Dormancy-associated gene expression in pea axillary buds.

Pea (Pisum sativum L. cv. Alaska) axillary buds can be stimulated to cycle between dormant and growing states. Dormant buds synthesize unique proteins and are as metabolically active as growing buds. Two cDNAs, PsDRM1 and PsDRM2, were isolated from a dormant bud library. The deduced amino acid sequence of PsDRM1 (111 residues) is 75% identical to that of an auxin-repressed strawberry clone. PsDRM2 encodes a putative protein containing 129 residues, which includes 11 repeats of the sequence [G]-GGGY[H][N] (the bracketed residues may be absent). PsDRM2 is related to cold- and ABA-stimulated clones from alfalfa. Decapitating the terminal bud rapidly stimulates dormant axillary buds to begin growing. The abundance of PsDRM1 mRNA in axillary buds declines 20-fold within 6 h of decapitation; it quickly reaccumulates when buds become dormant again. The level of PsDRM2 mRNA is about three fold lower in growing buds than in dormant buds. Expression of PsDRM1 is enhanced in other non-growing organs (roots root apices; fully-elongated stems >elongating stems), and thus is an excellent “dormancy” marker. In contrast, PsDRM2 expression is not dormancy-associated in other organs. Received: 10 December 1997 / Accepted: 23 January 1998     

Two novel transcripts expressed in pea dormant axillary buds

To elucidate the molecular mechanism of apical dominance, the expression patterns of genes that are preferentially expressed in dormant axillary buds of pea (Pisum sativum L. cv. Alaska) seedlings were investigated. We isolated two cDNA clones, cPsAD1 and cPsAD2 whose corresponding genes were named PsAD1 and PsAD2, from a cDNA library of dormant axillary buds using the differential display method. The deduced amino acid sequence of PsAD1 contains 87 residues and is rich in glycine residues in the amino terminal region. A search of the protein databases failed to find any sequences similar to PsAD1 protein except for the glycine-rich region. Northern blot analyses showed that PsAD1 mRNA mainly accumulated in dormant axillary buds and that its amount rapidly decreased after decapitation of the terminal bud. In situ hybridization analyses indicated that PsAD1 mRNA was localized in the apical meristem, procambia, and leaf primordia in dormant axillary buds that were competent to grow out but whose growth was temporarily suspended. That is, the expression of the PsAD1 gene is closely associated with the dormancy of axillary buds. The deduced amino acid sequence of PsAD2 contains 98 amino acid residues and is not similar to those of previously characterized proteins. PsAD2 mRNA accumulated in dormant axillary buds, roots, mature leaflets and elongated stems, suggesting that PsAD2 is involved in not only the dormancy of axillary buds but also the non-growing state in various tissues.     

PsRBR1 encodes a pea retinoblastoma-related protein that is phosphorylated in axillary buds during dormancy-to-growth transition

In intact plants, cells in axillary buds are arrested at the G1 phase of the cell cycle during dormancy. In mammalian cells, the cell cycle is suppressed at the G1 phase by the activities of retinoblastoma tumor suppressor gene (RB) family proteins, depending on their phosphorylation state. Here, we report the isolation of a pea cDNA clone encoding an RB-related protein (PsRBR1, Accession No. AB012024) with a high degree of amino acid conservation in comparison with RB family proteins. PsRBR1 protein was detected as two polypeptides using an anti-PsRBR1 antibody in dormant axillary buds, whereas it was detected as three polypeptides, which were the same two polypeptides and another larger polypeptide 2 h after terminal decapitation. Both in vitro-synthesized PsPRB1 protein and lambda protein phosphatase-treated PsRBR1 protein corresponded to the smallest polypeptide detected by anti-PsRBR1 antibody, suggesting that the three polypeptides correspond to non-phosphorylated form of PsRBR1 protein, and lower- and higher-molecular mass forms of phosphorylated PsRBR1 protein. Furthermore, in vivo labeling with [³²P]-inorganic phosphate indicated that PsRBR1 protein was more phosphorylated before mRNA accumulation of cell cycle regulatory genes such as PCNA. Together these findings suggest that dormancy-to-growth transition in pea axillary buds is regulated by molecular mechanisms of cell cycle control similar to those in mammals, and that the PsRBR1 protein has an important role in suppressing the cell cycle during dormancy in axillary buds.     

