Joel P. Stafstrom

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.

Dormancy-associated gene expression in pea axillary buds. Cloning and expression of PsDRM1 and PsDRM2

Abstract. 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.

Molecular cloning and expression of a MAP kinase homologue from pea

The cdc2 kinases are important cell cycle regulators in all eukaryotes. MAP kinases, a closely related family of protein kinases, are involved in cell cycle regulation in yeasts and vertebrates, but previously have not been documented in plants. We used PCR to amplify Brassica napus DNA sequences using primers corresponding to amino sequences that are common to all known protein kinases. One sequence was highly similar to KSS1, a MAP kinase from Saccharomyces cerevisiae. This sequence was used to isolate a full-length MAP kinase-like clone from a pea cDNA library. The pea clone, called D5, shared approximately 50% amino acid identity with MAP kinases from yeasts and vertebrates and about 41% identity with plant cdc2 kinases. An expression protein encoded by D5 was recognized by an antiserum specific to human MAP kinases (ERKs). Messenger RNA corresponding to D5 was present at similar levels in all tissues examined, without regard to whether cell division or elongation were occurring in those tissues.

Axillary Bud Development in Pea: Apical Dominance, Growth Cycles, Hormonal Regulation and Plant Architecture

Apical meristems of the shoot and root are responsible for building the vegetative plant body. Precise patterns of cell division and differentiation at the shoot apex result in the iterative formation of modules or phytomers comprised of a leaf, an axillary bud, a node and an internode (Sussex, 1989). Axillary bud meristems have the same potential for growth and development as the terminal meristem; however, most buds remain dormant and never realize this potential. As any gardener or keeper of house plants knows, removing the terminal bud promotes the growth of dormant axillary buds and gives rise to bushier plants. Control of axillary bud growth by the terminal bud is called apical dominance.

Development of supernumerary buds from the axillary meristem of pea, Pisum sativum (Fabaceae)

Leaf axils of higher plants commonly contain vegetative axillary buds, which are derived from an axillary meristem. The persistence and continued organogenic activity of the axillary meristem has been studied experimentally in only two species. Pea (Pisum sativum L.) leaf axils contain up to four preformed axillary buds. Decapitating plants above Node 5 promoted the development of preformed buds at all nodes. Buds at each node were removed as soon as they began to grow. These manipulations eventually led to the growth of all the preformed buds and promoted the development of supernumerary buds in the axil of each leaf. Adventitious buds were not observed anywhere on these plants. Examination of leaf axil anatomy and external morphology indicated that new buds were derived from the axillary meristem. The axillary meristem at Node 2 was capable of forming at least six supernumerary buds. ‘Alaska’ plants (Rms-2) exhibited strong apical dominance whereas WL5951 plants (rms-2) contained branches at several nodes. Patterns of bud organogenesis and development were similar in both cultivars.

EXPRESSION PATTERNS OF ARABIDOPSIS DRG GENES: PROMOTER-GUS FUSIONS, QUANTITATIVE REAL-TIME PCR, AND PATTERNS OF PROTEIN ACCUMULATION IN RESPONSE TO ENVIRONMENTAL STRESSES

DRGs are very highly conserved GTP‐binding proteins. All eukaryotes contain DRG1 and DRG2 orthologs. Arabidopsis has three DRGs: AtDRG1 (At4g39520), AtDRG2 (At1g17470), and AtDRG3 (At1g72660). DRG2 and DRG3 encode proteins that are 95% identical; identity between DRG1 and DRG2/3 is 55%. The focus of this article is expression of Arabidopsis DRGs. DRG1 and DRG2 promoter‐GUS constructs showed similar spatial expression in seedlings and mature organs, but gene‐specific differences were noted. Quantitative real‐time PCR experiments indicated similar levels of DRG1 and DRG2 mRNA accumulation in most tissues. DRG3 transcripts were very low in all tissues. Heat stress at 37°C led to a 10‐fold increase in DRG1 transcripts and a 1000‐fold increase in DRG3 transcripts. DRG1 antibodies recognized a 43‐kD protein, and DRG2 antibodies recognized bands at 30, 43, and 45 kD. Plants were exposed to stresses (salt, heat, cold, UV light, osmotic, and other stresses) and examined by Western blotting. Only heat stress caused detectable changes. Heat did not affect DRG1, but DRG2 and a 72‐kD protein recognized by DRG2 antibodies both increased. The modest changes in DRG mRNA and protein levels seen here suggest that other types of regulation, such as altered subcellular localization, may be important for their cellular functions.

