Studies On Biological Functions Of Arabinoglactan Protein Gene AtFLA3 In Arabidopsis Thaliana

Arabinogalactan-proteins (AGPs) are glycoproteins that widely distributed in plant kingdom. And AGPs have been implicated in a variety of plant development processes including sexual plant reproduction. According to AGPs structure characteristics, there are existing approximately 85 AGPs genes in Arabidopsis annotated genome. In present paper, bioinformatical analysis, semi quantitative and quantitative PCR, RNA interference, overexpression, promoter fusion, subcellular localization and floral-dip transformation of Arabidopsis combine with other molecular and cytological methods were employed to investigate the biological functions of AtFLA3 in Arabidopsis. Main results were described as follows:1. The result of bioinformatic analysis indicated that AtFLA3 has an 843-bp-long open reading frame encoding 280 amino acids. And the most abundant residues of the deduced protein are Pro (10.71%), Ala (10.71%), Ser (10.36%) and Thr (6.79%). And it has four distinct regions including:an N-terminal signal sequence for secretion, a central hydrooxyproline/proline rich AGP domain, a fasciclin-like domain and a C-terminal hydrophobic sequence considered as a GPI addition signal. The position between 24 and 25 amino acids has the max cleavage site probability for N-Signal peptide cleavage. The potential C-terminal GPI-Modification site is the 256 amino acid "S". The results of Arabidopsis Affymetrix GeneChip ATH1 and MPSS indicated AtFLA3 mainly expressed in inflorescence.2. The results of quantitative PCR showed that AtFLA3 is specifically expressed in floral organs and regulated by light. Some web tools for prediction cis-acting regulatory DNA elements were employed to analysis the CRE sequence in the promoter region of AtFLA3. The promoter of AtFLA3 contains many CREs homolog to eukaryotes such as:light responsive elements, hormone responsive elements, abiotic stress responsive elements and so on. Especially, there are existing three "POLLEN1LELAT52" and one "GTGANTG10" elements which were discovered in late pollen genes LAt52 and G1O respectively. The pAtFLA3::GUS fusion vector was constructed and transformed into Arabidopsis via floral-dip method. And then the homozygous pAtFLA3::GUS transgenic lines were obtained after resistant selection and PCR confirmation. GUS staining results indicated that AtFLA3 specifically expressed in anthers, mature pollens and pollen tubes, while no signal can be detected in other parts of the flowers. And this specific expression started at floral stage 10 and reached maximum at flower stage 13. At floral stage 13, GUS signal was seen in the stigma and transmitting tract and these singals might come from pollen tubes rather than female organs. Semi-thin section of the GUS stained floral organs further indicated AtFLA3 specifically expressed in pollen grains.3. We constructed AtFLA3 and eGFP reporter gene fusion vector eGFPm-AtFLA3 and eGFPm-AtFLA3AGPI respectively, then transformed into Arabidopsis via floral-dip method. Hypocotyl cells of the homozygous transgenic seedlings were used for study the subcellular localization pattern under confocal microscopy. The results indicated that AtFLA3 exhibits cell membrane localization, and GPI-anchoring signal sequence may affect its localization.4. Analysis of SALK₀16582 mutant showed that the T-DNA region insert into the 3'untranslated region of AtFLA3. Homozygous mutants of SALK₀16582 were testified by PCR using genomic DNA as template. Further investigation indicated that no reduction of AtFLA3 expression can be detected in homozygous lines. Meanwhile, no obvious differences can be observed compared with wild-type plants.5. RNA interference and overexpression methods were utilized to reveal the biological function of AtFLA3 in Arabidopsis. So we constructed AtFLA3 RNAi and overexpression vectors respectively, and then transformed into Arabidopsis. Phenotypical observation showed that both RNAi and overexpression plants were sterility. The RNAi transgenic lines had many small, shrunken, wrinkled pollen grains. Malformed pollen grains of RNAi plants showed no viability seen by FDA and I2-KI staining. The results of genetic crosses further confirmed the pollen defection was the only cause for sterility in RNAi plants. Further observations indicated that the intine layer of aborted pollens in RNAi plants is not well defined, and this abnormality occurs during the transition from uninucleate stage microspore to bicellular stage pollen. Calcofluor white staining showed that abnormal cellulose distribution in aborted pollen, which may incur the defection in pollen intine layer and finally lead to pollen sterility in RNAi plants. To sum up, AtFLA3 plays important role in microspore development, especially in pollen intine formation.However, the OE pollens were as normal as wild-type pollens. The defection of stamen filaments elongation and female organs were the two main reasons for sterility in OE plants.