Studies on Cytokinins in Watermelon Seeds

Extracts of immature seeds of watermelon contain three cytokinins which are adsorbed on Dowex 50W ion exchange resin. The Rf of the fastest moving factor is similar to that of zeatin. The slowest moving factor is insoluble in n-butanol and shows some resemblance to zeatin ribotide. The chromatographic properties of the second factor are, however, different from those of any of the known cytokinins and is believed to be new to literature.     

The Biosynthesis of Cytokinins in Germinating Lupin Seeds

Imbibed intact seeds, and excised embryos and cotyledons ofyellow lupin (Lupinus luteus L. cv. Weiko III) have been incubatedwith [¹⁴C]-adenine to investigate cytokinin biosynthesis duringthe early stages of germination. Following incubation the tissueswere extracted and purified by solvent partition and chromatographyon cellulose phosphate, diethylaminoethyl cellulose and SephadexLH-20 columns. Using a variety of thin layer chromatographic,high performance liquid chromato-graphic and chemical procedures,incorporation of ¹⁴C into dihydrozeatin riboside and its nucleotidewas demonstrated in extracts of intact embryos, intact cotyledonsand excised embryos. However, radioactivity was not found associatedwith cytokinins in fractions derived from the isolated cotyledons.This is the first direct demonstration of cytokinin biosynthesisin germinating seeds and the results indicate that the capacityfor cytokinin biosynthesis is probably confined to the embryonicaxes. If this is so, the levels of [¹⁴CJ-dihydrozeatin ribosideassociated with intact embryo and intact cotyledon fractionsindicate that the synthesized cytokinin is transported to andaccumulates in the cotyledons. Key words: Lupinus luteus, cytokinin biosynthesis, seed germination     

The Location of Cytokinins and Gibberellins in Wheat Seeds

Wheat seeds (Triticum aestivum L. cvs. Yorkstar and Sirius) were cut transversely into embryoless and embryo-containing (embryonated) halves and the content of endogenous cytokinins and gibberellins in both halves determined before and after the seeds imbibed water for 12–15 h at 22°C in the light. Dry seeds contained little ethyl acetate-extractable gibberellin activity as measured by bioassay but n-butanol-soluble cytokinins were detected mainly in the embryoless halves. Dry, embryonated half-seeds contained water-soluble gibberellins which could be extracted into acidic ethyl acetate after treatment of the aqueous extract with either alkaline phosphatase, β-glucosidase or a crude pectolytic enzyme preparation. When half-seeds were allowed to imbibe water for 12 h and then extracted, cytokinin activity was largely lost from the embryoless halves and completely from the embryonated halves and water-soluble gibberellins were also lost from the embryonated halves. However, ethyl acetate-soluble gibberellins were present in the latter suggesting that “bound’ gibberellins were released during imbibition. The hormones present in normal and naturally embryoless dry grains of cv. Yorkstar were also determined. Both gibberellin and cytokinin activity was higher in normal grains suggesting that the presence of an embryo is essential for synthesis or accumulation of these hormones in the grain during development.     

The Sodium and Potassium Exchange between Intact Frogs and Their Environment

Intact living frogs (Rana pipiens) were partially immersed in dilute salt solution labeled with K⁴² or Na²⁴ or, alternatively, injected with Ringer’s fluid containing the appropriate isotope and then partially immersed in unlabeled dilute salt. Before isotopic equilibrium, the animals were sacrificed and specific activities of K⁴² and Na²⁴ were determined for medium, skin, plasma, and other tissues. With Na²⁴, entering from the medium or escaping to the medium, specific activities of the skin approach that of the plasma. For K⁴², entering from the medium, the specific activity exceeds that of the plasma. The results are interpreted as indicating that the exchange rate for Na is greater plasma to skin than medium to skin, with the reverse situation for K. Values are given for average Na, K, and Cl contents of the various organ systems.     

