Flooding of the apoplast is a key factor in the development of hyperhydricity.

The physiological disorder hyperhydricity occurs frequently in tissue culture and causes several morphological abnormalities such as thick, brittle, curled, and translucent leaves. It is well known that hyperhydric shoots are characterized by a high water content, but how this is related to the abnormalities is not clear. It was observed that water accumulated extensively in the apoplast of leaves of hyperhydric Arabidopsis seedlings and flooded apoplastic air spaces almost completely. In hyperhydric Arabidopsis seedlings, the volume of apoplastic air was reduced from 85% of the apoplast to only 15%. Similar results were obtained with hyperhydric shoots of statice. The elevated expression of hypoxia-responsive genes in hyperhydric seedlings showed that the water saturation of the apoplast decreased oxygen supply. This demonstrates a reduced gas exchange between the symplast and its surroundings, which will consequently lead to the accumulation of gases in the symplast, for example ethylene and methyl jasmonate. The impairment of gas exchange probably brings about the symptoms of hyperhydricity. Interestingly, stomatal aperture was reduced in hyperhydric plants, a previously reported response to injection of water into the apoplast. Closure of the stomata and the accumulation of water in the apoplast may be the reasons why seedlings with a low level of hyperhydricity showed improved acclimatization after planting into soil.

Comparative analysis of LEA-like 11-24 gene expression and regulation in related plant species within the Linderniaceae that differ in desiccation tolerance.

The resurrection plant Craterostigma plantagineum is able to withstand desiccation of its vegetative tissues and is found in areas with variable water availability. The closely related species Lindernia brevidens and Lindernia subracemosa are both endemic to montane rainforests of coastal Africa, but remarkably L. brevidens is tolerant to desiccation. We studied the regulation of the desiccation-related LEA-like 11-24 gene at multiple levels in closely related species in order to investigate the conservation of mechanisms involved in desiccation tolerance. The dehydration-responsive transcription of the LEA-like 11-24 gene is differentially regulated in these plants. Comparison of the LEA-like 11-24 core promoter regions revealed that promoters have different activities, but some functional cis-acting elements are conserved between species. Upon dehydration, LEA-like 11-24 proteins are phosphorylated at different levels and phosphorylation sites are not conserved among the three LEA-like 11-24 proteins. Differences in the regulation of the LEA-like 11-24 gene in the studied plant species appear to be the result of mutations that occurred during evolution. We postulate that L. brevidens will eventually lose the ability to survive vegetative desiccation, given that this trait appears not to be essential for survival.

Lindernia brevidens: a novel desiccation-tolerant vascular plant, endemic to ancient tropical rainforests.

A particular adaptation to survival under limited water availability has been realized in the desiccation-tolerant resurrection plants, which tend to grow in a habitat with seasonal rainfall and long dry periods. One of the best-studied examples is Craterostigma plantagineum. Here we report an unexpected finding: Lindernia brevidens, a close relative of C. plantagineum, exhibits desiccation tolerance, even though it is endemic to the montane rainforests of Tanzania and Kenya, where it never experiences seasonal dry periods. L. brevidens has been found exclusively in two fragments of the ancient Eastern Arc Mountains, which were protected from the devastating Pleistocene droughts by the stable Indian Ocean temperature. Analysis of the microhabitat reveals that L. brevidens is found in the same habitat as hygrophilous plant species, which further indicates that the plant never dries out completely. The objective of this investigation was to address whether C. plantagineum and L. brevidens have desiccation-related pathways in common, or whether L. brevidens has acquired novel pathways. A third, closely related, desiccation-sensitive species, Lindernia subracemosa, has been included for comparison. Mechanisms that confer cellular protection during extreme water loss are well conserved between C. plantagineum and L. brevidens, including the interconversion of 2-octulose to sucrose within the two desiccation-tolerant species. Furthermore, transcriptional control regions of desiccation-related genes belonging to the late embryogenesis abundant (LEA) protein family are also highly conserved. We propose that L. brevidens is a neoendemic species that has retained desiccation tolerance through genome stability, despite tolerance being superfluous to environmental conditions.

Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression.

Aquaporins facilitate the uptake of soil water and mediate the regulation of root hydraulic conductivity (Lp(r)) in response to a large variety of environmental stresses. Here, we use Arabidopsis (Arabidopsis thaliana) plants to dissect the effects of salt on both Lp(r) and aquaporin expression and investigate possible molecular and cellular mechanisms of aquaporin regulation in plant roots under stress. Treatment of plants by 100 mm NaCl was perceived as an osmotic stimulus and induced a rapid (half-time, 45 min) and significant (70%) decrease in Lp(r), which was maintained for at least 24 h. Macroarray experiments with gene-specific tags were performed to investigate the expression of all 35 genes of the Arabidopsis aquaporin family. Transcripts from 20 individual aquaporin genes, most of which encoded members of the plasma membrane intrinsic protein (PIP) and tonoplast intrinsic protein (TIP) subfamilies, were detected in nontreated roots. All PIP and TIP aquaporin transcripts with a strong expression signal showed a 60% to 75% decrease in their abundance between 2 and 4 h following exposure to salt. The use of antipeptide antibodies that cross-reacted with isoforms of specific aquaporin subclasses revealed that the abundance of PIP1s decreased by 40% as early as 30 min after salt exposure, whereas PIP2 and TIP1 homologs showed a 20% to 40% decrease in abundance after 6 h of treatment. Expression in transgenic plants of aquaporins fused to the green fluorescent protein revealed that the subcellular localization of TIP2;1 and PIP1 and PIP2 homologs was unchanged after 45 min of exposure to salt, whereas a TIP1;1-green fluorescent protein fusion was relocalized into intracellular spherical structures tentatively identified as intravacuolar invaginations. The appearance of intracellular structures containing PIP1 and PIP2 homologs was occasionally observed after 2 h of salt treatment. In conclusion, this work shows that exposure of roots to salt induces changes in aquaporin expression at multiple levels. These changes include a coordinated transcriptional down-regulation and subcellular relocalization of both PIPs and TIPs. These mechanisms may act in concert to regulate root water transport, mostly in the long term (> or =6 h).

Production of bifunctional proteins by Aspergillus awamori: llama variable heavy chain antibody fragment (V(HH)) R9 coupled to Arthromyces ramosus peroxidase (ARP).

The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5'- or 3'-terminal ends of the gene encoding llama variable heavy chain antibody fragment V(HH) R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which produced up to 30 mgl(-1) fusion protein in the culture medium. Both fusion proteins showed peroxidase activity in an ABTS activity test. Considerable amounts of fusion protein were detected intracellularly, suggesting that the fungus encounters problems in secreting these kind of proteins. ELISA experiments showed that ARP-R9 was less able to bind its antigen, the azo-dye RR6, as compared to R9-ARP. Furthermore, in contrast to R9-ARP, ARP-R9 bound to RR6 did not show peroxidase activity anymore. These results indicate that fusion of ARP to the C-terminus of the antibody fragment V(HH) R9 (R9-ARP) is the preferred orientation.