Molecular and morphological evaluation of transgenic Persian walnut plants harboring Fld gene under osmotic stress condition.

BACKGROUND: Soil drought stress is a limiting factor of productivity in walnut (Juglans regia L). Ferredoxin (Fd) level decreases under adverse environmental stress. Functional replacement of decreased Fd by Fld (Flavodoxin) had been shown to have protective effect under abiotic stress condition. This study aimed to evaluate four transgenic lines (L3, L4, L13 and L17) along with non-transgenic line under three osmotic stresses levels (0, 10 and 12% PEG). METHODS AND RESULTS: This experiment carried out based on a completely randomized design with four replications. To confirm that the Fld gene is successfully integrated into the walnut genome, PCR and dot blot analysis were carried out. The transgenic lines of walnut expressing Fld displayed increased tolerance to osmotic stress at 10 and 12% PEG condition. Lines expressing Fld exhibited increasing tolerance to drought stress and maintained health of plants under osmotic conditions. Results of real time PCR showed that expression level of Fld gene in L4 was higher than the others. Among transgenic lines, L4 was more tolerant than other lines under osmotic stress. CONCLUSIONS: These findings indicate that expression of Fld gene can increase tolerance to osmotic stress in Persian walnut and is useful tool for walnut production in arid and semi-arid regions.

Valorisation of walnut shell and pea pod as novel sources for the production of xylooligosaccharides.

According to the high interest in agro-industrial waste reutilisation, underutilised lignocellulosic materials, such as walnut shell (WS) and pea pod (PP), come in focus. The aim of this paper was to evaluate WS and PP as sources for the production of xylooligosaccharides (XOS). Hemicelluloses from WS and PP were recovered by combining varying parameters of delignification and alkaline extraction. At optimal recovery conditions, the fractions were further hydrolysed to XOS using GH11 endo-xylanase, by varying time and enzyme concentration. Xylose was predominant in the monomeric composition of the obtained hemicelluloses, building low-branched (arabino)glucuronoxylan, in WS exclusively, while in PP some xyloglucan as well. Delignification was essential for high recovery of total xylose from the materials, up to at least 70 %. High xylan conversions were obtained for 24 h hydrolysis, resulting in xylobiose and xylotriose when using low enzyme concentration, while in xylose and xylobiose with high enzyme concentration.

Quantitative phenotyping of shell suture strength in walnut (Juglans regia L.) enhances precision for detection of QTL and genome-wide association mapping.

Walnut shell suture strength directly impacts the ability to maintain shell integrity during harvest and processing, susceptibility to insect damage and other contamination, and the proportion of kernel halves recovered during cracking. Suture strength is therefore an important breeding objective. Here, two methods of phenotyping this trait were investigated: 1) traditional, qualitative and rather subjective scoring on an interval scale by human observers, and; 2) quantitative and continuous measurements captured by a texturometer. The aim of this work was to increase the accuracy of suture strength phenotyping and to then apply two mapping approaches, quantitative trait loci (QTL) mapping and genome wide association (GWAS) models, in order to dissect the genetic basis of the walnut suture trait. Using data collected on trees within the UC Davis Walnut Improvement Program (n = 464), the genetic correlation between the texturometer method and qualitatively scored method was high (0.826). Narrow sense heritability calculated using quantitative measurements was 0.82. A major QTL for suture strength was detected on LG05, explaining 34% of the phenotypic variation; additionally, two minor QTLs were identified on LG01 and LG11. All three QTLs were confirmed with GWAS on corresponding chromosomes. The findings reported in this study are relevant for application towards a molecular breeding program in walnut.

Analysis of genetic diversity and structure in a worldwide walnut (Juglans regia L.) germplasm using SSR markers.

