Transcriptomic and Metabolomic Investigation on Leaf Necrosis Induced by ZmWus2 Transient Overexpression in Nicotiana benthamiana.

WUSCHEL (WUS) is a crucial transcription factor in regulating plant stem cell development, and its expression can also improve genetic transformation. However, the ectopic expression of WUS always causes pleiotropic effects during genetic transformation, making it important to understand the regulatory mechanisms underlying these phenomena. In our study, we found that the transient expression of the maize WUS ortholog ZmWus2 caused severe leaf necrosis in Nicotiana benthamiana. We performed transcriptomic and non-target metabolomic analyses on tobacco leaves during healthy to wilted states after ZmWus2 transient overexpression. Transcriptomic analysis revealed that ZmWus2 transformation caused active metabolism of inositol trisphosphate and glycerol-3-phosphate, while also upregulating plant hormone signaling and downregulating photosystem and protein folding pathways. Metabolomic analysis mainly identified changes in the synthesis of phenylpropanoid compounds and various lipid classes, including steroid synthesis. In addition, transcription factors such as ethylene-responsive factors (ERFs), the basic helix-loop-helix (bHLH) factors, and MYBs were found to be regulated by ZmWus2. By integrating these findings, we developed a WUS regulatory model that includes plant hormone accumulation, stress responses, lipid remodeling, and leaf necrosis. Our study sheds light on the mechanisms underlying WUS ectopic expression causing leaf necrosis and may inform the development of future genetic transformation strategies.

SaRCC1, a Regulator of Chromosome Condensation 1 (RCC1) Family Protein Gene from Spartina alterniflora, Negatively Regulates Salinity Stress Tolerance in Transgenic Arabidopsis.

A regulator of chromosome condensation 1 (RCC1) family protein has been functionally characterized to be involved in various cellular processes. In this study, one RCC1 gene named SaRCC1 was cloned from the full-length cDNA library of Spartinaalterniflora. The open reading frame (ORF) of SaRCC1 was 1440 bp, and it encoded 479 amino acids with a calculated molecular mass of 51.65 kDa. Multiple amino acid sequence alignments showed that SaRCC1 had high identity with other plant RCC1s, and the phylogenetic analysis indicated that SaRCC1 had a closer affinity to Zea mays RCC1 family protein (ZmRCC1). SaRCC1 gene was induced under salt stress conditions, and its encoded protein was located in peroxisome. In order to further investigate the function of SaRCC1, transgenic Arabidopsis plants ectopically both sense-overexpressing and antisense-overexpressing SaRCC1 were generated. SaRCC1-overexpressing lines exhibited an increased salt and ABA hypersensitivity and reduced resistance to salinity stress. On the other hand, the transcripts of some stress-responsive genes in the SaRCC1 transgenic plants were affected in response to salinity stress. Our results provide evidence for the involvement of SaRCC1, negatively regulating salt stress responses by affecting stress-related gene expression in Arabidopsis.

Pollen semi-sterility1 encodes a kinesin-1-like protein important for male meiosis, anther dehiscence, and fertility in rice.

In flowering plants, male meiosis produces four microspores, which develop into pollen grains and are released by anther dehiscence to pollinate female gametophytes. The molecular and cellular mechanisms regulating male meiosis in rice (Oryza sativa) remain poorly understood. Here, we describe a rice pollen semi-sterility1 (pss1) mutant, which displays reduced spikelet fertility (~40%) primarily caused by reduced pollen viability (~50% viable), and defective anther dehiscence. Map-based molecular cloning revealed that PSS1 encodes a kinesin-1-like protein. PSS1 is broadly expressed in various organs, with highest expression in panicles. Furthermore, PSS1 expression is significantly upregulated during anther development and peaks during male meiosis. The PSS1-green fluorescent protein fusion is predominantly localized in the cytoplasm of rice protoplasts. Substitution of a conserved Arg (Arg-289) to His in the PSS1 motor domain nearly abolishes its microtubule-stimulated ATPase activity. Consistent with this, lagging chromosomes and chromosomal bridges were found at anaphase I and anaphase II of male meiosis in the pss1 mutant. Together, our results suggest that PSS1 defines a novel member of the kinesin-1 family essential for male meiotic chromosomal dynamics, male gametogenesis, and anther dehiscence in rice.

