OsBBX19-OsBTB97/OsBBX11 module regulates spikelet development and yield production in rice.

Spikelet and floral-related organs are important agronomic traits for rice grain yield. BTB (broad-complex, tram track, and bric-abrac) proteins control various developmental functions in plants; however, the molecular mechanism of BTB proteins underlying grain development and yield production is still unknown. Here, we evaluated the molecular mechanism of a previously unrecognized functional gene, namely OsBTB97 that regulates the floral and spikelet-related organs which greatly affect the final grain yield. We found that the knockdown of the OsBTB97 gene had significant impacts on the development of spikelet-related organs and grain size, resulting in a decrease in yield, by altering the transcript levels of various spikelet- and grain-related genes. Furthermore, we found that the knockout mutants of two BBX genes, OsBBX11 and OsBBX19, which interact with the OsBTB97 protein at translation and transcriptional level, respectively, displayed lower OsBTB97 expression, suggesting the genetic relationship between the BTB protein and the BBX transcription factors in rice. Taken together, our study dissects the function of the novel OsBTB97 by interacting with two BBX proteins and an OsBBX19-OsBTB97/OsBBX11 module might function in the spikelet development and seed production in rice. The outcome of the present study provides promising knowledge about BTB proteins in the improvement of crop production in plants.

OsMBTB32, a MATH-BTB domain-containing protein that interacts with OsCUL1s to regulate salt tolerance in rice.

MATH-BTB proteins are involved in a variety of cellular processes that regulate cell homeostasis and developmental processes. Previous studies reported the involvement of BTB proteins in the development of various organs in plants; however, the function of BTB proteins in salt stress is less studied. Here, we found a novel MATH-BTB domain-containing OsMBTB32 protein that was highly expressed in leaf, root, and shoot. The up-regulation of the OsMBTB32 transcript in 2-week-old seedlings under salt stress suggests the significant role of the OsMBTB32 gene in salinity. The OsMBTB32 transgenic seedlings (OE and RNAi) exhibited significant differences in various phenotypes, including plumule, radical, primary root, and shoot length, compared to WT seedlings. We further found that OsCUL1 proteins, particularly OsCUL1-1 and OsCUL1-3, interact with OsMBTB32 and may suppress the function of OsMBTB32 during salt stress. Moreover, OsWRKY42, a homolog of ZmWRKY114 which negatively regulates salt stress in rice, directly binds to the W-box of OsCUL1-1 and OsCUL1-3 promoters to promote the interaction of OsCUL1-1 and OsCUL1-3 with OsMBTB32 protein in rice. The overexpression of OsMBTB32 and OsCUL1-3 further confirmed the function of OsMBTB32 and OsCUL1s in salt tolerance in Arabidopsis. Overall, the findings of the present study provide promising knowledge regarding the MATH-BTB domain-containing proteins and their role in enhancing the growth and development of rice under salt stress.MATH-BTB proteins are involved in a variety of cellular processes that regulate cell homeostasis and developmental processes. Previous studies reported the involvement of BTB proteins in the development of various organs in plants; however, the function of BTB proteins in salt stress is less studied. Here, we found a novel MATH-BTB domain-containing OsMBTB32 protein that was highly expressed in leaf, root, and shoot. The up-regulation of the OsMBTB32 transcript in 2-week-old seedlings under salt stress suggests the significant role of the OsMBTB32 gene in salinity. The OsMBTB32 transgenic seedlings (OE and RNAi) exhibited significant differences in various phenotypes, including plumule, radical, primary root, and shoot length, compared to WT seedlings. We further found that OsCUL1 proteins, particularly OsCUL1-1 and OsCUL1-3, interact with OsMBTB32 and may suppress the function of OsMBTB32 during salt stress. Moreover, OsWRKY42, a homolog of ZmWRKY114 which negatively regulates salt stress in rice, directly binds to the W-box of OsCUL1-1 and OsCUL1-3 promoters to promote the interaction of OsCUL1-1 and OsCUL1-3 with OsMBTB32 protein in rice. The overexpression of OsMBTB32 and OsCUL1-3 further confirmed the function of OsMBTB32 and OsCUL1s in salt tolerance in Arabidopsis. Overall, the findings of the present study provide promising knowledge regarding the MATH-BTB domain-containing proteins and their role in enhancing the growth and development of rice under salt stress.

