Improving rice tolerance to potassium deficiency by enhancing OsHAK16p:WOX11‐controlled root development

Potassium (K) deficiency in plants confines root growth and decreases root‐to‐shoot ratio, thus limiting root K acquisition in culture medium. A WUSCHEL‐related homeobox (WOX) gene, WOX11, has been reported as an integrator of auxin and cytokinin signalling that regulates root cell proliferation. Here, we report that ectopic expression of WOX11 gene driven by the promoter of OsHAK16 encoding a low‐K‐enhanced K transporter led to an extensive root system and adventitious roots and more effective tiller numbers in rice. The WOX11‐regulated root and shoot phenotypes in the OsHAK16p:WOX11 transgenic lines were supported by K‐deficiency‐enhanced expression of several RR genes encoding type‐A cytokinin‐responsive regulators, PIN genes encoding auxin transporters and Aux/IAA genes. In comparison with WT, the transgenic lines showed increases in root biomass, root activity and K concentrations in the whole plants, and higher soluble sugar concentrations in roots particularly under low K supply condition. The improvement of sugar partitioning to the roots by the expression of OsHAK16p:WOX11 was further indicated by increasing the expression of OsSUT1 and OsSUT4 genes in leaf blades and several OsMSTs genes in roots. Expression of OsHAK16p:WOX11 in the rice grown in moderate K‐deficient soil increased total K uptake by 72% and grain yield by 24%–32%. The results suggest that enlarging root growth and development by the expression of WOX11 in roots could provide a useful option for increasing K acquisition efficiency and cereal crop productivity in low K soil.     

The peu‐miR160a−PeARF17.1/PeARF17.2 module participates in the adventitious root development of poplar

Deep roots give rise to flourishing leaves, and the two complement each other. However, the genetic mechanisms underlying adventitious rooting for forest trees have remained elusive. In this study, we verified that peu‐miR160a targets six poplar genes AUXIN RESPONSE FACTORS (ARFs), PeARF10.1, PeARF16.1, PeARF16.2, PeARF16.3, PeARF17.1 and PeARF17.2, using 5’RLM‐RACE. Interaction experiments with peu‐miR160a and PeARFs in poplar protoplasts further confirmed that peu‐miR160a targets and negatively regulates the six PeARFs. Peu‐miR160a and its target genes exhibited obvious temporal expression in different stages of adventitious root development, and they could also be induced by IAA and abscisic acid. Peu‐miR160a‐overexpressing lines exhibited a significant shortening of adventitious root length, an increase in the number of lateral roots, severe dwarfing and shortened internodes. In addition, the overexpression of PeARF17.1 or mPeARF17.2 (peu‐miR160a‐resistant version of PeARF17.2) significantly increased the number of adventitious roots. Furthermore, PeARF17.1‐overexpressing lines had multiple branches with no visible trunk, although the adventitious root length of the PeARF17.1‐overexpressing lines was significantly increased. Our findings reveal that the peu‐miR160a ? PeARF17.1/PeARF17.2 module is an important regulator involved in the development of the adventitious roots of poplar.     

Transgenic modification of gai or rgl1 causes dwarfing and alters gibberellins, root growth, and metabolite profiles in Populus

In Arabidopsis and other plants, gibberellin (GA)-regulated responses are mediated by proteins including GAI, RGA and RGL1-3 that contain a functional DELLA domain. Through transgenic modification, we found that DELLA-less versions of GAI (gai) and RGL1 (rgl1) in a Populus tree have profound, dominant effects on phenotype, producing pleiotropic changes in morphology and metabolic profiles. Shoots were dwarfed, likely via constitutive repression of GA-induced elongation, whereas root growth was promoted two- to threefold in vitro. Applied GA₃ inhibited adventitious root production in wild-type poplar, but gai/rgl1 poplars were unaffected by the inhibition. The concentrations of bioactive GA₁ and GA₄ in leaves of gai- and rgl1-expressing plants increased 12- to 64-fold, while the C₁₉ precursors of GA₁ (GA₅₃, GA₄₄ and GA₁₉) decreased three- to ninefold, consistent with feedback regulation of GA 20-oxidase in the transgenic plants. The transgenic modifications elicited significant metabolic changes. In roots, metabolic profiling suggested increased respiration as a possible mechanism of the increased root growth. In leaves, we found metabolite changes suggesting reduced carbon flux through the lignin biosynthetic pathway and a shift towards allocation of secondary storage and defense metabolites, including various phenols, phenolic glucosides, and phenolic acid conjugates.     

