Takao Komatsuda¹, Guoxiong Chen², Jian Feng Ma³, Christiane Nawrath⁴, Mohammad Pourkheirandish¹, Akemi Tagiri¹, Yin-Gang Hu⁵, Mohammad Sameri¹,Xinrong Li², Xin Zhao², Yubing Liu², Chao Li², Xiaoying Ma², Aidong Wang², Sudha Nair¹, Ning Wang¹, Akio Miyao¹, Shun Sakuma¹, Naoki Yamaji³, Xiuting Zheng⁵, Eviatar Nevo⁶

¹Plant Genome Research Unit, NIAS, ²Cold and Arid Regions Environmental and Engineering Institute(China), ³Okayama University, ⁴University of Lausanne, ⁵Northwest Agriculture and Forestry University (China), ⁶University of Haifa

［Abstract］

A spontaneous mutation, eibi1.b , in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle.Map-based cloning revealed that Eibi1  encodes an HvABCG31  full size transporter.Two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves.

［Keywords］

ABC transporter, cuticular layer, drought resistance, eibi1

［Background］

Drought stress is one of most severe agricultural problems affecting plant growth and crop yield. The aerial surface of land plants is protected by the cuticle, a multilayered structure composed of polyester,cutin, and wax. The naturally occurring drought-hypersensitive wild barley (Hordeum spontaneum Koch )mutant eibi1.b  suffers from a particularly severe level of water loss and displays a defective cuticle. The high transpiration rate of eibi1  is not related to an abnormal stomatal density. The defective eibi1  cuticle not only enhances the plant’s sensitivity to drought stress, but also is associated with the rapid loss of water from detached leaves. eibi1  was isolated and its biological function was analyzed in this study.

［Results and Discussion］

1. eibi1.b  was isolated by a map-based approach (Fig. 1A). Eibi1 encoded an HvABCG31 full transporter, predicted to participate in the transport of secondary metabolites. eibi1.b  revealed a point mutation that resulted in a loss of function of HvABCG31. A de novo wild barley mutant named “eibi1.c” mimicked the behavior of the eibi1.b  mutant in its droughthypersensitivity and dwarfism (Fig. 1B,C).
2. A reverse genetic approach was applied to rice by taking advantage of a library of established transposon insertions. Two independent transposon Oryza sativa 17  (Tos17 ) events within OsABCG31  (osabcg31.b  and osabcg31.c ) were identified. The two mutants were both highly sensitive to drought stress.
3. Transmission electron microscopy showed that the thickness of the mutant’s cuticle was only 〜 25% of that of the wild type in all three zones of leaves. In addition, the quantity of the major cutin monomers of wild barley was reduced to 〜 50% in the eibi1.b  mutant, suggesting that the ABC transporter gene is specifically involved in the formation of cutin and retention of water.
4. The monocot rice and the dicot Arabidopsis ABCG31 shared 92% and 71% sequence identities with EIBI1, respectively, indicating the conservation of EIBI1 in monocot and dicot plants. The green alga Volvox carteri  also contains an EIBI1 homolog, but the sequence identity was 35%.One may infer that V. carteri  has a cutin-like layer, although experimental evidence is not available. It may be concluded that EIBI1 is highly conserved in the evolution of land plants.

1. This work underlines the importance of an intact cuticle and its structural polyester in protecting leaves from uncontrolled water loss and the crucial evolution of HvABCG31 homologs in colonization of land by plants, a major milestone in terrestrial evolution of life.

Fig.1. Barley drought-hypersensitive eibi1 mutant and its functional assignment (A) Exon/intron structure of barley Eibi1. The single-nucleotide difference between the wild-type and the eibi1.b  mutant sequence is indicated. The red dashed line indicates the 9-bp deletion in eibi1.c exon 10. (B) The irradiation-induced eibi1.c mutant and its wild type at the flowering stage. (C) Water-loss test of detached leaves from eibi1.c  and OUH602.

[Reference]

1. Chen G, Komatsuda T, Ma J.F, Nawrath C, Pourkheirandish M, Tagiri A, Hu Y-G, Sameri M, Li X, Zhao X, Liu Y, Li C, Ma X, Wang A, Nair S, Wang N, Miyao A, Sakuma S, Yamaji N, Zheng X, Nevo E (2011) An ATPbinding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice Proceedings of the National Academy of Sciences of the United States of America  108(30):12354-12359