Two similar but distinct second intron fragments from tobacco AGAMOUS homologs confer identical floral organ-specific expression sufficient for generating complete sterility in plants

Abstract

The carpel- and stamen-specific AtAGIP promoter derived from the Arabidopsis AGAMOUS (AG) second intron/enhancer is ideal for engineering complete sterility but it is highly host-specific. To ascertain whether a chimeric promoter with similar tissue specificity can be created for species other than Arabidopsis, we isolated two similar but distinct AG second intron/enhancers from tobacco (NtAGI-1 and NtAGI-2) and analyzed their ability to drive floral organ-specific expression in plants through the creation of forward- and reverse-oriented chimeric promoters, fNtAGIP1, rNtAGIP1, fNtAGIP2 and rNtAGIP2. Analyses of transgenic plants bearing each respective promoter fused to the beta-glucuronidase (GUS) reporter gene showed that all four promoters are able, like the AtAGIP, to drive very similar carpel- and stamen-specific expression without any leaky activity in vegetative tissues. These results indicate that unlike their counterparts in rice and maize, the tobacco NtAGI-1 and NtAGI-2 enhancers share a highly conserved regulatory function. Interestingly, all four promoters display additional tissue specificity in petals, and their activity is influenced by the orientation of the incorporated enhancer, with reverse-oriented enhancers exhibiting approximately double the effectiveness of forward-oriented enhancers. These properties are novel and have not been observed with the AtAGIP promoter in Arabidopsis. As expected, these highly specific promoters can also direct the expression of the DT-A cytotoxic gene exclusively in carpels, stamens and petals, resulting in complete sterility through the precise ablation of targeted floral organs. Further analyses demonstrated that the resulting trait is mitotically stable, which is critical for the long-term containment of seed-, pollen- and fruit-mediated gene flow in field conditions.

Highly interactive nature of flower‐specific enhancers and promoters, and its potential impact on tissue‐specific expression and engineering of multiple genes or agronomic traits

“…As expected, 94% of rNtAGIP1::DT‐A plants produced floral buds devoid of visible petals, stamens and carpels (Figures b and c,k) in comparison with normal vegetative and floral development exhibited in wild‐type ( Wt ) plants (Figure a,g,h), confirming the tissue specificity of this enhancer/promoter (Yang et al ., ). Also as expected, almost all (91%) of the pBASE ACT ‐transformed plants displayed normal vegetative growth but a self‐sterile phenotype (Figures b and b) with ablated stigmas (Figure i) and collapsed pollen (Figure j), consistent with previous findings in Arabidopsis (Liu et al ., ).…”

“…On the other hand, rNtAGIP1::DT‐A lines had produced sepals and petals but no visible stamens or carpels by stages 6–7 (Figure g). Petals that had emerged at this point had begun to turn brown by stages 8–9 (Figure h) and eventually died by stage 13 (Figure i), which is consistent with previous findings (Yang et al ., ). Those pAGI1F ACT lines with an activated ablation phenotype showed similar floral morphological defects as rNtAGIP1::DT‐A plants, without visible stamen and carpel primordia by stages 6–7 (Figure m,p).…”

“…The AG enhancer ( AGI ) from a variety of species can independently drive carpel and stamen expression (Liu and Liu, ; Yang et al ., , ) and can also interact with its cognate constitutive promoter to confine expression to stamens and carpels in Arabidopsis (Deyholos and Sieburth, ; Sieburth and Meyerowitz, ). To determine whether the AGI enhancer interferes with other promoters, we devised a novel phenotype‐based assay vector, pBASE ACT , which is composed of a nos promoter‐driven NPTII selectable marker cassette and a pollen‐ and stigma‐specific Pps promoter‐driven DT‐A cassette arranged in a tail‐to‐tail orientation and separated by 1.5 kb of intervening DNA (Figure a).…”

“…The enhancer/promoter‐activated ablation activity observed in this study generally fell within the range of the particular interfering enhancer/promoters’ inherent tissue specificity. In the case of NtAGI1 ‐activated ablation, petals, stamens and carpels were specifically targeted (Figures b and m–r), which is identical to the expression pattern driven by the rNtAGIP1 promoter alone (Figures b, c,k and g‐i; Yang et al ., ); however, NtAGI1 ‐activated ablation occurred one floral development stage behind rNtAGIP1 promoter‐directed ablation (Figure g–I,j‐m). Similarly, the retardation/ablation phenotype elicited by AGL8 promoter‐mediated activation (Figure o) is identical to that observed when DT‐A expression was driven by the AGL8 promoter alone (Figure n).…”

Engineered selective plant male sterility through pollen‐specific expression of theEcoRIrestriction endonuclease

“…pekinensis ) was observed to produce 50% aborted pollen and 50% normal pollen as expected (Kim et al ., ). In subsequent studies, the DT‐A gene was used to engineer male sterility for the purpose of transgene bioconfinement (Liu and Liu, ; Liu et al ., ; Skinner et al ., ; Yang et al ., , ). Although each of these studies reported a high degree of reproductive tissue ablation, only one study reported outcrossing results.…”