Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.
Phospholipase D (PLD) is involved in responses to abiotic stress and abscisic acid (ABA) signaling. To investigate the roles of two Arabidopsis (Arabidopsis thaliana) PLDs, PLDa1 and PLDd, in ABA signaling in guard cells, we analyzed ABA responses in guard cells using Arabidopsis wild type, plda1 and pldd single mutants, and a plda1 pldd double mutant. ABA-induced stomatal closure was suppressed in the plda1 pldd double mutant but not in the pld single mutants. The plda1 and pldd mutations reduced ABAinduced phosphatidic acid production in epidermal tissues. Expression of either PLDa1 or PLDd complemented the double mutant stomatal phenotype. ABA-induced stomatal closure in both plda1 and pldd single mutants was inhibited by a PLD inhibitor (1-butanol ), suggesting that both PLDa1 and PLDd function in ABA-induced stomatal closure. During ABA-induced stomatal closure, wild-type guard cells accumulate reactive oxygen species and nitric oxide and undergo cytosolic alkalization, but these changes are reduced in guard cells of the plda1 pldd double mutant. Inward-rectifying K + channel currents of guard cells were inhibited by ABA in the wild type but not in the plda1 pldd double mutant. ABA inhibited stomatal opening in the wild type and the pldd mutant but not in the plda1 mutant. In wild-type rosette leaves, ABA significantly increased PLDd transcript levels but did not change PLDa1 transcript levels. Furthermore, the plda1 and pldd mutations mitigated ABA inhibition of seed germination. These results suggest that PLDa1 and PLDd cooperate in ABA signaling in guard cells but that their functions do not completely overlap.
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