Role of Specific Phosphorylation Sites of <Emphasis Type="Italic">Arabidopsis</Emphasis> Brassinosteroid-Insensitive 1 Receptor Kinase in Plant Growth and Development

Brassinosteroid-insensitive 1 (BRI1), the receptor of brassinosteroids (BRs), is a dual-function serine/threonine/tyrosine protein kinase which initiates BR signaling and regulates plant growth via its protein kinase activity. Previous research has identified phosphorylation sites of Arabidopsis BRI1 in vivo and in vitro, but the significance of which to BR signaling and plant development has not been discussed comprehensively. To investigate this, we systematically characterized Arabidopsis BRI1 site-directed mutants in the weak bri1-5 background. For vegetative organ development regulation, we demonstrated that Thr-1039, Ser-1042, and Ser-1044 were critical for vegetative development because mutants with eliminated phosphorylation at these residues exhibited aberrant leaf growth, whereas Ser-1172 and Ser-1187 slightly inhibited leaf growth. For reproductive organ development regulation, first, the notion that Thr-1039, Ser-1042, and Ser-1044 were essential for normal plant height is supported by the evidence that mutations preventing phosphorylation at Thr-1039, Ser-1042, and Ser-1044 decreased plant height. Second, comparison of seed yield-related traits showed that unphosphorylated Ser-1168-Ala, Ser-1172-Ala, and Ser-1179-Ala+Thr-1180-Ala mutants reduced seed yield dramatically, whereas eliminating phosphorylation at Ser-1042 caused increased seed production. In addition, we found that Ser-1042 and Ser-1044 were essential for BR signaling. The unphosphorylated Ser-1042-Ala and Ser-1044-Ala mutants displayed hyposensitive phenotypes accompanied with decreased accumulation of dephosphorylated BRI1-EMS suppressor 1 (BES1) protein and increased Constitutive Photomorphogenesis Dwarf expression levels as well as limited inhibition of hypocotyl and root elongation under exogenous brassinolide. Taken together, our data suggest that BRI1 phosphorylation at specific sites differentially affects growth and development which may provide novel approaches to precisely regulate economic yield through modifying specific BRI1 phosphorylation sites in crop species.     

Brassinosteroid signaling modulates submergence-induced hyponastic growth in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The role of brassinosteroids (BRs) in hyponastic growth induced by submergence was investigated in Arabidopsis thaliana. Under flooding conditions, exogenously applied BRs increased hyponastic growth of rosette leaves. This hyponastic growth was reduced in a BR insensitive mutant (bri1-5), while it was increased in a BR dominant mutant (bes1-D). Further, expression of hypoxia marker genes, HRE1 and HRE2, was elevated in submerged bes1-D. These results indicate that BRs exert a positive action on hyponastic growth of submerged Arabidopsis leaves. Expression of ethylene biosynthetic genes, such as ACS6, ACS8 and ACO1, which are up-regulated by submergence, was also activated by application of BRs and in bes1-D. The enhanced hyponastic growth in submerged bes1-D was significantly reduced by application of cobalt ion, suggesting that BRs control hyponastic growth via ethylene, which seems to be synthesized by ACO6 and ACO8 followed by ACO1 in submerged leaves. A double mutant, bes1-Dxaco1-1, showed hyponastic growth activity similar to that seen in aco1-1, demonstrating that the BR signaling for regulation of hyponastic growth seems to be an upstream event in ethylene-induced hyponastic growth under submergence in Arabidopsis.     

Genome-wide identification and expression profiling analysis of brassinolide signal transduction genes regulating apple tree architecture

Brassinolide (BR) is crucial for regulating plant architecture. Apple dwarfing rootstocks are used to control apple tree size. However, information regarding the effects of BR on apple trees is limited. In addition, the molecular mechanism underlying the dwarfing of apple rootstocks is poorly understood. To elucidate the role of BR signal transduction genes in controlling apple tree architecture, five BR receptor kinase 1 (BRI1), nine BR-signaling kinase 1 (BSK1), two BRI1 KINASE INHIBITOR 1 (BKI1), and seven BR-insensitive 2 (BIN2) genes were analyzed. Bioinformatic analyses revealed that gene duplication events likely contributed to the expansion and evolution of the identified genes. Nine homologs between apple and Arabidopsis thaliana were also identified, and their expression patterns in different tissues were characterized. Exogenous BR treatments increased the primary shoot length and altered the expression of BR signal transduction genes (MdBRI1-5, MdBSK3-8, MdBKI1–2, MdBIN1–4, and MdBIN6/7). The scion of Fuji/Malling 9 (M.9) trees exhibited inhibited growth compared with that of Fuji/Fuji trees. The Fuji/M.9 trees had lower levels of the positive regulators of BR signaling (MdBRI1-5,MdBSK1, MdBSK4/7, and MdBSK6) and higher levels of the negative regulators (MdBIN5-7) compared with the Fuji/Fuji trees. Thus, the above-mentioned genes may help to regulate apple tree size in response to BR. In addition, MdBRI1–5, MdBSK1, MdBSK4/7, MdBSK6, and MdBIN5–7 have important roles in different grafting combinations. Our results may provide the basis for future analyses of BR signal transduction genes regarding their potential involvement in the regulation of plant architecture.     

