Double-stranded (ds)RNA interference (RNAi) is widely used for functional analysis of plant genes and is achieved via generating stable transformants expressing dsRNA in planta. This study demonstrated that RNAi can also be utilized to examine gene functions in protoplasts. Because protoplasts are nongrowing cells, effective RNAi-triggered gene silencing depends not only on a depletion of gene transcripts but also on turnover rates of corresponding polypeptides. Herein, we tested if transient RNAi in protoplasts would result in the depletion of a targeted polypeptide and, because protoplasts have a limited life span, if functional assays of RNAi knockout genes would be feasible in protoplasts. We showed that protoplasts transfection with an in vitro-synthesized dsRNA against Arabidopsis (Arabidopsis thaliana) g-glutamylcysteine synthase (ECS1), a key enzyme in the synthesis of glutathione, resulted in a 95% depletion of ECS1 transcript, a 72% decrease of ECS1 polypeptide, and a 60% drop in glutathione content. These results were comparable with those obtained upon analysis of Arabidopsis seedlings bearing the cad2-1 mutant allele of ECS1. We also improved the procedure for RNAi inactivation of several genes simultaneously. Finally, because we isolated protoplasts from tissues of 14-d-old seedlings instead of 1-monthold mature plants, the described procedure is rapid (as it only takes 20 d from seed planting to functional studies), suitable for analyzing multiple genes in parallel, and independent of cloning dsRNAs into plant expression vectors. Therefore, RNAi in protoplasts complements existing genetic tools, as it allows rapid, cost-and space-efficient initial screening and selection of genes for subsequent in planta studies.

Half-molecule ATP-binding cassette transporters of the HMT-1 (heavy metal tolerance factor 1) subfamily are required for Cd 2؉ tolerance in Schizosaccharomyces pombe, Caenorhabditis elegans, and Chlamydomonas reinhardtii. Based on studies of S. pombe, it has been proposed that SpHMT-1 transports heavy metal⅐phytochelatin (PC) complexes into the vacuolysosomal compartment. PCs are glutathione derivatives synthesized by PC synthases (PCS) in plants, fungi, and C. elegans in response to heavy metals. Our previous studies in C. elegans, however, suggested that HMT-1 and PCS-1 do not necessarily act in concert in metal detoxification. To further explore this inconsistency, we have gone on to test whether DmHMT-1, an HMT-1 from a new source, Drosophila, whose genome lacks PCS homologs, functions in heavy metal detoxification. In so doing, we show that heterologously expressed DmHMT-1 suppresses the Cd 2؉ hypersensitivity of S. pombe hmt-1 mutants and localizes to the vacuolar membrane but does not transport Cd⅐PC complexes. Crucially, similar analyses of S. pombe hmt-1 mutants extend this finding to show that SpHMT-1 itself either does not transport Cd⅐PC complexes or is not the principal Cd⅐PC/apoPC transporter. Consistent with this discovery and with our previous suggestion that HMT-1 and PCS-1 do not operate in a simple linear metal detoxification pathway, we demonstrate that, unlike PCS-deficient cells, which are hypersensitive to several heavy metals, SpHMT-1-deficient cells are hypersensitive to Cd 2؉ , but not to Hg 2؉ or As 3؉ . These findings significantly change our current understanding of the function of HMT-1 proteins and invoke a PC-independent role for these transporters in Cd 2؉ detoxification.The adverse health effects of heavy metals such as cadmium (Cd 2ϩ ), mercury (Hg 2ϩ ), and lead (Pb 2ϩ ) from food and air are well established (1-4). Despite this knowledge, exposure to heavy metals continues, and has even increased in some areas, due to their sustained production and emission into the environment. At the cellular level, the toxicity of heavy metals results from the displacement of endogenous cofactors from their cellular binding sites, the oxidation of essential enzymes and other proteins, and promotion of the formation of reactive oxygen species (3, 4). The variety of ways by which heavy metals exert their effects places demands on a wide range of distinct cellular detoxification mechanisms in which ATP-binding cassette (ABC) 3 transporters are clearly implicated (5-9). The ABC transporter family is one of the largest families of membrane proteins. Although 60 ABC transporter family members are known in Caenorhabditis elegans, 49 in humans, 57 in Drosophila, 103 in Arabidopsis, 30 in Saccharomyces cerevisiae, and 11 in Schizosaccharomyces pombe (10 -13), the exact role played by the many that are implicated in heavy metal detoxification remains to be determined. What is known is that ABC transporters mediate the Mg⅐ATP-energized transmembrane transport of a wide range of substrates, reside on ...

No abstract

No abstract

Yield of the perennial grass Miscanthus × giganteus has shown an inconsistent and unpredictable response to nitrogen (N) fertilizer, yet fertilization underpins the crop’s environmental and economic sustainability. The interactions among soil microbial communities, N availability, and M. × giganteus and management may explain changes in plant productivity. In this study, soil samples from different stand ages of M. × giganteus in a replicated chronosequence field trial were used to investigate the effects of stand age and N fertilizer rates on microbial community structure. We hypothesized that there is a definable M. × giganteus soil microbiome and that this community varies significantly with stand age and fertilization. Our results showed that the main phyla in soil microbial communities, regardless of plant age, are similar but microbial community structures are significantly different. The variation in observed microbial communities generally decreases in older stand ages. The amount of N fertilizer applied also affected the microbial community structure associated with different aged M. × giganteus. Specifically, the relative abundance of Proteobacteria (Alphaproteobacteria and Gammaproteobacteria) and Acidobacteria (Subgroup Gp1) increased shortly after fertilization and were more associated with younger M. × giganteus. Further, our results show a significant relationship between bacterial alpha diversity and fertilization rates and that this response is also impacted by stand age. Overall, our results emphasize linkages between microbial community structure, plant age, and fertilization in M. × giganteus.

Cropping history affects nodulation and symbiotic efficiency of Cropping history affects nodulation and symbiotic efficiency of distinct hairy vetch (distinct hairy vetch (Vicia villosa

Legume cover crops can play a valuable role in maintaining and increasing soil quality and nitrogen availability, but are infrequently grown in the Upper Midwest due to short growing seasons with minimal management windows; cold, wet springs; and harsh winters. This study was performed to assess the viability of winter annual legume species in northern climates as a potential source of nitrogen (N) fertility to a 75-day sweet corn (Zea mays convar. saccharata var. rugosa) cash crop in Lamberton and Grand Rapids, MN in 2016 and 2017. Treatments included medium red clover (Trifolium pratense), two coldhardy ecotypes of hairy vetch (Vicia villosa Roth), a cereal rye-hairy vetch biculture (Secale cereale L., Vicia villosa Roth), cereal rye as a non-legume control, and a fallow weed-free control. Legumes were split into rhizobia inoculated and non-inoculated treatments. Inoculation had no effect on nodulation, biomass production, or N fixation likely due to competition with endogenous rhizobia strains. The rye monoculture and biculture produced the most biomass at all site-years averaging 7.7 and 7.0 Mg ha -1 respectively while the two vetch ecotypes averaged 4.5 and 3.9 Mg ha -1 . Both vetch ecotypes contributed among the most nitrogen in all site-years, contributing up to 211 kg N ha -1 from aboveground biomass. Data from natural abundance isotopic approaches indicate that 75% of vetch tissue N in Grand Rapids and 59% of vetch tissue N in Lamberton was derived from atmospheric N fixation, with equal or higher percent fixation of vetch in biculture at all site-years. More studies should be performed to better understand controls on N fixation of legume cover crops in cold climates.

No abstract