-
The observation of cell-to-cell movement of large molecules initially arose from the micro-injection of fluorescent dye in plant tissues[21−24]. The first endogenous protein exhibiting the intercellular mobility is KNOTTED1 (KN1), a homeodomain protein essential for maintenance of the shoot apical meristem (SAM) in maize[25,26]. Recently, the ribosomal RNA-processing protein 44A (AtRRP44A) was shown to mediate the cell-to-cell trafficking of KN1[27]. Since then, a large number of transcription factors were identified in plants that can move between tissues and cells to provide positional instruction during plant development[21]. These mobile regulators can traffic across just a few cell layers to function locally or over a long distance to affect global developmental change.
One of the central questions in organogenesis is how to spatiotemporally maintain stem cells and specify cell fates. In SAM, WUSCHEL (WUS) is expressed in the organizing center of shoot apical meristem, but the protein moves to the layer1 and 2 (L1 & 2) of shoot apical meristem where WUS triggers CLAVATA 3 (CLV3) expression, which in turn inhibits WUS transcription in L1 and L2 layer[28,29]. With this WUS-CLV3 feedback loop, plants can maintain the stem cell population in proper size in SAM. With the similar strategy, plants maintain the root stem cell niche via WOX5-CLE40 loop, in which WOX5 traffics from quiescent center (QC) to columella stem cell (CSC) to repress the cell differentiation[30]. In Arabidopsis, SHOOT MERISTEMLESS (STM) and ARABIDOPSIS KNOTTED-LIKE (KNAT1)/BREVIPEDICELLUS (BP) are two homologs of the KN1 gene, previously described to be mobile in maize SAM. When driven by an L1 specific promoter, STM and KNAT1 were observed to move from the L1 layer into the inner cell layers of the SAM[31,32]. In addition, KNAT1 was able to pass the interface between cortex and epidermis in Arabidopsis when mis-expressed by a mesophyll specific promoter[33].
In embryogenesis, TARGET OF MONOPTEROS 7 (TMO7), encoding a bHLH transcription factor, is essential for hypophysis, the founder cell for forming root apex during post-embryonic growth. TMO7 is transcribed in embryonic cells while the TMO7-GFP fusion can be detected in the neighboring hypophysis, indicating a non-cell-autonomy of this regulator[34,35]. In post-embryonic growth, intercellular movement of transcriptional factors regulates a variety of developmental aspects ranging from root radial patterning to root hair and trichome initiation. These mobile regulators including SHORT-ROOT (SHR), CAPRICE (CPC), TRANSPARENT TESTA GLABRA 1 (TTG1), GLABRA 3 (GL3), ENHANCER OF TRY AND CPC 3 (ETC3)/ TRIPTYCHON (TRY), UBIQUITIN-SPECIFIC PROTEASE (UBP1) have been well reviewed previously[15,21]. A previous screen estimated that around 15% of transcriptional factors in roots can move between cells[36]. In contrast, we only have limited understanding of the functionality of these mobile proteins.
Recently, more mobile transcriptional factors have been identified (summarized in Table 1). Two closely related AT-hook family members, AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 3 (AHL3) and AHL4, were shown to interact in vivo and regulate the boundaries between the procambium and xylem[37]. Interestingly, their interaction seemed to be required for their intercellular trafficking. A SHR target, SCL23 displays a bidirectional radial spread and long-range movement into meristem in Arabidopsis roots. Through direct interaction, SCL23 controls movement of SHR and participate in endodermal specification in the root meristem[38].
Table 1. Summary of the mobile transcription factors identified in plants.
