Geupil Jang

Overexpression of the OsERF71 transcription factor Alters Rice Root Structure and Drought Resistance

OsERF71 alters root structure to enhance drought resistance. Plant responses to drought stress require the regulation of transcriptional networks via drought-responsive transcription factors, which mediate a range of morphological and physiological changes. AP2/ERF transcription factors are known to act as key regulators of drought resistance transcriptional networks; however, little is known about the associated molecular mechanisms that give rise to specific morphological and physiological adaptations. In this study, we functionally characterized the rice (Oryza sativa) drought-responsive AP2/ERF transcription factor OsERF71, which is expressed predominantly in the root meristem, pericycle, and endodermis. Overexpression of OsERF71, either throughout the entire plant or specifically in roots, resulted in a drought resistance phenotype at the vegetative growth stage, indicating that overexpression in roots was sufficient to confer drought resistance. The root-specific overexpression was more effective in conferring drought resistance at the reproductive stage, such that grain yield was increased by 23% to 42% over wild-type plants or whole-body overexpressing transgenic lines under drought conditions. OsERF71 overexpression in roots elevated the expression levels of genes related to cell wall loosening and lignin biosynthetic genes, which correlated with changes in root structure, the formation of enlarged aerenchyma, and high lignification levels. Furthermore, OsERF71 was found to directly bind to the promoter of OsCINNAMOYL-COENZYME A REDUCTASE1, a key gene in lignin biosynthesis. These results indicate that the OsERF71-mediated drought resistance pathway recruits factors involved in cell wall modification to enable root morphological adaptations, thereby providing a mechanism for enhancing drought resistance.

The NF-YA transcription factor OsNF-YA7 confers drought stress tolerance of rice in an abscisic acid independent manner.

The mechanisms of plant response and adaptation to drought stress require the regulation of transcriptional networks via the induction of drought-responsive transcription factors. Nuclear Factor Y (NF-Y) transcription factors have aroused interest in roles of plant drought stress responses. However, the molecular mechanism of the NF-Y-induced drought tolerance is not well understood. Here, we functionally analyzed two rice NF-YA genes, OsNF-YA7 and OsNF-YA4. Expression of OsNF-YA7 was induced by drought stress and its overexpression in transgenic rice plants improved their drought tolerance. In contrast, OsNF-YA4 expression was not increased by drought stress and its overexpression in transgenic rice plants did not affect their sensitivity to drought stress. OsNF-YA4 expression was highly induced by the stress-related hormone abscisic acid (ABA), while OsNF-YA7 was not, indicating that OsNF-YA7 mediates drought tolerance in an ABA-independent manner. Analysis of the OsNF-YA7 promoter revealed three ABA-independent DRE/CTR elements and RNA-seq analysis identified 48 genes downstream of OsNFYA7 action putatively involved in the OsNF-YA7-mediated drought tolerance pathway. Taken together, our results suggest an important role for OsNF-YA7 in rice drought stress tolerance.

The rice OsNAC6 transcription factor orchestrates multiple molecular mechanisms involving root structural adaptions and nicotianamine biosynthesis for drought tolerance

Summary Drought has a serious impact on agriculture worldwide. A plants ability to adapt to rhizosphere drought stress requires reprogramming of root growth and development. Although physiological studies have documented the root adaption for tolerance to the drought stress, underlying molecular mechanisms is still incomplete, which is essential for crop engineering. Here, we identified OsNAC6‐mediated root structural adaptations, including increased root number and root diameter, which enhanced drought tolerance. Multiyear drought field tests demonstrated that the grain yield of OsNAC6 root‐specific overexpressing transgenic rice lines was less affected by drought stress than were nontransgenic controls. Genome‐wide analyses of loss‐ and gain‐of‐function mutants revealed that OsNAC6 up‐regulates the expression of direct target genes involved in membrane modification, nicotianamine (NA) biosynthesis, glutathione relocation, 3′‐phophoadenosine 5′‐phosphosulphate accumulation and glycosylation, which represent multiple drought tolerance pathways. Moreover, overexpression of NICOTIANAMINE SYNTHASE genes, direct targets of OsNAC6, promoted the accumulation of the metal chelator NA and, consequently, drought tolerance. Collectively, OsNAC6 orchestrates novel molecular drought tolerance mechanisms and has potential for the biotechnological development of high‐yielding crops under water‐limiting conditions.

