Pablo A Manavella

Thomas Blein

Jingtang Chen
email: chenjingtang@126.com

Soulaiman Sakr
email: soulaiman.sakr@agrocampus-ouest.fr

Ming Wang

Lili Zang

Fuchao Jiao

Maria-Dolores Perez-Garcia

Laurent Ogé

Latifa Hamama

José Le Gourrierec

Sugar Signaling and Post-transcriptional Regulation in Plants: An Overlooked or an Emerging Topic?

Keywords: sugar, RNA-binding protein, post-transcriptional regulation, microRNA, mRNA stability

Plants are autotrophic organisms that self-produce sugars through photosynthesis. These sugars serve as an energy source, carbon skeletons, and signaling entities throughout plants' life. Post-transcriptional regulation of gene expression plays an important role in various sugar-related processes. In cells, it is regulated by many factors, such as RNA-binding proteins (RBPs), microRNAs, the spliceosome, etc. To date, most of the investigations into sugar-related gene expression have been focused on the transcriptional level in plants, while only a few studies have been conducted on post-transcriptional mechanisms. The present review provides an overview of the relationships between sugar and post-transcriptional regulation in plants. It addresses the relationships between sugar signaling and RBPs, microRNAs, and mRNA stability. These new items insights will help to reach a comprehensive understanding of the diversity of sugar signaling regulatory networks, and open onto new investigations into the relevance of these regulations for plant growth and development.

INTRODUCTION

As living organisms, plants need various compounds to meet the requirements of their global metabolism and to finely adapt to different external stimuli. In this process, one of the most essential compounds is sugar, which has both trophic and signaling activities during plant development -a high-energy-demanding and well-controlled process. Plants synthesize sugar from carbon dioxide and water through photosynthesis and finely tune their sugar status to avoid sugar starvation [START_REF] Stitt | Starch turnover: pathways, regulation and role in growth[END_REF][START_REF] Kanwar | Alterations in plant sugar metabolism: signatory of pathogen attack[END_REF][START_REF] Signorelli | Sugars and autophagy in plants[END_REF]. They have evolved a sophisticated machinery to sense different forms of sugars, including hexoses, sucrose, and various sugar phosphates (e.g., trehalose-6-phosphate), and elicit the appropriate responses. Some responses are sugar-type-specific [START_REF] Granot | Substantial roles of hexokinase and fructokinase in the effects of sugars on plant physiology and development[END_REF][START_REF] Figueroa | A tale of two sugars: trehalose 6-phosphate and sucrose[END_REF][START_REF] Li | Dynamic and diverse sugar signaling[END_REF]; Janse van Rensburg and [START_REF] Janse Van Rensburg | UDP-glucose: a potential signaling molecule in plants? Front[END_REF][START_REF] Wingler | Transitioning to the next phase: the role of sugar signaling throughout the plant life cycle[END_REF]. As a signaling entity, sugar can influence a diversity of physiological processes of the plant life cycle and operates on transcriptional, post-transcriptional, translational, and post-translational regulation. Most of the currently available knowledge is focused on sugar-dependent transcriptional regulation [START_REF] Lastdrager | Sugar signals and the control of plant growth and development[END_REF][START_REF] Li | Dynamic and diverse sugar signaling[END_REF][START_REF] Sakr | The sugar-signaling hub: overview of regulators and interaction with the hormonal and metabolic network[END_REF][START_REF] Wingler | Transitioning to the next phase: the role of sugar signaling throughout the plant life cycle[END_REF][START_REF] Jiao | Glucose signaling, AtRGS1 and plant autophagy[END_REF][START_REF] Rodriguez | TOR and SnRK1 signaling pathways in plant response to abiotic stresses: do they always act according to the "yin-yang" model?[END_REF][START_REF] Sami | Interaction of glucose and phytohormone signaling in plants[END_REF]. Post-transcriptional regulation of gene expression is a pivotal mechanism whereby plants rapidly reprogram their transcriptome and proteome in response to endogenous and environmental cues and involves many factors such as proteins [RNA-binding proteins (RBPs)], microRNAs (miRNAs), and the spliceosome [START_REF] Guerra | Post-transcriptional and post-translational regulations of drought and heat response in plants: a spider's web of mechanisms[END_REF][START_REF] Romanowski | Circadian rhythms and posttranscriptional regulation in higher plants[END_REF][START_REF] Zhang | MicroRNA: a new target for improving plant tolerance to abiotic stress[END_REF][START_REF] Kawa | Regulation of mRNA decay in plant responses to salt and osmotic stress[END_REF][START_REF] Samad | MicroRNA and transcription factor: key players in plant regulatory network[END_REF][START_REF] Marondedze | Changes in the Arabidopsis RNA-binding proteome reveal novel stress response mechanisms[END_REF][START_REF] Rigo | Alternative splicing in the regulation of plant-microbe interactions[END_REF]. RBPs are mainly cytosolic and nuclear proteins that govern the processing, cellular localization, and decay of cellular RNA. They contain RNA recognition motifs (RRMs) that allow their binding to a specific sequence in the target transcripts [START_REF] Chou | Multifunctional RNA binding protein OsTudor-SN in storage protein mRNA transport and localization[END_REF]Tian et al., 2018a;[START_REF] Lu | Genomewide identification and expression analysis of glycine-rich RNA-binding protein family in sweet potato wild relative Ipomoea trifida[END_REF][START_REF] Mahalingam | Genomic survey of RNA recognition motif (RRM) containing RNA binding proteins from barley (Hordeum vulgare ssp. vulgare)[END_REF]. miRNAs are small non-coding RNA molecules that function in RNA silencing via base-pairing with complementary sequences within mRNA molecules [START_REF] Bartel | MicroRNAs: genomics, biogenesis, mechanism, and function[END_REF][START_REF] Bartel | Metazoan micrornasmicroRNAs[END_REF], leading to mRNA cleavage; they shorten the poly(A) tail of mRNAs, or influence mRNA translation, altogether downregulating gene expression through the target transcript [START_REF] Zhang | MicroRNA: a new target for improving plant tolerance to abiotic stress[END_REF][START_REF] Meyers | MicroRNAs in plants: key findings from the early years[END_REF]. Alternative splicing -also termed alternative RNA splicing -is mediated by the spliceosome, a complex and large molecular machinery mainly located within the nucleus of eukaryotic cells [START_REF] Wilkinson | RNA splicing by the spliceosome[END_REF]. Splicing can also have other functions, like the generation of premature stop codons that recruit the nonsense-mediated decay (NMD) machinery [START_REF] Kesarwani | Multifactorial and species-specific feedback regulation of the RNA surveillance pathway nonsense-mediated decay in plants[END_REF][START_REF] Wilkinson | RNA splicing by the spliceosome[END_REF]. Consequently, the proteins translated from alternatively spliced mRNAs are expected to have different amino acid sequences, different protein structures, and even different biological functions. Sugar signaling-dependent regulation represents an intricate regulatory network that relies on highly diverse mechanisms that coordinate the appropriate use of available energy and sugar to sustain plant development and growth under the everchanging environment. Sugar-dependent post-transcriptional regulation could be one important mechanistic aspect of this network. Current knowledge in this topic is still fragmented and makes it very difficult to draw a comprehensive scheme and bring out new research questions. The present review addresses the relationship between sugar and post-transcriptional gene regulation in plants and provides first insights into the role of various important mechanisms of post-transcriptional regulation, i.e., RBPs, miRNAs, and mRNA decay/stability in sugar-related pathways. It underlines the physiological relevance of such regulation mechanisms in different biological contexts and raises questions for upcoming studies.

