<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.

     

Effect of Dwarfing Interstocks on Xylem Sap Composition in Apple Trees: Effect on Nitrogen, Potassium, Phosphorus, Calcium and Magnesium Content

This paper concerns nutrient concentrations in xylem sap collectedfrom above and below M.8, M.9, M.26 and M.7 dwarfing interstocks.The sap flowing from above an interstock had lower concentrationsof nutrients than that from the rootstock levels and these differencesincreased with the dwarfing effect of the interstock. There were indications that nutrient concentrations were enhancedbelow interstocks but were reduced in the sap flowing from theinterstocks into the scions. Generally these reductions weregreater the more dwarfing the effect of the interstock. Analyses of sap from above, mid-way along and below interstocksindicated that the changes in content were produced in, or closeto, the graft union between scion and interstock.     

Indole-3-acetic acid level in wood,bark and cambial sap of apple rootstocks differing in growth vigour

Free and conjugated IAA levels were determined in wood, bark and cambial sap of M.9, M.26 and MM.106 apple rootstock genotypes differing in growth vigour. The measurements were done on May 15^th, June 15^th and July 15^th. The level of free IAA in bark and wood of the tested trees varied from 27.0 to 52.7 ng·g⁻¹ f.w. while the conjugated hormone content averaged 3–5 times higher. In the bark and wood samples, the differences in auxin content between rootstock genotypes and the time of stem harvesting were insignificant. The level of free IAA in cambial sap was on average 10 to 20 times higher than in both bark and wood tissues, while the conjugated hormone level varied from none (below detection limit) to 37 ng·g⁻¹ f.w. Content of free IAA level in cambial sap from dwarf M.9 rootstock was significantly lower than that in either of the more vigorous genotypes. In both vigorous rootstocks IAA level in cambial sap remained at a similar level at all sampling dates but M.9 cambial sap showed a trend towards decreasing auxin content later in the growing season.     

Application of architectural analysis and AMAPmod methodology to study dwarfing phenomenon: the branch structure of 'Royal Gala' apple grafted on dwarfing and non-dwarfing rootstock/interstock combinations

Architectural analysis was applied to study branch development of 'Royal Gala' apple trees grafted with dwarfing and non-dwarfing rootstock/interstock combinations, which had been chosen to produce trees with a wide range of vigour. Using AMAPmod methodology, the structure of 3-year-old branches was described at four levels of representation: branch; annual shoot; growth unit; and node. Three types of growth units were distinguished: extension growth unit (vegetative unit with internode extension); vegetative spur with minimal internode extension; and fruiting spur or bourse. The aim of the analysis was to describe exactly how the rootstock/interstock combinations affected the structure building process. The number of extension growth units, vegetative spurs and fruiting spurs per annual shoot changed over the years, but this was not affected by rootstock/interstock combination. Compared with MM.106 rootstock, M.9 rootstock reduced the number of nodes per extension growth unit. In most cases, rootstock/interstock combination had no effect on the linear relationship between extension growth unit length and node number (R(2) = 0.88). Average internode length depended on unit node number, with internodes being shorter for units with fewer nodes. Thus the difference in apple branch size induced by the rootstock/interstock combinations was mainly due to a reduction in the length and number of neoformed nodes produced on extension growth units. As percentage budbreak of axillary buds on extension growth units was not affected by rootstock/interstock combination, differences in numbers of axillary annual shoots per branch were entirely due to differences in the total numbers of nodes extended during the previous year.     

Key flowering genes including FT-like genes are upregulated in the vasculature of apple dwarfing rootstocks

