Taproot promoters cause tissue specific gene expression within the storage root of sugar beet

The storage root (taproot) of sugar beet (Beta vulgaris L.) originates from hypocotyl and primary root and contains many different tissues such as central xylem, primary and secondary cambium, secondary xylem and phloem, and parenchyma. It was the aim of this work to characterize the promoters of three taproot-expressed genes with respect to their tissue specificity. To investigate this, promoters for the genes Tlp, His1-r, and Mll were cloned from sugar beet, linked to reporter genes and transformed into sugar beet and tobacco. Reporter gene expression analysis in transgenic sugar beet plants revealed that all three promoters are active in the storage root. Expression in storage root tissues is either restricted to the vascular zone (Tlp, His1-r) or is observed in the whole organ (Mll). The Mll gene is highly organ specific throughout different developmental stages of the sugar beet. In tobacco, the Tlp and Mll promoters drive reporter gene expression preferentially in hypocotyl and roots. The properties of the Mll promoter may be advantageous for the modification of sucrose metabolism in storage roots.     

Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz)

A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava’s starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.     

Comparative gene expression study to identify genes possibly related to storage root formation in cassava

Cassava storage roots result from swelling of adventitious roots by secondary growth. In the present study we aimed to gain insight into the molecular processes occurring during cassava storage root formation. We report a comparative gene expression study in adventitious and storage roots in order to identify genes possibly related to storage organ formation. Our results revealed five genes with higher expression levels in secondary xylem of storage roots than adventitious roots. Among them, the Mec1 gene coding for Pt2L4 glutamic acid-rich protein and a putative RING Zinc Finger and LEA protein genes were strongly induced in secondary xylem tissue.     

Comparative expression of β-glucuronidase with five different promoters in transgenic carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) root and leaf tissues

Tissue-specific patterns and levels of protein expression were characterized in transgenic carrot plants transformed with the β-glucuronidase (GUS) gene driven by one of five promoters: Cauliflower mosaic virus 35S (35S) and double 35S (D35S), Arabidopsis ubiquitin (UBQ3), mannopine synthase (mas2) from Agrobacterium tumefaciens or the rooting loci promoter (rolD) from A. rhizogenes. Five independently transformed carrot lines of each promoter construct were assessed for GUS activity. In leaves, activity was highest in plants with the D35S, 35S and UBQ3 promoters, while staining was weak in plants with the mas2 promoter, and only slight visual staining was present in the leaf veins of plants containing rolD promoter . Strong staining was seen in the lateral roots, including root tips, hairs and the vascular tissues of plants expressing the 35S, D35S and UBQ3. Lateral roots of plants containing the rolD construct also showed staining in these tissues while the mas2 promoter exhibited heightened staining in the root tips. Relatively strong GUS staining was seen throughout the tap root with all the promoters tested.. When GUS expression was quantified, the UBQ3 promoter provided the highest activity in roots of mature plants, while plants with the D35S and 35S promoter constructs had higher activity in the leaves. Although plants containing the mas2 promoter had higher levels of activity compared to the rolD plants, these two promoters were significantly weaker than D35S, 35S and UBQ3. The potential for utilization of specific promoters to target expression of desired transgenes in carrot tissues is demonstrated.     

Partition of 14C Assimilate between Organs and Fractions of Contrasting Varieties of Carrot during Initiation of the Storage Root

This work examines the differences in partition and activityof ¹⁴C in two varieties of carrot (Daucus carota L.) contrastingin shoot to storage root ratio at maturity. Plants were grownin a controlled environment of 20 ?C and 500 µmol m^–2s^–1. During initiation of the storage root (10–25d from sowing) plants were exposed to ¹⁴CO₂ for 1 h and theradioactivity in ethanol-soluble and -insoluble fractions ofshoots, storage and fibrous roots estimated at various timesup to 48 h after exposure. Between 35% and 40% of radioactivityinitially present in the plants was respired during the first24 h and 25–35% of that remaining after 24 h was foundin the roots, depending on age. The proportion found in thestorage region remained fairly constant between 15 and 25 dand was smaller than at 10 d. In the variety with a larger proportionof storage root at maturity (cv. Super Sprite), there was agreater proportion of label in both ethanol-soluble and -insolublefractions of the storage region soon after storage root initiationhad begun than in the variety with a smaller proportion of storageroot at maturity (cv. Kingston). There was no varietal differencein specific activities of the storage roots, but fibrous rootsof cv. Super Sprite showed a greater specific activity thanin cv. Kingston. Differences in shoot to storage root ratiomay thus be associated with characteristics of the fibrous roots.Partition and specific activities are discussed in relationto the initiation and development of the storage organ. Key words: Daucus carota, carrot, assimilate, partition, ¹⁴C, storage root     