SUGAR AND RNA-BINDING PROTEINS

RNA-binding proteins control nearly all aspects of eukaryotic post-transcriptional gene regulation and consequently determine the fate and expression of the plant transcriptome. Hundreds of RBPs have been identified in Arabidopsis. Most of them are plant specific, and could carry out specific functions in plant physiology [START_REF] Lorković | Role of plant RNA-binding proteins in development, stress response and genome organization[END_REF][START_REF] Reichel | In planta determination of the mRNA-binding proteome of Arabidopsis etiolated seedlings[END_REF]. They share one or more canonical RNA-binding domains including the RRM, the K-homology (KH) domain, the Pumilio/FBF (PUF) domain, the RRM and KH domains, DEAD/DEAH boxes, zinc-finger structures, the Piwi/Argonaute/Zwille (PAZ) domain, double-stranded RNA-binding domains (DS-RBD), pentatricopeptide-repeat (PPR) domains, etc. [START_REF] Silverman | Genomic era analyses of RNA secondary structure and RNA-binding proteins reveal their significance to post-transcriptional regulation in plants[END_REF][START_REF] Lee | Emerging roles of RNA-binding proteins in plant growth, development, and stress responses[END_REF]Wang et al., 2018a). The link between glucose signaling and RBP-mediated post-transcriptional regulation has been explored in the model plant Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing atRZ-1a, which encodes a zinc-finger-containing glycine-rich RNA-binding protein (GRP), exhibited delayed germination and seedling growth under abiotic stresses (dehydration or salt stress), and hypersensitivity to glucose and ABA, relatively to the wild type [START_REF] Kim | The role of a zinc finger-containing glycinerich RNA-binding protein during the cold adaptation process in Arabidopsis thaliana[END_REF]Kim et al., 2007b). Yet, the molecular function of atRZ1 in glucose-dependent post-transcriptional regulation of seedling establishment is still unknown. The RBP FLOWERING CONTROL LOCUS A (FCA) contains two RRM domains and one WW domain [START_REF] Jang | Survey of rice proteins interacting with OsFCA and OsFY proteins which are homologous to the Arabidopsis flowering time proteins, FCA and FY[END_REF]; Table 1) and operates as an inhibitor of FLOWERING LOCUS C (FLC; [START_REF] Macknight | FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains[END_REF][START_REF] Liu | The Arabidopsis RNA-binding protein FCA requires a lysine-specific demethylase 1 homolog to downregulate FLC[END_REF], one of the repressor integrators, tightly controls flowering signals [START_REF] Michaels | FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering[END_REF]. FLC is positively and transcriptionally regulated by the ABI5 transcription factor (ABA-insensitive 5, Figure 1), which is involved in ABA-mediated floral transition [START_REF] Wang | The inhibitory effect of ABA on floral transition is mediated by ABI5 in Arabidopsis[END_REF] and in integrating glucose (www.arabidopsis.org;[START_REF] Swarbreck | The Arabidopsis information resource (TAIR): gene structure and function annotation[END_REF][START_REF] Berardini | The Arabidopsis information resource: making and mining the "gold standard" annotated reference plant genome[END_REF].

Frontiers and ABA-signaling during early seedling development of Arabidopsis [START_REF] Arroyo | Three genes that affect sugar sensing (abscisic acid insensitive 4, abscisic acid insensitive 5, and constitutive triple response 1) are differentially regulated by glucose in Arabidopsis[END_REF][START_REF] Dekkers | Interaction between sugar and abscisic acid signalling during early seedling development in Arabidopsis[END_REF]. AtSOAR1 (SUPPRESSOR OF THE ABAR OVEREXPRESSOR 1) encodes a dual-localized (cytoplasm-nucleus) pentatricopeptide repeat (PPR) protein repeat [START_REF] Mei | Arabidopsis pentatricopeptide repeat protein SOAR1 plays a critical role in abscisic acid signalling[END_REF][START_REF] Jiang | Crucial roles of the pentatricopeptide repeat protein SOAR1 in Arabidopsis response to drought, salt and cold stresses[END_REF]. By binding to the mRNA of ABI5, it represses ABI5 translation in the regulatory cascade downstream of a putative ABA receptor (ABAR; [START_REF] Bi | Arabidopsis translation initiation factors eIF iso4G1/2 link repression of mRNA cap-binding complex eIF iso4F assembly with RNA-binding protein SOAR 1-mediated ABA signaling[END_REF]. At the transcriptional level, the transcription factor RAV1, a member of the RAV (Related to ABI3/VP1) subfamily [START_REF] Riechmann | Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes[END_REF][START_REF] Feng | Article 578096 profiling of developmental, hormonal or environmental responsiveness of the Arabidopsis AP2/EREBP transcription factor gene family[END_REF], binds directly to the ABI5 promoter and represses its expression, which is alleviated when RAV1 is phosphorylated by ABA-activated sucrose-non-fermenting-1-related protein kinase-2s (SnRK2s; [START_REF] Feng | Arabidopsis RAV1 transcription factor, phosphorylated by SnRK2 kinases, regulates the expression of ABI3, ABI4, and ABI5 during seed germination and early seedling development[END_REF]. SnRK2s is a central node that integrates plant growth and development with ABA signaling and environmental stresses [START_REF] Zheng | The protein kinase SnRk2.6 mediates the regulation of sucrose metabolism and plant growth in Arabidopsis[END_REF][START_REF] Zhang | SnRK2 acts within an intricate network that links sucrose metabolic and stress signaling in wheat[END_REF][START_REF] Shinozawa | SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment[END_REF], partially through dissociation and inhibition of the target of rapamycin (TOR) kinase complex (Wang et al., 2018b; Figure 1). TOR kinase is, itself, an evolutionary conserved master regulator that integrates nutrients, hormones, and energy to promote cell proliferation [START_REF] Dobrenel | TOR signaling and nutrient sensing[END_REF][START_REF] Rosenberger | To grow or not to grow: TOR and SnRK2 coordinate growth and stress response in Arabidopsis[END_REF][START_REF] Shi | TOR signaling in plants: conservation and innovation[END_REF]. Interestingly, TOR kinase can directly phosphorylate APUM2, APUM3, and APUM4, three PUF proteins in Arabidopsis (Figure 1), providing a direct link between the nutrient status and the activity of RBPs (Table 1; [START_REF] Van Leene | Capturing the phosphorylation and protein interaction landscape of the plant TOR kinase[END_REF].

Although their genuine activity is still unclear, APUM-1 to APUM-6 might act as regulators of stem cell maintenance in the shoot meristem [START_REF] Francischini | Molecular characterization of Arabidopsis thaliana PUF proteins-binding specificity and target candidates[END_REF], where TOR kinase signaling is required for integrating sugar, hormone, and environmental signals [START_REF] Li | Differential TOR activation and cell proliferation in Arabidopsis root and shoot apexes[END_REF]. The expression of Rosa hybrida PUF4 (RhPUF4, an ortholog of APUM2) is upregulated by sucrose before the onset of bud outgrowth and may contribute to the promotion of sugar-mediated shoot branching by binding to the 3'UTR of RhBRC1 (Wang et al., 2019a), a main repressor hub of shoot branching [START_REF] Wang | BRANCHED1: a key hub of shoot branching[END_REF]. Furthermore, pharmacological disruption of the oxidative pentose phosphate pathway (OPPP) alters sucrose-related RhPUF4 upregulation and RhBRC1 downregulation, suggesting a major role of the OPPP in this process (Wang et al., 2019a).

The fact that TOR kinase could mediate the upregulation of glucose-6-phosphate dehydrogenase (G6PD, one of key enzymes of the OPPP) and the activity of TOR kinase is probably under the positive regulation of NADPH, a product of the OPPP [START_REF] Corradetti | Upstream of the mammalian target of rapamycin: do all roads pass through mTOR?[END_REF][START_REF] Liu | TOR is a negative regulator of autophagy in Arabidopsis thaliana[END_REF], it would be noteworthy to investigate the crosstalk between these two pathways in this post-transcriptional process. In addition, although these findings suggest a plausible role of TOR kinase and the OPPP in sugar-mediated RBP-dependent post-transcriptional regulation, questions about whether additional sugar signaling could contribute to this regulation and the nature of the underlying molecular mechanisms are still open. RNA-binding-proteins can also directly regulate the sugar metabolism by triggering sugar metabolism-related enzymes. GLYCINE RICH PROTEIN 2 (GRP2), a cold-induced zincfinger-containing GRP [START_REF] Fusaro | AtGRP2, a cold-induced nucleo-cytoplasmic RNAbinding protein, has a role in flower and seed development[END_REF], negatively affects germination, in interaction with ABA and glucose (Kim et al., 2007a). GRP2 can interact with mitochondrial malate dehydrogenase (m-MDH) and citrate synthase (CS), two enzymes of the tricarboxylic acid cycle (TCA), probably leading to an adjustment of the sugar metabolism (Kim et al., 2007a; Figure 1). The response of GRP2 to other environmental cues and to endogenous factors, including sugars, deserves to be investigated to evaluate the physiological relevance of this regulation. Interestingly, sugar metabolism-related enzymes could both include RBPs and display metabolic activities. Based on an interactome capture technique in Arabidopsis cell cultures and leaves, [START_REF] Marondedze | The RNA-binding protein repertoire of Arabidopsis thaliana[END_REF] identified 18 RBPs involved in glycolysis, and 15 involved in the glyoxylate and dicarboxylate metabolism, while their respective target mRNAs are still unknown. A similar plausible dual function was also reported for RAFFINOSE SYNTHASE 6 (RS6), a metabolic enzyme, involved in the biosynthesis of the raffinose family oligosaccharides and ALDEHYDE DEHYDROGENASE 7B4 (ALDH7B4; [START_REF] Fujiki | Multiple signaling pathways in gene expression during sugar starvation. Pharmacological analysis of din gene expression in suspension-cultured cells of Arabidopsis[END_REF][START_REF] Hou | Comparative study of the aldehyde dehydrogenase (ALDH) gene superfamily in the glycophyte Arabidopsis thaliana and Eutrema halophytes[END_REF][START_REF] Reichel | In planta determination of the mRNA-binding proteome of Arabidopsis etiolated seedlings[END_REF][START_REF] Gilmonreal | Induction of the PDH bypass and upregulation of the ALDH7B4 in plants treated with herbicides inhibiting amino acid biosynthesis[END_REF][START_REF] Marondedze | Changes in the Arabidopsis RNA-binding proteome reveal novel stress response mechanisms[END_REF] in different biological contexts (Table 1). ALDH7B4 protein accumulates abundantly in response to abiotic stress and function as aldehyde-detoxifiying enzymes and ROS scavengers enzymes [START_REF] Kirch | Detailed expression analysis of selected genes of the aldehyde dehydrogenase (ALDH) gene superfamily in Arabidopsis thaliana[END_REF][START_REF] Zhao | The role of Arabidopsis aldehyde dehydrogenase genes in response to high temperature and stress combinations[END_REF]. [START_REF] Zhao | The ATAF1 transcription factor is a key regulator of aldehyde dehydrogenase 7B4 (ALDH7B4) gene expression in Arabidopsis thaliana[END_REF] demonstrated that ALDH7B4 is a direct target of the NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON (NAC) transcription factor ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR1 (ATAF1) that integrate carbon starvation responses and trehalose metabolism [START_REF] Garapati | Transcription factor Arabidopsis activating factor1 integrates carbon starvation responses with trehalose metabolism[END_REF]. These findings open onto new investigations on the functional role of their respective target mRNAs and their role in sugar signaling-dependent post-transcriptional regulation.

