<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.

     

Genome-wide identification and expression profiling analysis of brassinolide signal transduction genes regulating apple tree architecture

Brassinolide (BR) is crucial for regulating plant architecture. Apple dwarfing rootstocks are used to control apple tree size. However, information regarding the effects of BR on apple trees is limited. In addition, the molecular mechanism underlying the dwarfing of apple rootstocks is poorly understood. To elucidate the role of BR signal transduction genes in controlling apple tree architecture, five BR receptor kinase 1 (BRI1), nine BR-signaling kinase 1 (BSK1), two BRI1 KINASE INHIBITOR 1 (BKI1), and seven BR-insensitive 2 (BIN2) genes were analyzed. Bioinformatic analyses revealed that gene duplication events likely contributed to the expansion and evolution of the identified genes. Nine homologs between apple and Arabidopsis thaliana were also identified, and their expression patterns in different tissues were characterized. Exogenous BR treatments increased the primary shoot length and altered the expression of BR signal transduction genes (MdBRI1-5, MdBSK3-8, MdBKI1–2, MdBIN1–4, and MdBIN6/7). The scion of Fuji/Malling 9 (M.9) trees exhibited inhibited growth compared with that of Fuji/Fuji trees. The Fuji/M.9 trees had lower levels of the positive regulators of BR signaling (MdBRI1-5,MdBSK1, MdBSK4/7, and MdBSK6) and higher levels of the negative regulators (MdBIN5-7) compared with the Fuji/Fuji trees. Thus, the above-mentioned genes may help to regulate apple tree size in response to BR. In addition, MdBRI1–5, MdBSK1, MdBSK4/7, MdBSK6, and MdBIN5–7 have important roles in different grafting combinations. Our results may provide the basis for future analyses of BR signal transduction genes regarding their potential involvement in the regulation of plant architecture.     

The role of pre-symbiotic auxin signaling in ectendomycorrhiza formation between the desert truffle <Emphasis Type="Italic">Terfezia boudieri</Emphasis> and <Emphasis Type="Italic">Helianthemum sessiliflorum</Emphasis>

The ectendomycorrhizal fungus Terfezia boudieri is known to secrete auxin. While some of the effects of fungal auxin on the plant root system have been described, a comprehensive understanding is still lacking. A dual culture system to study pre mycorrhizal signal exchange revealed previously unrecognized root–fungus interaction mediated by the fungal auxin. The secreted fungal auxin induced negative taproot gravitropism, attenuated taproot growth rate, and inhibited initial host development. Auxin also induced expression of Arabidopsis carriers AUX1 and PIN1, both of which are involved in the gravitropic response. Exogenous application of auxin led to a root phenotype, which fully mimicked that induced by ectomycorrhizal fungi. Co-cultivation of Arabidopsis auxin receptor mutants tir1-1, tir1-1 afb2-3, tir1-1 afb1-3 afb2-3, and tir1-1 afb2-3 afb3-4 with Terfezia confirmed that auxin induces the observed root phenotype. The finding that auxin both induces taproot deviation from the gravity axis and coordinates growth rate is new. We propose a model in which the fungal auxin induces horizontal root development, as well as the coordination of growth rates between partners, along with the known auxin effect on lateral root induction that increases the availability of accessible sites for colonization at the soil plane of fungal spore abundance. Thus, the newly observed responses described here of the root to Terfezia contribute to a successful encounter between symbionts.     

Endophytic microbes <Emphasis Type="Italic">Bacillus</Emphasis> sp. LZR216-regulated root development is dependent on polar auxin transport in <Emphasis Type="Italic">Arabidopsis</Emphasis> seedlings

Key message

Endophytic microbes Bacillus sp. LZR216 isolated from Arabidopsis root promoted Arabidopsis seedlings growth. It may be achieved by promoting the lateral root growth and inhibiting the primary root elongation.

Abstract

Plant roots are colonized by an immense number of microbes, including epiphytic and endophytic microbes. It was found that they have the ability to promote plant growth and protect roots from biotic and abiotic stresses. But little is known about the mechanism of the endophytic microbes-regulated root development. We isolated and identified a Bacillus sp., named as LZR216, of endophytic bacteria from Arabidopsis root. By employing a sterile experimental system, we found that LZR216 promoted the Arabidopsis seedlings growth, which may be achieved by promoting the lateral root growth and inhibiting the primary root elongation. By testing the cell type-specific developmental markers, we demonstrated that Bacillus sp. LZR216 increases the DR5::GUS and DR5::GFP expression but decreases the CYCB1;1::GUS expression in Arabidopsis root tips. Further studies indicated that LZR216 is able to inhibit the meristematic length and decrease the cell division capability but has little effect on the quiescent center function of the root meristem. Subsequently, it was also shown that LZR216 has no significant effects on the primary root length of the pin2 and aux1-7 mutants. Furthermore, LZR216 down-regulates the levels of PIN1-GFP, PIN2-GFP, PIN3-GFP, and AUX1-YFP. In addition, the wild-type Arabidopsis seedlings in the present of 1 or 5 µM NPA (an auxin transport inhibitor) were insensitive to LZR216-inhibited primary root elongation. Collectively, LZR216 regulates the development of root system architecture depending on polar auxin transport. This study shows a new insight on the ability of beneficial endophytic bacteria in regulating postembryonic root development.