Calcium and EGTA Alleviate Cadmium Toxicity in Germinating Chickpea Seeds

Impact of exogenous calcium and ethylene glycol tetraacetic acid (EGTA) supplement on chickpea (Cicer arietinum L.) germinating seeds exposed to cadmium stress for 6 days was studied. Ca and EGTA late treatment (3 days) alleviated growth inhibition and decreased Cd accumulation as well as lipid peroxidation and protein carbonylation in both root and shoot cells. Exogenous effector application relieved Cd-induced cell death which was associated with a constant level of ATP, which was considered as an apoptotic-like process. Redox balance was examined through the study of the redox state of pyridine nucleotide couples NAD⁺/NADH and NADP⁺/NADPH as well as their related oxidative [NAD(P)H-oxidase] and dehydrogenase (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and malate dehydrogenase) enzyme activities. The present research illustrated an ameliorative effect of Ca and EGTA on growth of Cd-exposed chickpea seedlings that occurs through the protection of sensitive cell sites from Cd-induced oxidation, namely membrane lipids and proteins, rather than the improvement of recycling capabilities of the cellular reducing power.     

鹰嘴豆种子胰蛋白酶抑制剂的分离纯化与鉴定

为了寻找具有药物作用的天然胰蛋白酶抑制物,采用硫酸铵分级沉淀、离子交换层析(DEAE-纤维素52)及Sephadex G-100凝胶层析等方法, 从鹰嘴豆种子中分离出一种鹰嘴豆胰蛋白酶抑制剂（CPTI）. 研究表明：CPTI对胰蛋白酶有较强的抑制作用,抑制率达80%,而对胰凝乳蛋白酶抑制作用较弱,抑制率为32%, 对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用; 用SDS-PAGE测得CPTI近似分子质量为25.7 kD; CPTI具有较高的热稳定性,在100 ℃下加热60 min,对胰蛋白酶活性仍保持78%抑制率; Lineveaer-Burk作图得知该抑制剂属竞争性抑制类型. 动力学测定显示,来自鹰嘴豆中的CPTI对胰蛋白酶的抑制作用常数(Ki)为3.99×10^-7 mol/L.     

Thermodormancy in Celery Seeds and its Removal by Cytokinins and Gibberellins

Imbibition of celery (Apium graveolens L.) seeds at 32°C for up to 96 h lowered the upper temperature limit for germination. If this high temperature treatment was given in the light, these seeds germinated slightly earlier than those treated in the dark although the final percentage germination was similar for both treatments. The inhibitory effect of the high temperature treatment was completely removed by allowing the seeds to imbibe in a mixture of the gibberellins A₄ and A₇ (GA_4/7) and partially removed by the cytokinin N⁶-benzylaminopurine (BA). GA_4/7 was less effective when added before rather than after the high temperature treatment, whereas the opposite was true of BA. At constant temperatures more GA_4/7 was required to promote germination as the temperature was raised but addition of BA reduced the concentration of GA_4/7 required. A model is proposed for the control of celery seed germination by light and temperature through the action of endogenous cytokinins and gibberellins.     

Dynamics of Cytokinins in Apical Shoot Meristems of a Day-Neutral Tobacco during Floral Transition and Flower Formation

This study considered cytokinin distribution in tobacco (Nicotiana tabacum L.) shoot apices in distinct phases of development using immunocytochemistry and quantitative tandem mass spectrometry. In contrast to vegetative apices and flower buds, we detected no free cytokinin bases (zeatin, dihydrozeatin, or isopentenyladenine) in prefloral transition apices. We also observed a 3-fold decrease in the content of cytokinin ribosides (zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) during this transition phase. The group concluded that organ formation (e.g. leaves and flowers) is characterized by enhanced cytokinin content, in contrast to the very low endogenous cytokinin levels found in prefloral transition apices, which showed no organogenesis. The immunocytochemical analyses revealed a differing intracellular localization of the cytokinin bases. Dihydrozeatin and isopentenyladenine were mainly cytoplasmic and perinuclear, whereas zeatin showed a clear-cut nuclear labeling. To our knowledge, this is the first time that this phenomenon has been reported. Cytokinins do not seem to act as positive effectors in the prefloral transition phase in tobacco shoot apices. Furthermore, the differences in distribution at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.     

Cytokinins in Populus×robusta: Qualitative Changes during Development

Qualitative changes of cytokinins in leaves of different ages from Populus x robusta (Schneid.) have been determined, together with seasonal changes in cytokinin activity in mature leaves and xylem sap. Chromatography on Sephadex LH-20 has shown that total cytokinin activity and diversity are at a maximum in expanding leaves. As leaves age, the amount and number of cytokinins decrease, with yellow senescent leaves having only one detectable cytokinin, thought to be a glucoside. Seasonal changes were followed by chromatography of the extracts on paper in butan-2-ol: 25 % NH₄OH (4:1). Maximum cytokinin levels, due to Fraction Z (Rf 0.5–0.8), in leaves and xylem sap were found in mid-summer. Prior and subsequent to cessation of shoot elongation growth, fraction Z decreased and fraction N (Rf 0–0.2) increased to predominate in senescent leaves. Removal of the apex resulted in an increase of fraction N in leaves from decapitated plants when compared to similar leaves from intact plants. It is suggested that, once apical sink activity has ceased, cytokinins in the xylem sap are diverted into leaves and converted to a cytokinin glucoside, possibly a storage form of the hormone.     

