Comparative Study On The Physiological And Molecular Mechanism Of The Mutagenesis Of The Yellow And Green Leaf Tissue In Chrysanthemum Yellow-Green Leaf Mutant

Chrysanthemum（Chrysanthemum x morifolium （Ramat.） Kitamura）, has a long history in China, and is one of the ten most famous flowers in China, which is used as the potted flower, cut flower, and applied as the ground-cover plant. Chrysanthemum is valuable for its ornamental character, and is very important for the flower production. Nowdays the study on chrysanthemum is mostly focused on its colors and types of flowers, but little study on its leaf color. So the study on leaf color will help to increase its ornamental value. In this study, chloroplast microstructure and ultrastructure> SDS-PAGE and spectra anlysis of thylakoid membrane protein^chlorophyll fluorescence characteristics of green leaf tissue and yellow leaf tissue in yellow-green mutant of chrysanthemum were studied. Then suppression subtractive hybridization （SSH） technique was used to identify different genes between the green leaf and yellow leaf tissue of the mutant, the two genes related to the mutation of the leaf color were screened, the expression of the two genes and the physiological difference in the green leaf tissue and yellow leaf tissue were also studied, all these research is signifcant to reveal the mechanism of the formation of leaf color and genetic improvement. The main results are as follows:1. The contents of chlorophyll, carotenoid, anatomic structures of green leaf tissue and yellow leaf tissue of chrysanthemum yellow-green leaf mutant ’NAU04-1-31’ were studied by optical and electron microscopy. The results showed that chlorophyll and carotenoid contents in the yellow leaf tissue were lower than that in green leaf tissue. The ratio of chlorophyll a and chlorophyll b in yellow leaves was equivalent to that in green leaf tissue. In the yellow leaf tissue, there were some abnormal chloroplasts, in which vacuolated structures, a clustered plastoglobuli and no clear starch grains could be observed in the disrupted chloroplasts. In the green leaf tissue, chloroplasts in the green leaf tissue looked normal in their formation of thylakoids and granal stacks, many plump starch grains could be clearly observed, just few disperse plastoglobuli were contained in the green leaf tissue. The analysis of thylakoid membrane protein gel:at least15polypeptides could be isolated from the green leaf tissue, just4polypeptides could be isolated from the yellow leaf tissue. Especially, the polypeptides of PS II in yellow leaf tissue decreased greatly than that in green leaf tissue. The thylakoid membranes of the green and yellow leaf tissue were used to study on their room temperature absorption spectra, chlorophyll emission and excitation fluorescence spectra. Compared to the green leaf tissue, the absorption spectra and fluorescence spectra of the yellow leaf tissue decreased significantly, the peak position red-shifted and blue-shifted, the structure of thylakoid might be destroyed, the efficiency of light harvest decreased, Chla rather than Chlb in the yellow leaf tissue is responsible for harvesting light and being excited by light.Chlorophyll fluorescence characteristics showed:compared to green leaf tissue in the mutant, the yellow leaf tissue had higher Fo, but lower Fv/Fm、Fv’/Fm’、Φ PS Ⅱ、 and so on, which indicated the structure of photosystem might be destroyed; the yellow leaf tissue decreased greatly in photochemical reaction, but greatly increased in heat excitation. The ql component of NPQ in the yellow leaf tissue is higher than in green leaf tissue of the mutant, which indicated the yellow leaf tissue is sensitive to photoinhibition. Chlorophyll fluorescence kinetic parameters indicated PS II electron transport was apparently blocked and oxygen-evolving complex was destroyed, density of reaction centers decreased in yellow leaf tissue, which caused lower performance index on absorption. Compared to the green leaf tissue, the photochemical efficiency of the yellow leaf tissue decreased, the yellow leaf tissue was sensitive to photoinhibition, which increased the heat dissipation to prevent the damage from overmuch energy.2. In order to identify the expression differences between the green leaf tissue and the yellow leaf tissue, SSH was used to reveal the differentially expressed genes. A total of339（positive） cDNA clones （44.1%） were selected with the dot blot hybridization technology,157clones were isolated from yellow leaf tissue,182clones were isolated from green leaf tissue. After the removal of vector sequences and poor quality sequences,293ESTs of high quality sequences were consequently obtained. Of the293clones,93clones （62%） were singletons and the remaining200ESTs were clustered into57（38%） contigs with occurrence of the common gene ranging from2to23times, and a total of150unigenes were ultimately obtained. Classification of gene function for the150unigenes using a BlastX homology search revealed that107unigenes （71.3%） could be identified or assigned to the putative functions of known genes,27unigenes （18%） were classified to be no significant homology or unknown function, and16unigenes （10.6%） had no matches in public databases. All the unigenes were classified into16primary functional categories, according to the putative function of their homologous genes in the databases. The largest group of genes （16%） was assigned to energy production and conversion. Genes involved in translation, ribosomal structure and biogenesis formed the second （14%）, carbohydrate transport and metabolism and secondary metabolites biosynthesis, transport and catabolism together formed the third （7.3%） largest group, respectively. While