Comparison of four constitutive promoters for the expression of transgenes in the tropical nitrogen-fixing tree Allocasuarina verticillata

Allocasuarina verticillata is an actinorhizal tree that lives in symbiotic association with a nitrogen fixing actinomycete called Frankia. In the search for promoters that drive strong constitutive expression in this tropical tree, we studied the organ specificity of four different constitutive promoters (CaMV 35S, e35S, e35S-4ocs and UBQ1 from Arabidopsis thaliana) in stably transformed A. verticillata plants. The ß-glucuronidase (gus) gene was used as a reporter and expression studies were carried out by histochemical analyses on shoots, roots and actinorhizal nodules. While the 35S promoter was poorly expressed in the shoot apex and lateral roots, both the e35S and e35S-4ocs were found to drive high constitutive expression in the transgenic non-nodulated plants. In contrast, the UBQ1 promoter was very poorly expressed and appeared unsuitable for A. verticillata. We also showed that none of the promoters studied were active in the nodule infected cells, whatever the developmental stage studied.     

Transfer and Expression of an Artificial Storage Protein (ASP1) Gene in Cassava (Manihot Esculenta Crantz)

In order to increase the nutritional quality of cassava storage roots, which contain up to 85% starch of their dry weight, but are deficient in protein, a synthetic ASP1 gene encoding a storage protein rich in essential amino acids (80%) was introduced into embryogenic suspensions of cassava via Agrobacterium-mediated gene transfer. Transgenic plants were regenerated from suspension lines derived from hygromycin-resistant friable embryogenic callus lines. Molecular analysis showed the stable integration of asp1 in cassava genome and its expression at RNA level in transformed suspension lines. PCR and Southern analyses proved the transgenic nature of the regenerated plant lines. The expression of asp1 at RNA level was demonstrated by RT-PCR. The ASP1 tetramer could be detected in leaves as well as in primary roots of cultured transgenic plants by western blots. These results indicate that the nutritional improvement of cassava storage roots may be achieved by constitutive expression of asp1 in transgenic plants.     

Allele exchange at the EPSPS locus confers glyphosate tolerance in cassava

Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and nonhomologous end‐joining (NHEJ) DNA repair pathways, we precisely introduced the best‐performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS‐edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T‐DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava.     

MORPHOLOGY OF HUMULUS LUPULUS. II. SECONDARY GROWTH IN THE ROOT AND SEEDLING VASCULARIZATION

Miller , Robert H. (U. Nevada, Reno.) Morphology of Humulus luppulus. II. Secondary growth in the root and seedling vascularization. Amer. Jour. Bot. 46(4): 269–277. Illus. 1959.—In the primary state the roots of Humulus lupulus L. have a diarch xylem plate with 2 strands of primary phloem lying on either side of the primary xylem. Secondary histogenesis is described for the primary root. Fibrous and fleshy storage roots are developed by the hop plant and their respective developmental and anatomical structures are described. Lateral roots are initiated in the pericycle opposite the protoxylem poles. The architecture of these secondary roots is similar to that of the primary root. The seedling develops a fleshy storage organ through secondary growth of the primary root and the hypocotyl. The hypocotyl eventually resembles a fleshy taproot throughout most of its extent. The vascular cambium differentiates large amounts of parenchymatous tissues. A relatively smaller amount of tracheary tissue is formed. The secondary phloem comprises a high percentage of phloem parenchyma and ray cells containing numerous large starch grains, and constitutes the larger portion of the fleshy storage root. Numerous thick-walled lignified fibers occur throughout the secondary vascular tissues. Resin and tannin cells are abundantly distributed. A phellogen is differentiated from the pericycle and develops a persistent periderm on the outer surface of the fleshy storage organ. A relatively short transition region occurs in the upper part of the hypocotyl. The transition takes place from a radially alternate arrangement of the vascular tissues in the root to a collateral arrangement in the cotyledons.     

