Tomato HAIRY MERISTEM4, expressed in the phloem, is required for proper shoot and fruit development

Abstract

The HAIRY MERISTEM (HAM) gene family encodes Type I and II GRAS domain transcriptional regulators in plants. Type II HAMs, predominantly expressed in meristems and regulated by microRNA171, are essential for maintaining undifferentiated meristems, a role conserved across various species. Conversely, the functions of Type I HAMs have been less characterized. In this study, we investigated the role of SlHAM4, a Type I HAM in tomato. CRISPR-induced SlHAM4 loss-of-function mutations (slham4 ^CR ) resulted in shoot and fruit abnormalities, which were fully reversed by reintroducing SlHAM4, driven by its native promoter, into the mutant background. Mutant abnormalities included simpler leaves and increased anthocyanin pigmentation in the leaf and sepal primordia, reminiscent of phenotypes observed in certain Arabidopsis mutants with compromised phloem. In addition, slham4 ^CR plants produced significantly smaller fruits with a subset developing catface-like scars, attributed to tears that occurred in the pericarp of setting fruits. Using a GUS reporter gene driven by the native SlHAM4 promoter, we found that SlHAM4 is predominantly expressed in phloem tissues. Consistent with this, transcriptome analysis of mutant anthesis ovaries revealed specific downregulation of genes implicated in phloem development and function, particularly those expressed in companion cells. However, histological analysis showed no obvious abnormalities in phloem vasculature. Taken together, our data suggest that SlHAM4 plays a role in shoot and fruit development likely by regulating genes essential for phloem function.

Figure 1

CRISPR/Cas9-mediated mutagenesis of the SlHAM4 gene. (A) Schematic of the SlHAM4 gene structure with gRNA target sites marked. Expanded views show gRNA target sequences with PAM motifs (red); arrowheads indicate expected Cas9 cleavage sites. (B) Alignment of CRISPR mutant alleles (slham4^CR) with the wild-type SlHAM4 sequence. gRNA target sequences are highlighted in red, and the PAM motif is noted. Numbering is from the start codon. (C) Representation of SlHAM4 protein architecture. The GRAS domain, key sequence motifs and regions, are shown. The conserved Leu²⁷¹ location is marked with an arrowhead. (D) Alignment of mutant and wild-type SlHAM4 protein sequences. Amino acids in the GRAS domain conserved across species are highlighted in red, according to the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/cdd/). Numbering starts from the start codon, with an asterisk (*) indicating a premature stop codon.

Figure 2

Comparison of M82 (wild-type) and slham4^CR shoot and flower phenotypes. (A) Seedlings at 14 days post-germination. (B) Mature plants cultivated in standard greenhouse conditions. (C) A representative fully expanded leaf. (D) Representative young leaves and inflorescences at the sympodial shoot. YL, young leaf; FB, flower bud. Note the increased purple pigmentation of mutant young leaves and sepals. (E) Anthesis flowers. (F) Detached sepals from anthesis flowers. (G) Representative flowers not subjected to fertilization, displaying senescence; mutant sepals exhibit noticeable dark brown areas. Scale bars = 1 cm (C) and 5 mm (D–G).

Figure 3

Characterization of M82 (wild-type) and slham4^CR fruits. (A) Isolated pistils from anthesis flowers. (B) Young immature green fruits. Arrows mark the pericarp ruptures in mutants. (C and D) Red ripe fruits and their longitudinal sections, respectively. Arrows point to catface-like scars. Scale bars = 2 mm (A and B) and 1 cm (C and D). (E and F) Cross-sections of anthesis ovaries and 4-mm stage young immature green fruits, respectively, stained with safranin and fast-green, where the red staining delineates xylem vasculature. The middle and bottom panels show close-up views of respective upper panel. p, pericarp; cl, columella; pl, placenta; vs, vascular tissue; xy, xylem; ph, phloem. In F, arrows mark the indentation in the mutant fruit pericarp. (G) Young immature and mature green wild-type fruits developed from anthesis ovaries with manually crushed pericarp, resulting in a ~1 mm indentation. The resulting catface-like scars are indicated by arrows. (H) Diagram illustrating the prevalence of catface formation in fruits from wild-type, heterozygous (slham4^CRΔ4−/+), and homozygous (slham4^CRΔ4) plants, with each row representing an individual plant. Plants were cultivated in greenhouse nested plots. (I) Average fruit weight of indicated genotypes. The median and average are indicated by a line and +, respectively; n = number of fruits; Different letters indicate significance (P < 0.01) as determined by Tukey–Kramer multiple comparison test.