Enhancement of hypocotyl elongation by LOV KELCH PROTEIN2 production is mediated by auxin and phytochrome-interacting factors in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Key message

Auxin and two phytochrome-interacting factors, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, play crucial roles in the enhancement of hypocotyl elongation in transgenic Arabidopsis thaliana plants that overproduce LOV KELCH PROTEIN2 (LKP2).

Abstract

LOV KELCH PROTEIN2 (LKP2) is a positive regulator of hypocotyl elongation under white light in Arabidopsis thaliana. In this study, using microarray analysis, we compared the gene expression profiles of hypocotyls of wild-type Arabidopsis (Columbia accession), a transgenic line that produces green fluorescent protein (GFP), and two lines that produce GFP-tagged LKP2 (GFP-LKP2). We found that, in GFP-LKP2 hypocotyls, 775 genes were up-regulated, including 36 auxin-responsive genes, such as 27 SMALL AUXIN UP RNA (SAUR) and 6 AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, and 21 genes involved in responses to red or far-red light, including PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5; and 725 genes were down-regulated, including 15 flavonoid biosynthesis genes. Hypocotyls of GFP-LKP2 seedlings, but not cotyledons or roots, contained a higher level of indole-3-acetic acid (IAA) than those of control seedlings. Auxin inhibitors reduced the enhancement of hypocotyl elongation in GFP-LKP2 seedlings by inhibiting the increase in cortical cell number and elongation of the epidermal and cortical cells. The enhancement of hypocotyl elongation was completely suppressed in progeny of the crosses between GFP-LKP2 lines and dominant gain-of-function auxin-resistant mutants (axr2-1 and axr3-1) or loss-of-function mutants pif4, pif5, and pif4 pif5. Our results suggest that the enhancement of hypocotyl elongation in GFP-LKP2 seedlings is due to the elevated level of IAA and to the up-regulated expression of PIF4 and PIF5 in hypocotyls.

     

Interaction of gibberellic acid and 24-epibrassinolide in the regulation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seedling scotomorphogenesis

The effects of GA₃, 24-epibrassinolide (EBL), and their combination on morphogenesis of Arabidopsis thaliana (L.) Heynh seven-day-old seedlings were studied. Four plant lines were analyzed: wild type Ler and ga4-1 mutant, belonging to the Landsberg erecta ecotype and wild type Col and det2 mutant, both of the Columbia ecotype. In ga4-1 and det2, GA_4/1-and brassinosteroid-deficient mutants, the highest hypocotyl growth response to the lack of hormones was noted. The cotyledon shape and size were dependent on EBL, and the root length was both GA₃-and EBL-regulated, indicating organ specificities in the responses to these hormones. Simultaneous treatment of dark-grown plants with GA₃ and EBL exerted an additive stimulatory effect on the root growth of det2, reduced the inhibitory effect of EBL on hypocotyl elongation of ga4-1, and enhanced the effect of EBL on hypocotyl and cotyledon elongation of det2.     

A mutant in the <Emphasis Type="Italic">CsDET2</Emphasis> gene leads to a systemic brassinosteriod deficiency and <Emphasis Type="Italic">super compact </Emphasis>phenotype in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Key message

A novel dwarf cucumber mutant, scp-2, displays a typical BR biosynthesis-deficient phenotype, which is due to a mutation in CsDET2 for a steroid 5-alpha-reductase.

