Two flowering regulators help limit woody tissue in tomato stems

Tomato is not a tree, but its stems still produce substantial amounts of secondary xylem: woody tissue that transports water and provides mechanical support. A bioRxiv preprint by Kailash Pandey and colleagues identifies two flowering regulators, FUL2 and STM3, as important restraints on that production. The study has not been certified by peer review. Its findings point to a developmental control system whose effects vary along the stem, rather than a simple switch thrown when flowering begins.

Mapping the stem’s active genes

Secondary growth increases a stem’s width. A layer of dividing cells called the vascular cambium produces secondary xylem inward and secondary phloem outward. The researchers studied the tomato cultivar Moneyberg under greenhouse conditions, combining tissue anatomy, gene-expression measurements, previously generated CRISPR/Cas9 mutants and chemical analysis of stem material.

First, they used laser capture microdissection to isolate tiny tissue regions from the middle of 60-day-old stems. RNA sequencing compared four fractions: cambium, xylem, phloem with cortex, and cortex with epidermis. Under their filtering criteria, the team identified 147 cambium-specific genes and 322 xylem-specific genes.

The profiles recovered tomato counterparts of established vascular regulators and showed enrichment of genes related to poplar wood formation. This supports shared features of secondary-growth regulation between herbaceous and woody plants, although matching expression patterns alone does not establish identical functions for every gene.

Among known flowering regulators, FUL2 stood out for its association with xylem. STM3 was strongly expressed more broadly across the inner vascular tissues. These patterns led the researchers to investigate whether the two genes also regulate stem thickening.

Removing the brakes

At 120 days after germination, a quadruple mutant lacking FUL2, STM3 and two related genes, MBP20 and TM3, had roughly twice the secondary xylem area of wild-type plants at the basal internode. Among single mutants, loss of either FUL2 or STM3 produced the strongest increases. Combining mutations generally did not further enhance secondary xylem area, although the xylem-to-phloem ratio was slightly higher in the quadruple mutant than in either single mutant.

That largely non-additive pattern suggests FUL2 and STM3 act in a shared pathway. Together with previously reported physical interaction between their proteins, overlapping expression and similar gene-expression responses, it supports the authors’ proposal that they work as partners in a protein complex. It does not, by itself, prove the complex’s composition or activity inside stem cells.

Chemical measurements showed another consequence. In the basal half of 120-day-old stems, the stm3 mutant had the highest lignin concentration: 16.3%, compared with 11.8% in wild type, on an extractives-free dry-weight basis. Lignin is a structural component of secondary cell walls. The ful2 and quadruple mutants had intermediate numerical increases, while total structural carbohydrate content remained comparable to wild type.

The anatomical changes emerged late. Secondary xylem area was broadly similar between mutants and wild type at 60 days; at 30 days, only the quadruple mutant showed a slight increase. By 120 days, mid-height mutant stems also showed increased xylem-to-phloem ratios, even though extensive xylem accumulation was most evident at the base.

Local development matters more than the calendar alone

RNA sequencing of mid-height stems revealed 641 differentially expressed genes shared by ful2 and stm3 at 60 days, before detectable anatomical differences. Candidate regulators included members of the NAC and MYB transcription-factor families, associated with xylem differentiation and secondary wall formation. At 120 days, a largely different response included NAC16 and MYB58. Many earlier responses had weakened or disappeared.

Follow-up quantitative PCR added an important qualification: genes identified as responding at 120 days in the middle of the stem were already responding at the base of 60-day-old plants. The base had reached a more advanced state of secondary growth. Thus, chronological age alone could not explain the changing responses.

Nor did the results show that flowering immediately activates these stem functions. Under the study’s conditions, the floral transition occurred around day 21, but FUL2 and STM3 expression remained low at day 30 and rose later. Reporter staining also became prominent in vascular ray cells at later stages, although the functional significance remains unresolved.

Several mechanistic questions remain open. Potential DNA-binding motifs in candidate gene promoters suggest possible direct regulation, not demonstrated binding. At 120 days, changes in tissue proportions could themselves account for some expression differences. The quadruple mutant also flowered much later, complicating developmental comparisons. The study therefore supports a role for FUL2 and STM3 in limiting tomato secondary xylem, while leaving their direct targets and relationship to flowering signals to be established.