PHANTASTICA regulates development of the adaxial mesophyll in Nicotiana leaves

Abstract

Initiation and growth of leaf blades is oriented by an adaxial/abaxial axis aligned with the original axis of polarity in the leaf primordium. To investigate mechanisms regulating this process, we cloned the Nicotiana tabacum ortholog of PHANTASTICA (NTPHAN) and generated a series of antisense transgenics in N. sylvestris. We show that NSPHAN is expressed throughout emerging blade primordia in the wild type and becomes localized to the middle mesophyll in the expanding lamina. Antisense NSPHAN leaves show ectopic expression of NTH20, a class I KNOX gene. Juvenile transgenic leaves have normal adaxial/abaxial polarity and generate leaf blades in the normal position, but the adaxial mesophyll shows disorganized patterns of cell division, delayed maturation of palisade, and ectopic reinitiation of blade primordia along the midrib. Reversal of the phenotype with exogenous gibberellic acid suggests that NSPHAN, acting via KNOX repression, maintains determinacy in the expanding lamina and sustains the patterns of cell proliferation critical to palisade development.

Figure 1.

Alignment of PHAN Orthologs. Amino acid alignment of PHAN proteins. Identical residues are shaded, and dashed lines were added to maximize alignment. Solid lines show the two imperfect N-terminal MYB repeats of 55 and 51 residues.

Figure 2.

NSPHAN Expression Analysis. Expression patterns for NSPHAN were examined in situ with digoxygenin-labeled probes ([A] to [C]), by RT-PCR (D), and in RNA gel blots (E). Bars in (A), (B), and (C) = 60, 80, and 100 μm, respectively. (A) In situ hybridization with an antisense RNA probe in a juvenile shoot apex shows NSPHAN mRNA is undetectable in the central zone of the meristem (cz) but is present throughout P1 and P2 leaf primordia. (B) In stages P4 to P5, NSPHAN is expressed throughout emerging leaf blades (lb) and in leaf midveins (mv). (C) In the expanding lamina, NSPHAN is localized to middle mesophyll (mm) and lateral veins (lv). mv, midveins. (D) RT-PCR amplification of NSPHAN and NTH23 as control from total RNA samples shows expression in leaves up through the 10-cm stage and in the stems. (E) RNA gel blot analysis with an NSPHAN-specific probe (pNAM5.2.3) shows similar mRNA levels at the tip and base of juvenile wild-type leaves (NSWT) and undetectable levels in three antisense transgenics (AF6J.4, AF34.1, and AF18B.1) relative to a ubiquitin control (pUBI). Expression levels of the antisense transgene were analyzed with the pRH81 probe.

Figure 3.

Juvenile Antisense Phenotype. In the juvenile vegetative phase, antisense NSPHAN transgenics produce leaves with normal adaxial/abaxial polarity but with a specific disruption in the adaxial domain, leading to formation of ectopic leaf blades on the lateral flanks of vascular ribs. Bars = 40 μm in (B), 20 μm in (C), 100 μm in (D), and 80 μm in (E) to (I). (A) Ectopic leaf blades (EB) emerge at the junction between the primary blade (PB) and the midrib and show a fixed polarity, the adaxial surface facing away from the rib. (B) Transverse sections of ectopic blades show differentiation of palisade (ectopic blade palisade, ebp) and lateral veins (ectopic blade vein, ebv). (C) and (D) Scanning electron micrograph of transgenic leaf primordia in (C) shows a flattened adaxial surface at the P2 stage, and longitudinal sections in (D) show normal adaxial/abaxial differentiation, indicating that leaf polarity is normal in the absence of NSPHAN. ad, adaxial; abr, abaxial rib; adb, adaxial blade; mv, midvein. (E) The primary disruption in transgenic leaves is abnormal proliferation and lack of palisade differentiation in the upper mesophyll beginning at stage P5, which forms a continuous pavement over the midvein (mv) and lateral veins (lv). um, upper mesophyll. (F) and (G) Corresponding wild-type leaves have palisade initials at this stage in the upper mesophyll (um), which form a single cell layer over lateral veins (lv). mv, midvein. (H) Where the abnormal upper mesophyll (um) of transgenics abuts adaxial cortex (adc) over the midvein (mv), periclinal cell divisions produce ectopic blade (eb) primordia. (I) Vascularized blade primordia (ectopic blade vein, ebv) emerge vertically on either side of the midrib, with an adaxial surface (ectopic blade adaxial, ebad) that is contiguous with the upper mesophyll of the primary blade (umpb), thus facing away from the rib.