SUGARS AND MICRORNAs

miRNAs are small non-coding RNA molecules that participate in RNA silencing and post-transcriptional regulation of gene expression [START_REF] Song | MicroRNAs and their regulatory roles in plant-environment interactions[END_REF]. miRNA genes are transcribed by RNA polymerase II in the nucleus. This generates long primary transcripts of miRNA (primary miRNAs, pri-miRNA in short), which are converted into a precursor miRNA (pre-miRNA) by endonuclease DICER-like1 (DCL1). After a complex processing involving the C2H2-zinc finger protein SERRATE (SE), DCL1 and the double-stranded RBP HYPONASTIC LEAVES1 (HYL1), the miRNA is loaded onto ARGONAUTE1 (AGO1) to integrate the RNA-induced silencing complex (RISC; [START_REF] Voinnet | Origin, biogenesis, and activity of plant microRNAs[END_REF][START_REF] Rogers | Biogenesis, turnover, and mode of action of plant microRNAs[END_REF]. Several studies support a direct link between sugar signaling and miRNAs in a variety of physiological processes in plants. [START_REF] Duarte | Involvement of microRNA-related regulatory pathways in the glucose-mediated control of Arabidopsis early seedling development[END_REF] shown that Arabidopsis mutants disrupted in miRNA biosynthesis (hyl1-2 and dcl1-11) and miRNA activity (ago1-25) exhibited a glucose-hyposensitive phenotype at the early seedling stage, and the expression of several miRNA target genes was deregulated, mainly via hexokinase-independent pathway. miRNA156 is one of the best characterized miRNAs in terms of sugar-dependent regulation. It is conserved in land plants and contributes to diverse physiological processes such as leaf development, heat stress memory, developmental transition, apical dominance, and flowering [START_REF] Kim | The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module regulates ambient temperature-responsive flowering via FLOWERING LOCUS T in Arabidopsis[END_REF][START_REF] Bhogale | MicroRNA156: a potential graft-transmissible microRNA that modulates plant architecture and tuberization in Solanum tuberosum ssp. andigena[END_REF][START_REF] Yu | Identification of differentially expressed microRNA in the stems and leaves during sugar accumulation in sweet sorghum[END_REF][START_REF] Zhang | MicroRNA: a new target for improving plant tolerance to abiotic stress[END_REF][START_REF] Gao | miR156/SPL10 modulates lateral root development, branching and leaf morphology in Arabidopsis by silencing AGAMOUS-LIKE 79[END_REF][START_REF] Kumar | PcG proteins MSI1 and BMI1 function upstream of miR156 to regulate aerial tuber formation in potato[END_REF]. The biological function of miRNA156 implies the repression of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKEs (SPLs; [START_REF] Wang | miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana[END_REF][START_REF] Wahl | Regulation of flowering by trehalose-6-phosphate signaling in Arabidopsis thaliana[END_REF][START_REF] Xu | Developmental functions of miR156-regulated SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes in Arabidopsis thaliana[END_REF][START_REF] Wei | Control of chrysanthemum flowering through integration with an aging pathway[END_REF]Zheng et al., 2019a,b;[START_REF] Hu | Transcriptome profiling of the flowering transition in saffron (Crocus sativus L.)[END_REF][START_REF] Jiao | Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice[END_REF][START_REF] Ponnu | The trehalose 6-phosphate pathway impacts vegetative phase change in Arabidopsis thaliana[END_REF]. A direct link between sugar and miRNA156 abundance is based on the ability of exogenous glucose or sucrose supply to cause the levels of mature miRNA156 to drop and thereby accelerate the vegetative-reproductive phase transition, along with the juvenile-to-adult phase transition. Conversely, defoliation and a reduced photosynthetic rate delay plant developmental transitions [START_REF] Yang | Sugar promotes vegetative phase change in Arabidopsis thaliana by repressing the expression of MIR156A and MIR156C[END_REF][START_REF] Yu | Sugar is an endogenous cue for juvenile-to-adult phase transition in plants[END_REF]. The glucoseinduced repression of miRNA156 is dependent on the hexokinase 1-signaling pathway [START_REF] Yang | Sugar promotes vegetative phase change in Arabidopsis thaliana by repressing the expression of MIR156A and MIR156C[END_REF], while trehalose-6-phosphaste regulates developmental transition through a distinct mechanism [START_REF] Wahl | Regulation of flowering by trehalose-6-phosphate signaling in Arabidopsis thaliana[END_REF][START_REF] Ponnu | The trehalose 6-phosphate pathway impacts vegetative phase change in Arabidopsis thaliana[END_REF]. miRNA156 also targets a variety of mRNAs that encode regulatory proteins involved in various physiological processes in plants [START_REF] Wang | The miR156/SPL module, a regulatory hub and versatile toolbox, gears up crops for enhanced agronomic traits[END_REF]. Like miRNA156, miRNA399 was determined to be sucroseresponsive through a microRNA array assay and high levels of sucrose inhibited the accumulation of microRNA399 family under phosphate starvation conditions in Arabidopsis (Tian et al., 2018b). miRNA398, that is associated with the adaptive plant response to biotic, abiotic, and nutrient stresses and could be involved in sugar-signaling pathway [START_REF] Sunkar | Post-transcriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance[END_REF][START_REF] Dugas | Sucrose induction of Arabidopsis miR398 represses two Cu/Zn superoxide dismutases[END_REF][START_REF] Jia | Differential and dynamic regulation of miR398 in response to ABA and salt stress in Populus tremula and Arabidopsis thaliana[END_REF][START_REF] Feng | Highthroughput deep sequencing reveals that microRNAs play important roles in salt tolerance of euhalophyte Salicornia europaea[END_REF]. miRNA398 accumulation is repressed by carbon depletion [START_REF] Pant | Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing[END_REF], while sucrose supply induces its accumulation through the SPL7 transcription factor that directly recognizes the GTAC boxes located in the miRNA398 promoter [START_REF] Yamasaki | SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis[END_REF]. In line with this, spl7 knockdown mutants consistently accumulate lower levels of miRNA398 under normal conditions [START_REF] Ren | Identification of sucrose-responsive microRNAs reveals sucrose-regulated copper accumulations in an SPL7-dependent and independent manner in Arabidopsis thaliana[END_REF]. Targets of miR398a include two ROS-scavenging enzymes (COPPER/ZINC SUPEROXIDE DISMUTASE, CSD1 and CSD2) necessary for detoxification of stress-dependent reactive oxygen species stimulation [START_REF] Farooq | Acquiring control: the evolution of ROS-induced oxidative stress and redox signaling pathways in plant stress responses[END_REF] and this sugar-mediated regulation of miRNA398 would be an appropriate response to nutrient stress. The miRNA398 binding site of CSD1 can be eliminated by alternative splicing in peanut and Arabidopsis, resulting in different tolerance levels to abiotic stress [START_REF] Park | Bypassing miRNA-mediated gene regulation under drought stress: alternative splicing affects CSD1 gene expression[END_REF], indicating how alternative splicing processes influence plant response through interactions with miRNAs. Microarray analyses have also shown responsiveness to sucrose from other mature miRNAs in Arabidopsis (Figure 2), including miRNA408 (involved in the response to iron deficiency and in photosynthesis; [START_REF] Pan | Overexpression of microRNA408 enhances photosynthesis, growth, and seed yield in diverse plants[END_REF][START_REF] Carrió-Seguí | The altered expression of microRNA408 influences the Arabidopsis response to iron deficiency[END_REF], miRNA319 (involved in leaf development; [START_REF] Koyama | Roles of miR319 and TCP transcription factors in leaf development[END_REF], and miRNA160 (involved in heat tolerance; [START_REF] Lin | MicroRNA160 modulates plant development and heat shock protein gene expression to mediate heat tolerance in Arabidopsis[END_REF]. The levels of miRNA319 and miRNA408 are enhanced by sucrose supply, while the levels of miRNA160 are reduced. Moreover, the induction of miRNA408 by sucrose is associated once again with SPL7 (Ren and Tang, 2012), which may play a prominent role in sugar-mediated regulation of miRNA biosynthesis (Figure 2). In sweet sorghum (Sorghum bicolor), the expression levels of nine known mature miRNAs and 12 novel mature miRNAs have been found influenced by sugar abundance in the stem [START_REF] Yu | Identification of differentially expressed microRNA in the stems and leaves during sugar accumulation in sweet sorghum[END_REF]. Although the targets of these mature miRNAs exhibit functions related to shoot apical meristem specification, polar specification of the adaxial/ abaxial axis, bilateral symmetry determination, and transcriptional regulation [START_REF] Yu | Identification of differentially expressed microRNA in the stems and leaves during sugar accumulation in sweet sorghum[END_REF], the genuine participation of sugar sensing and signaling in this regulatory network remains to be elucidated. A link between sucrose transporters (SUTs, H + /Suc symporters) and miRNAs exists in plants. SUTs are key players in sucrose phloem loading and sugar allocation within plants [START_REF] Braun | Understanding and manipulating sucrose phloem loading, unloading, metabolism, and signalling to enhance crop yield and food security[END_REF][START_REF] Milne | Sucrose transporter localization and function in phloem unloading in developing stems[END_REF][START_REF] Doidy | Sugar transporters in Fabaceae, featuring SUT MST and SWEET families of the model plant Medicago truncatula and the agricultural crop Pisum sativum[END_REF]. The half-life of SUT mRNAs ranges between 60 and 130 min and is tightly related to miRNA regulation [START_REF] He | Phloem mobility and stability of sucrose transporter transcripts[END_REF][START_REF] Liesche | Sucrose transporter regulation at the transcriptional, post-transcriptional and post-translational level[END_REF]. Interestingly, the relationship between miRNA biosynthesis and the cellular energy status is also supported by the fact that the transient overexpression of the energysensing SnRK1 in protoplasts leads to the repression of a variety of miRNAs [START_REF] Confraria | miRNAs mediate SnRK1-dependent energy signaling in Arabidopsis[END_REF]. These include miRNA159B (involved in leaf senescence, [START_REF] Huo | Identification of miRNAs associated with dark-induced senescence in Arabidopsis[END_REF], miRNA161 (induces the expression of PPR genes, [START_REF] Cai | The disease resistance protein SNC1 represses the biogenesis of microRNAs and phased siRNAs[END_REF], miRNA775A (no function reported to date), and miRNA824A (involved in flowering time regulation, [START_REF] Hu | miR824-regulated AGAMOUS-LIKE16 contributes to flowering time repression in Arabidopsis[END_REF] and might be involved in SnRK1-dependent energy signaling. However, the molecular regulatory network involved in this SnRK1-dependent miRNA biosynthesis remains an open question.

