Interaction of Phytohormones in Regulating the Axillary Bud Growth in Pea

In the present study the interactions of GR24, a synthetic analog of newly discovered plant hormones strigolactones (SLs), with cytokinin (CK), benzyladenine (BA), auxin naphthaleneacetic acid (NAA), gibberellic acid (GA₃) and abscisic acid (ABA) in the regulation of axillary bud growth in pea (Pisum sativum L.) were investigated. The hormones were applied directly to buds at node 1 and 2 and the dose-response experiments were performed on 8–10-day-old SL-deficient rms1 and rms5–1 mutants, branching SL-sensitive rms2–1 mutants and wild-type plants. In the mutant plants the treatment with 5 μM GR24 completely inhibited bud growth while BA up to 100 μM stimulated it. The combined application of GR24 and BA showed that GR24 efficiency to reduce bud outgrowth constantly declines as CK-stimulated bud growth increased, with the inhibiting effect of GR24 abolished at 100 μM BA applied. GA₃ accelerated bud outgrowth, but did not interfere with GR24 inhibitory action. NAA reduced bud growth in intact SL-sensitive rms2–1 mutant and also in SL-insensitive rms3–2 and rms4–1 mutants. The NAA effect was observed already at 0.5 μM, however, even at a response saturating concentration of 500 μM its inhibiting effect was inferior to that of 5 μM GR24. At combined treatment the effectiveness of GR24 in suppressing bud growth decreased with a decrease in NAA-inhibited bud growth, suggesting that auxin might act as a suppressor of SL action. ABA strongly inhibited the bud outgrowth and, like CK and auxin, reduced the inhibitory effectiveness of GR24. The revealed ability of CK, ABA and auxin to suppress bud response to SLs is supposed to result from phytohormone signaling crosstalks.     

Response to strigolactone treatment in chrysanthemum axillary buds is influenced by auxin transport inhibition and sucrose availability

Axillary bud outgrowth is regulated by both environmental cues and internal plant hormone signaling. Central to this regulation is the balance between auxins, cytokinins, and strigolactones. Auxins are transported basipetally and inhibit the axillary bud outgrowth indirectly by either restricting auxin export from the axillary buds to the stem (canalization model) or inducing strigolactone biosynthesis and limiting cytokinin levels (second messenger model). Both models have supporting evidence and are not mutually exclusive. In this study, we used a modified split-plate bioassay to apply different plant growth regulators to isolated stem segments of chrysanthemum and measure their effect on axillary bud growth. Results showed axillary bud outgrowth in the bioassay within 5 days after nodal stem excision. Treatments with apical auxin (IAA) inhibited bud outgrowth which was counteracted by treatments with basal cytokinins (TDZ, zeatin, 2-ip). Treatments with basal strigolactone (GR24) could inhibit axillary bud growth without an apical auxin treatment. GR24 inhibition of axillary buds could be counteracted with auxin transport inhibitors (TIBA and NPA). Treatments with sucrose in the medium resulted in stronger axillary bud growth, which could be inhibited with apical auxin treatment but not with basal strigolactone treatment. These observations provide support for both the canalization model and the second messenger model with, on the one hand, the influence of auxin transport on strigolactone inhibition of axillary buds and, on the other hand, the inhibition of axillary bud growth by strigolactone without an apical auxin source. The inability of GR24 to inhibit bud growth in a sucrose treatment raises an interesting question about the role of strigolactone and sucrose in axillary bud outgrowth and calls for further investigation.     

Effects of endogenous hormones on variation of shoot branching in a variety of non-heading Chinese cabbage and related gene expression

