How It All Begins: Molecular Players of the Early Graviresponse in the Non-elongating Part of Flax Stem

doi:10.21203/rs.3.rs-4790400/v1

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How It All Begins: Molecular Players of the Early Graviresponse in the Non-elongating Part of Flax Stem

https://doi.org/10.21203/rs.3.rs-4790400/v1

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Plants have developed two major approaches to adjust their position in accordance with the direction of the gravity vector: differential cell growth at the opposing sides of elongating regions and complex processes in non-elongating stem parts, like reaction wood development. Gravistimulation of flax plants induces the formation of gravitropic curvature in non-elongating stem parts, largely associated with modifications in phloem and xylem fibers. To gain knowledge about key “triggers” and “forward players” that induce negative gravitropic reactions, transcriptome profiling of the phloem fibers and xylem tissues from pulling and opposite stem sides 1 and 8 hours after gravistimulation was carried out. As the first reaction, activation of the processes associated with RNA synthesis and protein folding in both tissues and both stem sides was detected; later, activation of kinases and transferases occurred. Among the major induced changes, transcriptomic data indicate rapid and substantial shifts in chloroplast metabolism in all analyzed tissues, including temporal activation of the branched-chain amino-acid pathway, adjustment of light-harvesting complexes, and jasmonic acid biosynthesis. Auxin transporter genes were activated only in the xylem, whereas other auxin-related genes were barely upregulated 1 hour after stem inclination in any analyzed sample. The asymmetric changes between stem sides included the sharp activation of ethylene-related genes in the phloem fibers of the opposite stem side, as well as tertiary cell wall deposition in both the phloem and xylem fibers of the pulling stem side during later stages of graviresponse. The obtained results provide informative insights into the graviresponse mechanisms.

flax

gravitropism

phloem fibers

xylem

transcriptome

tertiary cell wall

Plants, being rooted in soil, have to survive in the place they grew up. In such a situation, of special importance for them is the ability to control and modify the position of their organs in space in accordance with the environmental factor vector. The movement, like turning or curving, of the plant part in response to the direction of external stimuli is called tropism. There are various kinds of tropisms defined by the type of inducing factor, for example, phototropism, thigmotropism, and gravitropism. The effects of gravity attract special attention due to the permanent presence of the inducing factor throughout the lifetime of organisms on Earth and also in view of the ideas to colonize other planets and space where gravity would be considerably modified or absent (Palme et al. 2018; Chin and Blancaflor 2022). The upward growth of shoots and downward growth of roots are determined by gravity direction, as is the restoration of vertical position by inclined stems or branches.

Plants have developed two major approaches to regulating organ position according to the gravity vector. The best-known one, described almost two centuries ago and often the only one considered (Palme et al. 2018; Levernier et al. 2021), is the differential growth of cells located at the opposite sides of the stem or root. This leads to the formation of a curvature in the direction of a side with less pronounced cell elongation. This type of graviresponse can be developed only in young tissues within the zone of an organ that contains cells able to elongate. Another type of graviresponse occurs in non-elongating parts of an organ and relies mainly on the very distinctive properties of cell walls deposited in cells at specific sides of the stem or branch. The renowned examples of the latter approach are tension wood and compression wood, which are developed in angiosperms and gymnosperms, respectively (Moulia and Fournier 2009; Groover 2016). Tissues with modified cell walls develop forces able to restore the vertical position of a trunk or branch.

Gravitropism is described as consisting of three sequential steps: gravity perception, gravity signal transduction, and gravity response (Chin and Blancaflor 2022). One of the comprehensive approaches that allows to form a general picture of cellular gene expression programs in living tissues is transcriptomics. The first changes in transcript levels of several genes in gravistimulated young Arabidopsis thaliana root are detected within 2 min; however, the pronounced changes are not observed before 30 min (Kimbrough et al. 2004). So, gravity perception in the roots and shoots of young plants is considered to occur within seconds and minutes, signal transduction within tens of minutes, and gravity response within hours and days (Weise and Kiss 1999; Sato et al. 2015; Dalal et al. 2016). The time course of similar sequential steps in more mature, non-elongating plant parts is more extended, with gravitropic responses taking days and weeks (Gerttula et al. 2015; Ibragimova et al. 2017).

Since the gravitropic response leads to asymmetrical events on the opposite sides of the stem or root, in order to understand the involved mechanisms, it is necessary to compare the reactions occurring at the upper and lower sides of the curvature being formed. Moreover, it is important to understand the input of different tissues into the joint effect of changing the position of an organ. There are not many model plants and not many effective approaches that allow conducting such studies. The studies of gravitropic reactions in elongating parts of plant organs often take stem or root zones as a whole without separating them into opposite halves, or, more so, into different tissues (Kimbrough et al. 2004; Zhang et al. 2018). The rare exceptions, in which the analyzed organ was separated into upper and lower flanks, are the experiments with elongating basal internodes of rice (Oryza sativa) (Hu et al. 2013), A. thaliana inflorescence stems (Taniguchi et al. 2014), and Brassica oleracea hypocotyls (Esmon et al. 2006). Transcriptomic studies of tension wood development are mainly concentrated only on the xylem part and analyze rather extended time periods, starting with days after inclination (Andersson-Gunneras et al. 2006; Liu et al. 2021). The early (30 min) transcriptional response to gravistimulation was characterized in the xylem of young poplar plants (Lopez et al. 2020).

