Seedling growth analyses were performed on one-quarter-strength Murashige and Skoog (MS) basal medium (Sigma) with 1 g/liter Mes, 0.8% agar, and the indicated concentrations of heavy metals. Plant Material, Growth Conditions, and Metal Stress Treatments. T2 seeds with 3:1 segregation on kanamycin plates were used for homozygote screening. Both constructs were transformed into the PC-deficient Arabidopsis mutant cad1-3 by direct Agrobacterium tumefaciens-mediated transformation using the floral dip technique ( 21). The fusion sequence was then digested with BamHI and SacI and subcloned into the binary expression vector pBI121, either with the cauliflower mosaic virus 35S or the Arabidopsis alcohol dehydrogenase ( Adh) promoter ( 20). All PCR products were confirmed by sequencing (Retrogen, La Jolla, CA). The subcloned myc fragment was recovered with SpeI and SacI enzymes and then fused to the 3′ end of the TaPCS1 ORF in the pBluescript II SK(+) vector at the SpeI site. A 3× myc tag DNA sequence was constructed by PCR recovery from a plasmid containing c- myc and then subcloned into the pGEM-T Easy vector (Promega). The TaPCS1 PCR fragment was subcloned into pBluescript II SK(+) vector. Two restriction enzyme sites, BamHI and SpeI, were introduced into the wheat TaPCS1 cDNA ( 9) by PCR. The presented findings show that ( i) transgenic expression of TaPCS1 suppresses the heavy-metal sensitivity of cad1-3, ( ii) PCs can be transported from roots to shoots, and ( iii) transgenic expression of the TaPCS1 gene increases long-distance root-to-shoot Cd 2 + transport and reduces Cd 2 + accumulation in roots.ĭNA Constructs and Plant Transformation. The reduced Cd 2 + accumulation in roots and enhanced root-to-shoot Cd 2 + transport in transgenic plants were abrogated by l-buthionine sulfoximine. Unexpectedly, transgenic expression of TaPCS1 reduced Cd 2 + accumulation in roots compared with cad1-3. Inductively coupled plasma atomic emission spectrometer analyses showed that either root-specific or ectopic expression of TaPCS1 significantly enhanced long-distance Cd 2 + transport into stems and rosette leaves. However, in Adh::TaPCS1/ cad1-3 plants, PCs were detected in roots and in rosette leaves and stems. Fluorescence HPLC analyses showed that under Cd 2 + stress, no PCs were detectable in cad1- 3. Northern blot, RT-PCR, and Western blot analyses showed Adh promoter-driven TaPCS1 expression only in roots and thus demonstrated lack of long-distance TaPCS1 mRNA and protein transport in plants. Adh::TaPCS1/ cad1-3 and 35S::TaPCS1/ cad1-3 complemented the cadmium, mercury, and arsenic sensitivities of the cad1-3 mutant. Here, wheat TaPCS1 cDNA expression was either targeted to Arabidopsis roots with the Arabidopsis alcohol dehydrogenase ( Adh) promoter (Adh::TaPCS1/ cad1-3) or ectopically expressed with the cauliflower mosaic virus 35S promoter (35S::TaPCS1/ cad1-3) in the PC-deficient mutant cad1-3. However, phytochelatins (PCs) are generally considered to function as intracellular heavy-metal detoxification mechanisms, and whether long-distance transport of PCs occurs during heavy-metal detoxification remains unknown. "The result is a surprising delight that feels at once vintage and vogueish.Phytochelatin synthases (PCS) mediate cellular heavy-metal resistance in plants, fungi, and worms. "It's effortlessly merged Ice Cold Beer with lessons learned from modern masterpieces like Spelunky," I wrote in our TumbleSeed review. #4PEAKS FOR PC PC#We recommended TumbleSeed in May, when it launched on Switch, PS4, PC and Mac. #4PEAKS FOR PC PRO#"Controls can be two Joy-Cons, a pro controller and a set of Joy-Cons or whatever you can dream up," the developer noted. It's a fascinating read.Īs far as the new Battle Mode goes, the developer described it on Twitter as "a one on one King of the Hill style competitive mode" in which players vie to stay in a highlighted zone the longest. #4PEAKS FOR PC UPDATE#In an excruciatingly detailed and insightful postmortem, developer Greg Greg Wohlwend examined every regret he had with TumbleSeed at launch and how the Four Peaks update should make everything better. The basic gist is that it adds new non-procedurally-generated training stages, a Weekly Challenge mode, and unlockable persistent perks. And we made a 2p battle only on Switch! /d1eA2ouAPW- TumbleSeed AugTo see this content please enable targeting cookies. The TumbleSeed 4 Peaks Update is live on Switch!!Ģ.
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