PROJECT RESULTS

Enhancing Germination Vigor in Cereals and Alfalfa

Applicant: 

Dr. Robert D. Hill
Department of Plant Science
University of Manitoba
Winnipeg, Manitoba  R3T 2N2  Canada

Table of Contents:

• Background and Objective
• Procedure and Project Activities
• Results and Discussion
• Conclusions

ARDI Project:

#98-088

Total Approved:

$30,000

Date Approved:

Project Status:

Completed October, 2001

Background and Objective:

Our laboratory discovered the existence of a class of hemoglobins (phytoglobins) that are widespread in the plant kingdom. They have unique chemical properties in that they bind oxygen very tightly. They are normally present in low concentrations, but their presence is increased when plant cells are starved for oxygen. We have shown that plant cells expressing phytoglobin perform better than non-expressing cells when exposed to oxygen starvation. They, therefore, may have a function in plants under conditions of flooding stress or in early germination when oxygen supply may be limiting. The intent of the project was to determine whether phytoglobins influenced the germination vigor of cereals. The approach was to prepare transgenic plants that either overexpressed phytoglobins or suppressed the expression of the native phytoglobin, and to study these plants in relation to their vigor under conditions such as flooding where oxygen was limiting.

Procedure and Project Activities:

In the early stages of the project, it became evident that attempting to transform cereals would unduly delay progress towards the goals of the project. We, thus, turned to transformation of alfalfa, which proved to be much more amenable to transformation. Alfalfa (Medicago sativa cv. Regen SY) stems were transformed using Agrobacterium rhizogenes as the transformation vector to overexpress and underexpress barley phytoglobin. This vector produces roots on the stems, which can easily be maintained as a root culture for experimentation. Furthermore, whole plants can be generated from the roots and further propagated, clonally. The results in this report describe work with transformed alfalfa roots. The Natural Sciences and Engineering Research Council and the Canadian Seed Growers Association also contributed funding to this project.

Results and Discussion:

Three critical pieces of information relating to the response of roots to flooding were obtained:

1. Alfalfa roots expressing phytoglobin maintain their growth under flooding stress.

Two lines underexpressing phytoglobin (Hb^-), two lines constitutively expressing phytoglobin (Hb⁺) and an untransformed line (WT) were exposed to three oxygen (O₂) concentrations over a period of 5 days and the growth rate of the roots measured. Under 40% oxygen, there was no significant difference in the growth rates. Under 20% and 3% oxygen, the growth rates of the Hb^- lines and the WT were significantly reduced relative to the 40% treatment. The growth rates of the Hb⁺ lines were not significantly different at any of the oxygen treatments.

These results, in conjunction with our earlier findings that cells expressing phytoglobin maintain their energy status when exposed to flooding stress, suggest that phytoglobin is an essential component in the short term survival mechanism to flooding of roots. From a crop production standpoint, the development of lines that respond to low oxygen conditions by strong, early expression of phytoglobin may lead to crops that withstand flooding stress more effectively. Furthermore, since metabolism during germination depletes oxygen reserves, the efficient expression of phytoglobin in the germinating seed may hasten germination and seedling establishment.

2. The degree of aerenchyma (air channel) formation in alfalfa roots varies in relation to phytoglobin expression.

Aerenchyma form in roots exposed to flooding stress. They are air channels that permit movement of air from the shoots to the roots under prolonged flooding stress. They are believed to be an adaptive mechanism allowing plant survival and are formed very early in the development of flooding tolerant species (e.g. rice). We have examined aerenchyma formation in alfalfa root lines during exposure to low oxygen conditions.

Cross sections of Hb^- and Hb⁺ alfalfa root lines exposed to 3 and 40% oxygen for 5 days were examined. Only in the Hb^- line under 3% oxygen is there evidence of aerenchyma. This is apparent by the lack of symmetry in the cell structure of the Hb^-, 3% oxygen treatment as compared to the other treatments.

Potentially, the results could lead to methods for manipulating aerenchyma formation in root systems to better adapt to flooding stress in crop plants. It would certainly be an advantage if, for example, barley could adapt to flooding stress in a similar manner to species of Echinochloa (Barnyard grass relatives) that are flooding tolerant. At this stage, the application of this information relative to a production situation is not clear and more research is required on the causative factors involved in aerenchyma formation. The next section provides a possible link to the mechanisms involved.

3. Nitric oxide (NO) is produced in alfalfa roots exposed to hypoxia.

All known hemoglobins react with NO. We hypothesized that one of the roles of phytoglobin may be to react with NO. We examined maize cells and found that NO was produced in these cells when exposed to low oxygen concentrations. Studies in this laboratory indicate that NO is also formed in alfalfa roots only under hypoxic conditions. Furthermore, the level of NO found is related to the phytoglobin content of the roots. Hb⁺ lines, with higher phytoglobin content, have lower NO levels than Hb^- lines. The results strongly suggest that phytoglobin reacts with NO to regulate the NO levels in flooded roots.

Aerenchyma formation is a consequence of selective death of cells within the root. Such a death process is generally considered to be a programmed cell death. NO, in addition to being chemically toxic to cells, is known to trigger a series of metabolically controlled events leading to programmed cell death. We believe that NO and phytoglobin are essential agents in the process or aerenchyma formation. An understanding of how they interact to cause aerenchyma formation will provide a means to adapt crop plants to better withstand flooding stress.

Conclusions:

The results that have been obtained on the relationship of phytoglobins and nitric oxide to flooding stress are a major advance in the understanding of crop adaptation to flooding stress. For the first time, evidence is available of changes in content (i.e. phytoglobin and nitric oxide) of roots that effect the viability and growth of the organ under flooding stress. Furthermore, the observation that nitric oxide is involved in this process clarifies a number of unanswered questions on adaptation to flooding stress. The basic requirements for modifying crops to handle flooding stress is in place. It is a matter of testing a whole plant system to determine whether detrimental effects have been created as a result of the transformations.

The first set of Hb^- and Hb⁺ transgenic plants are currently growing in soil in growth chambers. Enough plant material to undertake whole plant studies will be available in the summer of 2002. Seed from these plants should be available by the summer of 2003, at which time seedling vigor trials will be undertaken.

If the results of the whole plant studies indicate that the transgenic alfalfa lines have potential in protecting against flooding stress, marketing of the transformed lines would be the next step in the development of the project.

Acknowledgments:

This work was made possible by grants from: the governments of Manitoba and Canada through the Canada-Manitoba Agri-Food Research and Development Initiative, the Canadian Seed Growers Association, and the Natural Sciences and Engineering Research Council of Canada.

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