Chloroplast-Like Organelles Were Found in Enucleate Sieve Elements of Transgenic Plants Overexpressing a Proteinase Inhibitor

Abstract

SaPIN2a, a plant proteinase inhibitor from nightshade (Solanum americanum), was located to the enucleate sieve elements (SEs) of phloem. The expressed SaPIN2a in transgenic lettuce showed inhibition of plant endogenous trypsin- and chymotrypsin-like activities, suggesting that SaPIN2a can regulate proteolysis in plant cells. To further investigate the physiological role of SaPIN2a, we produced transgenic nightshade and lettuce plants overexpressing SaPIN2a from the cauliflower mosaic virus (CaMV) 35S promoter using Agrobacterium-mediated transformation. Overexpression of SaPIN2a in transgenic plants was demonstrated by northern blot and western blot analysis. SaPIN2a-overexpressing transgenic nightshade plants showed significantly lower height than wild-type plants. Transmission electron microscopy analysis showed that chloroplast-like organelles with thylakoids, which are not present in enucleate SEs of wild-type plants, were present in the enucleate SEs of SaPIN2a-overexpressing transgenic plants. This finding is discussed in terms of the possible role played by SaPIN2a in the regulation of proteolysis in SEs.

Serine Protease Inhibitors Specifically Defend Solanum nigrum against Generalist Herbivores but Do Not Influence Plant Growth and Development

“…Moreover, we could not observe any other PIdependent developmental phenotype that had been reported for other species. There was no significant difference in flower diameter (Sin et al, 2006), number of reproductive units (Zavala et al, 2004a), plant growth (Zavala et al, 2004a;Xie et al, 2007), or trichome morphology and density (Liu et al, 2006;Luo et al, 2009). As a consequence, we infer that the developmental importance of SPI2a and SPI2b differs between species.…”

“…However, in S. nigrum, we did not observe any effect on flower size and found that only 0.7 to 2.8% of the seeds were aborted or defective (see Supplemental Figure 3 online). Zavala et al (2004a) and Xie et al (2007) reported that silencing or ectopic overexpression of SPI genes can affect plant growth. In comparative growth experiments, we found no differences in plant height between wild-type and SPI-silenced plants (see Supplemental Figure 3 online).…”

“…The increased accumulation of SPI2a and SPI1 in stem and midribs suggests a function in vascular tissues. Sa PIN2a, a homolog of Sn SPI2a from S. americanum, is localized in the phloem (Xu et al, 2001), and ectopic overexpression of this gene results in chloroplast formation in enucleate sieve elements (Xie et al, 2007). However, it cannot be ruled out that PIs accumulate in these tissues also as a defense response against phloem sap-feeding insects.…”

“…There is evidence that plant PIs protect specific tissues, act as storage proteins, regulate the activity of proteases, and direct their release (Ryan, 1973;Mosolov and Valueva, 2005). Plants altered in the expression of endogenous PIs display changes in growth rate (Zavala et al, 2004a;Xie et al, 2007), flower morphology and seed development (Sin et al, 2006), phloem structure (Xie et al, 2007), and nectar protein composition (Bezzi et al, 2010). These alterations in fitness-relevant traits showed that members of the potato inhibitor type II family (PI-II), formerly associated with defense, can have other physiological functions.…”

Plant Bioreactors for Pharmaceuticals

“…Zavala et al (2004) have shown that constitutive overexpression of trypsin PI (TPI) in transgenic Nicotiana attenuata incurred large fitness costs, resulting in a large reduction in growth rate, plant height and seed production. Overexpression of a sieve element-localized PI of Solanum americanum, SaPIN2a, also caused growth retardation in transgenic plants, which might be due to the impaired differentiation of sieve elements in transgenic plant phloem (Xie et al, 2007).…”