CROSSTALK BETWEEN SUGAR-RELATED RNA-BINDING PROTEINS AND MICRORNAs

Post-translational modifications have been reported as a key regulator of the miRNA-biogenesis machinery. In Arabidopsis, HYL1 activity is controlled by its phosphorylation state through complex mechanisms. The Protein Phosphatase 4 (PP4)/ Suppressor of MEK 1 (SMEK1) complex and C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 and 2 (CPL1 and CPL2) dephosphorylate and activate HYL1, while Mitogen-activated Protein Kinase (MPK) phosphorylates and inactivates it [START_REF] Manavella | Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1[END_REF][START_REF] Su | The protein phosphatase 4 and SMEK1 complex dephosphorylates HYL1 to promote miRNA biogenesis by antagonizing the MAPK cascade in Arabidopsis[END_REF][START_REF] Meng | Sucrose signaling regulates anthocyanin biosynthesis through a MAPK cascade in Arabidopsis thaliana[END_REF], Wang et al., 2019c). MPK might bridge a gap between miRNA biosynthesis and sugar signaling, based on its transducing role the WALL-ASSOCIATED KINASE (WAK)-dependent regulation of vacuolar invertase, a driver of cell expansion and growth and a player of sugar signaling [START_REF] Kohorn | An Arabidopsis cell wall-associated kinase required for invertase activity and cell growth[END_REF][START_REF] Kohorn | Pectin activation of MAP kinase and gene expression is WAK2 dependent[END_REF]; Figure 1). SnRK2s can also affect the phosphorylation status of HYL and SE [START_REF] Yan | The SnRK2 kinases modulate miRNA accumulation in Arabidopsis[END_REF], and it will be very interesting to explore the sensitivity of these two proteins to the kinase activity of SnRK1 and, thereby, its relevance in SnRK1-dependent miRNA biosynthesis regulation [START_REF] Confraria | miRNAs mediate SnRK1-dependent energy signaling in Arabidopsis[END_REF]. Beyond this, the basic helix-loop-helix (bHLH) transcription factor phytochrome-interacting factor 4 (PIF4) interacts directly with DCL1 and HYL1 to promote their destabilization and regulate the processing of primary miRNAs during the dark-to-red-light transition [START_REF] Sun | Coordinated regulation of Arabidopsis microRNA biogenesis and red light signaling through Dicer-like 1 and phytochrome-interacting factor 4[END_REF]. PIF4 is also controlled through the trehalose-6-phosphate pathway and SnRK1 to modulate Arabidopsis hypocotyl elongation in response to high temperature [START_REF] Delatte | Growth arrest by trehalose-6-phosphate: an astonishing case of primary metabolite control over growth by way of the SnRK1 signaling pathway[END_REF][START_REF] Hwang | Trehalose-6phosphate signaling regulates thermoresponsive hypocotyl growth in Arabidopsis thaliana[END_REF], so it might be seen as a hub integrating sugar signaling and environmental cues to modulate the regulation of miRNA biogenesis through the DCL1-SE-HYL complex.

Many previous studies indicate that various RBPs participate in miRNA homeostasis. For instance, the WD-40 protein PLEIOTROPIC REGULATORY LOCUS1 (PRL1) is required for miRNAs and small siRNAs to accumulate, by stabilizing pri-miRNAs through its RNA-binding activity and enhancing DCL1 activity [START_REF] Zhang | PRL1, an RNA-binding protein, positively regulates the accumulation of miRNAs and siRNAs in Arabidopsis[END_REF]. Beyond this function, PRL1 acts as a global regulator of sugar, stress, and hormone responses, partly through SnRK1 repression [START_REF] Flores-Pérez | Pleiotropic regulatory locus 1 (PRL1) integrates the regulation of sugar responses with isoprenoid metabolism in Arabidopsis[END_REF]. However, additional investigations are required to elucidate the molecular connections between these different PRL1-dependent regulatory mechanisms. miRNA172 is a downstream component of the regulatory cascade involved in the regulation of flowering time by sugar-dependent miRNA156 repression [START_REF] Wu | The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis[END_REF][START_REF] Martin | The microRNA156 and microRNA172 gene regulation cascades at post-germinative stages in Arabidopsis[END_REF], in which miRNA172 acts as an inducer of FLOWERING LOCUS T (FT) expression. FCA, an RBP, binds to the flanking sequences of the stem-loop within primary miRNA172 transcripts (pri-mRNA172) via the RRM, and promotes its accumulation in response to ambient temperature (Figure 1). FCA also binds to the primary transcripts of other temperatureresponsive miRNAs, such as miRNA398 and miRNA399 [START_REF] Jung | Arabidopsis RNAbinding protein FCA regulates microRNA172 processing in thermosensory flowering[END_REF]. The RBP TOUGH (TGH) contributes to the pri-miRNA-HYL1 interaction (Ren et al., 2012), while MOS2 (MODIFIER OF SNC1, 2) is involved in pri-miRNA processing [START_REF] Wu | A role for the RNA-binding protein MOS2 in microRNA maturation in Arabidopsis[END_REF]. Many other examples exist, including EMBRYO DEFECTIVE 2793 (EMB2793, THO2), MOS4-ASSOCIATED COMPLEX 7 (MAC7), and REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) which participate in the regulation of miRNA biogenesis by interacting with HYL1 [START_REF] Francisco-Mangilet | THO 2 , a core member of the THO/TREX complex, is required for micro RNA production in Arabidopsis[END_REF][START_REF] Karlsson | KH domain protein RCF3 is a tissue-biased regulator of the plant miRNA biogenesis cofactor HYL1[END_REF][START_REF] Jia | The Arabidopsis MOS4-associated complex promotes microRNA biogenesis and precursor messenger RNA splicing[END_REF]. However, whether other core components of miRNA processing are dependent on RBPs and the way sugar signaling could contribute to this regulatory network still remains unclear.

