Evidence for a Role for NAD(P)H Dehydrogenase in Concentration of CO2 in the Bundle Sheath Cell of Zea mays.

Prior studies with Nicotiana and Arabidopsis described failed assembly of the chloroplastic NDH [NAD(P)H dehydrogenase] supercomplex by serial mutation of several subunit genes. We examined the properties of Zea mays leaves containing Mu and Ds insertions into nuclear gene exons encoding the critical o- and n-subunits of NDH, respectively. In vivo reduction of plastoquinone in the dark was sharply diminished in maize homozygous mutant compared to normal leaves but not to the extreme degree observed for the corresponding lesions in Arabidopsis. The net carbon assimilation rate (A) at high irradiance and saturating CO2 levels was reduced by one-half due to NDH mutation in maize although no genotypic effect was evident at very low CO2 levels. Simultaneous assessment of chlorophyll fluorescence and A in maize at low (2% by volume) and high (21%) O2 levels indicated the presence of a small, yet detectable, O2-dependent component of total linear photosynthetic electron transport in 21% O2 This O2-dependent component decreased with increasing CO2 level indicative of photorespiration. Photorespiration was generally elevated in maize mutant compared to normal leaves. Quantification of the proportion of total electron transport supporting photorespiration enabled estimation of the bundle sheath cell CO2 concentration (Cb) using a simple kinetic model of ribulose bisphosphate carboxylase/oxygenase function. The A versus Cb relationships overlapped for normal and mutant lines consistent with occurrence of strictly CO2-limited photosynthesis in the mutant bundle sheath cell. The results are discussed in terms of a previously reported CO2 concentration model [Laisk A, Edwards GE (2000) Photosynth Res 66: 199-224].

Evolutionarily conserved repressive activity of WOX proteins mediates leaf blade outgrowth and floral organ development in plants.

The WUSCHEL related homeobox (WOX) genes play key roles in stem cell maintenance, embryonic patterning, and lateral organ development. WOX genes have been categorized into three clades--ancient, intermediate, and modern/WUS--based on phylogenetic analysis, but a functional basis for this classification has not been established. Using the classical bladeless lam1 mutant of Nicotiana sylvestris as a genetic tool, we examined the function of the Medicago truncatula WOX gene, STENOFOLIA (STF), in controlling leaf blade outgrowth. STF and LAM1 are functional orthologs. We found that the introduction of mutations into the WUS-box of STF (STFm1) reduces its ability to complement the lam1 mutant. Fusion of an exogenous repressor domain to STFm1 restores complementation, whereas fusion of an exogenous activator domain to STFm1 enhances the narrow leaf phenotype. These results indicate that transcriptional repressor activity mediated by the WUS-box of STF acts to promote blade outgrowth. With the exception of WOX7, the WUS-box is conserved in the modern clade WOX genes, but is not found in members of the intermediate or ancient clades. Consistent with this, all members of the modern clade except WOX7 can complement the lam1 mutant when expressed using the STF promoter, but members of the intermediate and ancient clades cannot. Furthermore, we found that fusion of either the WUS-box or an exogenous repressor domain to WOX7 or to members of intermediate and ancient WOX clades results in a gain-of-function ability to complement lam1 blade outgrowth. These results suggest that modern clade WOX genes have evolved for repressor activity through acquisition of the WUS-box.

STENOFOLIA regulates blade outgrowth and leaf vascular patterning in Medicago truncatula and Nicotiana sylvestris.

Dicot leaf primordia initiate at the flanks of the shoot apical meristem and extend laterally by cell division and cell expansion to form the flat lamina, but the molecular mechanism of lamina outgrowth remains unclear. Here, we report the identification of STENOFOLIA (STF), a WUSCHEL-like homeobox transcriptional regulator, in Medicago truncatula, which is required for blade outgrowth and leaf vascular patterning. STF belongs to the MAEWEST clade and its inactivation by the transposable element of Nicotiana tabacum cell type1 (Tnt1) retrotransposon insertion leads to abortion of blade expansion in the mediolateral axis and disruption of vein patterning. We also show that the classical lam1 mutant of Nicotiana sylvestris, which is blocked in lamina formation and stem elongation, is caused by deletion of the STF ortholog. STF is expressed at the adaxial-abaxial boundary layer of leaf primordia and governs organization and outgrowth of lamina, conferring morphogenetic competence. STF does not affect formation of lateral leaflets but is critical to their ability to generate a leaf blade. Our data suggest that STF functions by modulating phytohormone homeostasis and crosstalk directly linked to sugar metabolism, highlighting the importance of coordinating metabolic and developmental signals for leaf elaboration.

MicroRNA-directed cleavage of Nicotiana sylvestris PHAVOLUTA mRNA regulates the vascular cambium and structure of apical meristems.

Leaf initiation in the peripheral zone of the shoot apical meristem involves a transition to determinate cell fate, but indeterminacy is maintained in the vascular cambium, a tissue critical to the continuous growth of vascular tissue in leaves and stems. We show that the orientation of cambial growth is regulated by microRNA (miRNA)-directed cleavage of mRNA from the Nicotiana sylvestris ortholog of PHAVOLUTA (NsPHAV). Loss of miRNA regulation in semidominant phv1 mutants misdirects lateral growth of leaf midveins and stem vasculature away from the shoot, disrupting vascular connections in stem nodes. The phv1 mutation also expands the central zone in vegetative and inflorescence meristems, implicating miRNA and NsPHAV in regulation of meristem structure. In flowers, phv1 causes reiteration of carpel initiation, a phenocopy for loss of CARPEL FACTORY/DICER LIKE1, indicating that miRNA is critical to the termination of indeterminacy in floral meristems. Results point to a common role for miRNA in spatial and temporal restriction of HD-ZIPIII mediated indeterminacy in apical and vascular meristems.

PHANTASTICA regulates development of the adaxial mesophyll in Nicotiana leaves.

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.