Persian or English walnut (Juglans regia L.), the walnut species cultivated for nut production, is one of the oldest food sources known and is grown worldwide in temperate areas. France is the 7th leading producer as of 2016 with 39 kt. Deciphering walnut genetic diversity and structure is important for efficient management and use of genetic resources. In this work, 253 worldwide accessions from the INRA walnut germplasm collection, containing English walnut and several related species, were genotyped using 13 SSR (Single Sequence Repeat) markers selected from the literature to assess diversity and structure. Genetic diversity parameters showed a deficiency of heterozygotes and, for several SSRs, allele-specificities among the accessions tested. Principal Coordinate Analysis (PCoA) showed the 253 accessions clustered in largely in agreement with the existing botanical classification of the genus. Among the 217 J. regia accessions, two main clusters, accessions from Eastern Europe and Asia, and accessions from Western Europe and America, were identified using STRUCTURE software. This was confirmed by Principal Coordinate Analysis and supported by Neighbor-Joining tree construction using DARwin software. Moreover, a substructure was found within the two clusters, mainly according to geographical origin. A core collection containing 50 accessions was selected using the maximum length sub-tree method and prior knowledge about their phenotype. The present study constitutes a preliminary population genetics overview of INRA walnut genetic resources collection using SSR markers. The resulting estimations of genetic diversity and structure are useful for germplasm management and for future walnut breeding programs.

Root order-based traits of Manchurian walnut & larch and their plasticity under interspecific competition.

Manchurian walnut and larch are key timber species of northeast China but information on (fine) root traits of both species is scarce. Plasticity of root traits in mixed plantations has been studied rarely although this could give important insights into mechanisms of root competition. This study examined root traits by branching order in 30-yr-old monocultures and their plasticity in mixed plantations. In monocultures, Manchurian walnut and larch differed in key fine root traits. Larch roots hold more absorptive root orders, larger diameter and lower specific root length/area. Walnut root orders featured greater cortex:stele ratios, N-concentrations and respiration rates. Under interspecific competition, the proportion of walnut root tips increased, the biomass/length of larch root orders 1-3 decreased. Larch possessed a greater morphological and anatomical plasticity of terminal root orders than walnut. Mycorrhizal colonization rates of walnut were reduced. Both species differed fundamentally in their fine root properties. Absorptive fine root orders reacted plastic under interspecific competition while traits of higher root orders remained unchanged. In mixture, larch roots possessed a greater plasticity in traits related to resource uptake (efficiency) than walnut roots whose reaction norm is suggested to be predominantly based on interference competition via juglone exudation.

Cultivated walnut trees showed earlier but not final advantage over its wild relatives in competing for seed dispersers.

Little is known about seeding regeneration of cultivated trees compared to wild relatives in areas where seed dispersers are shared. Here, we investigated the differences in seed fates of cultivated walnut (Juglans regia) and wild Manchurian walnut (Juglans mandshurica) trees under rodent predation and dispersal. J. regia seeds have higher nutritional value (large size, mass and kernel mass) and lower mechanical defensiveness (thin endocarp) than J. mandshurica seeds. We tracked seeds of J. regia and J. mandshurica under both enclosure and field conditions to assess differences in competing for seed dispersers of the two co-occurring tree species of the same genus. We found that rodents preferred to harvest, eat and scatter-hoard seeds of J. regia as compared to those of J. mandshurica. Seeds of J. regia were removed and scatter-hoarded faster than those of J. mandshurica. Caches of J. regia were more likely to be rediscovered by rodents than those of J. mandshurica. These results suggest that J. regia showed earlier dispersal fitness but not the ultimate dispersal fitness over J. mandshurica in seeding regeneration under rodent mediation, implying that J. regia has little effect on seeding regeneration of J. mandshurica in the field. The effects of seed traits on seed dispersal fitness may vary at different dispersal stages under animal mediation.

Root tip morphology, anatomy, chemistry and potential hydraulic conductivity vary with soil depth in three temperate hardwood species.

Root traits in morphology, chemistry and anatomy are important to root physiological functions, but the differences between shallow and deep roots have rarely been studied in woody plants. Here, we selected three temperate hardwood species, Juglans mandshurica Maxim., Fraxinus mandschurica Rupr. and Phellodendron amurense Rupr., in plantations in northeastern China and measured morphological, anatomical and chemical traits of root tips (i.e., the first-order roots) at surface (0-10 cm) and subsurface (20-30 cm) soil layers. The objectives of this study were to identify how those traits changed with soil depth and to reveal potential functional differences. The results showed that root diameters in deep root tips were greater in J. mandshurica and F. mandschurica, but smaller in P. amurense. However, root stele diameter and the ratio of stele to root diameter in the subsurface layer were consistently greater in all three species, which may enhance their abilities to penetrate into soil. All deep roots exhibited lower tissue nitrogen concentration and respiration rate, which were possibly caused by lower nutrient availability in the subsurface soil layer. Significant differences between shallow and deep roots were observed in xylem structure, with deep roots having thicker stele, wider maximum conduit and greater number of conduits per stele. Compared with shallow roots, the theoretical hydraulic conductivities in deep roots were enhanced by 133% (J. mandshurica), 78% (F. mandschurica) and 217% (P. amurense), respectively, indicating higher efficiency of transportation. Our results suggest that trees' root tip anatomical structure and physiological activity vary substantially with soil environment.

