PtABI3 impinges on the growth and differentiation of embryonic leaves during bud set in poplar

Abstract

The Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3) protein plays a crucial role during late seed development and has an additional function at the vegetative meristem, particularly during periods of growth-arresting conditions and quiescence. Here, we show that the ABI3 homolog of poplar (PtABI3) is expressed in buds during natural bud set. Expression occurs clearly after perception of the critical daylength that initiates bud set and dormancy in poplar. In short-day conditions mimicking natural bud set, the expression of a chimeric PtABI3::beta-glucuronidase (GUS) gene occurred in those organs and cells of the apex that grow actively but will undergo arrest: the young embryonic leaves, the subapical meristem, and the procambial strands. If PtABI3 is overexpressed or downregulated, bud development in short-day conditions is altered. Constitutive overexpression of PtABI3 resulted in apical buds with large embryonic leaves and small stipules, whereas in antisense lines, bud scales were large and leaves were small. Thus, PtABI3 influences the size and ratio of embryonic leaves and bud scales/stipules that differentiate from the primordia under short-day conditions. These observations, together with the expression of PtABI3::GUS in embryonic leaves but not in bud scales/stipules, support the idea that wild-type PtABI3 is required for the relative growth rate and differentiation of embryonic leaves inside the bud. These experiments reveal that ABI3 plays a role in the cellular differentiation of vegetative tissues, in addition to its function in seeds.

Figure 1.

Seasonal Expression of PtABI3, PtABI1b, and PtACT in Poplar. Expression was analyzed in seeds (7 and 27 days after pollination), in buds (from August until March), at bud break (BB), in young leaves (L; harvested in April), and in xylem (X). Fragments of the genes were amplified by reverse transcriptase–mediated (RT)–PCR using gene-specific primers, hybridized with ³²P-labeled probes of the respective genes, and visualized through autoradiography. Actin fragments could be amplified from all samples, indicating that cDNA was generated successfully, although the amount of actin transcripts varied and was particularly low in January. This experiment was repeated with similar results with independent bud material.

Figure 2.

P. tremula × P. alba Plants during Short Day–Induced Bud Set (8 h/Day Photoperiod). (A) Close-up of the apex of an actively growing wild-type plant under long-day conditions. Two stipules are formed together with each leaf but are too small to be seen at this stage. (B) Close-up of the apex of a bud-setting wild-type plant with formation of the first bud scales after 4 weeks under short-day conditions. Arrows point to the initiated bud scales. (C) Close-up of the apical bud of a wild-type plant grown for 6 weeks under short-day conditions. Arrows point to the bud scales that have enlarged and started to encase the embryonic leaves. (D) Apical part of a wild-type plant grown under long-day conditions. (E) Apical part of a wild-type plant grown for 6 weeks under short-day conditions. The arrow marks the last mature leaf below the bud. The inset shows an enlargement of the apex. (F) Apical part of a PtABI3-overexpressing plant grown for 6 weeks under short-day conditions. The arrow marks the last mature leaf below the bud. The inset shows an enlargement of the apex. Bars in (A) to (C) = 1 mm.

Figure 3.

Schemes of the Fate of Successive Primordia during Short Day–Induced Bud Set in Poplar and of the Morphology of the Bud at Accomplished Bud Set. (A) Dormancy induction is illustrated with an apex at the perception of the critical daylength (top left) and a bud after bud set has been completed (top right and bottom). The planes of sectioning for longitudinal (L) and radial (R) sections are indicated by solid lines, and the apical meristem is indicated by the dashed line through the bud. At the perception of the critical daylength, the apex of the plant already contains committed primordia that will develop into leaves (primordia 5 to 0). Only those primordia initiated after the perception of the critical daylength will change morphogenetic fate (primordium −1 and younger ones). After bud set has been completed, the following units of organs are found inside a P. tremula × P. alba bud (in successive order from outside to inside): one abortive bud scale leaf, consisting of two sometimes fused bud scales (primordium −1; absl); one incipient bud scale leaf, consisting of two bud scales and a small embryonic leaf lamina (primordium −2; ibsl [not always found]); approximately seven embryonic leaves sensu stricto, consisting of two stipules and an embryonic leaf lamina (primordia −3 to −9; el); and approximately two not yet developed leaf primordia (primordia −10 and −11; lp). (B) Morphology of the bud after bud set has been completed. As a result of the helical phyllotaxis of the bud, radial sections (R) reveal approximately one-third more organs than longitudinal sections (L). The plane of radial sections is situated just above the apical meristem (see bud in [A]); therefore, the enlarged stipules contributing to bud scales (primordia 1 and 0) usually are not revealed. In the schemes of radial and longitudinal sections, embryonic leaf laminae are dark gray and stipules and bud scales are light gray. In the radial section, organs 0 and −1 out of primordia 0 and −1 are numbered (to conform with the primordium numbers in [A]) to illustrate the fact that at the end of bud formation, a different number of organs may persist from different primordia.

Figure 4.

