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Regulation of human trophoblast syncytialization by histone demethylase LSD1

A successful pregnancy is critically dependent upon proper placental development and function. During human placentation, villous cytotrophoblast (CTB) progenitors differentiate to form syncytiotrophoblasts (SynTBs), which provide the exchange surface between the mother and fetus and secrete hormones to ensure proper progression of pregnancy. However, epigenetic mechanisms that regulate SynTB differentiation from CTB progenitors are incompletely understood. Here, we show that lysine-specific demethylase 1 (LSD1; also known as KDM1A), a histone demethylase, is essential to this process. LSD1 is expressed both in CTB progenitors and differentiated SynTBs in first-trimester placental villi; accordingly, expression in SynTBs is maintained throughout gestation. Impairment of LSD1 function in trophoblast progenitors inhibits induction of endogenous retrovirally encoded genes SYNCYTIN1/endogenous retrovirus group W member 1, envelope (ERVW1) and SYNCYTIN2/endogenous retrovirus group FRD member 1, envelope (ERVFRD1), encoding fusogenic proteins critical to human trophoblast syncytialization. Loss of LSD1 also impairs induction of chorionic gonadotropin α (CGA) and chorionic gonadotropin β (CGB) genes, which encode α and β subunits of human chorionic gonadotrophin (hCG), a hormone essential to modulate maternal physiology during pregnancy. Mechanistic analyses at the endogenous ERVW1, CGA, and CGB loci revealed a regulatory axis in which LSD1 induces demethylation of repressive histone H3 lysine 9 dimethylation (H3K9Me2) and interacts with transcription factor GATA2 to promote RNA polymerase II (RNA-POL-II) recruitment and activate gene transcription. Our study reveals a novel LSD1–GATA2 axis, which regulates human trophoblast syncytialization.

Human pregnancy is associated with a hemochorial placentation (1,2) in which placental trophoblasts are directly exposed to the maternal blood to establish the exchange surface for nutrients and gases at the maternal-fetal interface. This exchange surface is established via formation of multinucleated SynTBs 5 (1,2). During human placentation, two distinct SynTBs develop from two different progenitors. At the onset of human placentation, a zone of invasive primitive syncytium forms from trophectoderm cells at the blastocyst implantation site (2)(3)(4). The primitive syncytium invades the endometrium and takes up nutrients from the maternal environment to ensure further embryonic development. The primitive syncytium also produces hCG that rescues the corpus luteum to maintain progesterone production required for pregnancy (5). Afterward, columns of CTB progenitors penetrate the primitive syncytium to form primary villi. The primary villi eventually branch and mature to form villous placenta, containing two types of matured villi: (i) anchoring villi, which anchor to maternal tissue, and (ii) floating villi, which float in the maternal blood of the intervillous space (2)(3)(4). The proliferating CTBs within anchoring and floating villi adapt distinct differentiation fates during placentation (6). In anchoring villi, CTBs establish a column of proliferating CTB progenitors known as column CTBs (6), which differentiate to invasive extravillous trophoblasts. In contrast, CTB progenitors of floating villi (villous CTBs) differentiate and fuse to form the outer multinucleated SynTB layer, which is bathed in maternal blood. The villous CTB-derived SynTBs establish the nutrient, gas, and waste exchange surface, produce hormones; and promote immune tolerance to fetus throughout gestation (7)(8)(9)(10)(11)(12).
CTB to SynTB differentiation is associated with cell cycle termination and alteration of gene expression programs, including activation of retroviral genes ERVW1 and ERVFRD1. ERVW1 and ERVFRD1 function as fusogen to induce CTB fusion (13)(14)(15). In addition, CTB to SynTB differentiation is also associated with the induction of CGA and CGB, which encode ␣ and ␤ subunits of hCG, an essential hormone for maintenance of human pregnancy (8 -10). Interestingly, a single CGA gene on human chromosome 6 encodes the hCG␣, whereas the hCG␤ can be produced by a cluster of six paralogous genes (CGB (also known as CGB3), CGB1, CGB2, CGB5, CGB7, and CGB8) located on chromosome 19 (16 -20).
Our understanding of molecular mechanisms of human SynTB differentiation mostly came from in vitro cell culture studies involving primary CTBs, which are isolated from term placenta (term CTBs) (21)(22)(23), and BeWo cells, a cell line derived from human choriocarcinoma (24,25). The term CTBs spontaneously differentiate to SynTBs, whereas activation of the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway in BeWo cells stimulates SynTB differentiation (26 -28). The SynTB differentiation in these cells can be tested by measuring induction of ERVW1, ERVFRD1, CGA, and CGB expressions; loss of E-Cadherin expression; and induction of cell fusion. Studies on term CTBs and BeWo cells have increased our understanding of signaling pathways (22,29,30) and transcriptional mechanisms, which are involved in CTB to SynTB differentiation (31,32). These studies also identified several evolutionarily conserved transcription factors, including glial cell missing 1 and GATA2 as regulators of ERVW1, ERVFRD1, CGA, and CGB transcription during SynTB differentiation (33)(34)(35). However, epigenetic mechanisms that coordinate with conserved transcription factors in response to signaling pathways to induce SynTB differentiation are poorly understood.
A study (36) with mutant mouse models and mouse trophoblast stem cells (TSCs) has implicated LSD1, the first identified histone demethylase (37), as an important regulator of trophoblast stem and progenitor cell differentiation. LSD1 is unique in its mechanism and can demethylate mono-and dimethyl groups on both histone H3 lysine 4 (H3K4) and histone H3 lysine 9 (H3K9) residues to promote either repression or activation of gene transcription (38,39). LSD1 interacts with the corepressor to the transcription factor REST (CoREST) and the nucleosome remodeling and deacetylase (NuRD) chromatinremodeling complexes (40 -44), which are involved in transcriptional repression. As a part of these repressive complexes, LSD1 specifically mediates demethylation of mono-and dimethylated H3K4, leading to transcriptional repression of a gene. On the contrary, LSD1 changes its substrate specificity from the H3K4 to H3K9 residue when it interacts with certain transcription factors such as androgen (AR) and estrogen (ER) nuclear hormone receptors (45)(46)(47)(48) and cMYB (49). In those contexts, LSD1 functions as an activator of gene transcription as demethylation of H3K9 residues is often associated with gene activation. Thus, depending on the cellular context and interaction with other factors, LSD1 can promote either activation or repression of a gene.
Mouse embryos with global deletion of Lsd1 gene die around embryonic day 7.5 (36). Major morphological defects and embryonic lethality are observed in trophoblast-specific Lsd1-knockout embryos. Furthermore, LSD1 has been implicated in promoting stemness (36,50) and in preventing senescence (51) in mouse TSCs. These studies reveal LSD1 as an important regulator of trophoblast development during placentation in mouse and in utero survival of a developing mouse embryo.
Both mouse and human undergo hemochorial placentation; however, the trophoblast cell types and the placentation process in these two species differ significantly (52). For example, mouse trophoblast syncytialization is not dependent on ERVW1 or ERVFRD1 as different endogenous retrovirally encoded proteins are involved in that process (53). Furthermore, mouse placentation is not associated with induction of CGA and CGB expressions (52). Thus, despite this importance in the context of mouse placentation, the role of LSD1 in human trophoblast development is yet to be defined. In addition, it is also unknown how histone modification dynamics is regulated by LSD1 during human trophoblast development and whether LSD1 functions as a coactivator or a corepressor during that process.
Given the conserved nature of LSD1 and the phenotype of the Lsd1-mutant mice, in this study, we investigated LSD1 expression at different stages of human placentation. We found that LSD1 is highly expressed in SynTBs throughout gestation. Therefore, we tested the importance of LSD1 via loss-of-function analyses and confirmed an essential role of LSD1 in human SynTB differentiation.