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways.     

Isolation of Fungi from Heterodera glycines and in vitro Bioassays for Their Antagonism to Eggs

Twenty fungi were assayed in vitro for antagonism to eggs of Heterodera glycines. Eight of the fungi were isolated from cysts or eggs of H. glycines during the current study, one was isolated from Panagrellus redivivus, and eleven were obtained from other researchers or collections. The bioassays were conducted on eggs from nematodes that had been grown monoxenically on excised root tips. Phoma chrysanthemicola, one strain of Verticillium chlamydosporium, and one strain of V. lecanii caused a decrease (P < 0.01, P < 0.05, P < 0.05, respectively) in the number of viable eggs, although no hyphae were observed colonizing live eggs. Trichoderma polysporum infected live eggs but enhanced (P < 0.05) egg survival. Acremonium bacillisporum, Chaetomium sp., Drechmeria coniospora (two strains), Epicoccum sp., Exophiala jeanselmei, Fusarium sp., Neocosmospora vasinfecta, Scytalidium fulvum, Trichoderma harzianum (two strains), V. chlamydosporium (one strain), V. lecanii (three strains), and an unidentified fungus did not measurably affect egg viability, even though hyphae of five of these fungi were seen in live eggs. The bioassay provides a useful step in the selection of a biological control agent for this major nematode pest.     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: Investigations of Homologous Recombination Pathways and Their Regulation

The DNA double-strand break (DSB), arising from exposure to ionizing radiation or various chemotherapeutic agents or from replication fork collapse, is among the most dangerous of chromosomal lesions. DSBs are highly cytotoxic and can lead to translocations, deletions, duplications, or mutations if mishandled. DSBs are eliminated by either homologous recombination (HR), which uses a homologous template to guide accurate repair, or by nonhomologous end joining (NHEJ), which simply rejoins the two broken ends after damaged nucleotides have been removed. HR generates error-free repair products and is also required for generating chromosome arm crossovers between homologous chromosomes in meiotic cells. The HR reaction includes several distinct steps: resection of DNA ends, homologous DNA pairing, DNA synthesis, and processing of HR intermediates. Each occurs in a highly regulated fashion utilizing multiple protein factors. These steps are being elucidated using a combination of genetic tools, cell-based assays, and in vitro reconstitution with highly purified HR proteins. In this review, we summarize contributions from our laboratory at Yale University in understanding HR mechanisms in eukaryotic cells.     

Regulatory Roles of Cytokinins and Cytokinin Signaling in Response to Potassium Deficiency in Arabidopsis

Potassium (K) is an important plant macronutrient that has various functions throughout the whole plant over its entire life span. Cytokinins (CKs) are known to regulate macronutrient homeostasis by controlling the expression of nitrate, phosphate and sulfate transporters. Although several studies have described how CKs signal deficiencies for some macronutrients, the roles of CKs in K signaling are poorly understood. CK content has been shown to decrease under K-starved conditions. Specifically, a CK-deficient mutant was more tolerant to low K than wild-type; however, a plant with an overaccumulation of CKs was more sensitive to low K. These results suggest that K deprivation alters CK metabolism, leading to a decrease in CK content. To investigate this phenomenon further, several Arabidopsis lines, including a CK-deficient mutant and CK receptor mutants, were analyzed in low K conditions using molecular, genetic and biochemical approaches. ROS accumulation and root hair growth in low K were also influenced by CKs. CK receptor mutants lost the responsiveness to K-deficient signaling, including ROS accumulation and root hair growth, but the CK-deficient mutant accumulated more ROS and exhibited up-regulated expression of HAK5, which is a high-affinity K uptake transporter gene that is rapidly induced by low K stress in ROS- and ethylene-dependent manner in response to low K. From these results, we conclude that a reduction in CK levels subsequently allows fast and effective stimulation of low K-induced ROS accumulation, root hair growth and HAK5 expression, leading to plant adaptation to low K conditions.     

