Os01g0615050

Oryza sativa chymotrypsin inhibitor-like 1 (OCPI1) is a member of serine PI family.

Annotated Information

Function

• OCPI1 might potentially be useful in the genetic improvement of drought resistance in rice. OCPI1 promoter has a bidirectional stress-inducible activity. Over-expression OCPI1 had significant effect on improving drought resistance at the reproductive stage of rice.

• Protease inhibitors play important roles in stress and developmental responses of plants. Rice genome contains 17 putative members in chymotrypsin protease inhibitor (ranging in size from 7.21 to 11.9 kDa) gene family with different predicted localization sites.

GO assignment(s): GO:0004867,GO:0009611

OCPI2 and OCPI1

• Another putative chymotrypsin inhibitor-like gene (OCPI2), located at the immediate upstream of the OCPI1 promoter fragment with reverse transcription direction to that of OCPI1 gene, was predicted in the genome annotation database. The OCPI2 gene is supported by a full-length cDNA and is also induced by drought and salt stress based on cDNA microarray profiling data.

• A vector that had GFP and GUS reporter genes in opposite orientations driven by 1881 bp intergenic sequence between the OCPI2 and OCPI1 (encompassing the region between the translation initiation sites of the two genes) was constructed and shot in onion epidermal cells by particle bombardment.

Mutation

• The full-length cDNA for OCPI1 under the control of CaMV 35S promoter was transformed into rice Zhonghua 11.
• RNA-blot analysis showed that more than 50% transgenic plants had obviously higher level of OCPI1 transcript than WT. The OCPI1-overexpresed transgenic plants contained one to several copies of the transgene based on Southern-blot analysis.
• Drought resistance testing:
  • three independent single copy plants
    • TL-4
    • TL-20
    • TL-25
  • a non-overexpression transgenic family
  • TL-21
• The positive transgenic plants had significantly higher grain yield and seed setting rate than the wild type and the negative transgenic control(no over-expression of the transgene) under the severe drought stress conditions, whereas the potential yield of transgenic plants under normal growth conditions was not affected.
• Chymotrypsin-inhibitor activity assay showed that the crude protein of the positive transgenic plants had stronger inhibitory activity than the negative control. Transgenic plants had less decrease of total proteins than the wild type under drought stress.

Expression

Figure 1. Northern-blot analysis of OCPI1 expression level under difierent abiotic stresses.(from reference ).

• The expression of OCPI1 was strongly induced by dehydration stresses (such as drought and salinity) and was responsive to ABA(Fig. 1):
  • In the drought treatment, very strong induction of OCPI1 was detected in the partially rolled leaves and its expression was decreased in the fully rolled leaves.
  • When the plants were re-watered for 1 day, the expression level of OCPI1 dropped to the level similar as in the non-stressed leaves.
  • The OCPI1 transcript level was rapidly increased shortly after salt treatment and maintained at high level of induction throughout the development of stress.
  • In the treatment of ABA, the transcript level of the gene was increased shortly after the treatment and peaked at 12 h.
• By histochemical assay, slight GUS expression was detected in callus, leaf, root, stem, sheath, ligule, auricle, glume, rachilla, pistil, and stamen of transgenic rice, suggesting that the endogenous OCPI1 gene may express in these tissues or organs with relatively low level under normal growth conditions. GUS activity of the crude protein extract from drought-stressed and salt-stressed transgenic leaves was significantly higher than the non-stressed transgenic samples and the stressed control plants. In other words, the expression of beta-glucuronidase(GUS) reporter gene under the control of OCPI1 promoter transformed into rice was strongly induced by drought and salt stresses.

Evolution

• sequence identity:
  • Protein sequence of OCPI1 showed 27–80% identity with various plant serine-proteinase inhibitors including the potato inhibitor I family.
  • The cDNA sequence of OCPI1 showed 98.5% identity with the OsSCI3(unpublished).
  • Using the protein sequence of OCPI1 to do BLASTP search against the rice annotation database , at least 16 putative chymotrypsin inhibitor were browsed.
  • Phylogenetic analysis of putative rice chymotrypsin inhibitors and a few chymotrypsin inhibitors from other species suggested that plant chymotrypsin inhibitors were largely diversified.
• OCPI1 belongs to the serine PI family.

Knowledge Extension

• Proteinase inhibitors (PI) constitute a large and complex group of plant proteins and have an enormous diversity of function by regulating the proteolytic activity of their target proteinases, resulting in the formation of a stable protease inhibitor complex.
• PIs were classified into non-specific and class-specific superfamilies and the later was subcategorized into several families including serine proteinase inhibitor, aspartic proteinase inhibitor, metalloproteinase inhibitor, and cysteine proteinase inhibitor. Genes encoding for PIs have been cloned and characterized from a varied range of plant species.
• Primarily, PIs are considered important in endogenous as well as exogenous defense against various pathogenic organisms. Some insects and many of the phyto-pathogenic microorganisms produce enzymes causing proteolytic digestion of host proteins. Plants fight against these pathogens through PIs that act against the proteolytic enzymes. Also, plant PIs have been shown to be involved in various physiological and developmental responses.

Labs working on this gene

• National Center of Plant Gene Research (Wuhan), National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
• Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021, India