The Malling 9 (M.9) dwarfing rootstock is widely used in apple breeding and commercial cultivation to shorten the juvenile period, reduce vegetative growth and increase flowering of the scion. A segment of M.9 stem (interstock) or M.9 bark grafted into a compound tree can cause significant dwarfing of the scion, suggesting that the dwarfing signal may be vascular derived. To better understand how the M.9 rootstock alters the growth and development of the scion, we compared gene expression in vascular-enriched tissue from dwarfing and vigorous rootstocks. RNA sequencing indicated that key flowering genes were upregulated in M.9 relative to a vigorous rootstock, Malling 793 (M.793). An in-depth analysis of the apple FT/TFL1 gene family identified four new members: MdMFTa, MdMFTb, MdBFTa and MdBFTb. Quantitative RT-PCR analysis confirmed the higher expression of MdFT1/2, MdBFTa/b, MdCO, MdGI, and MdSOC1 in two different dwarfing rootstocks (M.9 and Malling 27 (M.27)) relative to M.793. Both MdFT1/2, and MdBFTa/b were expressed at higher levels in multiple dwarfing rootstock accessions relative to more vigorous genotypes. In perennial species, FT promotes flowering, and has additional roles in accelerating the transition from juvenility to maturity, and regulating cycles of seasonal growth and termination. Apple dwarfing rootstocks reduce the juvenile phase and promote both flowering and early shoot termination. Our work supports a role for MdFT in promoting flowering and earlier shoot termination. We suggest that upregulation of a suite of flowering genes including MdFT, and possibly MdBFT, in the vasculature is part of the underlying mechanism of apple dwarfing rootstocks. Genes involved with response to biotic and abiotic stress and disease were also upregulated in the M.9 rootstock, suggesting that stress, possibly mediated by JA and ABA signalling, also plays a role in the M.9-induced phenotype.     

The influence of dwarfing interstocks on the distribution and metabolism of xylem-applied [h]gibberellin a(4) in apple

The influence of an interstock of the dwarfing cultivar M9 and the nondwarfing cultivar MM115 on the distribution and metabolism of labeled gibberellic acid A₄ ([³H]GA₄) of high specific radioactivity (5.18 × 10¹⁰ becquerel per millimole) applied to the xylem of the rootstock in grafted apple (Malus × domestica Borkh.) trees was compared. Free [³H] GA-like metabolites of [³H]GA₄, including putative GA₁, GA₂, GA₃, and GA₃₄, as well as various ³H-putative GA glucosyl conjugates were detected in stem segments from both cultivars. M9 interstocks reduced the total uptake of [³H]GA₄ and decreased the proportion of ³H metabolites transported to the shoots and leaves of scions. The M9 interstock tissue and adjacent rootstock and scion tissue retained a much greater amount and a higher proportion of the label than did comparable tissue of the nondwarfing MM115 interstock. In addition, the amount and proportion of free [³H]GAs was higher, and the proportion of putative [³H]GA glucosyl conjugates lower, in M9 interstocks compared to MM115. These effects of the dwarfing interstock on GA distribution and metabolism indicate a significant role for GAs in any satisfactory explanation of the dwarfing mechanism in apple.     

GC-MS-SIM analysis of abscisic acid and indole-3-acetic acid in shoot bark of apple rootstocks

Concentrations of abscisic acid and indole-3-acetic acid were measured by GC-MS-SIM in the shoot bark of clonal apple rootstocks (M.27, M.9, MM.106 and MM.111) when the rootstocks were growing actively in the UK. These rootstocks are known to exhibit a wide range of control of tree size when grafted to a common scion. Shoot bark of the dwarfing rootstocks (M.27 and M.9) contained higher concentrations than the more vigorous rootstocks (MM.106 and MM.111) of ABA. Concentrations of ABA increased from May to July, followed by a decline in August. Only the month of sampling showed any significant influence on the concentration of IAA in shoot bark; however, there was a general increase, although not significant statistically, in IAA concentration with the increasing invigoration-capacity of the rootstock. At each sampling date the dwarfing rootstocks showed greater ratios of ABA:IAA than the invigorating rootstocks and generally the ratio for each rootstock increased from May to July, except for M.27 which showed the smallest ratio in June and the largest ratio in August. The results are discussed in relation to the generally accepted control exerted by the rootstocks on tree size and the possible influence of ABA on polar auxin transport.     

Natural variation in cytokinin maintenance improves salt tolerance in apple rootstocks

Plants experiencing salt‐induced stress often reduce cytokinin levels during the early phases of stress‐response. Interestingly, we found that the cytokinin content in the apple rootstock “robusta” was maintained at a high level under salt stress. Through screening genes involved in cytokinin biosynthesis and catabolism, we found that the high expression levels of IPT5b in robusta roots were involved in maintaining the high cytokinin content. We identified a 42 bp deletion in the promoter region of IPT5b, which elevated IPT5b expression levels, and this deletion was linked to salt tolerance in robusta×M.9 segregating population. The 42 bp deletion resulted in the deletion of a Proline Response Element (ProRE), and our results suggest that ProRE negatively regulates IPT5b expression in response to proline. Under salt stress, the robusta cultivar maintains high cytokinin levels as IPT5b expression cannot be inhibited by proline due to the deletion of ProRE, leading to improve salt tolerance.     