Mobile TFs Function Moves from:to Reference HY5 Root growth and N uptake Shoot-to-root Chen et al. (2016)[41] DWARF14 Regulate the development of AMs Through phloem into axillary meristems (AMs) Kameoka et al. (2016)[139] BdMUTE BdMUTE is required for subsidiary cell formation GMCs to neighboring cell files Raissig et al. (2017)[97] SPCH Stomatal cell fate Cell-to-cell diffusion in the leaf epidermis of chorus Guseman et al. (2010)[96] AN3 Leaf development From the mesophyll to the epidermis in leaves Kawade et al. (2013)[140] WUS Meristem maintenance From the organizing centre to L1, L2 layers Yadav et al. (2011)[28] KN1/STM Meristem maintenance Broadly in the SAM Kim et al. (2003)[31], 2005[32] PLT2 Longitudinal root zonation Longitudinally from the root meristem forming a gradient Mahonen et al. (2014)[141]; Galinha et al. (2007)[142] SHR Root radial patterning and RAM maintenance Within Stele; Stele into endodermis, QC, CEI and CED Koizumi et al. (2011)[44], Nakajima et al. (2001)[78] AHL3/AHL4 Xylem specification From procambium cells to the xylem Zhou et al. (2013)[37] WOX5 Stem cell maintenance QC to CSC Pi et al. (2015)[30] TMO7 Recruitment of the hypophysis Embryo into the upper cell of suspensor Schlereth (2010)[34]; Lu et al. (2018)[35] Cyp1 Root growth From leaves to root in tomato Spiegelman et al. (2015)[143] UBP1 Transition from cell division to elongation Stele and LRC to cells into transition/elongation zone Tsukagoshi et al. (2010)[144] SCL23 Endodermal cell fate Bidirectional radial spread and movement into meristem Long et al. (2015)[38] TTG1 Trichome patterning Atrichoblasts into trichome initials CPC Trichome patterning, root hair initiation Trichome initials into Atrichoblasts; non-root hair cell into root hair cell Wester et al. (2009)[90] GL3/EGL3 Root hair initiation Root hair cell into non-root hair cell Kang et al. (2013)[91] Besides the local regulation, transcriptional factors were also found to traffic long-distance between organs to direct global developmental transition in plants in Fig 1. An early example is the detection of FLOWERING LOCUS T (FT) trafficking from leaves where it is synthesized in response to day length, to the
SAM to trigger flowering[39,40]. Recently, a light-activated transcriptional factor, ELONGATED HYPOCOTYL 5 (HY5) was shown to move via phloem from shoot-to-root. This translocation of HY5 was proposed to mediate light-activated root growth and N uptake from the soil to balance photosynthetic carbon fixation in the leaf[41]. Figure 1.
Mobile proteins and RNAs in plant development and stress response. The mobile regulators participate widely in the development of different organs (as illustrated). They can travel short-range to regulate local tissue patterning or long-distance to transduce systemic signaling. Gray arrow: phloem-based long-distance movement. WUS and STM regulate SAM maintenance; SPCH, BdMUTE, AN3, TTG1, GL3 and CPC are involved in epidermal patterning. In roots, PLT2, SHR, AtDof4.1, AHL3/AHL4, WOX5, TMO7, UBP1 and SCL23 govern a variety of processes including cell division, radial patterning, stem cell maintenance and developmental transition. Long-distance signaling regulators such as FT and HY5 can traffic from leaves to SAM to promote flowering, and from shoot to root to regulate root growth and nitrate uptake respectively. Environmental stresses can induce PD closure. Small RNAs including miR399d, 827 and 2111 move from aerial parts to roots in response to phosphate starvation.
Considering the size of transcriptional factors, PD seems to be the most possible way for the intercellular translocation. With an iclas3m system (described in detail in a later part of this review) that blocked the PD between stele and endodermis, SHR intercellular transport was terminated[3]. Another piece of evidence supporting PD transport of transcriptional factors is the blocked movement of TMO7 from meristematic cells into the root cap in the cals3-2d, a mutant in which PD is restricted by over-accumulated callose[35]. To get access to PD, transcriptional factors could exploit intracellular apparatus including microtubules and endomembrane delivery system[42,43]. Besides, an unknown function protein named SHR INTERACTING EMBRYONIC LETHAL (SIEL) was shown to interact with a number of mobile transcriptional factors and the mutation of this gene seemed to reduce SHR intercellular movement[44]. As SIEL partially localized to endosomes, it was proposed that this protein could function as a 'shuttle' to facilitate delivery of mobile transcriptional factors. In addition, some facilitating proteins have also been identified. After passing through PD, a few mobile proteins including APS KINASE 1 (KN1), SHOOT MERISTEMLESS (STM) and TRANSPARENT TESTA GLABRA 1 (TTG1) were discovered to associate with a group of type II chaperonin complexes consisting of CHAPERONIN CONTAINING T-COMPLEX POLYPEPTIDE-1 SUBUNIT 7 and 8 (CCT7 & CCT8), which facilitate the movement possibly by promoting the protein refolding after the PD cross-over[27].
