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In cucumber seedlings, gravitropism interferes with hydrotropism, which results in the nearly complete inhibition of hydrotropism under stationary conditions. However, hydrotropic responses are induced when the gravitropic response in the root is nullified by clinorotation. Columella cells in the root cap sense gravity, which induces the gravitropic response. In this study, we found that removing the root tip induced hydrotropism in cucumber roots under stationary conditions. The application of auxin transport inhibitors to cucumber seedlings under stationary conditions suppressed the hydrotropic response induced by the removal of the root tip. To investigate the expression of genes related to hydrotropism in de-tipped cucumber roots, we conducted transcriptome analysis of gene expression by RNA-Seq using seedlings exhibiting hydrotropic and gravitropic responses. Of the 21 and 45 genes asymmetrically expressed during hydrotropic and gravitropic responses, respectively, five genes were identical. Gene ontology (GO) analysis indicated that the category auxin-inducible genes was significantly enriched among genes that were more highly expressed in the concave side of the root than the convex side during hydrotropic or gravitropic responses. Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis revealed that root hydrotropism induced under stationary conditions (by removing the root tip) was accompanied by the asymmetric expression of several auxin-inducible genes. However, intact roots did not exhibit the asymmetric expression patterns of auxin-inducible genes under stationary conditions, even in the presence of a moisture gradient. These results suggest that the root tip inhibits hydrotropism by suppressing the induction of asymmetric auxin distribution. Auxin transport and distribution not mediated by the root tip might play a role in hydrotropism in cucumber roots.

Roots of land plants show gravitropism and hydrotropism in response to gravity and moisture gradients, respectively, for controlling their growth orientation. Gravitropism interferes with hydrotropism, although the mechanistic aspects are poorly understood. Here, we differentiated hydrotropism from gravitropism in cucumber roots by conducting clinorotation and spaceflight experiments. We also compared mechanisms regulating hydrotropism and auxin-regulated gravitropism. Clinorotated or microgravity (μG)-grown cucumber seedling roots hydrotropically bent toward wet substrate in the presence of moisture gradients, but they grew straight in the direction of normal gravitational force at the Earth's surface (1G) on the ground or centrifuge-generated 1G in space. The roots appeared to become hydrotropically more sensitive to moisture gradients under μG conditions in space. Auxin transport inhibitors significantly reduced the hydrotropic response of clinorotated seedling roots. The auxin efflux protein CsPIN5 was differentially expressed in roots of both clinorotated and μG-grown seedlings; with higher expression in the high-humidity (concave) side than the low-humidity (convex) side of hydrotropically responding roots. Our results suggest that roots become hydrotropically sensitive in μG, and CsPIN5-mediated auxin transport has an important role in inducing root hydrotropism. Thus, hydrotropic and gravitropic responses in cucumber roots may compete via differential auxin dynamics established in response to moisture gradients and gravity., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Cucumber (Cucumis sativus) seedlings grown in a horizontal position develop a specialized protuberance (or peg) on the lower side of the transition zone between the hypocotyl and the root. This occurs by suppressing peg formation on the upper side via a decrease in auxin resulting from a gravitational response. However, the gravity-stimulated mechanism of inducing asymmetric auxin distribution in the transition zone is poorly understood. The gravity-sensing tissue responsible for regulating auxin distribution in the transition zone is thought to be the endodermal cell. To characterize the gravity-stimulated mechanism, the auxin efflux facilitator PIN-FORMED1 (CsPIN1) in the endodermis was identified and the localization of CsPIN1 proteins during the gravimorphogenesis of cucumber seedlings was examined. Immunohistochemical analysis revealed that the accumulation pattern of CsPIN1 protein in the endodermal cells of the transition zone of cucumber seedlings grown horizontally differed from that of plants grown vertically. Gravistimulation for 30 min prompted changes in the accumulation pattern of CsPIN1 protein in the endodermis as well as the asymmetric distribution of auxin in the transition zone. Furthermore, 2,3,5-triiodobenzoic acid inhibited the differential distribution of auxin as well as changes in the accumulation pattern of CsPIN1 in the endodermis of the transition zone during gravistimulation. These results suggest that the altered pattern of CsPIN1 accumulation in the endodermis in response to gravistimulation influences lateral auxin transport through the endodermis, resulting in asymmetric auxin distribution in the transition zone.

