Serpins in fruit and vegetative tissues of apple (Malus domestica): Expression of four serpins with distinct reactive centres and characterisation of a major inhibitory seed form, MdZ1b

Most serpins irreversibly inhibit serine proteinases of the chymotrypsin family using a suicide-substrate-based mechanism. Serpins are present in all domains of life, but physiological functions in the plant kingdom are yet to be elucidated. Inhibitory properties of many abundant cereal grain serpins are well characterised, but serpins have not been identified in eudicot seeds. In apple (Malus domestica Borkh.), the origin of 88 serpin expressed sequence tags (ESTs) identified among 160 000 ESTs from 30 cultivar-, tissue- and time-specific libraries showed that serpin genes are expressed in a wide variety of tissues, including developing and mature fruits, seeds and vegetative buds as well as developing, mature and senescing leaves. Analysis of 46 sequences, most full-length, identified serpins with four distinct reactive centres belonging to two subfamilies (MdZ1 and MdZ2) with ∼85% amino acid sequence identity. MdZ1 included three molecular forms with identical reactive centre loop (RCL) sequences except for three different, but related, residues at P₂ (Asp, Asn or Glu). A major seed serpin, MdZ1b, with P₂-P ₁′ Glu-Arg-Arg was purified from decorticated seeds and characterised kinetically. MdZ1b was a fast inhibitor of bovine and porcine trypsin (second-order association rate constant k_a ∼4 × 10^-6 M^-1 s^-1 and stoichiometry of inhibition SI = 1). Human plasmin and urokinase-type plasminogen activator (u-PA), but not thrombin, were inhibited at lower rates (k_a ∼10⁴ M^-1 s^-1). Chymotrypsin was inhibited at the same site (k_a ∼4 × 10³ M^-1 s^-1), but a significant part of MdZ1b was cleaved as substrate (SI > 2). Unexpectedly, the MdZ1b-trypsin complex was relatively short-lived with a first-order dissociation rate constant k_d in the order of 10^-4s ^-1. The bulk of mature seed MdZ1b was localised to the cotyledons. The content of MdZ1b in ripe apples was 5-26 μg per seed, whereas MdZ1b could not be detected in the cortex or skin. Localisation and inhibitory specificity of serpins in monocot and eudicot plants are compared and putative functions are discussed.