Arabidopsis thaliana vegetative storage protein (VSP) genes: gene organization and tissue-specific expression

We have previously identified two cDNAs encoding vegetative storage proteins (VSPs) in Arabidopsis thaliana. Unlike soybean in which VSPs accumulate at high levels in leaves, A. thaliana VSP mRNAs are abundant in flowers. To understand tissue-specific expression and possible roles of VSPs on reproductive organ development, genes corresponding to VSPs (Vsp1 and Vsp2) and their putative promoters were characterized in this study. Genomic sequence analysis revealed that Vsp1 and Vsp2 resemble each other except in their introns, and that these two genes were organized in a tandem array with an interval of 6 kb in a region. The expression patterns of Vsp1 and Vsp2 were examined using transgenic A. thaliana plants carrying a promoter from Vsp1 or Vsp2 fused to a bacterial -glucuronidase (GUS) reporter gene. The promoter from Vsp1 expressed its effect in gynoecia, especially in styles, the basal and distal ends of ovaries and in siliques, whereas the promoter from Vsp2 showed its activity in vegetative shoots, petioles, peduncles and receptacles of floral organs. These results suggest that expression of Vsp1 and Vsp2 may be developmentally regulated in A. thaliana. In the transgenic plants, the GUS activity was induced by wounding in an area around the mid-rib of leaves. Therefore, Vsp1 and Vsp2 promoters appear to have elements required for both tissue specificity and wounding.     

Rol genes alter hormonal requirements for protoplast growth and modify the expression of an auxin responsive promoter

Summary Growth characteristics of tobacco protoplasts containing rolA linked to its own promoter, or the rolB, or rolC genes of Agrobacterium rhizogenes linked to the Cauliflower Mosaic Virus 35S RNA promoter were compared with those from untransformed plants. RolA protoplasts require auxin and cytokinin for callus formation. Protoplasts overexpressing rolB and C form callus in the absence of exogenously applied auxin and cytokinin, respectively. Long term callus growth requires auxin, but the requirement for cytokinin is not critical. Optimal transient expression of an auxin responsive promoter element occurred at lower external levels of auxin in rolB and rolC protoplasts compared with untransformed protoplasts. Addition of putrescine was required for auxin responsive transient gene expression in rolA protoplasts suggesting that polyamines, or their products affect gene expression in rolA plants.Abbreviations T-DNA transferred DNA - TL-DNA left transferred DNA - NAA naphthalene acetic acid - PEG polyethylene glycol - GUS glucuronidase - CaMV cauliflower mosaic virus     

Polar transport of kinetin in tissues of radish

Polar transport of kinetin-8-¹⁴C occurred in segments of petioles, hypocotyls, and roots of radish (Raphanus sativus L.). The polarity was basipetal in petioles and hypocotyls and acropetal in roots. In segments excised from seedlings with fully expanded cotyledons, indole-3-acetic acid was required for polarity to develop. In hypocotyl segments isolated at this stage, basipetal and acropetal movements were equal during the first 12 hours of auxin treatment after which time acropetal movement declined. Pretreatment with auxin eliminated this delay in the appearance of polarity. In hypocotyl segments excised from seedlings with expanding cotyledons, exogenous auxin was unnecessary for polarity. Potassium cyanide abolished polarity at both stages of growth by allowing increased acropetal movement. The rate of accumulation of kinetin in receiver blocks was greater than the in vivo increase in cytokinin content of developing radish roots.     

Phosphatidic acid activates a wound-activated MAPK in Glycine max

Many plant species demonstrate a systemic increase in phosphatidic acid (PA) levels after being wounded (Lee et al., 1997). To understand the role of PA in wound signal transduction, we investigated if PA can activate protein kinases in soybean (Glycine max L.). We found that a MAPK is activated in soybean seedlings in both wounded and neighboring unwounded leaves. The wound-activated soybean kinase is specifically recognized by an antibody against the alfalfa MAPK, SIMK. When PA production is inhibited with n-butanol, an inhibitor of phospholipase D, the wound-induced activation of the MAPK is suppressed, suggesting that an elevation in PA levels is essential for its activation. Supporting this is the observation that exogenous PA activates the MAPK in suspension-cultured soybean cells. Activation of the 49 kDa MAPK occurs almost exclusively by PA, as other lipids are unable to or can only weakly activate the kinase. PA-induced activation of the MAPK is not a direct effect on the kinase but is mediated by upstream kinases. Our results suggest that PA acts as a second messenger in wound-induced MAPK signaling in plants.     