Shoot branching (tillering) primarily determines plant shoot architecture and has been studied in many plants. Shoot branching is an important trait in non-heading Chinese cabbage (Brassica rapa ssp. chinensis Makino). The B. rapa ssp. chinensis var. multiceps exhibits unique and multiple shoot branching characteristics. Here, we analyzed the variation in shoot branching between ‘Maertou,’ with multiple shoot branching, and ‘Suzhouqing,’ a common variety. The levels of endogenous indole-3-acetic acid (IAA), zeatin riboside and active gibberellins in the shoot meristem tissues of the two cultivars were quantified by enzyme-linked immunosorbent assay during the vegetative growth stage. High levels of IAA maintained axillary bud dormancy and repressed axillary bud outgrowth allowing shoot branching to form in the vegetative stage in ‘Suzhouqing.’ In contrast, low levels of IAA did not inhibit axillary buds in ‘Maertou,’ while a high level of cytokinin promoted axillary bud growth and branch shoot development. Exogenous hormone (rac-GR24 and 6-benzylaminopurine) treatment showed that ‘Maertou’ was relatively sensitive to cytokinin, because the fold changes of cytokinin-responsive genes in ‘Maertou’ were significantly more frequent than those in ‘Suzhouqing’. Cytokinin was the direct regulator for axillary bud growth of ‘Maertou’. Compared with ‘Suzhouqing’, ‘Maertou’ was sensitive to cytokinin and this weakened the strigolactone–cytokinin branching pathway.     

Hormone-Induced Gene Expression During Gravicurvature of <Emphasis Type="Italic">Brassica</Emphasis> Roots

To investigate the spatial and temporal dependence of hormonal regulation during gravitropism, we compared the effects of root cap application of indole-3-acetic acid (IAA) and abscisic acid (ABA) with gene expression changes occurring naturally during gravitropic reaction of Brassica rapa roots. The expression of auxin, ABA, and metabolism-related genes in the tip, elongation zone, and maturation zone varied with time, location, and hormone concentration and characterized polar auxin transport. IAA was transported readily shootward and inhibited growth more than ABA. Expression of PIN3 and IAA5 in the elongation zone showed downregulation on the convex but upregulation on the concave side. Both PIN7 and IAA5 responded near maximally to 10^?8 M IAA within 30 min, suggesting that auxin activates its own transport system. Ubiquitin 1 (UBQ1) responded after a lag time of more than 1 h to IAA. The metabolic control gene Phosphoenolpyruvate carboxylase 1 (PEPC1) was more sensitive to ABA but upregulated by high concentrations of either hormone. The time course and duration of gene activation suggests that ABA is not involved in gravitropic curvature, differential elongation is not simply explained by IAA-induced upregulation, and that reference genes are sensitive to auxin.     

Actions of strigolactone GR24 and <Emphasis Type="Italic">DRM1</Emphasis> gene expression on Arabidopsis root architecture

Strigolactones are mostly known for their influence on apical dominance, but new insights suggest that they may be involved in many other biological events including root development. DRM1 gene is ubiquitary expressed in plants but its role is not well known. In our experiments, the strigolactone analogue GR24 stimulated the expression of DRM1.1, DRM1.2, DRM1.4 splicing variants and inhibited root branching in 5-day-old Arabidopsis thaliana (L.) Heynh. seedlings. On the other hand, the expression of these splicing variants was lower in 10-day-old GR24-treated roots. DRM1.6 gene expression differently responded to GR24 than other DRM1 splicing variants, however, there was no clear relationship between DRM1.6 expression and root length. Our results suggest that strigolactones and the expression of DRM1 gene play interactive roles in root branching.     

<Emphasis Type="Italic">MdPIN1b</Emphasis> encodes a putative auxin efflux carrier and has different expression patterns in BC and M9 apple rootstocks

Key message

Lower promoter activity is closely associated with lower MdPIN1b expression in the M9 interstem, which might contribute to the dwarfing effect in apple trees.

