Expression, activation and processing of a novel plant milk-clotting aspartic protease in Pichia pastoris.

Galium verum, also known as Lady's Bedstraw or Cheese Rennet, is an herbaceous perennial plant traditionally used in cheese-making. We used RACE PCR to isolate novel enzymes from Galium verum with the ability to clot milk. This approach generated two cDNA sequences (named preprogaline A and B) encoding proteins displaying the typical plant aspartic protease primary structure. Preprogaline B was expressed in the yeast Pichia pastoris, after deleting and replacing its original signal peptide with the yeast α-factor signal peptide from Saccharomyces cerevisiae. The secreted recombinant protein was obtained by growing P. pastoris in YPD medium and had the ability to clot milk. The mature form of progaline B is a heterodimeric glycosylated enzyme, with a molecular weight of approximately 48 kDa, that contains a heavy (30.7 kDa) and a light (13.5 kDa) polypeptide chains linked by disulfide bonds. Western blot analysis revealed that progaline B is activated by the acidification of the yeast culture medium and that enzymatic activation requires two steps. First the precursor protein is cleaved into two polypeptide chains by partial removal of the plant-specific insert (PSI) present in plant aspartic proteases; this is later followed by propeptide removal. By altering the pH of the P. pastoris culture medium, we were able to obtain either active or inactive forms of the enzyme. Recombinant progaline B displayed a κ-casein hydrolysis pattern analogous to those produced by the animal and microbial coagulants currently used in the dairy industry, but it exhibited a different digestion profile on α- and ß-caseins. The plant protease progaline B displays milk-clotting activities suitable for the production of novel dairy products.

Indole-3-acetic acid and auxin herbicides up-regulate 9-cis-epoxycarotenoid dioxygenase gene expression and abscisic acid accumulation in cleavers (Galium aparine): interaction with ethylene.

Interaction between auxin and auxin-induced ethylene was suggested in previous work to up-regulate abscisic acid (ABA) biosynthesis in cleavers (Galium aparine) through stimulated cleavage of xanthophylls to xanthoxin, catalysed by 9-cis-epoxycarotenoid dioxygenase (NCED). Here, the effects of auxin on NCED gene expression were studied in relation to changes in ethylene synthesis and ABA levels. A gene from G. aparine shoot tissue was cloned based on sequence similarity to cloned NCED genes from tomato (LeNCED1), potato, Phaseolus, and Arabidopsis. When the roots of G. aparine plants were treated with 0.5 mM indole-3-acetic acid (IAA), IAA concentrations increased from 0.2 microM to 65 microM IAA in the shoot tissue after 3 h. Transient increases in GaNCED1 mRNA levels were detectable as early as 1 h after treatment and reached maximum values of 40-fold, relative to the control, after 3 h. Increases in GaNCED1 mRNA preceded increases in 1-aminocyclopropane-1-carboxylic acid and ethylene. Levels of ABA began to increase more slowly and, significantly, with a lag phase of 2 h, and reached levels 24-fold higher than those in controls after 24 h. GaNCED1 gene expression was also stimulated by auxin herbicides. The ethylene-releasing compound ethephon induced GaNCED1 transcript levels only moderately. In accordance with this, aminoethoxyvinylglycine and cobalt ions, which inhibit ethylene synthesis, only slightly affected the increase in GaNCED1 transcript levels by IAA. However, both ethylene inhibitors decreased IAA-induced ABA accumulation by up to 70%. This suggests that auxin and auxin-induced ethylene are involved in ABA accumulation. While auxin is the primary trigger for NCED gene expression, ethylene appears to enhance ABA biosynthesis, possibly by up-regulation of NCED activity post-transcriptionally.