SUGAR AND MRNA DECAY/STABILITY

In plants, mRNA decay/stability is an important control point in the regulation of gene expression and can discard potentially deleterious errors in mRNA synthesis [START_REF] Nagarajan | RNA degradomes reveal substrates and importance for dark and nitrogen stress responses of Arabidopsis XRN4[END_REF]. The mRNA decay/stability of many sugar-metabolism-related enzymes is controlled through post-transcriptional regulation. This holds true for the maize cell wall invertase gene (Incw1) that displays two transcripts -Incw1-S (small) and Incw1-L (large) -according to the respective lengths of its 3'untranslated regions (UTR; [START_REF] Cheng | Sugars modulate an unusual mode of control of the cell-wall invertase gene (Incw1) through its 3' untranslated region in a cell suspension culture of maize[END_REF]. Since sucrose and D-glucose appear to be associated with the increased steady-state abundance of Incw1-S mRNA and cell wall invertase activity, these authors suggested that the 3'UTR of the Incw1 gene was a regulatory sensor of carbon starvation and acted as a link between translation activity and the sink metabolism in plants. The 3'UTRs of OsVIN1 and AtvacINV2, encoding vacuolar invertases in rice and Arabidopsis, respectively, are involved in this process. Downstream regulatory elements or a motif that participates in the rapid degradation of mRNAs, e.g., small auxin-up RNAs (SAUR; [START_REF] Feldbrügge | Comparative analysis of the plant mRNA-destabilizing element, DST, in mammalian and tobacco cells[END_REF][START_REF] Van Mourik | Divergent regulation of Arabidopsis SAUR genes: a focus on the SAUR10-clade[END_REF], may be involved too [START_REF] Huang | Regulation of invertase: a 'suite' of transcriptional and post-transcriptional mechanisms[END_REF]. The expression of α-amylase, an endo-amylolytic enzyme that catalyzes starch degradation in plants, is induced by sucrose starvation and suppressed by sucrose availability in rice. Sugar repression of α-amylase 3 (αAMY3) expression in rice suspension cells involves controlling both its transcription rate and mRNA stability [START_REF] Sheu | Control of transcription and mRNA turnover as mechanisms of metabolic repression of α-amylase gene expression[END_REF] Frontiers in Plant Science | www.frontiersin.org 6 November 2020 | Volume 11 | Article 578096 [START_REF] Chan | The 3' untranslated region of a rice α-amylase gene functions as a sugar-dependent mRNA stability determinant[END_REF]). An analysis of reporter mRNA half-lives indicated that two subdomains of the αAMY3 3'UTR contained the UAUAUAUGUA motif required for the sugar-dependent destabilization of αAMY3 mRNA [START_REF] Sheu | Control of transcription and mRNA turnover as mechanisms of metabolic repression of α-amylase gene expression[END_REF][START_REF] Chan | The 3' untranslated region of a rice α-amylase gene functions as a sugar-dependent mRNA stability determinant[END_REF]. The same motif might also be involved in sugarmediated post-transcriptional downregulation of RhBRC1 in R. hybrida (Wang et al., 2019a), and could be conserved in angiosperms. Such sugar-dependent regulation of mRNA stability is required for the rapid adjustment of gene expression in response to the sugar status of the cell. In Arabidopsis cell cultures, the stability of 224 mRNAs was repressed by sucrose limitation, concomitantly with a drop in the cell metabolic activity [START_REF] Nicolai | Large-scale analysis of mRNA translation states during sucrose starvation in Arabidopsis cells identifies cell proliferation and chromatin structure as targets of translational control[END_REF]. The mRNA half-lives of actin (ACT), alcohol dehydrogenase 2 (ADH2), glyceraldehyde 3-phosphate dehydrogenase (G3PD), and sucrose synthase P-2 (SSP2) were consistently 1.6-to 2.6-fold longer in sucrosesupplied rice cells [START_REF] Ho | Sugar coordinately and differentially regulates growth-and stress-related gene expression via a complex signal transduction network and multiple control mechanisms[END_REF]. In line with this, the mRNA stability of the bZIP63 transcription factor, an important mediator of the adaptive response induced by SnRK1 during energy or sugar depletion [START_REF] Baena-González | A central integrator of transcription networks in plantstress and energy signaling[END_REF] decreased following exogenous glucose supply in Arabidopsis seedlings [START_REF] Matiolli | The Arabidopsis bZIP gene AtbZIP63 is a sensitive integrator of transient abscisic acid and glucose signals[END_REF]. The involvement of bZIP63 as a hub integrating the sugar and energy statuses and mRNA stability deserves to be addressed. Low-β-amylase1 (lba1) is a missense mutation of UP-FRAMESHIFT 1 (UPF1) RNA helicase, involved in nonsense-mediated mRNA decay (NMD). Its Arabidopsis mutant exhibited lower sugar induction of the AtβAmy transcript, which was restored by complementation of the lba1 mutation with wild type UPF1, further supporting the link between sugar signaling and the fate of the mRNA [START_REF] Yoine | The lba1 mutation of UPF1 RNA helicase involved in nonsense-mediated mRNA decay causes pleiotropic phenotypic changes and altered sugar signalling in Arabidopsis[END_REF]. All these findings clearly indicate a relationship between the sugar status and mRNA stability in a variety of biological contexts, opening the avenue for deciphering the sugar sensing and signaling mechanisms. In line with that, mRNA stability might also be important for the diurnal regulation of mRNA levels of sucrose transporters and in turn in sugar allocation at the whole plant level. For example, a sucrose transporter (SUT1) displayed a quick turnover rate in leaves of tomato (Lycopersicon esculentum), potato (Solanum tuberosum), and tobacco (Nicotiana tabacum; [START_REF] Kühn | Macromolecular trafficking indicated by localization and turnover of sucrose transporters in enucleate sieve elements[END_REF][START_REF] Kühn | Sucrose transporters of higher plants[END_REF]. The mRNA levels of StSUT2 and StSUT4 may be regulated by putative RBPs [START_REF] He | Phloem mobility and stability of sucrose transporter transcripts[END_REF]. Two AUUUA motifs exist in the 3'UTR or CDS region of StSUT2 and StSUT4 mRNA [START_REF] He | Phloem mobility and stability of sucrose transporter transcripts[END_REF]; they have been characterized as the binding sites of proteins involved in mediating mRNA degradation [START_REF] Chen | AU-rich elements: characterization and importance in mRNA degradation[END_REF][START_REF] He | Phloem mobility and stability of sucrose transporter transcripts[END_REF][START_REF] Liesche | Sucrose transporter regulation at the transcriptional, post-transcriptional and post-translational level[END_REF]. However, the nature of these proteins is still unknown.