Cell cycle regulation during growth-dormancy cycles in pea axillary buds

Accumulation patterns of mRNAs corresponding to histones H2A and H4, ribosomal protein genes rpL27 and rpL34, MAP kinase, cdc2 kinase and cyclin B were analyzed during growth-dormancy cycles in pea (Pisum sativum cv. Alaska) axillary buds. The level of each of these mRNAs was low in dormant buds on intact plants, increased when buds were stimulated to grow by decapitating the terminal bud, decreased when buds ceased growing and became dormant, and then increased when buds began to grow again. Flow cytometry was used to determine nuclear DNA content during these developmental transitions. Dormant buds contain G₁ and G₂ nuclei (about 3:1 ratio), but only low levels of S phase nuclei. It is hypothesized that cells in dormant buds are arrested at three points in the cell cycle, in mid-G₁, at the G₁/S boundary and near the S/G₂ boundary. Based on the accumulation of histone H2A and H4 mRNAs, which are markers for S phase, cells arrested at the G₁/S boundary enter S within one hour of decaptitation. The presence of a cell population arrested in mid-G₁ is indicated by a second peak of histone mRNA accumulation 6 h after the first peak. Based on the accumulation of cyclin B mRNA, a marker for late G₂ and mitosis, cells arrested at G₁/S begin to divide between 12 and 18 h after decapitation. A small increase in the level of cyclin B mRNA at 6 h after decapitation may represent mitosis of the cells that had been arrested near the S/G₂ boundary. Accumulation of MAP kinase, cdc2 kinase, rpL27 and rpL34 mRNAs are correlated with cell proliferation but not with a particular phase of the cell cycle.     

Acropetal disappearance of PsAD1 protein in pea axillary buds after the release of apical dominance

We recently isolated PsAD1 cDNA from pea (Pisum sativum L. cv. Alaska) seedlings, whose mRNA abundantly accumulated in dormant axillary buds and disappeared after decapitation [Madoka and Mori (2000) Plant Cell Physiol. 41: 274]. To further elucidate the function of PsAD1, we investigated the temporal and spatial distribution patterns of PsAD1 protein using Western blot and immunocytochemical analyses. Western blot analyses showed that accumulation patterns of PsAD1 protein in axillary buds after decapitation and in response to IAA and 6-benzyladenine were the same as those of PsAD1 mRNA. Immunocytochemical analyses showed that (1) PsAD1 proteins were localized in the procambia, leaf primordia, apical meristem, and secondary axillary buds in the dormant axillary bud, and this distribution was the same as that of PsAD1 mRNA, (2) PsAD1 proteins acropetally disappeared after decapitation, and (3) the growth of axillary buds occurred in the same manner. These acropetal changes occur in a manner similar to the way in which the procambium differentiates into vascular tissue. These results suggest that PsAD1 plays some role in the inhibition of growth and differentiation, or in the maintenance of the dormant state in axillary buds.     

Integrant characteristics in the deflexion of biserial buds in pea seedlings

The main epicotyledonary axis inPisum sativum exhibits a very fluctuating either left- or right-handed deflexion of its first-order laterals, but the biserial buds on the laterals of various further order reveal in this respect certain regularities according to their position on the seedling, their deflexion being very often adaxial in the vicinity of cotyledons, however, in more distant and therefore later produced laterals, as a rule, abaxial. The integrant characteristics of these relationships may become clear from their experimental shift with the aid of exogenous auxin increasing inhibitions as well as of triiodobenzoic acid blocking the transport of endogenous auxin participant also in these embryonic-growth correlations.     

Competitive canalization of PIN-dependent auxin flow from axillary buds controls pea bud outgrowth

Shoot branching is one of the major determinants of plant architecture. Polar auxin transport in stems is necessary for the control of bud outgrowth by a dominant apex. Here, we show that following decapitation in pea (Pisum sativum L.), the axillary buds establish directional auxin export by subcellular polarization of PIN auxin transporters. Apical auxin application on the decapitated stem prevents this PIN polarization and canalization of laterally applied auxin. These results support a model in which the apical and lateral auxin sources compete for primary channels of auxin transport in the stem to control the outgrowth of axillary buds.     

