Characterization Of The Arabidopsis Acs7-1Mutant And Functional Analyses Of ACS-like Genes In Physcomitrella Patens

The phytohormone ethylene plays essential roles in plant growth and development, and is involved in multiple biotic and abiotic stress responses.The ethylene biosynthesis pathway has been well documented in vascular plants. The key enzyme is1-Amino-cyclopropane-l-carboxylate (ACC) synthase (ACS), which catalyzes the formation of ethylene precursor, ACC. ACS is encoded by a multi-gene family. In the model plant Arabidopsis thaliana, there are nine authentic ACS genes. The specific functions and regulatory mechanisms of each gene in plant development and stress adaptation were still not well described.Bryophytes, the early diverging land plants, represent a lineage distinct from vascular plants in the evolutionary history of the plant kingdom, because they are non-vascular. Results of sequence alignments show that the genome of the model plant Physcomitrella patens (P. patens) codes for two putative ACS-like (PpACLs) proteins. However, it is still unclear whether these two PpACLs function as ACC synthases; and further, whether the ethylene biosynthesis pathway conserved in vascular plants exists in P. patens.This study comprises two parts.In the first part, the T-DN A insertion mutants of multiple Arabidopsis ACS genes were characterized. We have previously shown that loss of A CS2,4,8, or9weaken abiotic stress tolerance, while loss of ACS7enhances heat and salt stress tolerance in Arabidopsis. In this study, we tried to further investigate the underlying molecular mechanisms. It was found that loss of ACS7inhibited the expression of ACS2,4, ACS5and ACS6in acs7-1mutant, resulting in a much lower ethylene emission than its wild-type control. Consistently, the transcript levels of ethylene-responsive genes, including ERF1, ERF2and AtEBP/ERF72, were reduced in the acs7mutant. Heat shock proteins (HSPs), induced by heat shock transcription factors (HSFs), are molecular chaperones that can protect cellular proteins against irreversible heat-induced denaturation and help in the refolding of heat-damaged proteins. Transcript analyses revealed that the higher and earlier induction of HSFs and HSPs under heat stress contributed to the enhanced thermo-tolerance of acs7-1. Compared to its wild-type control, acs7-1had elevated transcript levels of stress-responsive genes involved in the ABA-dependent pathway and higher salt induced ABA accumulation following salt treatment, indicating the important functions of ABA synthesis and signaling in enhanced salt stress tolerance of acs7. When treated with ABA biosynthesis inhibitor or ABA analogue, acs7became even more sensitive to salt than its wild-type control, which was consistent with the positive role of ethylene in salinity adaptation. Combined, these results suggested that ACS7acts as a negative regulator of ABA accumulation and sensitivity under abiotic stress and appears as a node in the cross-talk between ethylene and ABA. Besides, ACS7is involved in seed germination, leaf expansion and primary root elongation in Arabidopsis.Phenotypic analyses of other ACS knockout mutants, including acs2-2, acs4-3, acs8-3and acs9-1, showed that ACS8positively regulated the IAA-mediated lateral root formation, and both ACS4and ACS8inhibited ABA-mediated inhibition of seed germination. In addition, a SAIL T-DNA insertion line of A CS5, named acs5-3, was found to produce more ethylene than its wild-type control. While expression of the full-length ACS5gene was not detected, a5’-truncated ACS5was activated in acs5-3, leading to increased ethylene production and enhanced ethylene responses. The etiolated seedlings of acs5-3showed inhibited root elongation while the light-grown seedlings had more root hairs and shorter primary root than its wild-type control. The adult mutant displayed accelerated bolting, flowering and leaf senescence phenotypes. The size of rosette leaves was not affected in acs5-3, however, slightly downward-curled leaves were observed. Overexpression of the truncated ACS5rendered hypersensitivity to exogenous auxin. Upon auxin treatment, the acs5-3mutant exhibited more lateral root formation than its wild type control. All these phenotypes could be reversed by AVG, suggesting that the N-terminal end of ACS5is not essential for its catalytic activity.In the second part, we investigated the functions of two PpACLs. Based on the genome information of database Phytozome, the full-length cDNAs of PpACLl and PpACL2were clone form the leafy gametophores of P. patens. To investigate whether PpACLl has ACS activity in vitro, His-tagged PpACL1was expressed in E. coli train BL21(DE3)pLysS. The empty vector and His-tagged AtACS7were used as negative and positive control, respectively. It was found that the level of extracellular ACC excreted by E.coli harboring His-tagged PpACLl was not increased. The purified PpACLl protein also could not convert AdoMet into ACC in vitro. To investigate whether PpACL1has ACS activity in vivo, PpACLl gene was introduced into Arabidopsis under control of the CaMV35S promoter. Neither increases in ethylene production nor ethylene-induced triple-response was found in the35S::PpACL1transgenic etiolated seedlings. These results suggested that PpACL1had no ACS activity.The PLP-dependent AAT-like proteins, including ACS, aminotransferase and CS lyase, have high sequence similarity. Some of them were incorrectly annotated in the past. Thus, we further analyzed the aminotransferase and CS lyase activities of the purified recombinant PpACL1protein. The enzymatic activity assays in vitro revealed that PpACLl had no aminotransferase activity, but acted as a CS lyase, catalyzing the cleavage of L-cysteine and L-cystine.Furthermore, a preliminary analysis of the function of PpACL2was carried out. Expression of His-tagged PpACL2in E.coli could not increase the ACC release, implying that PpACL2was not a ACC synthase either. futher studies are needed to characterize the function of PpACL2gene.Based on the observation that both PpACLl and PpACL2had no ACS activity, we postulated that the ethylene biosynthesis pathway, if existed in P. patens, might be different from that in vascular plants.