Expression of HMA4 cDNAs of the zinc hyperaccumulator Noccaea caerulescens from endogenous NcHMA4 promoters does not complement the zinc-deficiency phenotype of the Arabidopsis thaliana hma2hma4 double mutant

Noccaea caerulescens (Nc) exhibits a very high constitutive expression of the heavy metal transporting ATPase, HMA4, as compared to the non-hyperaccumulator Arabidopsis thaliana (At), due to copy number expansion and altered cis-regulation. We screened a BAC library for HMA4 and found that HMA4 is triplicated in the genome of a N. caerulescens accession from a former Zn mine near La Calamine (LC), Belgium. We amplified multiple HMA4 promoter sequences from three calamine N. caerulescens accessions, and expressed AtHMA4 and different NcHMA4 cDNAs under At and Nc HMA4 promoters in the A. thaliana (Col) hma2hma4 double mutant. Transgenic lines expressing HMA4 under the At promoter were always fully complemented for root-toshoot Zn translocation and developed normally at a 2-mu M Zn supply, whereas the lines expressing HMA4 under Nc promoters usually showed only slightly enhanced root to shoot Zn translocation rates in comparison with the double mutant, probably owing to ectopic expression in the roots, respectively. When expression of the Zn deficiency responsive marker gene ZIP4 was tested, the transgenic lines expressing AtHMA4 under an NcHMA4-1-LC promoter showed on average a 7-fold higher expression in the leaves, in comparison with the double hma2hma4 mutant, showing that this construct aggravated, rather than alleviated the severity of foliar Zn deficiency in the mutant, possible owing to expression in the leaf mesophyll

Analysis of Arabidopsis thaliana HKT1 and Eutrema salsugineum/botschantzevii HKT1;2 Promoters in Response to Salt Stress in Athkt1:1 Mutant

Soil salinity imposes a serious threat to the productivity of agricultural crops. Among several other transporters, high-affinity K + transporter (HKT)’s play an important role in reducing the phytotoxicity of Na + . Expression of Eutrema salsugineum (a halophyte) HKT1;2 is induced upon salt exposure. To elucidate the role of its promoter, we compared the sequences of HKT1;2 promoters from E. salsugineum (1822 bp) and E. botschantzevii (1811 bp) with Arabidopsis thaliana HKT1;1 (846 bp) promoter. In silico analysis predicted several cis-acting regulatory elements (GT-1 elements, core motifs of DRE/CRT, MYC/MYB-recognition sites and ACGT elements). Activities of the three promoters were analyzed by measuring HKT1;1 and/or HKT1;2 transcript level in the Athkt1;1 mutant plants. NaCl tolerance of the transgenics was also assessed. Our results depicted that expressing either AtHKT1;1 or EsHKT1;2 coding regions under the control of AtHKT1;1 promoter, almost reversed the hypersensitivity of the mutant for salt, on contrarily, when AtHKT1;1 coding sequence expressed under either Es or EbHKT1;2 promoters did not. Changes in shoot Na + /K + concentrations under salt exposure is significantly consistent with the complementation ability of the mutant. The transcript concentration for genes under the control of either of Eutrema promoters, at control level was very less. This may suggest that either an important upstream response motif is missed or that A. thaliana misses a transcriptional regulator that is essential for salt-inducible HKT1 expression in Eutrema

Lysyl oxidase-like 3b is critical for proliferation of chondrogenic progenitor cells during Zebrafish craniofacial development

Vertebrate craniofacial development requires coordinated morphogenetic interactions between the extracellular matrix (ECM) and the differentiating chondrocytes essential for cartilage formation. Recent studies reveal a critical role for specific lysyl oxidases in ECM integrity required for embryonic development. We now demonstrate that loxl3b is abundantly expressed within the head mesenchyme of the zebrafish and is critically important for maturation of neural crest derived cartilage elements. Histological and ultrastructural analyses of cartilage elements in loxl3b morphant embryos reveal abnormal maturation of cartilage and altered chondrocyte morphology. Spatiotemporal analysis of craniofacial markers in loxl3b morphant embryos shows that cranial neural crest cells migrate normally into the developing pharyngeal arches but that differentiation and condensation markers are aberrantly expressed. We further show that the loxl3b morphant phenotype is not due to P53 mediated cell death but likely to be due to reduced chondrogenic progenitor cell proliferation within the pharyngeal arches. Taken together, these data demonstrate a novel role for loxl3b in the maturation of craniofacial cartilage and can provide new insight into the specific genetic factors important in the pathogenesis of craniofacial birth defects. © 2011 International Society of Matrix Biology