Adventitious root formation 'in vitro' in apple rootstocks (Malus pumila) II. Uptake and distribution of indol-3yl-acetic acid during the auxin-sensitive phase in M.9 and M.26

During the auxin-sensitive phase of root initiation, rates of 3-indolyl- [2-¹⁴C] acetic acid (IAA) uptake into the 1 cm bases of shoots of the apple rootstock M.9 ( Malus pumila Mill.) 'in vitro' were not significantly affected by the presence of 10⁻³ M phloroglucinol (PG) using either liquid or agar-solidified media. The use of a liquid medium did however reduce rates of uptake over a 10-day period of auxin application. The distribution of labelled IAA between the 1-cm base and the shoot remainder was not affected by PG.
Exposure of shoots of the difficult-to-root M.9 and the easy-to-root M.26, to 2.8 × 10⁻⁵ M IAA containing [2-¹⁴C] IAA revealed no positive correlation between the amount of label taken up by the 1-cm base and rooting performance. M.9 bases absorbed almost twice as much label as M.26 after 9 days but had produced only one-third as many roots. Measurements of label distribution between the 1-cm base and the shoot remainder showed that less than 10% of the label moved to the shoot remainder over a 6-day period of auxin application. Dose-response curves of IAA and rooting over the range 1 × 10⁻⁵ M and 3 × 10⁻³ M showed that root number in M.9 was at an optimum at 1 × 10⁻³ M IAA after 6 days whilst M.26 required only 1 × 10⁻⁴ M for a similar response. These data support the hypothesis that differences in rooting of the two rootstocks reflect differences in the endogenous metabolism of exogenous IAA and not differences in its rates of uptake or distribution in the shoots.     

Relationship of indole-3-butyric acid and adventitious rooting in M.26 apple (Malus Pumila mill.) shoots cultured in vitro

The role of indole-3-butyric acid (IBA) in adventitious root formation was studied by analyzing the uptake and subsequent metabolism of IBA in shoots of M.26 apple (Malus pumila Mill.) rootstock grown in vitro. Roots were induced by exposing shoots to 4 M IBA and [³H]IBA for 5 days in the dark and then transferring them to plant growth regulator (PGR)-free medium in the light until roots formed. Approximately 50% of the total radioactivity applied was taken up from the agar medium by the shoots during the 5-day incubation period in IBA. Indole-3-butyric acid metabolism was studied by extraction and high-performance liquid chromatographic (HPLC) separation of [³H]IBA and metabolites from the basal sections of treated shoots. The major [³H]IBA metabolite co-eluted with authentic [¹⁴C]indole-3-acetic acid (IAA) suggesting that IBA was converted to IAA in the shoots. The proportion of newly synthesized IAA present as conjugates was higher at the end of the 5-day IBA treatment period than after 13 days in PGR-free medium. There appeared to be no conjugation of IBA at any time.     

Almond brown line and decline: a new disease probably caused by a mycoplasma-like organism

Young almond (Prunus dulcis, cvs Carmel, Peerless and Price) orchards established on the plum rootstock Marianna 2624 (P. cerasifera×P. munsoniana) contained trees that exhibited poor terminal shoot growth and wilted, chlorotic leaves. The scion/rootstock graft union showed an external splitting of the bark and an internal line of necrotic bark tissues that extended into the woody cylinder of the union, which was deeply pitted. Affected trees declined. The disease was named almond brown line and decline (ABLD). Incidence of ABLD ranged up to 55% per cultivar in some orchards. Numerous attempts to graft-transmit orchard collections of ABLD to healthy almond/Marianna 2624 indicators failed. Also, ABLD does not appear to be soil-borne. However, ABLD was serendipitiously determined to be bud-perpetuated when infected scion buds from an apparently healthy appearing Peerless almond/peach tree located in a foundation orchard were grafted onto healthy rooted cuttings of Marianna 2624 to produce yearling trees. Also, graft-inoculations on the almond scion portion of healthy trees, but not the plum rootstock portion, with the peach yellow leafroll mycoplasma-like organism (PYLR-MLO) caused symptoms resembling ABLD. Laboratory and glasshouse assays of several symptomatic trees did not detect tomato ringspot virus and two ilarviruses. These results suggest that an MLO, possibly PYLR-MLO, may be the causal agent of ABLD and that Marianna 2624 is probably resistant to the PYLR-MLO.     