LSD1 is highly expressed in SynTBs of a human placenta
Studies with gene-knockout mouse models and mouse TSCs indicated that LSD1 is essential to prevent premature differentiation of mouse TSCs and is also required for directing their differentiation fate (36). A search of published studies related to LSD1 function in human trophoblasts revealed only three studies with human trophoblast cell lines (HTR-8/SVneo, JEG3, and JAR cells), implicating LSD1 as a part of the gene regulatory machinery downstream of long noncoding RNAs (54 -56). However, LSD1 expression patterns in primary trophoblast cells during human placentation and the functional importance of LSD1 in the context of human trophoblast differentiation have never been studied. Hence, we started our study by looking at LSD1 expression during human placentation. We tested LSD1 protein expression in both first-trimester and term human placentae. In a first-trimester human placental villus, two trophoblast cell layers exist: a basal layer of mononuclear CTB progenitors and an outer multinucleated SynTB layer. We found that in a first-trimester human placenta, LSD1 is abundantly expressed in both CTB progenitors and differentiated SynTBs (Fig. 1A). In contrast, LSD1 expression is very low in nontrophoblast stromal cells. We also detected a high level of LSD1 expression within the SynTBs in term human placentae (Fig. 1B). Thus, our expression analyses revealed that LSD1 expression is maintained in human SynTBs throughout the gestational time period.

LSD1 is important for CTB to SynTB differentiation
We tested the importance of LSD1 in human trophoblast syncytialization by using BeWo cells (Clone B30) as a model LSD1 regulates human trophoblast syncytialization system. BeWo cells have been widely used to study trophoblast syncytialization as they recapitulate the cellular behavior and gene expression patterns of syncytiotrophobast development in response to PKA activators like forskolin and 8-bromo-cAMP (8-Br-cAMP) (24 -28). In the standard culture condition, BeWo cells are unable to spontaneously differentiate and maintain a cellular state, equivalent to undifferentiated CTB progenitors of a third-trimester human placenta (27). However, following exposure to 8-bromo-cAMP (BeWo-differentiating condition), BeWo cells undergo morphological changes (Fig. 1C) and are a good model for testing the human SynTB differentiation process. This syncytialization process in BeWo cells could be monitored by transcriptional induction of ERVW1/ERVFRD1, CGA, and CGB (Fig. 1D).
We found that LSD1 is highly expressed in both undifferentiated and differentiated BeWo cells (Fig. 1, E and F) and localizes in the nuclei (Fig. 1E), which is consistent with its function in chromatin modification. Notably, the LSD1 protein expression level is not altered upon BeWo cell differentiation (Fig. 1F).
To test the importance of LSD1 in SynTB differentiation, we depleted LSD1 in BeWo cells via RNAi using lentiviral shRNAs. The shRNA-mediated knockdown of LSD1 resulted in a nearly 90% loss in LSD1 mRNA expression ( Fig. 2A) and appeared just as efficient when LSD1 protein expression was visualized by Western blotting (Fig. 2B). Also, the knockdown efficiency was similarly maintained in LSD1-depleted BeWo cells (LSD1KD BeWo cells) when they were cultured in the differentiating culture condition with 8-Br-cAMP (Fig. 2B). However, mRNA expressions of several other histone demethylases such as KDM1B, KDM2A, KDM2B, and KDM3A were not affected in LSD1KD BeWo cells (Fig. S1A), indicating that shRNA-mediated knockdown was specific to LSD1. Also, the depletion of LSD1 did not alter the proliferation rate of undifferentiated BeWo cells (data not shown), indicating that LSD1 is not essential for survival of BeWo cells. However, loss of LSD1 strongly impaired 8-Br-cAMP-induced mRNA expressions of SYNCYTINs, CGA, and CGB genes (Fig. 2C). Interestingly, expressions of ITGA6 and HLA-G, which are known to be expressed in human trophoblast cells (57), including the BeWo cells (58,59) were not altered in LSD1KD BeWo cells (Fig. S1, B and C), indicating that LSD1 loss did not induce a pleotropic effect on gene expression in BeWo cells. We also analyzed E-Cadherin expression in LSD1KD BeWo cells and found that loss of LSD1 maintained E-Cadherin expression and inhibited cell-cell fusion in differentiating BeWo cells (Fig. 2D). Collectively, the impaired induction of SynTB differentiation-associated genes, the maintenance of E-Cad-herin expression, and the impaired cell-cell fusion in LSD1depleted BeWo cells indicated that LSD1 might be an important regulator for SynTB differentiation in primary CTBs. Therefore, we performed a loss-of-LSD1-function study using a human primary CTB differentiation model.
In vitro culturing of primary CTBs from term human placenta results in their spontaneous differentiation to SynTBs (23). Therefore, we depleted LSD1 in term CTBs (LSD1KD CTBs) via RNAi (Fig. 2E) and cultured them to test efficiency of SynTB differentiation. We noticed efficient cell syncytialization (Fig. 2F) and induction of ERVW1, CGA, and CGB genes ( Fig.  2G) when term CTBs were differentiated to SynTBs. However, similar to our findings in BeWo cells, cell syncytialization and induction of ERVW1, CGA, and CGB genes were impaired in LSD1KD CTBs (Fig. 2, F and G). Thus, from our two-pronged

LSD1 regulates human trophoblast syncytialization
analyses in BeWo cells and in primary term CTBs, we inferred that LSD1 mediates an essential function to ensure SynTB differentiation from CTB progenitors.