One of the most convenient model objects to study the development of gravitropic response in the non-elongating part of an organ is the flax plant, which has a rather uniformly built, non-branched stem able to restore the vertical position after plant inclination by making a curvature in the non-elongating stem part (Fig. 1). In flax, both phloem (bast) and xylem fibers are involved in such a gravitropic response, as demonstrated by microscopy combined with immunocytochemistry or microspectroscopy (Ibragimova et al. 2017; Petrova et al. 2021; Beaugrand et al. 2022; Blervacq et al. 2023) and transcriptomic studies (Gorshkov et al. 2018; Mokshina et al. 2018; Petrova et al. 2021). The driving force for plant movement is associated with tertiary cell walls (TCW) (Gorshkova et al. 2018). The phloem fibers involved in the gravitropic response in the non-elongating stem part have already developed the TCW since it is deposited constitutively in the course of normal plant development, while in xylem fibers, the same as in tension wood, the TCW is induced from zero by gravistimulation (Ibragimova et al. 2017; Petrova et al. 2021). Bundles of flax phloem fibers can be quickly isolated from the developing stem, giving the important possibility of analyzing individual cell types and comparing the observed effects with the xylem stem part.

To figure out the early events after stem gravistimulation, to compare the changes in fibers with pre-formed and induced TCWs, as well as to check the role of different tissues located at pulling and opposite stem sides, we have performed transcriptomic analysis specifically for phloem fibers and xylem stem parts in the course of the developing graviresponse, including its early stages. The obtained results permit the realization of the input of different tissues in the joint effort to restore the vertical position of the inclined stem and to compare the sequence of events and major players with those involved in the elongating parts of the gravistimulated organ.

Plant material

Plants of long-fibered flax Linum usitatissimum L. cultivar Mogilevsky were grown in open air in boxes with a 50-cm soil layer in natural daylight and daily watering. The experiments started in the second half of June, 20–30 days after sowing, when the plants were at a fast growth period of development and the plant height was 25–30 cm.

For gravistimulation, plants were inclined by gently attaching the lower part of the stem in the region of cotyledon leaves to the soil with a metal staple so that the stem got the horizontal position. Segments (5 cm long) of the lower part of the flax stem (3 cm above cotyledon leaves) that had ceased elongation long in advance of the experiment were collected 1 and 8 h after plant inclination, long before the first anatomical changes in phloem and xylem fibers could be detected in the region of stem curvature forming to return the inclined plants to vertical position, as described previously (Ibragimova et al. 2017; Petrova et al. 2021). Each stem segment was cut along into halves and separated into PULLING (PUL) and OPPOSITE (OPP) sides, collectively designated as GRAVI (Fig. 1). CONTROL (CTR) samples were taken from the non-inclined plants at the same stem level and at the same time as for the inclined plants. Fiber-enriched stem peels, separated from xylem tissues, were washed in 80% ethanol in a mortar several times to obtain isolated phloem fibers. All collected samples were frozen in liquid nitrogen and stored at -80°C until analysis. Samples for analysis were collected from control (non-inclined) or inclined stems in four biological replicates, each containing portions from five plants.

RNA extraction and sequencing

Total RNA samples in four biological replicates were obtained using ExtractRNA reagent (Evrogen, Russia) combined with RNeasy Plant Mini Kit and RNeasy Plus Micro Kit columns (Qiagen, Germany), removing residual DNA using the DNA-free DNA Removal Kit (Ambion, USA). Qualitative and quantitative evaluation of RNA samples was performed using the NanoPhotometer NP-80 Touch (Implen, Munich, Germany). Further manipulations with RNA samples, including determination of RNA integrity number (RIN ≥ 7), cDNA library generation, and mRNA sequencing, were performed at Novogene Corporation (China; https://en.novogene.com/) using the Illumina NovaSeq 6000 platform in the paired-end read mode with a 150 nucleotide read length. The data in the form of raw reads was added to BioProject number PRJNA631357 in the Sequence Read Archive (SRA).

RNA-Seq data processing and gene expression analysis

The clean reads after removal of contaminating sequences (rRNAs, tRNAs, snRNAs, and snoRNAs) by BBDuk algorithms of BBTools v38.73 [BBTools, https://jgi.doe.gov/data-and-tools/software-tools/bbtools/] were used as input data for assembly, mapping, and count of expression values using HISAT2 v2.1 (Kim et al. 2015) and StringTie v2.0 (Pertea et al. 2016) with parameters for paired-end reads. The flax genome as a multi-fasta file and the annotation as a gff3 file were downloaded from Phytozome v12 (https://phytozome.jgi.doe.gov/). Data normalization and differential expression analysis were performed using the package DESeq2 and related libraries in the R environment (Love et al. 2016, https://www.r-project.org). The relative level of gene expression was assessed in normalized TGR values (total gene reads), formed as the sum of all reads mapped to the coding part of a gene. A gene was considered expressed if the normalized TGR value in at least one sample was ≥ 16 (SEQC/MAQC-III Consortium 2014). A gene was considered differentially expressed if there was a two-fold or more change in TGR value with padj (adjusted p-value) ≤ 0.01 in pairwise comparisons of samples at each time point according to the stem fragment (pulling or opposite) and tissue type (phloem fibers or xylem).

The online resources Morpheus (https://software.broadinstitute.org/morpheus/) and ShinyGO v0.77 (Ge et al. 2020, http://bioinformatics.sdstate.edu/go/) were used to cluster genes using the K-means algorithm and to perform the over-representation analysis of Gene Ontology (GO) terms. For clustering, the following parameters were used: TGR ≥ 16 at least in one sample, padj ≤ 0.01 at least for one pair (GRAVI/CTR), and 2 ≤ GRAVI/CTR ≤ 0.5. For clustering within one type of tissue, TGR ≥ 50 was used. In ShinyGO, the relationship between enriched pathways in clusters was built for Arabidopsis homologues based on the GO Molecular Function. Two pathways (nodes) are connected if they share 20% (default) or more genes. Darker nodes have more significantly enriched gene sets. Bigger nodes represent larger gene sets. Thicker edges represent more overlapped genes. The distribution of differentially expressed flax genes in the metabolic pathways was visualized using the KEGG Mapper Color tool (https://www.kegg.jp/kegg/mapper/color.html) built for A.thaliana.