Agrobacterium-mediated transformation of the apple rootstock Malus micromalus Makino with the RolC gene

Summary Malus micromalus Makino was genetically engineered with the rolC gene via Agrobacterium-mediated transformation. The antibiotics carbenicillin, hygromycin, and kanamycin (Km) inhibited shoot regeneration from in vitro leaf explants. The leaf segments were infected with Agrobacterium tumefaciens strain LBA4404 harboring one of the three different plasmids. Shoots were regenerated only from leaf segments infected with LBA4404 harboring the plasmid with the introncontaining neomycin phosphotransferase II gene as the selectable marker. Preculture of leaf segments on regeneration medium for 2 d before Agrobacterium treatment reduced formation of Km-resistant calluses. Transformation was confirmed by a histochemical β-glucuronidase (GUS) assay, amplification of the rolC gene by polymerase chain reaction (PCR), and by Southern blot analysis. The rolC-transformed M. micromalus shoots showed an increased rooting ability without auxin treatment, and reduced height, internode length and leaf areas. This research shows the potential application of using the rolC gene for developing dwarf apple rootstocks.     

The Anatomy of the Developing Bud Union and its Relationship to Dwarfing in Apple

Light microscopy has been used to study the effect of dwarfingand semi-dwarfing apple rootstocks on the early developmentof bud-unions with 'Gala', and the anatomy of 2-year-old bud-unionsbetween 'Bramley' and the same rootstocks. The bridging of thecut edges of the cambia of bud and rootstock was achieved bydifferentiation of callus formed at an early stage in budding.New cambial cells were aligned at right angles to the pre-existingcambia, with their long axes horizontal. Subsequently-formedxylem adopted this arrangement, so that fibres and vessels werearranged obliquely to the axis of the stem. At the interfacebetween the bud and dwarfing rootstocks vessels with smallerthan normal diameter were formed, indicating the presence ofelevated levels of auxin in this region. In addition, littlexylem was produced in the adjacent rootstock tissue. In thecase of semi-dwarfing rootstocks, the rootstock produced normalxylem after a brief interruption. We suggest that failure ofauxin to cross the bud-union interface in the case of the dwarfingrootstocks leads to reduced rootstock xylem formation, and hencea poor supply of water and minerals to the scion, and this underliesthe dwarfing effect.Copyright 1994, 1999 Academic Press Apple, budding, dwarfing, anatomy, graft union     

黄土丘陵区不同土地利用方式下土壤水分变化特征

选择黄土丘陵区砖窑沟流域不同土地利用方式为研究对象,在2016年6月至11月对0—300 cm土层土壤含水量进行监测,分析刺槐林、草地、柠条灌木林、小叶杨林、海红林和撂荒地6种土地利用方式下土壤含水量的垂直剖面分布特征、土壤贮水量的季节变异特征。结果表明:(1)土壤含水量随深度的变化自上而下均呈"S"状分布,随着土层深度的增加,土壤含水量呈先增加后减小的趋势,具有明显的垂直变异特征。(2)不同土地利用方式具有不同的土壤湿度剖面,土壤水分活跃层、次活跃层、相对稳定层的深度范围不同。(3)6种土地利用方式下各土层的土壤贮水量均具有明显的季节变化特征,海红林的土壤贮水量最大,为258.21 mm,然后依次为小叶杨林、撂荒地、草地和刺槐林,柠条灌木林样地最小;监测期内土壤贮水量随时间呈增长趋势,在11月达到最大值。土壤含水量的变异系数均随着土层深度的增加逐渐递减,在100 cm以下土壤深层季节变异趋于稳定。研究认为,乔灌林消耗更多深层的土壤水分,柠条灌木林易引起土壤干燥化,海红林的土壤水分条件较好,撂荒地和草地土壤水分条件相对稳定。     

Relationship between Indole-3-Acetic Acid Levels in Apple (Malus pumila Mill) Rootstocks Cultured in Vitro and Adventitious Root Formation in the Presence of Indole-3-Butyric Acid