LSD1 eliminates repressive H3K9Me2 modification at the SynTB-associated genes
In association with repressive complexes such as Co-REST and NuRD, LSD1 demethylates mono-and dimethylated H3K4 residues, thereby promoting formation of a compact, repressive chromatin structure (Fig. 3A). In contrast, upon interacting with certain transcription factors like AR and ER (41)(42)(43)(44), LSD1 demethylates mono-and dimethylated H3K9 residues, leading to formation of an open, transcriptionally active chromatin (Fig. 3A). Therefore, we investigated the histone demethylase properties of LSD1 in the context of SynTB differentiation. To that end, we focused on dimethylated H3K4 (H3K4Me2), a histone modification specifically enriched at the promoter regions of transcriptionally active genes as well as genes that are poised for future expression during cellular differentiation (60 -62), and H3K9Me2 modification, a histone mark associated with the promoter regions of repressed genes that are predominantly localized within the euchromatin region rather than within the constitutive pericentromic heterochromatin region (63,64).
We noticed that global levels of dimethylated H3K4Me2 and H3K9Me2 are not altered in LSD1KD BeWo cells (Fig. 3B). Also, quantitative ChIP analyses showed that enrichment of the H3K4Me2 mark at the promoter regions of ERVW1/ERVFRD1, CGA, and CGB3 genes was not significantly altered in BeWo cells upon induction of syncytialization with 8-Br-cAMP or in response to the loss of LSD1 expression (Fig. 3C). Rather, we noticed that SynTB differentiation of BeWo cells is associated with strong loss of H3K9Me2 modification at the ERVW1/2, CGA, and CGB3 promoter regions (Fig. 3D). However, 8-Br-cAMP treatment in LSD1KD BeWo cells was unable to reduce
We also tested incorporation of the H3K9Me2 mark at the promoter regions of ERVW1, CGA, and CGB3 genes in primary term CTBs. We found that differentiation of CTBs is also associated with significant loss of the H3K9Me2 mark at those promoters regions, and this process is impaired in LSD1KD CTBs. (Fig. 3E). Collectively, these findings indicated that during SynTB differentiation, LSD1 functions as a transcriptional coactivator by removing the repressive H3K9Me2 mark at key gene loci.

LSD1 complexes with GATA2 and promotes RNA polymerase II (Pol-II) recruitment at the SynTB-associated genes
The coactivator function of LSD1, in which it promotes activation of gene transcription by H3K9 demethylation, depends upon its interaction with specific transcription factors like AR, ER, and cMYB. However, we noticed that these factors are either expressed at very low levels or not expressed in BeWo cells and primary CTBs. Consequently, we reasoned that other transcription factors might interact with LSD1 to promote its coactivator function during SynTB differentiation. To that end, we focused on the GATA family transcription factor GATA2, which is known to interact with LSD1 in a different cellular context (65) and is implicated in trophoblast gene regulation, including induction of ERVW1 and CGA transcription in human trophoblast cells (34,35). In an earlier study (66), we showed that GATA2 is selectively expressed in the trophoblast cells (both in CTBs and SynTBs) of a first-trimester human placenta. GATA2 is also abundantly expressed in the SynTBs of a term human placenta (Fig. 4A) and in undifferentiated and differentiated BeWo cells (Fig. 4B). Interestingly, GATA2 expression was maintained in LSD1KD BeWo cells both in undifferentiated and differentiating culture conditions (Fig.  4B), indicating that GATA2 expression is regulated in BeWo cells via an LSD1-independent mechanism.
As GATA2 expression is maintained in differentiating BeWo cells with and without LSD1 depletion, we asked whether GATA2 chromatin recruitment is impaired at certain gene loci in LSD1KD BeWo cells. Earlier studies with reporter genes implicated GATA2 in transcriptional regulation of ERVW1 (34) and CGA (35) genes. However, GATA2 chromatin recruitment at the endogenous ERVW1 and CGA loci has never been tested. Therefore, we tested GATA2 chromatin occupancy at these two gene loci and at the CGB3 locus in control and LSD1KD BeWo cells.
GATA factors selectively occupy canonical W(A/T)GAT-AR(A/G) motifs at a chromatin locus to directly regulate gene expression program (66 -68). The human CGA locus contains multiple conserved WGATAR motifs within the region spanning ϩ1 to Ϫ1 kb of the transcription start site (Fig. 4, C and D), whereas the human CGB3 locus contains a single conserved WGATAR motif at Ϫ350 bp upstream of the transcription start site (Fig. 4, C and D). In addition, the Ϫ250 bp region of the human ERVW1 locus harbors a canonical W/(A)-GATA-R/(A) motif and a W(A)-GATAC motif (Fig. 4E), which were earlier implicated for transcriptional activation of ERVW1 (34). Therefore, we tested GATA2 chromatin occupancy at theses GATA motifs in undifferentiated and differentiated BeWo cells. We found that GATA2 occupies multiple GATA motifs at the CGA locus, the Ϫ350 bp region of the CGB3 locus and the Ϫ250 bp region of the ERVW1 locus only in differentiated BeWo cells (Fig. 4F). We also found that loss of LSD1 impairs GATA2 chromatin occupancy at the CGA, CGB, and ERVW1 loci during BeWo cell differentiation (Fig. 4F). We made a similar observation in differentiated primary CTBs in which a significant loss in GATA2 occupancy at the CGA, CGB3, and ERVW1 loci was observed upon loss of LSD1 (Fig. 4G).
Our findings that LSD1 removes the repressive histone H3K9Me2 mark and promotes GATA2 recruitment at the SynTB-associated genes indicated that an LSD1-GATA2 transcriptional activation complex could be a part of the transcriptional machinery during SynTB differentiation. As GATA2 facilitates recruitment of RNA-POL-II at its target genes (69,70), we also hypothesized that the LSD1-GATA2 complex promotes RNA-POL-II binding to induce transcription at GATA2 target genes during SynTB differentiation. Therefore, we next asked whether LSD1, GATA2, and RNA-POL-II physically interact at the endogenous CGA, CGB3, and ERVW1 loci and whether loss of LSD1 impairs that interaction, leading to loss of RNA-POL-II recruitment to those genes during SynTB differentiation. To that end, we performed sequential ChIP assays with differentiated BeWo cells and confirmed both LSD1-GATA2 and GATA2-RNA-POL-II interactions at the CGA, CGB3, and ERVW1 gene loci (Fig. 5A). We also confirmed loss of RNA-POL-II recruitment at the CGA, CGB3, and ERVW1 gene promoters in 8-Br-cAMP-treated LSD1KD BeWo cells (Fig. 5B) and LSD1-depleted primary CTBs (Fig. 5C). Collectively, these results provided evidence for formation of an LSD1-GATA2 complex, which recruits RNA-POL-II at key gene loci to activate their transcription during CTB to SynTB differentiation.

