Effects Of Water-deficit Stress On The Genes Expression Of Developing Immature Ear And Tassel, And Cloning And Ascertainment Of Glutamate Decarboxylase Gene In Maize

Maize(Zea mays L.)is not only an important economic crop in the world,but also a vital resource for forage and food industry.In our country about 60%of maize area is under water-deficit stress,which causes 20%-30%of reduction every year and affects the development of national economy and the normal life of people in the arid field.The extent to which crop productivity is affected depends largely on the development stage at which the plants encounter stresses.Among these stages,reproductive development from meiosis in the spore mother cells to fertilization and early seed establishment is extremely sensitive to various stresses, such as drought.Two peaks of sensitivity are encountered within this period.The first is centered on the period from meiosis to tetrad break-up in anthers.The second peak occurs during anthesis and initial stages of grain development.Stresses in this period cause a variety of abnormalities in floral organs which interfere with pollination or fertilization,and induce abscission of flowers or abortion of newly formed grains.Understanding the mechanism of plant tolerance to environmental stresses and the molecular basis of plant responses to water stress,especially at extremely sensitive stage might provide new strategies to improve the stress tolerance of agriculturally important plants.Understanding maize responses to water stress requires a comprehensive evaluation of stress-induced changes in gene expression and is expected to advance our insight into crop improvement.Microarray provides an analytical tool by which thousands of genes can be studied at one time.cDNA or Oligo microarray has recently been used to monitor global gene expression in response to several abiotic stresses in higher plants especially in Arabidopsis and rice.Recently,this technology has been applied to analyze maize gene expression profiles under various stress conditions.The leaves and roots of maize seedlings had different expression profiles under drought treatment,and an ethylene signaling pathway might be involved in the maize response to this stress.The response of the transcriptome of maize roots to salt stress was rapid and transient,leading to a burst changes after three-hour salt treatment.Of the salt induced ESTs,genes involved in the transport and signal transduction pathways were prominent.The results of gene expression profiles in ears before and after silking under shade stress showed that genes concerned in the ABA signal pathway were up-regulated,and genes involved in starch synthesis were down-regulated while genes of starch degradation didn't have significant changes.Oligonucleotide microarrays were used to examine genes expression at 4 days after silking and 8 days after pollination in maize ear and kernel in response to water-deficit stress.The result showed that the sensitivity to the extent of stress were different in different tissues.Only 17 genes,involved in many responses,expressed differentially in all the tissues.During water- and salt-stress treatments of developing kernels at 15 days after pollination,the genes involved in various stress responses(abiotic,wounding and pathogen attack)were up-regulated and the gene predominantly involved in energy generation were down-regulated in both stresses.Collectively,those researches were focused on the maize seedling and reproductive stage at the second peak of sensitivity to abiotic stress.However,the mechanistic bases of cellular response at the first sensitive peak to water deficit are still not been reported previously.To advance our understanding of the response to water deficit stress in the maize reproductive organs at the first peak sensitive to water deficit,we monitored gene expression in the developing immature tassels and ears under water deficit stress using oligo microarray slides containing 57,452 transcripts.After 1 day and 7 days of stress,immature tassels and ears differed considerably in their transcriptional responses,and the majority of changes were organ specific.By 1 day of stress,70 transcripts were up-regulated and 89 transcripts(56%of differential expression transcripts)were down-regulated in the ears;191 transcripts were up-regulated and 681 transcripts(78%of differential expression transcripts)were down-regulated in the tassels.By 7 days of stress,169 transcripts(84%of differential expression transcripts)were up-regulated and 33 transcripts were down-regulated in the ears;939 transcripts(62%of differential expression transcripts)were up-regulated and 574 transcripts were down-regulated in the tassels.There were obvious differences between the expression profiles of the ears and tassels.Only 73 transcripts were up-regulated and 17 were down-regulated in the two organs.Most of these transcripts have not been previously reported to be associated with water-deficit stress and are involved in a broad range of cell metabolisms,predominately in sucrose, trehalose and raffinose metabolism,and in cell wall metabolism and signal transduction in the tassels.These transcripts may be the genes response to water-deficit stress.A cDNA macroarray containing 514 unique ESTs in the subtracted cDNA library from maize leaves under water-deficit stress was constructed and used to analyze their expression profiles in maize immature ears and tassels during water-deficit stress.The results indicated that 58 ESTs by 1 day of stress and 106 ESTs by 7 days of stress were significantly up-regulated in the ears under water stress. Correspondingly,79 and 151 ESTs were significantly up-regulated in the tassels by 1 day and 7 days of stress.All of 222 ESTs,found to be up-regulated for at least one time-course point in either maize ears or tassels,were used for the hierarchical cluster analysis,and the results suggested that most ESTs were involved in cell metabolism, which was a good agreement with the microarray result.When the 222 ESTs were aligned with the sequences represented by 70 mer oligo on microarray using BLASTN program,184 ESTs were shown with good matched sequences.About 68%of these 184 ESTs were co-upregulated in the microarray and macroarray experiments,which showed a good agreement between them.A full-length cDNA,putative glutamate decarboxylase designated ZmGAD1, induced by water stress both in the microarry and macroarry experiments,has been isolated by the combination of bioinformatics and PCR based approaches.The entire cDNA ORF coding for the ZmGAD1 protein was inserted into the pET30a(+) expression vector.The recombinant protein showed the activity of glutamate decarboxylase.Sequence analysis showed that this protein had two conserved domains of glutamate decarboxylase,a pyridoxal-5'-phosphate(PLP)binding domain in the middle region of the peptides and a calmodulin(CaM)-binding domain at the carboxyl terminus.Southern blotting analysis suggested that the ZmGAD1 was a single-copy gene in the maize genome.The genomic sequence of ZmGAD1 was cloned,showed that ZmGAD1 was located on Chromosome 1 and had 7 exons and 6 introns.Based on a short mRNA sequence,we cloned another maize GAD gene in silico,designated ZmGAD2,which also had an entire ORF.Sequence analysis showed that ZmGAD2 didn't have the CaM site.ZmGAD2 was located on Chromosome 2 and had 4 exons and 3 introns.A homology tree of GADs from various plant species revealed ZmGAD1 had a closer evolutionary relationship with rice OsGAD1,and ZmGAD2 with OsGAD2,...