Histochemical Localization of PtABI3::GUS Expression in P. tremula × P. alba after 22 to 42 days under Short-Day Conditions (See Text). (A) to (C) PtABI3::GUS expression in apical buds under bright-field optics. The arrowhead in (B) points to a group of subapical cells expressing PtABI3::GUS. (D) PtABI3::GUS expression in an apical bud under dark-field optics. (E) PtABI3::GUS expression in an axillary bud under dark-field optics. (F) to (H) PtABI3::GUS expression in the first internode beneath the apical bud under dark-field optics. Arrowheads in (H) point to single PtABI3::GUS–expressing cells containing oxalate crystals. The scheme in Figure 3B indicates the plane of sectioning through the bud as well as the type and order of organs seen in longitudinal sections. Similar results were obtained with seven independent PtABI3::GUS lines (see Methods). bs, bud scales; cz, cambial zone; el, embryonic leaves; phl, phloem; ps, procambial strand; st, stipules; xyl, xylem. Bars = 100 μm.

Figure 5.

Morphology of Apical Buds of P. tremula × P. alba after 6 Weeks of Short-Day Treatment in Wild-Type, PtABI3-Overexpressing, and PtABI3 Antisense Lines. (A) Wild-type line, longitudinal section. (B) PtABI3-overexpressing line, longitudinal section. (C) PtABI3 antisense line, longitudinal section. (D) Actively growing wild-type plant (long-day plant) for comparison, radial section. (E) Wild-type line, radial section. (F) PtABI3-overexpressing line, radial section. (G) PtABI3 antisense line, radial section. Resinous inclusions give rise to greenish blue staining in the bud scales of antisense lines and are highlighted by arrowheads. Bud scales and stipules can be distinguished from embryonic leaves by their lighter blue staining with toluidine blue and by their morphology. For an easier interpretation of the micrographs, corresponding schemes with identical lowercase letters ([a] to [g]) indicate the types of organs seen in the sections. Bud scales and stipules are shown in light gray, and embryonic leaves and leaf primordia are shown in dark gray. Similar results were obtained with five PtABI3-overexpressing and four PtABI3 antisense lines. AS, PtABI3 antisense; LD, long day; OE, PtABI3 overexpression; SD, short day; wt, wild type. Bars = 100 μm.

Figure 6.

Number of Leaf-Like and Stipule/Scale-Like Organs in Apical Buds of P. tremula × P. alba after 6 Weeks of Short-Day Treatment in Wild-Type, PtABI3-Overexpressing (S8, S11, and S16), and Antisense (AS3, AS18, AS22, and AS29) Lines. The number of organs irrespective of size was counted in serial microscopic sections of a bud and is expressed as average number ± se. The number of buds analyzed per line is given in brackets. wt, wild type. (A) Number of leaf-like (embryonic leaves; dark gray) and stipule/scale-like (bud scales and stipules; light gray) organs per bud, given with decreasing number of leaf-like organs. (B) Index of stipules per leaf as calculated by dividing the number of stipules by the number of embryonic leaves, given with decreasing index.

Figure 7.

Transmission Electron Microscopy of Embryonic Leaves and Bud Scales in Apical Buds of P. tremula × P. alba after 6 Weeks of Short-Day Treatment in Wild-Type and PtABI3-Overexpressing Lines. (A) Plastids with thylakoid membranes in embryonic leaves of the wild-type line. (B) Small plastids with thylakoid membranes in stipule/scale-like organs of the wild-type line. Phenolic compounds give rise to the dark staining in the vacuole. (C) Amyloplasts with thylakoid membranes in embryonic leaves of a PtABI3-overexpressing line. (D) Amyloplasts with thylakoid membranes in stipule/scale-like organs of a PtABI3-overexpressing line. Similar results were obtained with two PtABI3-overexpressing lines. cw, cell wall; nucl, nucleus; pl, plastid; st, starch; vac, vacuole. Bars = 1 μm.

Figure 8.

Morphology of the First Internode Subtending the Bud after 6 Weeks of Short-Day Treatment of Wild-Type and PtABI3-Overexpressing P. tremula × P. alba Plants. (A) and (C) Wild-type plants. (B) and (D) PtABI3-overexpressing P. tremula × P. alba plants. Five individual plants of the wild type, two PtABI3-overexpressing plants, and two PtABI3 antisense plants (sections not shown) were investigated. fib, phloem fibers; phl, phloem; ves, xylem vessels; xyl, xylem. Bars = 100 μm.

Figure 9.

Expression of Endogenous PtABI3 and Transgenes in Apical Buds of P. tremula × P. alba Grown for 4 Weeks under Short Days of 8 h/Day Photoperiod. Fragments were amplified by RT-PCR with specific primers and ³³P-labeled dATP. Products were separated in denaturing polyacrylamide gels and visualized by autoradiography. Primers for PtABI3 amplify both the endogenous and transgene transcripts. Primers for PtABI3 (s) and PtABI3 (as) amplify the sense and antisense transgene transcripts, respectively (see Methods). WT, wild type.

Figure 10.

Evolution of ABA (A) and IAA (B) Concentrations in the Apical Bud (Circles, Top Line) and the First Leaf Subtending the Bud (Rectangles, Bottom Line) during Short Day–Induced Bud Set. Values given are means ± se of three individual plants. This experiment was repeated twice with similar results. Although the magnitude of ABA concentration was different, a similar increase was observed after 24 days.