Abstract

Brassinosteroids (BRs) are a group of plant hormones that play important roles in the development of plant architecture, and extreme dwarfism is a typical outcome of BR-deficiency. Most cucumber (Cucumis sativus L.) varieties have an indeterminate growth habit, and dwarfism may have its value in manipulation of plant architecture and improve production in certain production systems. In this study, we identified a spontaneous dwarf mutant, super compact-2 (scp-2), that also has dark green, wrinkle leaves. Genetic analyses indicated that scp-2 was different from two previously reported dwarf mutants: compact (cp) and super compact-1 (scp-1). Map-based cloning revealed that the mutant phenotype was due to two single nucleotide polymorphism and a single-base insertion in the CsDET2 gene that resulted in a missense mutation in a conserved amino acid and thus a truncated protein lacking the conserved catalytic domains in the predicted steroid 5α-reductase protein. Measurement of endogenous hormone levels indicated a reduced level of brassinolide (BL, a bioactive BR) in scp-2, and the mutant phenotype could be partially rescued by the application of epibrassinolide (EBR). In addition, scp-2 mutant seedlings exhibited dark-grown de-etiolation, and defects in cell elongation and vascular development. These data support that scp-2 is a BR biosynthesis-deficient mutant, and that the CsDET2 gene plays a key role in BR biosynthesis in cucumber. We also described the systemic BR responses and discussed the specific BR-related phenotypes in cucumber plants.

     

Conjugative plasmid transfer from <Emphasis Type="Italic">Escherichia coli</Emphasis> is a versatile approach for genetic transformation of thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Geobacillus</Emphasis> species

We previously demonstrated efficient transformation of the thermophile Geobacillus kaustophilus HTA426 using conjugative plasmid transfer from Escherichia coli BR408. To evaluate the versatility of this approach to thermophile transformation, this study examined genetic transformation of various thermophilic Bacillus and Geobacillus spp. using conjugative plasmid transfer from E. coli strains. E. coli BR408 successfully transferred the E. coli–Geobacillus shuttle plasmid pUCG18T to 16 of 18 thermophiles with transformation efficiencies between 4.1 × 10^?7 and 3.8 × 10^?2/recipient. Other E. coli strains that are different from E. coli BR408 in intracellular DNA methylation also generated transformants from 9 to 15 of the 18 thermophiles, including one that E. coli BR408 could not transform, although the transformation efficiencies of these strains were generally lower than those of E. coli BR408. The conjugation was performed by simple incubation of an E. coli donor and a thermophile recipient without optimization of experimental conditions. Moreover, thermophile transformants were distinguished from abundant E. coli donor only by high temperature incubation. These observations suggest that conjugative plasmid transfer, particularly using E. coli BR408, is a facile and versatile approach for plasmid introduction into thermophilic Bacillus and Geobacillus spp., and potentially a variety of other thermophiles.     

Interaction of jasmonic acid and blue light in the regulation of arabidopsis morphogenesis

The effects of blue light (BL) and jasmonic acid (JA) on morphogenesis of Arabidopsis thaliana (L.) Heynh seedlings of genotypes Col and Ler and their mutants, namely, axr1-3 and jar1-1 mutants resistant to IAA and JA, respectively, and a CRY1 photoreceptor-deficient mutant hy4 were studied. Both 1 μM JA and BL exposure retarded hypocotyl growth of Ler, Col, and jar1-1 seedlings, whereas JA had no effect on hypocotyl growth of axr1-3, but the suppression of hypocotyl growth of this mutant by BL was even more noticeable than that of Ler, Col, and jar1-1. JA and BL applied simultaneously inhibited hypocotyl growth of axr1-3 and especially of Ler, Col, and jar1-1 more than either of factors applied separately. The hy4 mutant did not respond to BL, whereas JA stimulated its hypocotyl growth. JA did not change the cotyledon size of Col, axr1-3, and jar1-1 and reduced the cotyledon size of Ler and hy4. BL enhanced the cotyledon growth of all wild-type and mutant plants used in the study. The cotyledon sizes of all plants except Ler were also increased when JA and BL were applied together. Some of the growth responses correlated with the endogenous IAA and ABA contents. Thus, for example, the hypocotyl and cotyledon growth retardation of Ler seedlings in the presence of JA correlated with a reduced level of free IAA and a considerable increase in the free ABA level in plants grown both in darkness and in BL. Under other growth conditions, no correlation between the endogenous IAA and ABA levels and A. thaliana seedling growth was noted. The interaction between the signal transduction pathways triggered by BL and JA at the early stages of arabidopsis morphogenesis is discussed on the basis of Col, Ler, axr1-3, and jar1-1 hypocotyl growth responses.     