Abstract

Apple trees grafted onto dwarfing rootstock Malling 9 (M9) produce dwarfing tree architecture with high yield and widely applying in production. Previously, we have reported that in Malus ‘Red Fuji’ (RF) trees growing on M9 interstem and Baleng Crab (BC) rootstock, IAA content was relatively higher in bark tissue of M9 interstem than that in scion or rootstock. As IAA polar transportation largely depends on the PIN-FORMED (PIN) auxin efflux carrier. Herein, we identify two putative auxin efflux carrier genes in Malus genus, MdPIN1a and MdPIN1b, which were closely related to the AtPIN1. We found that MdPIN1b was expressed preferentially in BC and M9, and the expression of MdPIN1b was significantly lower in the phloem of M9 interstem than that in the scion and rootstock. The distinct expression of MdPIN1b and IAA content were concentrated in the cambium and adjacent xylem or phloem, and MdPIN1b protein was localized on cell plasma membrane in onion epidermal cells transiently expressing 35S:MdPIN1b-GFP fusion protein. Interestingly, an MdPIN1b mutant allele in the promoter region upstream of M9 exhibited decreased MdPIN1b expression compared to BC. MdPIN1b over-expressing interstem in tobacco exhibited increased polar auxin transport. It is proposed that natural allelic differences decreased promoter activity is closely associated with lower MdPIN1b expression in the M9 interstem, which might limit the basipetal transport of auxin, and in turn might contribute to the dwarfing effect. Taken together, these results reveal allelic variation underlying an important apple rootstock trait, and specifically a novel molecular genetic mechanism underlying dwarfing mechanism.

     

Strigolactone Acts Downstream of Auxin to Regulate Bud Outgrowth in Pea and Arabidopsis

During the last century, two key hypotheses have been proposed to explain apical dominance in plants: auxin promotes the production of a second messenger that moves up into buds to repress their outgrowth, and auxin saturation in the stem inhibits auxin transport from buds, thereby inhibiting bud outgrowth. The recent discovery of strigolactone as the novel shoot-branching inhibitor allowed us to test its mode of action in relation to these hypotheses. We found that exogenously applied strigolactone inhibited bud outgrowth in pea (Pisum sativum) even when auxin was depleted after decapitation. We also found that strigolactone application reduced branching in Arabidopsis (Arabidopsis thaliana) auxin response mutants, suggesting that auxin may act through strigolactones to facilitate apical dominance. Moreover, strigolactone application to tiny buds of mutant or decapitated pea plants rapidly stopped outgrowth, in contrast to applying N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, which significantly slowed growth only after several days. Whereas strigolactone or NPA applied to growing buds reduced bud length, only NPA blocked auxin transport in the bud. Wild-type and strigolactone biosynthesis mutant pea and Arabidopsis shoots were capable of instantly transporting additional amounts of auxin in excess of endogenous levels, contrary to predictions of auxin transport models. These data suggest that strigolactone does not act primarily by affecting auxin transport from buds. Rather, the primary repressor of bud outgrowth appears to be the auxin-dependent production of strigolactones.     

Activation of gibberellin 20-oxidase 2 undermines auxin-dependent root and root hair growth in NaCl-stressed <Emphasis Type="Italic">Arabidopsis</Emphasis> seedlings

Although salt stress mainly disturbs plant root growth by affecting the biosynthesis and signaling of phytohormones, such as gibberellin (GA) and auxin, the exact mechanisms of the crosstalk between these two hormones remain to be clarified. Indole-3-acetic acid (IAA) is a biologically active auxin molecule. In this study, we investigated the role of Arabidopsis GA20-oxidase 2 (GA20ox2), a final rate-limiting enzyme of active GA biosynthesis, in IAA-directed root growth under NaCl stress. Under the NaCl treatment, seedlings of a loss-of-function ga20ox2-1 mutant exhibited primary root and root hair elongation, altered GA₄ accumulation, and decreased root Na⁺ contents compared with the wild-type, transgenic GA20ox2-complementing, and GA20ox2-overexpression plant lines. Concurrently, ga20ox2-1 alleviated the tissue-specific inhibition of NaCl on IAA generation by YUCCAs, IAA transport by PIN1 and PIN2, and IAA accumulation in roots, thereby explaining how NaCl increased GA20ox2 expression in shoots but disrupted primary root and root hair growth in wild-type seedlings. In addition, a loss-of-function pin2 mutant impeded GA20ox2 expression, indicating that GA20ox2 function requires PIN2 activity. Thus, the activation of GA20ox2 retards IAA-directed primary root and root hair growth in response to NaCl stress.     

