Ancient duons may underpin spatial patterning of gene expression in C
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            leaves

Reyna-Llorens, Ivan; Burgess, Steven J.; Reeves, Gregory; Singh, Pallavi; Stevenson, Sean Ross; Williams, Bruce; Stanley, Susan E.; Hibberd, Julian M.

doi:10.1073/pnas.1720576115

Cited by 54 publications

(48 citation statements)

References 59 publications

(64 reference statements)

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“…For example, Reyna‐Llorens et al . () have recently identified two cis ‐elements, BSM1a and BSM1b, that are present in the coding sequence of multiple BS‐preferentially expressed genes in Gynandropsis gynandra (C 4 ), and are also present in the Arabidopsis thaliana orthologues, suggesting that BSM1a and BMS1b were co‐opted during the evolution of C 4 photosynthesis. The identification of OsbHLH112 and its maize homologues, ZmbHLH80 and ZmbHLH90, binding to the same ZmPEPC1 upstream region may provide the evidence for co‐option of existing C 3 TFs during C 4 photosynthesis evolution.…”

Section: Discussionmentioning

confidence: 99%

ZmbHLH80 and ZmbHLH90 transcription factors act antagonistically and contribute to regulatePEPC1cell‐specific gene expression in maize

et al. 2019

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Compartmentation of photosynthetic reactions between mesophyll and bundle sheath cells is a key feature of C 4 photosynthesis and depends on the cell-specific accumulation of major C 4 enzymes, such as phosphoenolpyruvate carboxylase 1. The ZmPEPC1 upstream region, which drives light-inducible and mesophyllspecific gene expression in maize, has been shown to keep the same properties when introduced into rice (C 3 plant), indicating that rice has the transcription factors (TFs) needed to confer C 4 -like gene expression. Using a yeast one-hybrid approach, we identified OsbHLH112, a rice basic Helix-Loop-Helix (bHLH) TF that interacts with the maize ZmPEPC1 upstream region. Moreover, we found that maize OsbHLH112 homologues, ZmbHLH80, and ZmbHLH90, also interact with the ZmPEPC1 upstream region, suggesting that these C 4 regulators were co-opted from C 3 plants. A transactivation assay in maize mesophyll protoplasts revealed that ZmbHLH80 represses, whereas ZmbHLH90 activates, ZmPEPC1 expression. In addition, ZmbHLH80 was shown to impair the ZmPEPC1 promoter activation caused by ZmbHLH90. We showed that ZmbHLH80 and ZmbHLH90 bind to the same cis-element within the ZmPEPC1 upstream region either as homodimers or heterodimers. The formation of homo-and heterodimers with higher oligomeric forms promoted by ZmbHLH80 may explain its negative effect on gene transcription. Gene expression analysis revealed that ZmbHLH80 is preferentially expressed in bundle sheath cells, whereas ZmbHLH90 does not show a clear cell-specific expression pattern. Altogether, our results led us to propose a model in which ZmbHLH80 contributes to mesophyll-specific ZmPEPC1 gene expression by impairing ZmbHLH90-mediated ZmPEPC1 activation in the bundle sheath cells.ZmbHLH80 and ZmbHLH90 antagonistically regulate ZmPEPC1 275 Transactivation assays in rice protoplastsFor the transient expression assay in pL2V-pZMUbi-HYG_pZm-PEPC321-GUS-17244.b rice protoplasts, we used the p2GW7:: OsbHLH112 effector plasmid. To construct this plasmid, the

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Section: Discussionmentioning

confidence: 99%

ZmbHLH80 and ZmbHLH90 transcription factors act antagonistically and contribute to regulatePEPC1cell‐specific gene expression in maize

et al. 2019

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“…Although these techniques can be used to assess gene and protein function in a relatively short amount of time, they do not allow analysis in cells of intact rice tissues. Microparticle bombardment has successfully been employed to transform individual cells in whole leaves of several plant species (Brown et al, 2011; Reyna-Llorens et al, 2018). For rice, even though particle bombardment has been used for transient expression in epidermal cells of the leaf sheath (Dangol et al, 2017; Meng et al, 2014; Wang et al, 2018), there are very few reports of successful microparticle bombardment of the mature rice leaf blade (Jia et al, 2000).…”