In vitro plant regeneration of Aster scaber via somatic embryogenesis

We established an in vitro plant regeneration system via somatic embryogenesis of Aster scaber, an important source of various biologically active phytochemicals. We examined the callus induction and embryogenic capacities of three explants, including leaves, petioles, and roots, on 25 different media containing different combinations of α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The optimum concentrations of NAA and BA for the production of embryogenic calli were 5.0 μM and 0.05 μM, respectively. Media containing higher concentrations of auxin and cytokinin (such as 25 μM NAA and 25 μM BA) were suitable for shoot regeneration, especially for leaf-derived calli, which are the most readily available calli and are highly competent. For root induction from regenerated shoots, supplemental auxin and/or cytokinin did not improve rooting, but instead caused unwanted callus induction or retarded growth of regenerated plants. Therefore, plant growth regulator-free medium was preferable for root induction. Normal plants were successfully obtained from calli under the optimized conditions described above. This is the first report of the complete process of in vitro plant regeneration of A. scaber via somatic embryogenesis.     

Effect of BA,NAA, and 2,4-D on red maple callus growth

Established red maple (Acer rubrum L.) callus was cultured on media varying in auxin (NAA or 2,4-D) and cytokinin (BA) concentrations. Callus growth was positively affected by the presence of both an auxin and cytokinin in the medium. Optimal growth depended on the ratio of cytokinin/auxin as well as the total amount of plant growth regulators in the medium.Abbreviations (NAA) naphthaleneacetic acid - (2,4-D) 2,4-dichlorophenoxyacetic acid - (BA) and 6-benzylaminopurine     

The contrasting role of auxin in submergence-induced petiole elongation in two species from frequently flooded wetlands

The involvement of auxin in the submergence-induced petiole elongation has been investigated in Rumex palustris and Ranunculus sceleratus. Both wetland species are capable of enhanced petiole elongation upon submergence or treatment with exogenous ethylene (5μl l⁻¹). Treatment of intact Rumex palustris plants with 1-naphthalene acetic acid (NAA) at 10⁻⁴ M enhanced petiole elongation, while treatment with N -1-naphthylphthalamic acid (NPA) had no effect on petiole elongation. The elongation response after NAA or NPA treatment was comparable for plants in both submerged and drained conditions. Pre-ageing of detached petioles of Rumex palustris for 3 h in light or in dark conditions had no effect on the submergence-induced elongation. In comparison to intact plants, detached petioles of Rumex palustris , with or without lamina, did not show significant differences in responsiveness to IAA between drained or submerged conditions. This was in contrast to Ranunculus sceleratus where submergence caused a clear increase in responsiveness towards IAA. Removal of the lamina, the putative source of auxin, or treatment with NPA did not hinder the submergence-induced elongation of detached Rumex palustris petioles, but severely inhibited elongation of detached Ranunculus sceleratus petioles. This inhibition could be restored by application of NAA, suggesting the specific involvement of auxin in the submergence response of Ranunculus sceleratus. It is concluded that, in contrast to Ranunculus sceleratus , auxin is probably not involved in the submergence-induced petiole elongation of Rumex palustris.     

Micropropagation of seed-derived plants ofCynara scolymus L., cv. ‘Green Globe’

Growth of Cymbidium kanran rhizome was enhanced by higher NAA:BAP ratios in modified Murashige & Skoog (MS) media. Only vegetative shoots resulted from rhizomes cultured in vitro when lower NAA:BAP ratios were used. The rhizomes were induced from the axils of leaves when shoots were explanted to medium containing higher concentrations of NAA. Root formation of C. kanran was inhibited by the addition of either auxin or cytokinin to the culture media. Differentiation of the rhizomes into plantlets occurred when the concentrations of ammonium nitrate and potassium nitrate in MS medium wewe reduced. The modified MS medium containing lesser amounts of potassium nitrate and ammonium nitrate than those of the original MS media, and was optimal for the production of plantlets from rhizomes of C. kanran without addition of auxin and cytokinin.Abbreviations NAA -naphthaleneacetic acid - BAP N⁶-benzylaminopurine - MS medium Murashige & S Skoog medium     

Methyl jasmonate induces the formation of secondary abscission zone in stem of Bryophyllum calycinum Salisb

