Os06g0612800

OsiSAP8, is a member of stress Associated protein(SAP) gene family from rice characterized by the presence of A20 and AN1 type Zinc finger domains.

Annotated Information

Function

• OsiSAP8(Os06g0612800) is composed of three exons of 89, 62 and 1,088 bp separated by two introns of 176 and 739 bp, respectively. The introns were found in the 50UTR region and the coding region is continuous without any intron and is encoded exclusively by exon 3.

• This kind of upregulation of OsiSAP8 within 30 min upon exposure to various stresses like salinity, drought, desiccation, cold, submergence, wounding, heavy metals and ABA suggests that its product might be required during early phase of stress response. It may be argued that overexpression of OsiSAP8 both in homologous and heterologous systems conferred tolerance to high salt, drought and cold stresses equally.

• It is found to be coding for a cytoplasmic zinc finger protein that might act early in the signal transduction of various stress responses. Its overexpression in both homologous (rice) and heterologous (tobacco) system lead to an increase in stress tolerance, as determined by salt-, drought-, and cold-tolerance assays. It was found that the chlorophyll retention, percentage germination, fresh weight, root and shoot elongation and leaf development were much better in rice transgenic lines, as compared to control plants, under stress and recovery conditions. However, the transgenic plants showed a yield penalty of 50% under unstressed conditions and the same yield was maintained under salt and drought stresses during anthesis, which is much better compared to no seed set in control stressed plants.

GO assignment(s): GO:0003677, GO:0008270

Mutation

Transgenic lines:

• T₀
• T₁

It was evident that salinity-induced loss of chlorophyll was lower in OsiSAP8 over expressing lines compared with those from the control plants (Fig. 3C). The results indicated that the transgenic tobacco and rice plants overexpressing OsiSAP8 have better ability to tolerate salinity stress than control (untransformed) plants.

Expression

Figure 1. Expression pattern of OsiSAP8 after different stresses to rice seedlings.(from reference ).

• Expression pattern of OsiSAP8 after different stresses to rice seedlings:
  • The transcript levels increased to higher level within 2 h after salt stress to seedlings and the level continued to increase till 12 h and declined thereafter. However, even at 24 h the mRNA level was more than that of control (Fig. 1A).
  • In case of drought stress, transcript levels peaked within 2 h and the level continued till 24 h (Fig. 1B).
  • With desiccation stress the transcript levels increased to higher level within 2 h and declined after 12 h (Fig. 1C).
  • The transcript levels were increased gradually with the increase in time of exposure of seedlings to heat stress (Fig. 1D).
  • The gene was induced within 2 h and maintained higher transcript levels till 24 h, however with a slight decline at 6 h (Fig. 1E). Similar results were observed with OsiSAP1.
  • The transcript level was increased to very high levels within 2h of wounding stress and was maintained till 4h, after which its mRNA levels drastically decreased to the level of control at 6 h(Fig. 1F).
  • OsiSAP8 transcript levels was increased to higher level within 2 h after cold stress to seedlings and the level remained same till 6 h and started declining thereafter. However, even at 12 and 24 h the transcript level was more than that of control (Fig. 1G).
  • The treatment of seedlings with membrane rigidifier DMSO significantly increases the OsiSAP8 expression (Fig. 1H).
  • Manganese and Lithium salts had only marginal effect on mRNA level. Mercury salt reduced the mRNA level possibly because of the toxic effect of mercuric chloride(Fig. 1I).
  • However, with the increasing concentration of ABA, the steady-state transcript level was maintained for a longer duration as the mRNA level declined at 12 h with 10 lM ABA and at 24 h with 100 lM ABA (Fig. 1J)

• The productivity was not affected in transgenic rice lines under drought and salt stresses compared to unstressed transgenic lines.

Evolution

• The MtSAP1 protein sequence shared a strong homology with AtSAP7 and OsiSAP8 (54 and 62 %, respectively).

Figure 2. Phylogenetic tree of rice and Arabidopsis SAP gene families encoded A20/AN1 zinc-Wnger proteins.(from reference ).

• Phylogenetic tree of rice and Arabidopsis SAP gene families encoded A20/AN1 zinc-Wnger proteins 18 rice and 14 Arabidopsis protein sequences were aligned using ClustalX multiple alignment program. An unrooted phylogenetic tree was constructed for Arabidopsis and rice using the neighbor-joining method and viewed using Treeview. The rice and Arabidopsis SAP family members formed six distinct groups (Fig. 2). This included two Arabidopsis specific groups, group IV (AtSAP3, 4 and 6) and group V (AtSAP1, 7, 8, 9, 10) and two rice specific groups, group II (OsSAP10, OsSAP18, OsSAP12, OsSAP7 and OsSAP14) and VI (OsSAP3, 5). Of the remaining two groups containing both rice and Arabidopsis proteins, group I contained majority of proteins with only the AN1 zinc-Wnger domain (Fig. 2).

Subcellular localization

Kanneganti et al. performed transient expression of green fluorescent protein (GFP) fused with OsiSAP8 coding region, in the epidermal cells of N. benthamiana. When expressed alone, GFP was distributed uniformly in the cells (Fig. 7A1–A4). GFP-SAP8 fusion protein was specifically localized to cell cytoplasm, indicating that unlike many zincfinger containing proteins, OsiSAP8 is a cytoplasmic protein.

Knowledge Extension

• In the recent past, a new family of genes termed as SAP(Stress Associated protein) gene family was studied in rice for its role in abiotic stress conditions by expression profiling under those conditions. SAP gene family members are characterized by the presence of A20/AN1 domain in their putative encoded proteins. The majority was found to have both the A20 zinc-finger domain (present at the N-terminus) and the AN1 zinc-finger domain (present at the C-terminus). This is consistent with the previous finding from animal systems that the A20 and AN1 zinc-finger domains are usually found associated with each other.

• All the members of the rice SAP genenfamily present in the rice genome showed inducibility to one or the other abiotic stresses. It would be important to define the relative function of the members of this gene family in the life of the rice plant.

• Based on the phylogenetic analysis of the AN1 zincfinger domains, Jin et al. recently divided all A20/AN1 zincfinger-containing SAP genes into two groups: Type I and Type II. Type I genes contain the traditional pattern of cysteine- and histidine-rich motifs, whereas Type II SAP genes contain the expanded domain CX4CX2CX9-12CX1-2CX4CX2HX5HXC where X represents any amino acid. Most Type I genes lack introns and contain one intact A20 type domain and/or one AN1 type zincfinger domain; most Type II genes have a single intron but do not contain an A20 domain.

• SAP gene family members, as in the case of animal systems may be involved in downregulating the pathway associated with abiotic stress injuries such as cell death by ubiquitinylating the key proteins and hence targeting them to degradation.

Labs working on this gene

• Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai 625021, TamilNadu, India
• Department of Genetics, Agricultural Research Organization, The Volcani Center, Post Box 6, Bet Dagan 50250, Israel
• Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India