Discussion
Epigenetic regulators, including histone-modifying enzymes, play critical roles in chromatin remodeling and influence gene expression patterns during cellular differentiation. LSD1 has been widely studied in influencing genome processes in various cellular contexts, including embryonic pluripotency, zygotic genome induction, cellular reprogramming, stem and progenitor cell differentiation, mammalian development, and cancers (43,49,(71)(72)(73)(74)(75)(76)(77)(78)(79)(80)(81). The importance of LSD1 in the context of mouse trophoblast stem cell differentiation and mouse placentation has also been defined (36); however, its role in human trophoblast development and the placentation process remained largely unexplored. Our findings in this study establish LSD1 as an essential epigenetic regulator for human SynTB differentiation. We also identified that LSD1 could function as a transcriptional coactivator in human trophoblast cells by promoting H3K9Me2 demethylation at key SynTB gene loci.
One of our interesting findings is the synchronous existence of both H3K4Me2 and H3K9Me2 modifications at the transcriptionally repressed ERVW1, CGA, and CGB loci in undifferentiated CTBs. Bivalent histone modifications, in which functionally opposite histone modifications coexist at a chromatin region, are a common feature at the transcript start site of LSD1 regulates human trophoblast syncytialization many developmentally regulated genes. Most of the known bivalent genes are associated with coexistence of repressive his-toneH3lysine27trimethylation(H3K27Me3)andH3K4trimethylation (H3K4Me3) modifications (82), which keep those genes at a transcriptionally "poised" state so they can readily be activated in response to the appropriate signal. Nevertheless, the presence of both H3K4Me2 and H3K9Me2 modifications at key SynTB gene loci indicates that these genes are at a poised bivalent state and LSD1-medited H3K9 demethylation is a key regulatory step to instigate their activation in response to SynTB differentiation cues. These data also indicate the possibility that the presence of the H3K4M2/H3K9Me2 bivalency and LSD1-mediated activation is a common regulatory mechanism at several other SynTB-associated genes during human placentation.
Our study implicated a LSD1-GATA2 gene regulatory axis that directly controls RNA-POL-II recruitment at GATA2 target genes to promote transcriptional activation during SynTB differentiation (Fig. 6). Our findings also support a recent study, which showed that CTB to SynTB differentiation is associated with increased RNA-POL-II binding to promoters of a subset of genes (83). However, we found that GATA2 is also expressed in the undifferentiated BeWo cells.
Thus, the question arises of what prevents GATA2 chromatin occupancy at the ERVW1/CGA/CGB gene loci in undiffer-

LSD1 regulates human trophoblast syncytialization
entiated BeWo cells. One of the possibilities is the inaccessibility of GATA motifs due to the presence of the repressive H3K9Me2 modifications at those chromatin loci in undifferentiated BeWo cells. We predict that the induction of LSD1-mediated H3K9Me2 demethylation allows accessibility of GATA motifs, thereby allowing GATA2 binding and RNA-POL-II recruitment (Fig. 6).
Zhu et al. (36) indicated that loss of LSD1 in mouse TSCs promotes SynTB differentiation, which is different from our findings in the context of human CTBs in which LSD1 is essential for the differentiation process. An explanation for this differential outcome could be the differences in the placentation process in humans versus mice. Both human and mouse develop hemochorial placentae and are regulated by common conserved factors. However, the placentation processes in these two species are fundamentally different. Thus, it is possible that LSD1 mediates its function in trophoblast progenitors in a species-specific manner. As LSD1 can interact with multiple chromatin regulators, the differential interactions of LSD1 with other protein partners could lead to species-specific function during placentation. An alternative explanation is the improper SynTB differentiation efficiency of mouse TSCs. The mouse TSCs show a spontaneous propensity to differentiate to trophoblast giant cells and only partially recapitulate the SynTB differentiation process (84,85). Hence, future gene-knockout studies in which LSD1 is selectively deleted in SynTB progenitors during mouse placentation could definitively indicate whether or not LSD1 mediates a conserved role in SynTB development in both mouse and human placentation.
An interesting finding of this study is the consistent expression of LSD1 in CTBs and SynTBs during the first-trimester human placentation process. These expression patterns indicate that LSD1 function could contribute to SynTB differentiation during the first trimester of pregnancy. Defective trophoblast development during first-trimester placentation is a major reason for early pregnancy loss or contributes to pregnancy-associated disorders during late gestation (86 -89).
Thus, it is important to understand whether impairment of LSD1 function during first-trimester placentation contributes to placental defects. Unfortunately, the villous CTB progenitors of a first-trimester placenta are distinct from CTBs of a term placenta as they do not spontaneously differentiate to SynTBs in culture. Thus, studying the importance of LSD1 for SynTB differentiation in first-trimester CTBs is technically challenging. Also, the BeWo cells are not a proper cell system to model the first-trimester CTBs. However, recently developed human trophoblast stem cells are described to recapitulate the properties of CTB progenitors of a first-trimester placenta as those cells can be efficiently differentiated to both SynTBs and extravillous trophoblasts (90). Thus, human TSCs could be utilized to model the importance of LSD1 in early human trophoblast development.
The high-level expression of LSD1 in CTBs within the firsttrimester placenta also raises the question of its importance in undifferentiated CTBs. As discussed earlier, LSD1 can also act as a corepressor by interacting with CoREST-histone deacetylase or NuRD complex. Our unpublished studies 6 revealed that RCOR1, which encodes CoREST, and components of the NuRD complex, including MBD2/3, CHD4, and RBBP4, are highly

LSD1 regulates human trophoblast syncytialization
expressed in first-trimester CTBs. Thus, it is attractive to hypothesize that the corepressor function of LSD1 plays an important role in regulating the biology of undifferentiated CTBs during first-trimester placentation.
In summary, in this study, we have provided experimental evidence to define LSD1 as an important regulator of CTB to SynTB differentiation. Based on its expression patterns during human placentation, we also propose that LSD1 is an important epigenetic regulator to orchestrate gene expression patterns in undifferentiated CTB progenitors. Given the dual mode of function as a coactivator and a corepressor, LSD1 could be an important therapeutic target to manipulate the gene expression program to manage pregnancy-associated disorders that originate from defective trophoblast development and function.

Human placental tissues and isolation of CTBs from human term placenta
Studies with human placentae were performed after obtaining approval from the Institutional Review Board of the University of Kansas Medical Center. Formaldehyde-fixed sections of first-trimester and term human placentae were obtained from Research Centre for Women's and Infants' Health Biobank of Mount Sinai Hospital, Toronto, Canada. For isolation of CTBs, normal term placentae were obtained at the University of Kansas Medical Center following cesarean sections at birth. CTBs were isolated following an established protocol (91) with some modifications. Placental tissue was washed using one time with saline and digested with trypsin, Dispase, and DNase in Hanks' balanced salt solution buffered with HEPES. Digestion was done in a shaker incubator at 37°C for 1 h. Additional trypsin and DNase were added after the first 30 min. The contents were then filtered into 50 ml of fetal bovine serum and subjected to a series of centrifugations. Pellets were resuspended in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and added to a discontinuous Percoll gradient followed by centrifugation. Trophoblasts were identified by density and extracted for use in experiments.