In vitro rooting response and indole-3-acetic acid (IAA) levels were examined in two genetically related dwarfing apple (Malus pumila Mill) rootstocks. M.26 and M.9 were cultured in vitro using Linsmaier-Skoog medium supplemented with benzyladenine (BA), indole-3-butyric acid (IBA), and 1,3,5-trihydroxybenzoic acid (PG). Rooting response was tested in Lepoivre medium supplemented with IBA and PG. IBA concentrations of 12.0 and 4.0 micromolar induced the maximum rooting percentages for M.9 and M.26, respectively. At these concentrations rooting response was 100% for M.26 and 80% for M.9. Free and conjugated IAA levels were determined in M.26 and M.9 shoots prior to root inducing treatment by high performance liquid chromatography with fluorescence detection and validated by gas chromatography-mass spectrometry using ¹³[C₆]IAA as internal standard. Basal sections of M.26 shoots contained 2.8 times more free IAA than similar tissue in M.9 (477.1 ± 6.5 versus 166.6 ± 6.7 nanograms per gram fresh weight), while free IAA levels in apical sections of M.26 and M.9 shoots were comparable (298.0 ± 4.4 versus 263.7 ± 9.3 nanograms per gram fresh weight). Conjugated IAA levels were significantly higher in M.9 than in M.26 indicating that a greater proportion of total IAA was present as a conjugate in M.9. These data suggest that differences between M.26 and M.9 rooting responses may be related to differences in free IAA levels in the shoot base.     

Shifts in xylem vessel diameter and embolisms in grafted apple trees of differing rootstock growth potential in response to drought

We investigated responses of plant growth rate, hydraulic resistance, and xylem cavitation in scion-rootstock-combinations of Malus domestica L. cv. Honeycrisp scions grafted onto a high-shoot vigor (HSV) rootstock, (semi-dwarfing Malling111), or onto a low-shoot vigor (LSV) rootstock, (dwarfing Budagovsky 9), in response to substrate moisture limitation. Adjustments in xylem vessel diameter and frequency were related to hydraulic resistance measurements for high- versus low- vigor apple trees. We observed a greater tolerance to water deficit in the high-shoot compared to the low-shoot vigor plants under water deficit as evidenced by increased growth in several plant organs, and greater scion anatomical response to limited water availability with ca. 25% increased vessel frequency and ca. 28% narrower current season xylem ring width. Whereas water limitation resulted in greater graft union hydraulic resistance of high-shoot vigor trees, the opposite was true when water was not limiting. The graft union of the low-shoot vigor rootstock exhibited higher hydraulic resistance under well-watered conditions. Scions of high-shoot vigor rootstocks had fewer embolisms at low plant water status compared to scions of low-shoot vigor rootstocks, presumably as a result of large differences in xylem vessel diameter. Our results demonstrated that anatomical differences were related to shifts in hydraulic conductivity and cavitation events, a direct result of grafting, under limited soil water.     

红桔基砧/锦橙中间砧/纽荷尔脐橙晚实原因探讨

通过连续两年田间试验,比较纽荷尔脐橙高接在两个不同基砧/中间砧组合上的产量表现,并探讨其原因。结果表明,红桔/锦橙组合投产显著地晚于枳/国庆一号温州蜜柑组合(对照);其晚实原因在于:红桔/锦橙组合比对照枝条加长生长旺、加粗生长弱、枝条较直立,树冠郁闭;红桔/锦橙组合树体光合能力受抑制;红桔/锦橙组合叶片可溶性糖含量低于对照,而淀粉含量高于对照;红桔/锦橙组合叶片N、P含量比对照低,Ca含量、C/N水平比对照高。     

A Study of the graft union in in vitro micrografted apple

Light microscopy was used to study graft union formation in in vitro micrografts of tissue cultured apple (Malus domestica. Borkh). Micrografts were constructed using horizontal incisions to form the grafting surface, and placing the cut ends of rootstock and scion into sterile silicon tubing to permit graft formation to occur.The outer morphological and histological development was similar for different stock-scion micrograft combinations but graft union formation was slower in heterografts than in autografts. Initial leaf expansion at the scion shoot apex occurred in all micrografted plantlets within 1–4 days and was not indicative of graft success. Progressive scion growth and development could be used as an indication of graft success by ten to fourteen days after grafting and probably was related to establisment of cell to cell contact at the graft interface. Microscopy showed initiation of callus proliferation in the vascular cumbium and the pith ten days after grafting. Differentiation was observed subsequently and this was reflected in scion development. Longitudinally orientated cambial cells began to differentiate between twenty and forty days after grafting, and formed a bridge between the vascular cylinders of scion and rootstock. The scions at this stage had as many as eight newly expanded leaves and micrografts were strong enough to permit silicon sleeve removal without damage. Continuity of new vascular elements in rootstock and scion was established around forty days. New vascular elements curved slightly in towards the pith to form a c shaped bridge across the graft union. Vascular development continued until it reached completion after six months.