Although no specific domain has been identified that accounts for intercellular mobility, the cell-to-cell transport of transcriptional factors seemed to be protein sequence-dependent. Homeodomain (HD) and the helical domains have been shown to be necessary and sufficient for PD-mediated transport of KN1. Unlike this, three conserved domains (HD, WUS-box, and EAR-like domain) in WUS
are not required for its movement. Instead, WUS mobility seems to be controlled by a non-conserved sequence between the HD domain and WUS-box[29]. Despite triple GFP Tag impaired TMO7 movement, protein size did not seem to be the primary determinant of intercellular transport. Instead, TMO7 was found to move in a sequence-dependent manner, and both nuclear residence and protein modification are important for TMO7 mobility[35]. In two other mobile transcriptional factors, CPC and SHR, the mobility relied on multiple regions within the proteins. In addition, the mobility of these two proteins seemed to be associated with the subcellular distribution in both the cytoplasm and the nucleus. In addition to transcriptional factors, small RNAs also participate in transcriptional regulation of diverse developmental and physiological events in plants. Small RNAs are 21−24 nt long and can be generally divided into siRNAs and miRNAs[45]. Small RNAs function either through degrading target genes by near-perfect complementarity, or via transcriptional silencing by histone modification and DNA methylation[46−50]. Small RNAs were often regarded as the long-distance signals as the initial efforts dissecting their mobility exploited the grafting system in which mutants defective in small RNAs biogenesis were included. Facilitated by high-throughput sequencing techniques, researchers identified a large number of mobile siRNAs that can traffic from shoot to root presumably via phloem. Besides siRNA, a large number of miRNAs were discovered to traffic in phloem exudates over long distance. Low-phosphate induced miR399s exhibited a shoot-to-root movement to repress downstream targets including PHO2 in the root[51]. Similarly,miR399d, miR827 and miR2111 were all found in grafting experiments to relocate from aerial parts to roots in response to phosphate starvation[52]. During rhizobial infection, miR2111 functioned as long-distance signals to post-transcriptionally regulate symbiosis suppressor TOO MUCH LOVE in roots[53]. miR395 can also translocate from wild-type scions to rootstocks of the miRNA processing mutant hen1-1 to target the APS gene[54]. In addition, both miR156 and miR172 have been confirmed as potentially phloem-mobile miRNAs that regulate tuber formation[55−57].
In grafting system, only small RNAs transporting from shoot-to-root via phloem could be analyzed. Other approaches that allow for the comparison between the expression areas and in situ RNA distribution patterns may help the identification of small RNAs acting locally as non-cell autonomous signals. To establish adaxial–abaxial leaf polarity, a member of Trans-acting small interfering RNA (ta-siRNA) family forms a gradient across the leaves by intercellular diffusion. This diffusion-driven pattern of ta-siRNA shapes the expression pattern of AUXIN RESPONSE FACTOR3 (ARF3), an abaxial determinant gene. Another small RNA, miR390 was proved to regulate the leaf polarity by the cell-to-cell movement from vasculature and pith region below the shoot apical meristem to the vegetative apex[54]. In addition, miRNA165/166 were discovered to move from the endodermis into the stele to regulate the xylem cell fate[58]. Moreover, miR394 was shown to regulate stem cell maintenance in SAM by the PD-mediated movement from L1 to inner cell layers to repress LEAF CURLING RESPONSIVENESS (LCR) expression[59].
In addition to siRNA and miRNA, mRNAs have also been found to travel beyond the cells in which they are expressed in Fig 1. In addition to the early example of mobile mRNAs of KN1, potato sucrose transporter SUC1 mRNA was also confirmed to be mobile. In grafting experiments, a number of mRNAs were found to travel, such as FT, FVE and AGL24 in Arabidopsis[60], Aux/IAA in melon and Arabidopsis[61], PP16 and NACP in pumpkin[62,63], BEL5 and POTH1 in potato, SLR/IAA14 in apple[64], PFP-T6 and PS in tomato[65] (summarized in Table 2). Recently, Luo et al. developed a fluorescence-based mRNA labeling system to identify mobile mRNAs targeted to PD[66]. Their analyses revealed that only mobile rather than not non-mobile mRNAs were selectively targeted to PD, providing further evidence for PD mediated transport of mRNAs. Interestingly, using a Nicotiana benthamiana/tomato heterograft system, Xia et al. found some mRNAs have bidirectional mobility between shoots and roots. In addition, forced expression of non-mobile mRNAs in the companion cells did not confer the mobility[67−71]. Thus, the movement of mRNA is likely an actively regulated process. Moreover, a large number of graft-transmissible mRNAs have been identified by high throughput sequencing in a variety of species including Arabidopsis, tobacco, grape, cucumber and tomato[67−72].
Table 2. List of mobile RNAs with functions in organ development.