pTAC10, a Key Subunit of Plastid-Encoded RNA Polymerase, Promotes Chloroplast Development

pTAC10, a key component of the plastid-encoded RNA polymerase complex, interacts with other components through its C-terminal region downstream of the S1 RNA-binding domain. Regulation of photosynthetic gene expression by plastid-encoded RNA polymerase (PEP) is essential for chloroplast development. The activity of PEP largely relies on at least 12 PEP-associated proteins (PAPs) encoded in the nuclear genome of plant cells. A recent model proposed that these PAPs regulate the establishment of the PEP complex through broad PAP-PEP or PAP-PAP interactions. In this study, we identified the Arabidopsis (Arabidopsis thaliana) seedling-lethal mutant ptac10-1, which has defects in chloroplast development, and found that the mutant phenotype is caused by the suppression of PLASTID S1 RNA-BINDING DOMAIN PROTEIN (pTAC10/PAP3). Analysis of the heterozygous mutant and pTAC10-overexpressing transgenic plants indicated that the expression level of pTAC10 is tightly linked to chloroplast development. Characterization of the interaction of pTAC10 with PAPs revealed that pTAC10 interacts with other PAPs, such as FSD2, FSD3, TrxZ, pTAC7, and pTAC14, but it does not interact with PEP core enzymes, such as rpoA and rpoB. Analysis of pTAC10 interactions using truncated pTAC10 proteins showed that the pTAC10 carboxyl-terminal region downstream of the S1 domain is involved in the pTAC10-PAP interaction. Furthermore, overexpression of truncated pTAC10s lacking the C-terminal regions downstream of the S1 domain could not rescue the ptac10-1 mutant phenotype and induced an abnormal whitening phenotype in Columbia-0 plants. Our observations suggested that these pTAC10-PAP interactions are essential for the formation of the PEP complex and chloroplast development.

Cytokinin-dependent secondary growth determines root biomass in radish (Raphanus sativus L.)

Highlight Comparative studies using Arabidopsis and radish (Raphanus sativus) found that cytokinin-mediated regulatory programmes in the cambium are important for the radial growth of radish roots and its variations.

Antagonistic interaction between jasmonic acid and cytokinin in xylem development

Developmental flexibility under stress conditions largely relies on the interactions between hormones that mediate stress responses and developmental processes. In this study, we showed that the stress hormone jasmonic acid (JA) induces formation of extra xylem in the roots of wild-type Arabidopsis thaliana (Col-0). JA signaling mutants such as coronatine insensitive1-1 and jasmonate resistant1-1 did not form extra xylem in response to JA, but the JA biosynthesis mutant oxophytodienoate-reductase3 did form extra xylem. These observations suggested that the JA response promotes xylem development. To understand the mechanism, we examined the regulatory interaction between JA and cytokinin, a negative regulator of xylem development. JA treatment reduced cytokinin responses in the vasculature, and exogenous cytokinin nullified the effect of JA on formation of extra xylem. A time-course experiment showed that suppression of cytokinin responses by JA does not occur rapidly, but the JA-mediated xylem phenotype is tightly linked to the suppression of the cytokinin response. Further analysis of arabidopsis histidine phosphotransfer protein6-1 and myc2-3 mutants revealed that the JA-responsive transcription factor MYC2 regulates the expression of AHP6 in response to JA and expression of AHP6 is involved in the JA-mediated xylem phenotype.

Intercellular trafficking of transcription factors in the vascular tissue patterning

Throughout life cycles, plants grow in an indeterminate manner by adding new cells and organs with specialized functions. Newly emerging cells acquire their identities depending on their positions relative to the neighboring cells. Exchanging positional signals between cells is critical in this process. Recent studies showed that many transcription factors move between cells or between organs in forms of proteins and messenger RNA (mRNA). Some of these were found to be important positional signals for cell type patterning. Cell type patterning in the vascular system is no exception from this. In this review, we describe recent discoveries of mobile transcription factors that function as positional signals for vascular tissue patterning and propose how these transcription factors integrate with other forms of signals.