Section: Resultsmentioning

confidence: 99%

Fluorescent reporters for functional analysis in rice leaves

Luginbuehl

El‐Sharnouby

Wang

et al. 2019

Preprint

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Fluorescent reporters have facilitated non-invasive imaging in multiple plant species and thus allowed analysis of processes ranging from gene expression and protein localization through to cellular patterning. However, in rice, a globally important crop and model species, there are relatively few reports of fluorescent proteins being used in leaves. Fluorescence imaging is particularly difficult in the rice leaf blade, likely due to a high degree of light scattering in this tissue. To address this, we investigated approaches to improve deep imaging in mature rice leaf blades. We found that ClearSee treatment, which has previously been used to visualise fluorescent reporters in whole tissues of plants, led to improved imaging in rice. Removing epidermal and subtending mesophyll cell layers was faster than ClearSee, and also reduced light scattering such that imaging of fluorescent proteins in deeper leaf layers was possible. To expand the range of fluorescent proteins suitable for imaging in rice, we screened twelve whose spectral profiles spanned most of the visible spectrum. This identified five proteins, mTurquoise2, mClover3, mNeonGreen, mKOκ and tdTomato that are robustly expressed and visible in mesophyll cells of stably transformed plants. Using microparticle bombardment, we show that mTurquoise2 and mNeonGreen can be used for simultaneous multicolour imaging of different sub-cellular compartments. Overall, we conclude that mTurquoise2, mClover3, mNeonGreen, mKOκ and tdTomato are suitable for high resolution live imaging of rice leaves, both after transient and stable transformation. Along with the rapid microparticle bombardment method, which allows transient transformation of major cell types in the leaf blade, these fluorescent reporters should greatly facilitate the analysis of gene expression and cell biology in rice.One sentence summaryWe report five fluorescent reporters suitable for functional analysis in rice leaves.

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“…6a, b ). Exonic transcription factor binding sites, although rare, are found in both animals and plants, and frequently link to developmental regulation 29, 30 . We identified three putative bZIP binding sites in LFY exon two: an evolutionarily conserved G-box and two partially conserved C-boxes ( Fig.…”

Section: Mainmentioning

confidence: 99%

Florigen family chromatin recruitment, competition and target genes

Zhu

Klasfeld

et al. 2020

Preprint

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17Plants monitor seasonal cues, such as day-length, to optimize life history traits including 18 onset of reproduction and inflorescence architecture 1-3 . Florigen family transcriptional 19 co-regulators TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) 20 antagonistically regulate these vital processes 4-6 yet how TFL1 and FT execute their 21 roles and what the mechanism is for their antagonism remains poorly understood. We 22 Main 35Plant development occurs after embryogenesis and is plastic, this allows modulation of 36 the final body plan in response to environmental cues to enhance growth and 37 reproductive success 7,8 . Of particular importance for species survival is the timing of the 38 formation of flowers that give rise to seeds which occurs in response to the seasonal 39 cues photoperiod and temperature 1,3,9 . For example, in plants that flower only once, like 40Arabidopsis and most crops, an early switch to flower formation allows rapid completion 41 of the life-cycle in a short growing season, but reduces total seed set or yield 10-12 . By 42 contrast, delaying flower formation supports formation of more seeds, but extends the 43 time to seed set. In many plant species, these alternative developmental trajectories are 44 tuned in response to daylength in antagonistic fashion by two members of the florigen 45 family of proteins 12,13 . FT promotes onset of the reproductive phase and flower 46 formation (determinacy), while TFL1 promotes vegetative development and branch fate 47 (indeterminacy) 4-6,13 . In Arabidopsis, which flowers in the spring, FT accumulates only 48 when the daylength exceeds a critical threshold, while TFL1 is present in both short-day 49 and long-day conditions 1,3,14 . A key unanswered question is how FT and TFL1 50 modulate plant form -what are the downstream processes they set in motion and what 51 is molecular basis for their antagonism? 52 53Accumulating evidence points to roles of FT and TFL1, which lack ability to bind DNA, 54 in transcriptional activation and repression, respectively, by forming complexes with a 55 bZIP transcription factor, FLOWERING LOCUS D (FD) 12,15-19 , although non-nuclear 56 functions for both proteins have also been described 20,21 . Mechanistic insight into 57 florigen activity has been hampered by their low protein abundance. To overcome this 58 limitation and to test the role of TFL1 in the nucleus, we conducted TFL1 chromatin 59 immunoprecipitation followed by sequencing (ChIP-seq). Towards this end, we first 60 generated a biologically active, genomic GFP-tagged version of TFL1 (gTFL1-GFP tfl1-61 1) (Supplementary Fig. 1a, b). Next, we enriched for TFL1 expressing cells by isolating 62 shoot apices from 42-day-old short-day-grown inflorescences just prior to onset of 63 flower formation (Fig. 1a). Finally, we optimized low abundance ChIP by combining 64 eight individual ChIP reactions per ChIP-seq replicate. We conducted FD ChIP-seq in 65 4 analogous fashion using a published 22 , biologically active ( Supplementary Fig. 1c), 66 genomic...

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Ancient duons may underpin spatial patterning of gene expression in C ₄ leaves

Cited by 54 publications

References 59 publications

ZmbHLH80 and ZmbHLH90 transcription factors act antagonistically and contribute to regulatePEPC1cell‐specific gene expression in maize

ZmbHLH80 and ZmbHLH90 transcription factors act antagonistically and contribute to regulatePEPC1cell‐specific gene expression in maize

Fluorescent reporters for functional analysis in rice leaves

Florigen family chromatin recruitment, competition and target genes

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Ancient duons may underpin spatial patterning of gene expression in C 4 leaves

Cited by 54 publications

References 59 publications

ZmbHLH80 and ZmbHLH90 transcription factors act antagonistically and contribute to regulatePEPC1cell‐specific gene expression in maize

ZmbHLH80 and ZmbHLH90 transcription factors act antagonistically and contribute to regulatePEPC1cell‐specific gene expression in maize

Fluorescent reporters for functional analysis in rice leaves

Florigen family chromatin recruitment, competition and target genes

Contact Info

Product

Resources

About

Ancient duons may underpin spatial patterning of gene expression in C ₄ leaves