Degradation of chloroplast proteins - identification and characterization of senescence associated cysteine proteases
Final Report Abstract
The objective of this project was to identify proteases involved in degradation of chloroplast proteins during senescence of barley leaves. Two different approaches were combined. In the first approach, a microarray was used for analyses of gene expression in senescent and control flag leaves collected from barley field plots fertilized either with standard nitrogen supply (SN) or with a threefold higher nitrogen supply (HN). Members of several groups of proteases were found to be upregulated during senescence. Most of the upregulated genes were found to encode cysteine proteases. Among the 10 differentially expressed cysteine protease genes, HvPAP14 showed highest expression in senescent flag leaves collected from fields with standard nitrogen supply. In a second approach, active cysteine proteases were enriched from extracts of non-senescent and senescent barley leaves by their affinity towards DCG-04, a biotin-coupled analogue of E64 which binds to the active sites of cysteine proteases. Mass spectrometry revealed that senescent leaves contained active HvPAP14 and the well known senescence marker HvSAG12. The sequence of HvPAP14 has an N-terminal signal peptide and a C-terminal ER retention sequence. To investigate its subcellular localization, the HvPAP14 sequence was fused to the sequence of the fluorescent reporter protein RFP. After transient transformation of protoplasts, the fluorescent protein was mainly detected in the ER and in vesicles. However, when the same construct was overexpressed in barley plants, fluorescence was found to be associated mainly with vesicles and chloroplasts. To further investigate the subcellular localization of HvPAP14, antibodies were raised towards two peptides deduced from the N-terminal inhibitor region as well as the C-terminal part containing the active site of the enzyme. Immunoblot analyses with the antibody detecting all forms of the enzyme confirmed localization in chloroplasts. There, the processed protein was found to be associated with thylakoids. Immunoblot analyses with the antibody detecting only forms including the inhibitory region, revealed that the precursor, accumulating in senescing leaves, is mainly located outside of chloroplasts. Immunogold labeling studies confirmed the association of the proenzyme and processed forms of HvPAP14 with thylakoids and moreover detected labeling inside vesicles. To investigate the properties of HvPAP14 in more detail, the sequence was overexpressed in E. coli. In vitro assays with the recombinant protein showed that activation by cleavage of the inhibitory domain occurred at a pH of 4.5-5 whereas the activated enzyme is capable to cleave artificial substrates over a broad range of pH values. Activation by low pH is in accordance with the detection of the proenzyme in association with thylakoids. While senescence-associated upregulation of the gene encoding HvPAP14 was largely independent of the nitrogen supply, immunoblot analyses revealed that accumulation of the proenzyme occurs only under nitrogen-limiting conditions and is suppressed under high nitrogen conditions. This indicates that accumulation of the proenzyme is regulated by nitrogen at the posttranscriptional/translational level. Activation occurs only in senescing leaves. Transgenic barley plants overexpressing the HvPAP14 gene are currently prepared. It is expected that the putative substrate/s of the protease will be degraded faster during senescence only at nitrogen-limiting conditions. It is expected that a higher level of HvPAP14 will increase protein degradation in chloroplasts.
Publications
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(2012) A new strategy of the dismantling of chloroplasts during leaf senescence in a high yield variety of barley. Physiol Plant 144:189-200
Krupinska K, Mulisch M, Hollmann J, Tokarz K, Zschiesche W, Kage H, Humbeck K, Bilger W
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(2012) A putative role for amino acid permeases in sinksource communication of barley tissues uncovered by RNA-seq. BMC Plant Biol 12: 154
Kohl S, Hollmann J, Blattner FR, Radchuk V, Andersch F, Steuernagel B, Schmutzer T, Scholz U, Krupinska K, Weber H, Weschke W
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(2012) The Tr-cp 14 cysteine protease in white clover (Trifolium repens) is localized to the ER and associated with programmed cell death during development of tracheary elements. Protoplasma 250:623-629
Mulisch M, Asp T, Krupinska K, Hollmann J, Holm PB
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(2013) Plant senescence and crop productivity. Plant Mol Biol 82:603-622
Gregersen P, Culetic A, Boschian L, Krupinska K
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(2014) Identification of predominant genes involved in regulation and execution of senescence-associated nitrogen remobilization in flag leaves of field grown barley. J Exp Bot 65: 3963-3973
Hollmann J, Gregersen PL, Krupinska K
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(2015) Generation of reactive oxygen species in thylakoids from senescing flag leaves of the barley varieties Lomerit and Carina, Planta 241:1497-1508
Krieger-Liszkay A, Trösch M, Krupinska K
