Barley cysteine protease PAP14 plays a role in degradation of chloroplast proteins.

Chloroplast protein degradation is known to occur both inside chloroplasts and in the vacuole. Genes encoding cysteine proteases have been found to be highly expressed during leaf senescence. However, it remains unclear where they participate in chloroplast protein degradation. In this study HvPAP14, which belongs to the C1A family of cysteine proteases, was identified in senescing barley (Hordeum vulgare L.) leaves by affinity enrichment using the mechanism-based probe DCG-04 targeting cysteine proteases and subsequent mass spectrometry. Biochemical analyses and expression of a HvPAP14:RFP fusion construct in barley protoplasts was used to identify the subcellular localization and putative substrates of HvPAP14. The HvPAP14:RFP fusion protein was detected in the endoplasmic reticulum and in vesicular bodies. Immunological studies showed that HvPAP14 was mainly located in chloroplasts, where it was found in tight association with thylakoid membranes. The recombinant enzyme was activated by low pH, in accordance with the detection of HvPAP14 in the thylakoid lumen. Overexpression of HvPAP14 in barley revealed that the protease can cleave LHCB proteins and PSBO as well as the large subunit of Rubisco. HvPAP14 is involved in the normal turnover of chloroplast proteins and may have a function in bulk protein degradation during leaf senescence.

Identification of predominant genes involved in regulation and execution of senescence-associated nitrogen remobilization in flag leaves of field grown barley.

The transcriptomes of senescing flag leaves collected from barley field plots with standard or high nitrogen supply were compared to identify genes specifically associated with nitrogen remobilization during leaf senescence under agronomically relevant conditions. In flag leaves collected in field plots with high nitrogen supply, the decline in chlorophyll content was delayed. By comparing changes in gene expression for the two nitrogen levels, it was possible to discriminate genes related to nitrogen remobilization during senescence and genes involved in other processes associated with the late development of leaves under field conditions. Predominant genes that were more strongly upregulated during senescence of flag leaves from plants with standard nitrogen supply included genes encoding the transcription factor HvNAC026, serine type protease SCPL51, and the autophagy factors APG7 and ATG18F. Elevated expression of these genes in senescing leaves from plants with standard nitrogen supply indicates important roles of the corresponding proteins in nitrogen remobilization. In comparison, the genes upregulated in both flag leaf samples might have roles in general senescence processes associated with late leaf development. Among these genes were the transcription factor genes HvNAC001, HvNAC005, HvNAC013, HvWRKY12 and MYB, genes encoding the papain-like cysteine peptidases HvPAP14 and HvPAP20, as well as a subtilase gene.

The Tr-cp 14 cysteine protease in white clover (Trifolium repens) is localized to the endoplasmic reticulum and is associated with programmed cell death during development of tracheary elements.

Cysteine proteases are known to be associated with programmed cell death, developmental senescence and some types of pathogen and stress-induced responses. In the present study, we have characterized the cysteine protease Tr-cp 14 in white clover (Trifolium repens). Tr-cp 14 belongs to the C1A family of cysteine proteases with homology to XCP1 and XCP2 from Arabidopsis thaliana and p48h-17 from Zinnia elegans, which previously have been reported to be associated with tracheary element differentiation. The proform as well as the processed form of the protein was detected in petioles, flowers and leaves, but the processed form was more abundant in leaves and petioles than in flowers. The Tr-cp 14 protein was localized to differentiating tracheary elements within the xylem, indicating that the cysteine protease is involved in protein re-mobilization during tracheary element differentiation. Immunogold studies suggest that the protease prior to the burst of the vacuole was associated to the ER cisternae. After disruption of the tonoplast, it was found in the cytoplasm, and, in later stages, associated with disintegrating material dispersed throughout the cell.

A putative role for amino acid permeases in sink-source communication of barley tissues uncovered by RNA-seq.

BACKGROUND: The majority of nitrogen accumulating in cereal grains originates from proteins remobilised from vegetative organs. However, interactions between grain filling and remobilisation are poorly understood. We used transcriptome large-scale pyrosequencing of flag leaves, glumes and developing grains to identify cysteine peptidase and N transporter genes playing a role in remobilisation and accumulation of nitrogen in barley. RESULTS: Combination of already known and newly derived sequence information reduced redundancy, increased contig length and identified new members of cysteine peptidase and N transporter gene families. The dataset for N transporter genes was aligned with N transporter amino acid sequences of rice and Arabidopsis derived from Aramemnon database. 57 AAT, 45 NRT1/PTR and 22 OPT unigenes identified by this approach cluster to defined subgroups in the respective phylogenetic trees, among them 25 AAT, 8 NRT1/PTR and 5 OPT full-length sequences. Besides, 59 unigenes encoding cysteine peptidases were identified and subdivided into different families of the papain cysteine peptidase clade. Expression profiling of full-length AAT genes highlighted amino acid permeases as the group showing highest transcriptional activity. HvAAP2 and HvAAP6 are highly expressed in vegetative organs whereas HvAAP3 is grain-specific. Sequence similarities cluster HvAAP2 and the putative transporter HvAAP6 together with Arabidopsis transporters, which are involved in long-distance transfer of amino acids. HvAAP3 is closely related to AtAAP1 and AtAAP8 playing a role in supplying N to developing seeds. An important role in amino acid re-translocation can be considered for HvLHT1 and HvLHT2 which are specifically expressed in glumes and flag leaves, respectively. PCA and K-means clustering of AAT transcript data revealed coordinate developmental stages in flag leaves, glumes and grains. Phloem-specific metabolic compounds are proposed that might signal high grain demands for N to distantly located plant organs. CONCLUSIONS: The approach identified cysteine peptidases and specific N transporters of the AAT family as obviously relevant for grain filling and thus, grain yield and quality in barley. Up to now, information is based only on transcript data. To make it relevant for application, the role of identified candidates in sink-source communication has to be analysed in more detail.