A simple, low‐cost technique for in situ measurement of leaf P concentration in field‐grown rice

Yamane, Koji; Mabesa-Telosa, Ranee C.; Tajima, Ryosuke; Banayo, Niño P.M.C.; Kato, Yoichiro

doi:10.1002/jpln.201800187

Cited by 2 publications

(1 citation statement)

References 6 publications

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“…The CDSs of the HSP90.6 and ehsp90.6 were ligated into pET-32a, which were subsequently transformed into BL21 (DE3) competent cells for protein expression and purification. ATPase activity was measured by the molybdenum blue method with minor modifications (Rodriguez et al, 1994; Yamane et al, 2019). We incubated 75 μL of reaction solution (30 mM Tris at pH 7.5, 8 mM MgCl 2 , 100 mM NaCl, 1 mM dithiothreitol (DTT), 4% (w/v) sucrose, 30 μg/ mL bovine serum albumin (BSA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1.5 μL of 100 nM ATP and 100 nM of prokaryotic purified HSP90.6/ hsp90.6 at 30°C for 10 s/30 s/1 min/ 2 min/4 min/8 min/16 min and then immediately added 175 μL of color developing solution (0.35% (m/v) (NH 4 ) 2 MoO 4 .4H 2 O, 0.86 mol/L H 2 SO 4 , 1.4% (w/v) ascorbic acid).…”

Section: Methodsmentioning

confidence: 99%

HSP90.6 is involved in grain filling via carbon and nitrogen metabolism in maize

Yang

Fei

et al. 2022

Preprint

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Carbon and nitrogen are the two most abundant nutrients in all living things, and their metabolism maintains normal plant growth. However, the molecular mechanism underlying carbon and nitrogen metabolism remains largely unknown. Here, we found that HSP90.6 is involved in the metabolism of carbon and nitrogen. We performed gene cloning and functional characterization of a maize EMS mutant ehsp90.6, whose kernels were small. HSP90.6 encodes heat shock protein 90.6, which has a single-amino acid mutation within its HATPase_c domain. Transcriptome profiling showed that the expression of amino acid biosynthesis- and carbon metabolism-related genes was significantly downregulated in hsp90.6. HSP90.6 is involved in the 26S proteasome degradation pathway, which affects nitrogen recycling to regulate amino acid synthesis; this occurs by interactions between HSP90.6 and the 26S proteasome subunits RPN6 and PBD2 (PRC2). The loss of HSP90.6 significantly reduced the activity of the 26S proteasome, resulting in the accumulation of ubiquitinated proteins and defects in nitrogen recycling. Moreover, HSP90.6 interacted with the 14-3-3 protein GF14-6 to participate in carbon metabolism. Together, these findings revealed that HSP90.6 regulates nutrient metabolism in maize seeds by affecting 26S proteasome-mediated nitrogen recycling and GF14-6-mediated carbon metabolism.One sentence summaryHSP90.6 is involved in nutrient metabolism via 26S proteasome-mediated protein degradation to promote nitrogen recycling and GF14-6 protein-mediated carbon metabolism.The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is Weibin Song (songwb@cau.edu.cn).HighlightsHATPase_c is necessary for HSP90.6 to regulate maize kernel development.HSP90.6 is involved in nitrogen recycling via the 26S proteasome degradation pathway.HSP90.6 interacts with the 14-3-3 protein GF14-6 to affect carbon metabolism.IN A NUTSHELLBackgroundSeeds are the main harvested organs of maize. Understanding the regulatory mechanism of grain filling is helpful to cultivate high-quality and high-yield maize. In the past few years, the regulatory network of grain filling has been explored through multiple means, including transcriptomic, proteomic and functional genomic techniques. Many genes that control grain filling through different mechanisms have been cloned, such as CTLP1 (Choline Transporter-like Protein 1), OS1 (Opaque Endosperm and Small Germ 1), and MN6 (Miniature Seed6). To identify new genes involved in maize grain filling, ethyl methanesulfonate (EMS) was used to induce mutations, and the ehsp90.6 mutant, which exhibited abnormal kernel development, was isolated by bulked segregant analysis RNA sequencing (BSR).QuestionWhy does the single-amino acid mutation of HSP90.6 affect grain size, and how does the loss of HSP90.6 affect grain filling?FindingsA single-amino acid mutant (ehsp90.6) and knockout mutant (hsp90.6) were obtained. We found that HSP90-6 was involved in the regulation of maize grain filling. A single-single amino acid mutation in the HATPase_c domain reduced the ATPase activity of HSP90.6, resulting in smaller grains. The functional loss of HSP90.6 resulted in the expression of amino acid biosynthesis- and carbon metabolism-related genes being significantly downregulated in hsp90.6. We indicated that HSP90.6 is involved in the 26S proteasome degradation pathway, which affects nitrogen recycling to regulate amino acid synthesis by interacting with the 26S proteasome subunits RPN6 and PBD2 (PRC2). Moreover, HSP90.6 was found to interact with the 14-3-3 protein GF14-6 to participate in carbon metabolism.Next stepsTo further verify that the interaction between HSP90.6 and 26S proteasome subunits and GF14-6 affects grain filling, knockout validation of RPN6, PBD2 (PRC2) and GF14-6 will be performed. In addition, since GF14-6 interacts with the phosphorylated proteins, we will determine the phosphorylation site of HSP90.6. Due to the important role of HSP90 family proteins in plant development, there are other regulatory pathways that need to be explored.

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Section: Methodsmentioning

confidence: 99%

HSP90.6 is involved in grain filling via carbon and nitrogen metabolism in maize

Yang

Fei

et al. 2022

Preprint

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HEAT SHOCK PROTEIN 90.6 interacts with carbon and nitrogen metabolism components during seed development

Yang

Fei

et al. 2023

Plant Physiology

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Carbon and nitrogen are the two main nutrients in maize (Zea mays L.) kernels, and kernel filling and metabolism determine seed formation and germination. However, the molecular mechanisms underlying the relationship between kernel filling and corresponding carbon and nitrogen metabolism remain largely unknown. Here, we found that HEAT SHOCK PROTEIN 90.6 (HSP90.6) is involved in both seed filling and the metabolism processes of carbon and nitrogen. A single-amino acid mutation within the HATPase_c domain of HSP90.6 led to small kernels. Transcriptome profiling showed that the expression of amino acid biosynthesis- and carbon metabolism-related genes was significantly downregulated in the hsp90.6 mutant. Further molecular evidence showed strong interactions between HSP90.6 and the 26S proteasome subunits REGULATORY PARTICLE NON-ATPASE6 (RPN6) and PROTEASOME BETA SUBUNITD2 (PBD2). The mutation of hsp90.6 significantly reduced the activity of the 26S proteasome, resulting in the accumulation of ubiquitinated proteins and defects in nitrogen recycling. Moreover, we verified that HSP90.6 is involved in carbon metabolism through interacting with the 14-3-3 protein GENERAL REGULATORY FACTOR14-4 (GF14-4). Collectively, our findings revealed that HSP90.6 is involved in seed filling and development by interacting with the components controlling carbon and nitrogen metabolism.

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A simple, low‐cost technique for in situ measurement of leaf P concentration in field‐grown rice

Cited by 2 publications

References 6 publications

HSP90.6 is involved in grain filling via carbon and nitrogen metabolism in maize

HSP90.6 is involved in grain filling via carbon and nitrogen metabolism in maize

HEAT SHOCK PROTEIN 90.6 interacts with carbon and nitrogen metabolism components during seed development

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