RNAi
Plasmids expressing shRNAs targeting human LSD1/ KDM1A were obtained from Sigma. Lentiviral supernatants were prepared in HEK293T cells following a procedure described previously (92), and viral particles were concentrated by centrifugation. Undifferentiated BeWo cells and term CTBs were transduced with lentiviral particles, and cells were se- Figure 6. The LSD1-GATA2 gene regulatory axis during SynTB differentiation. The model illustrates an LSD1-GATA2 gene regulatory axis that directly controls RNA-POL-II recruitment at GATA2 target genes to promote transcriptional activation during SynTB differentiation. From our experimental data, we propose that in the undifferentiated CTBs, SynTB-associated genes such as CGA harbor both H3K4Me2 and H3K9Me2 modifications, leading to a relatively compact chromatin inaccessible to GATA2 binding. The lack of GATA2 binding in turn results in impaired RNA-POL-II recruitment and suppression of transcription. In response to cell signaling like PKA signaling, a LSD1-GATA2 complex forms at the SynTB-associated genes, which leads to H3K9Me2 demethylation by LSD1 and instigates RNA-POL-II recruitment and gene transcription.

Western blot analysis
Whole-cell lysates were extracted with a lysis buffer. Electrophoresis was performed using a 10 -12% polyacrylamide gel in Tris-glycine running buffer (pH 8.3). Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA). Blocking was performed with 5% nonfat dry milk (Difco) in 20 mM Tris-HCl (pH 7.6) (TBS with Tween). Primary antibody incubation, washing, secondary antibody incubation, and development of chemiluminescent signals were done following a protocol published previously (93).

Quantitative ChIP and sequential ChIP
Cells were collected, and protein-DNA cross-linking was achieved by treating the cells with 1% formaldehyde (Sigma) for 10 min at room temperature with gentle rocking. 125 mM glycine was added to quench the reaction. ChIP was completed using antibodies against LSD1, GATA2, RNA-POL-II, H3K4Me2, and H3K9Me2 following protocols established previously (94,95). Mouse IgG1 was used as a negative control for antibody binding.
Sequential ChIP analyses were performed using a protocol described earlier (96). Cross-linked chromatin fragments were prepared from differentiated BeWo cells and immunoprecipitated with anti-GATA2 antibody. Eluted chromatin was reincubated with anti-LSD1, anti-RNA-POL-II, and mouse IgG1 for overnight at 4°C in the presence of 50 g/ml yeast tRNA. After immunoprecipitation, chromatins were reverse cross-linked, digested with proteinase K (Sigma), and purified. Amplification by real-time PCR was used to quantify precipitated DNA. Primers for ChIP studies were designed based on gene sequences available in human genome assembly GRCh38.p12 (available in the Ensemble genome browser). The conserved GATA motifs at human CGA locus were identified via genome alignment in ECR Browser (97) (https://ecrbrowser.dcode.org). 7 Sequences of ChIP primers are available on request.

Immunofluorescence
The immunofluorescence procedure was adapted from a protocol published previously (66). Cells were washed with PBS and fixed on glass coverslips with 4% paraformaldehyde (Sigma) in PBS. Cells were permeabilized in 0.25% Triton X-100 in PBS and blocked with 10% fetal bovine serum in PBS with 1% Triton X-100. Primary antibody incubation was completed in 1 h followed by washing with 0.05% Triton X-100 in PBS and then secondary antibody (conjugated with Alexa Fluor 488 (green)) incubation for 30 min while shielded from light. Next, the cells were counterstained with 4Ј,6-diamidino-2-phenylindole (Invitrogen) and observed under a fluorescence microscope (Leica, Buffalo Grove, IL). All incubations took place at room temperature.

Immunohistochemistry
Human placental sections were deparaffinized and rehydrated using Histoclear (National Diagnostics, Atlanta, GA) followed by decreasing ethanol gradient and distilled water. Antigen retrieval occurred using Reveal Decloaker in a Decloaking Chamber (Biocare Medical, Pacheco, CA) at 90°C for 15 min. Peroxidase blocking was done using 3% hydrogen peroxide for 10 min at room temperature followed by blocking for nonspecific binding with 10% normal goat serum (Invitrogen) for 30 min. Primary antibody and secondary antibody incubations were done for 30 min using antibody dilution buffer (1% BSA, 0.3% Tween 20 in PBS (pH 8.0)) for secondary antibody (biotinylated anti-rabbit or -mouse IgG (heavy ϩ light) (vector) with 0.05% normal human serum). Tissues were incubated with horseradish peroxidase-streptavidin (Life Technologies) for 10 min followed by 3,3Ј-diaminobenzidine chromogen solution (Dako, Carpinteria, CA) for color change and Mayer's hematoxylin solution (Sigma) for 5 min. Hematoxylin color change was achieved using warm, running tap water for ϳ2 min. Dehydration of the tissue was achieved using distilled water, increasing ethanol gradient, and xylene. Stained tissues were imaged using a brightfield microscope (Nikon).


extfieldsofstudy:
Environmental Science
Medicine

text:
In Vivo Phenotyping for the Early Detection of Drought Stress in Tomato

Drought stress imposes a major constraint over a crop yield and can be expected to grow in importance if the climate change predicted comes about. Improved methods are needed to facilitate crop management via the prompt detection of the onset of stress. Here, we report the use of an in vivo OECT (organic electrochemical transistor) sensor, termed as bioristor, in the context of the drought response of the tomato plant. The device was integrated within the plant's stem, thereby allowing for the continuous monitoring of the plant's physiological status throughout its life cycle. Bioristor was able to detect changes of ion concentration in the sap upon drought, in particular, those dissolved and transported through the transpiration stream, thus efficiently detecting the occurrence of drought stress immediately after the priming of the defence responses. The bioristor's acquired data were coupled with those obtained in a high-throughput phenotyping platform revealing the extreme complementarity of these methods to investigate the mechanisms triggered by the plant during the drought stress event.

