GENETIC ACCLIMATION FOR FREEZING TOLERANCE

GENETIC ACCLIMATION FOR FREEZING TOLERANCE
Kan Wang

A late spring cold wind-current or early fall frost can cause severe damage to crop yield. A number of strategies using recombinant DNA technology and genetic transformation has been utilized to enhance crop freezing tolerance in recent years1,2. These approaches include the overexpression of biosynthetic enzymes for osmoprotectants (such as mannitol, proline, treholose, or glycine betaine), the constitutive expression of stress-induced proteins (such as late embryogenesis abundant [LEA] proteins or heat shock proteins [HSPs]), altering the enzyme activity of antioxidants (such as superoxide dismutase [SOD] or glutathione S-transferase [GST]) that are involved in the detoxification of active oxygen species (AOS) accumulated during the stress environment, and expression of transcriptional factors (such as dehydration-responsive element [DRE] or CRT-binding factors [CBFs]) that bind to water deficit and cold responsive genes. Recently, it was reported that overexpression of a plasma membrane-associated phospholipase Dδ could enhance freezing tolerance in Arabidopsis3. While most work utilizes Arabidopsis as a model system, we carried out experiments on maize, a frost-sensitive crop plant originated from subtropical regions. The freezing tolerance enhancement described here involves the constitutive expression of a protein kinase in an oxidative stress signaling pathway4.

Many plant species increase their tolerance to cold or freezing temperature after they are exposed to a sub-optimal temperature. This process is called cold acclimation. Exposure to acclimation temperature causes many changes, including mild oxidative stress in plants, which can consequently induce chilling tolerance. At the molecular level, extensive alteration in gene expression has been observed during this process. Oxidative stress generates and accumulates active oxygen species such as H2O2 in plants, which triggers the activation of a mitogen-activated protein kinase (MAPK) cascade. The activation of the MAPK pathway induces production of a number of stress responsive proteins, such as heat shock proteins (HSPs), which in turn protect plants from stresses.

The MAPK signal transduction pathway is conserved among different organisms. It is usually activated only upon stress conditions. However, in some cold stress conditions, plants may be severely damaged before they even get a chance to turn on their protective mechanisms. Our hypothesis was then, if a plant was acclimated genetically, namely, its stress-induced pathways or proteins were turned on without first being stressed, could it withstand sudden severe stress such as subzero freezing temperatures?

We introduced a tobacco MAP kinase kinase kinase gene (NPK1) into maize through Agrobacterium-mediated transformation. The cDNA fragment encoding a 268-amino acid catalytic domain of NPK1, which is under a constitutive CaMV 35S promoter, was shown previously to enhance tolerance to freezing, salt, and heat stresses in transgenic tobacco5. Two dozen transgenic NPK1 maize lines were generated. According to the NPK1 gene transcript levels in R1 plants, we categorized maize lines into high, medium, and low expressers. Two events, A4-9 and A4-15, representing the medium and high levels of gene expression, respectively, were used for freezing analysis.

We performed two types of freezing tests for these transgenic plants: graduated freezing and constant freezing. In the graduated freezing test, the temperature of the growth chamber was set at –1oC and continually decreased at the rate of 1ºC per hour until it reached a temperature of –6oC, while in the constant freezing test, the temperature was set at –5ºC. Maize plants were grown under normal growth conditions (25ºC, 14 hr day length) to the three-leaf stage before they were subjected to freezing treatments. Cellular damage of treated seedlings due to freeze-induced membrane lesions was estimated by measuring electrolyte leakage (EL) from the leaves of treated plants. The higher the EL, the more severe the damage to the plant membrane, thus indicating that samples had less tolerance to freeze challenging. Considering the possibility of genetic variation among these transgenic events, we used the null segregants from each transgenic event as the negative control. We observed that leaf EL increased with a decrease in environmental temperature. When the temperature dropped to –4ºC, the EL of negative segregants increased extensively, indicating that severe membrane damage had been caused by freezing stress. The EL of transgenic plants of A4-9 and A4-15, on the other hand, did not increase until the temperature dropped to –5ºC and –6ºC, respectively. This result indicates that these transgenic maize events were able to tolerate up to 2ºC lower freezing temperature than their negative control siblings.

The increased freezing tolerance in transgenic plants of events A4-15 and A4-9 were confirmed in the constant freezing test. The EL of A4-15 transgenic plants did not increase until 5 hours at –5ºC temperature. The EL of A4-15 negative control siblings, however, increased to 57% and 90% after 3 and 4 hours of –5ºC treatment, respectively. This result indicates that transgenic A4-15 plants can survive 1–2 more hours than their negative control siblings at a temperature of –5ºC. A similar difference in freezing tolerance between transgenic plants and their non-transgenic siblings was observed in event A4-9 in which transgenic plants survived for 3 hours at –5ºC, while the negative siblings survived for 1 hour.

