AIR1-CT92-0205 Engineering Stress Tolerance in Maize (ESTIM)

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AIR Cluster X - Inputs for Non-Food Crops : Biotechnology : Plant Genetics

	Proposal No:	AIR1-CT92-0205
	Date Prepared:	April 1998
	Source:	Project Summary - Progress Report

Engineering stress tolerance in maize

Introduction
The general objective of this project is the engineering of stress tolerance in maize. The approach is based on the modulation of expression of genes encoding superoxide radical and hydrogen peroxide scavenging enzymes. To this end, gene products targeted to the chloroplasts are designed and introduced in maize. Genes of interest within this project are superoxide dismutases and genes coding for enzymes from the ascorbate-glutathione cycle.

Objectives

• Specific objectives for the reporting period included:
• Cloning of additional genes coding for the target enzymes.
• Transformation of tobacco and maize with gene constructs coding for the target enzymes.
• Physiological and biophysical evaluation of transgenic plants.

Activities
The following transformations were carried out (SOD = superoxide dismutase, GR = glutathione reductase).

1. MnSOD transformants
   1. 62 MnSOD-maize transformants obtained.
   2. Transcriptional analysis identified 2 maize lines expressing MnSOD.
   3. Enzymatic analysis demonstrated extra SOD isozyme activity in both lines.
   4. Immunofluorescence localisation/ 2d-protein analysis.
2. Transformation
   1. 82 FeSOD-maize transformants obtained.
   2. Tobacco transformed with GR, gshI and gshII.
   3. 45 GR-maize transformants obtained.
3. Characterisation of maize anti-oxidant genes
   1. Cytosolic APx cDNA sequenced and characterised.
   2. GR cDNA identified and partially sequenced.
   3. FeSOD cDNA sequenced and characterised.
4. Physiological evaluation
   1. MV assay optimised.
   2. Antioxidant levels and MV-tolerance in maize determined.
   3. SOD levels and MV-tolerance in MnSOD-overproducing maize determined.
   4. Characterisation of ascorbate-glutathione cycle in maize leaves in progress.
   5. Growth and temperature tolerance of different maize lines in several locations studied.
5. Seed production
   1. H99 and transgenic lines produced in sufficient quantities for physiological experimentation.

Results
The following vector constructs containing chimeric genes have been designed and made.

Selectable marker: pDE110 CaMV35S-bar-3'nos

Superoxide dismutases:

MnSOD: N. Plumbaginifolia

pHW165 CaMV35S-tp-MnSOD-3'g7, pGSJ3780A

FeSOD: Arabidopsis

pKA4 CaMV35S-tp-FeSOD-3'g7,

FeSOD maize

pCO127 CaMVV35S-FeSOD-3'35S

Hydrogen peroxide scavenging enzymes:

ascorbate peroxidase:

pGV6 pAPAT1

glutathione reductase:

pGR42, dCaMV35S-GR-3'35S

catalase

glutathione peroxidase

Glutathione synthesis genes

gshI: pGSH105, pGSH106

gshII: pGSH208, pGSH209

Transformed maize containing either SOD or a hydrogen peroxide scavenging enzyme has been obtained. The procedure followed for transformation is based on co-transferring two independent plasmid vectors carrying the selectable marker gene and the chimeric genes of interest respectively. As selectable marker gene, the CaMV35S-bar gene construct was used. The bar gene codes for phosphinothricin acetyl transferase that confers resistance to the herbicidal compound phosphinothricin. The gene constructs are preferentially transformed in the corn line H99; more recently Pa91 and the BC1 (Pa91xH99)xH99 have been used. Several hundred primary transformants have been obtained of which, so far, 11 have been shown to have acceptable expression level.

Scavenging enzymes have been analysed in nontransformed maize. cDNA has been identified, partly sequenced and characterised for Apx cDNA, GR cDNA and FeSOD cDNA.

Expression levels in the primary transformants have been determined. Transformed maize has been obtained and molecular analysis of the primary transformants has been carried out for MnSOD, FeSOD and GR. Techniques used include Western PAGE, Northern blotting, assay of the enzyme levels and the use of immunofluorescence localisation, together with 2D-protein analysis and amino-terminal sequence determination.

Rapid screen assay for physiological analysis has been established. The methyl viologen (MV)-assay has proven to be useful for the detection of differences in oxygen radical scavenging capacity between transgenic (SOD-overproducing, and also Apx-overproducing) and non-transgenic plants. The assay can also be applied in maize, although the test seems to be less selective for light-induced oxygen radical stress. Conditions with respect to leaf age and part of the leaf to be used have been standardised.

Successful transformant has been selected in rapid screen. Plants transformed with MnSOD showed an increase in SOD activity compared with untransformed plants. Plants transformed with MnSOD were at least as susceptible to chilling damage to PSII and CO₂ assimilation as untransformed plants. These experiments were carried out on a limited number of plants and transformants. Further work will be required to ascertain the validity of the conclusions.

Continuation
The consortium decided to introduce the use of transgenic tobacco plants in order to facilitate the screening for gene constructs of potential interest. This has become especially relevant in light of the poor expression levels which have been obtained with the initial gene constructs. The expression levels of the transgenes remained below expectations. It was therefore decided to evaluate new gene constructs first in tobacco and only after that in maize. It is anticipated that this strategy will improve the changes for identifying the best working gene constructs.