Introduction
Drought is one of the most frequently occurring and damaging abiotic constraints compromising plant growth and crop yield [1,2]. The increasing demand for water, driven by the need to feed a growing world population, will be exacerbated by reductions in soil water reserves driven by the predicted rise in global temperature. Maintaining crop yields in an environment where drought is even more prevalent than at present is recognized as an urgent priority [3]. Despite this, however, the genetic and physiological basis of a crop yield under conditions of water insufficiency remains inadequately understood [3,4], largely because simulating a stress which varies both in time and in intensity is very difficult. The plant response to drought stress depends heavily on the duration and severity of the stress but is also influenced by the plant's genotype and its developmental stage [2,3]. Nevertheless, it is clear that the main consequences of the stress are to reduce the rate of cell division and expansion, which results in the plants forming smaller leaves, shorter stems, and a reduced root system. In a drying soil, nutrient uptake is compromised by the altered physiochemical status and flux of the xylem sap [5].
A number of phenotyping platforms have been explored to characterize the plant drought stress response. These include the use of optical sensors designed to monitor the plants' photosynthetic activity [6][7][8], growth status [9,10], and overall water content [11]. While the major focus has remained on the aerial part of the plant, the importance of phenotyping the root system has also been recognized [12]. Continuous monitoring in real time, however, remains the exception rather than the rule, and most commonly, the plant's physiological status is measured only indirectly [13,14]. A recent development has featured a graphene sensor able to monitor in real time the transport of water from a plant's roots to its leaves [15], while an integrated electrochemical chip-on-plant was used to detect gene expression under stress condition in tobacco leaves [16]. A different class of sensor, referred to as a "bioristor," has been shown to be able to detect, in vivo and in real time, the changes in the composition of the plant sap in a growing tomato (Solanum lycopersicum) plant [17], without interfering with plant functions. The bioristor is based on an organic electrochemical transistor (OECT) realized on textile thread [18,19] and enables measuring the changes in ion concentration in the plant sap. The research activities regarding OECT are very active, and several reviews have already been published on OECTs [20][21][22] or on OECTs applied to biology [23][24][25]. The choice of tomato, a crop which is produced and consumed worldwide, reflects its particular sensitivity to drought stress, especially during its flowering and fruit enlargement phases [1,26]. Here, a demonstration is given of how the bioristor concept can be exploited as a tool to achieve the early detection of drought stress in tomato applicable also as a complemental tool for plant phenotyping.

Experiment
2.1. Tomato Plants and the Imposition of Drought Stress. Two experiments in controlled conditions have been performed. A pilot experiment, carried out in Parma (Italy), was set up to demonstrate the ability of the bioristor to respond to drought stress. The seeds were kindly furnished by ALSIA Metapontum Agrobios Research Center. Seven cv. Red Setter plants were grown up to the stage of 5 th fully expanded leaves in 1.5 dm 3 soil-filled pots under controlled conditions, namely, a constant temperature of 24°C, a relative humidity of 50%, and a 16 h photoperiod. The plants were kept fully irrigated until their fifth true leaf had fully expanded, after which a bioristor was inserted in the stem of each plant (Figure 1(a)). After 3 d, four of the plants were exposed to drought stress by withholding watering for 14 d (DSI); the plants were then irrigated over 2 d (RE), and, finally, a 6 d stress episode was imposed by withholding water (DSII) (Figure 1(a)). A set of four plants was kept fully watered as the control.
On the basis of the results obtained in the pilot experiment, the main experiment was performed in the ALSIA plant phenomics facility (Metaponto, Italy). The trial was based on eight cv. Ikram plants, available at the ALSIA Metapontum Agrobios Research Center, grown in 3 dm 3 pots exposed to a 12 h photoperiod (light intensity 180 μmol m -2 s -1 ) with a daytime temperature of 24°C and a nighttime one of 18°C; the relative humidity ranged from 50 to 60%. When the plant reached the stage of 5 th fully expanded leaves, the sensors were integrated and 1 d after the implantation of the bioristor, watering was withheld from four of the plants for 16 d (DSI) and then restored for a further 7 d (RE) (Figure 1(b)). A limited irrigation 50 cm 3 was supplied to the 3 dm 3 pots to maintain the plant turgor and allow for the acquisition of images (day 8). The remaining four plants were kept fully watered as the control.

Bioristor
Measurements. The sensors were prepared, inserted into the plant stems, and connected to a computer, following Coppedè et al. [17] (Figure 2, Supplementary  Fig. 1). A constant voltage (V ds ) was applied across the main transistor channel (a textile fiber functionalized with PEDOT:PSS), along with a positive voltage at the gate (V g ); the resulting currents (I ds ) were monitored continuously (for 26 d in the pilot experiment and for 23 d in the main experiment). The sensor response parameter (R) was given by the expression ðI dS − I dS0 Þ/I dS0 , where I ds0 represented the current across the channel when V g = 0.
The normalized sensor response (NR) was shown as the bioristor output and calculated as the ratio between R of stressed and nonstressed plants. The sensor response parameter R was transformed into a normalized sensor response (NR) from the ratio between R of stressed and nonstressed plants.
In vitro analyses of the bioristor ability to monitor concentrations in the changes of main cations involved in the drought stress response have been performed. Transfer characteristics of the sensor response were measured using different concentrations of sodium (Na + ), potassium (K + ), White blocks indicate days during which full watering was provided following the bioristor's implantation (SI), grey blocks indicate days during which watering was withheld (DSI, DSII), and the shaded blocks indicate the recovery phase (RE). Arrows indicate the timing of the Scanalyzer readings and image acquisition, while the black star shows when the emergency irrigation was provided.
Na + K + Ca 2+ Figure 2: The bioristor setup. The device was inserted into the stem of each tomato plant and connected to a readout system and a computer.
calcium (Ca 2+ ), and magnesium (Mg 2+ ) salts expressed as the ratio ðI dS − I dS0 Þ/I dS0 representing the sensor response, where I ds is the off current (measured for gate voltages, V g ≠ 0 V) and I 0 is the on current (measured for V g = 0 V).

Leaf Stomatal
Conductance. Leaf stomatal conductance was measured from two fully expanded leaves per plant (fourth and fifth leaf) of the cv. Ikram plants, using an SC-1 leaf porometer (Decagon Devices, Pullman, WA, USA).

Physiological Analyses.
Four controls and four stressed plants have been analyzed for the relative water content (RWC) as reported by Barrs and Weatherley [27], by taking the fully expanded leaf as the sample (two replicates for each plant). Chlorophyll content measurements were performed by using the SPAD 502 meter (Konica Minolta, Ramsey, USA). Measurements from 10 leaves of each plant of varying age and colour were selected for measurements made under diffuse lighting. The relative SPAD value was considered. All data were analyzed statistically applying Student's t-test, and the standard error was calculated between replicates.

Nondestructive
Phenotyping. Images were captured at 2 d intervals from cv. Ikram plants following Petrozza et al. [9], using a Scanalyzer 3D device (LemnaTec GmbH, Aachen, Germany). The imaging, initiated prior to the imposition of drought stress, involved three mutually orthogonal vantage points, using near-infrared (NIR) and white (RGB) illumination. The NIR images were used to evaluate the plants' water content; the RGB ones were for the assessment of both the plants' state of health (green: healthy tissue, yellow: chlorotic tissue, and brown: necrotic tissue) and for morphological measurements; these data were used to calculate the plants' biovolume and height; their biovolume (a parameter proportional to the aerial mass of the plant) was calculated from the expression ½Σ pixel sideview 0°+ Σ pixel sideview 90°+ log 10 Σ pixel sideview /3, following Eberius and Lima-Guerra [28] and Petrozza et al. [9]. Plant compactness, which describes how much of the hull area is covered by leaves, was calculated as object area/convex hull area [29]. The NIR index was calculated as weighted mean from the pixel intensities of greyscale NIR images divided into 128 bins, representing a range in leaf water content, while the green index that expresses the fraction of green colour detected in the leaves was calculated in accordance with Casadesús et al. [30].