Methyl jasmonate (JA-Me) at a concentration of 0.5 % induced the formation of secondary abscission zone and senescence in several types of stem explants (only internode segment, internode segment with nodes and without leaves, internode segment with nodes and debladed petioles) of Bryophyllum calycinum when it was applied in various places of the stem or the debladed petiole as lanolin paste. In the presence of small leaves in stem explants methyl jasmonate also induced the formation of secondary abscission zone and senescence but the presence of larger leaves completely inhibited methyl jasmonate-induced processes. Auxin, (indole-3-acetic acid, IAA), at a concentration of 0.1 % extremely prevented the formation of secondary abscission zones and senescence in the stem tissues induced by methyl jasmonate. Similar relationship between auxin and methyl jasmonate to induce the formation of secondary abscission zone and senescence was found in decapitated shoot of the intact plant. Mechanisms of the formation of secondary abscission zone are also discussed in terms of the interaction of methyl jasmonate with auxin.     

An increase in ethylene sensitivity is associated with jasmonate-promoted senescence of detached rice leaves

The role of ethylene in jasmonate-promoted senescence of detached rice leaves was investigated. Ethylene production in methyl jasmonate-treated leaf segments of rice was lower than in the control leaves. Treatment of leaf segments with silver nitrate or/and silver thiosulfate, inhibitors of ethylene action, inhibited methyl jasmonate-, jasmonic acid-, linolenic acid-, and abscisic acid-promoted senescence of detached leaves. We suggest that an increase in ethylene sensitivity, but not ethylene level, is the initial event triggering the enhanced senescence by jasmonates of detached rice leaves.Abbreviations JA jasmonic acid - MJ methyl jasmonate - STS silver thiosulfate - ABA abscisic acid     

Auxins and cytokinins exuded during formation of roots by detached primary leaves and stems of dwarf French bean (Phaseolus vulgaris L.)

Summary Hypocotyls of detached stems standing in culture solution produced adventitious roots sooner than did petioles of detached primary leaves. An auxin, probably indol-3-ylacetic acid, appeared in the solutions before the hypocotyls or petioles produced roots. After attaining a maximum, the amounts of auxin in the solutions decreased as fewer roots were formed. Two cytokinins were found in the culture solutions; one had a similar R_f to zeatin, the other ran more slowly on chromatograms. The amounts of cytokinin in the solutions were associated with root formation. Stems soon died unless their hypocotyls formed roots, but the primary leaves survived without roots forming provided a callus formed on the petiole. Hence adventitious roots, or callus tissues, may have produced cytokinins that replaced those produced by the original roots, found in sap exuded from the stem stumps, and were essential for survival of the stems and leaves.     

APICAL DOMINANCE,POLARITY, AND ADVENTITIOUS GROWTH IN MARCHANTIA POLYMORPHA

Benzylaminopurine, indoleacetic acid, and the auxin inhibitors transcinnamic acid, triiodobenzoic acid, and dinitrophenol were employed to elucidate the role of apical dominance and hormones during regeneration of thalli and gemmae of Marchantia polymorpha L. The cytokinin suppressed normal gemma germination and led to the development of nodular, callus-like growths. When removed from the influence of benzylaminopurine, the site and magnitude of normal thallus outgrowths varied with the length of time that the tissue had remained on the cytokinin-containing medium. This aberrant germination was not influenced by the incorporation of indoleacetic acid into the medium. Exogenous auxin neither accelerated nor inhibited the regeneration of normal thallus growth on excised vegetative discs. Transcinnamic acid and dinitrophenol inhibited regeneration. Auxin reversed this suppression. Triiodobenzoic acid did not significantly affect regeneration. Autoradiographs demonstrated a pronounced accumulation of labelled auxin in the midribs and the acropetal regions of excised thallus discs. This evidence suggests that there is an endogenous, basipetal auxin gradient in Marchantia; that the maintenance of this gradient is vital to normal growth and regeneration of the thallus; and that high endogenous concentrations of cytokinin destroy this polarity by increasing the auxin-synthesizing capacity of the tissue.     