Gravity exerts a profound influence on plant growth. Proteomics may be the most promising technique to identify the proteins that are induced, repressed, or post-transcriptionally modified during gravity response in plants. In this research, the model plant, Arabidopsis grown in stationary and clino-rotated condition for 7 to 10 days and cucumber plant with the unique gravimorphogenesis were used. Namely, cucurbit seedlings develop a peg on the transition zone between the hypocotyls and root. Cucumber seedlings grown in a horizontal position on the ground suppress the development of the peg (negatively-controlled) on the upper side of the transition zone in response to gravity. Then, we analyzed gravity-regulated proteins expression by two-dimensional (2-D) gel electrophoresis.

Cucurbit seedlings potentially develop a peg on each side of the transition zone between the hypocotyl and root. Seedlings grown in a horizontal position suppress the development of the peg on the upper side of the transition zone in response to gravity. It is suggested that this suppression occurs due to a reduction in auxin levels to below the threshold value. We show in this study that the free indole-3-acetic acid (IAA) content is low, while IAA conjugates are significantly more abundant in the upper side of the transition zone of gravistimulated seedlings, compared to the lower side. A transient increase in mRNA of the auxin-inducible gene, CS-IAA1, was observed in the excised transition zone. The result suggests that the transition zone is a source of auxin. Cucumber seedlings treated with auxin-transport inhibitors exhibited agravitropic growth and developed a peg on each side of the transition zone. Auxin-transport inhibitors additionally caused an increase in CS-IAA1 mRNA accumulation at the transition zone, indicating a rise in intracellular auxin concentrations due to a block of auxin efflux. To study the involvement of the auxin transport system in peg formation, we isolated the cDNAs of a putative auxin influx carrier, CS-AUX1, and putative efflux carrier, CS-PIN1, from cucumber (Cucumis sativus L.) plants. Both genes (CS-AUX1 in particular) were auxin-inducible. Accumulation of CS-AUX1 and CS-PIN1 mRNAs was observed in vascular tissue, cortex and epidermis of the transition zone. A reduced level of CS-AUX1 mRNA was observed in the upper side of the gravistimulated transition zone, compared with the lower side. It is therefore possible that a balance in the activities of auxin influx and efflux carriers controls intracellular auxin concentration at the transition zone, which results in lateral placement of a peg in cucumber seedlings.

Cucumber seedlings display not only gravitropism but also peg formation in response to gravity. Gravimorphogenesis is mediated by auxin distribution. As first step to reveal the mechanism that regulates auxin distribution by auxin efflux, we isolated five partial cDNAs of auxin efflux carriers by RT-PCR method. In addition, we isolated two full-length cDNAs (CsPIN2, CsPIN3) from a cucumber cDNA library. CsPIN2, AtPIN3, AtPIN4 and AtPIN7 fall within the same clade. CsPIN3, AtPIN1 and CsPIN1 fall within the same clade. CsPIN5, CsPIN6 and AtPIN2 fall within the same clade. Our phylogenetic analysis of PIN in cucumber and Arabidopsis indicates that cucumber may diversify CsPIN protein compared with AtPIN protein.

Cucumber (Cucumis sativus L.) seedlings grown in microgravity developed a peg on each side of the transition zone between hypocotyl and root, whereas seedlings grown in a horizontal position on the ground developed a peg on the concave side of the gravitropically bending transition zone. The morphological features of the space-grown seedlings were similar to those of seedlings grown in a vertical position on the ground with their radicles pointing down: both became two-pegged seedlings. Morphogenesis of cucumber seedlings is thus inhibited by gravity. Analysis by in-situ hybridization of an auxin-inducible gene, CS-IAA1, showed that its mRNA accumulated to a much greater extent on the lower side of the transition zone in the horizontally placed seedlings on the ground just prior to and during the initiation period of peg formation. On the other hand, when seedlings were grown in microgravity or in a vertical position on the ground, accumulation of CS-IAA1 mRNA occurred all around the transition zone. Accumulation of CS-IAA1 mRNA in horizontally grown seedlings appreciably decreased on the upper side of the transition zone and increased on the lower side upon gravistimulation, compared with the two-pegged seedlings. Application of IAA to seedlings in a horizontal position caused the development of a peg on each side of the transition zone, or a collar-like protuberance, depending on the concentration used. These results suggest that upon gravistimulation the auxin concentration on the upper side of the horizontally placed transition zone is reduced to a level below the threshold value necessary for peg formation. Space-grown seedlings of cucumber might develop two pegs symmetrically because the auxin level in the entire transition zone is maintained above the threshold. This spaceflight experiment verified for the first time that auxin does not redistribute in microgravity.