Results
3.1. The Bioristor Output. The monitoring over 26 d of the behaviour of four drought-stressed and three well-watered cv. Red Setter plants confirmed the bioristor's capacity to record day/night changes in the xylem sap's composition [17]. The R parameter (analyzed with MATLAB (https://uk .mathworks.com/) and Microsoft Excel 2016) fell during the daytime and rose during the nighttime, as was expected (data not shown). When each 24 h cycle of data was averaged to generate a mean NR value, four distinct phases (defined by a slope change) were recognized ( Figure 3): the first phase (PI) covered the first 3 d following the sensor's implantation; PII was initiated 24 h after the withholding of water and was characterized by a decrease in NR over a 6 d period, followed by a short 2 d innate recovery (DA). PIII reflected the plants' recovery following rewatering, during which time NR rose back to its prestress level; finally, during PIV, when the plants were once again deprived of water, NR fell, this time more rapidly than it did during PII. Measurements of both leaf chlorophyll content (as estimated using a SPAD device, Figure 4(a)) and relative water content (RWC, Figure 4(b)) confirmed that the plants were experiencing drought stress (Figure 4(b)). The SPAD value in the stressed plants significantly increases during the drought stress (p ≤ 0:05) although only two points have been acquired, and, as expected, the RWC consistently decreases by about 23% (p ≤ 0:05) during the drought stress and is completely restored when the recovery occurred (Figure 4(b)).
The validation of the bioristor as a tool permitting the early detection of drought stress allowed a more detailed analysis conducted in the main experiment, in which four drought-stressed and four well-watered cv. Ikram plants were continuously monitored over a period of 23 d under controlled conditions ( Figure 5). Once again, the slope of the NR parameter was used to define a number of phases. The PI phase followed the implantation of the sensor; during PII, there was an initial (days 2-4) sharp fall in NR as the intensity of the drought stress increased, but over the subsequent 4 d, NR recovered somewhat (DA) but with higher extent as for the Red Setter cultivar. The stress treatment had a strong effect on stomatal conductance, a widely used indicator of drought stress [31], over the period 7-14 d after the withholding of water reducing it by 4-5-fold in comparison to the well-watered plants ( Figure 6). Reductions in stomatal conductance not only reduce transpirational water loss from the leaf but also constrain their photosynthetic activity, due to the limitation imposed on gas exchange [32]. An equivalent, although less pronounced, stomatal conductance response was, in retrospect, also recognized in the cv. Red Setter plants monitored in the pilot experiment, suggesting the existence of genotypic variation for the response. However, this defence mechanism failed as the stress period was prolonged, as was shown by the resumed fall in NR over days 7-9, after which the parameter remained at a low but stable level through to 23 d. A slight increase in the NR was observed corresponding to the limited irrigation operated (days 10-16). PIII was initiated upon the plants' rewatering, during which period NR increased to a level which was maintained through to the end of the experiment. This level was, however, much lower than the baseline obtained at the start of the treatment, reflecting a degree of irreversible damage caused by the stress (Figure 7(d)). Overall, the conclusion was that a stress response was detectable through the negative trend of the NR parameter already within the first 30 h following the withholding of water.
The ability of the bioristor to monitor physiological mechanisms strictly related to the changes of ion concentration in the plant sap in relation to the transpiration stream is strongly supported by the correlation analysis performed. In fact, a strong and highly significant correlation between the sensor response (R) and the stomatal conductance (SC) (r = 0:82, p value < 0.001; Figure 8) was observed, highlighting also a clear separation of the stressed and nonstressed samples indicating a strong influence of DS on the R value.
Moreover, to further demonstrate the ability of the bioristor in revealing changes in the concentration of the ions identified as the main players in the drought stress response, the transfer characteristics of the sensor response were measured, in vitro, using different concentrations of sodium (Na + ), potassium (K + ), calcium (Ca 2+ ), and magnesium (Mg 2+ ) salts. The analysis of the sensor response confirms the ability of the bioristor to detect changes in all the tested ions ( Supplementary Fig. 2).

The Automated Monitoring of the Phenotypic Response to
Stress. In the main experiment, imaging was used to monitor the phenotypic response to the stress treatment (Figures 7(a)  and 7(b)). The chosen indices involved four based on RGB images (digital biovolume (Figure 9(a)), plant height (Figure 8(b)), plant compactness (Figure 9(c)), and green index (Figure 9(d))) and one based on NIR images (hydration index (Figure 9(e)). The growth of the plants, as indicated by their biovolume, height, and compactness, their greenness, and their hydration status were all strongly affected by the drought treatment. Changes in plant compactness and height   were clearly visible, while the green and NIR-based indices first became detectable after 6-8 d (Figure 9). Biovolumes fell markedly over the first 8 d of the stress and were not recovered following rewatering (Figure 9(a)). The stress triggered severe wilting (Figures 7(b) and 7(d)); within 6 d of the withholding of water, the height of the stressed plants was 15% lower than that of the well-watered plants (Figure 9(b)). Plant compactness differed significantly between the treated and control plants within 4 d of the withholding of water; the stressed plants lost turgor, which steadily increased their compactness over the period 4-14 d; a gradual recovery occurred following rewatering and the compactness reached the level shown by the controls at the end of the experiment (Figures 7(d) and 9(c)). A similar trend was observed for the NIR intensity. For the control plants, the NIR-based index rose strongly over the first 6 d; this was also the case, although less markedly, for the stressed plants. For the latter, the index fell between days 7 and 10, until emergency irrigation was supplied to prevent plant death; the level was fully restored by rewatering carried out on day 16. The green index of the stressed plants fell slightly over the initial 12 d following the withholding of water, then remained steady, in contrast to the response of the control plants, which comprised a continuous fall over the whole measurement period (Figure 9(d)).
To confirm the suitability of the bioristor to complement the image-based high-throughput phenotyping techniques, we performed a Pearson correlation analysis between the digital biovolume as the index of drought stress [33] and the in vivo bioristor sensor response.
A good and significant correlation (r = 0:66, p value < 0.001; Figure 10) was observed between the two variables, firstly confirming the occurrence of the drought stress and secondly supporting the suitability of the bioristor to monitor in vivo the drought stress profile in plants.
A comprehensive correlation analysis of all image-based index and manual-based measurements allowed us to observe a high correlation between the sensor response and those parameters linked with the transpiration process and the water use efficiency (SC and DB) and to exclude, at least in this experiment conditions, a direct correlation with the NIR intensity (NI), the green index (GI), and compactness (C, Supplementary Fig. 3).
In addition, to evaluate the overall phenotypic profile and distinguish plants of different agronomic groups, we performed a principal component analysis (PCA) using the "prcomp" function of R (https://cran.r-project.org/doc/ FAQ/R-FAQ.html#Citing-R). Data were represented as a biplot (R package factoextra [34]), evaluating the compactness (C), green index (GI), stomatal conductance (SC), sensor response (R), NIR intensity (NI), and digital biovolume (DB) as variables.
The first two components explain 71.1% of the variability ( Figure 11). The first PC (PC1) explains almost a half (49.1%) of the phenotypic variation, which perfectly separated stressed plants from control plants. Stomatal conductance (SC) and digital biovolume (DB) have large positive loading on the PC1. The regularly irrigated controls and the drought-stressed plants are well separated in the biplot indicating the efficacy of the drought treatment.