Could rapid diameter changes be facilitated by a variable hydraulic conductance?

Adequate radial water transport between elastic bark tissue and xylem is crucial in trees, because it smoothens abrupt changes in xylem water potential, greatly reducing the likelihood of suffering dangerous levels of embolism. The radial hydraulic conductance involved is generally thought to be constant. Evidence collected about variable root and leaf hydraulic conductance led us to speculate that radial hydraulic conductance in stem/branches might also be variable and possibly modulated by putative aquaporins. We therefore correlated diameter changes in walnut (Juglans regia L.) with changes in water potential, altered by perfusion of twig samples with D-mannitol solutions having different osmotic potentials. Temperature and cycloheximide (CHX; a protein synthesis inhibitor) treatments were performed. The temperature response and diameter change inhibition found in CHX-treated twigs underpinned our hypothesis that radial hydraulic conductance is variable and likely mediated by a putative aquaporin abundance and/or activity. Our data demonstrate that radial water transport in stem/branches can take two routes in parallel: an apoplastic and a cell-to-cell route. The contribution of either route depends on the hydraulic demand and is closely linked to a boost of putative aquaporins, causing radial conductance to be variable. This variability should be considered when interpreting and modelling diameter changes.

Flow-induced pruning of branched systems and brittle reconfiguration.

Whereas most plants are flexible structures that undergo large deformations under flow, another process can occur when the plant is broken by heavy fluid-loading. We investigate here the mechanism of such possible breakage, focusing on the flow-induced pruning that can be observed in plants or aquatic vegetation when parts of the structure break under flow. By computation on an actual tree geometry, a 20-yr-old walnut tree (Juglans Regia L.) and comparison with simple models, we analyze the influence of geometrical and physical parameters on the occurrence of branch breakage and on the successive breaking events occurring in a tree-like structure when the flow velocity is increased. We show that both the branching pattern and the slenderness exponent, defining the branch taper, play a major role in the breakage scenario. We identify a criterion for branch breakage to occur before breakage of the trunk. In that case, we show that the successive breakage of peripheral branches allows the plant to sustain higher flow forces. This mechanism is, therefore, similar to elastic reconfiguration, and can be seen as a second strategy to overcome critical events, possibly a widespread solution in plants and benthic organisms.

Contrasting the morphology, anatomy and fungal colonization of new pioneer and fibrous roots.

Not all roots born as first-order branches are the same and this has important consequences for overall function. We hypothesized that, compared with fibrous roots, pioneer roots are built to live longer at the expense of absorptive capacity. We tested this hypothesis by investigating pioneer and fibrous roots in their first 14 d of life in the arbuscular mycorrhizal tree species: Acer negundo, Acer saccharum, Juglans nigra, Liriodendron tulipifera and Populus tremuloides. Root observations were made with root-access boxes that allowed roots to be sampled at known ages in field-grown trees. Compared to fibrous roots, pioneer roots had larger diameter, lower specific root length, greater average length and a lack of mycorrhizal or nonmycorrhizal fungal colonization. Pioneer roots < 14 d old had more layers of hypodermis with a lower percentage of putative passage cells and more protoxylem groups than similar age fibrous roots. Our results suggest that pioneer roots are constructed for defense against biotic and abiotic challenges, exploration of soil distal to the stem, high fibrous root branching and secondary development with high axial hydraulic conductivity at the expense of mycorrhizal colonization and high absorptive capacity for water and nutrients.

Photosynthetic, hydraulic and biomechanical responses of Juglans californica shoots to wildfire.