Figure 4

Functional complementation of slham4^CRΔ4 mutation. (A and B) Images of representative senescing flowers (A) and red ripe fruits (B) from M82 (wild type) and indicated genotypes. Scale bars = 1 cm. (C) Schematic representation of catface development in fruits of indicated genotypes. Each column represents a single plant. (D) Quantitation of SlHAM4 transcript levels in M82 (wild-type) and 35S::SlHAM4–5 slham4^CRΔ4 anthesis ovaries, normalized to SlTIP41 as the reference gene. Error bars indicate ±SD over three biological replicates. The P-value as determined by Student’s t-test is shown.

Figure 5

Tissue-specific expression of SlHAM4. (A–O) Histochemical staining for GUS activity in M82 (wild type) and transgenic tomato plants expressing GUS driven by SlHAM4 native promoter (SlHAM4pro::GUS). GUS activity was visualized using the chromogenic substrate X-Gluc after ethanol clearing. (A) Mature embryos extracted from seeds 1 DAI. (B–E) Representative whole seedlings at indicated DAG. Insets in B–D show isolated cotyledons (adaxial side). (F) Adaxial side of the first leaf distal half. (G) Seedling stem. Scale bars = 200 μm (A and F), 500 μm (B), 1 mm (C), 2 mm (D, E, and G). Inset scale bars = 500 μm (B and C), 1 mm (D). cot, cotyledon; hyp, hypocotyl. (J–O) Manual longitudinal (top panel) and cross (bottom panel) sections of anthesis ovary (J) and fruits at indicated developmental stage (K–N). DPA, days post-anthesis. Arrows indicate GUS staining in representative vasculature. Scale bars 1 mm (J–L), 1 cm (M–O). (H, I, O, and P) Histological cross-sections of the SlHAM4pro::GUS-8 tissues stained with Ruthenium red. (H) Seedling stem shown in (G). (O) Pericarp vasculature of mature green fruit shown in (M). Scale bar = 100 μm. (I and P) Higher magnification of the histological sections outlined in (H) and (O) by a black box. Scale bar = 20 μm. p, parenchyma; ph, phloem; pi, internal phloem; pe, external phloem; xy, xylem; se, sieve element cell; cc, companion cell.

Figure 6

Global gene expression changes in slham4^CRΔ4 anthesis ovaries. (A) Principal components analysis of all expressed genes showing three distinct groups. (B) Total number of DEGs in slham4^CRΔ4 mutant and heterozygous (slham4^CRΔ4(−/+)) ovaries compared to wild type. (C) Venn diagram displaying specific and overlapping DEGs between slham4^CRΔ4 and slham4^CRΔ4(−/+) datasets. (D) Expression profile of cluster 24 cDEGs in fruit pericarp tissues based on TEA database data [14]. Cluster-wide average expression is plotted with solid lines, first and third quartiles by dashed lines, and maximum and minimum by dotted lines. The SlHAM4-specific profile is plotted with red solid line. (E and F) Overlap between DEGs and cDEGs (E) or Arabidopsis orthologs of cDEGs (F) with indicated published vascular datasets. To compensate for dataset size, the numbers of up and down DEGs and cDEGs were normalized relative to the total number of genes in the largest group dataset (Table S1f). Asterisks indicate statistical significance of overlap as calculated by http://nemates.org/MA/progs/overlap_stats.cgi. Ph, phloem; Vi, vascular initials; Xy, xylem.