The Barley Heavy Metal Associated Isoprenylated Plant Protein HvFP1 Is Involved in a Crosstalk between the Leaf Development and Abscisic Acid-Related Drought Stress Responses.

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.

The zinc-binding nuclear protein HIPP3 acts as an upstream regulator of the salicylate-dependent plant immunity pathway and of flowering time in Arabidopsis thaliana.

Biotic 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 ISOPRENYLATED 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 salicylate-dependent pathway of pathogen response and is also involved in abiotic stress responses and seed and flower development.

An alternative strategy of dismantling of the chloroplasts during leaf senescence observed in a high-yield variety of barley.

Changes in function and composition of the photosynthetic apparatus as well as the ultrastructure of chloroplasts in mesophyll cells were analyzed in flag leaves of the high-yield barley (Hordeum vulgare) variety cv. Lomerit during senescence under field conditions in two successive years. In contrast to previous results obtained with the elder variety cv. Carina photosystem II efficiency measured by F(v)/F(m) was found to be rather stable until a very late stage of senescence. Chlorophyll a fluorescence and P700 absorbance measurements revealed that the activities of the two photosystems declined in parallel. An increase in the chlorophyll a/b ratio at a late stage of senescence was observed to coincide with a decline in the level of the Lhcb1 apoprotein of the light harvesting complex (LHC) and the level of the corresponding transcript. Ultrastructural investigations revealed the presence of gerontoplasts that have long, single or pairwise thylakoids and lack large grana stacks. It is hypothesized that the early degradation of grana thylakoids harboring the major LHC reduced the risk of photoinhibition and might be causally related to the high yield of the barley variety cv. Lomerit.

Nuclear targeted AtS40 modulates senescence associated gene expression in Arabidopsis thaliana during natural development and in darkness.

The Arabidopsis thaliana AtS40-3 gene belongs to a group of genes sharing the conserved DUF548 sequence motif with up to now unknown function. One member of this group, the barley HvS40, was shown before to play a role in regulation of leaf senescence. Similar as the barley gene, AtS40-3 is induced during senescence and is also regulated in response to dark treatment, ABA, salicylic acid and pathogen attack. By localization of the GUS fusion protein, the AtS40-3 gene was shown to encode a nucleus targeted protein. The s40-3a mutant with a T-DNA insertion in the promoter region of the gene was observed to have a staygreen phenotype. By quantitative real-time PCR analyses expression of the AtS40-3 gene in this mutant was observed to be constitutive and not induced during senescence. This coincided with WRKY53 gene expression in nonsenescent leaves and lowered expression levels of WRKY53 and SAG12 at later stages of development. While in the wildtype expression of AtS40-3 was activated by darkness, in the s40-3a mutant the expression of AtS40-3 stayed at a low level. This coincided with lower expression of the SEN1 and SAG12 genes. In another promoter mutant with a T-DNA insertion further upstream of the coding sequence the levels of AtS40-3 and SAG12 transcripts increased in parallel both in a natural light-dark regime and in darkness. The data on gene expression in promoter T-DNA insertion mutants of the s40-3 gene indicate that AtS40 regulates senescence either by modulation of WRKY53 or by activation of SAG12 independent of WRKY53.

Stress induced and nuclear localized HIPP26 from Arabidopsis thaliana interacts via its heavy metal associated domain with the drought stress related zinc finger transcription factor ATHB29.

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.

Isolation of a novel barley cDNA encoding a nuclear protein involved in stress response and leaf senescence.

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.