Dissecting Plant Gene Functions Using CRISPR Toolsets for Crop Improvement.

The CRISPR-based gene editing technology has become more and more powerful in genome manipulation for agricultural breeding, with numerous improved toolsets springing up. In recent years, many CRISPR toolsets for gene editing, such as base editors (BEs), CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), and plant epigenetic editors (PEEs), have been developed to clarify gene function and full-level gene regulation. Here, we comprehensively summarize the application and capacity of the different CRISPR toolsets in the study of plant gene expression regulation, highlighting their potential application in gene regulatory networks' analysis. The general problems in CRISPR application and the optimal solutions in the existing schemes for high-throughput gene function analysis are also discussed. The CRISPR toolsets targeting gene manipulation discussed here provide new solutions for further genetic improvement and molecular breeding of crops.

The highly interactive BTB domain targeting other functional domains to diversify the function of BTB proteins in rice growth and development.

The BTB (broad-complex, tram track, and bric-abrac) proteins are involved in developmental processes, biotic, and abiotic stress responses in various plants, but the molecular basis of protein interactions is yet to be investiagted in rice. In this study, the identified BTB proteins were divided into BTB-TAZ, MATH-BTB, BTB-NPH, BTB-ANK, BTB-Skp, BTB-DUF, and BTB-TPR subfamilies based on the additional functional domains found together with the BTB domain at N- and C-terminal as well. This suggesting that the extension region at both terminal sites could play a vital role in the BTB gene family expansion in plants. The yeast two-hybrid system, firefly luciferase complementation imaging (LCI) assay and bimolecular fluorescence complementation (BiFC) assay further confirmed that BTB proteins interact with several other proteins to perform a certain developmental process in plants. The overexpression of BTB genes of each subfamily in Arabidopsis revealed that BTB genes including OsBTB4, OsBTB8, OsBTB64, OsBTB62, OsBTB138, and OsBTB147, containing certain additional functional domains, could play a potential role in the early flowering, branching, leaf, and silique development. Thus we concluded that the presence of other functional domains such as TAZ, SKP, DUF, ANK, NPH, BACK, PQQ, and MATH could be the factor driving the diverse functions of BTB proteins in plant biology.

Genome-wide DNA methylation analysis and biochemical responses provide insights into the initial domestication of halophyte Puccinellia tenuiflora.

KEY MESSAGE: Puccinellia tenuiflora was domesticated for two years by growing it under non-saline conditions, providing epigenetic and biochemical insights into the initial domestication of extreme halophytes. Some halophytes have economic value as crop species. The domestication of halophytes may offer hope in solving the problem of soil salinization. We domesticated a wild halophyte, Puccinellia tenuiflora, for two years by growing it under non-saline conditions in a greenhouse and used re-sequencing, genome-wide DNA methylation, biochemical, and transcriptome analyses to uncover the mechanisms underlying alterations in the halophyte's tolerance to saline following domestication. Our results showed that non-saline domestication altered the methylation status for a number of genes and transposable elements, resulting in a much higher frequency of hypomethylation than hypermethylation. These modifications to DNA methylation were observed in many critical salinity-tolerance genes, particularly their promoter regions or transcriptional start sites. Twenty-nine potassium channel genes were hypomethylated and three were hypermethylated, suggesting that the DNA methylation status of potassium channel genes was influenced by domestication. The accelerated uptake of potassium is a major salinity tolerance characteristic of P. tenuiflora. We propose that modifications to the DNA methylation of potassium channel genes may be associated with the development of salinity tolerance in this species. By assessing whether non-saline domestication could change the salinity tolerance of P. tenuiflora, we demonstrated that non-saline domesticated plants are less tolerant to saline, which may be attributable to altered sucrose metabolism. DNA methylation and transposable elements may, therefore, be integrated into an environment-sensitive molecular engine that promotes the rapid domestication of P. tenuiflora to enable its use as a crop plant.

Comparative Genomics and Transcriptomics of the Extreme Halophyte Puccinellia tenuiflora Provides Insights Into Salinity Tolerance Differentiation Between Halophytes and Glycophytes.

