Phytochelatin (PC) synthases are g-glutamylcysteine (g-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (g-Glu-Cys) n Gly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd 21 . A purified N-terminally hexahistidinyltagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd 21 or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes g-Glu-Cys acylation at two sites during the Cd 21 -dependent synthesis of PCs from GSH and is stimulated by free Cd 21 when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes g-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd 21 when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent g-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary g-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary g-Glu-Cys acylation.

Addressing the looming global food security crisis requires the development of high-yielding crops. In agricultural soils, deficiency in the micronutrient copper significantly decreases grain yield in wheat (Triticum aestivum), a globally important crop. In cereals, grain yield is determined by inflorescence architecture, flower fertility, grain size, and weight. Whether copper is involved in these processes, and how it is delivered to the reproductive organs is not well understood. We show that copper deficiency alters not only the grain set but also flower development in both wheat and its recognized model, Brachypodium distachyon. We then show that the Brachypodium yellow stripe-like 3 (YSL3) transporter localizes to the phloem, transports copper in frog (Xenopus laevis) oocytes, and facilitates copper delivery to reproductive organs and grains. Failure to deliver copper, but not iron, zinc, or manganese to these structures in the ysl3 CRISPR-Cas9 mutant results in delayed flowering, altered inflorescence architecture, reduced floret fertility, grain size, weight, and protein accumulation. These defects are rescued by copper supplementation and are complemented by YSL3 cDNA. This knowledge will help to devise sustainable approaches for improving grain yield in regions where soil quality is a major obstacle for crop production. Copper distribution by a phloem-localized transporter is essential for the transition to flowering, inflorescence architecture, floret fertility, size, weight, and protein accumulation in seeds.

35Addressing the looming global food security crisis requires the development of high yielding 36 crops. In this regard, the deficiency for the micronutrient copper in agricultural soils 37 decreases grain yield and significantly impacts a globally important crop, wheat. In cereals, 38 grain yield is determined by inflorescence architecture, flower fertility, grain size and weight. 39 Whether copper is involved in these processes and how it is delivered to the reproductive 40 organs is not well understood. We show that copper deficiency alters not only the grain set 41 but also flower development in both wheat and it's recognized model, Brachypodium 42 distachyon, We then show that a brachypodium yellow-stripe-like 3 (YSL3) transporter 43 localizes to the phloem and mediates copper delivery to flag leaves, anthers and pistils. 44 Failure to deliver copper to these structures in the ysl3 CRISPR/Cas9 mutant results in 45 delayed flowering, altered inflorescence architecture, reduced floret fertility, grain number, 46 size, and weight. These defects are rescued by copper supplementation and are complemented 47 by the YSL3 cDNA. This new knowledge will help to devise sustainable approaches for 48 improving grain yield in regions where soil quality is a major obstacle for crop production. 49 50Global food security and the demand for high-yielding grain crops are among the most urgent 51 drivers of modern plant sciences due to the current trend of population growth, extreme 52 weather conditions and decreasing arable land resources [1]. The grain yield is directly linked 53 to the crop and soil fertility. In this regard, it has been known for decades that the deficiency 54 for the micronutrient copper in alkaline, coarse-textured or organic soils that occupy more 55 than 30% of the world arable land, compromises crop fertility, reduces grain/seed yield and in 56 acute cases results in crop failure [2][3][4][5]. In accord with the essential role of copper in 57 reproduction, recent studies using synchrotron x-ray fluorescent (SXRF) microscopy 58 established that copper localizes to anthers and pistils of flowers in a model dicotyledonous 59 species, Arabidopsis thaliana, and failure to deliver copper to these reproductive organs 60 severely compromises fertility and seed set [6]. Although copper deficiency can be remedied 61 by the application of copper-based fertilizers, this approach is not environmentally friendly 62 and can lead to the build-up of toxic copper levels in soils [2,5,7]. Mineral nutrient 63 transporters have been recognized as key targets for improving the mineral use efficiency in 64 sustainable crop production [8]. Wheat is the world's third important staple crop after maize 65 (Zea mays) and rice (Oryza sativa); however, wheat grain yield remained relatively low under 66 marginal growing environments despite significant breeding efforts [9]. Wheat is also 67 regarded as the most sensitive to copper deficiency [2, 3,5]. How copper uptake and internal 68 transport is achieved in wheat and how it af...