Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases.

Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4COP1/SPAs, has been reported for plant CRYs so far. Here we show that Cul3LRBs is the second E3 ligase of CRY2 in Arabidopsis. We demonstrate the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2, at the CCE domain of CRY2, to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4COP1/SPAs and Cul3LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature.

The SUPPRESSOR of MAX2 1 (SMAX1)-Like SMXL6, SMXL7 and SMXL8 Act as Negative Regulators in Response to Drought Stress in Arabidopsis.

Drought represents a major threat to crop growth and yields. Strigolactones (SLs) contribute to regulating shoot branching by targeting the SUPPRESSOR OF MORE AXILLARY GROWTH2 (MAX2)-LIKE6 (SMXL6), SMXL7 and SMXL8 for degradation in a MAX2-dependent manner in Arabidopsis. Although SLs are implicated in plant drought response, the functions of the SMXL6, 7 and 8 in the SL-regulated plant response to drought stress have remained unclear. Here, we performed transcriptomic, physiological and biochemical analyses of smxl6, 7, 8 and max2 plants to understand the basis for SMXL6/7/8-regulated drought response. We found that three D53 (DWARF53)-Like SMXL members, SMXL6, 7 and 8, are involved in drought response as the smxl6smxl7smxl8 triple mutants showed markedly enhanced drought tolerance compared to wild type (WT). The smxl6smxl7smxl8 plants exhibited decreased leaf stomatal index, cuticular permeability and water loss, and increased anthocyanin biosynthesis during dehydration. Moreover, smxl6smxl7smxl8 were hypersensitive to ABA-induced stomatal closure and ABA responsiveness during and after germination. In addition, RNA-sequencing analysis of the leaves of the D53-like smxl mutants, SL-response max2 mutant and WT plants under normal and dehydration conditions revealed an SMXL6/7/8-mediated network controlling plant adaptation to drought stress via many stress- and/or ABA-responsive and SL-related genes. These data further provide evidence for crosstalk between ABA- and SL-dependent signaling pathways in regulating plant responses to drought. Our results demonstrate that SMXL6, 7 and 8 are vital components of SL signaling and are negatively involved in drought responses, suggesting that genetic manipulation of SMXL6/7/8-dependent SL signaling may provide novel ways to improve drought resistance.

Efficient genetic transformation of Sorghum using a visual screening marker.

To transform grain sorghum (Sorghum bicolor (L.) Moench) with a visual reporter gene (gfp) and a target gene (tlp), three genotypes (two inbreds, Tx 430 and C401, and a commercial hybrid, Pioneer 8505) were used. We obtained a total of 1011 fertile transgenic plants from 61 independent callus lines, which were produced from 2463 zygotic immature embryos via Agrobacterium-mediated transformation. The reporter gene, gfp, encoding green fluorescent protein (GFP), was used as a visual screening marker, and the target gene, tlp, encoding thaumatin-like protein (TLP), was chosen for enhancing resistance to fungal diseases and drought. Both genes were under the control of the maize ubi 1 promoter in the binary vector pPZP201. A total of 320 plants showing GFP expression, derived from 45 calli, were selected and analyzed by Southern blot analysis. There was a 100% correlation between the GFP expression and the presence of the target gene, tlp, in these plants. Transgenic plants showing strong TLP expression were confirmed by Western blotting with antiserum specific for TLP. The transgene segregated in various ratios among progeny, which was confirmed by examining seedlings showing GFP fluorescence. The progeny also showed different copy numbers of transgenics. This report describes the successful use of GFP screening for efficient production of stably transformed sorghum plants without using antibiotics or herbicides as selection agents.

Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system.

A dual-marker plasmid containing the selectable marker gene, manA, and the reporter gene, sgfp, was used to transform immature sorghum embryos by employing an Agrobacterium-mediated system. Both genes were under the control of the ubi1 promoter in a binary vector pPZP201. The Escherichia coli phosphomannose isomerase (PMI) gene, pmi, was used as the selectable marker gene and mannose was used as the selective agent. The sgfp gene encoding green fluorescence protein (GFP) was the reporter gene and served as a visual screening marker. A total of 167 transgenic plants were obtained from nine different embryogenic callus lines grown on a selection medium containing 1%-2% mannose. Embryoids and shoots regenerated via embryogenesis, that showed strong GFP fluorescence, were selected from two sorghum genotypes: C401, an inbred line, and Pioneer 8505, a commercial hybrid. The GFP accumulation in transgenic plants was observed with a dissecting stereomicroscope. The integration and expression of the manA gene was confirmed by Southern blot and Western blot analyses, and the feasibility of manA selection was demonstrated by the chlorophenol red (CPR) assay. Our results indicated that transgenes segregated in the Mendelian fashion in the T1 generation. The conversion of mannose to a metabolizable fructose carbon source is beneficial to plants. In addition, except in soybean and a few legumes, no endogenous PMI activity has been detected in plant species, indicating that PMI is useful in the transformation of sorghum. In addition, PMI has no sequence homology to known allergens. Optimization of this selection system for sorghum transformation provides an efficient way to produce transgenic plants without using antibiotic or herbicidal agents as selectable markers, and our results showed that the transformation efficiency reached 2.88% for Pioneer 8505 and 3.30% for C401, both values higher than in previously published reports.

