Multiple strategies for heat adaptation to prevent chalkiness in the rice endosperm

Wada, Hiroshi; Hatakeyama, Yuto; Onda, Yayoi; Nonami, Hiroshi; Nakashima, Taiken; Erra‐Balsells, Rosa; Morita, Satoshi; Hiraoka, Kenzo; Tanaka, Fukuyo; Nakano, Hiroshi

doi:10.1093/jxb/ery427

Cited by 54 publications

(60 citation statements)

References 44 publications

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“…After the first SWP measurement, the plant was laid down and mounted on a laboratory jack-stand placed on a vibration-free table in the system of the on-site cell metabolome analysis using picoPPESI-MS (see Supplemental Fig. S1 in Wada et al, 2019). A part of the bark tissue (9.4 mm in length 3 6.1 mm in width, the area was approximately 57.8 mm 2 ) at the middle position of the stem was quickly removed using a small chisel under humid conditions to make a tangential section, and thereafter the stem was gently fixed to the holder in two positions (Supplemental Fig.…”

Section: In Situ Cell Metabolomicsmentioning

confidence: 99%

“…Adopting the same system used by Wada et al (2019), in situ cell metabolome analysis using picoPPESI-MS (Nakashima et al, 2016) was carried out individually for parenchymal ray cells and tracheids of developing xylem in semi-intact plants, where a small part of bark tissue was removed. Quartz microcapillary tips were prepared by a micropipette puller and were ground by using a microgrinder to prepare approximately 2-mm internal diameter tips.…”

Section: In Situ Cell Metabolomicsmentioning

confidence: 99%

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Ray Parenchymal Cells Contribute to Lignification of Tracheids in Developing Xylem of Norway Spruce

et al. 2019

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A comparative transcriptomic study and a single-cell metabolome analysis were combined to determine whether parenchymal ray cells contribute to the biosynthesis of monolignols in the lignifying xylem of Norway spruce (Picea abies). Ray parenchymal cells may function in the lignification of upright tracheids by supplying monolignols. To test this hypothesis, parenchymal ray cells and upright tracheids were dissected with laser-capture microdissection from tangential cryosections of developing xylem of spruce trees. The transcriptome analysis revealed that among the genes involved in processes typical for vascular tissues, genes encoding cell wall biogenesis-related enzymes were highly expressed in both developing tracheids and ray cells. Interestingly, most of the shikimate and monolignol biosynthesis pathway-related genes were equally expressed in both cell types. Nonetheless, 1,073 differentially expressed genes were detected between developing ray cells and tracheids, among which a set of genes expressed only in ray cells was identified. In situ single cell metabolomics of semi-intact plants by picoliter pressure probe-electrospray ionization-mass spectrometry detected monolignols and their glycoconjugates in both cell types, indicating that the biosynthetic route for monolignols is active in both upright tracheids and parenchymal ray cells. The data strongly support the hypothesis that in developing xylem, ray cells produce monolignols that contribute to lignification of tracheid cell walls.

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Section: In Situ Cell Metabolomicsmentioning

confidence: 99%

Section: In Situ Cell Metabolomicsmentioning

confidence: 99%

Ray Parenchymal Cells Contribute to Lignification of Tracheids in Developing Xylem of Norway Spruce

et al. 2019

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“…We have used a newly developed on-site cell-specific analysis (see Fig. S1 in Wada et al 2019) 18 to make this analysis possible. This analytical method consists of single cell metabolomics, called 'picolitre pressure-probe-electrospray-ionization mass spectrometry (picoPPESI-MS)' 19 and accurate environmental control.…”

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confidence: 99%

“…This analytical method consists of single cell metabolomics, called 'picolitre pressure-probe-electrospray-ionization mass spectrometry (picoPPESI-MS)' 19 and accurate environmental control. The picoPPESI-MS technique has been successfully applied to several cell-specific research in different plant tissues, such as tomato trichomes 19 , rice endosperms 18 , and developing xylems in Norway spruce 20 . In this study, we have directly inserted the tip of a finely tapered quartz capillary tip into the developing pollen grains in intact plants growing under controlled high temperature environments to identify the internal metabolites.…”

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On-site single pollen metabolomics reveals varietal differences in phosphatidylinositol synthesis under heat stress conditions in rice