<Emphasis Type="Italic">CKB1</Emphasis> is involved in abscisic acid and gibberellic acid signaling to regulate stress responses in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Casein kinase II (CK2), an evolutionarily well-conserved Ser/Thr kinase, plays critical roles in all higher organisms including plants. CKB1 is a regulatory subunit beta of CK2. In this study, homozygous T-DNA mutants (ckb1-1 and ckb1-2) and over-expression plants (35S:CKB1-1, 35S:CKB1-2) of Arabidopsis thaliana were studied to understand the role of CKB1 in abiotic stress and gibberellic acid (GA) signaling. Histochemical staining showed that although CKB1 was expressed in all organs, it had a relatively higher expression in conducting tissues. The ckb1 mutants showed reduced sensitivity to abscisic acid (ABA) during seed germination and seedling growth. The increased stomatal aperture, leaf water loss and proline accumulation were observed in ckb1 mutants. In contrast, the ckb1 mutant had increased sensitivity to polyaluminum chloride during seed germination and hypocotyl elongation. We obtained opposite results in over-expression plants. The expression levels of a number of genes in the ABA and GA regulatory network had changed. This study demonstrates that CKB1 is an ABA signaling-related gene, which subsequently influences GA metabolism, and may play a positive role in ABA signaling.     

Molecular mapping of a rust resistance gene <Emphasis Type="Italic">R</Emphasis><Subscript><Emphasis Type="Italic">14</Emphasis></Subscript> in cultivated sunflower line PH 3

Sunflower, the fifth largest oilseed crop in the world, plays an important role in human diets. Recently, sunflower production in North America has suffered serious yield losses from newly evolved races of sunflower rust (Puccinia helianthi Schwein.). The rust resistance gene, designated R ₁₄ , in a germplasm line PH 3 originated from a wild Helianthus annuus L. population resistant to 11 rust races. PH 3 has seedling with an extraordinary purple hypocotyl color. The objectives of this study were to map both the R ₁₄ rust resistance gene and the purple hypocotyl gene-designated PHC in PH 3, and to identify molecular markers for marker-assisted breeding for sunflower rust resistance. A set of 517 mapped SSR/InDel and four SNP markers was used to detect polymorphisms between the parents. Fourteen markers covering a genetic distance of 17.0 cM on linkage group (LG) 11 were linked to R ₁₄ . R ₁₄ was mapped to the middle of the LG, with a dominant SNP marker NSA_000064 as the closest marker at a distance of 0.7 cM, and another codominant marker ORS542 linked at 3.5 cM proximally. One dominant marker ZVG53 was linked on the distal side at 6.9 cM. The PHC gene was also linked to R ₁₄ with a distance of 6.2 cM. Chi-squared analysis of the segregation ratios of R ₁₄ , PHC, and ten linked markers indicated a deviation from an expected 1:2:1 or 3:1 ratio. The closely linked molecular or morphological markers could facilitate sunflower rust-resistant breeding and accelerate the development of rust-resistant hybrids.     

Overexpression of <Emphasis Type="Italic">RcFUSCA3</Emphasis>, a B3 transcription factor from the PLB in <Emphasis Type="Italic">Rosa canina</Emphasis>, activates starch accumulation and induces male sterility in Arabidopsis

A homologue of the Arabidopsis gene FUSCA3 (FUS3), isolated from the protocorm-like body (PLB) of Rosa canina and designated RcFUS3, encodes 331 amino acid residues. It was shown that RcFUS3 is specifically expressed in the PLB of R. canina and that its subcellular localization is in the nucleus. The Arabidopsis fus3-3 mutant phenotype could be rescued by over-expression of RcFUS3, suggesting that RcFUS3 has a function similar to that of Arabidopsis FUS3. Over-expression of RcFUS3 in wild type Arabidopsis resulted in a decrease in endogenous GA and CTK levels, an increase in ABA and IAA contents, starch grain accumulation in the cotyledon and hypocotyl, failure of cotyledon extension and abnormal elongation of the hypocotyl, abnormal stomatal and pavement cells, an increase in branch numbers, prolongation of the growth cycle, and morphological changes in floral organs. Interestingly, over-expression of RcFUS3 in homozygous form resulted in premature degradation of the tapetum, indehiscent anthers and hypogenetic stamens, causing complete male sterility in Arabidopsis; this is the first observation that over-expression of a gene (RcFUS3) homologous to FUS3 can lead to male sterility and starch grain accumulation in Arabidopsis.