The boron efflux transporter ROTTEN EAR is required for maize inflorescence development and fertility

Abstract

Although boron has a relatively low natural abundance, it is an essential plant micronutrient. Boron deficiencies cause major crop losses in several areas of the world, affecting reproduction and yield in diverse plant species. Despite the importance of boron in crop productivity, surprisingly little is known about its effects on developing reproductive organs. We isolated a maize (Zea mays) mutant, called rotten ear (rte), that shows distinct defects in vegetative and reproductive development, eventually causing widespread sterility in its inflorescences, the tassel and the ear. Positional cloning revealed that rte encodes a membrane-localized boron efflux transporter, co-orthologous to the Arabidopsis thaliana BOR1 protein. Depending on the availability of boron in the soil, rte plants show a wide range of phenotypic defects that can be fully rescued by supplementing the soil with exogenous boric acid, indicating that rte is crucial for boron transport into aerial tissues. rte is expressed in cells surrounding the xylem in both vegetative and reproductive tissues and is required for meristem activity and organ development. We show that low boron supply to the inflorescences results in widespread defects in cell and cell wall integrity, highlighting the structural importance of boron in the formation of fully fertile reproductive organs.

Figure 1.

The rte Mutant Is Affected in Tassel and Ear Development. (A) to (D) Wild-type and rte-1 tassels in two different genetic backgrounds, Mo17 ([A] and [B]) and A619 ([C] and [D]). CS, central spike; TB, tassel branch. (E) Quantitative measurements of tassel defects in rte-1 in the Mo17 background. t test, *P < 0.0001; t test, **P < 0.05; n = 9. Error bars indicate sd. (F) Wild-type fertilized ear. Arrowheads point to mature silks. (G) to (I) rte-1 and rte-2 ears. The majority of the silks have been removed to show the overall ear architecture. In (G) and (H), two representative rte-1 ears in Mo17 and A619 backgrounds, respectively, are shown. In (I), rte-2 mutant ear in the A619 background showing a shriveled inflorescence tip and sterile florets.

Figure 2.

Scanning Electron Microscopy Analysis of Immature rte Ears. (A), (B), (G), and (H) In the early stages of ear primordia development, no visible differences are noted between wild-type ([A] and [B]) and rte ([G] and [H]) ears at 0.5 and 1.5 cm length. (C) to (F) and (I) to (L) Later in development (>2 cm), clear differences can be seen between the wild type ([C] to [F]) and the rte mutant ([I] to [L]). The rte phenotype is particularly evident in the ear tip where the inflorescence meristem collapses, floral meristems and floral organs appear malformed (I), and the overall tissue eventually wrinkles ([J] to [L]). FMs, floral meristems. Bars = 100μm.

Figure 3.

Positional Cloning of rte. (A) Schematic representation of the map-based cloning of the rte gene located on chromosome 1. (B) rte gene structure and location of mutant alleles. Rectangles represent exons, and the black arrow represents the transcription start site. [See online article for color version of this figure.]

Figure 4.

Rescue of the rte Phenotype by Exogenous Boric Acid Application. (A), (C), (E), and (J) Tassels of wild-type and rte-1 plants at different boric acid concentrations. (B), (D), and (F) Leaves of wild-type and rte-1 plants at different boric acid concentrations. (G) Successful fertilization of a wild-type ear using pollen from an rte-1 tassel treated with 100 μM boric acid. (H) and (I) Fertile pollen grains in pollen germination media from rte-1 plants rescued with 100 μM boric acid (H) and fertile pollen grains from the wild type (I). (K) and (L) Ears from rte-1 plants grown in different boric acid concentrations. (M) Successful cross obtained using an ear from a rte-1 plant treated with 200 μM boric acid and wild-type pollen. Bars = 2 cm.

Figure 5.

Bayesian Consensus Phylogram of 94 RTE-Like Proteins from Land Plants. Thick branches indicate ≥95% posterior probability. Black, eudicots; blue, monocots; red, lycophyte; green, mosses; orange, green alga. Taxonomic family abbreviations: BRAS, Brassicaceae; CARI, Caricaceae; CHLA, Chlamydomonaceae; CUCU, Cucurbitaceae; EUPH, Euphorbiaceae; FABA, Fabaceae; FUMA, Fumariaceae; PHYR, Phyrmaceae; POAC, Poaceae; SALI, Salicaceae; SELA, Selaginellaceae; VITA, Vitaceae. Maize Zm-RTE and Arabidopsis At-BOR1 are highlighted in bold, and the RTE/BOR1 clade is marked.

Figure 6.

RTE Rescues the Arabidopsis bor1 Mutant and Localizes to the Plasma Membrane. (A) to (D) The vegetative bor1-3 phenotype is rescued by the 35S_pro:rte construct but not by the 35S_pro:rte L361 construct. Arrowheads point to defective leaves. (E) to (H) The reproductive defects of bor1-3 (reduced apical dominance, floral defects, and infertility) are also rescued by the 35S_pro:rte construct. (F) is a close-up picture of bor1-3 flower buds. (I) to (K) Confocal images of RTE-YFP, RTE-L361-YFP, and YFP proteins expressed in tobacco cells. Arrowheads point to putative endosomal compartments.

Figure 7.

Expression Analysis of rte. (A) Quantitative RT-PCR of rte in different maize tissues. The y axis shows the fold change relative to leaf expression levels. Error bars in the figure represent se of the mean. (B) to (E) In situ hybridizations of rte in stems, leaves, and inflorescences of wild-type plants. (B) Cross section of a seedling stem showing rte expression in vascular cells. Inset: close-up of a vascular bundle; strong expression is observed in cells surrounding the xylem (arrowheads). (C) and (D) Cross sections of leaves hybridized with rte antisense (C) and negative control sense probes (D). (E) Longitudinal section of an immature ear. rte transcripts are localized to vasculature (arrowheads). Bars = 200 μm.

Figure 8.

TEM Analysis of rte Ears. Zoomed-out view of cells ([A], [B], [E], and [G]) and close-up views of cell walls ([C], [D], [F], and [H]) in wild-type and rte ears. Stars indicate abnormal and swollen cell walls; arrowheads indicate shrinking cells and plasma membranes. Bars = 2 μm in ([A], [B], [E], and [G]) and 0.5 μm in ([C], [D], [F], and [H]). N, nucleus; CW, cell wall.