| Oilseed rape, which needs more phosphorus than other Brassica crops, is one of the most important oil crops in China as well as in the world. Phosphorus (Pi) deficiency in soil would retard plant growth at the seedling stage, decrease branch number and finally reduce seed yield at mature stage. At low Pi, increase of root hair length and density would promote the uptake efficiency of Pi. In addition, most of root secretion is produced and released from the root hair, which could promote the utilization of sparingly soluble phosphorous (Pi) in soil.Arabidopsis thaliana and Brassica oilcrops are closely related species in Brassicae. Agar screening, nutrient solution culture and pot culture were used to study differences in root hair initiation, Pi uptake and plant growth between wild type Columbia-0 and wer mutant to reveal the contribution of root hair to Pi absorption. According to sequence information of WEREWOLF(WER), a transcription factor controlling initiation of root hair in Arabidopsis, its homologous genes in Brassica napus (B.napus), Brassica rapa (B.rapa) and Brassica olereacea (B.olereacea) were cloned. Expression analysises of three BnWER paralogs at different P levels were conducted. The main results are as follows:1. Root hair growth of wer mutant and its association with Pi uptake at different P levelsRoot hair number of wer mutant was more than that of WT and both at low and high P levels; moreover, at the same P level, wer mutant could produce more root hairs or protuberance than that of WT. Although there was no significant difference in shoot dry weight and Pi content between WT and wer mutant at Plevel with 1.0μM by hydroponic culture, secretion amount of oxalic, citric acid and phytase activity of wer mutant were more/higher than that of WT, which indicated that wer mutant has stronger ability to produce and excrete organic acid. As KH2PO4 in nutrient solution is a kind of available P resource, thus, these root secretion substance could not promote Pi uptake in solution. However, plant shoot dry weight and growth of wer mutant at low Pi was more/better than that of WT, which suggested that more root hairs and root secretion could promote the utilization of sparingly soluble phosphorous (Pi) in soil.A lot of citric acid was detected in the root secretion at low Pi, whereas none was found at high Pi, indicating that citric acid would be a kind of specific root excretes organic acid induced by Pi deficiency. Oxalic and malic acid secretion amount increased with decreasing of Pi content in growth medium. Both are root excretes related to Pi efficiency.2. Isolation of WER homologs in B.napus, B.rapa and B. oleracea and expression analysis of BNWER paralogous genes at different P levelsA total of 10 WER homologous genes were isolated in Brassica, four in B.napus (BNWER1 to BNWER4), two in B.rapa (BRWER1 and BRWER2) and four in B.oleracea (BOWER1 to BOWER4). BNWER3 and BNWER4 are two new paralogs found in B. napus. Sequence of BRWER1 is the same as that of one fragment obtained from BAC:AC189227. Phylogenic analysis of these WER homologous genes as well as WER in Arabidopsis showed that they could be devided into two groups:Cluster 1 (BNWER1, BNWER3, BNWER2, BRWER1, BOWERl and BOWER4) and Cluster 2 (BNWER2, BNWER4, BRWER2, BOWER2 and BOWER3), and each cluster has the homologous genes from all of the three Brassica species, suggesting that the evolution of A and C geneome are very similar and complex.Expression analysis of BNWER2, BNWER3 and BNWER4 with semi-quantitative PCR and quantitative PCR showed that BnWERs were up-regulated genes induced by Pi deficiency, whereas CAPRICE (CPC), another transcription factor controlling root hair initiation was more strongly up-regulated than BnWER at low Pi. Up-regulated of BNWERs could inhibit root hair initiation, thus, the mutation of this gene would produce more root hairs.These results will be helpful to screen and identify Pi-efficient BNWER mutants in B. napus with TILLING... |
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