Halophytes and glycophytes exhibit clear differences in their tolerance to high levels of salinity. The genetic mechanisms underlying this differentiation, however, remain unclear. To unveil these mechanisms, we surveyed the evolution of salinity-tolerant gene families through comparative genomic analyses between the model halophyte Puccinellia tenuiflora and glycophytic Gramineae plants, and compared their transcriptional and physiological responses to salinity stress. Under salinity stress, the K+ concentration in the root was slightly enhanced in P. tenuiflora, but it was greatly reduced in the glycophytic Gramineae plants, which provided a physiological explanation for differences in salinity tolerance between P. tenuiflora and these glycophytes. Interestingly, several K+ uptake gene families from P. tenuiflora experienced family expansion and positive selection during evolutionary history. This gene family expansion and the elevated expression of K+ uptake genes accelerated K+ accumulation and decreased Na+ toxicity in P. tenuiflora roots under salinity stress. Positively selected P. tenuiflora K+ uptake genes may have evolved new functions that contributed to development of P. tenuiflora salinity tolerance. In addition, the expansion of the gene families involved in pentose phosphate pathway, sucrose biosynthesis, and flavonoid biosynthesis assisted the adaptation of P. tenuiflora to survival under high salinity conditions.

Adaptive strategy of allohexaploid wheat to long-term salinity stress.

BACKGROUND: Most studies of crop salinity tolerance are conducted under short-term stress condition within one growth stage. Understanding of the mechanisms of crop response to long-term salinity stress (LSS) is valuable for achieving the improvement of crop salinity tolerance. In the current study, we exposed allohexaploid wheat seeds to LSS conditions from germination stage to young seedling stage for 30 days. To elucidate the adaptive strategy of allohexaploid wheat to LSS, we analyzed chloroplast ultrastructure, leaf anatomy, transcriptomic profiling and concentrations of plant hormones and organic compatible solutes, comparing stressed and control plants. RESULTS: Transcriptomic profiling and biochemical analysis showed that energy partitioning between general metabolism maintenance and stress response may be crucial for survival of allohexaploid wheat under LSS. Under LSS, wheat appeared to shift energy from general maintenance to stress response through stimulating the abscisic acid (ABA) pathway and suppressing gibberellin and jasmonic acid pathways in the leaf. We further distinguished the expression status of the A, B, and D homeologs of any gene triad, and also surveyed the effects of LSS on homeolog expression bias for salinity-tolerant triads. We found that LSS had similar effects on expression of the three homeologs for most salinity-tolerant triads. However, in some of these triads, LSS induced different effects on the expression of the three homeologs. CONCLUSIONS: The shift of the energy from general maintenance to stress response may be important for wheat LSS tolerance. LSS influences homeolog expression bias of salinity-tolerant triads.

Proteomic profiling sheds light on alkali tolerance of common wheat (Triticum aestivum L.).

Alkali (high-pH) stress is an important factor limiting agricultural production and has complex effects on plant metabolism. Transcriptomics is widely used in the discovery of stress-response genes, but it provides only a rough estimation for gene expression. Proteomics may be more helpful than transcriptomics for the discovery and identification of stress-response genes. In this study, wheat plants were treated with sodic alkaline stress (50 mM, NaHCO3: Na2CO3 = 1:1; pH 9.7), and then proteomic analysis was carried out on control and stressed plants. We detected 3,104 proteins, including 69 alkaline stress-response proteins. Five superoxide dismutases, three malate dehydrogenases, three dehydrin proteins, and one V-ATPase protein were upregulated in sodic alkaline-stressed wheat roots. We propose that these salinity response proteins may be important for ion homeostasis and osmotic regulation of sodic alkaline-stressed wheat. Additionally, two malic enzymes and many enzymes involved in the tricarboxylic acid cycle (TCA) were downregulated in the roots. The upregulation of malate dehydrogenase and the downregulation of TCA enzymes and malic enzymes may enhance the accumulation of malate in sodic alkaline-stressed wheat roots. Previous studies have demonstrated that the accumulation of malate in roots is a crucial adaptive mechanism of wheat to sodic alkaline stress. Herein, our proteomics results provided molecular insights into this adaptive mechanism.