Integrated transcriptomics and metabolomics revealed the mechanism of catechin biosynthesis in response to lead stress in tung tree (Vernicia fordii).

Lead (Pb) affects gene transcription, metabolite biosynthesis and growth in plants. The tung tree (Vernicia fordii) is highly adaptive to adversity, whereas the mechanisms underlying its response to Pb remain uncertain. In this work, transcriptomic and metabolomic analyses were employed to study tung trees under Pb stress. The results showed that the biomass of tung seedlings decreased with increasing Pb doses, and excessive Pb doses resulted in leaf wilting, root rot, and disruption of Pb homeostasis. Under non-excessive Pb stress, a significant change in the expression patterns of flavonoid biosynthesis genes was observed in the roots of tung seedlings, leading to changes in the accumulation of flavonoids in the roots, especially the upregulation of catechins, which can chelate Pb and reduce its toxicity in plants. In addition, Pb-stressed roots showed a large accumulation of VfWRKY55, VfWRKY75, and VfLRR1 transcripts, which were shown to be involved in the flavonoid biosynthesis pathway by gene module analysis. Overexpression of VfWRKY55, VfWRKY75, and VfLRR1 significantly increased catechin concentrations in tung roots, respectively. These data indicate that Pb stress-induced changes in the expression patterns of those genes regulate the accumulation of catechins. Our findings will help to clarify the molecular mechanism of Pb response in plants.

Type III Secretion System Repressor RhpR Induces GrlP, a Glycine-Rich Outer Membrane Lipoprotein with Functions in Regulating the Periplasmic Space and Pleiotropic Responses.

The two-component system RhpRS was initially identified as a regulator of genes encoding the type III secretion system (T3SS) in Pseudomonas syringae. Phosphorylated RhpR (P-RhpR) negatively regulates the T3SS genes by repressing the hrpR promoter, but directly activates the expression of a small gene named here as grlp. Here, we show that grlp is expressed higher in rich medium than in minimal medium in P. s. pv. tomato DC3000 and encodes a glycine rich lipoprotein (GrlP) located in the outer membrane (OM). The grlp gene has a pleiotropic effect on bacterial behaviors such as reductions in pathogenicity, swimming motility, biofilm formation, tolerance to various stresses and antibiotics, and long-term survival when overexpressed, but induces these responses when it is deleted in P. s. pv. tomato DC3000. Overexpression of grlp increases the size of periplasm while deletion of grlp decreases the periplasmic space. Further, GrlP interacts with OprI, the ortholog of E. coli OM lipoprotein Lpp, a key player in determining the size of periplasm and mechanic stiffness of the OM by tethering the OM to peptidoglycan (PG) in periplasm. As periplasmic space and OM mechanics play central roles in regulating bacterial physiology, we speculate that GrlP probably imposes its functions on bacterial physiology by regulating the periplasmic space and OM mechanics. These findings suggest that the T3SS gene regulation is closely coordinated with bacterial cell envelope properties by RhpRS in P. syringe. IMPORTANCE The OM of Gram-negative bacteria is the most front line in contact with extracellular milieu. OM is not only a protective layer, but also a structure that determines the envelope stiffness. Recent evidence indicated that components determining the periplasmic space and cross-links of lipopolysaccharide on the OM play key roles in regulating the mechanical properties of the OM. However, whether the OM composition and mechanical properties are coordinated with the expression of the T3SS genes is unknown. Here, we found that the two-component system (TCS) regulator P-RhpR, a direct repressor of the T3SS regulator hrpRS operon, directly activates the expression of the OM lipoprotein gene grlp bearing a function in regulating the periplasmic space. This finding suggests a coordination between the OM properties and the T3SS gene regulation and reveals a new target for control of the T3SS gene expression and bacterial pathogenicity.

The nuclear-encoded plastid ribosomal protein L18s are essential for plant development.

Plastid ribosomal proteins (PRPs) are necessary components for plastid ribosome biogenesis, playing essential roles in plastid development. The ribosomal protein L18 involved in the assemble of 5S rRNA and 23S rRNA, is vital for E. coli viability, but the functions of its homologs in plant plastid remain elusive. Here, we characterized the functions of the plant plastid ribosomal protein L18s (PRPL18s) in Arabidopsis and rice. AtPRPL18 was ubiquitously expressed in most of the plant tissues, but with higher expression levels in seedling shoots, leaves, and flowers. AtPRPL18 was localized in chloroplast. Genetic and cytological analyses revealed that a loss of function of AtPRPL18 resulted in embryo development arrest at globular stage. However, overexpression of AtPRPL18 did not show any visible phenotypical changes in Arabidopsis. The rice OsPRPL18 was localized in chloroplast. In contrast to AtPRPL18, knockout of OsPRPL18 did not affect embryo development, but led to an albino lethal phenotype at the seedling stage. Cytological analyses showed that chloroplast development was impaired in the osprpl18-1 mutant. Moreover, a loss-function of OsPRPL18 led to defects in plastid ribosome biogenesis and a serious reduction in the efficiency of plastid intron splicing. In all, these results suggested that PRPL18s play critical roles in plastid ribosome biogenesis, plastid intron splicing, and chloroplast development, and are essential for plant survival.

