The auxin response factor (ARF) transcription factors are a key component in auxin signaling and play diverse functions in plant growth, development, and stress response. ARFs are regulated at the transcript level and posttranslationally by protein modifications. However, relatively little is known regarding the control of ARF protein levels. We expressed five different ARFs with an HA (hemagglutinin) tag and observed that their protein levels under the same promoter varied considerably. Interestingly, their protein levels were affected by several hormonal and environmental conditions, but not by the auxin treatment. ABA (abscisic acid) as well as 4 °C and salt treatments decreased the levels of HA-ARF5, HA-ARF6, and HA-ARF10, but not that of HA-ARF19, while 37 °C treatment increased the levels of the four HA-ARFs, suggesting that the ARF protein levels are regulated by multiple factors. Furthermore, MG132 inhibited the reduction of HA-ARF6 level by ABA and 4 °C treatments, suggesting that these treatments decrease HA-ARF6 level through 26S proteasome-mediated protein degradation. It was also found that ABA treatment drastically increased HA-ARF6 ubiquitination, without strongly affecting the ubiquitination profile of the total proteins. Together, these results reveal another layer of control on ARFs, which could serve to integrate multiple hormonal and environmental signals into the ARF-regulated gene expression.

Soil salinity is a growing concern for global crop production and the sustainable development of humanity. Therefore, it is crucial to comprehend salt tolerance mechanisms and identify salt-tolerance genes to enhance crop tolerance to salt stress. Suaeda glauca, a halophyte species well adapted to the seawater environment, possesses a unique ability to absorb and retain high salt concentrations within its cells, particularly in its leaves, suggesting the presence of a distinct mechanism for salt tolerance. In this study, we performed de novo sequencing of the S. glauca genome. The genome has a size of 1.02 Gb (consisting of two sets of haplotypes) and contains 54 761 annotated genes, including alleles and repeats. Comparative genomic analysis revealed a strong synteny between the genomes of S. glauca and Beta vulgaris. Of the S. glauca genome, 70.56% comprises repeat sequences, with retroelements being the most abundant. Leveraging the allele-aware assembly of the S. glauca genome, we investigated genome-wide allele-specific expression in the analyzed samples. The results indicated that the diversity in promoter sequences might contribute to consistent allele-specific expression. Moreover, a systematic analysis of the ABCE gene families shed light on the formation of S. glauca’s flower morphology, suggesting that dysfunction of A-class genes is responsible for the absence of petals in S. glauca. Gene family expansion analysis demonstrated significant enrichment of Gene Ontology (GO) terms associated with DNA repair, chromosome stability, DNA demethylation, cation binding, and red/far-red light signaling pathways in the co-expanded gene families of S. glauca and S. aralocaspica, in comparison with glycophytic species within the chenopodium family. Time-course transcriptome analysis under salt treatments revealed detailed responses of S. glauca to salt tolerance, and the enrichment of the transition-upregulated genes in the leaves associated with DNA repair and chromosome stability, lipid biosynthetic process, and isoprenoid metabolic process. Additionally, genome-wide analysis of transcription factors indicated a significant expansion of FAR1 gene family. However, further investigation is needed to determine the exact role of the FAR1 gene family in salt tolerance in S. glauca.

Protein ubiquitination plays important roles in plants, including stress responses. The ubiquitin (Ub) E2 enzymes are required in the transfer of Ub to a substrate and are also important in determining the Ub-chain linkage specificity. However, for many of the 37 E2 genes in Arabidopsis thaliana, there is currently little or no understanding of their functions. In this study, we investigated three members of an E2 subfamily. The single, double, and triple mutants of UBC7, UBC13, and UBC14 did not show any phenotypic changes under normal conditions, but were more sensitive than the wild-type (WT) plants to multiple stress conditions, suggesting that the three genes are not critical for normal growth, but required in plant stress responses. The severity of the phenotypes increased from single to triple mutants, suggesting that the functions of the three genes are not completely redundant. The three E2s are closely related to the yeast Ubc7 and its homologs in animals and human, which are an important component of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. The stress sensitivity phenotypes of the mutants and shared evolutionary root with the Ubc7 homologs in yeast and metazoans suggest that UBC7, UBC13, and UBC14 may function in the plant ERAD pathway.

