Os07g0556800

Oryza sativa Ribosome-inactivating protein gene 18 (OSRIP18) may be potentially useful in further improving plant tolerance to various abiotic stresses by over-expression.

Annotated Information

Function

• OSRIP18(Os07g0556800) might play a role in increasing tolerance to drought and high salinity during panicle development but not in seedling stage. After overexpression, active expression can be detected in all tested developmental stages and as a result, the transgenic plants exhibited higher tolerance to both stresses in these stages. It means the gene may play a role as a member of natural defense system against various environmental conditions including drought and high salinity stresses.

• OSRIP18 may play a role in pollen development by sensing different environmental cues, the gene may play important roles as a member of natural defense system against varying environmental conditions during pollen development.

• OSRIP18 and other rice RIP genes may be potentially useful for developing new plant varieties with higher tolerance to various stresses.

GO assignment(s): GO:0017148, GO:0030598

Mutation

transgenic lines:

• UT66
• UT71
• UT3
• UT64

To investigate the biological functions of the OSRIP18 gene, it was ectopically expressed under the control of 35S promoter. Totally, 33 transgenic plants were generated with this construct. These plants were integrated 1–4 copies of the construct by southern blot analysis. Among them, 11 independent transgenic plants contained single copy insertion of the T-DNA and they were used for further investigation. All of them showed significantly increased tolerance to drought and high salinity stresses.

Expression

Figure 1.Phenotypic characterization of transgenic plants overexpressing OSRIP18.(from reference ).

• Transgenic plants overexpressing OSRIP18:
  • 11 independent transgenic plants with single copy of T-DNA insertion were fertile and the seeding rates had no co-relation with ectopic expression this gene. Some of the data were shown in Fig. 1a.
  • After treatment for 2 h under 30% PEG solution, transgenic plants still showed normal leaf phenotype while WT plants exhibited withered and curved leaves (Fig. 1b). The result suggested that ectopic expression of this gene in rice increased its tolerance to 30% PEG stress.
  • The increased tolerance to drought and high salinity was also observed during reproductive stage (Fig. 1c). Since we have carried out the northern blot analysis for three lines UT64, UT66 and UT71 (Fig. 1b), we further investigated the effect of expression abundance on the tolerance to both abiotic stresses.
    

The over-expressed OSRIP18 gene exhibits different expression abundance among different transgenic lines. However, these lines showed the similar tolerance to both PEG and high salinity stresses.

• Global gene expression changes by Microarray analysis showed that more than 100 probe sets were detected with up-regulated expression abundance while signals from only three probe sets were down-regulated after over-expression of OSRIP18. Most of them were not regulated by drought or high salinity stresses.

• Ectopic expression of this gene in rice increased its tolerance to 30% PEG stress.

• Transgenic plants showed significantly higher tolerance to high salinity stress.Two transgenic lines UT66 and UT71 showed that the integrated OSRIP18 were also expressed in all tested tissues with differential transcript abundance.

• Transgenic plants exhibited no significant differences in response to both biotic stresses (Megnaporthe grisea) and Xoo (Xanthomonas oryzae pv oryzae) as well as to cold stress when compared with WT plantsas.

• The transgenic plants over-expressing OSRIP18 showed no obvious phenotype difference during vegetative or reproductive development stages under normal growth conditions. This gene was induced by PEG and high salinity treatments during panicle development.

Figure 2.Phylogenetic analysis and classification of genes encoding RIP domain proteins in rice and other plants.(from reference ).

Evolution of the RIP family:

1. To classify the family members and explore their evolutionary relationships, the amino acid sequences from RIP domains of all rice members and some members from other species were used for sequence alignment and a phylogenetic tree was constructed based on these aligned sequences (Fig. 2).

• As shown in Figure 2, RIP genes from the same family usually clustered together. All RIP genes can be classified into 4 groups. The group I consisted of members from the family Poaceae, which contained all rice RIP genes and those from wheat, and barley and maize. This group can be subgrouped into 3 clades. All three non-rice RIP genes from Poaceae were grouped into the clade 2. The biological functions among these 3 clades may be different and both clade 1 and clade 3 may be specific for rice.
• The group II contained only one member, which was from Liliaceae since only one gene was selected from this family for phylogenetic analysis.
• The group III consisted of members from Caryophyllales, which contained 4 sub-families. The members of group IV were all from the family Cucurbitaceae.
• The group V belonged to bacterial members suggesting that RIP genes might have evolved in parallel to the evolution of their

corresponding species.

2. It seems that type 1 RIPs could generally be more abundant than type 2 RIPs(Girbes et al. 2004). Only a few reports described coexist of type 1 with type 2 RIPs in a same organism (Girbes et al. 2004). However RIP family has evolved together with the differentiation of a species and largescale genome duplications represent the major mechanism for the family expansion. 3. BLAST searches showed that OSRIP18 shared 100% homology at the nucleotide level with the RA39 gene previously reported by Ding et al.. This report showed that RA39 was expressed in tapetum specific manner by in-situ hybridization.

Knowledge Extension

Figure 3.Classification of RIPs.(from reference ).

• Only type 1 RIP genes are present in rice genome and some of them might have evolved into pseudogenes since they show no expression under normal growth conditions or various environmental stresses. Expression analyses showed that some members of rice RIP family might play important roles in biotic stressrelated biological processes and function as a regulator of various environmental cues and hormone signaling.

• Ribosome-inactivating proteins (RIPs) either single-chain (type 1) or two-chain (type 2) are frequent in plants, often in multiple forms. They are RNA N-glycosidases, have antiviral, antifungal and insecticidal activity. Their expression in plants is increased under stressful conditions.

RIPs are investigated for practical applications in medicine and in agriculture. In medicine, RIPs have been linked to, or fused with, appropriate antibodies or other carriers to form "immunotoxins" or other conjugates specifically toxic to the cells target of the carrier, with the aim of eliminating malignant or other undesired cells. In agriculture, it has been observed that an enhanced expression of RIPs confers to plants an increased resistance to viruses, fungi, insects, and also to drought and salinity.

• RIPs are classified into two main classes according to their structure, namely holo-RIPs and chimero-RIPs. This classification system is illustated in Figure 3. Holo-RIPs comprise the RIPs that consist of a N-glycosylase domain only and usually consist of one chain, while chimero-RIPs are proteins made up of two domains. The class of the holo-RIPs contains two types of RIPs: one-chain type-1 RIPs and two-chain type-1 RIPs.

Labs working on this gene

• Rice Functional Genomics Group, Temasek Life Sciences Laboratory, 1 Research Link, The National University of Singapore, Singapore 117604, Singapore
• Republic Polytechnic, 9 Woodlands Ave 9, Singapore 738964, Singapore