Control by phytochrome of C-sucrose incorporation into buds of etiolated pea seedlings

When etiolated pea epicotyls are excised immediately above the cotyledons and dipped basally into ¹⁴C-sucrose, their terminal buds respond to red light by increased growth (IG) and enhanced incorporation of sucrose (EIS). Both phenomena are phytochrome controlled, showing typical kinetics, reversal by far-red light, escape from photochemical control and limitation to leaf tissue. EIS is of greater magnitude, occurs more rapidly and is saturated by lower energies of red light than IG, suggesting its possible importance as a controlling reaction in phytochrome-mediated growth. Both IG and EIS are best shown in the presence of a long epicotyl derived from a 5 to 6-day-old seedling in the presence of about 0.1 m unlabeled sucrose in the medium.     

The antagonism of (2-Chlorethyl) trimethylammonium chloride and indole 3-acetic acid in epicotyl and axillary buds growth in the pea seedlings

The 0·03–0·06% indole-3-acetic acid (IAA) in lanoline paste smeared on the cut surface of decapitated epicotyl of the pea seedling inhibited the growth of the axillary buds of the cotyledons. 0·03–0·06% solution of (2-Chlorethyl) trimethylammonium chloride (CCC) considerably weakened the inhibitory effect of IAA when applied simultaneously to the roots. In a similar way CCC diminishes even the growth inhibition of intact pea epicotyls caused by 0·06% IAA lanoline paste. 0·03% solution of CCC administered to the roots of the decapitated pea seedlings significantly increased growth of the axillary buds. This effect may play a role in the demonstrated antagonism between the low concentration of CCC and IAA.     

The S7 ribosomal protein gene is truncated and overlaps a cytochrome c biogenesis gene in pea mitochondria

The pea mitochondrial genome contains a truncated rps7 gene lacking ca. 40 codons at its 5 terminus. This single-copy sequence is immediately downstream of and slightly overlapping an actively transcribed and edited reading frame of 744 bp (designated ccb248) homologous to the bacterial helC gene which encodes a subunit of the ABC-type heme transporter involved in cytochrome c biogenesis. This region of mitochondrial DNA appears recombinogenic, and the carboxy-termini of helC-type proteins are predicted to vary in sequence and length among plants. Sequences corresponding to the 5 coding region of rps7 were not detected elsewhere in the pea mitochondrial genome using wheat rps7 probes, and only a very short internal rps7 segment was observed in soybean mitochondrial DNA. The presence of rps7-homologous sequences in the nuclear genomes of pea and soybean is consistent with the recent transfer of a functional mitochondrial rps7 gene to the nucleus in certain plant lineages.     

Release of lateral buds from apical dominance by glyphosate in soybean and pea seedlings

Application of a sublethal dose of glyphosate (N-[phosphonomethyl]glycine) to the seedlings of soybean (Glycine max L. Merr. cv. Evans) and pea (Pisum sativum L. cv. Alaska) promoted growth of the cotyledonary and other lateral buds. The pattern of the glyphosate-induced lateral bud growth was different from that induced by decapitation. Under the experimental condition, glyphosate did not kill the apical buds. Feeding stem sections of the seedlings with radiolabeled indole-3-acetic acid ([2¹⁴C]IAA) and subsequent analysis of free [2-¹⁴C]IAA and metabolite fractions revealed that the glyphosate-treated plants had higher rates of IAA metabolism than the control plants. The treated pea plants metabolized 75% of [2-¹⁴C]IAA taken up in the 4-h incubation period compared to 46.5% for the control, an increase of 61%. The increase was small but consistent in soybean seedlings. As a result, the glyphosate-treated plants had less free IAA and ethylene than the control plants. The increase of IAA metabolism induced by glyphosate is likely to change the auxin-cytokinin balance and contribute to the release of lateral buds from apical dominance in these plants.     

