Functional characterization of a chloroplast-localized Mn2+(Ca2+)/H+ antiporter, ZmmCCHA1 from Zea mays ssp. mexicana L.

The annual Zea mays ssp. mexicana L. is a member of the teosinte group and a close wild relative of maize. Thus, Zea mays ssp. mexicana L. can be effectively used in maize breeding. AtCCHA1 is a Mn2+ and/or Ca2+/H+ antiporter localized in chloroplasts in Arabidopsis. In this study, its homolog from Zea mays ssp. mexicana L., ZmmCCHA1, was isolated and characterized. The transcriptional level of ZmmCCHA1 in Zea mays ssp. mexicana L. was repressed in response to excessive Ca2+ or Mn2+. Heterologous functional complementation assays using yeast mutants showed that ZmmCCHA1 mediates Ca2+ and Mn2+ transport. The ZmmCCHA1 protein was localized in the chloroplasts when expressed in tobacco leaves. Furthermore, ectopic overexpression of ZmmCCHA1 in the Arabidopsis ccha1 mutant rescued the mutant defects on growth and the photosynthetic proteins in the thylakoid membranes. Moreover, the photosynthetic phenotypes of Arabidopsis ccha1 mutant at steady-state were greatly but not completely complemented by the overexpression of ZmmCCHA1. In addition, overexpressing the ZmmCCHA1 rescued the sensitives of PSII in the Arabidopsis ccha1 mutant to Mn2+ deficiency or high Ca2+ condition. These results indicate that the isolated ZmmCCHA1 is the homolog of AtCCHA1 and plays a conserved role in maintaining the Mn2+ and/or Ca2+ homeostasis in chloroplasts which is critical for the function of PSII in photosynthesis.

RNA-seq Analysis of Cold and Drought Responsive Transcriptomes of Zea mays ssp. mexicana L.

The annual Zea mays ssp. mexicana L. is a member of teosinte, a wild relative of the Zea mays spp. mays L. This subspecies has strong growth and regeneration ability, high tiller numbers, high protein and lysine content as well as resistance to many fungal diseases, and it can be effectively used in maize improvement. In this study, we reported a Zea mays ssp. mexicana L. transcriptome by merging data from untreated control (CK), cold (4°C) and drought (PEG2000, 20%) treated plant samples. A total of 251,145 transcripts (N50 = 1,269 bp) and 184,280 unigenes (N50 = 923 bp) were predicted, which code for homologs of near 47% of the published maize proteome. Under cold conditions, 2,232 and 817 genes were up-regulated and down-regulated, respectively, while fewer genes were up-regulated (532) and down-regulated (82) under drought stress, indicating that Zea mays ssp. mexicana L. is more sensitive to the applied cold rather than to the applied drought stresses. Functional enrichment analyses identified many common or specific biological processes and gene sets in response to drought and cold stresses. The ABA dependent pathway, trehalose synthetic pathway and the ICE1-CBF pathway were up-regulated by both stresses. GA associated genes have been shown to differentially regulate the responses to cold in close subspecies in Zea mays. These findings and the identified functional genes can provide useful clues for improving abiotic stress tolerance of maize.

A novel Zea mays ssp. mexicana L. MYC-type ICE-like transcription factor gene ZmmICE1, enhances freezing tolerance in transgenic Arabidopsis thaliana.

The annual Zea mays ssp. mexicana L., a member of the teosinte group, is a close wild relative of maize and thus can be effectively used in maize improvement. In this study, an ICE-like gene, ZmmICE1, was isolated from a cDNA library of RNA-Seq from cold-treated seedling tissues of Zea mays ssp. mexicana L. The deduced protein of ZmmICE1 contains a highly conserved basic helix-loop-helix (bHLH) domain and C-terminal region of ICE-like proteins. The ZmmICE1 protein localizes to the nucleus and shows sumoylation when expressed in an Escherichia coli reconstitution system. In addition, yeast one hybrid assays indicated that ZmmICE1 has transactivation activities. Moreover, ectopic expression of ZmmICE1 in the Arabidopsis ice1-2 mutant increased freezing tolerance. The ZmmICE1 overexpressed plants showed lower electrolyte leakage (EL), reduced contents of malondialdehyde (MDA). The expression of downstream cold related genes of Arabidopsis C-repeat-binding factors (AtCBF1, AtCBF2 and AtCBF3), cold-responsive genes (AtCOR15A and AtCOR47), kinesin-1 member gene (AtKIN1) and responsive to desiccation gene (AtRD29A) was significantly induced when compared with wild type under low temperature treatment. Taken together, these results indicated that ZmmICE1 is the homolog of Arabidopsis inducer of CBF expression genes (AtICE1/2) and plays an important role in the regulation of freezing stress response.