QTLs controlling the production of transgenic and adventitious roots in Brassica oleracea following treatment with Agrobacterium rhizogenes

Brassica oleracea can be genetically engineered using Agrobacterium rhizogenes. The initial stage of this process is the production of transgenic (hairy) roots; shoots are subsequently regenerated from these roots. Previous work using gus and gfp reporter genes has shown that genotypes of B. oleracea vary in their performance for transgenic root production. Quantitative trait loci (QTLs) controlling this trait have been located in one mapping population. The current study provides evidence that performance for transgenic root production is associated with performance for adventitious (non-transgenic) root production in B. oleracea across a second mapping population. This is shown by regression analyses between performance for the two traits and the demonstration that QTLs controlling the two traits map to the same positions within the genome. Since the rate of adventitious root production does not differ significantly in the presence and absence of A. rhizogenes, there is no evidence that the expression of Agrobacterium genes induces adventitious root production. It is apparent that genotypes exhibiting high adventitious root production in the absence of A. rhizogenes will also tend to show high transgenic root production, thereby allowing the selection of lines that are more efficiently transformed.     

Relationship between genotype and soil environment during colonization of poplar roots by mycorrhizal and endophytic fungi

Poplars are among the few tree genera that can develop both ectomycorrhizal (ECM) and arbuscular (AM) associations; however, variable ratios of ECM/AM in dual mycorrhizal colonizations were observed in the roots of a variety of poplar species and hybrids. The objective of our study was to analyze the effect of internal and external factors on growth and dual AM and ECM colonization of poplar roots in three 12–15-year-old common gardens in Poland. We also analyzed the abundance of nonmycorrhizal fungal endophytes in the poplar roots. The Populus clones comprised black poplars (Populus deltoides and P. deltoides × Populus nigra), balsam poplars (Populus maximowiczii × Populus trichocarpa), and a hybrid of black and balsam poplars (P. deltoides × P. trichocarpa). Of the three sites that we studied, one was located in the vicinity of a copper smelter, where soil was contaminated with copper and lead. Poplar root tip abundance, mycorrhizal colonization, and soil fungi biomass were lower at this heavily polluted site. The total mycorrhizal colonization and the ratio of ECM and AM colonization differed among the study sites and according to soil depth. The influence of Populus genotype was significantly pronounced only within the individual study sites. The contribution of nonmycorrhizal fungal endophytes differed among the poplar clones and was higher at the polluted site than at the sites free of pollution. Our results indicate that poplar fine root abundance and AM and ECM symbiosis are influenced by environmental conditions. Further studies of different site conditions are required to characterize the utility of poplars for purposes such as the phytoremediation of polluted sites.     

Enhanced drought and salt tolerance by expression of AtGSK1 gene in poplar

A GSK3/shaggy-like kinase (AtGSK1) has been implicated in the regulation of drought and salt tolerance. We transferred AtGSK1 from Arabidopsis thaliana to a hybrid poplar (Populus alba × P. tremula var. grandulosa) to determine the effect of the transgene expression in the transgenic trees. The results from northern blot and RT-PCR analyses showed that the expression level varied among the transgenic lines. During their culture on tissue culture media, the transgenic poplars formed vigorous growing roots even in the presence of 125 mM NaCl and callus in the presence of 150 mM NaCl. When the transgenic poplars were growing in pots and provided with NaCl solution, they stayed much healthier than did nontransgenic poplars, showing higher rates of photosynthetic rates, stomatal conductance, and evaporation rates under the stress. Whereas the total level of leaf Na⁺ level increased dramatically in transgenic poplars under severe saline conditions (150 mM NaCl), that of leaf K⁺ decreased in the same plants under the same conditions. Total root Na⁺ level increased in nontransgenic poplars under severe saline conditions. In contrast, total root K⁺ level decreased in the same plants under the same conditions. The chloride content and relative electrical conductivity of the transgenic poplars after salt stress treatment were lower than those of nontransgenic poplars. The transgenic poplars were also tolerant to up to 20 % PEG remaining significantly healthy when compared with nontransgenic poplars with necrosis and chlorosis symptoms. Another dramatic feature of the transgenic poplars was wilting tolerance for prolonged drought treatment up to 2 weeks. The results provide evidence that the expression of AtGSK1 gene conferred drought and salt tolerance in the transgenic poplars.     

Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol

Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best‐characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild‐type poplar hybrid Populus tremula × P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding γ‐glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild‐type plants; soil contamination significantly decreased biomass accumulation in both wild‐type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two‐dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast surface/volume ratio, both at the control and the contaminated site. Chloroplast number per cell did not differ between wild and transgenic poplars at the control site. Soil contamination led to suppression of chloroplast replication in wild‐type plants. From these results, we assume that overexpressing the bacterial gsh1 gene in the cytosol interacts with processes in the chloroplast and that sequestration of heavy metal phytochelatin complexes into the vacuole may partially counteract this interaction in plants grown at heavy metal‐contaminated field sites. Further experiments are required to test these assumptions.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.