Ethylene Controls Adventitious Root Initiation Sites in <Emphasis Type="Italic">Arabidopsis</Emphasis> Hypocotyls Independently of Strigolactones

Adventitious root formation is essential for cutting propagation of diverse species; however, until recently little was known about its regulation. Strigolactones and ethylene have both been shown to inhibit adventitious roots and it has been suggested that ethylene interacts with strigolactones in root hair elongation. We have investigated the interaction between strigolactones and ethylene in regulating adventitious root formation in intact seedlings of Arabidopsis thaliana. We used strigolactone mutants together with 1-aminocyclopropane-1-carboxylic acid (ACC) (ethylene precursor) treatments and ethylene mutants together with GR24 (strigolactone agonist) treatments. Importantly, we conducted a detailed mapping of adventitious root initiation along the hypocotyl and measured ethylene production in strigolactone mutants. ACC treatments resulted in a slight increase in adventitious root formation at low doses and a decrease at higher doses, in both wild-type and strigolactone mutants. Furthermore, the distribution of adventitious roots dramatically changed to the top third of the hypocotyl in a dose-dependent manner with ACC treatments in both wild-type and strigolactone mutants. The ethylene mutants all responded to treatments with GR24. Wild type and max4 (strigolactone-deficient mutant) produced the same amount of ethylene, while emanation from max2 (strigolactone-insensitive mutant) was lower. We conclude that strigolactones and ethylene act largely independently in regulating adventitious root formation with ethylene controlling the distribution of root initiation sites. This role for ethylene may have implications for flood response because both ethylene and adventitious root development are crucial for flood tolerance.     

Analysis the role of arabidopsis <Emphasis Type="Italic">CKRC6/ASA1</Emphasis> in auxin and cytokinin biosynthesis

The crosstalk between auxin and cytokinin (CK) is important for plant growth and development, although the underlying molecular mechanisms remain unclear. Here, we describe the isolation and characterization of a mutant of Arabidopsis Cytokinin-induced Root Curling 6 (CKRC6), an allele of ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) that encodes the á-subunit of AS in tryptophan (Trp) biosynthesis. The ckrc6 mutant exhibits root gravitropic defects and insensitivity to both CK and the ethylene precursor 1-aminocyclopropane-1-carboxylicacid (ACC) in primary root growth. These defects can be rescued by exogenous indole-3-acetic acid (IAA) or tryptophan (Trp) supplementation. Furthermore, our results suggest that the ckrc6 mutant has decreased IAA content, differential expression patterns of auxin biosynthesis genes and CK biosynthesis isopentenyl transferase (IPT) genes in comparison to wild type. Collectively, our study shows that auxin controls CK biosynthesis based on that CK sensitivity is altered in most auxin-resistant mutants and that CKs promote auxin biosynthesis but inhibit auxin transport and response. Our results also suggest that CKRC6/ASA1 may be located at an intersection of auxin, CK and ethylene metabolism and/or signaling.     

Involvement of polar auxin transport in the inhibition of Arabidopsis seedling growth induced by Stenotrophomonas maltophilia

A wide range of microorganisms found in the rhizhosphere are able to regulate plant growth and development, but little is known about the mechanism by which epiphytic microbes inhibit plant growth. Here, an epiphytic bacteria Stenotrophomonas maltophilia, named as LZMBW216, were isolated and identified from the potato (Solanum tuberosum L. cv. Da Xi Yang) leaf surface. They could decrease primary root elongation and lateral root numbers in Arabidopsis seedlings. The inhibitory effects of LZMBW216 on plant growth were not due to a reduced indole-3-acetic acid (IAA) content, as exogenously applied IAA did not recover the inhibition. Furthermore, LZMBW216 did not affect the expression of DR5::GUS and CycB1;1::GUS. However, we found that LZMBW216 exhibited little effect on the primary root elongation in the pin2 mutant and on the lateral root numbers in the aux1-7 mutant. Moreover, LZMBW216 decreased expressions of AUX1 and PIN2 proteins. Together, these results suggest that root system architecture alterations caused by LZMBW216 may involve polar auxin transport.     

