SummaryPatterning of stomata, valves on the plant epidermis, requires the orchestrated actions of signaling components and cell-fate determinants. To understand the regulation of stomatal patterning, we performed a genetic screen using a background that partially lacks stomatal signaling receptors. Here, we report the isolation and characterization of chorus (chor), which confers excessive proliferation of stomatal-lineage cells mediated by SPEECHLESS (SPCH). chor breaks redundancy among three ERECTA family genes and strongly enhances stomatal patterning defects caused by loss-of-function in TOO MANY MOUTHS. chor seedlings also exhibit incomplete cytokinesis and growth defects, including disruptions in root tissue patterning and root hair cell morphogenesis. CHOR encodes a putative callose synthase, GLUCAN SYNTHASE-LIKE 8 (GSL8), that is required for callose deposition at the cell plate, cell wall and plasmodesmata. Consistently, symplastic macromolecular diffusion between epidermal cells is significantly increased in chor, and proteins that do not normally move cell-to-cell, including a fluorescent protein-tagged SPCH, diffuse to neighboring cells. Such a phenotype is not a general trait caused by cytokinesis defects. Our findings suggest that the restriction of symplastic movement might be an essential step for the proper segregation of cell-fate determinants during stomatal development.

SUMMARYIt has been known for more than a century that sieve plates in the phloem in plants contain callose, a b-1,3-glucan. However, the genes responsible for callose deposition in this subcellular location have not been identified. In this paper we examine callose deposition patterns in T-DNA insertion mutants (cs7) of the Callose Synthase 7 (CalS7) gene. We demonstrated here that the CalS7 gene is expressed specifically in the phloem of vascular tissues. Callose deposition in the phloem, especially in the sieve elements, was greatly reduced in cs7 mutants. Ultrastructural analysis of developing sieve elements revealed that callose failed to accumulate in the plasmodesmata of incipient sieve plates at the early perforation stage of phloem development, resulting in the formation of sieve plates with fewer pores. In wild-type Arabidopsis plants, callose is present as a constituent polysaccharide in the phloem of the stem, and its accumulation can also be induced by wounding. Callose accumulation in both conditions was eliminated in mature sieve plates of cs7 mutants. These results demonstrate that CalS7 is a phloem-specific callose synthase gene, and is responsible for callose deposition in developing sieve elements during phloem formation and in mature phloem induced by wounding. The mutant plants exhibited moderate reduction in seedling height and produced aberrant pollen grains and short siliques with aborted embryos, suggesting that CalS7 also plays a role in plant growth and reproduction.

Temperature is one of the most important environmental factors affecting the growth and survival of microorganisms and in light of current global patterns is of particular interest. Here, we highlight studies revealing how vitamin B 12 (cobalamin)-producing bacteria increase the fitness of the unicellular alga Chlamydomonas reinhardtii following an increase in environmental temperature. Heat stress represses C. reinhardtii cobalamin-independent methionine synthase (METE) gene expression coinciding with a reduction in METE-mediated methionine synthase activity, chlorosis and cell death during heat stress. However, in the presence of cobalamin-producing bacteria or exogenous cobalamin amendments C. reinhardtii cobalamin-dependent methionine synthase METHmediated methionine biosynthesis is functional at temperatures that result in C. reinhardtii death in the absence of cobalamin. Artificial microRNA silencing of C. reinhardtii METH expression leads to nearly complete loss of cobalamin-mediated enhancement of thermal tolerance. This suggests that methionine biosynthesis is an essential cellular mechanism for adaptation by C. reinhardtii to thermal stress. Increased fitness advantage of METH under environmentally stressful conditions could explain the selective pressure for retaining the METH gene in algae and the apparent independent loss of the METE gene in various algal species. Our results show that how an organism acclimates to a change in its abiotic environment depends critically on co-occurring species, the nature of that interaction, and how those species interactions evolve.

Pollination is essential for seed reproduction and for exchanges of genetic information between individual plants. In angiosperms, mature pollen grains released from dehisced anthers are transferred to the stigma where they become hydrated and begin to germinate. Pollen grains of wild-type Arabidopsis thaliana do not germinate inside the anther under normal growth conditions. We report two Arabidopsis lines that produced pollen grains able to in situ precociously germinate inside the anther. One of them was a callose synthase 9 (cs9) knockout mutant with a T-DNA insertion in the Callose Synthase 9 gene (CalS9). Male gametophytes carrying a cs9 mutant allele were defective and no homozygous progeny could be produced. Heterozygous mutant plants (cs9/+) produced approximately 50% defective pollen grains with an altered male germ unit (MGU) and aberrant callose deposition in bicellular pollen. Bicellular pollen grains germinated precociously inside the anther. Another line, a transgenic plant expressing callose synthase 5 (CalS5) under the CaMV 35S promoter, also contained abnormal callose deposition during microsporogenesis and displaced MGUs in pollen grains. We also observed that precocious pollen germination could be induced in wild-type plants by incubation with medium containing sucrose and calcium ion and by wounding in the anther. These results demonstrate that precocious pollen germination in Arabidopsis could be triggered by a genetic alteration and a physiological condition.

SummaryThe genetically tractable microalga Chlamydomonas reinhardtii has many advantages as a model for renewable bioproducts and/or biofuels production. However, one limitation of C. reinhardtii is its relatively low-lipid content compared with some other algal species. To overcome this limitation, we combined ethane methyl sulfonate mutagenesis with fluorescence-activated cell sorting (FACS) of cells stained with the lipophilic stain Nile Red to isolate lipid hyperaccumulating mutants of C. reinhardtii. By manipulating the FACS gates, we sorted mutagenized cells with extremely high Nile Red fluorescence signals that were rarely detected in nonmutagenized populations. This strategy successfully isolated several putative lipid hyperaccumulating mutants exhibiting 23% to 58% (dry weight basis) higher fatty acid contents than their progenitor strains. Significantly, for most mutants, nitrogen starvation was not required to attain high-lipid content nor was there a requirement for a deficiency in starch accumulation. Microscopy of Nile Red stained cells revealed that some mutants exhibit an increase in the number of lipid bodies, which correlated with TLC analysis of triacyglycerol content. Increased lipid content could also arise through increased biomass production. Collectively, our findings highlight the ability to enhance intracellular lipid accumulation in algae using random mutagenesis in conjunction with a robust FACS and lipid yield verification regime. Our lipid hyperaccumulating mutants could serve as a genetic resource for stacking additional desirable traits to further increase lipid production and for identifying genes contributing to lipid hyperaccumulation, without lengthy lipid-induction periods.