barren inflorescence2 Encodes a co-ortholog of the PINOID serine/threonine kinase and is required for organogenesis during inflorescence and vegetative development in maize

Abstract

Organogenesis in plants is controlled by meristems. Axillary meristems, which give rise to branches and flowers, play a critical role in plant architecture and reproduction. Maize (Zea mays) and rice (Oryza sativa) have additional types of axillary meristems in the inflorescence compared to Arabidopsis (Arabidopsis thaliana) and thus provide an excellent model system to study axillary meristem initiation. Previously, we characterized the barren inflorescence2 (bif2) mutant in maize and showed that bif2 plays a key role in axillary meristem and lateral primordia initiation in the inflorescence. In this article, we cloned bif2 by transposon tagging. Isolation of bif2-like genes from seven other grasses, along with phylogenetic analysis, showed that bif2 is a co-ortholog of PINOID (PID), which regulates auxin transport in Arabidopsis. Expression analysis showed that bif2 is expressed in all axillary meristems and lateral primordia during inflorescence and vegetative development in maize and rice. Further phenotypic analysis of bif2 mutants in maize illustrates additional roles of bif2 during vegetative development. We propose that bif2/PID sequence and expression are conserved between grasses and Arabidopsis, attesting to the important role they play in development. We provide further support that bif2, and by analogy PID, is required for initiation of both axillary meristems and lateral primordia.

Figure 1.

Cloning of bif2 by transposon tagging, A, Normal tassel with several long lateral branches at the base of the main spike. Spikelet pairs cover the branches and main spike. B, bif2 tassel with no long lateral branches. A few single spikelets are visible on the main spike. C, DNA gel-blot analysis of EcoRI-digested genomic DNA probed with Mu1. An 8.5-kb band (arrowed) is present in bif2 mutants (b) and absent in normal cousins (+). D, Schematic of the bif2 gene showing exons as large rectangles and UTRs as narrow rectangles. The 11 subdomains of the kinase catalytic domain are shaded. The positions of transposon insertions are indicated with triangles. Probes used in Southern and northern hybridization (0.7-kb EcoRI-NotI restriction fragment) and RNA in situ hybridization (0.6-kb PstI restriction fragment) are indicated as lines beneath the map. E, Multiple sequence alignment of PID (Arabidopsis), PsPK2 (pea), bif2 (maize), and Osbif2 (rice). The conserved kinase subdomains are indicated with a line over the alignment. The insertion domain required for localization of PID is indicated with a dotted line over the alignment. The amino acids shown to be important for activation of PID by PDK1 are indicated with asterisks.

Figure 2.

Phylogenetic analysis of bif2 homologs from grasses and dicots. Bayesian consensus phylogram of 39 bif2-like protein kinases from diverse angiosperms rooted using Physcomitrella patens PHOTOTROPIN1 (PpPHOTB1) and Adiantum capillus-veneris PHOTOTROPIN2 (Ac-vPHOT2). Thick branches are supported by 1.00 CC and medium thickness branches are supported by >0.95 CC. At = Arabidopsis (Brassicaceae); As = Avena sativa (Poaceae); Br = Brassica rapa (Brassicaceae); Cs = Cucumis sativus (Curcubitaceae); Lh = Lithachne humilus (Poaceae); Os = Oryza sativa (Poaceae); Pg = Pennisetum glaucum (Poaceae); Pm = Panicum miliaceum (Poaceae); Ps = Pisum sativum (Fabaceae); Pv = Phaseolis vulgaris (Fabaceae); Sb = Solanum berthaultii (Solanaceae); Se = Solanum (Lycopersicon) esculentum (Solanaceae); Si = Setaria italica (Poaceae); St = Solanum tuberosum (Solanaceae); Sv = Setaria viridis (Poaceae); Zm = Zea mays (Poaceae).