Drought stress promotes xylem differentiation by modulating the interaction between cytokinin and jasmonic acid

ABSTRACT Drought stress provokes jasmonic acid (JA) signaling, which mediates plant stress responses; moreover, growing numbers of studies suggest that JA is involved in the modulation of root development under drought stress. Recently, we showed that JA promotes differentiation of xylem from procambial cells in Arabidopsis roots. Further molecular and genetic approaches revealed that the effect of JA on xylem development is caused by suppression of cytokinin responses, suggesting that JA antagonistically interacts with cytokinin to modulate xylem development. Here, we showed that, similar to JA, drought stress promotes xylem development. This suggests that the antagonistic interaction between JA and cytokinin is involved in drought-mediated xylem development, a hypothesis supported by the observation that drought stress increases JA responses and decreases cytokinin responses. Based on these findings, we propose that drought stress promotes xylem development, and the antagonistic interaction between JA and cytokinin is deeply involved in this process.

Genetic chimerism of CRISPR/Cas9-mediated rice mutants

The CRISPR/Cas9 technology is useful for genome editing to generate targeted mutants. Based on this genome editing technology, it was attempted to generate the rice mutant which lacks JAZ9 activity to understand its function in stress response. The sequence of guide RNA for the recognition of target site was obtained from CRISPR-PLANT website (http://www.genome.arizona.edu/crispr) to minimize off-target effect and was recombined into the CRISPR/Cas9 binary vector pRGEB31. Embryonic calli regenerated from the mature seeds (O. sativa L. cv. Nakdong) were co-cultivated with transformed Agrobacterium tumefaciens LBA4404, and 26 individual transgenic plants were obtained through the hygromycin selection process. Nucleotide sequence analysis showed that most of T0 plants carried both edited and unedited wt sequence of JAZ9, suggesting genetic chimerism of T0 plants. Even though 2 individual lines carried homozygous mutation on JAZ9, they were also chimeric due to biallelic mutation. The relative ratio between edited and unedited wt sequence was variable among individual lines. Expression level of Cas9 is correlated with the frequency of genome editing frequency. In some plants, the enrichment ratio changed along with developmental stage. The nucleotide sequence analysis revealed that Cas9-mediated A/T addition occurred at -3 nucleotide position from protospacer adjacent motif (PAM), whereas G addition at -5 nucleotide position from the PAM. Further analysis of T1 transgenic plants showed that the genome editing patterns were similar between T0 plants and their T1 sibling plants. These suggested that earlier selection of T0 plants with homozygous mutation is an efficient way to obtain homozygous mutants in T1 generation.

CHLORIDE CHANNEL 1 promotes drought tolerance in rice, leading to increased grain yield

Plant chloride channels (CLCs) localize to the plasma and organellar membranes; these channels play pivotal roles in the modulation of ion homeostasis and cell turgor. Recent studies have shown that the expression of CLCs is involved in plant responses to environmental stress. Here, we examined the rice (Oryza sativa) tonoplast-localized channel OsCLC1. OsCLC1 is preferentially expressed in roots, and therefore, we generated transgenic rice with root-specific overexpression of OsCLC1 (RCc3::OsCLC1). We also identified a T-DNA mutant line that lacks expression of OsCLC1 (osclc1). We found that RCc3::OsCLC1 rice plants showed increased tiller number and grain yield, whereas the osclc1 plants exhibited decreased tiller number and grain yield, compared with wild type. These observations suggest that expression of OsCLC1 affects rice growth and productivity. Furthermore, RCc3::OsCLC1 plants showed enhanced drought tolerance, leading to increased grain yield compared to wild-type plants grown under the same conditions. By contrast, osclc1 mutants exhibited reduced drought tolerance and productivity compared to wild-type plants. When expression of OsCLC1 was analyzed in drought, jasmonic acid (JA) or abscisic acid (ABA)-treated rice, expression of OsCLC1 was preferentially upregulated in roots in response to drought and JA, and was preferentially upregulated in shoots in response to ABA. Together with the finding that expression of OsCLC1 is positively correlated both with expressions of OsDREB1A and OsbHLH148, key transcription factors in drought and JA responses, respectively, these results suggest that OsCLC1 regulates drought tolerance in rice and JA signaling is involved in this process.