Functional annotations of flax genes were borrowed both from the original annotation files and from open online sources and databases: Phytozome v13.0, TAIR (https://www.arabidopsis.org), iTAK (http://itak.feilab.net/cgi-bin/itak/index.cgi), and Uniprot (https://www.uniprot.org/) according to the description of Arabidopsis homologues, which can be found in Tables S1 and S2.

Transcriptome response of phloem fibers and xylem part: tissue factor

We performed transcriptome profiling of 12 samples in 4 biological replicates, each containing portions from five flax plants. The samples included the phloem fibers (f) and xylem tissues (x) collected from the pulling and opposite sides of the inclined stems at 1 hour and 8 hours after gravistimulation, as well as the control (non-inclined plants) sampled at the same time points (Fig. 1). A total of about 1.2 billion paired-end reads with a Q30 quality score of more than 92% were obtained. The average mapping rate to the flax genome was 96.5%. Principal component analysis indicated both good reproducibility between biological replicates (mean r > 0.95 ± 0.027). In the space of the first component (PC1 = 82%), transcriptome profiles were divided into 2 large groups corresponding to xylem and phloem localization of samples, whereas within the PC2 component (PC2 = 9%), each group was divided into three pools mainly corresponding to control samples, 1 h and 8 h samples (Fig. 2a). We compared the transcriptome profiles considering three main factors: different tissues, time course, and stem side (pulling or opposite).

The number of expressed genes (TGR ≥ 16 in at least one sample) in both fiber and xylem was 29806 and 29408 genes, respectively. The construction of a Venn diagram showed that of the 30980 expressed genes, 28286 genes were shared, while 1520 genes were specific to fiber samples (i.e., had TGR values ≥ 16 in at least one fiber sample and TGR˂16 in all xylem samples, Table S1) and 1122 genes were specific to xylem (Fig. 2b). Among the genes designated as xylem-specific, the highest expression levels were observed for players related to xylem cell differentiation processes and deposition of SCW, such as XCP2 (Lus10013204), TED6, 7 (Lus10028626 and Lus10018925), IRX9 (Lus10026487), and others, confirming the physiological relevance of the obtained transcriptomic data (Table S1).

We mainly considered differential gene expression and presented the results, comparing the tissues from gravistimulated plants to their own controls (Fig. 2c). This approach permits us to reveal the major molecular players in graviresponse and to minimize the effects of circadian rhythms and possible external factors on the effects of gravistimulation as such. In total, out of 30928 expressed genes (TGR ≥ 16), 7403 unigenes were differentially expressed relative to the corresponding control (log₂FC≥|1|, padj ≤ 0.01) (Fig. 2d).

Genes that were differentially expressed in xylem samples (xGRAVI) were grouped in cluster 7, while those differentially expressed in phloem fiber samples (fGRAVI) were in cluster 10 (Fig. 2d). Genes from cluster 7 mainly encode protein kinases, chaperons, and microtubule-related proteins (Fig. 3). The group of genes for chaperones was overwhelmed by genes for small heat shock proteins from the HSP20 family. These genes were also highly upregulated in fGRAVI, but the fold change was considerably lower. The most numerous group of genes from cluster 7 encodes various kinases; around half of which (25 genes) encode leucine-rich repeat receptor-like kinases (LRR-RLKs) (Table S1).

Cluster 7 also included the upregulated genes for different auxin transporters, both for influx (LAX2; Lus10034488, and Lus10025057), efflux (PIN1; Lus10042004; ABCB19; Lus10030674), and intracellular auxin accumulation at the endoplasmic reticulum (PILS7, PIN-LIKES 7; Lus10016688) (Table S1). Among other hormone-related genes, Lus10002547, encoding lipoxygenase that may be involved in jasmonic acid (JA) biosynthesis, was the most noticeable (Fig. 3).

The list of genes from cluster 10 that were upregulated in all phloem fiber samples of gravistimilated plants (fGRAVI/CTR padj ≤ 0.05) was not extended and included only 5 genes with TGR > 100 (Table S1). Lus10009351 for the transcriptional regulator, the homologue of which in Arabidopsis specifically binds DNA sequence 5'-AGAAnnTTCT-3' known as heat shock promoter elements (Uniprot), and Lus10034316 for the ethylene response factor (ERF118) were among them. In general, the statistically significant upregulation of gene expression in a given tissue through all GRAVI samples was rather rare, especially in phloem fiber samples.

Transcriptome response of phloem fibers and xylem part: time factor

Cluster 1 contains the genes whose products could be associated with a relatively early response: they were upregulated, as compared to control, both in phloem fibers and xylem tissues, but only 1 hour after plant inclination (Fig. 4). The highest fold change in expression values was observed for Lus10011905 encoding WUS-interacting protein 2 (WSIP2, other name TOPLESS-RELATED 4, TPR4). It was upregulated from nothing in CTR to quite substantial levels in 1 hour GRAVI samples; later, TGR values in GRAVI samples went down and, at the same time, increased in control plants 8 hours after gravistimulation to similar values as in 1 hour gravistimulated plants (Fig. 4c, Table S1).

In cluster 1, the genes for various chaperones were among the most numerous categories (Fig. 4c, Table S1). Several genes for HSP70, key chaperones that facilitate folding of de novo synthesized proteins, for HSP101 and HSP90.1, for chloroplastic HSP21, peroxisomal HSP20-like, and mitochondrion-localized HSP23.6 had the highest fold change of upregulation (Fig. 4c, Table S1). Besides, genes for calreticulins and calnexins were considerably upregulated in all GRAVI samples 1 hour after stem inclination, as were Lus10010133 and Lus10012648 for molecular chaperone regulator 6 from the BAG (B cell lymphorma 2-associated athanogene) family (Table S1).