To understand the mechanism of freezing tolerance in NPK1 transgenic maize plants, we measured total soluble sugar content (TSC). An increase in TSC was positively correlated with enhanced freezing tolerance in plants. It is believed that soluble sugars function as cryoprotectants and osmolytes that protect cells from freezing damage. In our study, NPK1-expressing transgenic plants had higher TSC both under non-acclimated (25ºC) and cold-acclimated (4ºC, 24 or 48 hrs) conditions compared to their negative non-transgenic siblings in all treatments. Cold acclimations significantly increased TSC levels in all plants. Under normal growth conditions (non-acclimated), event A4-9 contained significantly higher total soluble sugar content compared to its null segregants (P<0.02). It is interesting that the increase of TSC in transgenic plants was not tightly correlated with NPK1 transgene expression level or freezing tolerance performance in our case. It is possible that while the NPK1 transgene induced cold-acclimation-like biochemical processes that elevated TSC, factors other than the NPK1 transgene also affected sugar levels in the maize seedlings.

We also conducted microarray analysis to investigate whether enhanced freezing tolerance in NPK1 transgenic maize was due to the activation of MAPK cascades resulting from oxidative stress. Using a fiber-optic BeadArray™ technology, we compared the expression levels of several stress-induced genes between transgenic and non-transgenic plants with or without cold acclimation. The fiber-optic array uses randomly ordered, self-assembled arrays of beads for parallel analysis of complex biological samples6. Since the miniature fiber optic arrays that interrogate hundreds to over one thousand targets are built into a 96- or 384-array matrix that matches microtiter plates, it allows multiple assays to be carried out rapidly and efficiently. Twenty-eight maize EST sequences based on the protein sequences of putative stress-related Arabidopsis and tobacco orthologues, together with a housekeeping gene (18S rRNA) and the transgene NPK1, were chosen for analysis. The expression of three genes, GST (glutathione S-transferase), HSP17.8 (small heat shock protein), and PR1 (pathogenesis-related), was up-regulated (> 1.5) in NPK1 transgenic plants under either normal or cold-acclimated conditions. Two of these genes, GST and HSP17.8, are involved in oxidative signaling pathways5. While constitutive expression of transgene NPK1 up-regulated the gene expression of GST and HSP17.8, cold acclimation treatment (4ºC, 48 hr) did not additionally increase their transcript levels.

As discerned from array analysis, about 50% of stress-induced genes tested in our study showed no significant increase in NPK1 transgenic maize lines. One explanation is that the transgene NPK1 expression level may be too low to up-regulate these stress related genes. It is also possible that these genes were only transiently induced and our assay condition did not capture their expression at the right moments.

We have generated transgenic maize plants that constitutively express a tobacco MAP kinase kinase kinase gene (NPK1) with enhanced freezing tolerance. In field evaluation of agronomic performance of 22 events, we detected no significant differences in plant heights and leaf numbers between transgenic plants and their non-transgenic segregants, suggesting that expression of transgene NPK1 did not affect maize growth under normal field conditions. Our results demonstrate that maize freezing tolerance level could be enhanced through a genetic acclimation (instead of cold acclimation) process in which stress-induced proteins for plant protection is achieved upon the activation of the oxidative signaling pathway through manipulation of the MAPK cascade.

References

1. Cushman JC, & Bohnert HJ (2000) Genomic approaches to plant stress tolerance. Curr. Opin. Plant Biol. 3: 117-124

2. Iba K (2002) Acclimative response to temperature stress in higher plants: Approaches of gene engineering for temperature tolerance. Annul Rev. Plant Biol. 53: 225-245

3. Li W, Li M, Zhang W, Welti R, & Wang, X (2004) The plasma membrane-bound phospholipase Dδ enhances freezing tolerance in Arabidopsis thaliana. Nat. Biotechnol 22, 427-433

4. Shou H, Bordallo P, Fan J-B, Yeakley JM, Bibikova M, Sheen J, & Wang K (2004) Expression of an active tobacco MAP kinase kinase kinase enhances freezing tolerance in transgenic maize. Proc. Natl. Acad. Sci. (USA) 101, 3298-3303

5. Kovtun, Y, Chiu, WL, Tena, G, & Sheen, J (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc. Natl. Acad. Sci. 97, 2940-2945

6. Yeakley JM, Fan J-B, Doucet D, Luo L, Wickham E, Ye Z, Chee MS, & Fu XD. (2002) Profiling alternative splicing on fiber-optic arrays. Nat. Biotechnol 20, 353-358

Kan Wang
Department of Agronomy
Iowa State University
kanwang@iastate.edu