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.

RNA-Guided Cas9-Induced Mutagenesis in Tobacco Followed by Efficient Genetic Fixation in Doubled Haploid Plants.

Customizable endonucleases are providing an effective tool for genome engineering. The resulting primary transgenic individuals (T0) are typically heterozygous and/or chimeric with respect to any mutations induced. To generate genetically fixed mutants, they are conventionally allowed to self-pollinate, a procedure which segregates individuals into mutant heterozygotes/homozygotes and wild types. The chances of recovering homozygous mutants among the progeny depend not only on meiotic segregation but also on the frequency of mutated germline cells in the chimeric mother plant. In Nicotiana species, the heritability of Cas9-induced mutations has not been demonstrated yet. RNA-guided Cas9 endonuclease-mediated mutagenesis was targeted to the green fluorescent protein (GFP) gene harbored by a transgenic tobacco line. Upon retransformation using a GFP-specific guide RNA/Cas9 construct, the T0 plants were allowed to either self-pollinate, or were propagated via regeneration from in vitro cultured embryogenic pollen which give rise to haploid/doubled haploid plants or from leaf explants that form plants vegetatively. Single or multiple mutations were detected in 80% of the T0 plants. About half of these mutations proved heritable via selfing. Regeneration from in vitro cultured embryogenic pollen allowed for homozygous mutants to be produced more efficiently than via sexual reproduction. Consequently, embryogenic pollen culture provides a convenient method to rapidly generate a variety of genetically fixed mutants following site-directed mutagenesis. The recovery of a mutation not found among sexually produced and analyzed progeny was shown to be achievable through vegetative plant propagation in vitro, which eventually resulted in heritability when the somatic clones were selfed. In addition, some in-frame mutations were associated with functional attenuation of the target gene rather than its full knock-out. The generation of mutants with compromised rather than abolished gene functionality holds promise for future approaches to the conclusive functional validation of genes which are indispensible for the plant.