The notable gene set encoding the chloroplast-localized enzymes that synthesize branched-chain amino acids (BCAAs, i.e., leucine, isoleucine, and valine) was revealed in cluster 1. These genes were significantly upregulated 1 hour after inclination and reached the TGR values of many thousands in the samples of gravistimulated plants: Lus10032040, Lus10035207, and Lus10016751, all encoding acetohydroxy acid synthase responsible for the first step of the BCAA biosynthesis from pyruvate; Lus10011918 and Lus10022849, both for ketol-acid reductoisomerase; Lus10043132 and Lus10033370, both for dihydroxy-acid dehydratase; and Lus10011604 for branched-chain aminotransferase 3 (Fig. S1, Table S1). Lus10003316 and Lus10003317, encoding threonine synthase 1, which is involved in the formation of 2-oxobutanoate, another precursor of isoleucine, were also highly upregulated (Table S1).

Cluster 1 also contained genes for transcription factors, e.g., C2H2 (Lus10004840), HY5 (Lus10002900, light response), ERF023 (Lus10005967), GATA (Lus10029863 and Lus10020684), bHLH (Lus10000484), and MYB55 (Lus10038022) (Table S1), as well as genes for proteins involved in various signaling pathways, including Lus10039672 and Lus10027171 encoding glutamate receptor-like proteins (GLRs). Similar expression patterns were revealed for Lus10039315 and Lus10040276 for receptor-like kinases with lectin domains, Lus10008885 for ralf-like 32 signaling peptide, the same as Lus10034592, encoding multiprotein bridging factor 1c (MBF1c) that stimulates transcriptional activity by bridging regulatory proteins and TATA-box binding factor, and Lus10006140 for chloroplastic “pseudoprotease” called ethylene-dependent gravitropism-deficient and yellow-green-like 3 (EGY3), which mediates ROS homeostasis and promotes retrograde signaling in response to stress.

Eight hours after inclination (cluster 8), the overwhelming pathways activated both in xylem tissues and in phloem fibers were those involving various kinases (Fig. 5, Table S1). Together with that, genes for several transcription factors were at the top of the upregulated gene list, for example, bZIP18 (Lus10030531) and LBD12 (lateral organ boundaries (LOB) domain family protein, Lus10017431). Also, genes for several HXXXD-type acyl-transferases were activated in both samples (Fig. 5c); their exact substrates are unknown.

Reaction of phloem fibers on the gravistimulation: stem side-factor

To analyze the differences between the gravistimulated plant tissues located at the pulling and opposite stem sides, we analyzed the fold changes of TGR values for corresponding samples. The number of genes with statistically significant differences between xPUL and xOPP 1 hour after stem inclination was minimal and raised considerably after 8 hours (Fig. 6). In phloem fiber samples collected from the opposing stem sides, the differences in gene expression levels were already well pronounced 1 hour after stem inclination.

Out of 95 genes upregulated more than twofold in fPUL1 as compared to fOPP1 (log₂(fPUL1/fOPP1) ≥ 1, padj ≤ 0.01, TGR ≥ 100 in fPUL1), at least 20% were the genes for various chloroplastic proteins. These included genes for several members of the chlorophyll A-B binding family, like Lus10006621 for early light-induced protein 1 (ELIP1) and Lus10039379 for ELIP2 (Fig. 7a), as well as Lus10025676 for nonphotochemical quenching 4 (NPQ4), Lus10033499 and Lus10020878 for cryptochrome 3, etc. (Table S2). Some of the mentioned genes were also activated in xylem tissues (Fig. 7a).

The groups of genes for transcription factors as well as for cell wall-related proteins were upregulated more than twofold in fPUL1 as compared to fOPP1 (padj ≤ 0.01). Four of the transcription factors belonged to the TALE family, including BHL2 (Lus10016790, Fig. 7a), two to the bZIP family (Lus10002900 and Lus10002028), and the others to the C2H2, Dof, and AP2 families (Table S2). The list of activated genes for cell wall-related proteins included Lus10007975, Lus10035540, and Lus10027753 for the isoforms of RG-I rhamnosyltransferases (RRT, Wachananawat et al. 2020) and, to a lesser extent, Lus10011175 and Lus10015438 for RG-I:galaturonosyltransferase (RGGAT1, Amos et al. 2022) (Table S1). Besides, Lus10029245 and Lus10007296 for the cellulose synthases LusCESA8, Lus10031972 and Lus10035131 for COBRA-like 4 (COBL4), considered as the cofactors of cellulose synthesis, and Lus10002979 for FASCICLIN-like arabinogalactan-protein 12 (FLA12) were upregulated in fPUL1 (Table S2). The above proteins are known to participate in TCW formation in flax phloem fibers (Gorshkova et al. 2023).

Data for differential expression of various hormone-related genes between GRAVI samples and corresponding controls are summarized as violin plots (Tang et al. 2023) in Fig. 8 (ethylene, jasmonic acid, and auxin), Fig. S2, and Table S3 (gibberellins, brassinosteroids, cytokinins, and abscisic acid). Hormone-related genes were poorly represented among those upregulated in fPUL1 (log₂FC ≥ 1, padj ≤ 0.01) as compared to fOPP1. Among those, Lus10011260 encoding transport inhibitor response 1 (TIR1), known as the auxin receptor, was the only auxin-related gene (Table S2).