Further insight into the role of <Emphasis Type="Italic">KAN1</Emphasis>, a member of <Emphasis Type="Italic">KANADI</Emphasis> transcription factor family in rice

The rice EMS-derived mutant leaf adaxialized 1 (lad1) was isolated based on its upward rolling leaf phenotype. Besides the adaxially rolled leaf, many other agronomic traits were also compromised in lad1. The rolling trait was characterized by a noticeable alteration of bulliform cells in the adaxial side of the leaves. Map-based cloning showed a single nucleotide substitution in the promoter region of the KAN1 gene in lad1 mutant. Further, over-expressing and CRISPR/cas9-edited knockdown transgenic plants confirmed that KAN1 was responsible for the mutant phenotype of lad1. Yeast two-hybrid and bimolecular fluorescence complementation assay demonstrated that KAN1 can interact with the auxin response factors ARF3, ARF7 and ARF15. Physiologically, the contents of auxin (IAA), abscisic acid (ABA), jasmonic acid (JA) and gibberellin (GA) were all significantly increased in the lad1 mutant. Moreover, the GA3 content dramatically decrease in wild-type, but increased in lad1 under IAA induction. Additionally, the expression levels of several IAA and GA biosynthesis and responsive-related genes and genes involved in leaf polarity determination were altered in lad1. Therefore, we hypothesized that KAN1/ARFs protein complexes act as auxin-dependent regulatory units that play a conserved role in leaf development.     

Jasmonic acid modulates xylem development by controlling polar auxin transport in vascular tissues

Development of xylem cells is affected by environmental stresses such as drought and oxidative stress, and recent findings suggested that jasmonic acid (JA) mediates this process through interaction with other phytohormones such as cytokinin. In this study, we showed that polar auxin transport regulated by PIN3 and PIN7 is involved in the JA-mediated xylem development in vascular tissues. The mutant plants that lack the activity of PIN3 and PIN7 responsible for the auxin transport developed extra xylems in vascular tissues such as the JA-treated wild-type plants. Visualization of auxin response and xylem development in the roots treated with NPA, an inhibitor of polar auxin transport, suggested that disruption of polar auxin transport is involved in the xylem phenotype of pin3 pin7 double mutants. We also found that cytokinin increases expressions of PIN3 and PIN7 responsible for the auxin transport while JA decreases only PIN7. These suggested that PIN7-mediated polar auxin transport system modulates xylem development in response to JA. The finding that JA affects auxin distribution in root vascular tissues further supported this. Collectively, these suggest that JA promotes xylem development by disrupting auxin transport in vascular tissues, and the auxin efflux genes, more especially PIN7 whose expression is suppressed by JA mediates this process.     

MIR166a Affects the Germination of Somatic Embryos in <Emphasis Type="Italic">Larixleptolepis</Emphasis> by Modulating IAA Biosynthesis and Signaling Genes

The somatic embryogenic regeneration system is an ideal model system to study the regulation of early developmental processes and morphogenesis in gymnosperms. We have previously generated five larch (Larix leptolepis) LaMIR166a overexpression cell lines. The germination rates of mature somatic embryos in transgenic and wild-type (WT) lines were calculated and the results showed that overexpression of the miR166a precursor (LaMIR166a) markedly enhanced germination, especially in the a-3, a-4, and a-5 lines. The relative expression of LaMIR166a and miR166a in the LaMIR166a overexpression lines was higher than in the WT control line during the germination process, whereas the expression levels of LaHDZ31–34 increased markedly throughout germination, potentially as a result of feedback regulation of miR166. The effect of miR166a on auxin biosynthesis and signaling genes was also studied. During germination, mRNA levels of Nitrilase (LaNIT), Auxin response factor1 (LaARF1), and LaARF2 were markedly higher in LaMIR166a overexpressing lines. These results indicated that indole-3-acetic acid (IAA) synthesis is required for germination in L. leptolepis. Further exogenous application of IAA at different concentrations showed that 2 mg L^?1 IAA clearly promoted germination, resulting in a 56% germination rate for L. leptolepis somatic embryos. This shows that IAA plays a vital role in controlling the germination ability of someatic embryos in L. leptolepis. Our results suggest that miR166a and LaHDZ31–34 have important roles in auxin biosynthesis and signaling during the germination of somatic embryos in L. leptolepis.