Bioinformatics, gene functional and germplasm physiology feature studies of Zea may ssp. mexicana L. under cold and other environmental stresses

The annual Zea mays ssp. mexicana L., a member of teosinte, has strong growth and regeneration ability, high tiller numbers, high protein and lysine content as well as resistance to many fungal diseases. It is a close wild relative of maize thus it can be effectively used in maize improvement. Most lines of Zea mays ssp. mexicana L. originated at high altitude in northern and central Mexico with a large spikelet and are adapted to acid soil. Since the best temperature for growing Zea mays ssp. mexicana L. ranges from 25 °C to 35 °C, it is considered to be more sensitive to cold/drought than to high temperature, especially during germination and early autotrophic growth stages. This project set out to detect the cold and/or drought tolerance of Zea mays ssp. mexicana L., using bioinformatics, gene functions and physiological features contributing to both stresses. The aims were to provide an effective approach to understand the responses of Zea mays ssp. mexicana L. to low temperature and drought. The understanding of molecular mechanisms for cold/drought tolerance can also help distinguish Zea mays ssp. mexicana L. from other tropical or temperate plants as well as provide useful references in improving abiotic stress tolerance of maize. In a transcriptome study, we generated a seedling plant transcriptome at a sequencing size of 51.78 Gb of Zea mays ssp. mexicana L. under stress free, cold or drought stress conditions. A total of 414,232,462 high quality clean reads were used to conduct de novo assembly and annotation of genes without reference genome information. All of these reads were assembled into 251,145 transcripts (N50 = 1,269 bp) and 184,280 unigenes (N50 = 923 bp). A total of 3,504 up-regulated and 1,220 down-regulated genes were detected under cold stress and 532 up-regulated and 82 down-regulated genes were detected under drought stress. A total of 208 genes were affected by both cold and drought stresses. Examination of these genes suggested that the ABA dependent pathway, trehalose synthetic pathway and the CBF6 gene of the ICE1-CBF pathway may play important roles in both stress tolerances of Zea mays ssp. mexicana L. Compared with the maize transcriptome database, GAs of Zea mays ssp. mexicana L. showed significant differences in regulating the responses to cold. These important functional genes identified in this study and the information on molecular mechanisms for cold and drought tolerance can provide useful references in improving abiotic stress tolerance of maize. In gene functional studies, ZmmICE1 and ZmmSIZ2, two important cold related genes, were cloned and isolated from a cDNA library of RNA-Seq from cold-treated seedling tissues of Zea mays ssp. mexicana L. ZmmICE1, a novel MYC-type ICE-like transcription factor gene, enhanced freezing tolerance in transgenic Arabidopsis thaliana. As a novel transcription factor, the ZmmICE1 protein localized in the nucleus and showed sumoylation when expressed in an Escherichia coli reconstitution system. ZmmICE1 showed ICE1-family characteristics, such as a highly conserved basic helixloop-helix (bHLH) domain and a C-terminal region of ICE-like proteins. The other important gene ZmmSIZ2, a SUMO E3 ligase, was also isolated from the same treated plants. The biochemistry experiment showed sumoylation of the ZmmSIZ2 protein when expressed in an Escherichia coli reconstitution system. The structure analysis indicated that the deduced protein ZmmSIZ2 contained a highly conserved MIZ/SP-RING zinc finger domain for SUMO E3 ligase activity and bound to SUMO E2, a helix-extended loop-helix SAP (scaffold attachment factors SAF-A/B, Acinus, PIAS) domain for DNA binding and PHD domain. In physiological studies, non-destructive chlorophyll fluorescence imaging technology was used to evaluate photosynthesis responses of three varieties of Zea mays ssp. mexicana L. under different cold treatments. Varieties showed significant differences in the damage caused by 5 °C and 15 °C treatments and the damage was dependent on treatment time. Additionally chlorophyll a and b showed the same tendency with imaging data. This study provided useful information on bioinformatics, molecular biological and physiological mechanisms of Zea mays ssp. mexicana L. tolerance of cold and drought stresses. The results could be used in maize genetic research and improving maize for cold and drought tolerance as well as reference data in NCBI database for other researchers.