In fOPP1, a very notable upregulation of hormone-related genes occurred. Specific activation of transcription in fOPP1 was observed for Lus10011565 and Lus10039647 encoding synthase ACS8, which catalyzes the synthesis of 1-aminocyclopropane-1-carboxylate (ACC), a direct precursor of ethylene (Fig. 7b). The difference in TGR values between fOPP1 and fPUL1 accounted for many hundredfold. Expression of Lus10001108 and Lus10027586 encoding ACC-oxidase (ACO, designated as 2OG Fe(II) oxygenase), the enzyme for the second step of ethylene biosynthesis, was also increased in fOPP1, the same as Lus10042557 for ERF1 (Table S2). ERF1 acts downstream of EIN3 (ethylene-insensitive3), which can be regulated by the EIN3-binding F-box protein EBF1_2; the corresponding flax gene, Lus10002165, was upregulated in fOPP1 (Table S2). Thus, there is a clear specificity in ethylene metabolism and signaling in fibers from the opposite stem side at an early stage of graviresponse, while in xylem tissues, significant differences in expression of genes for ethylene biosynthesis were not observed (Fig. 7b). Several genes for negative regulators of auxin signaling from the AUX/IAA family, Lus10039488 encoding IAA4, Lus10039413 for IAA3 (SHY2), and Lus10039487 for IAA14 were upregulated in fOPP1 (Fig. 8; Table S2).

To summarize the changes in the transcription of different hormone-related genes 1 h after stem inclination, the most pronounced ones were the sharp asymmetric upregulation of ethylene-related genes in phloem fibers of the opposite stem side and the activation of JA-related genes common for all GRAVI samples. At the same time, the transcription of some auxin-related genes was activated at the early stage of graviresponse; however, the fold-changes were rather moderate, as were the expression levels (Fig. 8, Table S3).

Other genes highly upregulated in fOPP1 included Lus10017061 (PP2C.D2) and Lus10015047 (PP2C.D4) for protein phosphatases 2C (Fig. 7b, Table S2). PP2C.D2 interacts with SAUR19, dephosphorylates, and represses plasma membrane H⁺-ATPases, negatively influencing plant growth (Ren et al. 2018). Among genes for cell wall-related proteins, notable activation was detected only for Lus10040121 and Lus10030923, both encoding xyloglucan endotransglucosylase/hydrolase 5 (Table S2). Lus10026611 and Lus10030452, encoding the MYB105 transcription factor that is involved in organ patterning, were also upregulated in fOPP1 (Fig. 7b). same as Lus10002191, Lus10034319, and Lus10040796 encoding 3-ketoacyl-CoA synthase involved in the biosynthesis of very-long-chain fatty acids; Lus10025636 and Lus10018200 for phosphatidylinositol 4-phosphate 5-kinase 1 (PIP5K1); and Lus10015046 for the E3 ligase SHOOT GRAVITROPISM9 (SGR9), which modulates the interaction between statoliths and F-actin. (Table S2).

At 8 h after gravistimulation, the set of genes differentially expressed in phloem fibers collected from the opposing stem sides was quite distinguishable from that in 1 h samples. The number of activated genes for chloroplastic proteins dropped (Table S2). In fPUL8, transcription of genes for various ions’ transporters went up, including Lus10039312 for the slow, weak voltage-dependent S-type anion efflux channel SLAH3 and Lus10017657 homologous to the AT1G53210 encoding sodium/calcium exchanger (Fig. 9a), as well as Lus10033052 and Lus10015687 for inward (KT1) and outward (SKOR) rectifying potassium channels (Table S2).

Among the genes for transporters whose expression was highly up-regulated in fPUL8 (Table S2), Lus10032073 and Lus10014606 encode lysine histidine transporter 1 (LHT1), which is involved in the transport of a wide range of amino acids but also of ACC (Shin et al. 2015). Besides, Lus10030975 for aluminum-activated malate transporter 12 (ALMT12) got increasingly activated in fPUL8 as compared to fPUL1. The increased accumulation of malate in the fibers from the pulling stem side has been detected by metabolome analysis at a later stage (96 h) of flax plant graviresponse (Mokshina et al. 2018).

In the top-list of genes upregulated in fPUL8, a high proportion belonged to those encoding cell wall proteins, namely FLA11, FLA12, and FLA7; the expansin-like protein EXPL1; the protein core (APAP1) of plant cell wall proteoglycan, glycosyl hydrolase from the GH9 family able to hydrolyze (1–4)-β-d-glucosidic linkages, and COBL4 (Fig. 9a). Besides, the mentioned genes for the RRT1 involved in RG-I biosynthesis (Lus10013503 and Lus10007975) increased their activity as compared to fPUL1. In addition, a number of genes encoding enzymes involved in the sugar interconversion and biosynthesis of substrates for cell wall polysaccharides were activated in fPUL8: Lus10037096, Lus10038911, Lus10010957, etc. (Table S2).

Genes for transcription factors from the TALE (Lus10016790 and Lus10022485), bZIP (Lus10017656), and bHLH (Lus10015902) families were specifically upregulated in fPUL8. Sharp activation was also detected for the genes encoding several intrinsically disordered proteins acting as chaperones (Hsiao, 2024), including the ERD10 and ERD14 proteins from the dehydrin family (Lus10005652 and Lus10021240), and late embryogenesis abundant protein LEA14 (Lus10036402). Genes for several SAM-dependent methyltransferases were upregulated, indicating the importance of methylation processes in the fPUL8 sample (Table S2).

The most notable groups of genes that were specifically upregulated in fOPP8 (Fig. 9b, Table S2) included those encoding several lipid transfer proteins and 3-ketoacyl-CoA synthases. They are required for the elongation of fatty acids and contribute to wax and suberin biosynthesis.

Reaction of xylem part on the gravistimulation: stem side-factor

The number of genes whose expression distinguished xylem tissues at pulling and opposite sides 1 h after stem inclination, was quite limited (Figs. 6, 10). There were no genes upregulated in xOPP1 as compared to xPUL1, at least with the chosen thresholds, while genes upregulated in xPUL8 relative to xOPP8 consisted mainly of genes whose products are involved in TCW formation.

