Regulation of shoot meristem integrity during Arabidopsis vegetative development

Abstract

Shoot growth and development is mediated by the activity of the shoot meristem, which initiates leaves and axillary meristems. Meristem maintenance is achieved by a poorly understood process that functions to sustain the balance of stem cell perpetuation in the central zone (CZ) and organogenesis in the peripheral zone (PZ). A recent study showed that two related homeodomain transcription factors, PENNYWISE (PNY) and POUND-FOOLISH (PNF), regulate meristem maintenance by controlling the integrity of the CZ. The non-flower producing phenotype displayed by pny pnf plants can be rescued by genetically increasing the size of the shoot meristem. In this addendum, we show that augmenting the size of the central region of pny pnf shoot meristems partially rescues the meristem termination phenotype that occurs during early stages of vegetative development. Thus, regulation of CZ integrity by PNY and PNF is crucial for vegetative and reproductive development.

Homeodomain Proteins Function to Regulate the Maintenance of the Shoot Meristem

Small self-organizing structures located at the tips of shoots, called shoot meristems, mediate the growth and development of the aerial parts of the plant.¹ Shoot meristems are subdivided into discrete functioning zones.² The apical stem cells are located and maintained in the central zone (CZ). Stem cells supply the morphogeneic regions of the shoot meristem, the peripheral (PZ) and rib meristem (RM), with cells that are eventually incorporated into axillary meristems, leaves and stems. The CLV-WUS negative feedback loop functions in part to maintain a stable population of stem cells in the CZ.³ The enlarged shoot meristems displayed in clv1 and clv3 plants are due to a lateral expansion of CZ/stem cells.^4,5 In contrast, the failure to maintain shoot growth in wus is due to the inability of the shoot meristem to sustain the stem cell population.⁶ In addition, the interplay between WUS and cytokinin (CK) signaling is thought to maintain region of high CK response in the core of the shoot meristem.^7–9

Genetic studies indicate that meristem maintenance involves the balance of stem cell perpetuation in the central apical cells in the CZ and organogenesis in the PZ.¹⁰ Although the mechanism(s) of meristem maintenance is poorly understood, studies in maize and Arabidopsis indicate that two orthologous homeodomain transcription factors KNOTTED1 (KN1) and SHOOT MERISTEMLESS (STM), respectively, regulate this process.^11,12 The expression patterns for STM and KN1, suggest these homeodomain proteins function in CZ, PZ and RZ. Moreover, in the appropriate genetic background, the shoot meristem terminally differentiates in plants harboring null alleles of kn1 and stm. Interestingly, shoot growth can be partially rescued in stm plants by increasing the size of the CZ/stem-cell population, indicating that STM may regulate the integrity of the CZ.¹³

STM and KN1 interact with members of the BELL1-like Homeodomain (BLH) family of transcription factors.^11,14 In Arabidopsis, the expression patterns described for some BLH genes in the shoot meristem suggest that STM regulates distinct meristem maintenance pathways in association with specific BLH proteins.¹⁵ PENNYWISE (PNY: also known as BELLRINGER, REPLUMLESS, VAAMANA and BLH9) and POUND-FOOLISH (PNF) encode two related BLH genes that associate with STM.¹⁴ During early stages of vegetative development, the shoot apical meristem in pny pnf plants frequently terminates after 3–5 leaves are initiated.^16,17 Subsequently, axillary shoots grow out from the axils of rosette leaves. However, the mature shoots of pny pnf plants fail to initiate axillary and floral meristems; therefore, pny pny shoots display a non-flower producing phenotype. The role of PNY and PNF in meristem maintenance was recently investigated and results show that these homeodomain proteins regulate the integrity of the CZ.¹⁸ In addition, increasing the size of the CZ in pny pnf plants by creating pny pnf clv triple mutants allows these plants to enter the reproductive phase of growth. In this addendum, results showed that the early vegetative shoot meristem termination phenotype displayed in pny pnf plants is only partially restored in pny pnf clv3 plants.

Loss of CLV3 Function Partially Rescues the Shoot Meristem Termination Phenotype in pny pnf Plants

In this analysis, growth from the main shoot was analyzed during vegetative development. Results show that the shoot apical meristems of wild-type and clv3 plants sequentially initiated leaves until these plants transitioned to reproductive growth (Fig. 1A and B). In contrast to wild-type, 78% of pny pnf shoot apical meristems terminated after the production of 3–5 leaves (Fig. 1C).^16,17 Subsequently, shoot development was restored by the outgrowth of axillary shoots from the axils of rosette leaves (data not shown).^16,17 However, the remaining 22% of pny pnf plants maintained shoot growth. In pny pnf clv3, 58% of the central shoots maintained vegetative growth, while 42% of the shoots terminated growth after 3–5 leaves were produced (Fig. 1E and F). These results indicate that loss of clv function in pny pnf plants partially restores the maintenance of the shoot apical meristem, during early stages of vegetative growth.

Figure 1.

Analysis of vegetative shoot growth under long-day growth conditions. (A) Wild-type and (B) clv3 plants maintained shoot growth during the vegetative phase of growth. However, (C) 78% of pny pnf shoots terminated after 3–5 leaves were produced (arrow points at terminated shoot in which leaf formation ceased), while (D) 22% of pny pnf shoots maintained shoot growth from the main axis. (E) 58% of pny pnf clv3 maintained vegetative shoot growth, while (F) the remaining 42% of the shoots terminated growth after 3–5 leaves were produced (arrow points at the terminated shoot).

Maintaining the Integrity of the CZ in Shoot Meristem

A recent study showed that PNY and PNF maintain the integrity of the CZ, which is crucial for regulating the CLV3 and WUS expression domains in the shoot apical meristem.¹⁸ Augmenting the size of the CZ by creating pny pnf clv mutants reestablishes the CLV3 and WUS expression domains and rescues the non-flower producing phenotype. In this addendum, we determined whether loss of clv3 function could suppress the pny pnf shoot meristem termination phenotype, which is displayed during early stages of vegetative growth. Experimental evidence shows that the pny pnf shoot meristems are considerably smaller than wild type. In addition, only 22% of pny pnf shoot meristems maintain growth during early stages of vegetative development. It was speculated that the small size of pny pnf shoot meristems combined with the loss of CZ integrity contribute the meristem termination phenotype during vegetative growth.¹⁸ During early stages of shoot development, 58% of pny pnf clv3 shoot meristems maintain an incessant pattern of growth, indicating the loss of CLV function in pny pnf plants partially restores meristem maintenance. In contrast, all pny pnf clv3 plants produce reproductive shoots. To explain these differences, we propose that the increased CZ in clv3 vegetative shoot meristems may not be sufficient to fully rescue the vegetative meristem termination phenotype produced in plants with reduced PNY and PNF function. For example, clv3 the embryonic shoot meristem is only 1.4 times larger than wild type.⁵ However, during later stages of clv3 shoot development, the inflorescence meristems can increase up to 1,000-fold larger than wild type. Therefore, the complete rescue reproductive shoot formation in pny pnf clv3 and pny pnf clv1 plants is likely caused by the excess production of meristematic cells, which enables these shoot meristems to respond to floral inductive cues and produce flower-like structures.

Addendum to: Ung N, Lal S, Smith HM. The role of PENNYWISE and POUND-FOOLISH in the maintenance of the shoot apical meristem in Arabidopsis. Plant Physiol. 2011;15:605–614. doi: 10.1104/pp.110.171462.