Discussion
Plants commonly experience periods of moisture deficiency in the course of their life cycle [35]. Drought stress therefore represents a critical constraint over the productivity   of crops [3]. An ability to detect drought stress before it causes irreversible damage is important for crop management and water savings and would also be valuable as a means of selecting varieties more resilient to the stress. Conventional phenotypic assays for drought tolerance are both labour intensive and imprecise, so the development of automated phenotyping platforms represents a promising advance [14]. Current platforms combine the robotic handling of plants with sensors and high-end computing to capture high-resolution, highly precise data in a highthroughput mode [14]. Their ability to collect data in real time in a nondestructive manner allows for potential insights to be gained into the temporal response of large numbers of plants to a particular treatment [9]. Moreover, an image-based index (RGB, NIR, and FLUO) is increasingly used to study the plant defence response mechanisms upon drought stress both in controlled conditions [3,9,29,33,[36][37][38] and in the open field [39]. The development of both proximal and remote effective sensors has been accelerating of late, but there remains a lack of simple-tooperate and affordable sensors which can be implanted within the plant. The bioristor utilized here represents a In addition, its capacity to monitor the plants' physiological status on a continuous basis, rather than relying on sampling at a series of discrete time points, should greatly enhance the data's value in the context of understanding how plants respond to drought stress. The breeding of crop varieties able to cope with the predicted changes in climate demands detailed knowledge of the mechanisms underlying the tolerance of plants to water deficiency and their ability to escape the stress. Changes in the composition of the xylem sap are thought to represent a major component of the drought defence machinery. Moisture deficiency inevitably reduces a plant's water content, thereby altering both the concentration of solutes in its transpiration stream and the mass of solutes exported from its roots at a given time [40].
Several reports have described the changes in nutrient and ion uptake and transport in plants during the occurrence of drought stress. In particular, a reduction in nutrient uptake by the roots partially due to the reduction in soil moisture was observed, which causes a decreased rate of nutrient diffusion from the soil matrix to the absorbing root surface [41] and translocation to the leaves [42,43]. During stress, a reduction in the general mineral accumulation [43] in the plant tissues together with a general low nutrient availability in the soil and lower nutrient transport in plants was also reported [5,41]. Stomatal closure is also a known mechanism of drought resilience that reduces transpiration, the nutrient transport from the roots to the shoot, and causes an imbalance in active transport and membrane permeability, resulting in a reduced absorption power in the roots [41,[44][45][46]. A direct measurement of the plant sap content during the drought stress also demonstrated that the amount of K + ions gradually decreased in the xylem sap of maize plants [5].
The bioristor has been designed to detect the movement and concentration of electrolytes through the vascular tissues. Its application in the context of tomato plants exposed to drought stress has shown that changes in both the composition and the concentration of key solutes occurred within 30 h of the withholding of water. Xylem flux, transpiration rate, solubilisation, and translocation of solutes are all negatively affected by drought stress in a range of plant species [1,5,[47][48][49], including tomato [1,50]. The present data support the hypothesis that, as a result of the forced reduction in the transpiration rate, the early phase of the plant defence response includes a reduction in the concentration of ions in the xylem sap [5]. The integration of the bioristor data with image-based analyses has provided insights into the timing of the tomato phenotypic and physiological response, as well as suggesting that at the start of the drought stress response, a change in the transport, allocation, and production of metabolites and ions occurs within the plant, which acts as a signal for stomatal closure and the subsequent decrease in transpiration.
The high correlation coefficient between the sensor response (R), stomatal conductance (SC), and digital biovolume (DB) together with the PCA analysis confirms the hypothesis that the bioristor is able to detect ions and molecules related to the drought stress and, in particular, those dissolved and transported through the transpiration stream, thus efficiently detecting the occurrence of drought stress immediately after the priming of the defence responses.
A difference in the extent and timing of a possible drought avoidance (Figures 2 and 4) was observed between the two cultivars tested, opening new perspectives for the use of a bioristor as a tool for genotype selection in prebreeding.

Conclusions
The present experiments represent a breakthrough in the use of in vivo sensing technology in tomato. The data generated have shown how dynamic changes in the chemical composition of the sap in the xylem occur in drought-stressed tomato plants, with these changes becoming detectable within the first 30 h of water deprivation. The key characteristics of the bioristor, namely, its ability to continuously monitor aspects of the plant physiological status, its minimal invasiveness [17], its low cost, and its ease of generating readable data, suggest that the bioristor would represent a valuable tool in the context of precision agriculture and high-throughput drought response phenotyping. Moreover, its potential ability to differentiate the response of two cultivars differing from one another with respect to drought tolerance could be exploited as a selection tool for breeding more droughtresilient tomato cultivars. Among our current research priorities is the extension of the bioristor's ability to detect specific compounds of particular relevance to the plant's drought response. Much of the climate change impact on agriculture is mediated through water, and considering that agriculture is currently withdrawing 70% of the fresh water available in the planet, the application of the bioristor in open fields may represent a real innovation for increasing the water balance efficiency in agriculture addressing one of the UN sustainable development goals.

Data Availability
The bioristor raw data and the images acquired with the Scanalizer platform are available upon request to the authors.

Disclosure
Nicola Coppede should be considered a joint first author.

Conflicts of Interest
The authors declare no competing interests.

Authors' Contributions
MJ initiated the project and drafted the manuscript. NC was responsible for the device's implantation. BM designed the acquisition software. AP and FC planned and supervised the main phenotyping platform experiment. SS and AP interpreted the phenotypic data. FV provided technical assistance. DD supervised the statistical analyses, and NM, DP, and SI aided in the writing of the manuscript. MJ and AZ coordinated the project. All authors have read and approved the final version of the manuscript. Michela Janni and Nicola Coppede equally contributed to the work.