The E3 Ubiquitin Ligase cIAP1 Binds and Ubiquitinates Caspase-3 and -7 via Unique Mechanisms at Distinct Steps in Their Processing

Inhibitor of apoptosis (IAP) proteins are widely expressed throughout nature and suppress cell death under a variety of circumstances. X-linked IAP, the prototypical IAP in mammals, inhibits apoptosis largely through direct inhibition of the initiator caspase-9 and the effector caspase-3 and -7. Two additional IAP family members, cellular IAP1 (cIAP1) and cIAP2, were once thought to also inhibit caspases, but more recent studies have suggested otherwise. Here we demonstrate that cIAP1 does not significantly inhibit the proteolytic activities of effector caspases on fluorogenic or endogenous substrates. However, cIAP1 does bind to caspase-3 and -7 and does so, remarkably, at distinct steps prior to or following the removal of their prodomains, respectively. Indeed, cIAP1 bound to an exposed IAP-binding motif, AKPD, on the N terminus of the large subunit of fully mature caspase-7, whereas cIAP1 bound to partially processed caspase-3 in a manner that required its prodomain and cleavage between its large and small subunits but did not involve a classical IAP-binding motif. As a ubiquitin-protein isopeptide ligase, cIAP1 ubiquitinated caspase-3 and -7, concomitant with binding, in a reaction catalyzed by members of the UbcH5 subfamily (ubiquitin carrier protein/ubiquitin-conjugating enzymes), and in the case of caspase-3, differentially by UbcH8. Moreover, wild-type caspase-7 and a chimeric caspase-3 (bearing the AKPD motif) were degraded in vivo in a proteasome-dependent manner. Thus, cIAPs likely suppress apoptosis, at least in part, by facilitating the ubiquitination and turnover of active effector caspases in cells.Apoptosis is a programmed form of cell death that is generally executed through the activation of caspases,² cysteine proteases that exhibit an almost absolute preference for cleavage after aspartate residues. Caspases are synthesized as single-chain zymogens, containing a prodomain, as well as large and small subunits that include residues required for substrate recognition and cleavage (1). During death receptor or mitochondria-dependent apoptosis, the long prodomain-containing initiator caspase-8/10 and -9 are recruited via their adapter proteins, Fas-associated death domain and apoptotic protease-activating factor-1 (Apaf-1), to multimeric caspase-activating complexes known as the death-inducing signaling complex and the apoptosome, respectively (1, 2). In the latter case, mitochondrial outer membrane permeabilization (MOMP) is required to mediate the release of cytochrome c from the intermembrane space into the cytosol, where it stimulates dATP/ATP-dependent oligomerization of Apaf-1 into the apoptosome (2). Once recruited, all initiator caspases are concentrated within their respective complexes and are thought to be activated as a result of dimerization, with concomitant autocatalytic cleavage of the activation loops that separate their large and small subunits (1). However, unlike caspase-8 and -10, caspase-9 must remain bound to the apoptosome to exhibit significant catalytic activity, so that in addition to promoting dimerization, the apoptosome may also induce conformational changes in caspase-9 that are necessary for its activation (3–6).In contrast to initiator caspases, effector caspases, such as caspase-3 and -7, contain short prodomains and exist normally as latent dimers, wherein their activation loops sterically hinder substrate access and hold the substrate binding pocket in an inactive conformation (1). Effector caspases are directly activated by caspase-8, -9, and -10, and following cleavage of caspase-3 between its large and small subunits, the two-chain p20/p12 form becomes a catalytically active heterotetramer and undergoes subsequent autocatalytic processing between its prodomain and large subunits to generate the fully mature p17/p12 form of the enzyme (7). Similarly, procaspase-7 is also activated following cleavage of its activation loop to generate its two-chain p22/p12 form; however, it remains unclear whether removal of its prodomain in cells (to generate its p19/p12 form) is accomplished primarily via autocatalysis, active caspase-3, or perhaps by serine proteases at a non-aspartate residue (8, 9). Caspase-3 and -7 exhibit significant sequence and structural homology, differing primarily in their short prodomains. Despite this fact, caspase-3 processes a wider array of protein substrates during apoptosis and is largely responsible for dismantling the cell (10). Thus, interesting questions remain regarding the physiological roles of caspase-7, whether caspase-7 activity is differentially regulated compared with caspase-3, and what structural features determine (and in some cases limit) its substrate specificity.Given the devastating consequences of unfettered caspase activation, cells have evolved mechanisms to regulate caspase activity. For example, IAPs, originally identified in baculoviruses, possess one or more baculovirus IAP repeat (BIR) domains, and at least one of the eight family members, XIAP, selectively inhibits the activities of caspase-9, -3, and -7 (1, 11). Mechanistically, the BIR3 domain in XIAP binds to an exposed IBM on the N terminus of the small subunit of processed caspase-9, situated directly above the active site, and limits the access of substrates (12, 13). By contrast, the linker region (located between the BIR1 and BIR2 domains in XIAP) lies across the active sites of caspase-3 and -7 and binds in a reverse orientation to substrates, thereby preventing cleavage of the linker while simultaneously preventing the access of substrates (14, 15). The BIR2 domain then stabilizes the linker-caspase-3 (and linker-caspase-7) interactions further by binding to an exposed IBM on the N terminus of the small subunit in the adjacent caspase dimer (14, 16). Importantly, IAP antagonists, such as Smac/DIABLO and Omi/HtrA2, are normally sequestered to the intermembrane space of mitochondria and are released (along with cytochrome c) into the cytoplasm during apoptosis. As IAP antagonists also possess IBMs, they bind to BIR domains and prevent or relieve the inhibition of caspases by IAPs (1).Previously, two additional IAP family members, cIAP1 and cIAP2, were also thought to inhibit caspases, but more recent studies suggest that these IAPs bind but do not inhibit caspases (17–19). Nevertheless, various studies have shown that cIAPs can protect cells from apoptosis, are overexpressed or mutated in some cancers, and can promote tumorigenesis (20–25), raising questions as to how these IAPs inhibit cell death or whether they have additional functions (26). XIAP, cIAP1, and cIAP2 possess C-terminal RING zinc finger domains with E3 ubiquitin (Ub) ligase activities capable of catalyzing the ubiquitination and subsequent proteasomal degradation of cellular targets, including themselves (27, 28). Moreover, cIAPs have been shown to ubiquitinate several factors, including TNF receptor-associated factor 2, the serine/threonine kinase NIK, receptor-interacting protein 1, and the IAP antagonist Smac (29–34). However, although there is some evidence to support a direct role for ubiquitination in the regulation of effector caspases by XIAP (35, 36), the role of cIAPs in this process remains unclear, particularly in vivo. We demonstrate herein that cIAP1 binds to caspase-3 and -7 at unique steps in their processing, prior to or following the removal of their prodomains, respectively. Moreover, rather than directly inhibiting these effector caspases, cIAP1 ubiquitinates them and targets them for proteasome-dependent degradation, thereby suppressing apoptosis.     

