HIPP26 from Arabidopsis thaliana belongs to a novel class of plant proteins, characterized by a heavy metal associated domain and an additional isoprenylation motif. It is induced during cold, salt and drought stress. The nuclear localization of HIPP26, predicted by a NLS motif, could be confirmed in onion epidermal cells overexpressing GFP-HIPP26. Experiments with modified HIPP26 indicate that the isoprenylation plays a role in the spatial distribution in the nucleus. Using promoter-GUS constructs, a tissue specific expression pattern of HIPP26 could be shown, with high expression in the vascular tissue. By a yeast-two-hybrid approach a strong interaction of HIPP26 with the zinc finger homeodomain transcription factor ATHB29, which is known to play a role in dehydration stress response could be detected. This was confirmed by GST pull-down assays. When using a modified HIPP26 lacking the two central cysteines of the heavy metal associated domain, ATHB29 was not bound in the GST pull-down assay, indicating that this structure is necessary for the interaction. Further yeast-two-hybrid analyses testing interaction of different members of the HIPP family with related zinc finger transcription factors revealed a specific interaction of ATHB29 with several HIPP proteins. A functional relationship between HIPP26 and ATHB29 is also indicated by experiments with mutants of HIPP26 showing altered expression levels of such genes known to be regulated by ATHB29.

SummaryBiotic and abiotic stress responses of plants are linked to developmental programs. Proteins involved in different signaling pathways are the molecular basis of this concerted interplay. In our study, we show that Arabidopsis thaliana HEAVY METAL-ASSOCIATED ISOPRENYLAT-ED PLANT PROTEIN3 (HIPP3; At5g60800) acts as an upstream regulator of stress-and development-related regulatory networks.Localization, metal-binding and stress-responsive gene expression of HIPP3 were analyzed via microscopy, protein and inductively coupled plasma (ICP)-MS analyses and quantitative real-time PCR. In addition, transcriptome and phenotype analyses of plants overexpressing HIPP3 were used to unravel its function.Our data show that HIPP3 is a nuclear, zinc-binding protein. It is repressed during drought stress and abscisic acid (ABA) treatment and, similar to other pathogen-related genes, is induced after infection with Pseudomonas syringae pv. tomato. HIPP3 overexpression affects the regulation of > 400 genes. Strikingly, most of these genes are involved in pathogen response, especially in the salicylate pathway. In addition, many genes of abiotic stress responses and seed and flower development are affected by HIPP3 overexpression. Plants overexpressing HIPP3 show delayed flowering.We conclude that HIPP3 acts via its bound zinc as an upstream regulator of the salicylatedependent pathway of pathogen response and is also involved in abiotic stress responses and seed and flower development.

In order to isolate genes involved in the early acclimation of winter barley (Hordeum vulgare L. cv. Trixi) to a combined cold and light stress of 2 degrees C and 600 micromol m(-2) s(-1) restriction fragment differential display-polymerase chain reaction was performed. Impact of the cold-treatment on the leaves was characterized by measuring chlorophyll content and photosystem II efficiency. By this approach several cDNAs of genes that quickly and transiently up-regulated during early stages of the stress were identified. One of these genes (HvFP1) includes sequence motifs representing a heavy metal associated domain (HMA), nuclear localization signals (NLS) and a farnesylation motif. This gene is also induced at drought stress, during leaf senescence and after exposure to abscisic acid. Analysis of its spatial expression patterns in barley plants either grown at 21 or 2 degrees C showed that in contrast to the situation in leaves transcript level of this gene is high not only in cold-treated plants but also in controls kept at 21 degrees C in plant compartments enriched in meristematic tissues. The nuclear localization of the protein was confirmed by confocal laser scanning microscopy of epidermal onion cells after particle bombardment with chimeric HVFP1-GFP constructs. Using a construct with a modified farnesylation motif yielded a different pattern of nuclear distribution of the chimeric protein.

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Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed.

The heavy metal associated isoprenylated plant proteins (HIPPs) are characterized by at least one heavy metal associated (HMA) domain and a C-terminal isoprenylation motif. Hordeum vulgare farnesylated protein 1 (HvFP1), a barley HIPP, is upregulated during drought stress, in response to abscisic acid (ABA) and during leaf senescence. To investigate the role of HvFP1, two independent gain-of-function lines were generated. In a physiological level, the overexpression of HvFP1 results in the delay of normal leaf senescence, but not in the delay of rapid, drought-induced leaf senescence. In addition, the overexpression of HvFP1 suppresses the induction of the ABA-related genes during drought and senescence, e.g., HvNCED, HvS40, HvDhn1. Even though HvFP1 is induced during drought, senescence and the ABA treatment, its overexpression suppresses the ABA regulated genes. This indicates that HvFP1 is acting in a negative feedback loop connected to the ABA signaling. The genome-wide transcriptomic analysis via RNA sequencing revealed that the gain-of-function of HvFP1 positively alters the expression of the genes related to leaf development, photomorphogenesis, photosynthesis and chlorophyll biosynthesis. Interestingly, many of those genes encode proteins with zinc binding domains, implying that HvFP1 may act as zinc supplier via its HMA domain. The results show that HvFP1 is involved in a crosstalk between stress responses and growth control pathways.

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