[Genetic analysis and molecular mapping of a high-tillering mutant (ht1) in rice.].

A rice (Oryza sativa L.) mutant with a high-tillering capacity, designated as ht1, was found from a japonica rice variety Xindao18. This high-tillering mutant phenotype was stably expressed through successive five self-crossed generations, and the number of tillers in mutants was three times more than that in wild-type rice. Genetic analysis showed that the phenotype of ht1was controlled by a single dominant nuclear gene, temporarily designated as HT1. By means of molecular marker technique, the HT1 gene was mapped to an interval between two SSR markers RM25435 and RM25552 on chro-mosome 10. Through high-resolution linkage analysis, the HT1 gene was further restricted to a 0.1 cM region flanked by two SSR markers RM25523 and RM25532. The physical distance between these two markers is about 130kb.

Sugar accumulation is associated with leaf senescence induced by long-term high light in wheat.

During the grain filling stage, high light (HL) usually results in premature leaf senescence and significant yield loss in wheat. To explore the responses of sugar metabolism and the association of sugar accumulation and leaf senescence in HL, the activity and gene expression of sugar metabolism-related enzymes were analyzed when two wheat cultivars Triticum aestivum L. Xiaoyan 54 (XY54, HL tolerant) and Jing 411 (J411, HL sensitive) were transferred from low light (LL) to HL for 28 d. The results showed that the CO2 assimilation rate, quantity of Rubisco and chlorophyll binding proteins decreased substantially for both cultivars in HL. However, the content of fructose, sucrose, and starch increased dramatically. In addition, the activity of hexokinase, pyruvate kinase, sucrose phosphate synthase, sucrose synthase, and alkaline/neutral invertase increased significantly while the expression of most of the sugar metabolism-related genes were repressed by long-term HL. Correlation analysis revealed that sugar content and sucrose phosphate synthase activity were negatively while the expression of most sugar metabolism-related genes were positively correlated with chlorophyll content during HL treatment. Comparatively, the HL tolerant cultivar XY54 accumulated less sugars than the HL sensitive cultivar J411, suggesting that sugar metabolism may be the regulation target for wheat improvement to cope with HL stress.

Fine mapping of pss1, a pollen semi-sterile gene in rice (Oryza sativa L.).

During routine seed increase procedures in rice, semi-sterile plants are common; however, such semi-sterility mutants in rice varieties have been only rarely analyzed genetically. W207-2 is a semi-sterile selection from the japonica rice variety Nipponbare. In this report, we found the female gamete of W207-2 was normal, and its semi-sterility was unaffected by growth duration but was conditioned by a recessive nuclear gene whose action leads to pollen semi-sterility and anther indehiscence, and the gene was named as pss1 (pollen semi-sterile). Using an F(2) population derived from the two parents W207-2 and Dular and a pooled DNA strategy, pss1 was mapped to an interval on chromosome 8 defined by the two SSR loci RM6356 and RS41. The position of pss1 was confirmed in another F(2) population derived from the cross W207-2 x Nipponbare. Over 2,000 homozygous pss1 segregants from the large W207-2 x Dular F(2) population were used to fine map pss1 to a 0.04 cM segment flanked by a CAPs marker L2 and a dCAPs L3 marker. Sequences for both markers are present on a single PAC clone, and the physical distance between them is about 28 kb. Analysis of the PAC sequence predicts the presence of five open reading frames, they are as follows: putative ribonuclease PH, putative avr9 elicitor response protein, kinesin1-like protein, putative protein RNP-D precursor and putative 40S ribosomal protein S13. This result would be helpful in cloning the pss1 gene.