Ubiquitous and tissue-specific gus expression in transgenic roots conferred by six different promoters in one coniferous and three angiosperm species

The activity of six different promoter-gus (uidA) binary plasmid constructs has been analysed in transgenic roots of Pinus contorta, Nicotiana tabacum, Lycopersicon esculentum and Arabidopsis thaliana. Transgenic roots were induced by infection with Agrobacterium rhizogenes strain LBA9402, harbouring a binary plasmid construct that contained one of the following promoters: Ubi-1 from Zea mays, 35S from CaMV, cdc2a and sam-1 from A. thaliana, HRGPnt3 from N. tabacum and RSI-1 from L. esculentum. Promoters of broad tissue specificity (cdc2a, Ubi-1 and 35S) showed GUS staining in most cell types of all the species. The other three promoters were expressed specifically in lateral root primordia. The studies of gene activity in primary transgenic roots allowed the screening of candidate promoters related to lateral and adventitious root formation within 3–6 weeks after inoculation in the angiosperm species and 2–3 months in P. contorta. Received: 30 September 1998 / Revision recieved: 10 November 1998 / Accepted: 30 November 1998     

Effects of prometryn and acetochlor on arbuscular mycorrhizal fungi and symbiotic system

Prometryn and acetochlor are common herbicides widely used to control weeds in agricultural systems. The impacts of the two herbicides on spore germination, hyphal elongation, the biomass and malondialdehyde content of carrot hairy roots were investigated using a strict in vitro cultivation system associating the Ri T‐DNA‐transferred carrot hairy roots with Glomus etunicatum. Alternatively, root colonization, daughter spore production and the proportion of hyphae with succinate dehydrogenase (SDH) and alkaline phosphatase (ALP) activities were also investigated. No significant impact on spore germination was noted in the presence of acetochlor at all three concentrations tested, while a significant decrease was observed with prometryn only at the highest concentration. Moreover, an inverse correlation was identified between herbicides concentrations and G. etunicatum root colonization and spore production as well as hyphal SDH and ALP activity, with a positive correlation identified among these four factors. Both herbicides exerted negative effects on the arbuscular mycorrhizal (AM) fungus and symbiosis at increasing concentrations, with prometryn apparently more toxic than acetochlor. Furthermore, the AM symbiotic system was shown to improve biomass, reduce malondialdehyde accumulation and ease lipid peroxidation in carrot hairy roots and decrease damage in host plants, thus enhancing plant tolerance to adverse conditions.

Significance and Impact of the Study

In this study, the effect of prometryn and acetochlor on the physiology and metabolic activities of the AM fungus Glomus etunicatum were investigated. Our findings demonstrate for the first time, the impact of the two herbicides at three concentrations (0·1, 1 and 10 mg l^?1) on transformed carrot hairy roots/AM fungus association under strict in vitro culture conditions, which may guide the application of the two herbicides in modern agriculture.     

DcMYB113, a root‐specific R2R3‐MYB,conditions anthocyanin biosynthesis and modification in carrot

Purple carrots, the original domesticated carrots, accumulate highly glycosylated and acylated anthocyanins in root and/or petiole. Previously, a quantitative trait locus (QTL) for root‐specific anthocyanin pigmentation was genetically mapped to chromosome 3 of carrot. In this study, an R2R3‐MYB gene, namely DcMYB113, was identified within this QTL region. DcMYB113 expressed in the root of ‘Purple haze’, a carrot cultivar with purple root and nonpurple petiole, but not in the roots of two carrot cultivars with a purple root and petiole (Deep purple and Cosmic purple) and orange carrot ‘Kurodagosun’, which appeared to be caused by variation in the promoter region. The function of DcMYB113 from ‘Purple haze’ was verified by transformation in ‘Cosmic purple’ and ‘Kurodagosun’, resulting in anthocyanin biosynthesis. Transgenic ‘Kurodagosun’ carrying DcMYB113 driven by the CaMV 35S promoter had a purple root and petiole, while transgenic ‘Kurodagosun’ expressing DcMYB113 driven by its own promoter had a purple root and nonpurple petiole, suggesting that root‐specific expression of DcMYB113 was determined by its promoter. DcMYB113 could activate the expression of DcbHLH3 and structural genes related to anthocyanin biosynthesis. DcUCGXT1 and DcSAT1, which were confirmed to be responsible for anthocyanins glycosylation and acylation, respectively, were also activated by DcMYB113. The WGCNA identified several genes co‐expressed with anthocyanin biosynthesis and the results indicated that DcMYB113 may regulate anthocyanin transport. Our findings provide insight into the molecular mechanism underlying root‐specific anthocyanin biosynthesis and further modification in carrot and even other root crops.     