The list of genes upregulated in xOPP8 as compared to xPUL8 contained 517 genes from diverse categories (Fig. 10c, Table S2). The most represented ones were LRR-RLKs (23 genes) and kinesins, microtubule motor proteins (27 genes). Quite a number of upregulated genes for proteins, which take part in chromatin rearrangement and cell cycle regulation, were detected: 12 genes for various histones, 14 genes for various cyclins, cyclin-dependent kinase B2 (Lus10021611), etc. (Table S2). Also, Lus10015843 for metacaspase 1 that acts as a positive regulator of cell death, Lus10015684 for microtubule-associated protein MAP65/ASE1 essential for cytokinesis, several genes for the transcription factors from the GRAS family were upregulated in xOPP8, as were genes for various transporters, like Lus10035860, Lus10025802, and Lus10037217 for nitrate transporters, Lus10008183 and Lus10022436 for sugar transporters, and Lus10014706 for sulfate transporter (Table S2).

Most of the genes upregulated in xOPP8 samples were also highly expressed in phloem fiber samples. Among the genes that had at least twice higher TGR values in xOPP8 than in fOPP8, the most noticeable categories included membrane-trafficking-related genes: Lus10023378 for Sec14p-like phosphatidylinositol transfer family protein, Lus10040872 for exocyst subunit EXO70H7, Lus10041836 and Lus10028383, both encoding regulators that mediate the transport between the trans-Golgi network and vacuoles (Table S2). Among the cell wall-related genes, significant upregulation in xOPP8 as compared to xPUL8 was detected for Lus10035580 and Lus10008646 encoding beta-mannan synthase (CSLA9).

Deep rearrangements take place in all analyzed stem tissues of gravistimulated flax plants

The study of graviresponse in non-elongating stem parts with the involvement of the TCW by microscopy mainly reveals the changes at the pulling side of the formed curvature, while the opposite side seems relatively unaltered as compared to the non-inclined control stem, both in fiber crops (Ibragimova et al. 2017; Petrova et al. 2021) and in trees with tension wood (Jourez et al. 2001). However, from the analysis of the transcriptomes, it is obvious that the processes providing graviresponse development involve all analyzed tissues, including those located on the opposite stem side.

Usually, only some special cell types, called statocytes, are suggested to contain statoliths, like columella cells or endodermis cells in elongating roots and shoots, respectively (Konstantinova et al. 2021; Chin and Blancaflor 2022), or secondary phloem in non-elongating wooden stem parts (Gerttula et al. 2015). The drastic changes detected in gene expression in different tissues of flax stem and the presence of a similar gene set highly and temporally activated in all GRAVI samples soon after gravistimulation, indicate that the signals provided by changing the position relative to the gravity vector can be quickly realized or maybe even perceived within phloem fibers and xylem tissues.

An alternative to the statolith and statocyte hypothesis of gravisensing is the hypothesis of gravitational pressure (Staves et al. 1997). It considers the whole protoplast as a kind of statolith and states that gravitational forces cause a tension between the plasma membrane and the cell wall at the cell top and a compression between them at the cell bottom (Staves 1997; Weise and Kiss 1999). These stresses affect the transmembrane proteins or other sensors of mechanical forces. Such sensors would detect the changes in cell orientation along the gravity vector due to the redistribution of tension and compression sites. The support for this hypothesis came from the study of the large internodal cells of the green algae Chara, which do not contain statoliths but sense gravity to control the polarity of cytoplasmic streaming (Staves 1997).

Flax phloem fibers belong to the longest plant cells, being 2.0 cm in average (Snegireva et al. 2010). The change in their position relative to the gravity vector may lead to effects similar to those in Chara internodal cells. The hypothesis of gravitational pressure can actually be true for other cell types, and we can assume it also works for the living cells of xylem. This may also be valid for the rather recently proposed “position sensor hypothesis”, which states that the gravity sensor detects an inclination and not a force (Pouliquen et al. 2017). Numerous lectin receptor-like kinases, the gene expressions of which are adjusted in the course of graviresponse in xylem tissues and phloem fibers, may be responsible for sensing mechanical stress redistribution (Shih et al. 2014). Phloem fibers, due to their peculiar cell shape, can be “the leader” in gravitropic response, as indicated by the sharpest and most pronounced upregulation of many genes, but the general mechanism of gravity perception seems to work in all analyzed tissues.

The role of plastid metabolism in graviresponse development

Plastids are considered key actors in the realization of gravity sensing mechanisms in plants since starch-containing amyloplasts may serve as the major statoliths, i.e., the “heavy” compact structures that sediment in the direction of the gravitational vector, initiating gravity perception (Kiss 2000; Kolesnikov et al. 2016). Here, we detected that the plastids themselves undergo substantial rearrangements in all analyzed tissues, both from the pulling and opposite stem sides of the gravistimulated flax stem. This is indicated by the quite high number of differentially expressed genes for various chloroplastic proteins while comparing the tissues of the inclined plants to the control ones. Together with genes for various chloroplastic chaperones, the genes for the enzymes of the metabolic pathway that operates in chloroplasts and leads to the formation of BCAAs were quite significantly upregulated in all GRAVI samples, irrespective of the stem side, and specifically at the early stages of gravitropic reaction (1 h). We suggest that the changes in BCAA metabolism in chloroplasts are coupled to their movement at the stage of gravity perception and may belong to the metabolic signals that lead to further development of graviresponse. BCAAs are known to contribute to signaling by TOR (target of rapamycin) kinase, an evolutionarily conserved master regulator of nutrient sensing, which causes reorganization of the actin cytoskeleton and actin-associated endomembranes (Cao et al. 2019). Together with that, BCAAs, particularly isoleucine, conjugate JA and transform it into the physiologically active form (Fonseca et al. 2009).