Fatty acids in callus cultures: Influence of growth factors on fatty acid composition of total lipids in callus cells

Callus tissues were derived from cotyledon segments of Cucumis melo and Cucumis sativus. Four combinations of growth factors, i.e., naphthalene acetic acid (NAA) plus coconut water (CW); NAA plus kinetin; NAA plus 6-benzylaminopurine (BAP) and indole 3-butyric acid plus BAP, were incorporated in the medium for callus initiation as well as for growth of excised callus in culture for six passages. The proportion of total saturated to unsaturated acids and the ratio of linoleic to linolenic acid was influenced by the change in the type of auxin and cytokinin in the combinations used. A many fold increase of myristic acid was recorded for the indole 3-butyric acid plus BAP combination.     

Cytokinin inhibits a subset of diageotropica-dependent primary auxin responses in tomato

Many aspects of plant development are regulated by antagonistic interactions between the plant hormones auxin and cytokinin, but the molecular mechanisms of this interaction are not understood. To test whether cytokinin controls plant development through inhibiting an early step in the auxin response pathway, we compared the effects of cytokinin with those of the dgt (diageotropica) mutation, which is known to block rapid auxin reactions of tomato (Lycopersicon esculentum) hypocotyls. Long-term cytokinin treatment of wild-type seedlings phenocopied morphological traits of dgt plants such as stunting of root and shoot growth, reduced elongation of internodes, reduced apical dominance, and reduced leaf size and complexity. Cytokinin treatment also inhibited rapid auxin responses in hypocotyl segments: auxin-stimulated elongation, H(+) secretion, and ethylene synthesis were all inhibited by cytokinin in wild-type hypocotyl segments, and thus mimicked the impaired auxin responsiveness found in dgt hypocotyls. However, cytokinin failed to inhibit auxin-induced LeSAUR gene expression, an auxin response that is affected by the dgt mutation. In addition, cytokinin treatment inhibited the auxin induction of only one of two 1-aminocyclopropane-1-carboxylic acid synthase genes that exhibited impaired auxin inducibility in dgt hypocotyls. Thus, cytokinin inhibited a subset of the auxin responses impaired in dgt hypocotyls, suggesting that cytokinin blocks at least one branch of the DGT-dependent auxin response pathway.     

Hormone interactions and regulation of PsPK2::GUS compared with DR5::GUS and PID::GUS in Arabidopsis thaliana

The putative pea PINOID homolog, PsPK2, is expressed in all growing plant parts and is positively regulated by auxin, gibberellin, and cytokinin. Here, we studied hormonal regulation of PsPK2::GUS expression compared with DR5::GUS and PID::GUS in Arabidopsis. PsPK2::GUS, DR5::GUS, and PID::GUS expression in Arabidopsis shoots is mainly localized in the stipules, hydathodes, veins, developing leaves, and cotyledons. Unlike DR5::GUS, PsPK2::GUS, and PID::GUS are weakly expressed in root tips. Both DR5::GUS and PsPK2::GUS are induced by different auxins and are more sensitive to methyl indole acetic acid, 4-chloro-indole acetic acid, and α-naphthalene acetic acid than others. GA(3) has no significant effect on GUS activity in DR5::GUS-transformed seedlings compared to the control, but induction by auxin and gibberellin in combination is synergistic. Cytokinin increases auxin transport in Arabidopsis seedlings. Auxin, gibberellin, and cytokinin all increase GUS activity in shoots of PsPK2::GUS transformed plants compared to the control. However, only auxin and gibberellin increase GUS activity in PID::GUS shoots. In conclusion, auxin, gibberellin, and cytokinin positively regulate PsPK2 expression in shoots, but not in roots. Auxin and gibberellin also upregulate AtPIN1 and LEAFY expression, which is similar to PsPIN1 and Uni in pea. With minor exceptions, the orthologous genes from both species are regulated similarly.     

Histological analysis of somatic embryogenesis induced in leaf explants ofHelianthus smithii Heiser

Summary According to the hormonal conditions, after one month of culture shoots or somatic embryos could be obtained from leaf explants ofHelianthus smithii. Well-shaped embryos developed on media containing a combination of auxin and cytokinin, while on media containing only cytokinin shoots were observed. The primary leaves of these shoots resembled cotyledons. A detailed histological study of the regeneration process on three media, containing only cytokinin or auxin, or a combination of both, allowed the origin and development of the observed structures to be determined. All three conditions induced somatic embryos, which then developed differently and, within one month, finally gave rise to the two types of structures which were initially observed.Abbrevations BAP 6-benzylaminopurine - MES 2-(N-morpholino)ethane-sulfonic acid - MS medium of Murashige and Skoog - NAA 1 -naphthaleneacetic acid - TDZ thidiazuron