Nucleotide Sequence of Four Ribosomal Protein L27 cDNAs from Growing Axillary Buds of Pea

The small and large subunits of the eukaryotic ribosome contain a total of three to four rRNA molecules and 70 to 80 ribosomal proteins, which are required in stoichiometric amounts (Mager, 1988). Expression of ribosomal protein genes in plants has been correlated with active growth and cell division in a variety of tissues and organs, including auxin-treated soybean hypocotyls (Gantt and Key, 19851, developing maize embryos (Larkin et al., 1989), cytokinintreated soybean cell cultures (Crowell et al., 19901, and tomato shoot apical meristems (Flemming et al., 1993). Ribosomal protein genes in plants and other organisms generally are expressed coordinately (Gantt and Key, 1985; Mager, 1988). Some ribosomal proteins are encoded by single-copy genes (Hwang and Goodman, 1993), whereas otliers are encoded by small gene families. For example, the Brussica rpSl5a (for ribosomal protein S15a) gene family contains two expressed members that encode identical polypeptides (Bonham-Smith et al., 1992). Based on Southern blot analysis, the maize rpS14 gene family may contain as many as six members, three of which have been shown to be expressed; the two rpS14 cDNA clones that have been sequenced encode peptides that differ in size and sequence (Larkin et al., 1989). We show here that the pea (Pisum sativum) rpL27 family contains at least five expressed members, the largest ribosomal protein gene family from any plant for which sequence data are available. We previously isolated an rpL27 cDNA from growing axillary buds of pea (rpL27-1; Stafstrom and Sussex, 1992). When compared to a rat gene, rpL27-1 lacked a single nucleotide at the 5’ end of the coding sequence (TG instead of ATG). To identify the complete coding sequence corresponding to this gene, two different cDNA libraries were screened using rpL27-1 as a probe. Four additional clones were isolated; DNA sequences of these clones were distinct from each other and from rpL27-1 (Table I). Three of these clones (rpL27-3, rpL274, and rpL27-5) contain an ATG at the expected position. In addition, rpL27-3 and rpL27-5 contain an in-frame stop codon at position -12. Nucleotide sequence identity among the five rpL27 clones is greater than 96%. The deduced amino acid sequences of the four (nearly) full-length clones differ from each other at one to three residues (rpL27-2, which encodes only the C-terminal49 amino acids,

TCA MICROSATELLITE REPEATS IN THE 59UTR OF THE Sat5 GENE OF WILD AND CULTIVATED ACCESSIONS OF PISUM AND OF FOUR CLOSELY RELATED GENERA 1

PsSat5, a cDNA clone from Pisum sativum cv. Alaska, contained a microsatellite consisting of 15 TCA repeats within the 5′UTR. This SSR microsatellite was immediately upstream of the presumptive ATG start codon. PCR amplification of genomic DNA from cv. Alaska yielded an identical sequence. This repeat region was analyzed from 10 additional wild and cultivated accessions of Pisum and from four closely related genera (Cicer, Lathyrus, Lens, and Vicia). All of the sequences were generally quite similar, with the exception of the number of TCA repeats (region 3) and a short domain immediately upstream of the repeats (region 2). Pisum humile‐northern and Lathyrus each contained four TCA repeats (the fewest number observed). Similar to P. sativum‐Alaska and other cultivated peas, Lens contained 15 repeats, the largest number observed. The number of TCA repeats does not appear to correspond to the established phylogeny of these accessions, so the cellular events that generated variable numbers of repeats probably have occurred repeatedly and have involved both expansions and contractions in the number of repeats. The mRNA corresponding to PsSat5 was found in all tissues of P. sativum‐Alaska that were examined, but its abundance in leaves and sepals was low. The level of expression was similar in growing and nongrowing stems, roots, and axillary buds. Northern blot analysis of stems and leaves of all 15 accessions showed similar levels of expression. Therefore, there is not a clear correlation between the number of TCA repeats in the 5′UTR and the level of Sat5 expression.

Association of DRG1 and DRG2 with Ribosomes from Pea, Arabidopsis and Yeast

DRGs are highly conserved GTP binding proteins. All eukaryotes examined contain DRG1 and DRG2 orthologs. The first experimental evidence for GTP binding by a plant DRG1 protein and by DRG2 from any organism is presented. DRG1 antibodies recognized a single ∼43‐kDa band in plant tissues, whereas DRG2 antibodies recognized ∼45‐, 43‐, and 30‐kDa bands. An in vitro transcription and translation assay suggested that the 45‐kDa band represents full‐length DRG2 and that the smaller bands are specific proteolytic products. Homogenates from pea roots and root apices were used to produce fractions enriched in cytosolic and microsomal monosomes and polysomes. DRG1 and the 45‐ and 43‐kDa DRG2 bands occurred in the cytosol and associated with cytosolic monosomes. In contrast, the 30‐kDa form of DRG2 was strongly enriched in polysome fractions. Thus, DRG1 and the larger forms of DRG2 may be involved in translational initiation, and the 30‐kDa form of DRG2 may be involved in translational elongation. DRG1 and the 45‐ and 43‐kDa forms of DRG2 can reassociate with ribosomes in vitro, a process that is partially inhibited by GTP‐γ‐S. Cells expressing FLAG‐tagged ribosomal proteins from transgenic lines of Arabidopsis and yeast also demonstrated DRG‐ribosome interactions.