Wada

Hatakeyama

Nakashima

et al. 2020

Sci Rep

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Although a loss of healthy pollen grains induced by metabolic heat responses has been indicated to be a major cause of heat-induced spikelet sterility under global climate change, to date detailed information at pollen level has been lacking due to the technical limitations. In this study, we used picolitre pressure-probe-electrospray-ionization mass spectrometry (picoPPESI-MS) to directly determine the metabolites in heat-treated single mature pollen grains in two cultivars, heat-tolerant cultivar, N22 and heat-sensitive cultivar, Koshihikari. Heat-induced spikelet fertility in N22 and Koshihikari was 90.0% and 46.8%, respectively. While no treatment difference in in vitro pollen viability was observed in each cultivar, contrasting varietal differences in phosphatidylinositol (PI)(34:3) have been detected in mature pollen, together with other 106 metabolites. Greater PI content was detected in N22 pollen regardless of the treatment, but not for Koshihikari pollen. In contrast, there was little detection for phosphoinositide in the single mature pollen grains in both cultivars. Our findings indicate that picoPPESI-MS analysis can efficiently identify the metabolites in intact single pollen. Since PI is a precursor of phosphoinositide that induces multiple signaling for pollen germination and tube growth, the active synthesis of PI(34:3) prior to germination may be closely associated with sustaining spikelet fertility even at high temperatures.

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“…This minimally invasive approach has been used to quantify cell-to-cell variation in water relations in-situ (Nonami and Schulze 1989). The CPP has also been used to collect cellular fluids for metabolite analyses in order to reveal spatial variations in cellular metabolism using enzymatic assays (Koroleva et al 1997) and more recently mass spectrometry-assisted cell metabolomics (Nakashima et al 2016; Wada et al 2019).…”

Section: Introductionmentioning

confidence: 99%

Minimally invasive, pressure probe based sampling allows for in-situ gene expression analyses in plant cells

Wada

Castellarin

Matthews

et al. 2019

Preprint

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24Background: Gene expression analyses are conducted using multiple approaches and 25 increasingly research has been focused on assessing gene expression at the level of a 26 tissue or even single-cells. To date, methods to assess gene expression at the single-cell in 27 plant tissues have been semi-quantitative, require tissue disruption, and/or involve laborious, 28 possibly artifact-inducing manipulation. In this work, we used grape berries (Vitis vinifera L. 29 Zinfandel) as a model in order to examine the validity and reproducibility of an in-situ gene 30 expression analysis method combining a cell pressure probe (CPP) with quantitative PCR 31 (qPCR).32 Results: We developed a method to directly assess gene expression levels via qPCR from 33 cellular fluids sampled in-situ with a CPP. Cellular fluids, with volumes in the picoliter range, 34 were collected from intact berries with a CPP at various depths across skin and mesocarp 35 tissues. The expression of a key anthocyanin biosynthetic gene, UDP-glucose: flavonoid 3-36 O-glucosyltransferase (VviUFGT), was analyzed as a test case since its expression is 37 restricted to cells producing anthocyanins in grape berry skins during ripening. The method 38 identifies samples contaminated with significant levels of genomic DNA by amplifying a 39 region of VviUFGT that spans an intron. Therefore false positives were discarded which 40 occurred in 28% of the samples tested. Shallow probing of skin cells showed high VviUFGT 41 expression as expected while deeper probing of mesocarp cells resulted in no VviUFGT 42 expression. 43 Conclusions: The clear correspondence of VviUFGT expression to the targeted cell 44 samples suggests that the in-situ gene expression analysis using a CPP is reliable and does 45 not result in contamination as the probe moves through tissues. This method can be paired 46 to single-cell transcriptomic analyses in the future. We conclude that this technique 47 235 exons flanking the intron. The expression of VviUFGT was analysed by these primer pairs: 236 VviUFGT-forward (5'-GCAGGGCCTAACTCACTCTC-3'), VviUFGT-reverse (5'-237 AAATTGAGCAGCTCGTCTTCA-3'), and VviUbi was analyzed according to (Castellarin et 238 al. 2007a). The final PCR products obtained from the VviUFGT amplification were analyzed 239 9 in 1.5 % agarose gel. The samples that amplified two bands, 178bp (VviUFGT gDNA) and 240 101bp (VviUFGT mRNA) bands, were discarded because of the gDNA comtamination. The 241 samples with only a 101bp band were considered to be positive samples and included in the 242 data analysis. VviUFGT gene expression is reported relative to VviUbi. 243 244 Declarations 245

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Multiple strategies for heat adaptation to prevent chalkiness in the rice endosperm

Abstract: This study revealed the cellular dynamics for heat-induced chalky formation and nitrogen-enhanced heat adaptive mechanism by conducting a newly developed on-site cell-specific analysis coupled with time-course analysis via microscopy.

Cited by 54 publications

References 44 publications

Ray Parenchymal Cells Contribute to Lignification of Tracheids in Developing Xylem of Norway Spruce

Ray Parenchymal Cells Contribute to Lignification of Tracheids in Developing Xylem of Norway Spruce

On-site single pollen metabolomics reveals varietal differences in phosphatidylinositol synthesis under heat stress conditions in rice

Minimally invasive, pressure probe based sampling allows for in-situ gene expression analyses in plant cells

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