Understanding the Role of Defective Invertases in Plants: Tobacco Nin88 Fails to Degrade Sucrose  

Abstract

Cell wall invertases (cwINVs), with a high affinity for the cell wall, are fundamental enzymes in the control of plant growth, development, and carbon partitioning. Most interestingly, defective cwINVs have been described in several plant species. Their highly attenuated sucrose (Suc)-hydrolyzing capacity is due to the absence of aspartate-239 (Asp-239) and tryptophan-47 (Trp-47) homologs, crucial players for stable binding in the active site and subsequent hydrolysis. However, so far, the precise roles of such defective cwINVs remain unclear. In this paper, we report on the functional characterization of tobacco (Nicotiana tabacum) Nin88, a presumed fully active cwINV playing a crucial role during pollen development. It is demonstrated here that Nin88, lacking both Asp-239 and Trp-47 homologs, has no invertase activity. This was further supported by modeling studies and site-directed mutagenesis experiments, introducing both Asp-239 and Trp-47 homologs, leading to an enzyme with a distinct Suc-hydrolyzing capacity. In vitro experiments suggest that the addition of Nin88 counteracts the unproductive and rather aspecific binding of tobacco cwINV1 to the wall, leading to higher activities in the presence of Suc and a more efficient interaction with its cell wall inhibitor. A working model is presented based on these findings, allowing speculation on the putative role of Nin88 in muro. The results presented in this work are an important first step toward unraveling the specific roles of plant defective cwINVs.

Multifunctional fructans and raffinose family oligosaccharides

“…These residues are essential to stabilize the Glc part of Suc in the active site of GH32 Suc splitting enzymes (CWINV, VI, 1-SST, 6-SFT; Van den Ende et al, 2009), and they are absent in enzymes that use fructans as donor substrates (FEH, 1-FFT, 6G-FFT). This was confirmed by site directed mutagenesis experiments on invertase, defective invertase, FEH and 6G-FFT (Le Roy et al, 2007, 2008, 2013; Lasseur et al, 2009). However, the presence of an Asp/Lys or Asp/Arg is not sufficient; this couple needs to be in the right 3D configuration as well (Schroeven et al, 2009).…”

“…However, a recent breakthrough paper (Le Roy et al, 2013) shows that Nin88, an apoplastic defective invertase from tobacco lacking FEH side activities, acts as indirect activator of active cell wall invertases (CWIs) which are crucial players in overall plant development, especially seed and fruit setting (Ruan et al, 2012). Although the exact underlying regulatory mechanisms require further research, data indicate that Nin88 interacts with cell walls in such a way that active CWIs bind to the cell wall in a more productive way (Le Roy et al, 2013). This fits nicely within the emerging concept that dead enzymes are very common in all kingdoms of life and that many of them fulfil crucial biological roles, as reviewed in a recent Science paper (Leslie, 2013).…”

“…Intriguingly, defective invertases are never affected in their catalytic triad, but rather in a neighboring “Asp/Lys” or “Asp/Arg couple” (present in a flexible loop in the proximity of the acid/base catalyst) and in some Trp residues (Le Roy et al, 2007, 2013). These residues are essential to stabilize the Glc part of Suc in the active site of GH32 Suc splitting enzymes (CWINV, VI, 1-SST, 6-SFT; Van den Ende et al, 2009), and they are absent in enzymes that use fructans as donor substrates (FEH, 1-FFT, 6G-FFT).…”

“…Within the basal eudicots, a type I VI developed into a pFT in Pachysandra terminalis ( Figure 2 ) and this is considered as a rather “recent” evolutionary event (Van den Ende et al, 2011a). On the contrary, defective invertases and FEHs evolved from CWIs within GH32 (Le Roy et al, 2007, 2013). It can be speculated that the loss or alteration of the above-mentioned “couple” is an early evolutionary event that led to the formation of defective invertases with cell wall localization and a high iso-electric point (pI) for interaction with the cell wall.…”

Identification of metabolites involved in heat stress response in different tomato genotypes

“…Koch 2004 reviewed the role of acid invertase in plants; Three different acid invertases can be identified according to their location: vacuolar invertase (VIN), cytoplasmic invertase (CIN) and cell wall invertase (CWIN). The function of acid invertases in reproductive tissues has been intensely studied in different species such as lily Knox 1984, Castro andClement 2007), tobacco (Goetz et al 2001, Le Roy et al 2013, tomato (Pressman et al 2012) and chilli pepper (García et al 2013). …”