Altogether, chloroplasts/plastids are quite important in the development of graviresponse, not only as passive, “stone-like” statoliths that mechanically affect the membranes but also as the source of the crucial signals and metabolites that temporally emerge soon after gravistimulation.

Dynamic cell wall rearrangements as part of graviresponse

The TCW deposition can be considered the motor acting in many plant species to change the position of the stem in accordance with the direction of the gravity vector (Gorshkova et al. 2018). Transcriptome analysis of flax stem tissues permits comparison of cell wall dynamics in phloem fibers with the preformed TCW and in xylem fibers, where this cell wall is induced after stem inclination. Differential expression of the genes for major TCW-related molecular players is summarized in Fig. 11.

The biosynthesis of cellulose is provided by cellulose synthase complexes that consist of numerous catalytic subunits named CESAs and work in combination with multiple protein cofactors (Pedersen et al. 2023). During constitutive deposition of TCWs and in the course of graviresponse, flax phloem fibers are known to hire both SCW-related CESAs (SCW CESAs) and primary cell wall-related CESAs (PCW CESAs) (Mokshina et al. 2017, 2022). In xylem tissues, which are a complex mixture of different cell types (parenchyma, vessels, and fibers) mostly depositing SCWs, the expression values of SCW CESAs overwhelm those of PCW CESAs (Fig. 11a, b, Table S3). The changes in CESA expression occur mainly from the pulling stem side and are rather moderate. In phloem fibers, genes for SCW CESAs are upregulated earlier, followed by PCW CESAs. Such differences in expression dynamics may be associated with the different rate of CESA turnover, which is higher for SCW CESAs as compared to PCW CESAs (Jacob-Wilk et al. 2006). Changes in CESA expression in the course of graviresponse development can be put together with the increase in the crystallinity of the cellulose, as well as changes in the proportion of cellulose allomorphs in phloem fibers isolated from pulling side as compared to fibers from control plants (Ibragimova et al. 2020).

The main non-cellulosic component of the TCW is RG-I with galactan side chains. In the current work, activation of genes for the known enzymes involved in RG-I backbone biosynthesis, RRT (Wachananawat et al. 2020) and RGGAT (Amos et al. 2022), was observed only in samples from the pulling stem side. For some of these genes, the activation was obvious already 1 h after stem inclination. The highest fold change values were detected in xylem tissues several hours later (Fig. 11, Table S3). Notably, the genes for RRT10 isoforms were more essentially activated in fGRAVI1, while RRT1s increased their expression later (Table S1). The genes for galactan synthase (GALS2, Liwanag et al. 2012), whose activity is necessary for the RG-I side chain elongation, were significantly upregulated only 8 hours after inclination (Table S2).

The expression patterns of other TCW-related genes encoding specific FLAs, RGL, and various cofactors of CESAs, like COBL4, had the highest fold changes of TGR values in xylem tissues from the pulling stem side 8 h after gravistimulation (Fig. 11c-e, Table S3). The rearrangement of cell-wall metabolism also involved the pronounced upregulation of genes for the enzymes that are involved in NDP-sugar interconversion and provide substrates for cell wall polysaccharides (Fig. 11f, Table S3). For many TCW-related genes, the upregulation in xylem samples occurred later than in phloem fibers; however, the set of upregulated cell wall-related genes in phloem fibers and xylem tissues, if located at the pulling stem side, was similar. This indicates that the mechanisms of TCW deposition in phloem and xylem fibers are largely the same, while their regulation differs.

In samples from the opposite stem side, where the fibers do not deposit tertiary cell wall, the number of differentially expressed cell wall genes was by far less than in samples from the pulling side. The major notable reaction was the moderate activation of genes for the enzymes involved in mannan synthesis in xOPP8 (Fig. 11g, Table S3) as well as in suberin deposition (Fig. 10b, Table S2).

Differentially expressed genes for cell wall-related proteins include members of several signaling pathways. Lus10009009, sharply and specifically upregulated in phloem fibers of GRAVI samples 1 h after gravistimulation (Table S1), encodes an oxidoreductase named Berberine bridge enzyme-like 8. The cell wall located BBE-like enzymes play a key role in the inactivation of biologically active oligosaccharides, fragments of pectin, and cellulose (Locci et al. 2019). Another gene for the Berberine-bridge enzyme, Lus10023375, is sharply down-regulated in xylem, also 1 h after gravistimulation in both GRAVI samples (Table S1). Genes for many receptor-like transmembrane kinases that may sense the situation in the cell wall and transmit the signal into the cytoplasm, are differentially expressed in various GRAVI samples. Additionally, signaling peptides, like rapid alkalinization factor ralf-like 32 with differential expression, modulate the activity of such cell surface receptors. Upregulation of many genes for cell-wall proteins involved in signaling pathways already 1 h after stem inclination indicated the involvement of the cell wall in signal formation and transduction in the course of graviresponse.

The sequence of events and the major molecular players in the development of flax stem graviresponse