Leaf gas exchange and stem xylem hydraulic and mechanical properties were studied for unburned adults and resprouting burned Juglans californica (southern California black walnut) trees 1 year after a fire to explore possible trade-offs between mechanical and hydraulic properties of plants. The CO(2) uptake rates and stomatal conductance were 2-3 times greater for resprouting trees than for unburned adults. Both predawn and midday water potentials were more negative for unburned adult trees, indicating that the stems were experiencing greater water stress than the stems of resprouting trees. In addition, the xylem specific conductivity was similar in the two growth forms, even though the stems of resprouting trees were less vulnerable to water-stress-induced embolism than similar diameter, but older, stems of adult trees. The reduced vulnerability may have been due to less cavitation fatigue in stems of resprouts. The modulus of elasticity, modulus of rupture and xylem density were all greater for resprouts, indicating that resprouts have greater mechanical strength than do adult trees. The data suggest that there is no trade-off between stem mechanical strength and shoot hydraulic and photosynthetic efficiency in resprouts, which may have implications for the success of this species in the fire-prone plant communities of southern California.

Occurrence of shallow bark canker of walnut (Juglans regia) in southern provinces of Iran.

From April 2001 to November 2002, samples of walnut branches and trunks with symptoms of shallow bark canker were collected from Fars and Kohgiluyeh-va-Boyerahmad provinces. Symptoms of the disease were small cracks in the bark of the trunk and scaffold branches of mature trees with dark watery exudates which stained the affected trunk or limb. By removal of phelloderm, extensive necrosis of the underlying tissues was observed. In some cases, necrosis extended to cambium and outer xylem. Sixty-one strains of a bacterium were isolated from infected tissues using EMB and YDC media. On the basis of standard biochemical and physiological tests the bacterium was identified as Brenneria nigrifluens. The pathogen was found to be wide-spread in the provinces. Isolates were compared by physiological and biochemical characters, antibiotic sensitivity and protein electrophoretic pattern. Most of the strains were fairly similar in phenotypic features and electrophoretic profiles ofwhole-cell proteins were similar to each other and to reference strain (B. nigrifluens 5D313). Inoculation of 1-2 years-old walnut seedlings in May and June produced blackening symptoms and the bacterium survived for long period in infected tissues. This is the first report of the shallow bark canker of walnut in southern Iran.

Walnut (Juglans).

Walnut species are important nut and timber producers in temperate regions of Europe, Asia, South America, and North America. Trees can be impacted by Phytophthora, crown gall, nematodes, and cherry leaf roll virus; nuts can be severely damaged by codling moth and Xanthomonas blight. The long-generation time of walnuts and an absence of identified natural resistance for most of these problems suggest biotechnological approaches to crop improvement. Described here is a somatic embryo based transformation protocol that has been used to successfully insert horticulturally useful traits into walnut. Selection is based on the combined use of the selectable neomycin phosphotransferase (nptII) gene and the scorable uidA gene. Transformed embryos can be germinated or micropropagated and rooted for plant production. The method described has been used to establish field trials of mature trees.

Flowering phenology and wind-pollination efficacy of heterodichogamous Juglans mandshurica (Juglandaceae).

BACKGROUND AND AIMS: Heterodichogamy differs from normal dichogamy, in that it involves two mating types (protogyny and protandry) that occur at a 1 : 1 ratio in a population. Flowering phases of the two mating types are synchronized and reciprocal, which was considered to ensure between-type outcrossing. This study aims to quantify the flowering pattern and pollination efficacy in Juglans mandshurica, a wind-pollinated heterodichogamous tree. METHODS: The pattern of flowering phenology was monitored within individual trees and pollen traps were used to measure air-borne pollen loads during the spring in 2003 and 2004. Pollen longevity was determined by staining technique. Also a pollen supplementation experiment was performed in 2004 to assess pollen limitation of fruit production. KEY RESULTS: There was no overlap between sexual functions within individual trees. Flowering periods of the two mating types were reciprocal and synchronous in both 2003 and 2004. Air-borne pollen loads were large, and protogynous and protandrous individuals each produced a high pollination peak, consistent with the two blooming periods. Maximum pollen longevity was about 4 h for protandrous individuals, and 3 h for protogynous individuals. Pollen supplementation did not increase fruit production in either protogynous or protandrous individuals. CONCLUSIONS: Heterodichogamous flowering in Juglans mandshurica effectively avoids selfing, promotes between-type outcrossing, and leads to efficient pollination in a natural population.

Nitrogen availability, local light regime and leaf rank effects on the amount and sources of N allocated within the foliage of young walnut (Juglans nigra x regia) trees.

