The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18∶1 to elongation reactions beginning with 18∶1 as substrate. These V2-based leaf assays produced ∼50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays.
Salinity affects plant growth by the osmotic stress of the salt around the roots as well as by toxicity caused by excessive accumulation of salt in leaves. The aim of this study was to determine whether there is significant genetic variation in tolerance to osmotic stress that can be useful in improving the salinity tolerance of crop plants. Durum wheat is a salt-sensitive crop whose yield is reduced by moderately saline soils. Genetic variation in tolerance to osmotic stress in durum wheat was examined in 50 international durum varieties and landraces by measuring the response of stomatal conductance to salt stress before salts built up in the leaf. Stomatal conductance is a sensitive indicator of the osmotic stress because it is reduced immediately with the onset of salinity, and is the initial and most profound cause of a decline in CO2 assimilation rate. Genetic differences of 2–3-fold were found in the magnitude of the response of stomatal conductance to salt-induced osmotic stress. Higher stomatal conductance in salt related to higher CO2 assimilation rate. There was a positive relationship between stomatal conductance and relative growth rate in salt. This study shows the potential for new genetic gains in salt tolerance in durum wheat.
Abstract. Nax1 and Nax2 are two genetic loci that control the removal of Na + from the xylem and thereby help to exclude Na + from leaves of plants in saline soil. They originate in the wheat ancestral relative Triticum monococcum L. and are not present in modern durum or bread wheat. The Nax1 and Nax2 loci carry TmHKT1;4-A2 and TmHKT1;5-A, respectively, which are the candidate genes for these functions. This paper describes the development of near-isogenic breeding lines suitable for assessing the impact of the Nax loci and their performance in controlled environment and fields of varying salinity. In young plants grown in 150 mM NaCl, Nax1 reduced the leaf Na + concentration by 3-fold, Nax2 by 2-fold and both Nax1 and Nax2 together by 4-fold. In 250 mM NaCl, Nax1 promoted leaf longevity and greater photosynthesis and stomatal conductance. In the uppermost leaf, the Na + -excluding effect of the Nax loci was much stronger. In the field, Na + in the flag leaf was reduced 100-fold by Nax1 and 4-fold by Nax2; however, Nax1 lines yielded 5-10% less than recurrent parent (cv. Tamaroi) in saline soil. In contrast, Nax2 lines had no yield penalty and at high salinity they yielded close to 25% more than Tamaroi, indicating this material is suitable for breeding commercial durum wheat with improved yield on saline soils.
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