The general notion of early event flow to initiate the graviresponse in higher plants, in a simplified way, can be summarized as statolith sedimentation, their action on membranes, the relocation of auxin transporters relative to gravity vector, and the concomitant changes in auxin distribution between and within tissues leading to asymmetric changes in metabolism at the opposing sides of an axial organ (Baldwin et al. 2013; Groover, 2016; Chin and Blancaflor 2022). The earliest steps in graviperception are the disturbance of intracellular membranes, disruption of the actin cytoskeleton network, and activation of mechanosensitive ion channels at the plasma membrane due to statolith sedimentation (Baldwin et al. 2013; Chin and Blancaflor 2022). Secondary messengers, such as inositol-1,4,5-tri-phosphate (InsP3), cytoplasmic calcium (Ca²⁺), and reactive oxygen species, get rapidly involved (Baldwin et al. 2013). Among the genes for proteins that could participate in this early step are those encoding glutamate receptor-like proteins (GLRs) that provide signaling between cells and tissues via alteration of surface potentials (Mousavi et al. 2013) or several receptor-like kinases localized at the plasma membrane and suggested to sense mechanical stresses (Shin et al. 2014). Additionally, the genes for SGR9-E3 ligase, which may promote detachment of plastids from actin filaments, allowing the statoliths to sediment (Nakamura et al. 2011), and for PIP5K1,– kinase involved in InsP3 formation, were significantly upregulated in phloem fibers located at the opposite stem side. Lus10037042, homologous to the Arabidopsis gene for LAZY1 involved in gravity signaling downstream of amyloplast displacement (Taniguchi et al. 2017), was highly expressed in all samples of phloem fibers but barely transcribed in xylem tissues, being activated only 8 h after inclination and only from the opposite stem side (Table S1).

Auxin redistribution is viewed as the next step in signal perception and transduction, at least in elongating organs. In accordance, transcriptome analysis of the early gravitropic response demonstrated numerous upregulated auxin-related genes, such as AUX/IAA and SAUR family genes (Kimbrough et al. 2004; Hu et al. 2013; Taniguchi et al. 2014) However, in non-elongation tissues, the role of this hormone is less substantiated: though the relocalization of PIN3 transporters toward the ground was demonstrated in proposed statocytes of the gravistimulated plants of hybrid aspen, suggesting lateral redistribution of auxin (Gerttula et al. 2015), differences in auxin balance during tension wood development were not revealed biochemically (Hellgren et al. 2004). In flax stem, the graviresponse in the non-elongating stem part was not coupled to a massive upregulation of auxin-related genes.

At the same time, genes for proteins involved in the metabolism of JA, as well as in the perception and transduction of its signal, were considerably upregulated, especially 1 h after plant inclination (Fig. 8). The increase in JA content in gravistimulated rice coleoptiles was demonstrated earlier by GC-MS/MS (Gutjahr et al. 2005). The synthesis of JA starts in chloroplasts, and thus its activation upon gravistimulation can be coupled to the discussed changes in the metabolism of these organelles. The changes in JA concentration may trigger numerous further reactions, including the activation of transcription factors and other regulators that may also involve other hormones. For example, MBF1c, a transcriptional coactivator involved in the ethylene-response signal transduction pathway, or TPR4, a corepressor of DELLA-binding transcription factor, which regulates gibberellin biosynthesis (Fukazawa et al. 2014), had the highest fold change values in all 1 h GRAVI samples. The extensive concomitant reorganization of protein metabolism is supported by the massive upregulation of genes for chaperones located in various cell compartments. Later, genes for numerous kinases became the most notable group of upregulated genes in all analyzed tissues.

Stem inclination immediately leads to an asymmetry in the quality of light reaching the tissues from the upper and lower stem sides. The design used in experiments is close to “real life” and does not separate gravitational, mechanical, and light stimuli. Each of them, or in combination, provides the asymmetry of reaction on the opposing stem sides and leads to the determination of the pulling and opposite sides. One hour after stem inclination, the number of genes with statistically significant differences between xPUL and xOPP was minimal and raised considerably after 8 hours. At the same time, the differences in gene transcription in phloem fibers located at pulling and opposite stem sides were already quite pronounced. They included the set of genes involved in ethylene biosynthesis and response (opposite side), as well as genes for several transcription factors and enzymes involved in remodeling of the previously deposited TCW (pulling side). The asymmetric reactions were also quite obvious in xylem tissues. In the tension wood, the deposition of TCW is induced in xylem fibers from the pulling side in parallel with some cell cycle-related processes in xylem tissues of the opposite stem side, which, to our knowledge, have not been studied yet. The summary of the events occurring in different tissues of the non-elongating part of the flax stem upon inclination is illustrated in Fig. 12. Altogether, we have characterized the early stages of graviresponse in herbal plant that hires TCW to restore vertical position, similar to tension wood formation. The crucial involvement of extraxylary fibers, together with the important, largely underestimated processes occurring in the tissues of the opposite stem side, were revealed. The outcoming similarities and differences with gravitropic reaction developed in elongating zones of axial organs provide informative insights into mechanisms of gravity perception and graviresponse development.

Conflict of interest

The authors declare there are no competing interests.

Funding

The work was supported by the Russian Science Foundation, project 19-14-00361П (RNA-Seq analysis). The part of work was performed at financial support from the government assignment for FRC Kazan Scientific Center of RAS (part of bioinformatic analysis, data interpretation).

Author contributions

TG, OG planned and designed the experiments, OG prepared samples and carried out the bioinformatics analysis, TG, OG, NM contributed to the conceptualization of the experiments and interpretation of data, drafted the manuscript. TG participated in supervision of the study. All co-authors have reviewed and approved the manuscript.

Data availability

The data presented in the study are deposited in the NCBI repository (accession number PRJNA631357).

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How It All Begins: Molecular Players of the Early Graviresponse in the Non-elongating Part of Flax Stem

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Version 1

Abstract

Figures

Introduction

Materials and Methods

Plant material

RNA extraction and sequencing

RNA-Seq data processing and gene expression analysis

Results

Transcriptome response of phloem fibers and xylem part: tissue factor

Transcriptome response of phloem fibers and xylem part: time factor

Reaction of phloem fibers on the gravistimulation: stem side-factor

Reaction of xylem part on the gravistimulation: stem side-factor

Discussion

Deep rearrangements take place in all analyzed stem tissues of gravistimulated flax plants

The role of plastid metabolism in graviresponse development

Dynamic cell wall rearrangements as part of graviresponse

Declarations

Conflict of interest

Funding

Author contributions

Data availability

References

Supplementary Files

Status:

Version 1