     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Temporal root responses in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> L. to chromate reveal structural and regulatory mechanisms involving the SOLITARY ROOT/IAA14 repressor for maintenance of identity meristem genes

The Arabidopsis root system is modified in response to stress generated by high concentrations of nonessential ions such as chromate [Cr(VI)]. In this work, the distribution of auxin and its transporters PIN1 and PIN7, as well as the expression of genes that maintain the identity of the root meristem, were analyzed in Arabidopsis thaliana wild-type (WT) seedlings and in a mutant affected in the SOLITARY ROOT (SLR1/IAA14) locus, which is required for root response to Cr(VI). We show that primary root inhibition, auxin transporter levels, and expression of meristem identity genes were maintained in the slr-1 mutants but not in WT plants in response to Cr(VI) in a time- and concentration-dependent manner. Notably, the outermost single cell layer of the lateral root cap, which normally dies and tends to peel off, remains viable and increases in size following exposure of WT plants, but not slr-1 mutants, to Cr(VI). Our results suggest that (1) the primary root tip senses Cr(VI), (2) the external lateral root cap may play a protective role during Cr(VI) exposure, and (3) Cr(VI) impacts cell division in root meristems via auxin redistribution and SLR1/IAA14 function, influencing the expression of root meristem genes.     

Morphological and Molecular Analysis of Isolated Cultures of Tobacco Adventitious Roots Obtained by the Methods of Biolistic Bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Transformation

Plant infection with Agrobacterium rhizogenes leads to the development of a hairy root disease notable for the rapid agravitropic growth of roots on hormone-free nutrient media. In order to look into the interaction of A. rhizogenes with plants and assess opportunities of practical application of hairy root culture, new approaches to their production are elaborated. A method of bacterium-free and plasmid-free production of genetically modified roots (hairy roots) by means of biolistic transformation of leaf explants with a DNA fragment (size of 5461 bp) consisting of genes rolA, rolB, rolC, and rolD are proposed. In most cases, such transformation resulted in the emergence of only adventitious roots with transient expression of rol-genes, and the growth of such roots on hormone-free media ceased in 2–3 months in contrast to genuine hairy roots capable of unrestricted growth. Molecular analysis of different systems of target genes’ expression showed an important role of transgene rolC and host gene of cyclin-dependent protein kinase CDKB1-1 in the maintenance of rapid growth of hairy roots in vitro (in isolated cultures).     

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.     

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.     

Abscisic acid,sucrose, and auxin coordinately regulate berry ripening process of the Fujiminori grape

The aim of this study was to examine the effect of abscisic acid (ABA), sucrose, and auxin on grape fruit development and to assess the mechanism of these three factors on the grape fruit ripening process. Different concentrations of ABA, sucrose, and auxin were used to treat the grape fruit, and the ripening-related indices, such as physiological and molecular level parameters, were analyzed. The activity of BG protein activity was analyzed during the fruit development. Sucrose, ABA, and auxin influenced the grape fruit sugar accumulation in different ways, as well as the volatile compounds, anthocyanin content, and fruit firmness. ABA and sucrose induced, but auxin blocked, the ripening-related gene expression levels, such as softening genes PE, PG, PL, and CELL, anthocyanin genes DFR, CHI, F3H, GST, CHS, and UFGT, and aroma genes Ecar, QR, and EGS. ABA, sucrose, and glucose induced the fruit dry weight accumulation, and auxin mainly enhanced fruit dry weight through seed weight accumulation. In the early development of grape, starch was the main energy storage; in the later, it was glucose and fructose. Sucrose metabolism pathway-related gene expression levels were significant for glucose and fructose accumulation. BG protein activity was important in the regulation of grape ABA content levels. ABA plays a core role in the grape fruit development; sucrose functions in fruit development through two pathways: one was ABA dependent, the other ABA independent. Auxin blocked ABA accumulation to regulate the fruit development process.     

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.