Mobile factor Function Moves from: to Reference mRNA KN1 SAM maintenance injected cell to neighbouring cells Lucas et al. (1995)[26] SUC1 Sucrose transport companion cells to sieve elements Kuhn et al. (1999)[145] FT1 Flowering induction Leaf to SAM Lu et al. (2012)[60] Aux/IAA18 Root development Leaf to root Notaguchi et al. (2012)[61] PP16 RNA transport Phloem to shoot apex Xoconostle-Cazares et al. (1999)[62] NACP Meristem maintenance Phloem to shoot apex Ruiz-Medrano et al. (1999)[146] StBEL5 Tuber formation Leaf to root Banerjee et al. (2009)[147] POTH1 Leaf development Leaf to root Mahajan et al. (2012)[148] SLR/IAA14 Lateral root formation Shoot to root Kanehira et al. (2010)[64] PFP-T6 Leaf development Leaf to leaf primordia Kim et al. (2001)[65] PS Pathogen resistance Shoot to root and vice versa Zhang et al. (2018)[149] GAI Leaf development host to parasite Roney et al. (2007) [150]; David-Schwartz et al. (2008)[151] ATC Floral initiation Leaf to flower apices Huang et al. (2012)[152] FVE floral regulators Root to SAM Yang and Yu (2010)[153] AGL24 floral regulators Root to SAM Yang and Yu (2010)[153] siRNA ta-siRNA Establishment of leaf polarity the adaxial to the abaxial side of the leaf Chitwood et al. (2009)[154] hc-siRNA DNA methylation Shoot to root Baldrich et al. (2016)[155] miRNA miR165/166 Xylem specification endodermis into the stele Carlsbecker et al. (2010)[58] miR390 Leaf polarity vasculature and pith region below the SAM to SAM Chitwood et al. (2009)[154] miR394 Meristem maintenance L1 to inner layers in the shoot meristem Knauer et al. (2013)[59] miR395 Sulfate homeostasis graft unions Buhtz et al. (2010)[54] miR399d Phosphate homeostasis shoot to root and vice versa Pant et al. (2008)[156]; Lin et al. (2008)[51] miR172 regulate tuber formation Leaf to root Martin et al. (2009)[55] miR2111 Phosphate homeostasis;
Rhizobial infection;shoot to root and vice versa Huen et al. (2017)[52];
Tsikou et al. (2018)[53]miR827 Phosphate homeostasis shoot to root and vice versa Huen et al. (2017)[52] -
Intercellular signaling across plasmodesmata plays crucial roles in a wide range of processes in plants. The currently identified signaling molecules across plasmodesmata are mainly transcription factors and RNAs. However, accumulating evidence suggests that many other signaling pathways including calcium signaling, redox signaling, phosphorylation signaling, and hormone signaling can also function in non-cell-autonamous manner[134−136]. As these pathways are often complex and interplay with each other, it is still difficult to unravel such non-cell-autonamous functions. With the advance in high-resolution imaging techniques, such as super-resolution microscopy, researchers will be able to visualize in vivo the action and mobility of the molecular players involved in intercellular signaling[137].
In addition to visualizing the intercellular mobility of molecules, it is crucial to precisely evaluate the phenotype with a specific intercellular signaling disrupted. Developing cell type specific approaches is the key step and thus identification of promoters with restricted expression in certain cell types is important. Furthermore, abolishing gene function in a specific cell type is a valuable tool for studying intercellular signaling. Previously, cell-specific RNAi was employed but the intercellular mobility of small RNAs prevents the precise evaluation of gene function. Recent rapid development of CRISPR-Cas9 technique has emerged as a powerful tool for this purpose. The combination of cell-specific expression of Cas9 with reporters that allows for visualizing the gene editing in different cells could greatly enhance our ability to precisely evaluate the function of mobile regulators.
Lastly, to gain a more comprehensive understanding of the plasmodesmata mediated intercellular signaling, it is important to integrate multiple approaches, such as high-resolution imaging, single-cell technique, multi-omics, and computational modeling. Although the cell-to-cell signaling often occurs locally, the impact could be systemic in plants. The complete assessment of plasmodesmata-mediated intercellular signalling, as well as derived tissue- or cell-type-specific techniques, will not only benefit the study of plant development, but also provide the opportunity for future biotechnological renovation of plants.
-
About this article
Cite this article
Li M, Niu X, Li S, Li Q, Fu S, et al. 2023. Intercellular signaling across plasmodesmata in vegetable species. Vegetable Research 3:22 doi: 10.48130/VR-2023-0022
Intercellular signaling across plasmodesmata in vegetable species
- Received: 20 December 2022
- Accepted: 10 May 2023
- Published online: 02 August 2023
Abstract: The formation of edible organs and stress adaption are two major focuses of the studies on vegetable species. The regulation of these two processes often involves cell-to-cell signaling. In most plants, including vegetable species, intercellular signaling can be delivered by mobile regulators that traffic through a channel called plasmodesmata connecting almost all cells. A large number of transcription factors and RNAs have been discovered to move across plasmodesmata (called the symplastic way) to travel a short-range or a long-distance. This symplastic transport of signaling molecules has emerged to be an important regulation of a wide range of developmental and physiological processes. Callose deposition to plasmodesmata is a key step controlling the plasmodesmata permeability in many cell types. Here we summarize the recent progress in our understanding of plasmodesmata-mediated signaling in plants.
-
Key words:
- Intercellular /
- Signaling /
- Plasmodesmata