Figure 4.

Adult Antisense Phenotypes. (A) and (B) Wild-type (A) and antisense NSPHAN transgenics (B) in the adult vegetative phase of growth, with axillary meristems (ax) on the adaxial surface of the petiole at the leaf/stem junction. Adult antisense leaves have radialized petioles lacking leaf blades except at the leaf tips. (C) Antisense leaves formed during the transition from juvenile to adult phase are radialized only at the base of the petiole (r) but are still bladeless in more distal regions where bilateral symmetry is retained (bi). ax, axillary meristem. (D) and (E) Adult antisense primordia show blade formation only at the leaf tip, in an abnormal position shifted toward the front of the primordium, in association with abnormal development of the midvein (mv) (E) in a closed U shape. (F) The base of the antisense primordium lacks blade formation and has a radialized midvein (mv) closed into a circular structure with phloem (p) surrounding xylem (x). (G) In situ hybridization with an antisense PHAVOLUTA probe in wild-type shoot apex shows expression throughout adult leaf primordia in the P2 stage. In P3 to P4 primordia, PHAV mRNA is localized to the midvein and adaxial regions of the leaf. (H) NSHAV shows a normal pattern of expression in the adaxial domain and in midveins at the base of radialized antisense leaves at the P5 stage. Bars = 20 μm in (D) and 80 μm in (E) to (H).

Figure 5.

NTH20 Expression Analysis. In situ hybridization of an NTH20 antisense probe in wild-type and antisense NSPHAN transgenics. Bars = 100 μm in (A) and (F), 80 μm in (B) and (C), 50 μm in (D), 60 μm in (E), and 2 cm in (G) and (H). (A) NTH20 mRNA is detected in the peripheral zone (pz) of the shoot meristem where leaf primordia (lp) are initiated but absent in the central zone of the meristem (cz). v, vascular. (B) and (C) NTH20 is not detectable in leaf primordia (B) in stages P2 to P5 but is present near the leaf/stem junction in P7 primordia (C) where axillary meristems (ax) are formed. mv, midvein. (D) and (E) Ectopic expression of NTH20 is observed in adult antisense primordia, both in the midvein and in the surrounding rib cortex. lb, leaf blade; mv, midvein. (F) Ectopic NTH20 is also observed in leaf primordia of juvenile antisense transgenics, particularly on the adaxial surface (ad) and in its normal location in vascular traces (v) in the adjoining stem. ab, abaxial. (G) and (H) The curled lamina and ectopic blade phenotype in juvenile leaves of heterozygous transgenics (G) is strongly suppressed by application of GA as shown in (H). Arrows show vestigial ectopic blades on the midrib and remnants of disorganization at the leaf tip.

Figure 6.

Blade Formation. (A) Adaxial leaf identity is a developmental state associated with expression of HD-ZIPIII genes (PHABULOSA, PHAVOLUTA, and REVOLUTA) in the meristem and on the adaxial surface of adjoining leaf primordia. Lateral outgrowth of blade primordia occurs where this adaxial tissue encounters the abaxial cortex on the lateral flank of the primordium in a process requiring the function of LAM1. NSPHAN is expressed throughout the primordium in this early phase. (B) The emerging lamina differentiates along an adaxial/abaxial axis of polarity, producing an adaxial palisade, an abaxial spongy parenchyma, and a vascularized middle mesophyll separating these domains. NSPHAN is initially expressed throughout emerging blade primordia and then becomes localized to the middle mesophyll during lamina expansion. NSPHAN via KNOX suppression maintains determinacy in the expanding lamina and sustains the regulated patterns of cell proliferation critical to palisade development in the upper mesophyll. (C) In the absence of NSPHAN, a polarized lamina is generated in the normal position, but the adaxial mesophyll appears suspended in an immature state, showing disorganized patterns of proliferation, delayed differentiation of palisade, and de novo reinitiation of polarized blade primordia along the flanks of the midrib. This is correlated with ectopic expression of the class I KNOX gene NTH20. GA suppression of the phenotype establishes a causal connection to ectopic KNOX and a requirement for GA in palisade development. ab, abaxial; ad, adaxial.