QTL analysis of lodging resistance and related traits in Italian ryegrass ( Lolium multiflorum Lam.).

Italian ryegrass ( Lolium multiflorum Lam.) is the most widely cultivated annual forage grass in Japan. Lodging damage reduces both harvested yield and forage quality. To identify the chromosomal regions controlling lodging resistance in Italian ryegrass, we analyzed seven quantitative characters--heading date, plant height, culm weight, culm diameter, culm strength, tiller number, and culm pushing resistance--and evaluated lodging scores in the field in a two-way pseudo-testcross F1 population. Significant correlations between most combinations of the traits examined were found. Seventeen QTLs for all traits except culm weight were detected on six of seven linkage groups by simple interval mapping using cross-pollination (CP) algorithm, and 33 independent QTLs were also detected by composite interval mapping from both male and female parental linkage maps. In addition, up to 18 QTLs for lodging scores evaluated at nine different times were detected on all linkage groups. The flanking markers of those QTLs will serve as a useful tool for marker-assisted selection of lodging resistance in Italian ryegrass.

Construction of a high-density linkage map of Italian ryegrass (Lolium multiflorum Lam) using restriction fragment length polymorphism, amplified fragment length polymorphism, and telomeric repeat associated sequence markers.

To construct a high-density molecular linkage map of Italian ryegrass (Lolium multiflorum Lam), we used a two-way pseudo-testcross F1 population consisting of 82 individuals to analyze three types of markers: restriction fragment length polymorphism markers, which we detected by using genomic probes from Italian ryegrass as well as heterologous anchor probes from other species belonging to the Poaceae family, amplified fragment length polymorphism markers, which we detected by using PstI/MseI primer combinations, and telomeric repeat associated sequence markers. Of the restriction fragment length polymorphism probes that we generated from a PstI genomic library, 74% (239 of 323) of randomly selected probes detected hybridization patterns consistent with single-copy or low-copy genetic locus status in the screening. The 385 (mostly restriction fragment length polymorphism) markers that we selected from the 1226 original markers were grouped into seven linkage groups. The maps cover 1244.4 cM, with an average of 3.7 cM between markers. This information will prove useful for gene targeting, quantitative trait loci mapping, and marker-assisted selection in Italian ryegrass.

Linkage of AFLP markers to lhd 1, a recessive heterochronic gene in Italian ryegrass.

A mutant, leafy head I (lhd 1), was discovered in Japan from the progeny of Italian ryegrass (Lolium multiflorum Lam.) 'Nioudachi' and local line 'Aichikei #3'. Compared with normal plants, the mutant plant is a dwarf with a larger number of intenodes per stem, shorter internodes, and smaller leaves. The plants also head later in the season. Aerial roots are usually produced from the stem nodes during rainy seasons. In characterizing lhd 1, it was found to have many branches with small leaves and many small panicles on the upper part of the plant. Panicle development was severely disturbed in lhd 1 mutants, and the number of leaves produced in the vegetative phase was nearly twice that produced in the wild-type counterpart. The lhd 1 mutant appears to be a heterochronic mutation that is able to extend the vegetative period during development. The frequency of mutants in segregating populations indicated that lhd 1 is a recessive allele. To determine the linkage relationship between the lhd 1 gene and AFLP markers,768 primer combinations were screened for polymorphisms using bulked segregant analyses in two populations with 316 and 30 plants, respectively. Five AFLP markers were linked to the lhd 1 locus. E3/M41-1 and E16/M14-2 cosegregated with lhd 1. E16/M14-1 and E30/M10-1 flanked the gene at a distance of 0.3 cM and E30/M14-2 was linked to lhd 1 at a distance of 0.6 cM.