Yao Xiao

In plants, the vascular network enables the transport of water, nutrients and metabolites. The vascular network formed in leaves is a striking example of tissue patterning, and leaf venation is a defining leaf feature. During patterning, veins arise from aligned, elongated procambial cells derived from undifferentiated ground meristem cells. The mechanisms behind procambial strand formation involving cell selection, polarization, alignment, elongation and division have remained elusive. Here we identify a previously uncharacterized role for the polar protein PAX in establishing coordinated cell and tissue polarity during this process. We uncover a novel, auxin-guided role for the polar PAX protein in establishing cell and tissue polarity that precedes and is independent of the polarization and activity of PIN auxin transporters. Our findings refine and redefine existing models of vascular patterning during vein network formation in plant leaves.

Plants use environmental cues to orient organ and plant growth, such as the direction of gravity or the direction, quantity, and quality of light. During the germination of Arabidopsis thaliana seeds in soil, negative gravitropism responses direct hypocotyl elongation such that the seedling can reach the light for photosynthesis and autotrophic growth. Similarly, hypocotyl elongation in the soil also requires mechanisms to efficiently grow around obstacles such as soil particles. Here, we identify KIPK (KINESIN-LIKE CALMODULIN-BINDING PROTEIN-INTERACTING PROTEIN KINASE) and the paralogous KIPKL1 (KIPK-LIKE1) as genetically redundant regulators of gravitropic hypocotyl bending. Moreover, we demonstrate that the homologous KIPKL2 (KIPK-LIKE2), which shows strong sequence similarity, must be functionally distinct. KIPK and KIPKL1 are polarly localized plasma membrane-associated proteins that can activate PIN-FORMED auxin transporters. KIPK and KIPKL1 are required to efficiently align hypocotyl growth with the gravity vector when seedling hypocotyls are grown on media plates or in soil, where contact with soil particles and obstacle avoidance impede direct negative gravitropic growth. Therefore, the polar KIPK and KIPKL1 kinases have different biological functions from the related AGC1 family kinases D6PK (D6 PROTEIN KINASE) or PAX (PROTEIN KINASE ASSOCIATED WITH BRX).

The 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a conserved master regulator of AGC kinases in eukaryotic organisms. pdk1 loss of function causes a lethal phenotype in animals and yeasts, but only mild phenotypic defects in Arabidopsis thaliana (Arabidopsis). The Arabidopsis genome contains two PDK1-encoding genes, PDK1 and PDK2. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to generate true loss-of-function pdk1 alleles, which, when combined with pdk2 alleles, showed severe developmental defects including fused cotyledons, a short primary root, dwarf stature and defects in male fertility. We obtained evidence that PDK1 is responsible for AGC1 kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX) activation by phosphorylation during vascular development, and that the PDK1 phospholipid-binding Pleckstrin Homology domain is not required for this process. Our data indicate that PDK1 regulates polar auxin transport by activating AGC1 clade kinases, resulting in PIN phosphorylation.

The 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) is a conserved and important master regulator of AGC kinases in eukaryotic organisms. pdk1 loss-of-function causes a lethal phenotype in animals and yeast. In contrast, only very mild phenotypic defects have been reported for the pdk1 loss-of-function mutant of the model plant Arabidopsis thaliana (Arabidopsis). The Arabidopsis genome contains two PDK1 genes, hereafter called PDK1 and PDK2. Here we show that the previously reported Arabidopsis pdk1 T-DNA insertion alleles are not true loss-of-function mutants. By using CRISPR/Cas9 technology, we created true loss-of-function pdk1 alleles, and pdk1 pdk2 double mutants carrying these alleles showed multiple growth and development defect, including fused cotyledons, a short primary root, dwarf stature, late flowering, and reduced seed production caused by defects in male fertility. Surprisingly, pdk1 pdk2 mutants did not phenocopy pid mutants, and together with the observations that PDK1 overexpression does not phenocopy the effect of PID overexpression, and that pdk1 pdk2 loss-of-function does not change PID subcellular localization, we conclude that PDK1 is not essential for PID membrane localization or functionality in planta . Nonetheless, most pdk1 pdk2 phenotypes could be correlated with impaired auxin transport. PDK1 is highly expressed in vascular tissues and YFP:PDK1 is relatively abundant at the basal/rootward side of root stele cells, where it colocalizes with PIN auxin efflux carriers, and the AGC1 kinases PAX and D6PK/D6PKLs. Our genetic and phenotypic analysis suggests that PDK1 is likely to control auxin transport as master regulator of these AGC1 kinases in Arabidopsis.