BREVIA

seven independent transgenic A. thaliana lines

producing the BOR4-GFP fusion under the con-

Plants Tolerant of High Boron Levels

Kyoko Miwa,1 Junpei Takano,1 Hiroyuki Omori,1 Motoaki Seki,2
Kazuo Shinozaki,2 Toru Fujiwara1,3*
oron (B) is an essential nutrient for plants
trol of cauliflower mosaic virus 35S RNA promoter.
Immunoblot analysis of a generated trans-
genic line showed that BOR4 accumulated in the
presence of a high B supply (Fig. 1A), suggesting
that BOR4 is exempt from the posttranslational
BOR1 degradation system.
We focused on AtBOR4 (The Arabidopsis The supply of 10 mM boric acid was sub-

B and animals, but in high concentrations it Information Resource code At1g15460 and
is toxic. Living organisms, including plants, GenBank code NM_101415), one of the six
must control the B distribution to maintain ade- BOR1 paralogs present in the A. thaliana
stantially lethal to wild-type plants, but much more
vigorous root and shoot growth with varying
degrees was observed in all the homozygous
quate levels of B in their cells. Reduced crop qual- genome. B transport activities of BOR4 and Pro35S-BOR4-GFP transgenic lines grown on
ity and yields in soils containing toxic levels of B BOR4–green fluorescent protein (GFP) fusion solid medium containing 10 mM boric acid
are a worldwide problem in food production, es- were confirmed in yeast (fig. S1). We generated (Fig. 1B and fig. S2). Accumulation of BOR4-
pecially in arid areas (1). GFP and tolerance of B were positively corre-
By manipulating B trans- lated (fig. S2). The B concentrations in the roots

Downloaded from www.sciencemag.org on November 16, 2010

port, we have generated and shoots of these transgenic plant lines were
plants that are tolerant of lower than that in the wild type in the presence of
high levels of B. 3 mM boric acid (Fig. 1C). Overall tracer B
B accumulation occurs uptake was also reduced in the transgenic line 4
both naturally and through (fig. S3). These results suggest that the overpro-
artificial means such as duction of BOR4-GFP improved growth under
irrigation. About five mil- conditions of B toxicity through B efflux.
lion ha of soils containing Furthermore, GFP fluorescence derived from
greater than 15 mg kg−1 B, BOR4-GFP was strongly detected in the plasma
above the threshold for membranes of the distal sides of epidermal cells
normal plant growth, exist in the elongation zone of roots of the transgenic
in southern Australia, lines carrying ProBOR4-BOR4-GFP (Fig. 1D).
corresponding to 30% of The distal localization of BOR4 is likely impor-
the region (www.dwlbc. tant for the directional export of B from the roots
sa.gov.au/land/topics/ to the soil to prevent the accumulation of B in the
rootzone/boron.html). Up xylem and growing cells. This enhanced B efflux
to 17% of the barley yield from the roots of crop plants is expected to result
loss in this area was esti- in improved crop productivity in the B-toxic soils
mated to be caused by B found in a number of regions of the world.
toxicity (2). B-tolerant
cultivars with reduced B References and Notes
uptake are known for 1. R. O. Nable, G. S. Bañuelos, J. G. Paull, Plant Soil 193,
181 (1997).
barley (3), but breeding 2. B. Cartwright, B. A. Zarcinas, A. H. Mayfield, Aust. J. Soil
programs have not yet Res. 22, 261 (1984)
produced a practical solu- 3. J. E. Hayes, R. J. Reid, Plant Physiol. 136, 3376 (2004).
tion for B tolerance. 4. J. Takano et al., Nature 420, 337 (2002).
5. J. Takano, K. Miwa, L. Yuan, N. von Wirén, T. Fujiwara,
Arabidopsis thaliana Proc. Natl. Acad. Sci. U.S.A. 102, 12276 (2005).
BOR1, an efflux-type bo- 6. K. Miwa, J. Takano, T. Fujiwara, Plant J. 46, 1084 (2006).
rate transporter, was the 7. This work was supported in part by grants from the
first B transporter identi- Japanese Society for the Promotion of Science to K.M. and
the Greentechno Project from the Ministry of Agriculture,
fied in a biological system
(4). BOR1 is required for Fig. 1. Improved growth in transgenic A. thaliana plants overproducing
a Grant-in-Aid for Scientific Research on Priority Areas
from the Ministry of Education, Culture, Sports, Science,
the transport of B from BOR4 under conditions of boron toxicity. (A) Immunoblot analysis of and Technology of Japan, and a 21st Century Center of
roots to shoots under con- transgenic plant line 4 carrying Pro35S-BOR4-GFP. Plants were exposed to Excellence project to T.F.
ditions of low B supply. 0.1 mM (–B), 30 mM (+B), or 3 mM (++B) boric acid for 3 days, and root Supporting Online Material
microsomal proteins were subjected to immunoblotting with an antibody www.sciencemag.org/cgi/content/full/318/5855/1417/DC1
BOR1 is capable of con-
against GFP. (B) Transgenic plants grown for 17 days on solid medium Materials and Methods
ferring high B tolerance
containing 10 mM boric acid. Scale bar indicates 10 mm. (Inset) An Figs. S1 to S3
to yeast by pumping bo- enlargement of the wild-type plant (scale bar, 1 mm). WT, wild-type Col-0;
18 June 2007; accepted 9 October 2007
ric acid out of the cell. L4, L5, L12, transgenic lines 4, 5, and 12, respectively, carrying the Pro35S- 10.1126/science.1146634
However, in plants under BOR4-GFP construct. (C) Total B concentrations in shoots and roots. Plants
high B conditions, BOR1 were grown on solid medium containing 3 mM boric acid for 18 days. Means
1
is degraded via endo- ± SD are shown (n = 5 to 6). Asterisks indicate significant difference (P < Biotechnology Research Center, University of Tokyo, Tokyo
cytosis (5), and overex- 0.001) by Student’s t test. DW, dry weight. (D) GFP fluorescence in roots of 113-8657, Japan. 2RIKEN Plant Science Center, Yokohama
230-0045, Japan. 3Solution-Oriented Research for Science
pression of BOR1 does transgenic plants carrying the ProBOR4-BOR4-GFP construct. Plants were and Technology, Japan Science and Technology Agency,
not improve plant growth grown on solid medium containing 30 mM boric acid for 10 days. Scale bars, Tokyo 103-0027, Japan.
in the presence of toxic 25 mm (left) and 100 mm (right). The left-hand image is a magnified view of *To whom correspondence should be addressed. E-mail:
levels of B (6). the area inside the box in the right-hand image. atorufu@mail.ecc.u-tokyo.ac.jp

www.sciencemag.org SCIENCE VOL 318 30 NOVEMBER 2007 1417