A sweetpotato SRD1 promoter confers strong root-, taproot-, and tuber-specific expression in Arabidopsis, carrot, and potato

Harvestable, starch-storing organs of plants, such as fleshy taproots and tubers, are important agronomic products that are also suitable target organs for use in the molecular farming of recombinant proteins due to their strong sink strength. To exploit a promoter directing strong expression restricted to these storage organs, we isolated the promoter region (3.0 kb) of SRD1 from sweetpotato (Ipomoea batatas cv. ‘White Star’) and characterized its activity in transgenic Arabidopsis, carrot, and potato using the β-glucuronidase (GUS) gene (uidA) as a reporter gene. The SRD1 promoter conferred root-specific expression in transgenic Arabidopsis, with SRD1 promoter activity increasing in response to exogenous IAA. A time-course study of the effect of IAA (50 μM) revealed a maximum increase in SRD1 promoter activity at 24 h post-treatment initiation. A serial 5′ deletion analysis of the SRD1 promoter identified regions related to IAA-inducible expression as well as regions containing positive and negative elements, respectively, controlling the expression level. In transgenic carrot, the SRD1 promoter mediated strong taproot-specific expression, as evidenced by GUS staining being strong in almost the entire taproot, including secondary phloem, secondary xylem and vascular cambium. The activity of the SRD1 promoter gradually increased with increasing diameter of the taproot in the transgenic carrot and was 10.71-fold higher than that of the CaMV35S promoter. The SRD1 promoter also directed strong tuber-specific expression in transgenic potato. Taken together, these results demonstrate that the SRD1 promoter directs strong expression restricted to the underground storage organs, such as fleshy taproots and tubers, as well as fibrous root tissues.     

栓皮栎根系解剖结构、水力特性及碳、氮含量研究

为阐明栓皮栎根系随径级的变化规律,探究其细根的合理划分标准。以1年生栓皮栎幼苗为研究对象,将其根系分为1、1～2、2～3、3～4 mm四个径级,分别制作石蜡切片观察解剖结构,比较木质部水力特性,测定碳氮含量及其比值,并采用主成分法对根系进行分类。结果表明:(1)随着径级增加,栓皮栎根系周皮、韧皮部和形成层组织厚度增加而占径比降低,木质部直径及其占径比均增加。(2)直径2 mm以上的栓皮栎根系木质部平均最大和最小导管直径、根比导水率和栓塞脆弱性指数增加显著; 而导管密度显著下降,导管面积与木质部面积之比变化不显著。(3)直径2 mm以上栓皮栎根系碳含量表现出显著增加,随着径级增加,根系氮含量下降、碳氮比升高。(4)主成分分析表明,13项根系结构和元素含量指标降维后,前2个主分量方差贡献率达62%,PCA双序轴显示栓皮栎根系可划分为2 mm以下的吸收根群和2 mm以上的运输根群。综上认为,以2 mm作为栓皮栎细根划分的标准兼顾了形态和功能的特点,更具有准确性。     

Transcriptome-based identification of genes revealed differential expression profiles and lignin accumulation during root development in cultivated and wild carrots

Key message

Carrot root development associates lignin deposition and regulation.

Abstract

Carrot is consumed worldwide and is a good source of nutrients. However, excess lignin deposition may reduce the taste and quality of carrot root. Molecular mechanisms underlying lignin accumulation in carrot are still lacking. To address this problem, we collected taproots of wild and cultivated carrots at five developmental stages and analyzed the lignin content and characterized the lignin distribution using histochemical staining and autofluorescence microscopy. Genes involved in lignin biosynthesis were identified, and their expression profiles were determined. Results showed that lignin was mostly deposited in xylem vessels of carrot root. In addition, lignin content continuously decreased during root development, which was achieved possibly by reducing the expression of the genes involved in lignin biosynthesis. Carrot root may also prevent cell lignification to meet the demands of taproot growth. Our results will serve as reference for lignin biosynthesis in carrot and may also assist biologists to improve carrot quality.

     

Evaluation of the effects of elevated CO2 concentrations on the growth of cassava storage roots by destructive harvests and ground penetrating radar scanning approaches

Cassava (Manihot esculenta Crantz) production will need to be improved to meet future food demands in Sub-Saharan Africa. The selection of high-yielding cassava cultivars requires a better understanding of storage root development. Additionally, since future production will happen under increasing atmospheric CO₂ concentrations ([CO₂]), cultivar selection should include responsiveness to elevated [CO₂]. Five farmer-preferred African cassava cultivars were grown for three and a half months in a Free Air CO₂ Enrichment experiment in central Illinois. Compared to ambient [CO₂] (~400 ppm), cassava storage roots grown under elevated [CO₂] (~600 ppm) had a higher biomass with some cultivars having lower storage root water content. The elevated [CO₂] stimulation in storage root biomass ranged from 33% to 86% across the five cultivars tested documenting the importance of this trait in developing new cultivars. In addition to the destructive harvests to obtain storage root parameters, we explored ground penetrating radar as a nondestructive method to determine storage root growth across the growing season.