SUGAR AND ALTERNATIVE SPLICING

Alternative splicing is a finely regulated process that takes place during gene expression and leads to a single gene coding for multiple proteins. Serine/arginine-rich 45 (SR45) is a serine/ arginine-rich splicing factor that participates in 5' and 3' splicing site selection of introns and can bridge the 5' and 3' components of the spliceosome. The SR45 splicing factor regulates glucose signaling during early seedling development in Arabidopsis [START_REF] Carvalho | The plant-specific SR45 protein negatively regulates glucose and ABA signaling during early seedling development in Arabidopsis[END_REF], more likely through the modulation of SnRK1-stability [START_REF] Carvalho | The Arabidopsis SR45 splicing factor, a negative regulator of sugar signaling, modulates SNF1-related protein kinase 1 stability[END_REF]. The sr45-1 knockout mutant indeed displays a high level of the energy-limitationsensing SnRK1 protein under glucose supply, which is in agreement with the upregulation of SnRK1-activated genes such as SENESCENCE-ASSOCIATED GENE1 (SEN1), GLUTAMINE-DEPENDENT ASPARAGINE SYNTHETASE1 (ASN1), and DORMANCY-ASSOCIATED GENE2 (DRM2). Moreover, the glucose hypersensitivity of the sr45-1 mutant is alleviated when SnRK1 is disrupted (Figure 1). SR45 controls the alternative splicing of 5-phosphatase 13 (5PTase13) in Arabidopsis, which encodes an inositol polyphosphate 5-phosphatase involved in regulating SnRK1 stability negatively in vitro [START_REF] Carvalho | The Arabidopsis SR45 splicing factor, a negative regulator of sugar signaling, modulates SNF1-related protein kinase 1 stability[END_REF]; Figure 1). This link between sugar signaling and RNA splicing has also been reported for the photomorphogenesisrelated alternative splicing shifts primarily controlled by a metabolic photosynthesis-derived signal and exogenous sucrose supply, correlated with the expression of dark-induced genes under the control of SnRK1 [START_REF] Hartmann | Alternative splicing substantially diversifies the transcriptome during early photomorphogenesis and correlates with the energy availability in Arabidopsis[END_REF]. AtTZF1/ AtCTH/AtC3H23 (a tandem-arrayed CCCH-type zinc finger motif involved in stress-and hormone-mediated growth), was also identified as a sugar-sensitive gene in Arabidopsis [START_REF] Qu | The Arabidopsis thaliana tandem zinc finger 1 (AtTZF1) protein in RNA binding and decay[END_REF]. AtTZF1 can traffick between the nucleus and cytoplasmic foci and bind both DNA and RNA in vitro; it may be involved in RNA regulation and under the control of sugar signaling [START_REF] Pomeranz | The Arabidopsis tandem zinc finger protein AtTZF1 traffics between the nucleus and cytoplasmic foci and binds both DNA and RNA[END_REF]. However, the basic molecular mechanisms behind this regulation have not been addressed to date.

CONCLUSION

Post-transcriptional regulation is an essential component of gene expression regulation in plants. Numerous findings have unveiled and characterized various factors involved in post-transcriptional regulation. The present review provides a first comprehensive picture of the relationship between sugar (metabolism and signaling) and post-transcriptional regulation factors in plants, including RBPs, miRNAs, and mRNA stability of sugar-related genes. More work needs to be carried out to figure out the functions and mechanisms related to the involvement of post-transcriptional regulation in sugar-related processes, e.g., whether regulatory mechanisms found in human cells or yeast are also conserved in plants. Considering the frequently observed connection between mRNA abundance and sugar, some recently developed technologies for RNA editing (CRISPR-Cas13), RNA binding (RNA interactome capture, photoactivatable ribonucleoside-enhanced crosslinking), and RNA folding (DMS-seq, SHAPE-seq) will support future studies. Besides, many aspects of RNA decay still need to be studied in depth, such as the spliceosome and the editosome (a large multi-protein complex that catalyzes RNA editing), which play a crucial role in post-transcriptional regulation. Although some reports about the interaction between sugar-related RBPs and miRNAs exist, further investigations are still required to gain a comprehensive understanding of the way sugar signaling operates through each of these post-transcriptional regulation

Frontiers

  in Plant Science | www.frontiersin.org 2 November 2020 | Volume 11 | Article 578096

FIGURE 1 |

 1 FIGURE 1 | The relationship between sugar and RNA-binding proteins (RBPs), the crosstalk between miRNA and sugar related RNA binding proteins, and alternative splicing in Arabidopsis. The green arrow means stimulation or positive effect, the red line means inhibitory effect, and the black line means protein interaction. GRP2, GLYCINE RICH PROTEIN 2; m-MDH, mitochondrial malate dehydrogenase; CS, citrate synthase; FCA, FLOWERING CONTROL LOCUS A; FLC, FLOWERING LOCUS C; ABI5, ABA-insensitive 5; SOAR1, SUPPRESSOR OF THE ABAR OVEREXPRESSOR 1; DRM2, DORMANCY-ASSOCIATED GENE2; SEN1, SENESCENCE-ASSOCIATED GENE1; ASN1, GLUTAMINE-DEPENDENT ASPARAGINE SYNTHETASE1; 5PTase13, 5-phosphatase 13; SR45, serine/ arginine-rich 45; PIF4, phytochrome-interacting factor 4; MPK3, MITOGEN-ACTIVATED PROTEIN KINASE 3; WAKs, WALL-ASSOCIATED KINASES.

FIGURE 2 |

 2 FIGURE 2 |The relationship between sugar and reported miRNA, and the function of the related miRNA. Sugar stimulates the transcription of miRNA398, miRNA408, and miRNA319 but inhibits that of miRNA160 and miRNA156. SnRK1 inhibits the transcription of miRNA824A, miRNA775A, and miRNA159B. HKX1, hexokinase 1; SPL, SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE.

TABLE 1 |

 1 The type and function of sugar related RBPs.

	Gene	RNA-binding protein	Function
		type
	AtRZ-1a	Zinc finger-containing	Involve in freezing
		glycine-rich RNA-binding	tolerance and cold stress
		protein
	GLYCINE RICH PROTEIN	RRM protein	Response to cold,
	2		osmotic stress, water
			deprivation, and seed
			germination
	FLOWERING CONTROL	RRM protein and contain	Involved in the promotion
	LOCUS A	WW domain	of the transition of the
			vegetative meristem to
			reproductive development
	HYPONASTIC LEAVES 1 No reports	Flowering, leaf, and root
			development
	APUM2, APUM3, and	Pumilio/FBF protein	No reports
	APUM4
	ALDH7B4	No reports	Aldehyde dehydrogenase
	RAFFINOSE SYNTHASE	No reports	Biosynthesis of the
	6		raffinose; response to
			cold, karrikin, and
			oxidative stress
	SOAR1	PPR protein	ABA responses, probably
			located upstream of an
			ABA-responsive
			transcription factor ABI5
	RhPUF4	Pumilio/FBF protein	Involved in bud outgrowth
			probably
	The function of Arabidopsis protein is from the annotation of TAIR database

ACKNOWLEDGMENTS

This work was supported by the program of China Scholarships Council (No. 201506320203), Talent Introduction Special Funds of Qingdao Agricultural University (663/1120070) and by the ANR (Agence Nationale de la Recherche) project Labcom, called ESTIM (Evaluation de STIMulateurs de vitalité des plantes; Projet ANR-15-LCV3-000).

mechanisms and how they crosstalk to regulate plant growth and development. The hub role of hexokinase, SnRK1, and/ or TOR kinase but also the relevance of the trehalose signaling pathway in the different post-transcriptional regulation networks could be two main future lines of research. This further knowledge will also pave the way for discovering a new and complex sugar regulatory network in plants.

AUTHOR CONTRIBUTIONS

All authors listed have made direct contribution to the work, and approved it for publication. MW, LZ, FJ, SS, and JC have written different part of the manuscript. M-DP-G, LO, and LH have contributed to the section sugar and RNA binding proteins and JL to the section of sugars and miRNA.

Conflict of Interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Timothée Baudequin

Cécile Legallais

Dr Fahmi Bedoui
email: fahmi.bedoui@utc.fr

In vitro bone cell response to tensile mechanical solicitations: is there an optimal protocol?

Keywords: Bone, mechanical solicitations, scaffolds, stretching, tensile loading ALP, alkaline phosphatase, MSC, Mesenchymal stem cell, PGE2, Prostaglandin E2

Bone remodelling is strongly linked to external mechanical signals. Such stimuli are widely used in vitro for bone tissue engineering by applying mechanical solicitations to cell cultures so as to trigger specific cell responses. However, the literature highlights considerable variability in devices and protocols. We reviewed here the major biological, mechanical and technical parameters implemented for in vitro tensile loading applications.

The objective was to identify which values were used most, and whether there was an optimal protocol to obtain a functional tissue-engineering construct.

First, we showed that a shift occurred from fundamental comprehension of bone formation, to its application in rebuilt tissues and clinical fields. Despite the lack of standardised protocols, consensual conditions relevant for in vitro bone development, in particular cell differentiation, could be highlighted. Culture processes were guided by physiological considerations, although out-of-range conditions were sometimes used without implying negative results for the development of rebuilt tissue. Consensus can be found on several parameters, such as strain frequency (1 Hz) or the use of rest periods, but other points have not yet been fully established, especially synergies with other solicitations. We believe the present work will be useful to develop new tissueengineering processes based on stretching.

Introduction

Bone is a living tissue constantly renewed by the combined actions of osteoclasts (responsible for bone resorption) and osteoblasts (bone formation) [1,2] . It is well-known that this remodelling is enhanced by mechanical solicitations. Wolff [3] first suggested in 1892 that there was a strong response from bone tissue to external mechanical loading in vivo, leading to resorption or an increase in formation. This behaviour was thus analysed in vitro and, in 1987, Frost proposed the principle of mechanostat [4] to explain the remodelling (Figure S1, Supporting data). In vivo, understanding of what is now mainly called "bone mechanotransduction" progressed year after year from "key early findings" [5] , based in particular on the work of Rubin and Lanyon [6][7][8][9] (Figure S1, Supporting data).