other categories were composed of a small number of expressed sequence tags （ESTs）, especially the cytoskeleton category, signal transduction mechanisms category, and coenzyme transport and metabolism were exiguous. The genes of CmChlH and CmFtsH were screened for further study due to its possiblity involved in the formation of the mutation.3. A full-length CmChlH cDNA （chrysanthemum large subunit of Mg-chelatase, AB543917） contains4,451bp with an open reading frame of4,149bp encoding1,383amino acids. The predicted isoelectric point （pI） and molecular weight （MW） of ChlH is5.95and154kDa, respectively. Chrysanthemum CmChlH has a pre-sequence of51N-terminal amino acids and was predicated to be targeted to the chloroplast. The deduced amino acid sequence of CmChlH shared85%,82%,67%and43%of amino acid residues with protein from AtChlH, Arabidopsis thaliana （NP₁96867）; OsChlH, Oryza sativa （ABF95686）, NpChlH, Nostoc punctiforme （YP₀01866414）, HmChlH, Heliobacterium modesticaldum Icel （YP₀01679881）. It is highly homologous to AtChlH （GUN5）, the large subunit of Mg-chelatase. Three conserved histidine residues （H666, H670and H815） are also contained in chrysanthemum putative amino acids of ChlH. These results indicated that CmChlH cDNA represents the large subunit of Mg-chelatase in higher plants.A full-length CmFtsH cDNA （Chrysanthemum, ATP-metalloprotease, AB542716） contains2,272bp with an open reading frame of2,094bp encoding698amino acids. The predicted isoelectric point （pI） and molecular weight（MW） of CmFtsH are5.99and74.67kDa, respectively. CmFtsH has a pre-sequence of57N-terminal amino acids and was predicated to be targeted to the chloroplast. Multiple sequence alignments revealed that consensus regions exist among FtsH homologs from chrysanthemum, Arabidopsis thaliana, Nicotiana tabacum and E.coli. The sequence of CmFtsH Protein contains conserved motifs for a Walker-type ATPase and metalloprotease, such as motifs A and B（Ⅰ, Ⅱ）, the second region of homology （SRH, Ⅲ）, and a zinc-binding domain （Ⅳ）. The deduced amino acid sequence of CmFtsH displayed84%,82%,53%and50%similarity with AtFtsH5（Arabidopsis, NP₅68604）; AtFtsH1（Arabidopsis, NP₅64563）; NtFtsH （Nicotiana tabacum, AAD17230）; EcFtsH （E.coli, P28691）.A phylogenetic analysis shows CmFtsH is highly homologous to AtFtsHl, AtFtsH5in Arabidopsis thaliana. Moreover sequence comparison showed that deduced amino acid sequence of CmFtsH was most homologous to AtFtsH5（VAR1）. Taken together, these results suggested that CmFtsH cDNA identified herein encoded a FtsH protease.4. Tissue specific expression of the two genes indicated:the expression of CmChlH in leaves was the highest, and the next was in stems, only weak expression in roots and flowers. Similarly, we found the expression of CmFtsH was the highest in leaves compared to the green leaf tissue, the expression of CmFtsH in stems was just inferior to that in leaves. Only low abundance mRNA of CmFtsH could be detected in roots and flowers. Following strong light irradiation, Fv/Fm in leaf recovered under dim light was detected. Fv/Fm declined slightly by0.11in the green leaf tissue of the mutant. But the value of Fv/Fm decreased greatly by0.45in the yellow leaf tissue of the mutant, which indicated that the yellow leaf tissue were highly sensitive to photoinhibition in PSII. Recovery of the photoinhibition in the green leaf tissue of the mutant was faster than that in the yellow leaf tissue of the mutant. Fv/Fm in the green leaf tissue of the mutant completely recovered after an overnight adaptation under dim light, While Fv/Fm in the yellow leaf tissue of the mutant did not show a recovery to static levels. Furthermore, their Fv/Fm decreased quickly to a lower value from3to20h. These results clearly indicated that the yellow leaf tissue of the mutant is very sensitive to photoinhibition in PSII, and that the damage caused at the given light intensity is irreversible.The expression of CmChlH to various light intensities was investigated using the green leaf tissue and the yellow leaf tissue in the mutant. In green leaf tissue, the abundance of CmChlH transcript increased steadily from10to150μmol/m2/s light exposure, then increased quickly from150to350μmol/m2/s light exposure, and peaked at450μmol/m2/s light exposure, but suppressed at600μmol/m2/s light exposure. In yellow leaf tissue, the transcript of CmChlH increased steadily from10to450μmol/m2/s, moreover, the transcript of CmChlH kept on increasing from450to600μmol/m2/s. Though there was a higher expression level of CmChlH in the light （0hours）, when placed in the dark, the expression of CmChlH in two types of leaf tissue were both down regulated to a low level, then maintained this level without great change from5to48hours in the dark, whose transcript level were great lower than the control in the light. These results indicated that the expression of CmChlH was induced by light. In the light treatment, we exposed the green leaf tissue and yellow leaf tissue to various light intensities for12h. The results showed that, in green leaf tissue, CmFtsH mRNAs increased slowly from10to450μmol/m2/s, peaked at450μmol/m2/s, then suppressed at600μmol/m2/s. In yellow leaf tissue, CmFtsH mRNAs kept on increasing from10to450μmol/m2/s and peaked at600μmol/m2/s.Similar to the light dependent expression, we found that CmFtsH transcript levels were decreased with the time in the dark. The results indicated that CmFtsH mRNA levels decreased about half of the control after5h in dark in two leaf types, and decreased quickly from12to48hours. Only trace mRNA expression of CmFtsH could be detected after48h in the dark. These results validated that CmFtsH expression is regulated by light condition at the transcript level,too.