Release of lateral buds from apical dominance by glyphosate in soybean and pea seedlings

Application of a sublethal dose of glyphosate (N-[phosphonomethyl]glycine) to the seedlings of soybean (Glycine max L. Merr. cv. Evans) and pea (Pisum sativum L. cv. Alaska) promoted growth of the cotyledonary and other lateral buds. The pattern of the glyphosate-induced lateral bud growth was different from that induced by decapitation. Under the experimental condition, glyphosate did not kill the apical buds. Feeding stem sections of the seedlings with radiolabeled indole-3-acetic acid ([2¹⁴C]IAA) and subsequent analysis of free [2-¹⁴C]IAA and metabolite fractions revealed that the glyphosate-treated plants had higher rates of IAA metabolism than the control plants. The treated pea plants metabolized 75% of [2-¹⁴C]IAA taken up in the 4-h incubation period compared to 46.5% for the control, an increase of 61%. The increase was small but consistent in soybean seedlings. As a result, the glyphosate-treated plants had less free IAA and ethylene than the control plants. The increase of IAA metabolism induced by glyphosate is likely to change the auxin-cytokinin balance and contribute to the release of lateral buds from apical dominance in these plants.     

Patterns of protein synthesis in dormant and growing vegetative buds of pea

Lateral buds on intact pea plants (Pisum sativum L. cv. Alaska) remain dormant until they are stimulated to develop by decapitating the terminal bud. Using two-dimensional gel electrophoresis, we have examined the protein content of terminal and lateral buds from intact plants and from plants at various times after decapitation. Silver-staining and in-vivo-labeling demonstrated very different sets of proteins. The level of expression of 18 stained and 25 labeled proteins was altered when growth was stimulated; this represents 3.4% and 9.1% of the total proteins detected by each method, respectively. Within 24 h of being stimulated, lateral buds doubled in length and their protein content was qualitatively nearly the same as that of terminal buds. Six hours after decapitation, before the onset of detectable growth, the overall pattern of protein synthesis in lateral buds was more like that of growing lateral buds or of terminal buds than that of dormant lateral buds. Direct application of N⁶-furfurylaminopurine (kinetin) to buds on intact plants stimulated their growth and resulted in the same pattern of protein synthesis as did decapitation. Inhibition of bud growth by addition of indole-3-acetic acid to the stumps of decapitated plants resulted in the synthesis of dormancy-related proteins. Lateral buds at all stages of development incorporated labeled amino acids at similar rates, indicating that metabolic activity is not a component of dormancy in these buds.Abbreviations IAA indole-3-acetic acid - IEF isoelectric focusing - KIN kinetin (N⁶-furfurylaminopurine) - SDS sodium dodecylsulfate - TCA trichloroacetic acid - 2D-PAGE two-dimensional polyacrylamide gel electrophoresis     

Purification and immunolocalization of an annexin-like protein in pea seedlings

As part of a study to identify potential targets of calcium action in plant cells, a 35-kDa, annexin-like protein was purified from pea (Pisum sativum L.) plumules by a method used to purify animal annexins. This protein, called p35, binds to a phosphatidylserine affinity column in a calcium-dependent manner and binds ⁴⁵Ca²⁺ in a dot-blot assay. Preliminary sequence data confirm a relationship for p35 with the annexin family of proteins. Polyclonal antibodies have been raised which recognize p35 in Western and dot blots. Immunofluorescence and immunogold techniques were used to study the distribution and subcellular localization of p35 in pea plumules and roots. The highest levels of immunostain were found in young developing vascular cells producing wall thickenings and in peripheral root-cap cells releasing slime. This localization in cells which are actively involved in secretion is of interest because one function suggested for the animal annexins is involvement in the mediation of exocytosis.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis The authors thank Dennis Brown (Cell Research Institute) for use of this research facilities and Zainab Ilahi and Collin Thomas for valuable technical assistance. Portions of this work were presented at the 1989 and 1990 meetings of the American Society for Cell Biology (Clark et al. 1989, 1990). This work was in part supported by a National Institute of Health Training Grant 1-T32-HD07296-01A3 and by a National Aeronautics and Space Administration grant NAGW 1519.     

Nucleoid proteins of pea chloroplasts: detection of a protein homologous to ribosomal protein.

Basic proteins were isolated from purified pea chloroplast nucleoids by acid extraction. Using RP-HPLC, the component composition of the basic proteins was studied. SDS-PAGE of major HPLC-fractions showed that the basic nucleoid proteins are heterogeneous with mol. masses of components from 17 to 30 kDa. One polypeptide with mol. mass of 28 kDa (P28) was obtained by RP-HPLC. The sequencing of three tryptic peptides of P28 (T6, T17, and T19) showed that they are homologous to the ribosomal protein L19 of Saccharomyces cerevisiae. The possible functional role of ribosomal proteins in chloroplast nucleoids is discussed.