Early season leaf growth depends largely on nitrogen (N) provided by remobilization from storage, and many studies have tested the effect of N availability to roots on the amount of N provided for new leaf development by remobilization. Although it is well known that the light regime experienced by a leaf influences the amount of N per unit leaf area (LA), the effect of the local light regime on the amount of N derived either directly from root uptake or from remobilization for early season leaf growth has never been tested at an intra- canopy scale. The objective of this study was to quantify the relative importance of (1) N availability to roots, (2) local light regime experienced by the foliage (at the shoot scale) and (3) leaf rank along the shoot, on the total amount of N allocated to leaves and on the proportions of N provided by remobilization and root uptake. To quantify the importance of N uptake and remobilization as sources of leaf N, potted hybrid walnut trees (Juglans nigra L. x regia L.) were grown outdoors in sand and fed with a labeled ((15)N) nutrient solution. By removing the apical bud, the trees were manipulated to produce only two shoots. The experimental design had two factors: (1) high (HN; 8 mol N m(-3)) and low (LN; 2 mol N m(-3)) N availability; and (2) high (HL; 90% of incident photosynthetically active photon flux (PPF)) and low (LL; 10% of incident PPF) light. Total leaf N per tree was unaffected by either N availability or irradiance. The HN treatment increased the amount of leaf N derived from root uptake at the whole-tree scale (typically around 8 and 2% in the HN and LN treatments, respectively). Nitrogen allocation within foliage of individual trees was controlled by the local light regime, which strongly affected individual leaf characteristics as leaf mass per unit LA and area- based amount of leaf (N(a)). Decreasing the light availability to a branch decreased the amount of N allocated to it, benefiting the less shaded branches. In contrast, shading of the lower branch did not affect the fraction of total leaf N remobilized for either the lower, shaded branch or the upper, unshaded branch. The relevance of these findings for tree growth modeling is discussed.

Periods of organogenesis in mono- and bicyclic annual shoots of Juglans regia L. (Juglandaceae).

The organogenetic cycle of shoots on main branches of 4-year-old Juglans regia trees was studied. Mono- and bicyclic floriferous and vegetative annual shoots were analysed. Five parent annual shoot types were sampled between October 1992 and August 1993. Organogenesis of summer growth units was monitored between 16 Jun. and 3 Aug. 1993. Variations over time in the number of nodes, cataphylls and embryonic green leaves of terminal buds were studied. The number of nodes of parent shoot buds was compared with the number of nodes of shoots derived from parent shoot buds. The spring growth units of mono- and bicyclic shoots consist exclusively of preformed leaves which were differentiated, respectively, during the spring flush of growth (mid-April until mid-May) or the summer flush of growth (mid-June until early August) in the previous growing season. Thus, winter buds may consist of flower and leaf primordia differentiated in two different periods during annual shoot extension. The summer growth units of bicyclic shoots consist of preformed leaves that were differentiated in spring buds during the spring flush of growth in the current growing season. Bud morphology is compared between spring and summer shoots.

Genotypic differences in branching pattern and fruiting habit in common walnut (Juglans regia L.).

Architectural analysis of 840 Slovenian walnut (Juglans regia L.) genotypes was performed to determine the most typical and frequent morphological types and to evaluate their vegetative and generative potential. Four branching and fruiting patterns (I-IV) were detected. A 3-year-old fruiting branch, consisting of a 3-year-old shoot plus corresponding 2-year-old and 1-year-old shoots, was used as a structural unit for quantitative analysis. In the intermediate fruit-bearing types with mesotonic and acrotonic branching pattern (types II and III), the total lengths of 3-, 2- and 1-year-old shoots were 385 and 380 cm, respectively, compared with 275 and 253 cm in the terminal and lateral-fruiting types (types I and IV). In type I, 1-year-old shoots had significantly fewer nodes than in other types. In addition, they had a thinner basal diameter than types III and IV, and their angles were the most erect (39 degrees ). Only 0.4 out of 3.6 1-year-old shoots were flowering with one mixed bud with 1.9 female flowers. In type IV, 2-year-old shoots had significantly more nodes and a larger basal diameter than other types. One-year-old shoots in type IV are thicker than those in other types. Ratios between the number of flowering and the total number of 1-year-old shoots were 0.7 in type IV, 0.6 in type III, 0.5 in type II and 0.1 in type I. On 1-year-old shoots in type IV, 1.7 mixed buds with a mean of three female inflorescences per bud were counted. Consequently, the generative potential is highest in type IV and lowest in type I. In types II and III, growth and the ability to bear fruits are more balanced.