Plant productivity in many regions of the world is limited primarily as a result of environmental stresses. High temperature and wounding caused by pest and pathogen infection are among the main factors accounting for unpredictable and often severe yield losses worldwide. These stresses, force the plants to alter then gene expression in order to adapt to the changed environment. Attempts were made in the study to isolate and characterise the differentially expressed heat shocked and wound-induced genes to understand the underlying molecular mechanism of heat shock and wounding response. The isolation of the promoters and their use to derive the tissue-specific and high expression of the linked coding sequences will be proved practically more significant. A cDNA clone designated LP 19 was isolated from a differential screening of a cDNA library prepared from lignifying pods of pea line L59. Sequence homology analysis showed that LP19 belongs to the hsp70 gene family. Northern analysis of RNA from pods from pea lines of different genotypes, showing the presence or absence of pod lignification, showed that LP 19 expression was specifically associated with lignification. Several cDNA species derived from transcripts of the LP 19 gene were subsequently isolated, which showed varying positions of poly (A) addition to the 3' untranslated region. Southern blotting of genomic DNA indicated the presence of single gene corresponding to LP 19.The pea hsp70 gene corresponding to LP 19 was isolated from a pea genomic library using LP 19 as a probe. The pea hsp70(LP19) gene predicts an open reading frame encoding a polypeptide of 648 amino acid residues. This sequence is similar to other plant hsp70 proteins. However, unlike most other plant hsp70 genes, the pea hsp70(LP19) gene lacks an intron. 1.8 kb of 5' flanking sequence of hsp70(LP19) gene was also sequenced. The promoter region contains 6 putative consensus heat shock elements (HSEs) as well as 4 A-T rich sites upstream from TATA box. Induction of gene expression of the pea hsp70(LP19) was observed in all organs of the plant after heat shock; the highest level of expression was observed in root, followed by stem and least in leaves. A similar expression pattern for a corresponding gene was observed in chickpea (Cicer arietinum L.). Other stress conditions such as salt stress and wounding failed to induce the expression of hsp70LP(19) gene both in pea and chickpea. The pea hsp70(LP19) promoter region, including 1.8 kb 5'-flanking sequence, and the first 18 amino acids of the coding region, was fused with coding sequence for P- glucuronidase (GUS). Tobacco plants were transformed with this chimaeric gene in order to study tissue specific and developmental expression of the hsp70(LP19) promoter. Histological staining of GUS activity in transgenic tobacco plants showed that protein was present predominantly in the phloem tissue in stem, root and petioles In addition, developmental expression of the hsp70(LP19) gene promoter, without heat shock, was observed in petals, pollen grains, developing seeds as well as in germinating seeds and seedlings at different stages of growth. Quantitative assay of GUS activity by fluorometric assay was used to follow the time course of protein accumulation. Activity was detected within few minutes of the start of heat shock and increased to a maximum after 6 hrs. A high level of GUS activity was observed only in the heat shocked parts of the plant; no endogenous signal that spread systemically from the heat shocked areas to the rest of the plant was observed.Pea and chickpea plants showed a transient increase of polyphenol oxidase (PPO) with maximum level at 48 hrs after wounding. No systemic induction of PPO was observed in unwounded parts in response to both wounding and MeJA treatment. In order to isolate transcripts expressed differentially in response to wounding, a pea subtractive cDNA library was made. 21 subtracted cDNA clones were partially sequenced. Most of the subtracted cDNA clones showed homology with wound or pathogen induced sequences. Northern analysis of the genes corresponding to the subtracted cDNA clones (SC3, SC7, SC12, SC33, SC57 and SC58), indicated differential expression in response to wounding. Full length or nearly full length cDNAs corresponding to 4 subtracted cDNA clones, designated SC10, SC15, SC57 and SC58, were isolated and sequenced. These cDNA clones will be further studied and efforts will be made to isolate their promoters. The tissue-specific expression will be carried out by using promoter-reporter system. These isolated cDNA clones were partially characterised and will be available for further studies to isolate their respective promoters. The tissue specific expression will be carried out by using promoter-reporter system.

The major digestive enzymes of larval Diabrotica undecimpunctata howardi, the southern corn rcotworm (SCR), have been investigated. A number of proteases have been identified, the majority cysteine proteases, some aspartic acid proteases are also present. In vitro assays of cysteine proteases showed that almost all activity could be arrested by E64 or chicken egg-white cystatin. This activity was also affected by TLCK, CpTI and thaumatin. Two inhibitory activities were demonstrated in protein extracts from rice seed. The first, oryzacystatin-I, caused marked inhibition of both the insect cysteine proteases and papain. The second produced strong inhibition of insect cysteine proteases but left papain virtually unaffected. Amino acid sequence data for this novel inhibitor was obtained, and significant homology demonstrated to the rice allergenic proteins (Izumi et a].,1992; Adachi et al., 1993). Oryzacystatin-I was expressed as a fully functional fusion protein (Rozc) in Escherchia coli, isolated, characterised and employed in feeding trials with larval SCR, a significant rise in mortality was demonstrated. Other protease inhibitors were also assayed in vivo, but none exhibited the efficacy of Rozc. A single iso-form of a-araylase was identified and characterised. In vitro assays with amylase inhibitors demonstrated the effectiveness of WAAI and the weak effect of BAAI. WAAI, employed in bioassay, produced a significant decrease in survival. Five lectins were tested by bioassay. WGA and GNA caused marked alterations in larval development, GNA was most effective. Saporin caused little effect when incorporated into artificial diet. WAAI, CpTI and GNA were employed in combination bioassays. An enhanced level of effectiveness was demonstrated with the double and triple combinations assayed. While further work is necessary, especially assaying protein efficacy in planta and establishing mechanisms of action, this project has successfully demonstrated the clear potential of plant antimetabolic proteins for use in the enhancement of inherent resistance of crop plants to insects, and of employing a number of proteins in a multi-mechanistic defence.