During the mechanotransduction process, a mechanical signal is translated into a biological one, with a strong role played by fluid motion in the lacuna-canalicular network [10] where osteocytes (mature osteoblasts stuck in the extracellular matrix) play the role of sensor cells [11] . Although not all of the mechanisms involved are perfectly understood yet, they have been reviewed many times [5,[11][12][13] . In particular, specific strain levels have been proposed as in vivo threshold values for triggering physiological and pathological responses [13] . This knowledge helps researchers in biotechnologies to adapt in vivo mechanisms to in vitro cell cultures, to refine understanding of bone behaviour but also to develop tissue-engineered substitutes [14][START_REF] Nair | Nanotechnologies Life Sci[END_REF][START_REF] Li | [END_REF] : cells of interest collected from the patient's body are cultured, mainly in bioreactors, on specific biomaterials ("scaffolds") to provide them with an appropriate environment for improving adhesion, proliferation or differentiation. The rebuilt tissue, a hybrid substitute made of both material and cells, can then be used to heal the same patient [17] . Differentiation relies on changes in the culture environment via the external signals (biochemical, mechanical) applied to the cells [START_REF] Khademhosseini | Micro and Nanoengineering of the Cell Microenvironment: Technologies and Applications[END_REF][START_REF] Hughes | [END_REF] . We will focus here on the specific requirements for in vitro culture steps, prior the implantation, to obtain a functional tissue-engineered construct.

There are different ways of applying mechanical solicitations [20] and many devices have been used and reviewed [20][21][22] . We have chosen here to focus on the application of tensile loading through deformation of the surface or scaffold on to which cells have adhered and tissue developed. Although compression (due to daily activity and body own weight) and fluid shear stress (due to osteocytes' sensibility in the lacuna-canalicular network [10] ) are physiological mechanical solicitations in vivo and can be mimicked in laboratory [23][24][25] , tensile stress remains a common and easy method to trigger the cell response in vitro. Over the years, several devices and bioreactors have been proposed to perform such solicitations, in particular by mounting the sample into clamps to stretch it, by bending the culture substrate with a 4-point system or negative pressure (Figure 1A,B,C). However, to date there has been no analysis of their optimal configuration for specific applications. Therefore, the present review focuses on tensile mechanical stimuli for in vitro bone cell cultures to analyse which ranges are the most used and whether an optimal protocol can be proposed to enhance tissue development. To the best of our knowledge, such a specific study analysing culture schedules and mechanical, biological and technical parameters has not yet been carried out.

Methodology

The first survey of the literature was performed using classical search engines (Pubmed, Scopus, Google Scholar), looking for the appearance in title or abstracts of the keywords: "bone, tensile, mechanical loading, in vitro", leading to around 200 occurrences.

It had been decided previously not to include articles from earlier than 1990 because Murray et al [26] was reported as the first publication studying the effect of mechanical load on in vitro cell culture [27] , following key early in vivo findings [6][7][8][9] . The publications were found to be spontaneously distributed over the last 3 decades (Figure 1D). Articles were further selected if they involved in vitro cell cultures, tensile loading and bone tissue, and were focused enough on these points to provide all the information requested (geometry of the mechanical device, frequency and magnitude of strain, etc) and a conclusion on the specific influence of solicitations. This screening led to the selection of 37 articles following the same trend over the years as the first general selection (Figure 1D). A systematic analysis of the in vitro culture steps presented in these papers was therefore performed to highlight major trends or variability, consensus, or processes still under discussion.

General analysis

Bone tissue engineering pillars

There are generally 3 components considered as essential for growing or building engineered tissue in vitro: cells, biomaterial and environment [28] . Classical 2D and new 3D culture approaches can be used, with various types of cell and different devices generating tensile loading as a specific dynamic environment. The relevant parameters from the above mentioned studies are summarised in Table 1.

Types of cell and relevant biological responses

Many different bone cell types have been used, from different species, lineages or differentiation states. The overall distribution is summarised in Figure 1E. This variety of cell sources can be partly explained by technical issues, such as the presence of animal facilities, ethical considerations, skills, costs, and time. Cell lines are therefore very popular, because of availability and stability, although they cannot claim to be completely the same as primary cells. In addition, there is no consensus on a specific line to study, and 8 different cell lines have been reported, the most common being MC3T3-E1 [29][30][31][32][33] .

Monocytes were also included when investigating osteoclastogenesis [34,35] . Primary fibroblasts were also mentioned in 2 cases, corresponding to a control group [29] for one and to the specific study of the bone/tendon interface [36] for the other.

For a very large number of articles, the biological analysis focused mainly on the functionality or differentiation state of the tissues or cell populations at the end of the culture period. Authors investigated differentiation states through gene expression analysis, production of specific proteins (such as osteocalcin or osteopontin), ECM deposition, alkaline phosphatase (ALP) activity, mineralisation, and bone nodule formation. In the case of co-culture studies or when the balance with other functions was explored, markers of other lineages were also analysed (for instance osteoclastogenesis markers [35] ).

In certain publications, the main objective was to highlight specific molecule signalling or specific pathways [26,29,31,34,[37][38][39][40][41][42][43][44][45][46] . The intention of these studies was not to obtain functional bone tissue-engineered constructs, but rather to have better fundamental understanding of bone response to solicitations.

In addition to differentiation, general cell properties were often investigated, such as cell viability, metabolic activity or proliferation [30,31,[47][48][49][50][51][52][53][54] . In particular, Weyts et al [53] proposed an extensive study of cell apoptosis with a cross-analysis of mechanical signals and differentiation. Cell morphology, more particularly alignment, was also a focus in several publications [48,49,[55][56][57] . Cell alignment could indeed improve bone growth and differentiation, an anisotropic tissue [58] . Finally, Struewer et al [36] specifically studied the influence of mechanical solicitations on cell detachment.

Biomaterials

We refer here to scaffolds when specific 2D or 3D biomaterials were used in addition to a standard substrate to perform and enhance cell cultures. The majority of the studies used only a standard culture surface (membrane, plate, bottom of a well or a dish), sometimes with a coating to improve cell adhesion (collagen or fibronectin, see for instance [43] ). When using scaffolds, degradation rate, overall biocompatibility and degradation products remain crucial parameters to ensure a successful integration in the body [59][60][61][62][63] .

Here, scaffolds were knitted [49] or electrospun [64] polymer fibres (respectively polyethylene terephthalate and polycaprolactone), fibrin or collagen gels [30,41,65,66] , a 3D

collagen structure [47] , or a nanostructure obtained by lithography [56] . Nevertheless, these biomaterials were not used as active culture structures capable of monitoring cell behaviour, and the effect of their morphology on the transmission of mechanical solicitations was not analysed. Mechanical solicitations remained the main way of monitoring cell development in these studies. Only Prodanov et al [56] studied the impact of a nanostructured surface on cell behaviour, concluding that synergy could appear between a relevant nanostructure and mechanical loading. Bruinink et al [49] compared the results of cell-seeded scaffolds with nude ones to estimate variations in its mechanical properties due to the presence of living tissue.

Environment: tensile loading application

Different devices were proposed to apply tensile loading at the cell scale through the deformation of samples. Regarding the culture substrate itself (besides an additional scaffold as seen above), it was possible to distinguish three cases: culture wells on elastic plates (16 papers), direct culture on dishes / plates / elastic membranes (15), or a specific culture chamber (6). Extensive reviews of the different mechanical loading devices and bioreactors have been proposed [14,20,21,67] . Here, the application of tensile loading to the living tissue were performed with 4-point bending (8 papers), by applying negative pressure to the substrate (13), by mounting the samples directly into clamps (11), or with specific designs (3). Overall, the geometry of the systems was not highly diversified, but to date there is no gold standard. Commercial devices may by a range of magnitude and frequency values or when specific scaffolds have to be used. The articles using scaffolds confirmed this, with 7 out of 8 publications using original or custom-made devices.

Main aims when applying tensile loading in vitro

All the publications aimed to analyse the influence of mechanical loading on cell behaviour, but more detailed objectives can also be highlighted, with two main trends (Figure 1G). On the one hand, articles focusing on understanding cell response mechanisms under mechanical solicitations mostly occurred in the early years (before 2001) [26,[40][41][42][43][44]50,[68][69][70] . On the other, the exploitation of the potential of these cell responses for clinical applications [45,66] and tissue engineering [36,38] started in the last decade, with the first publication in the eponymous journal [65] in 2010. 7 out of the 8 papers using a scaffold were published in the second half of the studied period (after 2001). This highlights a logical shift from fundamental comprehension of bone cell response to external solicitation, to its application for rebuilt tissues and clinical fields by using mechanical loading in vitro before implantation.

