Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate.

A key question in developmental biology is how cells exchange positional information for proper patterning during organ development. In plant roots the radial tissue organization is highly conserved with a central vascular cylinder in which two water conducting cell types, protoxylem and metaxylem, are patterned centripetally. We show that this patterning occurs through crosstalk between the vascular cylinder and the surrounding endodermis mediated by cell-to-cell movement of a transcription factor in one direction and microRNAs in the other. SHORT ROOT, produced in the vascular cylinder, moves into the endodermis to activate SCARECROW. Together these transcription factors activate MIR165a and MIR166b. Endodermally produced microRNA165/6 then acts to degrade its target mRNAs encoding class III homeodomain-leucine zipper transcription factors in the endodermis and stele periphery. The resulting differential distribution of target mRNA in the vascular cylinder determines xylem cell types in a dosage-dependent manner.

Mutations in the SLAC1 anion channel slow stomatal opening and severely reduce K+ uptake channel activity via enhanced cytosolic [Ca2+] and increased Ca2+ sensitivity of K+ uptake channels.

The Arabidopsis guard cell anion channel SLAC1 is essential for stomatal closure in response to various endogenous and environmental stimuli. Interestingly, here we reveal an unexpected impairment of slac1 alleles on stomatal opening. We report that mutations in SLAC1 unexpectedly slow stomatal opening induced by light, low CO(2) and elevated air humidity in intact plants and that this is caused by the severely reduced activity of inward K(+) (K(+)(in)) channels in slac1 guard cells. Expression of channels and transporters involved in stomatal opening showed small but significant reductions in transcript levels in slac1 guard cells; however, this was deemed insufficient to explain the severely impaired K(+)(in) channel activity in slac1. We further examined resting cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) and K(+)(in) channel sensitivity to [Ca(2+)](cyt) in slac1. These experiments showed higher resting [Ca(2+)](cyt) in slac1 guard cells and that reducing [Ca(2+)](cyt) to < 10 nM rapidly restored the activity of K(+)(in) channels in slac1 closer to wild-type levels. These findings demonstrate an unanticipated compensatory feedback control in plant stomatal regulation, which counteracts the impaired stomatal closing response of slac1, by down-regulating stomatal opening mechanisms and implicates enhanced [Ca(2+)](cyt) sensitivity priming as a mechanistic basis for the down-regulated K(+)(in) channel activity.

Ozone-triggered rapid stomatal response involves the production of reactive oxygen species, and is controlled by SLAC1 and OST1.

The air pollutant ozone can be used as a tool to unravel in planta processes induced by reactive oxygen species (ROS). Here, we have utilized ozone to study ROS-dependent stomatal signaling. We show that the ozone-triggered rapid transient decrease (RTD) in stomatal conductance coincided with a burst of ROS in guard cells. RTD was present in 11 different Arabidopsis ecotypes, suggesting that it is a genetically robust response. To study which signaling components or ion channels were involved in RTD, we tested 44 mutants deficient in various aspects of stomatal function. This revealed that the SLAC1 protein, essential for guard cell plasma membrane S-type anion channel function, and the protein kinase OST1 were required for the ROS-induced fast stomatal closure. We showed a physical interaction between OST1 and SLAC1, and provide evidence that SLAC1 is phosphorylated by OST1. Phosphoproteomic experiments indicated that OST1 phosphorylated multiple amino acids in the N terminus of SLAC1. Using TILLING we identified three new slac1 alleles where predicted phosphosites were mutated. The lack of RTD in two of them, slac1-7 (S120F) and slac1-8 (S146F), suggested that these serine residues were important for the activation of SLAC1. Mass-spectrometry analysis combined with site-directed mutagenesis and phosphorylation assays, however, showed that only S120 was a specific phosphorylation site for OST1. The absence of the RTD in the dominant-negative mutants abi1-1 and abi2-1 also suggested a regulatory role for the protein phosphatases ABI1 and ABI2 in the ROS-induced activation of the S-type anion channel.

Plant development. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation.

Photoassimilates such as sugars are transported through phloem sieve element cells in plants. Adapted for effective transport, sieve elements develop as enucleated living cells. We used electron microscope imaging and three-dimensional reconstruction to follow sieve element morphogenesis in Arabidopsis. We show that sieve element differentiation involves enucleation, in which the nuclear contents are released and degraded in the cytoplasm at the same time as other organelles are rearranged and the cytosol is degraded. These cellular reorganizations are orchestrated by the genetically redundant NAC domain-containing transcription factors, NAC45 and NAC86 (NAC45/86). Among the NAC45/86 targets, we identified a family of genes required for enucleation that encode proteins with nuclease domains. Thus, sieve elements differentiate through a specialized autolysis mechanism.

Synthesis of ketocarotenoids in the seed of Arabidopsis thaliana.

A cDNA coding for a gene necessary for synthesis of ketocarotenoids was cloned from the alga Haematococcus pluvialis and expressed in the seed of Arabidopsis thaliana. The expression of the algal beta-carotene-oxygenase gene was directed to the seed by use of the 2S, seed storage protein promoter napA. Extracts from seeds of the transgenic plants were clearly red because of accumulation of ketocarotenoids, and free and esterified forms of ketocarotenoids were found in addition to the normal carotenoid composition in the seed. The major ketocarotenoids in the transgenic plants were: 4-keto-lutein (3,3'-dihydroxy-beta-,epsilon-carotene-4-one), adonirubin (3-hydroxy-beta-,beta'-carotene-4,4'-dione) and canthaxanthin (beta-,beta'-carotene-4,4'-dione). 4-Keto-lutein differs from the more common adonixanthin only in the position of one double bond. To increase the substrate availability for the beta-carotene-oxygenase, these transformants were crossed with transgenic plants overexpressing a construct of an endogenous phytoene synthase gene, also under the control of the napA promoter. The resulting crossings gave rise to seeds with a 4.6-fold relative increase of the total pigment, and the three major ketocarotenoids were increased 13-fold compared to seeds of transgenic plants carrying only the beta-carotene-oxygenase construct.