Ontogeny of the maize shoot apical meristem

Abstract

The maize (Zea mays) shoot apical meristem (SAM) arises early in embryogenesis and functions during stem cell maintenance and organogenesis to generate all the aboveground organs of the plant. Despite its integral role in maize shoot development, little is known about the molecular mechanisms of SAM initiation. Laser microdissection of apical domains from developing maize embryos and seedlings was combined with RNA sequencing for transcriptomic analyses of SAM ontogeny. Molecular markers of key events during maize embryogenesis are described, and comprehensive transcriptional data from six stages in maize shoot development are generated. Transcriptomic profiling before and after SAM initiation indicates that organogenesis precedes stem cell maintenance in maize; analyses of the first three lateral organs elaborated from maize embryos provides insight into their homology and to the identity of the single maize cotyledon. Compared with the newly initiated SAM, the mature SAM is enriched for transcripts that function in transcriptional regulation, hormonal signaling, and transport. Comparisons of shoot meristems initiating juvenile leaves, adult leaves, and husk leaves illustrate differences in phase-specific (juvenile versus adult) and meristem-specific (SAM versus lateral meristem) transcript accumulation during maize shoot development. This study provides insight into the molecular genetics of SAM initiation and function in maize.

Figure 1.

SAM Formation and Organ Initiation. Toluidine blue O-stained embryos at key stages in embryo development (proembryo [A], transition stage [B], coleoptile stage [C], and L1 stage [D]) and the SAM (E) and lateral meristem (F) from 14-d-old seedlings. In situ hybridization showing transcript accumulation of KN1 during embryogenesis ([G] to [J]) and in the SAM (K) and lateral meristem (L) of a 14-d-old seedling. Before and after laser microdissection of maize embryos ([M] to [P] and [S] to [V]) and SAM ([Q] and [W]) and lateral meristem ([R] and [X]) from 14-d-old seedlings. The area selected for laser microdissection is outlined in blue or green. Arrows point to meristem. 1, leaf 1; c, coleoptile; esr, embryo-surrounding region; p, embryo proper; s, suspensor; sc, scutellum. Bars = 100 μm. (Figure 1E courtesy of Addie Thompson.)

Figure 2.

Genes Represented in Each of the Six RNA-Seq Data Sets. (A) All genes represented in each of the six samples (RPM ≥ 1). (B) Heat map based on the relative transcript accumulation of 226 candidate genes sorted according to implicated function. 1, L1 stage; 14, L14 stage; C, coleoptile stage; L, lateral meristem; P, proembryo; T, transition phase.

Figure 3.

Establishing a Meristem. (A) Venn diagram of all 4145 transcripts upregulated or present/absent in the premeristematic proembryo compared with the five samples with meristems. Transcripts in the overlapping region of the Venn diagram are present before and after the meristem forms, whereas transcripts in the nonoverlapping regions are present before but not after meristem formation or vice versa. (B) The 418 out of the 4145 transcripts that are implicated in transcriptional regulation are presented in a Venn diagram. (C) MapMan bin distribution of the 742 transcripts that are differentially accumulated after the meristem forms. CHO, carbohydrate. (D) The 123 transcripts implicated in transcriptional regulation present after the meristem forms sorted into their respective gene product families. In situ hybridizations using ZYB16 ([E] to [H], [J], and [L]) and KN1 ([I] and [K]) probes. Arrowheads point to transcript accumulation. col, coleoptile stage; pro, proembryo; tr, transition phase. Bars = 100 μm.

Figure 4.

Transcriptomic Comparisons of a Newly Formed Meristem to a Mature Meristem. (A) Venn diagram of the 1706 transcripts upregulated or present/absent when comparing the transition stage to the L14 stage. Transcripts in the overlapping region of the Venn diagram are present in both newly formed and mature meristems, whereas transcripts in the nonoverlapping regions are present before but not after meristem maturation or vice versa. (B) The 1706 upregulated transcripts sorted into modified MapMan categories. Total number of transcripts in each functional category is displayed in the bars. Darker bars in (B) and (D) designate the transition stage, lighter bars indicate the L14 stage. Asterisk after transcript number indicates enrichment of that functional category using Fisher’s exact test (P ≤ 0.01; FDR, 6%). CHO, carbohydrate. (C) The 192 out of 1706 transcripts upregulated or present/absent that are implicated in transcriptional regulation in a newly formed meristem and a mature meristem. (D) The 192 transcripts are distributed into their respective gene product family.

Figure 5.

Elaboration of the Scutellum, the Coleoptile, and the First Foliar Leaf. (A) Three main clusters that comprise 517 of 532 genes upregulated in the three-way comparison of the transition stage (T), coleoptile stage (C), and L1 stage (L1) embryos. Distribution of genes into functional categories according to MapMan. CHO, carbohydrate; TCA, tricarboxylic acid. (B) The 63 genes that are implicated in transcriptional regulation from the three main clusters distributed into gene families. The transition stage is shaded gray, the coleoptile stage is shaded black, and the L1 stage is shaded white. In situ hybridizations illustrate the accumulation of LEC1 transcripts during embryogenesis ([C] to [G]) and in 14-d-old seedlings ([H] and [I]). col, coleoptile stage; L1, leaf 1 stage; L14; 14-d-old seedling SAM; LM, lateral meristem; pro, proembryo; tr, transition phase. Bars = 100 μm.

Figure 6.

Differentially Accumulated Transcripts during the Elaboration of Embryonic Organs. In situ hybridizations illustrate transcript accumulation of TDL1 ([A] to [E]), RANBP2 ([F] to [J]), OLE ([K] to [O]), and LTP*1 ([P] to [T]). 24 DAP, embryo harvested 24 d after pollination; col, coleoptile stage; L1, leaf 1 stage; L14; 14-d-old seedling SAM; LM, lateral meristem; pro, proembryo; tr, transition phase. Bars = 100 μm.

Figure 7.

Developmental Markers Identified in RNA-Seq Analyses. In situ hybridizations illustrate transcript accumulation of ALOG*1 ([A] to [E]), UNK*1 ([F] to [J]), and UNK*2 ([K] to [O]). col, coleoptile stage; L1, leaf 1 stage; L14; 14-d-old seedling SAM; LM, lateral meristem; pro, proembryo; tr, transition phase. Bars = 100 μm.

Figure 8.

Initiation of Juvenile, Adult, and Husk Leaves. (A) A total of 12 clusters were generated based on the transcript accumulation of the 5103 upregulated genes when making the three-way comparison of the L1 stage, L14 stage, and lateral meristem. (B) Functional category enrichment of the 12 clusters. The −log₁₀P value and FDR 5% are included for significant enrichment. White indicates nonsignificant enrichment, whereas red indicates significant enrichment. Gray indicates that no genes are present in the category for that cluster. In situ hybridizations using the following probes: ALOG*2 ([C] to [E]), LTP*3 ([F] to [H]). L1, leaf 1; L14, leaf 14; LM, lateral meristem; TCA, tricarboxylic acid. Bars = 100 μm.