Culture conditions and loading protocol

Dynamic loading

There was a major consensus on the use of dynamic loading, in particular a sinusoidal signal with a 1-Hz frequency. It was often argued that this value was chosen as a physiological one for everyday life activities i.e. physiological walking [4,6,9,50,71,72] .

The remaining studies differed in frequency with lower values, from 0.05 Hz [70] , to 0.5 Hz [30,45,53] . Tanaka et al [30] was the only one to use a frequency above 1 Hz (3 Hz).

Bruinink et al [49] used a frequency of 0.01 Hz as a physiological value for rest periods, which was in fact close to the solicitation frequency chosen by Cillo et al [70] (0.05 Hz).

Significant results were nevertheless obtained with this low value.

In contrast with frequency, the maximum magnitude varied considerably from one study to another (reported either as a microstrain unit (µstrain) or an elongation percentage (1,000 µstrain = 0.1%)). When different strains were successively applied to the same sample during the experiment, only the maximum value was considered in the analysis. Magnitude distribution is shown in. Most of the articles used a magnitude of less than 25,000 µstrain (2.5% elongation), in particular from 1,000 to 5,000 µstrain (0.1 to 0.5%), which is indeed considered to be a physiological range [13,44,73] (Figure S2, supporting data). Bone resorption occurs at less than 200 µstrain, a physiological range is up to 2,500 µstrain (walking and running generate 400 and 950 µstrain in humans, respectively [7,9,50] ), an overused window with increasing formation is up to 5,000 µstrain and pathological overloading above [13] .

However, many studies used loading protocols outside of this physiological window. Bruinink et al [49] first specified that a 20,000 µstrain would be a "macroscopic strain", enabling the easy detection of the in vitro response to a magnitude greater than the usual in vivo response. Zhu et al [74] used the same strategy and multiplied the values obtained by finite element modelling by 20 before in vitro culture to ensure cell response.

With deformation over the physiological range, it may become possible to compare physiological and pathological behaviours [26] . Nevertheless, it remains important to limit magnitudes under a threshold that would lead to cell death and bone resorption [49] . The maximum magnitude could also be limited by the behaviour of the substrate itself: we could assume that plastic deformation of the scaffold under solicitations would have an impact on the culture and loading applied to the cells. It should therefore be avoided.

Most of the articles studying several magnitudes actually compared physiological and higher values. Cell response peaked around the physiological range in terms of prostaglandin E2 (PGE2) production [26] , proliferation [40,52] or ALP production [51] . However, an upturn sometimes appeared for the higher values, for instance a second peak of PGE2 production above 28,000 µstrain [26] or a fibrous response in addition to proliferation above 10,000 µstrain [40] . For Murray et al [26] , these results showed the repair mechanisms which may occur in the failure range. In other articles, the parameters analysed remained stable for any range of magnitude for cell viability [52] , proliferation [53] or apoptotic response [53] . Finally, some responses shifted, highlighting the difficulty in adapting in vivo principles to in vitro experiments. For instance, Thomas et al [68] found an ALP production peak for 230 µstrain (compared to 35 and 2500 µstrain), lower than expected [68] . The shift often occurred towards higher values, as found by Jagodzinski et al [75] (ALP, osteocalcin and Cbfa1 production for 80,000 µstrain), Koike et al [51] (proliferation, 150,000 µstrain), Zhu et al [74] (osteocalcin, collagen, runx2 production, 32,000 µstrain), Prodanov et al [56] (continuously increased alignment from 10,000 to 80,000 µstrain) and Matsugaki et al [55] (alignment peak, 40,000 µstrain).

Duration of the dynamic culture phase

The dynamic culture phase lasts from the very first mechanical load to the last cycle, even if rest periods occurred between these points. Most of the studied articles (around 40%) belong to two groups: a very short (<1 hour) or a long (1 day -7 days) dynamic culture period. Short times may be more suitable for studying the early cell response to tensile loading, with regular sampling, whereas long experiments make it possible to study overall tissue reconstruction.

Certain articles detailed a comparison between different durations, either for short times [57,70] , or several days or even weeks [48,54,64] . The results showed that some parameters, especially gene expressions, had non-monotonic variations with a peak at a specific duration [57,70] , while other values continuously increased or decreased (respectively mechanical properties [64] and metabolic activity [48] ). Connelly et al [65] suggested that these different behaviours may have occurred because of successive effects and different mechanism triggers, and thus noted the need for both short and long term studies. For instance, cell response began with an increase in the production of proteins and proteoglycans, then this production decreased and an increase in collagen production occured [65] .

As these non-monotonic variations sometimes occurred for a very short time, it was therefore interesting to use high sampling even for long term studies, because quick adaptive responses could be missed (for instance gradual or cyclic responses), just as the real period of a periodic signal could be lost if the sampling did not satisfy the Nyquist-Shannon theorem [76] , classically respected in electronics and signal acquisition and processing.

Rest periods

In vivo studies suggested that cell sensitivity to tensile loading could saturate, but is quickly recovered [6,77,78] . This means that cells are able to recover their capacity to translate mechanical signals into a specific biological response after a short or very short rest period [6,77,78] . Many studies mimicked this concept in vitro, using different rest periods schedules (Figure 1H) but several exceptions could be noticed. In particular, Baker et al [64] and Bruinink et al [49] did not use complete rest periods but left the samples respectively under agitation or with low-frequencies solicitations (0.01 Hz). However, they referred to these phases as "resting phase" [49] . We could assume that this method also mimicked in vivo behaviour, where cells never experience complete rest, due to muscle tone, body's own weight or normal micro-movements [79] .

Conclusion

To date, there is no standard protocol for applying tensile loading to in vitro cell cultures, nor any specific protocol depending on target applications or cell sources.

However, for certain technical parameters, optimized and consensual ranges of values could be highlighted and appeared to be both relevant and efficient (summary table S1).

The analyses of schedules and technical parameters allowed us to highlight "optimal" values, i.e. protocols which led to huge differences in the biological response compared to unstrained control groups while, in the best cases, bone differentiation was triggered. As an overall analysis, a 1-Hz sinusoidal signal in the physiological range seems relevant in most cases for preventing cell death and detachment while triggering bone cell differentiation, maturation and functions. Short dynamic phases separated by rest periods, for instance 600 cycles (10 minutes) of stretching every 6 hours, should allow cells to recover their sensitivity which can fade after a moderate number of cycles. A 24-hour static pre-culture expansion stage should also be considered, in particular when a scaffold is used, to ensure cell attachment.

In contrast, other parameters could be seen as irrelevant and showed decreases in cell viability or cell numbers. In particular, an out-of-range magnitude could lead to undesirable effects, such as no cell response below specific loading (300 µstrains in contrast with 3,000 µstrains and more [40] ) or a decrease in cell alignment [55] and proliferation [52] for high magnitudes.

The analysis was conducted here for tensile loading devices and bone tissue applications, and the main conclusions could be used as a tool to help in decision-making in the development of future experiments. The technical parameters were often guided by in vivo results or physiological values, but moving to out-of-range conditions did not necessarily have a negative impact, especially when the main objectives were to obtain a rebuilt tissue. It appeared furthermore that a slide from studies of fundamental mechanisms to tissue engineering applications may be occurring. We can expect this shift to be strengthened in the future as tissue engineering processes start to reach clinical applications. Moreover, there is now a growing interest for the regeneration of interfaces and composite tissues, complex hybrid systems [80] . Mechanical solicitations will require accurate optimisation to monitor the growth of several cell lineages simultaneously in addition to bone, such as ligament [81] .

Table 1. Detailed summary of the articles studied.

Table 1. Detailed summary of the articles studied

Figure 1 :

 1 Figure 1: Examples of cell tensile loading, with uniaxial stretching into clamps (A), 4-point bending (B) or gel matrix and vacuum (C). Adapted from Brown et al[20] and Flexcell® documentation. D: Cumulative distribution of the articles retained (blue line) and of the total number of publications with the keywords "bone tensile mechanical loading in vitro" according to Scopus (red line). Distribution of (E) cell sources regarding species, origins (primary cells / cell lines), (F) differentiation states and functions, (G) the main objectives as announced in the respective introductions (some studies announced several objectives) and (H) the different ways of applying rest periods to cell cultures in vitro.

Acknowledgement

Timothée Baudequin acknowledges the financial support of the CNRS and the Collegium INSIS-UTC.

Conflict of interest

The authors declare no conflict of interest.