Cell wall structural modifications through pectin cross-linkages between calcium ions and/or boric acid may be key to mitigating dehydration stress and fungal pathogens. Water loss was profiled in a pure pectin system and in vivo. While calcium and boron reduced water loss in pure pectin standards, the impact on Allium species was insignificant (p > 0.05). Nevertheless, synchrotron X-ray microscopy showed the localization of exogenously applied calcium to the apoplast in the epidermal cells of Allium fistulosum. Exogenous calcium application increased viscosity and resistance to shear force in Allium fistulosum, suggesting the formation of calcium cross-linkages (“egg-box” structures). Moreover, Allium fistulosum (freezing tolerant) was also more tolerant to dehydration stress compared to Allium cepa (freezing sensitive). Furthermore, the addition of boric acid (H₃BO₃) to pure pectin reduced water loss and increased viscosity, which indicates the formation of RG-II dimers. The Arabidopsis boron transport mutant, bor1, expressed greater water loss and, based on the lesion area of leaf tissue, a greater susceptibility to Colletotrichum higginsianum and Botrytis cinerea. While pectin modifications in the cell wall are likely not the sole solution to dehydration and biotic stress resistance, they appear to play an important role against multiple stresses.

The ubiquitin-conjugating enzyme (E2) is required for protein ubiquitination. Arabidopsis has 37 E2s grouped into 14 subfamilies and the functions for many of them are unknown. We utilized genetic and biochemical methods to study the roles of Arabidopsis UBC4, UBC5, and UBC6 of the E2 subfamily IV. The Arabidopsis ubc4/5/6 triple mutant plants had higher levels of glucose, sucrose, and starch than the control plants, as well as a higher protein level of a key gluconeogenic enzyme, cytosolic fructose 1,6-bisphosphatase 1 (cyFBP). In an in vitro assay, the proteasome inhibitor MG132 inhibited the degradation of recombinant cyFBP whereas ATP promoted cyFBP degradation. In the quadruple mutant ubc4/5/6 cyfbp, the sugar levels returned to normal, suggesting that the increased sugar levels in the ubc4/5/6 mutant were due to an increased cyFBPase level. In addition, the ubc4/5/6 mutant plants showed early leaf senescence at late stages of plant development as well as accelerated leaf senescence using detached leaves. Further, the leaf senescence phenotype remained in the quadruple ubc4/5/6 cyfbp mutant. Our results suggest that UBC4/5/6 have two lines of important functions, in sugar metabolism through regulating the cyFBP protein level and in leaf senescence likely through a cyFBP-independent mechanism.

Protein ubiquitination is important for the regulation of meiosis in eukaryotes, including plants. However, little is known about the involvement of E2 ubiquitin-conjugating enzymes in plant meiosis. Arabidopsis UBC22 is a unique E2 enzyme, able to catalyze the formation of ubiquitin dimers through lysine 11 (K11). Previous work has shown that ubc22 mutants are defective in megasporogenesis, with most ovules having no or abnormally functioning megaspores; furthermore, some mutant plants show distinct phenotypes in vegetative growth. In this study, we showed that chromosome segregation and callose deposition were abnormal in mutant female meiosis while male meiosis was not affected. The meiotic recombinase DMC1, required for homologous chromosome recombination, showed a dispersed distribution in mutant female meiocytes compared to the presence of strong foci in WT female meiocytes. Based on an analysis of F1 plants produced from crosses using a mutant as the female parent, about 24% of female mutant gametes had an abnormal content of DNA, resulting in frequent aneuploids among the mutant plants. These results show that UBC22 is critical for normal chromosome segregation in female meiosis but not for male meiosis, and they provide important leads for studying the role of UBC22 and K11-linked ubiquitination.