Downregulation of Solanum americanum genes encoding proteinase inhibitor II causes defective seed development

Abstract

SummaryProteinase inhibitor II proteins (PIN2) are serine proteinase inhibitors found in the Solanaceae. Here, we assign functions in seed development to two Solanum americanum genes, SaPIN2a and SaPIN2b, encoding proteinase inhibitor II. Their mRNAs and proteins have been previously localized to the reproductive tissues, including the inner cell layers of ovules in senescent flowers at the beginning of fruit development, suggestive of their endogenous roles in reproductive development. We have employed RNA interference (RNAi)-induced post-transcriptional gene silencing (PTGS) to further investigate the role of SaPIN2a and SaPIN2b during seed development. A SaPIN2a-derived construct that shared 83% nucleotide homology to SaPIN2b was used in PTGS to silence both genes. Northern blot analyses confirmed that the PIN2-RNAi transgenic plants contain small interfering RNAs (siRNAs) and exhibit reduced levels of SaPIN2a and SaPIN2b mRNAs at various stages of floral development. A reduction in seed set due to seed abortion was observed in PIN2-RNAi transgenic lines. Cytological and molecular analyses of these lines showed the lack of SaPIN2a and SaPIN2b mRNAs and proteins at the inner cell layers of the ovules in senescent flowers. Aborted seeds in transgenic fruits had an abnormal endothelium. The anomalous expansion of the endothelium prevented proper development of the endosperm and embryo, leading to seed abortion. Our observations indicate that SaPIN2a and SaPIN2b are essential for seed development and suggest that the endothelium may protect the embryo sac, allowing proper formation of the endosperm and embryo, as a result of its ability to produce proteinase inhibitors.

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.…”

“…Sin et al (2006) demonstrated that S. americanum silenced in the expression of PIN2a and PIN2b aborted around 80% of its seeds. Surprisingly, we found this effect to be very small in S. nigrum.…”

“…Lines transformed with the double RNAi construct irSnSPI1/2a+b were additionally silenced in SPI1 transcripts at levels of 16 and 31%. Sin et al (2006) reported an increase in flower size and an 80% seed abortion after silencing homologs of SPI2a and SPI2b with similar efficiency in S. americanum. 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).…”

“…Due to the focus on agricultural importance, most studies use insect herbivore feeding assays with transgenic crop plants ectopically overexpressing PIs or with artificial diet supplemented with isolated PIs. There are only a few publications that altered the expression of endogenous PIs in undomesticated native plants to study their relevance for plant defense in an ecological context (Zavala et al, 2004a(Zavala et al, , 2004bSin et al, 2006;Steppuhn and Baldwin, 2007;Steppuhn et al, 2008). In one study (Steppuhn et al, 2008), field experiments on the diverse natural herbivore community revealed that one PI is indeed an effective defense but only under specific circumstances and against particular insect species.…”

“…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.…”