We examined the effect of microgravity on the peg formation of cucumber seedlings for clarifying the mechanism of gravimorphogenesis in cucurbitaceous plants. The spaceflight experiments verified that gravity controls the formation of peg, hypocotyl hook and growth orientation of cucumber seedlings. Space-grown cucumber developed a peg on each side of the transition zone of the hypocotyl and root, indicating that on the ground peg formation is regulated negatively by gravity (Takahashi et al. 2000). It was found that the auxin-regulated gene, CS-IAA1, was strongly expressed in the transition zone where peg develops (Fujii et al. 2000). In the seedlings grown horizontally on the ground, CS-IAA1 transcripts were much abundant on the lower side of the transition zone, but no such differential expression of CS-IAA1 was observed in the space-grown cucumber (Kamada et al. 2000). These results imply that gravity plays a role in peg formation through auxin redistribution. By the negative control, peg formation on the upper side of the transition zone in the horizontally growing seedlings might be suppressed due to a reduction in auxin concentration. The threshold theory of auxin concentration accounted for the new concept, negative control of morphogenesis by gravity (Kamada et al. 2000). Anatomical studies have shown that there exists the target cells destined to be a peg and distinguishable at the early stage of the growth. Ultra-structural analysis suggested that endoplasmic reticulum develops well in the cells of the future peg. Furthermore, it was found that reorganization of cortical microtubules is required for the change in cell growth polarity in the process of peg formation. The spaceflight experiment with cucumber seedlings also suggested that in microgravity positive hydrotropic response of roots occurred without interference by gravitropic response (Takahashi et al. 1999b). Thus, this spaceflight experiment together with the ground-based studies has shown that cucumber seedling is an ideal for the study of gravimorphogenesis, hydrotropism and their interaction. Although peg formation is seen specifically in cucurbitaceous seedlings, it involves graviperception, auxin transport and redistribution and cytoskeletal modification for controlling cell growth polarity. This system could be a useful model for studying important current issues in plant biology.

The plant hormone auxin transcriptionally activates Aux/IAA genes. We have isolated three Aux/IAA cDNA from cucumber, two cDNAs (CS-IAA1 and CS-IAA2) containing the complete open reading frame (ORF), and one partial cDNA (CS-IAA3). Northern blotting analysis showed that Aux/IAA mRNAs were induced during the emergence of radicles from seed coats. After radicle emergence, their mRNAs accumulated in the basal part of the hypocotyl much more than in the apical part, and later in elongating region of hypocotyls. CS-IAAI and CS-IAA3 mRNA significantly accumulated in response to auxin, although the increment of the former mRNA accumulation by auxin application was much greater than that of the latter. CS-IAA2 did not show an apparent change by auxin treatment in our experiment. In horizontally germinating seedlings, the transition zone between hypocotyl and root curves was due to downward gravitropic growth. On the other hand, vertically germinating seedlings of cucumber do not curve in the early stage of seedling development. The CS-IAA1 mRNA accumulation in horizontally germinating seedlings was more than that in vertically germinating ones during radicle emergence. Furthermore, asymmetric distribution of CS-IAA1 mRNA was detected in the transition zone in in situ hybridization analysis. These results suggest that the CS-IAA1 gene product may be involved in the gravity response during early development of seedlings.

Seedlings of most cucurbitaceous plants develop a peg (protuberance caused by cell outgrowth) on the transition zone between the hypocotyl and root. The peg is necessary for removing the seed coat after germination. In our spaceflight experiments on the STS-95 space shuttle, Discovery, we found that cucumber (Cucumis sativus L.) seedlings grown under microgravity conditions developed two pegs symmetrically at the transition zone. Thus, cucumber seedlings potentially develop two pegs and do not require gravity for peg formation itself, but on the ground the development of one peg is suppressed in response to gravity. This may be considered as negative control of morphogenesis by gravity.

Seedlings of Cucurbitaceae plants form a protuberance, termed peg, on the transition zone between hypocotyl and root. Our spaceflight experiment verified that the lateral positioning of a peg in cucumber seedlings is modified by gravity. It has been suggested that auxin plays an important role in the gravity controlled positioning of a peg on the ground. Furthermore, cucumber seedlings grown in microgravity developed a number of the lateral roots that grew towards the water containing substrate in the culture vessel, whereas on the ground they oriented perpendicular to the primary root growing down. The response of the lateral roots in microgravity was successfully mimicked by clinorotation of cucumber seedlings on the three dimensional clinostat. However, this bending response of the lateral roots was observed only in an aeroponic culture of the seedlings but not in solid medium. We considered the response of the lateral roots in microgravity and on clinostat as positive hydrotropism that could easily be interfered by gravitropism on the ground. This system with cucumber seedlings is thus a useful model of spaceflight experiment for the study of the gravimorphogenesis, root hydrotropism and their interaction.