM and the organizing center (OC) in the SAM are known to be essential for the maintenance of meristematic activity. To understand the mechanism that maintains post-embryonic meristems, we isolated an Arabidopsis mutant, halted root (hlr). In this mutant, the cellular organization was disrupted in post-embryonic meristems both in the root and in the shoot, and their meristematic activity was reduced or became abnormal. We showed that the mutant RAM lost its QC identity after germination, which was specified during embryogenesis, whereas the identity of differentiated tissues was maintained. In the post-embryonic SAM, the expression pattern of a typical OC marker gene, WUSCHEL, was disturbed in the mutant. These observations indicate that the HLR gene is essential to maintain the cellular organization and normal nature of the RAM and SAM. The HLR gene encodes RPT2a, which is a subunit of the 26S proteasome that degrades key proteins in diverse cellular processes. We showed that the HLR gene was expressed both in the RAM and in the SAM, including in the QC and the OC, respectively, and that the activity of proteasomes were reduced in the mutant. We propose that proteasome-dependent programmed proteolysis is required to maintain the meristem integrity both in the shoot and in the root.

Production of Arachidonic and Eicosapentaenoic Acids in Plants Using Bryophyte Fatty Acid Δ6-Desaturase, Δ6-Elongase, and Δ5-Desaturase Genes

The liverwort Marchantia polymorpha L. synthesizes arachidonic (ARA) and eicosapentaenoic acids (EPA) from linoleic and α-linolenic acids respectively by a series of reactions catalyzed by Δ6-desaturase, Δ6-elongase, and Δ5-desaturase. Overexpression of the M. polymorpha genes encoding these enzymes in transgenic M. polymorpha plants resulted in 3- and 2-fold accumulation of ARA and EPA respectively, as compared to those in the wild type. When these three genes were introduced and co-expressed in tobacco plants, in which long-chain polyunsaturated fatty acids (LCPUFAs) are not native cellular components, ARA and EPA represented up to 15.5% and 4.9% respectively of the total fatty acid in the leaves. Similarly in soybean, C20-LCPUFAs represented up to 19.5% of the total fatty acids in the seeds. These results suggest that M. polymorpha can provide genes crucial to the production of C20-LCPUFAs in transgenic plants.

Arabidopsis RPT2a Encoding the 26S Proteasome Subunit is Required for Various Aspects of Root Meristem Maintenance, and Regulates Gametogenesis Redundantly with its Homolog, RPT2b

The 26S proteasome plays fundamental roles in the degradation of short-lived regulatory proteins, thereby controlling diverse cellular processes. In Arabidopsis, the essential RPT2 subunit is encoded by two highly homologous genes: RPT2a and RPT2b. Currently, only RPT2a has been reported to regulate various developmental processes, including the maintenance of the root apical meristem (RAM), although the roles of RPT2a in the RAM are still obscure. Here, we analyzed the cell type-specific requirement for RPT2a. When RPT2a was expressed locally in the rpt2a mutant, pleiotropic defects in the RAM, such as cell death and distorted cellular organization, were rescued differently, suggesting that RPT2a regulates various specific activities, which converge to maintain the RAM. On the other hand, the homologous RPT2b was also expressed in meristems, and the expression of RPT2b protein under the control of the RPT2a promoter complemented the rpt2a RAM defects, although the rpt2b mutant showed no obvious defect in all developmental aspects we examined. These results show that RPT2b might work in the RAM, but is dispensable for RAM maintenance in the presence of RPT2a. In contrast, the rpt2a rpt2b double mutant was lethal in male and female gametophytes, suggesting that RPT2a and RPT2b are redundantly required for gametogenesis. Furthermore, we showed that similar meristematic and gametophytic defects were caused by mutations in other subunit genes, RPT5a and RPT5b, suggesting that proper activity of the proteasome, not an RPT2-specific function, is required. Taken together, our results suggest that RPT2a and RPT2b contribute differently to the proteasome activity required for each developmental context.

Enhancement of Phosphate Absorption by Garden Plants by Genetic Engineering: A New Tool for Phytoremediation

Although phosphorus is an essential factor for proper plant growth in natural environments, an excess of phosphate in water sources causes serious pollution. In this paper we describe transgenic plants which hyperaccumulate inorganic phosphate (Pi) and which may be used to reduce environmental water pollution by phytoremediation. AtPHR1, a transcription factor for a key regulator of the Pi starvation response in Arabidopsis thaliana, was overexpressed in the ornamental garden plants Torenia, Petunia, and Verbena. The transgenic plants showed hyperaccumulation of Pi in leaves and accelerated Pi absorption rates from hydroponic solutions. Large-scale hydroponic experiments indicated that the enhanced ability to absorb Pi in transgenic torenia (AtPHR1) was comparable to water hyacinth a plant that though is used for phytoremediation causes overgrowth problems.

Identification and Characterization of Novel Nemophila menziesii Flavone Glucosyltransferases that Catalyze Biosynthesis of Flavone 7,4′-O-Diglucoside, a Key Component of Blue Metalloanthocyanins

The brilliant blue color of the Nemophila menziesii flower is derived from metalloanthocyanin, which consists of anthocyanin {petunidin 3-O-[6-O-(trans-p-coumaroyl)-β-glucoside]-5-O-[6-O-(malonyl)-β-glucoside]}, flavone [apigenin 7-O-β-glucoside-4-O-(6-O-malonyl)-O-β-glucoside] and metal ions (Mg2+, Fe3+). Although the two glucosyl moieties at the apigenin 7-O and 4-O positions are essential for metalloanthocyanin formation, the mechanism of glucosylation has not yet been clarified. In this study, we used crude protein extract prepared from N. menziesii petals to determine that apigenin is sequentially glucosylated by the catalysis of UDP-glucose:flavone 4-O-glucosyltrasferase (F4GT) and UDP-glucose:flavone 4-O-glucoside 7-O-glucosyltransferase (F4G7GT). We identified 150 contigs exhibiting homology with a UDP-glucose-dependent GT in the N. menziesii petal transcriptome and isolated 24 putative full-length GT cDNAs which were then subjected to functional analysis. Two GT cDNAs, NmF4GT and NmF4G7GT, which are highly expressed during the early stages of petal development and rarely in leaves, were shown to encode F4GT and F4G7GT activities, respectively. Biochemical characterization of the recombinant enzymes revealed that NmF4GT specifically catalyzed 4-glucosylation of flavonoids and that NmF4G7GT specifically catalyzed 7-glucosylation of flavone 4-O-glucosides and flavones. Apigenin 7,4-O-diglucoside was efficiently synthesized from apigenin in the presence of recombinant NmF4GT and NmF4G7GT. Transgenic tobacco BY-2 cells expressing NmF4GT and NmF4G7GT converted apigenin into apigenin 7,4-O-diglucoside, confirming their activities in vivo. Based on these results, we conclude that these two GTs act co-ordinately to catalyze apigenin 7,4-O-diglucoside biosynthesis in N. menziesii.