Figure 3.

bif2 is expressed in axillary meristems and lateral primordia in maize. A, RNA gel-blot analysis using 10 μg total RNA probed with the 0.7-kb EcoRI-NotI restriction fragment. bif2 is highly expressed in immature tassels (7 mm) and ears (7 mm) and declines in expression in older ears (10 and 20 mm). bif2 is not detectable in roots, coleoptile, immature pale green leaf (im leaf), mature dark green leaf (mat leaf), and vegetative apices (veg) at this level of detection. Ethidium bromide-stained gel is shown as a loading control. B to H, RNA in situ hybridization using DIG-labeled antisense (B,C, E–H) or sense (D) probe of bif2. B, bif2 expression in an immature tassel in spikelet pair meristems (spm) in the axil of bracts (br) and on the flanks of the inflorescence meristem (im) before spm arise. C, Tassel branch showing bif2 expression in spikelet pair meristems (spm) on the flanks of the branch meristem (bm). D, Control section showing a tassel branch probed with the sense probe of bif2 has no detectable signal. E, Ear spikelet showing bif2 expression in floral meristem (fm) of lower floret and floral organs (gynoecium [g]; stamens[st]) of upper floret. F, Older ear spikelet showing bif2 expression in developing floral organs of lower floret (lf) and in lemma (l), palea (p), gynoecium (g), and vasculature (v) of upper floret (uf). Note expression in younger inner glume (ig), which is absent from older outer glume (og). G, bif2 expression in a vegetative axillary meristem (axm) and vasculature (v) of the stem and leaves. H, Vegetative apical meristem showing bif2 expression in leaf primordia (lp), including the next arising leaf primordium at plastochron 0 (P₀) and vasculature (v) of young leaves. Scale bars in B to H = 100 μm.

Figure 4.

Osbif2 is expressed in axillary meristems and lateral primordia in rice. RNA in situ hybridization expression patterns for Osbif2. A, Vegetative meristem showing Osbif2 expression in leaf primordia (lp) including the next arising leaf primordia at plastochron 0 (P₀) and vascular (v) tissue. B, Young inflorescence showing Osbif2 expression in branch meristems (bm). C, Young spikelet meristem (sm) showing Osbif2 expression on the flanks of the meristem where organs of the fertile upper floret will arise. D, Older spikelet showing Osbif2 expression in floral meristems (fm) of two sterile lower florets. E, In florets, Osbif2 expression is visible in the tips of the stamens (st) in the gynoecium (g). F, In older florets, Osbif2 expression is visible in the ovule (o). Scale bars in A to E = 100 μm; F = 50 μm.

Figure 5.

bif2 plays a role in vegetative axillary meristems. A, bif2;tb1 double-mutant analysis. Normal (N) plant with no tillers and one visible ear (arrow), bif2 mutant plant with no tillers and no ears, tb1 mutant plant with many primary and secondary tillers (the ear shoot that is masculinized is indicated with an arrowhead), bif2;tb1 double mutant with two tillers and no ears. B, Quantitative analysis of all primary branches produced from the main stem (both primary tillers and ear shoots). bif2;tb1 double mutants have a statistically significant (P < 0.001) reduction in the number of tillers and ear shoots compared to tb1. C, Quantitative analysis showing primary tiller number only. bif2;tb1 mutants have a statistically significant (P < 0.001) reduction in the number of tillers compared to tb1. [See online article for color version of this figure.]

Figure 6.

Auxin transport and vasculature defects in bif2 inflorescence stems. A, Transport of [³H]IAA in normal and bif2 inflorescence stems. Bars showing transport in normal inflorescences are in dark gray and in bif2 are light gray. Acropetal transport is signified with hatched lines and basipetal transport with solid fill. B, Cross section of a normal inflorescence stem photographed in dark field. This is representative of the inflorescence stem tissue used in the auxin transport assay (below the first branch before the insertion of the stem into the flag leaf node). C, Cross section of bif2 inflorescence stem photographed in dark field. Because bif2 usually has no branches, sections used for the auxin transport assay were taken from the basal part of the inflorescence stem, 8 cm above the insertion of the stem into the flag leaf node. Scale bar in B and C = 1 mm. [See online article for color version of this figure.]