Changes in Polysomes and RNA in Developing Seeds of Chickpea (Cicer arietinum L.)

In the present investigation changes in polyribosomes and RNAs in the developing seeds of chickpea (Cicer arietinum L.) have been studied. The total polysome yield was higher in the early stages of development and declined at the later stages. The maximum level of polyribosomes was obtained at 18 days after flowering and a drastic decrease was noticed at maturity. The total RNA yield correlated with the polysomal yield. Northern hybridization with a heterologous probe (pea legumin cDNA) gave distinct hybridization with the mRNA coding for legumin proteins at different stages of seed development. Hybridization showed a direct relation between mRNA levels and seed weight accumulation.     

Galactosylononitol and Stachyose Synthesis in Seeds of Adzuki Bean : Purification and Characterization of Stachyose Synthase

Stachyose synthase (STS) (EC 2.4.1.67) was purified to homogeneity from mature seeds of adzuki bean (Vigna angularis). Electrophoresis under denaturing conditions revealed a single polypeptide of 90 kD. Size-exclusion chromatography of the purified enzyme yielded two activity peaks with apparent molecular masses of 110 and 283 kD. By isoelectric focusing and chromatofocusing the protein was separated into several active forms with isoelectric point values between pH 4.7 and 5.0. Purified STS catalyzed the transfer of the galactosyl group from galactinol to raffinose and myo-inositol. Additionally, the enzyme catalyzed the galactinol-dependent synthesis of galactosylononitol from d-ononitol. The synthesis of a galactosylcyclitol by STS is a new oberservation. Mutual competitive inhibition was observed when the enzyme was incubated with both substrates (raffinose and ononitol) simultaneously. Galactosylononitol could also substitute for galactinol in the synthesis of stachyose from raffinose. Although galactosylononitol was the less-efficient donor, the Michaelis constant value for raffinose was lower in the presence of galactosylononitol (13.2 mm) compared with that obtained in the presence of galactinol (38.6 mm). Our results indicate that STS catalyzes the biosynthesis of galactosylononitol, but may also mediate a redistribution of galactosyl residues from galactosylononitol to stachyose.     