The Ubiquitin E3 Ligase RHA2b Promotes Degradation of MYB30 in Abscisic Acid Signaling.

The phytohormone abscisic acid (ABA) is critical for plants encountering abiotic stress. We reported previously that the Arabidopsis (Arabidopsis thaliana) transcription factor MYB30 participates in ABA responses via SUMO ligase SAP-MIZ Domain-Containing SIZ1-mediated sumoylation. Here, we show that the RING-type ubiquitin E3 ligase RHA2b, which positively regulates ABA signaling, interacted with and ubiquitinated MYB30 to modulate MYB30 stability through the 26S proteasome pathway. The degradation rate of MYB30 was repressed significantly in the rha2b-1 mutant. Phenotypic analyses showed that MYB30 acts genetically downstream of RHA2b in ABA signaling. Substitutions of lysine-283 (K283) and K165 blocked ubiquitination, suggesting that these residues are sites of ubiquitination. K283 residue substitution significantly inhibited the degradation of MYB30 induced by ABA. The K165 site functioned additively with K283 in ABA-induced MYB30 degradation and ABA responses. At the same time, sumoylation protected MYB30 from degradation under cycloheximide and ABA treatment. Compared with MYB30, overexpression of MYB30-SUMO1 partially recovered the ABA sensitivity of siz1-2 But MYB30-SUMO1 exhibited similar localization with MYB30 in nuclei. Overall, our results suggest that RHA2b targets MYB30 for degradation to modulate ABA signaling. Considering that the K283 residue also is the major site for sumoylation, we propose that sumoylation and ubiquitination act antagonistically in the ABA response to regulate the stability of MYB30 by occupying the same residue.

MYB30 transcription factor regulates oxidative and heat stress responses through ANNEXIN-mediated cytosolic calcium signaling in Arabidopsis.

Cytosolic calcium signaling is critical for regulating downstream responses in plants encountering unfavorable environmental conditions. In a genetic screen for Arabidopsis thaliana mutants defective in stress-induced cytosolic free Ca2+ ([Ca2+ ]cyt ) elevations, we identified the R2R3-MYB transcription factor MYB30 as a regulator of [Ca2+ ]cyt in response to H2 O2 and heat stresses. Plants lacking MYB30 protein exhibited greater elevation of [Ca2+ ]cyt in response to oxidative and heat stimuli. Real-time reverse transcription-polymerase chain reaction (RT-PCR) results indicated that the expression of a number of ANNEXIN (ANN) genes, which encode Ca2+ -regulated membrane-binding proteins modulating cytosolic calcium signatures, were upregulated in myb30 mutants. Further analysis showed that MYB30 bound to the promoters of ANN1 and ANN4 and repressed their expression. myb30 mutants were sensitive to methyl viologen (MV) and heat stresses. The H2 O2 - and heat-induced abnormal [Ca2+ ]cyt in myb30 was dependent on the function of ANN proteins. Moreover, the MV and heat sensitivity of myb30 was suppressed in mutants lacking ANN function or by application of LaCl3 , a calcium channel blocker. These results indicate that MYB30 regulates oxidative and heat stress responses through calcium signaling, which is at least partially mediated by ANN1 and ANN4.

The Glycosyltransferase QUA1 Regulates Chloroplast-Associated Calcium Signaling During Salt and Drought Stress in Arabidopsis.

Cytoplasmic Ca2+ ([Ca2+]cyt) elevation induced by various signals is responsible for appropriate downstream responses. Through a genetic screen of Arabidopsis thaliana mutants defective in stress-induced [Ca2+]cyt elevation, the glycosyltransferase QUASIMODO1 (QUA1) was identified as a regulator of [Ca2+]cyt in response to salt stress. Compared with the wild type, the qua1-4 mutant exhibited a dramatically greater increase in [Ca2+]cyt under NaCl treatment. Functional analysis showed that QUA1 is a novel chloroplast protein that regulates cytoplasmic Ca2+ signaling. QUA1 was detected in chloroplast thylakoids, and the qua1-4 mutant exhibited irregularly stacked grana. The observed greater increase in [Ca2+]cyt was inhibited upon recovery of chloroplast function in the qua1-4 mutant. Further analysis showed that CAS, a thylakoid-localized calcium sensor, also displayed irregularly stacked grana, and the chloroplasts of the qua1-4 cas-1 double mutant were similar to those of cas-1 plants. In QUA1-overexpressing plants, the protein level of CAS was decreased, and CAS was readily degraded under osmotic stress. When CAS was silenced in the qua1-4 mutant, the large [Ca2+]cyt increase was blocked, and the higher expression of PLC3 and PLC4 was suppressed. Under osmotic stress, the qua1-4 mutant showed an even greater elevation in [Ca2+]cyt and was hypersensitive to drought stress. However, this sensitivity was inhibited when the increase in [Ca2+]cyt was repressed in the qua1-4 mutant. Collectively, our data indicate that QUA1 may function in chloroplast-dependent calcium signaling under salt and drought stresses. Additionally, CAS may function downstream of QUA1 to mediate these processes.