13 C-Metabolic flux analysis ( 13 C-MFA) has greatly contributed to our understanding of plant metabolic regulation. However, the generation of detailed in vivo flux maps remains a major challenge. Flux investigations based on nuclear magnetic resonance have resolved small networks with high accuracy. Mass spectrometry (MS) approaches have broader potential, but have hitherto been limited in their power to deduce flux information due to lack of atomic level position information. Herein we established a gas chromatography (GC) coupled to MS-based approach that provides 13 C-positional labelling information in glucose, malate and glutamate (Glu). A map of electron impact (EI)-mediated MS fragmentation was created and validated by 13 Cpositionally labelled references via GC-EI-MS and GC-atmospheric pressure chemical ionization-MS technologies. The power of the approach was revealed by analysing previous 13 C-MFA data from leaves and guard cells, and 13 C-HCO 3 labelling of guard cells harvested in the dark and after the dark-to-light transition. We demonstrated that the approach is applicable to established GC-EI-MS-based 13 C-MFA without the need for experimental adjustment, but will benefit in the future from paired analyses by the two GC-MS platforms. We identified specific glucose carbon atoms that are preferentially labelled by photosynthesis and gluconeogenesis, and provide an approach to investigate the phosphoenolpyruvate carboxylase (PEPc)derived 13 C-incorporation into malate and Glu. Our results suggest that gluconeogenesis and the PEPcmediated CO 2 assimilation into malate are activated in a light-independent manner in guard cells. We further highlight that the fluxes from glycolysis and PEPc toward Glu are restricted by the mitochondrial thioredoxin system in illuminated leaves.

Resumo -O objetivo deste trabalho foi avaliar a acurácia das regressões que definem os limites do intervalo hídrico ótimo e a validade do uso deste indicador ao se considerar a variabilidade espacial de lavouras sob sistema plantio direto. Foram coletadas 100 amostras de solo com estrutura preservada, em áreas com dimensões de 10x10 m, em Latossolo Vermelho distrófico, Nitossolo Vermelho distroférrico e Latossolo Vermelho aluminoférrico, manejados sob sistema plantio direto. Determinaram-se porosidade de aeração (0,10 m 3 m -3 ), capacidade de campo (potencial de -0,006 MPa), ponto de murcha permanente (potencial de -1,5 MPa) e resistência mecânica do solo à penetração crítica (2 MPa). As regressões que descreveram a capacidade de campo tiveram os menores coeficientes de determinação, enquanto as que descreveram a porosidade de aeração, o ponto de murcha permanente e a resistência mecânica do solo à penetração apresentaram os maiores valores. As regressões que definem o limite da capacidade de campo não são acuradas em descrever a variação na umidade volumétrica do solo por meio da densidade do solo, e os solos avaliados apresentam elevada variabilidade espacial para o intervalo hídrico ótimo.Termos para indexação: capacidade de campo, ponto de murcha permanente, porosidade de aeração, resistência mecânica do solo à penetração. Spatial variability of the least limiting water range of soils under a no-tillage systemAbstract -The objective of this work was to evaluate the regressions that define the least limiting water range limits and the validity of using this indicator considering the spatial variability of crops under a no-tillage system. One hundred undisturbed soil samples were collected in areas with dimensions of 10x10 m, in two different Oxisols and an Ultisol managed under no-tillage. Air-filled porosity (0.10 m 3 m -3), field capacity (potential of -0.006 MPa), permanent wilting point (potential of -1.5 MPa), and soil mechanical resistance (2 MPa) were determined. The regressions that described field capacity had the lowest coefficients of determination, whereas those that described air-filled porosity, permanent wilting point, and soil mechanical resistance to penetration showed the highest values. Regression adjustments that define field capacity limits are not accurate in explaining variation on volumetric soil water content through bulk density, and the evaluated soils show high spatial variability regarding the least limiting water range.

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13C-Metabolic flux analysis (13C-MFA) have greatly contributed to revealing the regulation of plant metabolism. However, mass spectrometry (MS) approaches have hitherto been limited in their power to deduce flux information due to lack of positional information. Here we established an MS-based 13C-positional isotopomer labelling approach and performed a multi-species/cell-types analysis based on previous 13C-MFA to compare flux modes through the tricarboxylic acid (TCA) cycle and associated pathways in mesophyll (MCs) and guard cells (GCs). Both cell types showed high 13C-enrichment in pyruvate. However, GCs and sink MCs, but not source MCs showed high 13C-incorporation into Glu/Gln following provision of 13C-sucrose. Only GCs showed higher 13C-enrichment in the carbon 1 atom of Gln, which is derived from PEPc-mediated CO2 fixation. Increased 13C-enrichment in the carbon 1 of Glu was also observed in both trxo1 and ntra ntrb mutants, but not in wild type Arabidopsis plants, following provision of 13C-glucose. Our results suggest that the mitochondrial thioredoxin system restricts the fluxes from PEPc and glycolysis to Glu in illuminated MCs and reveal that fluxes throughout the TCA cycle of GCs resemble those of sink MCs but operate different non-cyclic flux modes to support Gln synthesis in the light.