Interactions between Abscisic Acid, Gibberellins and Cytokinins in Grand Rapids Lettuce Seeds Germination

Experiments with Grand Rapids lettuce seeds (Lactuca sativa L.) maintained in darkness or irradiated with red light have shown that the inhibition of germination induced by low concentrations of ABA (2, 4, 6 μM) could be overcome by gibberellins (GA₃ or GA₄). The same results were obtained, although to a lesser extent, under the influence of two out of the four cytokinins tested (K and BAP) for seeds maintained in darkness. To suppress the block induced by higher concentrations of ABA (for example 8 μM), it was necessary to apply a cytokinin (K, BAP, Z or 2iP) and a gibberellin (GA₄ or GA₃) simultaneously, or a cytokinin following a red light treatment. Experiments conducted in darkness in which ABA (8 μM) was applied together with a cytokinin (BAP) and a gibberellin (GA₄) showed that the gibberellin and the cytokinin played similar roles towards each other and towards ABA.     

AtSOFL1 and AtSOFL2 Act Redundantly as Positive Modulators of the Endogenous Content of Specific Cytokinins in Arabidopsis

Background

Although cytokinins have been known for decades to play important roles in the regulation of plant growth and development, our knowledge of the regulatory mechanism of endogenous content of specific cytokinins remains limited.

Methodology/Principal Findings

Here, we characterized two SOB five-like (SOFL) genes, AtSOFL1 and AtSOFL2, in Arabidopsis (Arabidopsis thaliana) and showed that they acted redundantly in regulating specific cytokinin levels. Analysis of the translational fusion AtSOFL1:AtSOFL1-GUS and AtSOFL2:AtSOFL2-GUS indicated that AtSOFL1 and AtSOFL2 exhibited similar expression patterns. Both proteins were predominantly expressed in the vascular tissues of developing leaves, flowers and siliques, but barely detectable in roots and stems. Overexpression of either AtSOFL1 or AtSOFL2 led to increased cytokinin content and obvious corresponding mutant phenotypes for both transgenic seedlings and adult plants. In addition, overexpression and site-directed mutagenesis experiments demonstrated that the SOFL domains are necessary for AtSOFL2''s overexpression phenotypes. Silencing or disrupting either AtSOFL1 or AtSOFL2 caused no obvious developmental defects. Endogenous cytokinin analysis, however, revealed that compared to the wild type control, the SOFL1-RNAi62 sofl2-1 double mutant accumulated lower levels of trans-zeatin riboside monophosphate (tZRMP) and N⁶-(Δ²-isopentenyl)adenosine monophosphate (iPRMP), which are biosynthetic intermediates of bioactive cytokinins. The double mutant also displayed decreased response to exogenous cytokinin in both callus-formation and inhibition-of-hypocotyl-elongation assays.

Conclusions/Significance

Taken together, our data suggest that in plants AtSOFL1 and AtSOFL2 work redundantly as positive modulators in the fine-tuning of specific cytokinin levels as well as responsiveness.     

Discrimination Against 13CO2 in Leaves,Pod Walls,and Seeds of Water-stressed Chickpea

The rate of photosynthesis (P _N) in leaves and pods as well as carbon isotope content in leaves, pod walls, and seeds was measured in well-watered (WW) and water-stressed (WS) chickpea plants. The P _N, on an area basis, was negligible in pods compared to leaves and was reduced by water stress (by 26%) only in leaves. WS pod walls and seeds discriminated less against ¹³CO₂ than did the controls. This response was not observed for leaves as is usually the case. Pod walls and seeds discriminated less against ¹³CO₂ than did leaves in both WW and WS plants. Measurement of carbon isotope composition in pods may be a more sensitive tool for assessing the impact of water stress on long-term assimilation than is the instantaneous measurement of gas exchange rates.